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Instituto Nacional de Pesquisas da Amazônia -INPA
Programa de Pós-graduação em Genética, Conservação e Biologia Evolutiva
REVELANDO PADRÕES EVOLUTIVOS DO GÊNERO AMAZOPHRYNELLA
(ANURA: BUFONIDAE): UMA ANÁLISE INTEGRATIVA PARA REVELAR
PROCESSOS DE DIVERSIFICAÇÃO NA AMAZÔNIA
ROMMEL ROBERTO ROJAS ZAMORA
Manaus, Amazonas
Dezembro, 2018
iii
ROMMEL ROBERTO ROJAS ZAMORA
REVELANDO PADRÕES EVOLUTIVOS DO GÊNERO AMAZOPHRYNELLA
(ANURA: BUFONIDAE): UMA ANÁLISE INTEGRATIVA PARA REVELAR
PROCESSOS DE DIVERSIFICAÇÃO NA AMAZÔNIA
TOMAS HRBEK
Marcelo Gordo
Tese apresentada ao Instituto
Nacional de Pesquisas da
Amazônia como parte dos
requisitos para obtenção do título
de Doutor em Genética,
Conservação e Biologia Evolutiva
Manaus, Amazonas
Dezembro, 2018
iv
v
Z25 Zamora, Rommel Roberto Rojas
Revelando padrões evolutivos do gênero Amazophrynella
(Anura: Bufonidae): Uma análise integrativa para revelar
processos de diversificação na Amazônia / Rommel Roberto
Rojas Zamora --- Manaus : [s.n.], 2018. v, 180 f. : il. color.
Tese (Doutorado) --- INPA, Manaus, 2019.
Orientador : Tomas Hrbek.
Coorientador: Marcelo Gordo.
Área de concentração : Genética, Conservação e Biologia
Evolutiva.
1.Amazophrynella. 2.Biogeografia. 3. Bufonidae. I. Título.
CDD
597.8
Sinopse:
Estudou-se a sistemática, taxonomia e diversificação do
gênero Amazophrynella (Anura). Reconstruímos uma
hipótese filogenética do gênero, atualizou-se seu status
taxonômico e inferiu processos sobre sua diversificação.
Palavras-chave: Herpetologia, diversidade, biogeografia,
identificação
vi
Dedicatória
Dedico este trabalho aos meus pais, Roberto e Elvira, por cuidar com empenho e
bom exemplo sua descendência
vii
Agradecimentos
Imensa e extremamente agradecido aos meus Pais: Roberto e Elvira e aos
meus irmãos Rosa e Carlo pelo apoio sem condições até encontrar o meu próprio
caminho na vida;
Agradeço aos meus orientadores Tomas Hrbek e Marcelo Gordo pelos
constantes ensinamentos;
Professora Izeni Pires Farias por me abrir as portas do Laboratório de
Evolução e genética animal-LEGAL;
Vinicius Carvalho, Alexandre Almeida que me ajudaram no início de este
processo desde que cheguei ao Brasil e Maria Tereza no ultimo estagio desta tese;
Meus amigos do LEGAL: Fabio, Valeria, Joiciane, Luciana, Juliana, Priscila,
Carol, Luceia, Mario, Israela, Roberta, Jessica, Elciomar, Sandra, Guta, Fabricio,
Rodrigo e Igor do LABEbo;
Aos meus amigos do projeto Sauim, Leandro Sauim, Leandro Capitão,
Tainara S., Aline M., Raiclicia, Erika Marina Del valle, Daisuke, Edson mucura;
Aos meus amigos das Chepas: Marco, Andy, Cilnio, Tony, Piero, Giu, Chuck
Morris, Cristian, Josep y Carlos oruga pela constante ajuda e risos;
Aos cantantes e grupos que me acompanharam com sua música durante este
doutorado, principalmente The Doors, Los Prisioneiros, Los Destellos, Juaneco, Pink
Floyd, Los Ramones, Ozzy e The ventures;
Meus tios: Ronald, Michel, Marcos, Nora, Silvia, Coco, Pablo, Percy, Elda,
Helencith, Moises; Jomber, Karina.
Aos meus queridos vovós in memoriam: Melitón e Micaela e Jorge e Otília por
seu exemplo;
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior- CAPES pela
bolsa de estudo, Universidade federal do Amazonas-UFAM e Instituto Nacional de
Pesquisas da Amazônia- INPA.
viii
Epígrafe
“Quero dizer agora o oposto do que eu disse antes,
Eu prefiro ser essa metamorfose ambulante
Do que ter aquela velha opinião formada sobre tudo...”
Raul Seixas
ix
Resumo geral
A exploração irracional de recursos naturais, destruição de habitats,
aquecimento global, doenças entre outras ameaças naturais e antropogênicas são
responsáveis pela redução da diversidade do mundo. Assim, delimitar espécies e
identificar processos evolutivos que originaram a diversidade biológica na terra,
especialmente em regiões hotspot de diversidade como a Amazônia, é um dos
maiores desafios científicos da atualidade. Em este sentido, é sumamente
importante documentar a diversidade biológica dentro de um espaço e tempo
histórico antes que desapareça. Este doutorado se centrou em revelar a diversidade
de espécies do gênero Amazophrynella (Anura: Bufonidae) usando diferentes
caracteres evolutivos e reconstruir seus padrões filogenéticos e história
biogeográfica. Integramos características morfológicas (quantitativas e qualitativas),
genéticas (sequências de DNA), comportamentais (cantos de acasalamentos) e
ecológicas (valores ambientais) para delimitar espécies do gênero Amazophrynella.
Usamos as relações filogenéticas de genes mitocondriais e genômicos (ddrad-seq)
entre Amazophrynella (Amazônia) e seu gênero irmão Dendrophryniscus (Mata
Atlântica) para reconstruir a divergência histórica entre estas duas áreas e discutir
subsequentes eventos de diversificação na Amazônia. Nossos resultados
evidenciaram uma grande subestimação da riqueza de espécies do gênero
Amazophrynella, sendo descritas 5 novas espécies, assim também, descrevemos os
parâmetros espectrais e temporais dos cantos de acasalamento de 4 espécies e
redescrevemos a espécie tipo do gênero: A. minuta. As reconstruções
biogeográficas permitiram conhecer o tempo histórico de divergência entre a
Amazônia e Mata Atlântica (Eoceno), e detectar um padrão basal Leste-Oeste
(Mioceno) seguido por uma diversificação Norte-Sul dentro da Amazônia. Este
trabalho atualizou o status taxonômico e sistemática do gênero Amazophrynella e
propõe um modelo complexo de diversificação de anfíbios no neotrópico, dominado
por episódios de fragmentação de populações ancestrais por mecanismos de
vicariância e influência de fatores ecológicos em sua diversificação.
Palavras chave: Amazônia, biogeografia, conservação, sistemática, taxonomia
x
Abstract
Revealing evolutionary patterns of genus Amazophrynella (Anura: Bufonidae): an
integrating analysis to discover diversification processes in the amazon basin
Irrational exploitation of natural resources, habitat destruction, global warming,
diseases and other natural and anthropogenic threats are responsible for reducing
the diversity of the world. Thus, delimiting species and identifying evolutionary
processes that originated the biological diversity in the earth, especially in high
diversity areas of as the Amazon, is one of the greatest scientific challenges of the
present times. It is extremely important to document biological diversity within a
historical space and time before it disappears. This PhD focused on revealing the
diversity of species of the genus Amazophrynella (Anura: Bufonidae) using different
evolutionary traits and reconstructing their phylogenetic patterns and biogeographic
history. We integrated morphological characteristics (quantitative and qualitative),
genetic (DNA sequences), behavioral (mating songs) and ecological (environmental
values) to delimit species of the genus Amazophrynella. We used the phylogenetic
relationships of mitochondrial and genomic genes (ddrad-seq) between
Amazophrynella (Amazon) and its sister genus Dendrophryniscus (Atlantic Forest) to
reconstruct the historical divergence between these two areas and discuss
subsequent diversification events in the Amazon. The results showed a great
underestimation of the richness of species of the genus Amazophrynella, being
described 5 new species, we also describe the spectral and temporal parameters of
the mating songs of 4 species and we redraw the type species of the genus: A.
minuta. The biogeographic reconstructions allowed to know the historical time of
divergence between the Amazon and Atlantic Forest (Eocene), and to detect a basal
East-West (Miocene) pattern followed by a North-South diversification within the
Amazon. We updated the taxonomic and systematic status of the genus
Amazophrynella and proposes a complex model of diversification of amphibians in
the neotropics, dominated by episodes of fragmentation of ancestral populations by
mechanisms of vicariance and influence of ecological factors in their diversification.
Keywords: Amazon, biogeography, conservation, systematics, taxonomy.
xi
Sumario
INTRODUÇÃO GERAL ..................................................................................... 1
Estado da arte do gênero Amazophrynella ..................................................... 6
História natural ............................................................................................. 6
História taxonômica ..................................................................................... 6
Sistemática de Amazophrynella ................................................................. 12
Problemática ................................................................................................. 13
Hipóteses ...................................................................................................... 14
OBJETIVOS ..................................................................................................... 14
Geral ............................................................................................................. 14
Específicos .................................................................................................... 14
REFERÊNCIAS BIBLIOGRÁFICAS……………………………………………….15
CAPITULO I. Uncovering the diversity inside the Amazophrynella minuta complex:
integrative taxonomy reveals a new species of Amazophrynella (Anura, Bufonidae)
from southern Peru……………………………………………………...…...……….23
CAPITULO II. A Pan-Amazonian species delimitation: high species diversity within
the genus Amazophrynella (Anura: Bufonidae)………………………..………….64
CAPITULO III. Description of the advertisement call of four species of
Amazophrynella (Anura: Bufonidae)……………………………………………….149
CAPITULO IV. Redescription of the Amazonian tiny tree toad Amazophrynella
minuta (Melin, 1941) (Anura: Bufonidae) from its type Locality…………………157
CAPITULO V. Diversification in Amazonian through the historical biogeography of
“terra firme” tiny tree toads Amazophrynella (Anura: Bufonidae)……………….190
CONCLUSÕES GERAIS...................................................................................218
xii
Lista de Figuras
Figura 1. Número de espécies ao redor do mundo, nota-se a presença de uma
maior abundância na Amazônia...................................................................................1
FIGURA 2. Protocolo de delimitação de espécies pelo critério da taxonomia
integrativa. Imagem obtida de Padial et al. (2010).......................................................3
FIGURA 3. Distribuição geográfica do gênero Amazophrynella (Anura:
Bufonidae)....................................................................................................................4
FIGURA 4. Espécies nominais de Amazophrynella: A) A. amazonicola, B) A. matses;
C) A. minuta; D) A. bokermanni; E) A. vote; F) A. manaos......................................... 5
FIGURA 5. Padrões morfológicos de Amazophrynella minuta. A) Rio Curaray, Peru
(Foto: Gagliardi); B) Rio Tahuayo, Peru (Foto: Medina); C) Taracuá, Brasil; D) Rio
Imiri, Peru; E) Reserva Yasuni, Equador (Foto: Ron)..................................................7
FIGURA 6. Padrões morfológicos de Amazophrynella bokermanni. A) Juruti, Brasil
(Foto: ©Gordo); B) Juruti, Brasil (Foto: ©Gordo); C) Rio Xingu, Brasil (©Peloso); D)
Rio Tapajós, Brasil (© Oliveira)....................................................................................8
FIGURA 7. Padrões morfológicos de Amazophrynella vote. A) Município de
Aripuanã, Brasil; B) Parque Nascente do Lago Jari, Brasil; C) Igarapé Açuã, Brasil,
D) Fazenda São Nicolau, Brasil. Fotos: ©Ávila............................................................9
FIGURA 8. Padrões morfológicos de Amazophrynella manaos. A) Campus da
Universidade Federal do Amazonas-UFAM, Brasil; B) Reserva CEPEAM, Brasil; C)
Reserva de Ducke, Brasil; D) Presidente Figueiredo, Brasil......................................10
FIGURA 9. Distribuição esquemática das seis espécies nominais de
Amazophrynella. Azul: A. amazonicola; Amarelo: A. matses; Celeste: A. minuta;
Vermelho: A. manaos; Rojo: A. bokermanni; Laranja: A. vote. Espaços sem cores
não apresentam dados para o gênero.......................................................................11
FIGURA 10. Relações filogenéticas do gênero Amazophrynella obtidas de Rojas et
al., (2015). Análise de Máxima verossimilhança do gene 16S DNAmt
...................................................................................................................................12
1
INTRODUÇÃO GERAL
Estima-se a existência de 6.5 milhões espécies terrestres e 2.2 milhões de
espécies oceânicas e que quase 30% das espécies do mundo ainda não foram
descritas (Mora et al., 2011). A Amazônia é considerada a área com maiores
registros de espécies de flora e fauna do mundo (Jenkins et al., 2013) (Figura 1),
sendo que, explicar a origem da sua diversidade tem intrigado naturalistas e
evolucionistas ao longo do tempo (Ribas et al., 2012).
FIGURA 1. Número de espécies ao redor do mundo, nota-se a presença de uma
maior abundância na Amazônia.
Entender os padrões e processos que geraram a biodiversidade é essencial
para o desenvolvimento de estratégias de conservação (Xu et al., 2008) e fornece
pistas para o entendimento da distribuição da biodiversidade em diversos grupos
taxonômicos (Webb, 2000; Webb et al., 2002; Vitt et al., 1999). Alfred Russel
Wallace (Wallace, 1852) postulou que os padrões de diversidade de vertebrados são
gerados pelos rios Amazônicos, desde então diversas hipóteses alternativas tem
sido propostas, visando explicar a distribuição e origem da diversidade (Haffer, 1969;
2
Fjeldsa, 1995; Endler, 1997; Colinvaux, 2001; Marrgoi e Cerqueira, 1997; Nores,
1999).
Reconstruções geológicas da Amazônia sugerem uma grande dinâmica ao
longo do tempo (Hoorn et al., 2010). Diversos estudos sugerem que os padrões de
diversidade atual não surgiram pela influência de um único evento diversificador,
mas sim pela combinação de eventos históricos, tais como: a reorganização
geológica (Hoorn et al., 2010) e modificações paleogeográficas (Rull, 2008) durante
o Neógeno, e eventos ecológicos: como mudanças cíclicas climáticas do Pleistoceno
(Haffer e Prance, 2001).
É conhecido que a distribuição da fauna Amazônica não ocorre ao acaso e
segue um padrão geral em áreas de endemismo que são delimitadas pelos grandes
interflúvios (Silva et al., 2005). Essas áreas possuem congruência de táxons
espacial e filogeneticamente com um conjunto próprio de linhagens e com trajetórias
evolutivas diferenciadas (Silva et al., 2005). Smith et al. (2014) reconheceram nove
grandes áreas de endemismo para aves na Amazônia: Guiana, Imeri, Napo,
Inambari, Rondônia, Tapajós, Xingu, Belém e Huallaga, sendo reconhecida também
a área do interflúvio Rio Negro/Solimões- Jaú (Borges e Silva, 2012).
Processos como extinção, vicariância e dispersão são reconhecidos como
processos naturais de diversificação ao longo da evolução dos taxa (Wiens e
Donoghue, 2004). De maneira geral, a especiação simpatrica (competição entre
populações com posterior divergência de nicho ecológico) e alopátrica (presença de
uma barreira geográfica) são atribuídos como os tipos de especiação mais comuns
na Amazônia (Rull, 2008; Antonelli et al., 2010, 2018). Assim, com o rápido
crescimento do conhecimento filogenético, processos de diversificação e eventos
evolutivos (Donoghue, 2008; Antonelli et al., 2010; Smith et al., 2014) novas
percepções sobre os padrões de diversidade no neotrópico foram questionadas,
como por exemplo a existência de complexos de espécies.
Diferentes grupos taxonômicos de vertebrados distribuídos na Amazônia são
considerados como complexos de espécies: mamíferos (Galimberti et al., 2015);
aves (Fernandes et al., 2013); répteis (Miralles e Carranza, 2010); peixes (Machado
et al., 2018) e anuros (Gehara et al., 2014) caracterizados por apresentar várias
3
linhagens evolutivas independentes nomeadas como uma única espécie através de
sua distribuição geográfica. Levando em consideração que o número de espécies
reconhecidas para a Amazônia encontra-se atualmente subestimada (Fouquet et al.,
2007), delimitar limites entre espécies torna-se fundamental para a conservação da
diversidade biológica.
Atualmente a comunidade científica aceita o fato de que o processo de
delimitação de espécies precisa ser multicaracteres (Padial et al., 2010), usando
caracteres morfológicos: (ex. caracteres diagnósticos merísticos, morfométricos e
diferenças estatísticas); pre- zigóticos (análises dos parâmetros espectrais e
temporais de cantos de acasalamento); ecológicos (requerimentos ambientais,
sobreposição de nicho, comportamento) e genéticos (monofilia, técnicas de
coalescência como GMYC, BPTP, bGMYC, distâncias genéticas, caracteres
diagnósticos genéticos) entre outros (ex. Angulo e Reichle, 2008; Ortega-Andrade et
al., 2015; Zahng et al., 2013). A integração desses critérios operacionais, permite
uma estabilidade taxonômica em grupos morfologicamente conservados ou aqueles
que apresentam alta plasticidade fenotípica, evitando assim a subestimação ou
superestimação da diversidade de espécies (Hebert et al., 2003; Hebert et al., 2005;
Simões et al., 2008; Elmer et al., 2013; Rojas et al., 2016) (Figura 2).
FIGURA 2. Protocolo de delimitação de espécies pelo critério da taxonomia
integrativa. Imagem obtida de Padial et al. (2010).
Em comparação com outras Classes de vertebrados, os Anfíbios,
particularmente espécies da Ordem Anura, constituem um excelente grupo para
4
estudar a diversidade subestimada e inferir padrões biogeográficos por possuírem
uma diversidade críptica o pseudocriptica (Funk et al., 2012; Cornils e Held, 2014),
representam complexos de espécies com várias linhagens independentes (Gehara
et al., 2014), pouco explorados taxonomicamente, baixa capacidade de dispersão
vs. aves e mamíferos (Vences e Wake, 2007), são sensíveis as mudanças
ambientais e eventos históricos (Godinho e Da Silva, 2018), possuem alta taxa
reprodutiva, dependem da qualidade do habitat e possuem ambas fases de vida
(aquática e terrestre) (Vences e Wake, 2007).
Além de ser um grupo propicio para inferir padrões evolutivos e pouco
conhecido taxonomicamente , estima-se que 40% das espécies de Anuros se
encontram em perigo de extinção (Collins, 2010), e que, além da destruição de seus
habitats, o fungo Batrachochytrium dendrobatidis encontra-se diminuindo
progressivamente as populações nos Andes e Amazônia (Becker et al., 2016;
Catenazzi e von May, 2014).
Neste contexto encontra-se o gênero Amazophrynella, este gênero
compreende anfíbios anuros pertencentes à família Bufonidae. O gênero apresenta
distribuição Pan- amazônica, sendo distribuído nos territórios da Bolívia, Peru,
Equador, Colômbia, Venezuela, Brasil, Suriname e Guiana Francesa e Guiana
(Fouquet et al., 2012a; Rojas et al., 2018a) (Figura 3).
FIGURA 3. Distribuição geográfica do gênero Amazophrynella (Anura: Bufonidae).
5
O gênero Amazophrynella é irmão de Dendrophryniscus, que possui espécies
restritas à Mata Atlântica do Brasil. As espécies de Amazophrynella encontram-se
restritas em alturas que variam entre 50-708 metros desde o nível do mar. No início
deste doutorado eram reconhecidas 06 espécies ao longo de toda sua distribuição
geográfica, sendo que 06 novas espécies foram descritas no processo deste
trabalho (Rojas et al., 2016, 2018a) (Figura 4).
FIGURA 4. Espécies nominais de Amazophrynella: A) A. amazonicola, B) A. matses;
C) A. minuta; D) A. bokermanni; E) A. vote; F) A. manaos.
6
Estado da arte do gênero Amazophrynella
História natural
Amazophrynella significa “pequenos Amazônicos” (Fouquet et al., 2012a). As
espécies apresentam esse nome porque são de tamanhos pequenos e ocorrem na
Amazônia. Os machos adultos apresentam entre 13-16 milímetros (mm), enquanto
as fêmeas entre 15-25 mm. São espécies de terra firme, encontrados
preferencialmente na serapilheira de bosques secundários e primários, as espécies
apresentam dimorfismo sexual evidente, sendo as fêmeas maiores que os machos,
sua reprodução ocorre durante a época de chuvas (Novembro - Fevereiro) (Rojas et
al., 2014, 2018b).
Os machos cantam em frequências altas (> 3000 Hz) próximos de pequenas
poças d` água de pouca profundidade durante a manhã ou crepúsculo, sendo
possível encontrar abundância de indivíduos agrupados ao redor de pequenos
corpos d`água com abundantes folhas, galhos, árvores ou arbustos (comunicação
pessoal do autor). Os ovos são pigmentados e são depositados em raízes de
árvores e arbustos ou debaixo da serapilheira úmida (Ávila et al., 2012; Rojas et al.,
2015, 2016). Os girinos são pequenos e vivem em pequenos grupos, são apenas
descritos para duas espécies: A. siona e A. manaos.(Menin et al., 2014; Rojas et al.,
2018a).
História taxonômica
A primeira espécie do gênero descrita foi Amazophrynella minuta (Melin,
1941) como Atelopus minutus, a localidade tipo é Taracuá, São Gabriel da
Cachoeira, estado do Amazonas, Brasil. A descrição foi muito breve e a diagnose
morfológica generalizada (Melin, 1941; Rojas et al., 2018c). Posteriormente
McDiarmid (1971) mudou para Dendrophryniscus minutus. Historicamente o nome
de A. minuta foi usado para indivíduos distribuídos em toda a Amazônia durante
muito tempo como o Peru, Equador, Brasil, Guiana francesa (ex. Duellman 1978;
Zimmerman e Rodrigues 1990; Magnusson e Hero 1991; Rodrigues e Duellman
1993; Duellman e Mendelson 1995; Fouquet et al., 2012a) desde o ano 1941 até o
2012 (Figura 5).
7
FIGURA 5. Padrões morfológicos de Amazophrynella minuta. A) Rio Curaray, Peru
(Foto: Gagliardi); B) Rio Tahuayo, Peru (Foto: Medina); C) Taracuá, Brasil; D) Rio
Imiri, Peru; E) Reserva Yasuni, Equador (Foto: Ron).
Posteriormente foi descrita Amazophrynella bokermanni (Izecksohn, 1992)
como uma espécie do gênero Dendrophryniscus. Esta espécie possui o I dedo da
mão maior ou igual ao II, sendo uma característica das espécies dentro deste
complexo (Rojas et al., 2018a). Foram reportadas populações do rio Tapajós e Rio
trombetas, estado do Pará, Brasil (Àvila et al.,2012), Urucu, município de Coari,
Amazonas e no baixo rio Purus (Waldes et al., 2013), mas esta população foi
8
confundida com A. vote (observação pessoal do autor). Esta espécie apresenta
profundas divergências morfológicas (Figura 6).
FIGURA 6. Padrões morfológicos de Amazophrynella bokermanni. A) Juruti, Brasil
(Foto: ©Gordo); B) Juruti, Brasil (Foto: ©Gordo); C) Rio Xingu, Brasil (©Peloso); D)
Rio Tapajós, Brasil (© Oliveira)
No ano 2012 foi proposto o gênero Amazonella (Fouquet et al., 2012b) mas
foi substituído por Amazophrynella (Fouquet et al., 2012a) para as duas espécies de
Dendrophryniscus (D. minutus e D. bokermanni) distribuídos na Amazônia. As
espécies de Dendrophryniscus da Mata Atlântica do Brasil mantiveram sua
identidade taxonômica.
9
O 2012 ano foi descrita A. vote Ávila, Carvalho, Gordo, Ribeiro & Morais 2012.
Esta espécie distribui-se nos municípios de Cotriguaçu; Colniza, Aripuanã; Parque
Estadual do Guariba, Manicoré; Reserva Biológica de Jaru, Ji-Paraná; Lago Açaí,
Novo Aripuanã; Parque Estadual do Matupiri, Manicoré; Cachoeirinha, Manicoré;
Parque Nacional Nascentes do Lago Jari, Tapauá; Igarapé Açuã. O principal caráter
de identificação morfológica é o padrão de coloração ventral e o tamanho (Ávila et
al., 2012); mas além de sua descrição, não se conhece nada sobre seus padrões
ecológicos, variação morfológica, entre outras características. Esta espécie também
apresenta variações fenotípicas (Figura 7).
FIGURA 7. Padrões morfológicos de Amazophrynella vote. A) Município de
Aripuanã, Brasil; B) Parque Nascente do Lago Jari, Brasil; C) Igarapé Açuã, Brasil,
D) Fazenda São Nicolau, Brasil. Fotos: ©Ávila
10
Amazophrynella manaos Rojas, Carvalho, Gordo, Ávila, Pires & Hrbek 2014
(Figura 8) apresenta como localidade tipo o campus da Universidade Federal do
Amazona-UFAM, Brasil, e sua distribuição geográfica abrange as localidades de
Mineração Taboca, Reserva Florestal Adolpho Ducke, Presidente Figueiredo,
Reserva ZF-2, REBIO Uatumã, RDS Uatumã e Parque Estadual Rio Negro Setor
Sul, Rio Cuieiras. Estudos prévios indicam uma linhagem evolutiva independente
dentro de essa espécie localizada na Guiana Francesa (Fouquet et al., 2012a).
FIGURA 8. Padrões morfológicos de Amazophrynella manaos. A) Campus da
Universidade Federal do Amazonas-UFAM, Brasil; B) Reserva CEPEAM, Brasil; C)
Reserva de Ducke, Brasil; D) Presidente Figueiredo, Brasil.
11
Posteriormente foram descritas Amazophrynella amazonicola Rojas,
Carvalho, Ávila, Pires, Gordo & Hrbek 2015 e A. matses Rojas, Carvalho, Ávila,
Pires, Gordo & Hrbek 2015, estas duas últimas espécies encontram-se distribuídas
dentro do departamento de Loreto, Perú (Rojas et al., 2015). Se conhece pouco
sobre sua história natural e extensão de sua distribuição geográfica. Estas duas
espécies formam parte do complexo A. minuta.
FIGURA 9. Distribuição esquemática das seis espécies nominais de
Amazophrynella. Azul: A. amazonicola; Amarelo: A. matses; Celeste: A. minuta;
Vermelho: A. manaos; Rojo: A. bokermanni; Laranja: A. vote. Espaços sem cores
não apresentam dados para o gênero.
Entre os trabalhos realizados com o gênero em estudo, podemos citar
Duellman (1978), Rodrigues e Duellman (1994), Duellman e Mendelsom (1995) e De
la Riva et al. (2010). Mas estes trabalhos focam-se principalmente, em apresentar
pontos de distribuição geográfica de A. minuta e A. bokermanni. Com respeito as
outras espécies de Amazophrynella, não existem discussões taxonômicas e estudos
de variação populacionais dos seus aspectos morfológicos, genéticos e aspectos de
história natural.
12
Sistemática de Amazophrynella
A primeira hipótese filogenética do gênero reconheceu duas espécies
nominais: A. minuta e A. bokermanni, e duas linhagens evolutivas independentes: A.
aff. minuta “western Amazonian” e A. sp “Guianas” (Fouquet et al., 2012a). Uma
árvore mais completa (Rojas et al., 2014) mostrou a existência de um complexo de
espécies dentro de A. minuta e populações não descritas relacionadas com A.
manaos.
Na hipótese filogenética de Rojas et al. (2015) identifica-se unidades
evolutivas independentes pertencentes ao complexo de A. minuta e A. manaos,
sendo descritas A. amazonicola e A. matses (Figura 10). Posteriores estudos em
sistemática e taxonomia do gênero foram realizados durante o processo deste
trabalho, onde foram descritas A. javierbustamantei (Rojas et al., 2016- ver capitulo
I) e A. teko, A. siona, A. xinguensis e A. moisesii (Rojas et al., 2018- ver capitulo II).
FIGURA 10. Relações filogenéticas do gênero Amazophrynella obtidas de Rojas et
al., (2015). Análise de Máxima verossimilhança do gene 16S DNAmt baseado em
480 pares de base.
13
Uma das características atuais de Amazophrynella e a existência de
distâncias genéticas elevadas. Rojas et al. (2014) através do uso do gene
mitocondrial 16S RNA observaram elevadas divergências moleculares (≤4% e
≥15%) entre as espécies conhecidas.
As elevadas divergências moleculares, junto com as características de vida destas
pequenas espécies, assim como o conhecimento da existência de uma diversidade
subestimada neste gênero considerado como críptico (Fouquet et al., 2007, Fouquet
et al., 2012a, Rojas et al., 2014), podem supor que as populações das espécies de
Amazophrynella possam representar espécies novas.
Problemática
Com o advento de análises genéticas coalescentes do sequenciamento
mitocondrial e de nova geração (next generation sequences- ddrad) junto com o uso
do protocolo de delimitação de espécies da taxonomia integrativa e baixo o conceito
unificado de espécies (De Queiroz, 2007), uma nova era para taxonomia tornou-se
possível, já que com as relações sistemáticas do gênero Amazophrynella atualizada
será possível inferir hipóteses mais exatas da influência dos eventos históricos,
biogeográficos e ecológicos em linhagens corretamente delimitadas. Esta
abordagem tornará possível novas interpretações da história biogeográfica das
espécies e permitirá conhecer a diversidade escondida de anuros e outros taxas
Amazônicos.
Historicamente as principais dificuldades em conhecer a diversidade do
gênero Amazophrynella foi a diagnose morfológica generalizada da espécie tipo: A.
minuta (Melin, 1941), ausência de amostras para análises comparativas
morfológicas, genéticas e do conhecimento básico da sua história natural e
comportamento reprodutivo (ex. cantos de acasalamentos). Adicionando-se a
inexistência de uma hipótese filogenética completa do gênero que permita identificar
linhagens evolutivas independentes, abordar a sistemática molecular junto a
taxonomia integrativa e entender seus padrões biogeográficos para revelar
processos de diversificação na Amazônia.
14
Hipóteses
No presente estudo as hipóteses são de que (1) o gênero Amazophrynella
apresenta uma diversidade subestimada de espécies sendo que o número de
espécies atuais não reflete a sistemática atual; (2) os atuais padrões de distribuição
das linhagens de Amazophrynella apresentam uma radiação evolutiva influenciada
por eventos históricos e ecológicos do Neógeno (Mioceno-Plioceno).
OBJETIVOS
Geral
Revisar a sistemática e taxonomia do gênero Amazophrynella e estudar os
padrões e processos envolvidos na origem de sua diversidade na Amazônia.
Específicos
Reconstruir a filogenia de Amazophrynella e usar a taxonomia integrativa para
delimitar espécies e propor rearranjos taxonômicos com o intuito de revelar a
diversidade escondida de anuros neotropicais;
Investigar a história evolutiva e diversificação em Amazophrynella visando
entender a divergência entre Amazônia e Mata Atlântica e procurando padrões
biogeográficos de anuros na Amazônia.
15
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CAPITULO I
Uncovering the diversity inside the Amazophrynella minuta complex: integrative
taxonomy reveals a new species of Amazophrynella (Anura, Bufonidae) from
southern Peru. Rojas, R.R., Chaparro, J.C., Carvalho, V.T. De, Ávila, R.W., Farias,
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24
Uncovering the diversity inside the Amazophrynella minuta complex (Anura,
Bufonidae): integrative taxonomy reveals a new species from southern Peru
Rommel R. Rojas1,2*, Juan C. Chaparro3, Vinícius Tadeu De Carvalho2,4, Robson W.
Ávila5, Izeni Pires Farias2, Tomas Hrbek2 and Marcelo Gordo6
1 Programa de Pós-graduação em Genética Conservação e Biologia Evolutiva-
Instituto Nacional de Pesquisas da Amazônia-INPA, Av. André Araújo, 2936,
Manaus, Brazil, 2 Laboratório de Genética e Evolução Animal, Departamento de
Genética, ICB, Universidade Federal do Amazonas, Av. Gen. Rodrigo Octávio
Jordão Ramos, 3000, Manaus, Brazil, 3 Museo de Historia Natural, Universidad
Nacional de San Antonio Abad del Cusco, Peru, 4 Programa de Pós-Graduação em
Biodiversidade e Biotecnologia, Av. Gen. Rodrigo Octávio Jordão Ramos, 3000,
Manaus, Brazil, 5 Universidade Regional do Cariri, Centro de Ciências Biológicas e
da Saúde, Departamento de Ciências Biológicas, Campus do Pimenta, Rua Cel.
Antônio Luiz, 1161, Bairro do Pimenta, Crato, Brazil, 6 Departamento de Biologia,
ICB, Universidade Federal do Amazonas, Av. Gen. Rodrigo Octávio Jordão Ramos,
3000, Manaus, Brazil.
*Corresponding autor: [email protected]
25
Abstract.
A new species of the genus Amazophrynella (Anura, Bufonidae) is described from
the departments of Madre de Dios, Cusco and Junin in Peru. An integrative taxonomy
approach is used. A morphological diagnosis, morphometrics comparisons,
description of the advertisement call, and the phylogenetic rela- tionships of the new
species are provided. Amazophrynella javierbustamantei sp. n. differs from other
spe- cies of Amazophrynella by: intermediate body-size (snout-vent length 14.9 mm
in males, n = 26 and 19.6 mm in females, n = 20), tuberculate skin texture of body,
greatest hand length of the Amazophrynella spp. (3.6 mm in males, n = 26 and 4.6
mm in females, n = 20), venter coloration yellowish, tiny rounded black points
covering the venter, and thirteen molecular autapomorphies in the 16S RNA gene. Its
distribution varies from 215 to 708 m a.s.l. This discovery highlights the importance of
the remnant forest in preserving the biodiversity in Peru, and increase in seven the
species formally described in the genus Amazophrynella.
Resumen.
Describimos una nueva especie del género Amazophrynella (Anura, Bufonidae) del
Perú de los Departamen- tos de Madre de Dios, Cusco y Junin de Peru. Utilizamos
un método de taxonomía integrativa. Obtuvimos la diagnosis morfológica,
comparaciones morfométricas, descripción del canto de reproducción y las rela-
ciones filogenéticas de la nueva especie. A. javierbustamantei sp. n. difiere de las
otras Amazophrynella spp. por poseer tamaño medio (Hocico-cloaca en machos 16.9
mm, n = 26 y en hembras 19.6 mm, n = 20); textura de la piel tuberculada; tamaños
de las manos mayores (3.6 mm en machos, n = 26 y 4.6 mm en hembras, n = 20);
coloración ventral amarillento-pálida, pequeños puntos redondos de color negro en
el vientre y por trece autopomorfias moleculares en el gen 16S RNA. Su distribución
varía desde 215 m hasta 708 m a.s.n.m. Este descubrimiento resalta la importancia
de los remanentes de la selva Peruana en térmi- nos de conservación, e incrementa
en siete las especies formalmente descritas en del género Amazophrynella.
26
Resumo.
Descrevemos uma nova espécie do gênero Amazophrynella (Anura, Bufonidae) dos
departamentos de Madre de Dios, Cusco e Junin do Peru. Utilizamos um método de
taxonomia integrativa. Apresentamos a diagnose morfológica, comparações
morfométricas, descrevemos o canto de anúncio e geramos uma hipó- tese
filogenética da nova espécie. Amazophrynella javierbustamantei sp. n. difere das
outras Amazophrynella spp. por possuir tamanho médio (Comprimento rostro-cloacal
16.9 mm em machos, n = 26 e 19.6 mm em fêmeas, n=20); textura da pele
tuberculada; tamanhos das mãos maiores (3.6 mm em machos, n = 26 e 4.6 mm em
fêmeas, n = 20); coloração ventral amarelo-clara, coberta por pequenos pontos
redondos pretos e por treze autapomorfias moleculares no gene 16S RNA. Sua
distribuição varia entre os 215 m até os 708 m a.n.m. Nossa descoberta aumenta a
importância dos remanescentes da floresta Peruana em termos de conservação e
incrementa em sete as espécies formalmente descritas no gênero Amazophrynella.
Keywords
Amphibian, Tree Toad, conservation, South of Peru, integrative taxonomy
Palabras claves
Anfibios, Sapo del árbol, conservación, Sur del Perú, taxonomía integrativa
Palavras chaves
Anfíbios, Sapo do arvore, conservação, Sul do Peru, taxonomia integrativa
27
Introduction
Until 2012, two species of Amazophrynella were placed in the genus
Dendrophryniscus Jimenez de la Espada, 1868. Fouquet et al. (2012a) recognized
that species of Den- drophryniscus from the Amazon and Atlantic rainforests
represented morphologically and genetically deeply divergent lineages, and thus the
authors proposed a new genus: Amazophrynella Fouquet, Recorder, Texeira,
Cassimiro, Amaro, Camacho, Damace- no, Carnaval, Moritz & Rodrigues, 2012 for
the Amazonian species A. minuta and A. bokermanni.
In the following years, an additional four new species of the genus were
described: A. vote Ávila, Carvalho, Gordo, Ribeiro & Morais, 2012 and A. manaos
Rojas, Carvalho, Gordo, Ávila, Farias & Hrbek, 2014 based on morphology; A.
amazonicola and A. matses Rojas, Carvalho, Gordo, Ávila, Farias & Hrbek, 2015,
based on morphology and genetic data (Ávila et al. 2012; Rojas et al. 2014, 2015).
Species of the genus are distributed in nine South American countries: Bolivia, Peru,
Ecuador, Colombia, Venezuela, Guiana, French Guiana Brazil, and presumably in
Suriname (Frost et al. 2015).
Using a phylogenetic analysis based on mitochondrial and nuclear genes
(Fouquet et al. 2007, 2012a), the existence of three independent evolutionary
lineages was discovered within the nominal species A. minuta from Ecuador and
French Guianas; subsequently, other independent evolutionary lineages were
discovered from Brazil and Peru (Rojas et al. 2014, 2015). The difficulties in
delimiting species within the A. minuta species complex resides in the relatively
generalized diagnosis (see Melin 1941) and the poor geographic sampling. For these
reasons, historically, the name A. minuta has been used for individuals distributed
throughout the Amazonian biome (e.g. Du- ellman 1978; Zimmerman and Rodrigues
1990; Magnusson and Hero 1991; Rod- rigues and Duellman 1993; Duellman and
Mendelson 1995; Fouquet et al. 2012a).
Thus, taxonomy and systematics of populations that are currently part of the A.
minuta complex remains largely unresolved (Rojas et al. 2014), in turn limiting the
knowledge of the true taxonomic diversity of the genus (Ávila et al. 2012; Rojas et al.
2014, 2015). Given this scenario, herein is described an additional new species of
28
Amazophrynella from the departments of Madre de Dios, Cusco and Junin, Peru,
founded on the prin- ciples of integrative taxonomy. Morphological, morphometric,
bioacoustic and phyloge- netic relationships are provided as evidence for the
existence of the new taxon.
Material and methods
Morphology
Forty-eight specimens previously identified as Amazophrynella minuta (Melin,
1941), deposited at the Museo de Historia Natural del Cusco, Universidad Nacional
de San Antonio Abad del Cusco (MHNC) and Museo de Historia Natural de la
Universidad Nacional Mayor de San Marcos (MHNSM) were analyzed. This material
was com- pared with twenty preserved specimens of A. minuta from the type locality
(Taracuá mission, on the right bank of the Uaupés River, municipality of São Gabriel
da Cach- oeira, Brazil), deposited in the Collection of Amphibians and Reptiles of the
Instituto Nacional de Pesquisas da Amazônia–INPA, Brazil (INPA-H). Further
comparisons were made with three syntypes deposited at the Naturhistoriska
Museet, Göteborg, Sweden (NHMG), and the original description of the species
(Melin 1941).
Additionally five preserved specimens of Amazophrynella bokermanni
(Izecksohn, 1993) from near the type locality (Juruti, 30 Km from type locality), the
holotype and paratypes of A. manaos deposited in the Collection of Amphibians and
Reptiles of the Instituto Nacional de Pesquisas da Amazônia–INPA, Manaus,
Amazonas, Brazil (INPA- H), the holotype of Amazophrynella vote, deposited in the
Coleção Zoológica de Ver tebrados of the Universidade Federal de Mato Grosso–
UFMT, Cuiabá, Mato Grosso, Brazil (UFMT-A), seventeen paratypes deposited in the
Collection of Amphibians and Reptiles of the Instituto Nacional de Pesquisas da
Amazônia–INPA, Manaus, Amazo- nas, Brazil (INPA-H), and the holotype and
paratypes of A. amazonicola and A. matses, deposited at the Museo de Zoologia de
la Universidad Nacional de la Amazonia Peruana (MZUNAP) were analyzed (see
Appendix 1 listing all the revised specimens). Morphological character analyses were
carried out according to Cruz and Fussi- nato (2008) and Fouquet et al. (2012a). Sex
was determined by gonad analysis.
29
Measurements were carried out with a digital caliper following Kok and Kala-
mandeen (2008) and Duellman (1978). SVL (snout-vent length) from the tip of the
snout to the posterior edge of the cloaca; HL (head length) from the posterior edge of
the jaw to the tip of the snout; HW (head width), the greatest width of the head,
usually at the level of the posterior edges of the tympanum; ED (eye diameter); IND
(internari- nal distance), the distance between the edges of the nares; SL (snout
length) from the anterior edge of the eye to the tip of the snout; HAL (hand length)
from the proximal edge of the palmar tubercle to the tip of Finger III; UAL (upper arm
length) from the edge of the body insertion to the tip of the elbow; THL (thigh length)
from the vent to the posterior edge of the knee; TL (tibia length) from the outer edge
of the knee to the tip of the heel; TAL (tarsal length) from the heel to the proximal
edge of the inner meta- tarsal tubercle; FL (foot length) from the proximal edge of the
inner metatarsal tubercle to the tip of Toe IV. Diagnosis of characters follow Chaparro
et al. 2015.
Statistical analysis. We used a total of 80 adult males of the Amazophrynella minuta
species complex (numbers of individuals and populations of origin in parentheses): A.
minuta sensu stricto (n = 23, from Taracuá), A. amazonicola (n = 15, from Puerto
Almendras and Fazenda Zamora); A. matses (n = 13, from Nuevo Salvador) and the
new species of Amazophrynella (n = 29, from Tambopata, Nuevo Arequipa, Candamo
and Inambari). All morphometric measures were log10 transformed to conform to
requirements of normality (Hayek et al. 2001). The effect of size was removed from
all variables by regressing them against SVL and using the residuals of each variable
in a Principal Component Analysis (PCA). Significance of morphometric differences
was tested with Multivariate Analysis of Variance (MANOVA) with the two first
principal compo- nents being treated as dependent variables and species as
independent variables. The first two principal components were used since they
explained the majority of observed variation in shape. A Discriminant Function
Analysis (DFA) was performed to test classification of individuals in predicted groups.
All the statistical analysis were per- formed in R (R Development Core Team 2011)
adopting a 5% significance cut-off. PCA was used to detect groups representing
putative cryptic species and DFA was subsequently applied to identify the set of
characters that best diagnose those groups (Padial and De la Riva 2009).
30
Additionally we noted large size in the HAL of the new species of Amazophrynella,
and we used an Analysis of Variance (ANOVA) of the origi- nal data (from A. minuta,
A. matses, A. amazonicola and the new species) to statistically support this
hypothesis
Molecular data
Laboratory procedure. Total DNA was extracted from muscle tissue using standard
phenol/chloroform extraction (Sambrook et al. 1989). A 480 bp fragment of the 16S
rDNA was PCR amplified using the 16Sar and 16Sbr primers (Palumbi 1996). Ampli-
fication was carried out under the following conditions: 60 s hot start at 92 °C
followed by 35 cycles of 92 °C (60 sec), 50 °C (50 sec) and 72 °C (1.5 min). Final
volume of the PCR reaction was 12 μl and contained 4.4 μL ddH2O, 1.5 μL of 25 mM
MgCl 21.25 μL of 10 mM dNTPs (2.5mM each dNTP), 1.25 μL of 10x buffer (75 mM
Tris HCl, 50 mM KCl, 20 mM (NH42SO4), 1 μL of each 2 μM primer, 0.3 μL of 5
U/μLDNA Taq Polymerase (Biotools, Spain) and 1 μL of DNA (about 30 ng/μL).
Sequenc- ing reactions were carried out according to the manufacturer’s
recommendation for the ABI BigDye Terminator cycle sequencing mix, using 16Sa
primer and an annealing temperature of 50 °C. Sequencing reactions were
precipitated using standard EDTA/ EtOH protocol, and resolved in an ABI 3130xl
automatic sequencer.
Phylogenetic analysis. We obtained 16S rDNA sequence data from two specimens of
the new species (Accession numbers: KR905184, KR905185), two paratypes of A.
vote (Accession numbers: KF433970, KF433971), two specimens of A. bokermanni
(Accession numbers: KF433975, KF433976), two topotypic specimens of A. minuta
(Accession numbers: KF792834, KF792836), two paratopotypes of A. matses
(Acces- sion number: KP681688, KP681689), the holotype and one paratopotype of
A. ama- zonicola (Accession number: KP681868, KP681669) and two paratypes of
A. manaos (Accession number: KF433954, KF433957) deposited in the tissue
collection of the Laboratório de Evolução e Genética Animal of the Universidade
Federal do Amazonas (CTGA-ICB/UFAM). The dataset also included two sequences
of A. sp. aff. minuta (Accession number: AY326000, DQ158420) from Darst and
Canatella (2004), Pra- muk (2006) and two sequences of A. sp. aff. manaos
31
(Accession number: EU201057, JN867570) from Fouquet et al. (2007). As outgroups
we used species of the sister taxon Dendrophryniscus (see Table 2 for samples
information). Sequences were aligned using the Clustal W algorithm (Thompson et
al. 1996) implemented in BioEdit (Hall 1999) and alignment was adjusted as
necessary against the secondary structure of the 16S rDNA. The existence of
lineages in a phylogenetic tree-based context (Baum and Donoghue 1995) was
performed using Maximum Like- lihood analysis (Felsenstein 1981) in the program
Treefinder (Jobb 2008) using the GTR+I+G model of substitution, selected via Akaike
information criterion as imple- mented in Modeltest 3.7 (Posada 2006). Phylogenetic
support was assessed via 10 000 non-parametric bootstrap (Felsenstein 1985).
Additionally uncorrected pairwise genetic distances between linages identified by
phylogenetic inference of Amazophrynella were calculated in MEGA 5.05 (Tamura et
al. 2007).
Molecular species delimitation. Evolutionary lineages are diagnosed by
discontinuities in character variation among lineages, and correspond to phylogenetic
species. The existence of lineages is therefore a necessary and sufficient
prerequisite for inferring the existence of a species under the different
conceptualizations of the Phylogenetic Species Concept (PSC) (Cracraft 1983; Baum
and Donoghue 1995; De Queiroz 2007). The existence of lineages in a non-tree-
based context (Cracraft 1983) was inferred us- ing Population Aggregation Analysis
performed at the level of an individual (Davis and Nixon 1992; Rach et al. 2008)
using the dataset with the Amazophrynella minuta spe- cies complex: A. matses, A.
minuta, A. amazonicola and the new species. The analyses were performed in the
program R (R Development Core Team 2011).
Bioacoustics
We analyzed one advertisement call obtained from the CD of Frogs of
Tambopata, Peru (Macauly Library of Natural Songs and Cornell Laboratory of
Ornithology) by the authors Cocroft et al. (2001) from the Natural Reserve of
Tambopata, a locality of occurrence of the new species. The call was edit with the
software Audacity 1.2.2 for Windows (Free Software Foundation Inc. 1991). The
spectral and temporal pa- rameters of the recording were analyzed in the software
32
Raven Pro. 1.3 for Windows (Cornell Laboratory of Ornithology). The advertisement
call was obtained from one male in a temperature 25 °C (Crocoft et al. 2001). We
measured the following quan- titative parameters: call duration (seconds); pulses per
call; length of silence between calls (seconds); dominant frequency (kHz);
fundamental frequency (kHz) and time to peak at maximum frequency (seconds).
Results
Phylogenetic analysis and systematics
In the resulting phylogeny, the six nominal species of Amazophrynella were
recognized as monophyletic (Fig. 1). In the genus we can distinguish two
monophyletic groups: One clade (bootstrap support = 100) formed by the species: A.
manaos, A. bokermanni and A. vote and another represented by the species of the
A. minuta “species complex” (bootstrap support = 98): A. minuta, A. amazonicola, A.
matses and the new species described herein. In the first clade the Amazophrynella
species: A. manaos is sister taxon of the possi- ble new specie from the Guiana
Shield: A. sp. aff. manaos (bootstrap support= 91), and both are sister to A.
bokermanni (bootstrap support= 98). Amazophrynella vote is sister of A. bokermanni
+ (A. manaos + A. sp. aff. manaos) with a bootstrap support of 81. The second clade
corresponding to the A. minuta “species complex”, A. amazonicola is sister of A.
minuta + A. sp. aff. minuta from western Amazonia (bootstrap sup- port= 99). Our
analysis further highlighted the occurrence of a new monophyletic lin- eage (A.
javierbustamantei sp. n.) showing sister relationship with A. matses (bootstrap
support = 96), both being in turn sister group of A. amazonicola + (A. minuta + A. sp.
aff. minuta) with a bootstrap support of 99.
Smallest uncorrected 16S rDNA p-distances estimated between phylogenetic
linag- es was observed between A. minuta and A. sp. aff. minuta (= 3%). Greatest
interspecific distance (= 14%) was observed between Amazophrynella
javierbustamantei sp. n. and A. bokermanni and was comparable to divergence
observed between A. manaos and A. minuta. Within the “A. minuta” species complex,
the new species shows a high degree of genetic divergence from A. minuta (= 7%),
A. amazonicola (= 9%) and minor genetic distance with their sister taxon A. matses
(= 3%) (see all pairwise genetic distance values summarized in Table 3). According to
33
the Population Aggregation Analysis, the newly identified lineage was also
diagnosable by thirteen molecular autapomorphic characters (Table 4) leading us to
the conclusion that this lineage corresponds to a new species.
Morphometric analysis
Comparative analysis of quantitative morphological data allowed us to
distinguish Amazophrynella sp. n. from the other members of the A. minuta “species
complex”. The first two principal components extracted by the PCA account for
48.56% of the variation found in the dataset. The first component (PC1) explained
24.93% of total variation. In the first principal component axis, A. amazonicola is
distinguished from the other species due to its larger size (SVL = 14.9 ± 0.7 mm, see
Table 1), sharing relative size with A. minuta sensu stricto (SVL = 13.5 ± 0.6 mm, see
Table 1), the spe- cies A. matses is distinguish by having the smallest size of the
genus (SVL range= 12.1 ±0.6 mm, see Table 1), and shares this characteristic with
Amazophrynella sp. n. (SVL = 14.9 ± 0.9 mm, see Table 1) (Fig. 2). The second
component explains 23.63% of the variation. This axis represents a shape variation
vector; in this axis Amazophrynella javierbustamantei sp. n. is well distinguished from
the three formally described species, sharing more similarity with A. matses (Table 5).
All the species of the group are significantly different in shape (MANOVA,
F24.3, Pillae´s trace < 0.001). The discriminate function analysis (DFA) found
specimens correctly classified in 56.6% of cases and a moderate prior probability of
groups (A. minuta = 28.75%, A. amazonicola = 18.75%, A. matses = 16.25% and A.
javierbustamantei sp. n. = 36.25%). The variables that contributed most to the
classification were HAL, SVL and TAL (Table 6). The differences in HAL were
significant (ANOVA, F45.27, P < 0.001) among all the species of A. minuta “species
complex” (see Fig. 1) and reveals Amazophrynella javierbustamantei sp. n. as the
species with the largest HAL (Fig. 3).
Morphological description
Amazophrynella javierbustamantei sp. n.
http://zoobank.org/A946B949-1D1F-4FF5-B722-0B33435EE610
34
Holotype (Fig. 4). MHNC 8331 (Genbank 16S rRNA: KR905184). Adult male,
col- lected at Quebrada Guacamayo (12°54'24.5"S; 69°59'32.7"W, 215 m a.s.l.) km
105 of the highway Puerto Maldonado–Cusco City, District Inambari, Province
Tambopata, Department Madre de Dios, Peru, on 27 October 2009 by Juan C.
Chaparro and Oscar Quispe.
Paratypes (Fig. 5). Twenty-two specimens (males= 09, females= 13). MHNC
8363, MHNC 8245, MHNC 8238, adult males, MHNC 8316, MHNC 8484, MHNC
8362, MHNC 8354, adult females, collected with the holotype (12°28'25"S,
69°12'36"W, 205 m a.s.l.). MHNC 11001, adult male, MHNC 11002, MHNC 11003,
MHNC 11004, adult females collected by E. Aguilar on 17 May 2009, from La Pampa
km 107 highway Puerto Maldonado–Cusco City, Department Madre de Dios
(12°40'14.14"S, 72°27'30"W, 250 m a.s.l.). MHNSM 17993, adult male col- lected by
A. Angulo in 1999; from Province Manu, locality of Inambari, Depart- ment Madre de
Dios (13°02'29.28"S, 70°22'46.65"W, 306 m a.s.l.). MHNSM 25651, adult female,
collected by D. Rodriguez on April 2007, from Province La Convención, locality of
Camana, Department Cusco (12°05'9.25”S, 73°03'2.61”W, 680 m a.s.l.). MHNC
9939, MHNC 9940, adult females, collected by J. Delgado on 17 January 2010 from
Province La Convención, locality of Mapi, Department Cusco (11°31'19.17”S,
73°28'29.83”W, 708 m a.s.l.). MHNC 9387, adult male, collected by G. Estrada on 21
January 2010, from locality of Tambo Poyeni near Quebrada Mayapo, Department
Junin (11°19'29.9”S, 73°32'16.7"W, 388 m a.s.l.). MHNC 9754, MHNC 9756, adult
males, MHNC 9626, MHNC 9679, MHNC 9680, MHNC 9757, adult females,
collected by A. Pari on January 2010, from local- ity of Tsoroja, Department Junin
(11°18'56.06”S, 73°32'32.11”W, 399 m a.s.l. and 11°23'14.50”S , 73°29'43.00”W, 450
m a.s.l.).
Diagnosis. The new species is part of Amazophrynella based on molecular
phylogenetic relationships (Fig. 1) and morphological synapomorphies (Fouquet et al.
2012a). Amazophrynella javierbustamantei sp. n. is characterized by: (1) skin on
dorsum tuberculate, with many subconical tubercles disperse on arms, legs, head
and body; ventral skin coarsely areolate, throat and chest aerolate; (2) tympanic
membrane and tympanic annulus not apparent through the skin; (3) snout long,
subacuminated, pro- truding in lateral views; (4) upper eyelid with smaller tubercles,
35
cranial crests absent; (5) dentigerous process of vomers absent; (6) vocal sac, vocal
slits and nuptial pads ab- sent; (7) finger I shorter than finger II, tips of digits rounded;
(8) fingers lacking lateral fringes; (9) ulnar tubercles present; (10) heel bearing eight
or more small low tubercles, tarsus with small tubercles and lack of folds; (11) plantar
surfaces of feet bearing one metatarsal tubercle, the inner 2.5x larger than the outer,
outer subconical; supernumerary plantar tubercles round and low; (12) toes lacking
lateral fringes; webbing basal; toe III equal than toe V, tips of digits rounded; (13)
dorsally is dark brown to light brown, and gray to black in some, ventrally, cream with
yellow to orange marks, with black to dark brown spots; (14) SVL 16.39–22.25 mm in
females, 12.79–16.42mm in males; (15) hand length is the greatest of all species of
Amazophrynella: 3.6 mm in males (n= 26) and 4.6 mm in females (n=20), see Fig. 3;
(16) thirteen molecular autapomorphies in the 16S rDNA gene.
Comparison with other species. Amazophrynella javierbustamantei sp. n. (Figs
4, 5, 6) differs in the following character states (states of other species in
parentheses). From A. minuta (Fig. 6A) by having body skin texture tuberculate
(roughly granular); relative abundance of spiny granules on the forelimbs (prickly
warty skin on axillary region of the forelimbs); absence of large warts on dorsum
(presence of large warts); throat and chest cream-grayish (light brown); posterior side
of belly color pale orange yellowish with tiny rounded black or dark brown spots
(throat and the whole belly in- tensely orange yellowish); tiny rounded black spots
covering the belly (irregular black ocelli or blotches); metatarsal tubercle rounded
(oval). From A. bokermanni (Fig. 6B) relative size of fingers, with finger I shorter than
II (I>II); snout vent length smaller in males (15.8 mm) and females (22.25 mm) (A.
bokermanni with maximum 22 mm SVL in males and 28 mm SVL in females, see
Izecksohn 1993); smaller snout in males, with 2.2 mm SL, n = 26 (2.7 mm SL, n = 5;
see Table 1); posterior side of belly color pale orange yellowish with tiny rounded
black or dark brown spots (white coloration with small black dots). From to A. vote
(Fig. 6C) snout subacuminated in dorsal view (rounded); posterior side of belly color
pale orange yellowish with tiny rounded black or dark brown spots (ventral color
pattern reddish brown, with presence of small white dots). From A. manaos (Fig. 6D)
snout subacuminated (snout truncate); dorsal skin finely granular (dorsal surfaces
granular); throat and chest grayish (dark coloration); posterior side of belly color pale
36
orange yellowish with tiny rounded black spots (ven- ter cream with black spots or
stripes). From to A. matses (Fig. 6E) snout subacumi- nated (snout slightly truncate),
edges of nasal protrusion not dilated (dilated in ventral view); shape of palmar
tubercle rounded (palmar tubercles elliptical); finger tips unex- panded (expanded),
rounded tiny black spots covering the belly (medium-sized black ocelli or streaks);
coloration of the belly pale yellow (belly completely yellow). From A. amazonicola
(Fig. 6F) by the absence of small triangular protrusion on the tip of the snout in both
dorsal and ventral views (presence); body surface granular (finely granu- lar), dorsum
uncovered with medium-sized granules scattered irregularly (covered with medium-
sized granules scattered irregularly); posterior side of belly color pale orange
yellowish with tiny rounded black or dark brown spots (orange yellowish with dark red
and brown blotches).
Description of the holotype. Body slender, head triangular, slightly longer than
wide; head length 35.5% of SVL, head width 30.9% of SVL. Snout long, subacumi-
nate in dorsal view, protruding in lateral view; canthus rostralis straight and loreal
region vertical; without papilla; snout length 39.0% of head length; tympanic
membrane and tympanic annulus not apparent through the skin, skin of the tympanic
area covered by round sub-conical warts; vocal sac externally not visible, vocal slits
absent; eyes prominent 23.8% of head length; upper eyelid covered with small
tubercles; those close to the external margin aligned in a more or less distinct row;
nostril closer to snout than to eyes; internarial distance smaller than eye diameter;
presence of a line of small spiny granules from the outer edge of the mouth to upper
arm, choanas small and circular. Dorsal skin finely tuberculate with several large
tubercles scattered sub-conical tubercles on upper arm; texture of ventral skin
granular, covered by rounded granules. Dorsolateral surfaces, granular, with
presence of large rounded tubercles. Forelimbs slender, upper arm length 29.6% of
SVL; edges of lower arm and upper arm finely tu-berculate with several large sub-
conical and spiny granules; hand length 76.5% of upper arm length; fingers slender,
tips not expanded; relative length of fingers I<II<IV<III; supernumerary tubercles and
accessory palmar tubercles present, palmar large and round- ed, supernumerary
tubercles low, small rounded; subarticular tubercles rounded and small, one tubercle
on fingers I, II and IV and two on finger III; fingers I and II basally webbed; indistinct
37
nuptial pad. Hind limbs slender; ventral skin from thigh to tarsus covered by spiny
tubercles, foot length 66% of thigh length; relative length of toes I<II<V<III<IV: inner
metatarsal tubercle oval, 2.5× larger than outer; outer metatarsal tubercles small,
rounded; subarticular tubercles present, rounded, present one on fin- gers I, II, and
two on fingers III, V and three on finger IV; and tip of toes not expanded.
Measurements of the holotype (in millimeters). SVL 15.1; HW 4.6; HL 5.3; SL 2.1; ED
1.2; IND 1.0; UAL 4.4; HAL 3.4; THL 8.1; TAL 8.1; TL 4.5; FL 5.3.
Coloration of the holotype. In life: dorsum of the holotype mostly light brown
with dark brown in the dorsum; dorsolaterally creamish-brown with scattered black
blotches; dorsal surfaces of hands and feet creamish-brown, and gray on arms and
legs; belly creamish-gray with black dots, and the throat gray; fingers, toes and
plantar sur- faces reddish-black; groin with orange marks; iris with a bronze ring;
cloaca with orange flap, black pupil and bronze iris. In alcohol: dorsum brownish-
grey; venter cream with black and brown dots; orange surfaces turned cream, with a
white longitudinal stripe on upper jaw extending from nostril to forearm.
Variation. The new species is phenotypically variable. In some individuals (e.g.
MHNC 8245 and MHNC 11002, see Fig. 5) patterning on the dorsum varies, with
these specimens presenting brown chevrons extending from the head to the vent.
Some individuals showed a white line extending from the tip of the nose to the upper
arm. Another specimen (MHNC 9739, see Fig. 5) presented a yellow pale coloration
in the axillary region (in ventral view). In some individuals, the coloration of the throat
ex- tended onto the chest (e.g. MHNC 11002, MHNC 9739 and MHNC 8245, see Fig.
5). The pale yellow coloration of the belly surface may extend from thighs to the chest
or just to the middle of the belly (e.g. MHNC 8362, see Fig. 5 and Fig. 7B). In some
individuals, the thighs are abundantly covered by rounded tiny spots extending to the
shank (Fig. 7B). In preserved specimens the dorsum becomes light brown and the
belly coloration vary from white to yellow pale (e.g. MHNSM 31255 and MHNSM
17993, see Fig. 5). The color of the finger becomes pale red and in other individuals
the red coloration of the fingers became brown or orange (Fig. 5).
Bioacoustics. The following values are presented as: min-max (average ± SD,
number of notes). The call is a trill type call issued during continuous and regular in-
38
tervals (Fig. 8). Each note had a duration of between 0.03 to 0.08 seconds (0.05 ±
0.01 seconds, n = 20). The number of pulses varied between 8 to 18 pulses per note
(10.4 ± 2.6 pulses/note, n = 20). The silence between notes varied from 0.4 to 1.6
seconds (0.8 ± 0.3 seconds, n = 20). The dominant frequency varied from 3962.1 to
3789.8 kHz (3927.6 ± 70.7 kHz, n = 20), and coincides with the fundamental
frequency. Time to peak amplitude was around 0.014 to 0.04 seconds (0.02 ± 0.01
seconds, n = 20).
Distribution, ecology and conservation. Amazophrynella javierbustamantei sp.
n. is known from the Department of Cusco, in the lower Urubamba river basin and
De- partment of Madre de Dios (Inambari, Candamo and Nueva Arequipa) in Peru
(Fig. 9). Its distribution can vary from 215 m a.s.l. to 708 m a.s.l. Additional
specimens were recorded at Los Amigos Biological Station , Tapir Lodge, and
Explorers Inn, in Tambopata National Reserve. Individuals were active during the day,
jumping on leaf litter, at night they were sleeping on leaves around 30 cm above
ground. This species breeds close to the edges of permanent oxbow lakes, males
call during the day while perched above streams in tangles (Cocroft et al. 2001).
Three of the localities, km 105, 107 and 117 of the highway Puerto Maldonado–
Cusco, Department Madre de Dios, show evidence of serious environmental impacts
due to illegal gold mining activities, with forest and soil removed, and environmental
pollution via organic and inorganic chemicals and heavy metal (specially mercury)
poisoning. In addition, the new species is distributed inside of territories where oil
companies are operating. On the other hand, the species is present in two protected
areas, the Tambopata Natural Reserve and Machiguenga Communal Reserve. The
conservation status of this species remains unknow, but was listed in 2008 as Least
Concern on the IUCN red list (2015), because it was confused with Amazophrynella
minuta, and because Amazophrynella minuta s.l. had wide distribution at that time,
apparent tolerance of a certain degree of habitat modification, presumed large
population, and because it is unlikely to be declining, and thus did not qualify for
listing in a more threatened category. With recent studies the genus, the species
complex of Amazophrynella minuta, was split in five species, three of them are now
formally described for Peru (Amazophrynella matses, A. amazonicola and A.
javierbustamantei sp. n.). The recognition of these new species will require the
39
reevaluation of the conservation status of these species. It should also act as an
impetus for additional field and laboratory studies of Peruvian amphibians, in order to
under- stand the real conservation status of this fauna.
Etymology. The species is named after Dr. Javier Bustamante, a Peruvian
residing in United States, to whom we dedicate this species in recognition of his
friendship and support of herpetological taxonomy and systematics research and
amphibian conserva- tion in Peru.
Discussion
Taxonomic reviews of Amazonian amphibians suggests that morphological
characters are too conservative to permit delimiting species since closely related
species share sim- ilar morphologies, and amphibians in general are morphologically
conservative (e.g., Elmer et al. 2007; Fouquet et al. 2007c; Funk et al. 2011; Padial et
al. 2009). Thus, the use of integrative techniques in taxonomy is revolutionizing the
identification and delimitation of species based on independent lines of evolutionary
evidence (Dayrat 2005; Padial and De la Riva 2009). The use of an integrative
approach not only allows for the discovery and delimitation of new species, it also
helps us to understand the mechanism of species formation. Thus, integrative
taxonomy allows us to have a better understanding of the true scope of anuran
diversity in the Amazon, and it allows us to better understand the processes that
generated this biodiversity.
The taxonomic ambiguity surrounding the name A. minuta and to a lesser
extent A. bokermanni resulted in a severe underestimation of the taxonomic diversity
of this genus. Since the descriptions of A. minuta in 1941 and A. bokermanni in 1993,
the taxonomy of the genus has not been revised, leading to misdiagnoses of other
species as either A. minuta or A. bokermanni due to the relatively generalized
descriptions of these taxa. Three publications since 2012 (Ávila et al. 2012; Rojas et
al. 2014, 2015) described four new species, increasing the taxonomic diversity of the
genus by 200%. All four species were previously classified as populations of a single
species with a large distribution (A. minuta sensu lato). Although striking, the severe
underestimation of taxonomic diversity observed in Amazophrynella and the
existence of multiple lineages in Amazophrynella minuta is nothing particular to this
40
group. Examples of other Ama- zonian species complexes include Rhinella
margaritifera and Scinax ruber, Pristimantis ockendeni, Pristimantis fenestratus,
Engystomops petersi, Hybsiboas fasciatus, Dendropso- phus minutus and
Osteocephalus taurinus (Fouquet et al. 2007; Elmer and Canatella 2008; Padial et al.
2009; Funk et al. 2011; Caminer and Ron 2014; Gehara et al. 2014, Jungfer et al.
2013). The descriptions by Rojas et al. (2014, 2015) were based, in part, on
diagnostic characters observed in the 16S rDNA. This gene is widely used as a DNA
barcode for amphibians, for reliable species identification (Vences et al. 2005,
Fouquet et al. 2007), for evaluating monophyly of species and for discovering
divergent lineages (Pa dial et al. 2009, Crawford et al. 2010; Padial et al. 2010 and
Padial et al. 2012). Based on 16S rDNA analyses, we also have evidence that A.
bokermanni and A. vote represent species complexes (RRRZ, personal observation).
This observation is in addition to the existence of the two candidate species of
Amazophrynella already observed in previous analyses: one from the Guiana Shield
(A. sp. aff. manaos), sister taxon of A. manaos, and another from Ecuador (A. sp. aff.
minuta), sister taxon of A. minuta sensu stricto (Fig. 1).
Although the taxonomic status of these candidate species will need to be con-
firmed using morphological and bioacoustics data, it is clear that even with the recent
descriptions, the taxonomic diversity of the genus remains underestimated. While
part of our evidence for the existence of the new species as well as those de- scribed
previously by Rojas et al. (2014, 2015) comes from the use of molecular data, the
descriptions make use of other data types and non-molecular diagnoses. Thus these
undiscovered lineages were not truly cryptic (morphologically cryptic), but rather the
result of poor taxonomic knowledge of the group. In this respect, the genus Amazo-
phrynella again is not the exception, but rather the norm.
The species A. javierbustamantei sp. n. is clearly differentiated in multi- variate
morphometric space from the other members of the Amazophrynella minuta “species
group” (A. minuta, A. amazonicola and A. matses). Together with the de- scription of
Amazophrynella javierbustamantei sp. n. we also provide advertisement call.
Amazophrynella javierbustamantei sp. n. is only the second species of the ge- nus for
which an advertisement call is known and recorded (see Duellman 1978). Acoustics
can provide evidence of potentially new species with behavioral or pre- mating
41
isolating mechanisms (e.g. De la Riva et al. 1997; Gerhardt 1998; Simões et al. 2008,
Padial and De la Riva 2009; Padial et al. 2012), thus providing evidence of
evolutionary mechanisms that contributed to the species diversity of the genus
Amazophrynella.
The threats to the biological conservation of A. javierbustamantei sp. n. are
evident, with uncontrolled exploration for gold, illegal mining and the destruction of
habitat in the Departments of Madre de Dios and Cusco, probably causing a
significant reduc- tion in the population sizes of the species and fragmenting its
distribution. For these reasons is necessary to analyze the current population status
and trends of this and another amphibian species in this Department of southern
Peru.
Acknowledgements
RRRZ was supported by a fellowship from Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (Coordenação de Aperfeiçoamento de Pessoal de
Nível Su- perior - CAPES), RRRZ also thanks Nora M. Rojas Ruiz, Silvia Rojas,
Marco Rojas Ruiz for hospitality in Lima, Elda Zamora, José Armas, Micaela Perea,
Ronald Rojas and Miguel Rojas in Iquitos and Edson Caceres, Fababa and Douglas
Giardini in Puerto Maldonado, Peru. For allowing access to the respective
herpetological collec- tions under their care, we are grateful to Richard C. Vogt
(Instituto Nacional de Pes quisas da Amazonônia - INPA), Göran Nilson
(Naturhistoriska Museet, Göteborg - NHMG), Marcos A. Carvalho (Universidade
Federal de Mato Grosso - UFMT) and Juan Carlos Cusi (Museo de Historia Natural
de la Universidad Nacional Mayor de San Marcos - MUSM), Percy Yanque and Rocio
Orellana (Museo de Historia Natural del Cusco - MHNC). We are grateful to
Asociación para la Conservación de la Cuenca Amazonica (ACCA) which partially
funded the expedition to Madre de Dios, under the project Manu-Tambopata
Conservation Corridor,that resulted in the discovery of the new species. VTC was
supported by a fellowship from Fundação de Amparo a Pesquisa do Estado do
Amazonas (FAPEAM), IF and TH were support- ed by research productivity
fellowships from the Conselho Nacional de Desenvolvi- mento Cientifico e
Tecnológico (CNPq). Collecting permits in Peru were granted by Dirección General
42
Forestal y de Fauna Silvestre (DGFFS) del Ministerio del Me- dio Ambiente (MINAM-
N°-0320, RJ-RCM-N°-001-2009-SERNANP-RCM, RJ- RCA-N°-002-2009-
SERNANP-RCAS, RD-N°-435-2009-AG-DGFFS-DGEFFS and RD-Nº-038-2010-AG-
DGFFS-DGEFFS). We are grateful to Mario Nunes for his assistance with laboratory
work.
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Appendix 1
Specimens examined
Amazophrynella minuta—BRAZIL: Taracuá, Uaupés River: INPA-H 32725, INPA-H
32723, INPA-H 32729, INPA-H32730, INPA-H32736, INPA-H32731 (females) and
INPA-H 32724, INPA-H32728, INPA-H 32733, INPA-H 32735, INPA-H 32722, INPA-H
32738, INPA-H 32737, INPA-H 32739. INPA-H 32720, INPA-H 32732, INPA-H 32726,
INPA-H 32730, INPA-H 32740, INPA-H 32734, INPA-H 32721 (males).
Amazophrynella bokermanni—BRAZIL: Juriti, Pará: INPA-H 31861, INPA-H 31864,
INPA-H 31863, INPA-H 31862, INPA-H 31865, municipality of Juriti, Pará State, Brazil
(50 km from type locality).
Amazophrynella vote—BRAZIL: Fazenda São Nicolau, Cotriguaçu, Mato Grosso
(Holotype: UFMT-A 11138); Madeira River, Manicoré (Paratypes: INPA-H 12256,
12331, 12255, 12342, 12343, 12366, 12267); Aripuanã River, Novo Aripuanã
(Paratype: INPA-H 12326); Parque Estadual do Guariba: Manicoré (Paratypes: INPA-
H 21558); Parque Nacional Nascentes do Lago Jari, Tapauá: Amazonas (Paratypes:
INPA-H 27412, 27417-27419, 27421-27423, 27425-27426).
Amazophrynella manaos—BRAZIL: Campus da Universidade Federal do Amazonas,
Amazonas (Holotype: INPA-H 31866, paratypes: INPA-H 6983, INPA-H 6984, INPA-H
6987, INPA-H 7797); Presidente Figueiredo, Amazonas (Paratypes: INPA-H 29568,
INPA-H 29569, INPA-H 29571, INPA-H 29570, INPA-H 29572, INPA-H 20986; INPA-
H 21217, INPA-H 30577, INPA-H 30575, INPA-H 30573, INPA-H 30572, INPA-H
30576); Reserva Florestal Adolpho Ducke, Amazonas (INPA-H 21028, INPA-H
21170, INPA-H 21060, INPA-H 31866, INPA-H 21007, INPA-H 21008, INPA-H 21009,
INPA-H 21010, INPA-H 21011, INPA-H 21012, INPA-H 21013).
Amazophrynella amazonicola—PERU: Puerto Almendra San Juan Bautista, Loreto
(Holotype: MZUNAP 901, paratopotypes: MZUNAP 906; MZUNAP 915; MZUNAP
110; MZUNAP 907, MZUNAP 917; MZUNAP 889; MZUNAP 910; MZUNAP 911;
MZUNAP 916; MZUNAP 913; MZUNAP 914; paratypes: MZUNAP 906; MZUNAP
915; MZUNAP 110; MZUNAP 907, MZUNAP 917; MZUNAP 889; MZUNAP 910;
MZUNAP 911; MZUNAP 916; MZUNAP 913; MZUNAP 914); 58 km of Iquitos–Nauta
49
highway on Fundo Zamora, San Juan Bautista, Loreto (Paratypes: MZUNAP 908,
MZUNAP 924, MZUNAP 886, MZUNAP 900, MZUNAP 888, MZUNAP 919, MZUNAP
902, MZUNAP 887, MZUNAP 905, MZUNAP 920); Nauta, Maynas (Paratypes:
MZUNAP 918, MZUNAP 909); Fundo UNAP, Maynas, Loreto (Paratype: MZUNAP
242)
Amazophrynella matses—PERU: Nuevo Salvador, Requena, Loreto (Holotype:
MZUNAP 921, paratopotypes: MZUNAP 934, MZUNAP 955 MZUNAP 940, MZUNAP
948 MZUNAP 943, MZUNAP 952, MZUNAP 953, MZUNAP 958, MZUNAP 922,
MZUNAP 923, MZUNAP 925, MZUNAP 926, MZUNAP 927, MZUNAP 944, MZUNAP
938, MZUNAP 936); Jenaro Herrera, Requena, Loreto (Paratypes: MZUNAP 928,
MZUNAP 929, MZUNAP 930, MZUNAP 931, MZUNAP 933, MZUNAP 955, MZUNAP
935, MZUNAP 950, MZUNAP 937, MZUNAP 939, MZUNAP 941, MZUNAP 942,
MZUNAP 946, MZUNAP 947, MZUNAP 949).
50
Fig. 1. Maximum Likelihood tree of the Amazophrynella species based on the
GTR+I+G model, using 480 bp of 16S rDNA. Numbers below branches represent
bootstrap support with 1000 replications.
51
Fig. 2. Principal Component Analysis (PCA) of the Amazophrynella minuta species
complex. See Table 4 for character loadings on each component.
Fig. 3. Measurement comparison of the Hand Length (HAL) between species of the
Amazophrynella minuta complex.
52
Fig. 4. Holotype of Amazophrynella javierbustamantei sp. nov. (MHNC 8331); A)
dorsal view; B) ventral view; C) dorsolateral view; D) right hand; E) right foot.
53
Fig. 5. Dorsal and ventral view of some paratypes of Amazophrynella
javierbustamantei sp. nov. MHNC 8245; MHNSM 31255; MHNSM 17993 (Adult
males); MHNC 1102, MHNC 9739, MHNC 8362 (Adult females).
54
Fig. 6. Dorsal and ventral morphological comparison between the Amazophrynella
spp. (non-voucher specimens): A) A. javierbustamantei sp. nov.; B) A. minuta; C) A.
bokermanni; D) A. vote; E) A. manaos; F) A. matses; G) A. amazonicola.
55
Fig. 7. Dorsal and ventral variation of Amazophrynella javierbustamantei sp. nov.
(non-voucher specimens): A-C) Nueva Arequipa, Madre de Dios Dept., Peru; B)
Basin of Bajo Urubamba, Cusco Dept., Peru.
56
Fig. 8. Advisement call of Amazophrynella javierbustamantei sp. nov. from the
Tambopata Reserve, Madre de Dios, Peru (207 meters a.s.l.) (©Macauly Library of
Natural Songs and ©Cornell Laboratory of Ornithology) by the authors Crocoft,
Morales and Mc Diarmid (2007). A) Oscilogram and spectrogram by one note; B)
Oscilogram and spectrogram of notes from the advisement call.
57
Fig. 9. Distribution map of Amazophrynella javierbustamantei sp. nov. records in
Peru. Red squares represent paratypes and the star the type locality (where the
holotype has been collected.
58
Table 1. Measurements (mm) of adult male specimens (including the holotype) in the type series of Amazophrynella spp. Mean ±
standard deviation, with ranges in parentheses. Abbreviations are defined in Material and methods.
Variable A. minuta s.s.
(n=15)
A. manaos s.s.
(n=29)
A. bokermanni
(n=5)
A. vote
(n=14)
A. amazonicola
(n=15)
A. matses
(n=13)
A. javierbustamantei sp. nov.
(n=26)
SVL 13.5±0.6 (12.5-14.2)
14.2±0.7 (12.3-15.0)
16.8±1.4 (14.6-18.2)
13.1±0.7 (12.0-14.1)
14.5±0.7 (13.3-15.4)
12.1±0.6 (11.5-13.5)
14.9±0.9 (12.7-16.4)
HW 4.2±0.2 (4.0-4.3) 4.2±0.3 (3.7-4.7)
3.2±0.3 (2.5-3.3)
4.0±0.7 (3.3-4.4)
4.4±0.3 (4.2-4.6) 3.6 ±0.2 (3.1-3.8)
4.2 ±0.2 (3.5-4.7)
HL 4.9±0.2 (4.8-5.3) 5.3±0.3 (4.7-5.6)
3.4±0.4 (2.8-3.8)
4.6±0.3 (4.0-5.2)
5.2±0.3 (5.0-6.2) 4.3 ±0.3 (3.9-4.8)
5.1 ±0.3 (4.4-5.6)
SL 2.3±0.1 (2.2-2.5) 2.7±0.2 (2.3-2.7)
3.0±0.4 (2.2-3.1)
2.1±0.2 (1.9-2.6)
2.4±0.2 (2.2-2.5) 2.0 ±0.3 (1.6-2.3)
2.2 ±0.2 (1.7-2.6)
ED 1.4±0.1 (1.3-1.5) 1.3±0.1 (1.2-1.6)
1.8±0.2 (1.5-2.0)
1.3±0.1 (1.2-1.5)
1.2±0.1 (0.9-1.2) 1.1 ±0.1 (0.9-1.2)
1.3 ±0.1 (1.0-1.6)
IND 1.2±0.1 (1-1.3) 1.1±0.1 (1.0-1.4)
1.4±0.2 (1.0-1.5)
1.1±0.1 (1.0-1.3)
1.2±0.1 (1.0-1.3) 1.0 ±0.1 (0.8-1.2)
0.9 ±0.1 (0.8-1.2)
UAL 3.8±0.2 (3.2-4.1) 3.6±0.4 (2.9-4.1)
5.5±0.6 (5.0-5.6)
3.9±0.5 (2.8-3.9)
4.5±0.3 (4.2-5.3) 3.5 ±0.4 (2.9-4.2)
4.5 ±0.4 (3.8-5.7)
HAL 2.8±0.2 (2.6-3.0) 2.8±0.6 (1.9-2.9)
3.4±0.6 (2.8-4.2)
2.7±0.3 (2.3-3.2)
3.2±0.2 (2.8-3.3) 2.7 ±0.2 (2.3-3.1)
3.6 ±0.4 (2.5-4.5)
59
THL 6.8±0.2 (6.4-7.2) 6.7±0.3 (2.3-3.1)
8.7 ±1.4 (7.2-8.9)
6.5±0.7 (5.4-7.2)
7.7±0.6 (6.3-8.0) 6.2 ±0.4 (5.1-6.3)
7.6 ±0.7 (6.2-9.2)
TAL 6.7±0.3 (6.3-7.1) 6.9±0.6 (4.2-7.3)
8.3±1.0 (6.7-9.2)
5.7±0.7 (4.8-7.0)
7.2±0.6 (6.1-7.9) 5.8 ±0.3 (5.1-6.3)
7.6 ±0.7 (6.2-8.8)
TL 4.1±0.2 (3.8-4.6) 4.6±0.4 (4.3-6.3)
5.4±1.4 (2.9-6.2)
3.8±1.0 (4.2-7.0)
4.2±0.6 (6.3-8.0) 3.8 ±0.2 (3.6-4.3)
4.7 ±0.8 (3.9-8.7)
FL 4.8±0.4 (4.2-5.2) 5.2±0.5 (4.7-6.1)
6.3±1.3 (3.9-7.6)
4.4±0.6 (3.2-5.4)
5.1±0.4 (4.7-6.0) 4.3 ±0.4 (5.5-3.0)
5.7 ±0.6 (4.5-7.2)
60
Table 2. Uncorrected p-distances between Amazophrynella species and one representative of the sister genus Dendrophryniscus.
Molecular distances are based on the 480-bp fragment of 16S rDNA. We included A. minuta s.s. from its type locality and the two
candidate species Amazophrynella aff. manaos and A. aff. minuta from Fouquet et al. (2012a).
16S rDNA 1 2 3 4 5 6 7 8 9 10
1 A. amazonicola
2 A. matses 0.08
3 A. aff. minuta 0.06 0.07
4 A. minuta 0.05 0.08 0.03
5 A. javierbustamantei sp. nov. 0.09 0.03 0.06 0.07
6 A. vote 0.12 0.12 0.12 0.12 0.13
7 A. bokermanni 0.12 0.12 0.11 0.11 0.13 0.10
8 A. manaos 0.12 0.12 0.12 0.14 0.12 0.10 0.08
9 A. aff. manaos 0.12 0.11 0.12 0.13 0.12 0.10 0.07 0.04
10 D. leucomystax 0.19 0.21 0.17 0.18 0.20 0.22 0.18 0.20 0.20
61
Table 3. Species level diagnostic characters observed in the 16S rDNA gene of
Amazophrynella javierbustamantei sp. nov. and other species of Amazophrynella.
First line indicates position of the character within the 16S rDNA gene; (-) indicates a
deletion.
Species 213 232 271 276 470 471 473 474 476 477 478 479 480
A. manaos A C A C A T G T C A A A A
A. vote A T A C C C C T T A A A G
A. minuta C T A A C C C T T A A A G
A. bokermanni A T A C A T G T C A A A A
A. amazonicola C C A C C C C T T A A T G
A. javierbustamantei T G G T T G T G A G C C -
A. matses C T A C C C C T T A A T T
62
Table 4. Character loadings, eigenvalues, and percentages of explained variance for
Principal Components (PC) 1–2. The analysis was based on eleven morphometric
variables of adult males of the Amazophrynella minuta complex (A. minuta sensu
strictro; A. amazonicola; A. matses and A. javierbustamantei sp. nov.)
Variables PC1 PC2
HW 0.462 -0.146
HL 0.455 -0.104
SL 0.374 -0.244
ED 0.261 0.052
IND 0.369 -0.271
UAL 0.139 0.258
HAL -0.032 0.484
THL 0.311 -0.295
TAL 0.314 0.350
TL 0.116 0.364
FL 0.063 0.433
% of variation 24.93 23.63
% 24.93 48.56
63
Table 5. Character loadings of explained variance for Discriminant Function Analysis
(DFA). The analysis was based on twelve morphometric variables of adult males of
the Amazophrynella minuta complex (A. minuta sensu stricto; A. amazonicola; A.
matses and A. javierbustamantei sp. nov.)
Variables Discriminant Function
SVL 6.343
HW -7.628
HL 0.146
SL -5.479
ED -1.175
IND -6.015
UAL 1.313
HAL 5.744
THL -3.871
64
CAPITULO II
A Pan-Amazonian species delimitation: high species diversity within the genus
Amazophrynella (Anura: Bufonidae). Rojas, R.R., Fouquet, A., Ron, S., Hernandez,
E., Melo-Sampaio, P., Chaparro, J., Vogt, R., Carvalho, V., Pinheiro, L., Ávila, R.,
Pires, I., Gordo, M. & Hrbek, T. (2018). PeerJ, 6, e4941.
65
A Pan-Amazonian species delimitation: high species diversity within the genus
Amazophrynella (Anura: Bufonidae)
Rommel R. Rojas1*, Antoine Fouquet2, Santiago R. Ron3, Emil José Hernández-
Ruz4, Paulo R. Melo-Sampaio5, Juan C. Chaparro6,7, Richard C. Vogt8, Vinícius
Tadeu de Carvalho1, Leandra Cardoso Pinheiro9, Robson W. Ávila10, Izeni Pires
Farias1, Marcelo Gordo11, Tomas Hrbek1*
1Laboratory of Evolution and Animal Genetics (LEGAL), Department of Genetics,
ICB, Universidade Federal do Amazonas, Manaus, AM, Brazil
2 USR 3456 LEEISA - Laboratoire Ecologie, Evolution et Interactions des Systèmes
Amazoniens, Centre de recherche de Montabo, Cayenne, French Guiana.
3 Museo de Zoología, Escuela de Biología, Pontificia Universidad Católica del
Ecuador, Quito, Ecuador.
4 Laboratório de Zoologia, Faculdade de Ciências Biológicas, Campus Universitário
de Altamira, Universidade Federal do Pará, Altamira Pará, Brazil.
5 Departamento de Vertebrados, Museu Nacional, Rio de Janeiro, Brazil
6 Museo de la Biodiversidad del Peru, Cusco, Peru.
7 Museo de Historia Natural de la Universidad Nacional de San Antonio Abad del
Cusco, Peru.
8 CEQUA, Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da
Amazônia, Manaus, AM, Brazil.
9 Museu Paraense Emilio Goeldi, Belem, Pará, Brazil.
10 Departamento de Ciências Biológicas, Centro de Ciências Biológicas e da Saúde,
Universidade Regional do Cariri , Crato, Brazil.
11 Departamento de Biologia, ICB, Universidade Federal do Amazonas, Manaus, AM,
Brazil.
* Correspondence: [email protected]; [email protected]
66
Abstract
Amphibians are probably the most vulnerable group to climate change and
climate-change associate diseases. This ongoing biodiversity crisis makes it thus
imperative to improve the taxonomy of anurans in biodiverse but understudied areas
such as Amazonia. In this study, we applied robust integrative taxonomic methods
combining genetic (mitochondrial 16S, 12S and COI genes), morphological and
environmental data to delimit species of the genus Amazophrynella (Anura:
Bufonidae) sampled from throughout their pan-Amazonian distribution. Our study
confirms the hypothesis that the species diversity of the genus is grossly
underestimated. Our analyses suggest the existence of eighteen linages of which
seven are nominal species, three Deep Conspecific Lineages, one Unconfirmed
Candidate Species, three Uncategorized Lineages, and four Confirmed Candidate
Species and described herein. We also propose a phylogenetic hypothesis for the
genus and discuss its implications for historical biogeography of this Amazonian
group.
67
Introduction
Amphibians are undergoing a drastic global decline (Beebee & Griffiths,
2005). This decline is primarily attributable to habitat destruction, diseases (chytrid
fungus) and global climate change (Collins, 2010). In Amazonia the primary threat is
habitat destruction, although the chytrid fungus has reached the Amazon basin
(Valencia-Aguilar et al., 2015; Becker et al., 2016), and is starting to have an impact
on Amazonian and Andean anurans (Lötters et al., 2005, 2009; Catenazzi & von
May, 2014). Most Amazonian amphibians are thought to have broad, often basin
wide distributions, although their geographic distributions are generally poorly known.
More detailed analyses generally reveal the existence of multiple deeply divergent
lineages, suggesting cryptic diversity. Fouquet et al. (2007) estimated that amphibian
diversity of Amazonia is underestimated by 115%, while Funk et al. (2011) suggest
this underestimate is closer to 150–350%. But even without taking into account the
high levels of crypsis or pseudocrypsis (morphological differences apparent but
overlooked) in widespread Amazonian anurans, Amazonia has the highest diversity
of amphibians on this planet (Jenkins et al., 2013).
Delimiting species and their geographic distributions is therefore crucial for the
understanding of impacts on the biodiversity of Amazonian anurans, and for the
assessment of their conservation status (Angulo & Reichle, 2008). Previous studies
suggest a prevalent conservatism in the morphological evolution of anurans (eg.
Elmer et al., 2007; Robertson & Zamudio, 2009; Vences et al. 2010; Kaefer et al.,
2012; Rowley et al., 2015), thus, species delimitation based solely on morphological
characters may fail to differentiate among species. Conversely, delimiting species
solely based on molecular characters or genetic distances harbors potential pitfalls
that have been well documented (eg. Carstens et al., 2013; Sukumaran & Knowles,
2017). Environmental data also have the potential to provide important information to
taxonomy since species have distinct ecological requirements that determine their
occurrence in time and space (Soberón et al., 2005). Therefore, species delimitation
relying on a pluralistic approach seeking to unite several lines of evidence (Dayrat,
2005; Padial et al., 2010) generally provides robust and consensual taxonomic
hypotheses (eg. Padial & De La Riva, 2009) especially in morphologically conserved
68
groups, i.e. taxonomic groups harboring cryptic or pseudocryptic taxa (Cornils &
Held, 2014).
The frog genus Amazophrynella Fouquet, Recoder, Teixeira, Cassimiro,
Amaro, Camacho, Damasceno, Carnaval, Moritz, & Rodrigues 2012a is distributed
throughout Amazonia, and currently comprises seven small-sized (12.0–25.0 mm)
species (Fouquet et al., 2012b). All species inhabit the forest leaf litter (Rojas et al.,
2015), breed in seasonal pools and have diurnal and crepuscular habits (Fouquet et
al., 2012b; Rojas et al., 2014, 2016).
Until 2012, only two species were recognized: Amazophrynella minuta from
western Amazon and A. bokermanni from eastern Amazon (Fouquet et al., 2012b).
Since 2012 five additional species have been described from western Amazon (A.
vote, A. manaos, A. amazonicola, A. matses and A. javierbustamantei). The
taxonomy of the genus remains, however, far from being resolved (Rojas et al.,
2016). Although molecular phylogenetic analyses in Fouquet et al. (2012b) and Rojas
et al. (2015, 2016) provided evidence for the existence of multiple lineages, the
scarcity of material suitable for morphological and bioacoustic analyses prevented
the description of these lineages as new species.
In this study, we revisit the genus Amazophrynella, include specimens from
new localities, and reconstruct intra- and inter-specific phylogenetic relationships. We
delimit candidate species based on molecular data and subsequently seek support
for these lineages combining qualitative and quantitative morphological data and
environmental evidence. As a result of these analyses, we formally describe four new
species of Amazophrynella from Brazil, Ecuador, French Guiana and Peru, and
identify additional seven candidate species. Additionally, we provide new insights into
the overall phylogenetic relationships for the genus, and discuss biogeographic
history of this Amazonian group.
Material and methods
Protocol for species delimitation
We evaluated the status of populations of Amazophrynella, adhering to the
unified species concept proposed by De Queiroz (2007), that conceptualizes species
69
as lineages of ancestor-descendent populations which maintain their distinctness
from other such lineages and which have their own evolutionary tendencies and
historical fates. We followed the consensus protocol of integrative taxonomy
proposed by Padial et al. (2010). The concept of candidate species adopted in this
study follows the subcategories proposed by Vieites et al. (2009) in using: Confirmed
Candidate Species (CCS) for lineages that present high genetic distance and can be
differentiated by other traits (i.e. morphological data), Deep Conspecific Lineages
(DCL) for lineages that are genetically divergent but not supported by any other
character (these characters being available), Unconfirmed Candidate Species (UCS)
for lineages that are genetically divergent but no additional characters are available
to support this divergence (these characters not available) and Uncategorized
Lineages (UL) for lineages that do not corresponds to any of the above categories.
Focal species and morphological examination
Field work and visits to museum collections were carried out between 2011
and 2017. Field collection of specimens followed the technique of visual encounter
surveys and pitfall-barrier traps (Crump & Scott,1994). Museum acronyms are found
in Sabaj (2016) except for Museo de Biodiversidad del Peru (MUBI; this collection is
part of Museo de Historia Natural, Universidad Nacional de San Antonio Abad,
Cusco, Peru). Collecting permits in Peru were granted by Dirección General Forestal
y de Fauna Silvestre del Ministerio del Medio Ambiente (MINAN; No. AUT-IFS-2017-
055), in Ecuador by Ministerio del Ambiente (MA; 001-1-IC-FAU-DNB/MA) and in
Brazil by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio; No.
39792-1 and No. 32401). The material of Amazophrynella teko from Mitaraka
(French Guiana) was collected during the “Our Planet Reviewed” expedition,
organized by the MNHN and Pro-Natura International.
We examined topotypical material of Amazophrynella minuta deposited at the
collection of Amphibians and Reptiles of the Instituto Nacional de Pesquisas da
Amazônia–INPA (INPA–H) and three syntypes (NHMG 462, NHMG 463, NHMG 464)
deposited at the Göteborgs Naturhistoriska Museum, Sweden; five specimens of A.
bokermanni (Izecksohn, 1993) from near the type locality (c. 30 Km) deposited at the
INPA collection; the type series of A. vote (Ávila et al., 2012) deposited at the
70
Coleção Zoológica de Vertebrados of the Universidade Federal de Mato Grosso–
UFMT, Cuiabá, Mato Grosso, Brazil (UFMT–A) and INPA; A. manaos (Rojas et al.,
2014) deposited at the INPA; A. amazonicola and A. matses (Rojas et al., 2015)
deposited at the Museo de Zoología–Universidad Nacional de la Amazonia Peruana–
UNAP and A. javierbustamantei (Rojas et al., 2016) deposited at the Museo de
Biodiversidad del Peru (MUBI), Museo de Historia Natural de la Universidad Nacional
Mayor de San Marcos (MHNSM). List of examined specimens is found in Appendix
S1.
Qualitative morphological terminology was according to Kok & Kalamandeen
(2008). Morphological comparison between specimens were made through visual
inspection of diagnostic characters that include: dorsal skin texture, ventral skin
texture, head shape, shape of palmar tubercle, relative length of fingers and venter
coloration (Fouquet et al., 2012b, Rojas et al., 2014, 2015, 2017). We used ventral
incision to perform gonadal analyses . Developmental stages of tadpoles were
determined using Gosner's protocol (1960). Descriptive terminology, morphometric
variables and developmental stages of tadpoles follow Altig & McDiarmid (1999).
Spectral and temporal parameters of advertisement calls (when available) were
analyzed in the software Praat for Windows (Boersma & Weenink, 2006).
Bioacoustics terminology followed Köhler et al. (2017).
Morphological quantitative analyses
Quantitative measurements of body were obtained with a digital caliper (0.1
mm precision) following Kok & Kalamandeen (2008) with the aid of an ocular
micrometer in a Leica stereomicroscope. Measurements were taken from the right
side of specimens, and, if this was not feasible, from the left side. Measurements
were: SVL (snout-vent length) from the tip of the snout to the posterior margin of the
vent; HL (head length) from the posterior edge of the jaw to the tip of the snout; HW
(head width), the greatest width of the head, usually at the level of the posterior
edges of the tympanum; ED (eye diameter); IND (internarinal distance), the distance
between the edges of the nares; SL (snout length) from the anterior edge of the eye
to the tip of the snout; HAL (hand length) from the proximal edge of the palmar
tubercle to the tip of finger III; UAL (upper arm length) from the edge of the body
71
insertion to the tip of the elbow; THL (thigh length) from the vent to the posterior edge
of the knee; TL (tibia length) from the outer edge of the knee to the tip of the heel;
TAL (tarsal length) from the heel to the proximal edge of the inner metatarsal
tubercle; FL (foot length) from the proximal edge of the inner metatarsal tubercle to
the tip of toe IV. We rounded all measurements to first decimal place to avoid
pseudoprecision (Hayek, Heyer & Gascon, 2001).
Principal Component Analyses (PCA) were performed on residuals obtained
by linear-regressing each variable on SVL, thus removing the effects of size. We
used only males specimens because of absence of females in some lineages. The
PCA was used to detect groups representing putative species. We also performed a
discriminant Function Analysis (DFA) to identify morphometric variables that
contribute the most to species separation and to test the classification of specimens
into mtDNA lineages. For DFA we used morphometric size-free data set. To
determine the number of correct and incorrect assignments of specimens to each of
the mtDNA lineages, we jackknifed our data matrix. The significance of differences of
morphological variables among mtDNA lineages was tested using the Kruskal-Wallis
(KW) non-parametric test. All the statistical analyses (PCA, DFA and KW) were
performed in R v3.4.3 (R Development Core Team) using the stats package and
setting the significance cut–off at 5%.
DNA amplification
DNA extraction, gene amplification and sequencing was carried out using
standard protocols (Appendix S2). Sequence data were deposited in GenBank under
the accession numbers MH269714–MH270330 (Table S2a).
Phylogenetic analyses and species delimitation
We collected molecular data for 230 individuals of Amazophrynella from 35
localities, including topotypical material for all nominal species and encompassing the
entire distribution of the genus. We obtained a total of 1430 bp from three
mitochondrial loci [16S rRNA (16S), 480 bp; 12S rRNA (12S), 350 bp; and
Cytochrome oxidase subunit I (COI), 600 pb (see Appendix S4, Table S4a)]. The
edition and alignment of the sequences was performed using Geneious v.6.1.8.
(Kearse et al., 2012) and the Clustal W algorithm (Thompson et al., 2002). We used
72
only unique haplotypes for phylogenetic reconstruction. We concatenated all loci,
treating them as a single partition evolving under the same model of molecular
evolution. The best model of molecular evolution (GTR+G+I) was estimated in
JModelTest (Posada, 2008) and selected using the Akaike Information Criterion–AIC.
Phylogenetic analyses were performed using Bayesian Inference (BI) using MrBayes
3.2.1. (Huelsenbeck & Ronquist, 2001). We generated 107 topologies, sampling
every 1000th topology and discarding the first 10% topologies as burn-in. The
stationarity of the posterior distributions for all model parameters was verified in
Tracer v1.5 (Rambaut & Drummond, 2009). From the MCMC output, we generated
the final consensus tree-maximum clade credibility tree using Tree Annotator v1.6.2
(part of Beast software package). For visualization and edition of the consensus
maximum clade credibility tree, we used the program Figtree v.1.3. (Rambaut, 2009).
We used a Poisson tree processes (PTP) model (Zhang et al., 2013) to infer
the most likely number of species in our dataset, as implemented in the bPTP server
(http://species.h-its.org/ptp/). The PTP model is a simple, fast and robust algorithm to
delimit species using non-ultrametric phylogenies, ultrametricity is not required
because the algorithm models speciation rates by directly using the number of
substitutions. The fundamental assumption is that the number of substitutions
between species is significantly higher than the number of substitutions within
species. In a sense, this is analogous to the GMYC (General Mixed Yule Coalescent)
approach that seeks to identify significant changes in the rate of branching events on
the tree. However, GMYC uses time to identify branching rate transition points,
whereas, in contrast, PTP directly uses the number of substitutions (Zhang et al.,
2013). For input, we used a BI tree estimated by MrBayes. We ran the PTP analyses
using 10⁵ MCMC generations, thinning value of 100, a burn-in of 10%, and opted for
remove the outgroup to improve species delimitation. Convergence of MCMC chain
was confirmed visually. To ensure that the lineages detected using PTP presented
high genetic distance (>3.0%, sensu Fouquet et al., 2007) we calculated uncorrected
p-distance using the 16S mtDNA (Vences et al., 2005) in the program MEGA 7.0
(Kumar, Stecher & Tamura, 2016).
To generate a dated tree in Beast 2.0 (Drummond & Rambaut, 2007), we
selected one representative individual per species. We used a birth and death prior,
73
GTR+I+G evolution model and calibrated the tree using normal distribution following
the divergence time estimates of Fouquet et al. (2012a): crown age of Hyloidea
(mean = 77.0 ± 10 Ma); basal divergence time of Bufonidae (mean = 67.9 ± 12 Ma);
divergence of Atelopus + Oreophrynella vs. other Bufonidae (mean = 60.0 ± 11 Ma);
Nannophryne vs. other Bufonidae (mean = 47.0 ± 8 Ma); Rhaebo vs. other crown
Bufonidae (mean = 40.8 ± 7 Ma) and Dendrophryniscus vs. other crown Bufonidae
(mean = 52.1 ± 9). We generated 107 topologies, sampling every 1000th topology and
discarding the first 10% topologies as burn-in. Stationarity of the posterior
distributions for all model parameters was verified in Tracer v1.5 (Rambaut &
Drummond, 2009). From the MCMC output, we generated the final consensus
maximum clade credibility tree using Tree Annotator v1.6.2 (part of Beast software
package). For visualization and edition of the consensus tree, we used the program
Figtree v.1.3. (Rambaut, 2009).
Environmental analyses
The environmental analyses were undertaken in order to test if delimited
species occur in distinct climatic environments (Soberón et al. 2005). We retrieved
high resolution bioclimatic layers (30 arc–seconds ~ 1 km, present environmental
conditions) using the Community Climate System Model- (CCSM4) from the
WorldClim project (http://www.worldclim.org/) (Hijmans et al., 2005). To avoid
geographic pseudoocurrence of points, localities were filtered using the program
Geographic Distance Matrix Generator 1.2.3. (Ersts, 2014) considering a threshold of
1 km between localities. The localities of each lineage used for analyses are in
Appendix S3, Table S3a.
To identify environmental variables that were most informative and test the
classification of specimens into mtDNA lineages using ecological variables, we
performed Principal Component Analysis (PCA) and Discriminant Function Analysis
(DFA) separately for each lineages/species of the eastern and western clades. The
analyses were performed using the 19 BioClim environmental variables in WordClim.
Probability of correct assignment of individuals to lineages was tested using
jackknife.
Electronic publication of new zoological taxonomic names
74
The electronic version of this article in Portable Document Format (PDF) will
represent a published work according to the International Commission on Zoological
Nomenclature (ICZN), and hence the new names contained in the electronic version
are effectively published under that Code from the electronic edition alone. This
published work and the nomenclatural acts it contains have been registered in
ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life
Science Identifiers) can be resolved and the associated information viewed through
any standard web browser by appending the LSID to the prefix http://zoobank.org/.
The LSID for this publication is: urn:lsid:zoobank.org:pub:1C6046BE-CFC4-4060-
A1CA-0C9C9C1C7A0A. The online version of this work is archived and available
from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.
Results
Phylogenetic and species diversity
The concatenated data resulted in a strongly supported phylogeny (Fig. 1),
with high degree of divergence among putative and nominal species of
Amazophrynella. The PTP model of species delimitation detected a total of eighteen
lineages (posterior probability = 0.48–0.91) (Appendix S4, Fig. S4a) of which seven
are nominal species and 11 are candidate species.
The phylogeny of Amazophrynella recovered the presence of two clades
diverging basally, both strongly supported: one distributed in eastern and other in
western Amazonia (see Fig. 1A). The eastern clade was formed by two strongly
supported subclades, herein called northeastern (NE) and southeastern (SE) clades.
The northeastern clade included three lineages and the southeastern clade seven
lineages. The western clade was formed by two well supported subclades, herein
called northwestern (NW) and southwestern (SW) clades. Both subclades were
composed of four lineages (see Fig. 1A). Uncorrected p-distances for 16S mtDNA
between pairs of sister lineages are presented in Table 1. Each lineage presented
high genetic divergence (>3.0%) compared to its sister taxon and ranged between
3.0–3.2% (3.0 ± 0.1) to 4.0–6.0% (5.0 ± 0.1).
Our timetree recovered Dendrophryniscus as sister taxon of Amazophrynella
(see Appendix S5, Fig. S5a for complete timetree calibration), with a divergence time
75
estimated at 38.1 Ma (95% HPD: 49.0–29.0 Ma), an Eocene divergence, with strong
support (pp = 1.0, see Fig. 2). Within Amazophrynella the eastern/western
divergence was estimated at 24.8 Ma (95% HPD: 30.0–19.0 Ma), a Late Oligocene
to Early Miocene divergence. Within the eastern clade the SE and NE subclades
diverged during the Early Miocene (20.1 Ma, 95% HPD: 22.0–18.0 Ma). In the
western clade, the split between the NW and SW subclades was estimated at 16.5
Ma (95% HPD= 18.0–13.0 Ma), a Middle Miocene divergence. Divergence time
between each pair of lineages within each of the four above clades varied between
10.8 and 2.1 Ma.
Morphological analyses
A total of 468 specimens (adult males and females) were examined for
comparative morphological analyses (Table 2); these analyses did not include
Amazophrynella aff. matses sp, A.sp2 and A sp3 (see Fig. 1). Measurements of
males and females are presented in Table 3 and Table 4. For morphometric analyses
(Principal Components Analyses-PCA and Discriminant Function Analyses-DFA) we
used 237 adult male specimens (87 from the eastern clade and 148 from the western
clade). The specimens used in morphometric analyses are listed in Appendix S6.
The PCA of the eastern and western clades revealed a grouping of specimens
based on morphometric traits and allowed us to distinguish all the mtDNA lineages in
multivariate space (Fig. 3A and 3B). Character loadings, eigenvalues and percentage
of variance explained for PCA (PC I-II) for morphometric variables for the eastern and
western clades are provided in Appendix S7 and Table S7a-b.
In the eastern clade specimens of each lineage can be successfully separated
based on morphometric traits using PCA (Fig 3A). The first two principal components
extracted by the PCA account for 57.7% of the variation found in the dataset. The
first component (PC1) explained 37.48% of the total variation and the second
component (PC2) explained 20.29% of the variation. Using DFA a total of 80% of
specimens were correctly classified to phylogenetic groups. The number of
individuals correctly assigned to each clade by DFA are presented in Table 5.The
DFA showed that the variables that contributed the most to the morphometric
76
separation were snout length, tarsal length, and head width. Head measurement
traits (head width, head length, snout length, and intranasal distance) explained 93%
of the classification by the first two discriminant axes (Appendix S8, Fig. S8a-B).
Loadings and percentage of variance explained for discriminant axes (F1–2) of
morphometric variables in eastern clade are provided in Appendix S8 and Table
S8a).
In the western clade specimens of each lineage can be successfully separated
based on morphometric traits using PCA (Fig 3B). The first two principal components
extracted by the PCA account for 52.37% of the variation found in the dataset. The
first component (PC1) explained 33.2% of the total variation and the second
component (PC2) explained 19.17% of the variation. Using the DFA a total of 68% of
specimens were correctly assigned to phylogenetic groups. The number of
individuals correctly assigned to each clade by DFA are presented in Table 5. The
DFA showed that the variables that most contributed to the morphometric separation
were eye diameter, hand length, head width and foot length. Head traits (head
length, eye diameter and intranasal distance) and hand traits (hand length) were the
variables that explained 78% of the classification by the first two discriminant axes
(Appendix S8, Fig. S8a-A). Loadings and percentage of variance explained for
discriminant axes (FI–II) of morphometric variables in western clade are provided in
Appendix S8 and Table S8a.
Environmental analyses
We obtained a total of 90 unique localities for final analysis, 43 localities of the
eastern and 47 localities of the western clade, representing the occurrences of all
species but Amazophrynella aff. matses sp, A.sp2 and A sp3 (see Fig. 1). The list of
localities used for environmental analyses and discriminant function analyses are in
Appendix S3 and Table S3a.
The PCA of the eastern and western clades revealed a grouping of specimens
based on environmental traits and allowed us to distinguish all the mtDNA lineages in
the multivariate space (Fig 3C and 3D). Character loadings, eigenvalues and
percentage of variance explained for PCA (PC 1-2) analyses for environmental
77
variables for the eastern and western clades are provided in Appendix S7 and Table
S7c-d.
In the eastern clade specimens of each lineages can be successfully
separated based on environmental traits using PCA (Fig 3C). The first two principal
components extracted by the PCA account for 87.71% of the variation found in the
dataset. The first component (PC1) explained 73.28% of the total variation and the
second component (PC2) explained 14.43% of the variation. A total of 65% of
specimens were correctly classified to their lineage. The numbers of individuals
correctly assigned to each clade by DFA are presented in Table 6. The
environmental variables that most contributed to separating lineages were mean
temperature of the coldest quarter (bio11), maximum temperature of the warmest
month (bio5), mean diurnal temperature range (bio2) and isothermality (bio3)
(Appendix S8, Fig. S8a-C). Loadings and percentage of variance explained per
discriminant axes (F1–2) of environmental variables in the eastern clade are provided
in Appendix S8 and Table S8b.
In the western clade specimens of each lineages can be successfully
separated based on environmental traits using PCA (Fig 3D). The first two principal
components extracted by the PCA account for 95.55% of the variation found in the
dataset. The first component (PC1) explained 95.37% of the total variation and the
second component (PC2) explained 0.18% of the variation. A total of 81% of
specimens were correctly assigned to their candidate species. The numbers of
individuals correctly assigned to each clade by DFA are presented in Table 6. The
environmental variables that most contributed to group separation were annual mean
temperature (bio1), mean diurnal temperature range (bio2), mean temperature of the
warmest quarter (bio10) and mean temperature of the wettest quarter (bio8)
(Appendix S8 and Fig. S8a-D). Loadings and percentage of variance explained for
discriminant axes (F1–2) of environmental variables in the western clade are
provided in Appendix S8 and Table S8b.
Taxonomic decisions
Our data analysis of Amazophrynella suggest the existence of 18 linages of
which seven are nominal species, three Deep Conspecific Lineages, one
78
Unconfirmed Candidate Species, three Uncategorized Lineages and four Confirmed
Candidate Species (Table 2). The four CCSs presented at least one diagnostic
morphological character, monophyly with a strong phylogenetic support using the
standard DNA barcode 16S fragment (Vences et al., 2005) and divergence from its
sister taxon at environmental and morphometric data. Based on these results, herein
we described A. teko sp. nov., A. siona sp. nov. A. xinguensis sp. nov. and A.
moisesii sp. nov.
Species accounts
Amazophrynella teko sp. nov.
urn:lsid:zoobank.org:act:590F41D2-7138-42F8-8509-448602C2D040
Amazonella sp. Guianas (Fouquet et al. 2012a: 829, French Guiana [in part])
Amazophrynella sp. Guianas (Fouquet et al. 2012b: 68, French Guiana [in part])
Amazophrynella sp. Guianas (Rojas et al. 2015: 85, French Guiana [in part])
Amazophrynella sp1. (Fouquet et al. 2015: 365, French Guiana [in part])
Amazophrynella sp. aff. manaos (Rojas et al. 2016: 49, French Guiana [in part])
Holotype (Fig. 4). MNHN 2015.136, adult male, collected at Alikéné (3°13'07''N,
52°23'47''W), 206 m a.s.l., district of Camopi, French Guiana by J.P. Vacher on
March 21, 2015.
Paratypes. Twenty-six specimens (males = 13; females = 13). French Guiana:
District of Saint Laurent du Maroni: Mitaraka layon (2°14'09''N, 54°26' 57''W) 330 m
a.s.l., MNHN 2015.137, MNHN 2015.138, MNHN 2015.139, MNHN 2015.140 (adult
males), MNHN 2015.141, MNHN 2015.142, MNHN 2015.143 (adult females), A.
Fouquet and M. Dewynter between 23 and 28 February 2015; Pic Coudreau du Sud
(2°15'14''N, 54°21'04''W) 360 m a.s.l., MNHN 2015.152 (adult male), MNHN
2015.153 (adult female), M. Blanc on February 2015. Flat de la Waki (3°05'15'' N,
53°24'12''W) 173 m a.s.l., INPA–H 36598 (adult female), J.P. Vacher on April 04,
2014. District of Camopi: Mitan (2°37'42''N, 52°33'15''W) 110 m a.s.l., INPA–H
36596, MNHN 2015.144, MNHN 2015.145, MNHN 2015.146, MNHN 2015.147,
MNHN 2015.148 (adult males), MNHN 2015.149, MNHN 2015.150 (adult females),
79
A. Fouquet and P. Nunes between 20 and 24 March 2015. Alikéné (3°13'07''N,
52°23'47''W) 206 m a.s.l. District of Saint Georges: Saint Georges (3°58'03''N,
51°52'20''W) 76 m a.s.l., MNHN 2015.151 (adult male), A. Fouquet and E. Courtois
on February 2015; Mémora (3°18'47''N, 52°10'49''W) 77 m a.s.l., MNHN 2015.154
(adult male), MNHN 2015.155 (adult female), A. Fouquet and P. Nunes on March 18,
2015; Saut Maripa (3°48'22''N, 51°53'36''W) 51 m a.s.l., INPA–H 36597, INPA–H
36610, INPA–H 36599, INPA–H 36601, INPA–H 36600 (adult females), Antoine
Fouquet and E. Courtois on February 2012.
Diagnosis. An Amazophrynella with (1) SVL12.9–15.8 mm in males, 17.9–21.5 mm in
females (2) snout acute in lateral view; upper jaw, in lateral view, protruding beyond
lower jaw; (3) texture of dorsal skin granular; (4) cranial crest, vocal slits and nuptial
pads absent; (5) dorsum covered by abundant rounded granules; (6) abundance of
granules on tympanic area, on edges of upper arms and on dorsal surface of arms;
(7) ventral skin highly granular; (8) fingers slender, basally webbed; (9) finger III
relatively short (HAL/SVL 0.2–0.22 mm, n = 30); (10) finger I shorter than finger II;
(11) palmar tubercle protruding and elliptical; (12) hind limbs relatively short
(TAL/SVL 0.48–0.49, n = 30); (13) toes slender, basally webbed; in life: (14) venter
cream; small blotches on venter.
Comparison with other species (characteristics of compared species in parentheses).
Amazophrynella teko sp. nov. is morphologically most similar to A. manaos from
which it can be distinguished by: large SVL of males 12.9–15.8 mm, n = 13 (vs. 12.3–
15.0 mm, n = 27, Fig. 5, t = 2.04, df = 16.78, p–value = 0.02); snout acute in lateral
view (truncate); larger THL of males, 53% of SVL, n = 13 (vs. smaller THL, 47.2% of
SVL, n = 27); abundance of granules on tympanic area (absent); smaller hind limbs,
TAL/SVL 0.48–0.49, n = 30 (vs. 0.50–0.51, n = 56). From A. bokermanni by the
relative size of fingers: FI<FII (vs. FI>FII); thumb not large and robust (thumb large
and robust, Fig. 6A vs. 6D). From A. vote by larger SVL of males 12.9–15.8 mm, n =
13 (vs. 10.0–14.2 mm, n = 14, see Fig. 3, t = 4.93, df = 25.91, p–value = 0.001) and
females 17.9–21.5 mm, n = 17 (vs. 13.5–19.1 mm, n = 21); texture of dorsal skin
granular (tuberculate); longer UAL, 33% of SVL (vs. smaller UAL 29.8%); longer hind
limbs, TAL/SVL 0.48–0.49, n = 30 (vs. 0.43–0.44, n = 35); venter coloration cream
(red-brown, Fig. 7B vs. 7F). From A. minuta by snout acute in lateral view (pointed,
80
Fig. 8A vs. 8B); larger snout of males–50% of HL, n = 14 (vs. SL 46% of HL, n = 13);
palmar tubercle elliptical (rounded, Fig. 6A vs. 6G); venter cream (yellow-orange, Fig.
7A vs. 7B). From A. amazonicola by dorsal skin texture granular (finely granular);
absence of small triangular protrusion on the tip of the snout (present, Fig. 8A vs.
8H); palmar tubercle elliptical (rounded); venter coloration cream (venter yellow–
orange). From A. matses by smaller SVL of males 12.9–15.8 mm, n = 13 (vs. 11.4–
13.5 mm, n = 13, Table 3 and Fig. 3, t = 7.89, df = 21.34, p–value = 0.001) and
females 17.9–21.5 mm, n = 17 (vs. 15.6–19.0 mm, n = 18); snout profile acute in
lateral view (truncate); texture of dorsal skin granular (spiculate); venter cream
(venter pale yellow). Compared to A. javierbustamantei by shorter hand, HAL/SVL
0.2–0.22, n = 30 (vs. 0.23–0.24, n = 60); texture of dorsal skin granular (tuberculate);
venter cream (pale orange yellowish); tiny blotches on venter (tiny rounded points,
Fig. 7B vs. 7J). Compared to A. siona sp. nov. by large size SVL of adult males 12.9–
15.8 mm, n = 14 (vs. 11.5–14.7 mm, n = 27, Fig. 5, t = 6.15, df = 18.1, p–value =
0.001) and adult females 17.9–21.5 mm, n = 17, (vs. 16.1–20.0 mm, n = 35) and;
smaller hind limbs, TAL/SVL 0.48–0.49, n = 30 (vs. 0.5–0,52, n = 62); palmar
tubercle elliptical (rounded), venter cream (venter bright red). From A. xinguensis sp.
nov. by FI < FII (vs. FI ≥ FII, Fig. 6A vs. 6C); palmar tubercle rounded (ovoid). From
A. moisesii sp. nov. by venter cream (venter pale yellow); shorter hand, HAL/SVL
0.2–0.22, n = 30 (vs. 0.23–0.25, n = 28).
Description of the holotype. Body slender, elongate. Head triangular in lateral view
and pointed in dorsal view. Head longer than wide. HL 34.4% of SVL. HW 27.8% of
SVL. Snout acute in lateral view and triangular in ventral view. SL 50% of HL. Nostrils
slightly protuberant, closer to snout than to eyes. Canthus rostralis straight in dorsal
view. Internarial distance smaller than eye diameter. IND 33.3% of HW. Upper eyelid
covered with smaller pointed tubercles. Eyes wide, prominent, ED 30.7% of HW.
Tympanum not visible through the skin. Skin around tympanum covered by granules.
Vocal sac not visible. Texture of dorsal skin granular. Texture of dorsolateral skin
granular. Forelimbs slender. Edges of forelimbs with scattered granules, in dorsal
and ventral view. Upper arms robust. UAL 33.1% of SVL. Abundance of granules on
upper arm. HAL about 22.5% of UAL. Fingers basally webbed. Fingers slender, tips
unexpanded. Relative length of fingers: I<II<IV<III. Supernumerary tubercles and
81
accessory palmar tubercles rounded. Palmar tubercle small and rounded.
Subarticular tubercles rounded. Texture of gular region granular. Texture of ventral
skin highly granular. Small granules in the venter. Hindlimbs slender. Edges of the
thigh to tarsus covered by conical tubercles. THL 52.3% of SVL. TAL 45.6% of SVL.
Tarsus slender. TL 29.8% of SVL. FL 70.8%. Relative length of toes: I<II<III<V<IV.
Inner metatarsal tubercle oval. Outer metatarsal tubercles small and rounded.
Subarticular tubercles rounded. Toes slender and elongate. Tip of toes not
expanded, basally webbed. Cloacal opening slightly above midlevel of thighs.
Measurement of the holotype (in mm). SVL: 15.1; HW: 4.2; HL: 5.2; SL: 2.6; ED: 1.6;
IND: 1.4; UAL: 5.0; HAL: 3.4; THL: 7.9; TAL: 6.9; TL: 4.5; FL: 5.6.
Variation (Fig. 9). There is little variation among the examined specimens. Sexual
dimorphism was observed in SVL, with 12.9–15.8 mm (14.7 ± 0.8 mm, n = 13) in
males and 17.9–21.5 mm (19.2 ± 1.8 mm, n =17) in females. Specimens (MNHN
2015.137, MNHN 2015.138, MNHN 2015.139, MNHN 2015.140) present lesser
abundance of granules on arm insertion. In some individuals (MNHN 2015.143) the
ventral and the dorsolateral region present one to three large tubercles. Subarticular
tubercles more protruding and swollen in females. Blotches on belly display different
sizes (larger vs. small, see Fig. 10). In life, venter coloration between cream to off-
white .Palm and sole between light red and orange. In preserved specimens, the
palmar tubercle is more flattened than in life.
Coloration of the holotype (in life). Head black brown, in dorsal view. Dorsum brown.
Flanks brown. Scattered tubercles on flanks white. Dorsal surfaces of upper arm, arm
and hand black. Dorsal surfaces of thighs, tibia, tarsus and foot black. Ventral
surfaces of upper arm, arm and palm cream. Ventral surfaces of thighs cream,
mottled with black blotches. In dorsal view, tarsus and tibia creamy, sole light red.
Gular region brown. Belly cream with black tiny blotches. Posterior region of the thigh
and cloaca with black blotches. Longitudinal white stripe on upper jaw extending from
nostril to tympanum. Iris golden and pupil black.
Color in preservative (~70% ethanol, Fig. 10). Almost the same as color in life. We
noted the progressive loss of dorsal coloration which eventually becomes black. The
82
chest lost its coloration and became less intense. The dark blotches on venter
became less evident. The coloration of the fingers and toes became pale red.
Bioacoustics (Fig. 11). Lescure & Marty (2000) described the advertisement call of
Amazophrynella teko sp. nov. as the call of Dendrophryniscus minutus. We recorded
two individuals at Mitaraka (2°14'09''N, 54°26'57''W) and Alikéné (3°13'07''N,
52°23'47''W), French Guiana. All call parameters described by Lescure & Marty
(2000) show an overlap with our recorded calls. Call trill emitted at regular intervals.
Note duration 0.15–0.19 seconds (0.16 ± 0.01 seconds, n = 29). Fundamental
frequency between 2733.3–3555.3 Hz (3115.3 ± 263.7 Hz, n = 29). Dominant
frequency between 3993.3–4980.8 Hz (4638.4 ± 288.27 Hz, n = 29). Number of
pulses between 10–30 per call (25.5 ± 10.4 pulses/call, n = 29). Time to peak
amplitude between 0.06–0.13 seconds (0.08 ± 0.02 seconds, n = 29). The call has a
downward modulation, reaching its maximum frequency near its beginning.
Distribution and natural history (Fig. 1B). Amazophrynella teko sp. nov. have been
recorded from the district of Saint Laurent du Marioni, Saint Georges and Camopi,
French Guiana, the state of Amapá, Brazil and in the southern region of Suriname
(AF personal observation). It occurs at elevations ranging from 70 m a.s.l. to 350 m
a.s.l. The species is diurnal and crepuscular but is also active at night during peak
breeding period, which normally occurs at the beginning of the rainy season
(January–February). This species shows a conspicuous sexual dimorphism, with
males being much smaller than females. The conservation status of this species
remains unknown. The habitat destruction and pollution must affect their populations;
however, due to its abundance we believe that this species probably needs not be
classified above Least Concern category.
Etymology. The specific epithet is a noun in apposition and refers to the name of the
Teko Amerindians who occupy the southern half of French Guiana; the area
occupied by the Teko tribe also encompasses the type locality.
Amazophrynella siona sp. nov.
urn:lsid:zoobank.org:act:66224D58-8DE0-4D5B-950D-1206FFA4AC11
Atelopus minutus: (Duellman & Lynch 1969: 238, Sarayacu [Ecuador])
83
Dendrophryniscus minutus (Duellman 1978: 120, Santa Cecilia [Ecuador])
Dendrophryniscus minutus (Duellman & Mendelson III 1995: 336, vicinities of San
Jacilllo and Teniente Lopez [Peru])
Amazonela cf. minutus “western Amazonia” (Fouquet et al. 2012a: 829, “western
Amazonia”, Ecuador [in part])
Amazophrynella cf. minutus “western Amazonia” (Fouquet et al. 2012a: 68, “western
Amazonia”, Ecuador [in part])
Amazophrynella aff. minuta “western Amazonia” (Rojas et al. 2015: 84, “western
Amazonia”, Ecuador [in part])
Amazophrynella aff. minuta (Rojas et al. 2016: 49, “western Amazonia”, Ecuador [in
part])
Holotype (Fig. 12). QCAZ 27790, adult male, collected at Yasuni National Park,
(0°40'01"S, 76°26'33"W), 200 m a.s.l., Bloque 31, Apaika, Province of Orellana,
Ecuador, by F. Nogales on October 7 2000.
Paratypes. Sixty-six specimens (males = 17, females = 49), Ecuador: Provincia
Sucumbíos: Reserva de Producción Faunística Cuyabeno (0°00'58"S, 76°09'59"W),
203 m a.s.l., QCAZ 52433–34, S. R. Ron; Reserva de Producción Faunística
Cuyabeno (0°00'58"S, 76°09'59"W), 203 m a.s.l, QCAZ 37758–59, QCAZ 37761, L.
A. Coloma; Reserva de Producción Faunística Cuyabeno (0°00'58"S, 76°09'59"W),
203 m a.s.l., QCAZ 6071, QCAZ 6091, QCAZ 6095, QCAZ 6097, QCAZ 6105 (adult
females), QCAZ 6111 (adult males), QCAZ 6113, QCAZ 6118, QCAZ 6127, QCAZ
6128, J. P. Caldwell; Santa Cecilia (0°04'50"S, 76°59'24"W), 330 m a.s.l., QCAZ
4469, QCAZ 4472, M. Crump; Tarapoa (0°07'10"S, 76°20'23"W), 330 m a.s.l., QCAZ
36331, QCAZ 36336, QCAZ 36338, QCAZ 36357, E. Ponce. Provincia Pastaza:
Community of Kurintza (2°03'50"S, 76°47'53"W), 350 m a.s.l., QCAZ 56342 (adult
female), QCAZ 56354, QCAZ 56361 (adult males), D. Velalcázar; A. Villano
community, AGIP oil company (1°30'28"S, 77°30'41"W), 307 m a.s.l., QCAZ 38599,
QCAZ 38679, QCAZ 38722, Galo Díaz; Around Villano community, AGIP oil
company (1°30'28"S, 77°30'41"W), 307 m a.s.l. QCAZ 38642, Y. Mera; Community
of Kurintza (2°03'50"S, 76°47'53"W), 350 m a.s.l., QCAZ 38809 (adult females), F.
84
Varela; Community of Kurintza (2°03'50"S, 76°47'53"W), 350 m a.s.l., QCAZ 54213,
Yerka Sagredo; Bataburo Lodge (1°12'30" S, 76°42'59"W), 260 m a.s.l., QCAZ
39408 (adult female), S. D. Padilla; Lorocachi (1°37'17" S, 75°59'21"W), 229 m a.s.l.,
QCAZ 8902 (adult female), M. C. Terán; Lorocachi (1°37'17"S, 75°59'21" W), 229 m
a.s.l., QCAZ 56165 (adult male), S. R. Ron; Bloque 31 in Yasuni National Park,
(0°56'20"S, 75°50'20"W), 230 m a.s.l, QCAZ 11973, QCAZ 11979, QCAZ 11981
(adult males), G. Fletcher; Canelos (0°29'53"W, 76°22'26"S), 265 m a.s.l., QCAZ
52819, QCAZ 52823, D. Pareja; Canelos (0°29'53"W, 76°22'26"S), 265 m a.s.l.,
QCAZ 17391, L. A. Coloma. Provincia Orellana: Tambococha (0°58'42" S,
75°26'13"W), 194 m a.s.l., QCAZ 55345 (adult female), Fernando Ayala-Varela;
Yasuni National Park, scientific station of the Pontificia Universidad Católica del
Ecuador-PUCE, (0°56'31" S, 75°54'18"W), 203 m a.s.l., QCAZ 51068, E. Contreras;
Yasuni National Park, scientific station of the Pontificia Universidad Católica del
Ecuador-PUCE, (0°56'31" S, 75°54'18"W), 203 m a.s.l., QCAZ 21425, QCAZ 21431
(adult females), J. Santos; Garzacocha (0°45'28"S, 76°00'44"W), 230 m a.s.l., QCAZ
20504 (adult female), M. Díaz; Yuriti (0°33'26"S, 76°48'55"W), 220 m a.s.l., QCAZ
10526, (adult female), M. Read; Kapawi Lodge (2°32'19"S, 76°51'30"W), 257 m
a.s.l., QCAZ 8725, S. R. Ron; Kapawi Lodge (2°32'19"S, 76°51'30"W), 257 m a.s.l.,
QCAZ 25504 (adult males), QCAZ 25533 (adult female), K. Elmer; Fatima, 10 km
from Puyo (1°24'47"S, 77°59'56"W), 1000 m a.s.l., QCAZ 7135 (adult female), M.
Tapia; Provincia Morona Santiago: Pankints (2°54'07"S, 77°53'39"W), 320 m a.s.l.,
QCAZ 46430 (adult female), J. B. Molina. Peru: Department Loreto: Teniente Lopez
(2°35'30.90"S, 76°07'2.84"W), 255 m a.s.l., MUBI 7611, MUBI 7685, MUBI 7686,
MUBI 7698, MUBI 7699, MUBI 7700 (adult females), J. C. Chaparro on October 12,
2008; Jibarito (2°47'55.90"S, 76°0'21.51"W), 236 m a.s.l., MUBI 7786, MUBI 7809,
MUBI 7814 (adult female), J. Delgado on November 5, 2008; Shiviyacu
(2°29'30.92"S, 76°5'18.31"W), 226 m a.s.l., MUBI 14730 (adult female), M. Medina
on June 17, 2008; Jibarito (2°43'51.4"S, 76°01'7.48"W), near Corrientes River, 220 m
a.s.l., MUBI 6292 (adult female), G. Chavez on March 20, 2008.
Referred specimens. USNM 520898, 520900b–01 (adult males), USNM 520896–97,
520899, 520901, 520906 (adult females), collected at Lagarto Cocha River
(0°31'23"S, 75°15'25"W), Province of Loreto, Peru by S. W. Gotte on March 1994.
85
Diagnosis. An Amazophrynella with (1) SVL 11.5–14.7 mm in males, 16.1–20.0 mm
in females; (2) snout acute in lateral view; upper jaw, in lateral view, protruding
beyond lower jaw; (3) texture of dorsal skin finely granular; (4) cranial crests, vocal
slits and nuptial pads absent; (5) small granules from the outer edge of the mouth to
upper arm; (6) ventral skin granular; (7) tiny granules on ventral surfaces; (8) fingers
slender, basally webbed; (9) finger III relative short (HAL/SVL 0.20–0.21, n = 62);
(10) finger I shorter than finger II; (11) palmar tubercle rounded; (12) hind limbs
relatively large (TAL/SVL 0.5–0.52, n =62); (13) toes lacking lateral fingers; in life:
(14) venter reddish brown; yellow blotches on venter.
Comparison with other species (characteristics of compared species in parentheses).
Amazophrynella siona sp. nov. is most similar to A. amazonicola from which it can be
distinguished by (characteristics of compared species in parentheses): the snout
acute in lateral view (pointed, Fig. 8C vs. 8H), absence of protuberance on the tip of
the snout (present); fingers basally webbed (webbing between FI and FII); yellow
blotches on venter (dark blotches, Fig. 7C vs. 7H). From A. matses by the texture of
dorsal skin granular (spiculate); larger HL, 5.6–7.2 mm in adult males, n = 27 (vs.
4.4–6.2 mm, n = 26, t = 7.21, df = 20.1, p–value = 0.001); snout acute in lateral
(truncate); palmar tubercle rounded (elliptical, Fig. 6B vs. 6F); yellow blotches on
venter (black blotches). From A. minuta by texture of dorsal skin finely granular
(highly granular); small granules from the outer edge of the mouth to upper arm
(small warts); tiny granules cover the venter surfaces (absent); shorter HAL,
HAL/SVL 0.20–0.21, n = 62 (vs. 0.2–0.3, n = 20). Compared to A. javierbustamantei
by shorter hand, HAL/SVL 0.20–0.21, n = 62 (vs. 0.23–0.24 , n = 60); texture of
dorsal skin finely granular (finely tuberculate); snout acute in lateral view
(subacuminate). From A. bokermanni by the relative size of fingers with FI<FII
(FI>FII); thumb not large and robust (large and robust, Fig. 6B vs. 6D). From A. vote
by snout acute in profile (rounded); dorsal skin finely granular (tuberculate); dorsal
coloration light brown (brown); venter bright red (red-brown, Fig. 7C vs. 7F); yellow
blotches on venter (white tiny spots). From A. manaos by present rounded palmar
tubercle (elliptical); snout acute in profile (truncate); venter bright red (white, Fig. 7C
vs. 7G); yellow blotches on venter (black patches). Compared to A. teko sp. nov. by
small SVL of adult males 11.5–14.7 mm, n = 27 (12.9–15.8 mm, n = 14, = 6.15, df =
86
18.1, p–value = 0.001, Fig. 5) and adult females 16.1–20.0 mm, n = 35 (vs. 17.9–
21.5 mm, n = 17); tiny granules cover venter (absent); longer hind limbs, TAL/SVL
0.5–0.52, n = 62 (vs. 0.48–0.49, n = 30); palmar tubercle round (elliptical); venter
bright red (cream). From A. xinguensis sp. nov. by FI<FII (vs. FI ≥ FII, Fig. 6); palmar
tubercle rounded (ovoid); venter bright red (cream). From A. moisesii sp. nov. by
shorter hand, HAL/SVL 0.20–0.21, n = 30 (vs. 0.23–0.25, n = 28); venter bright red
(pale yellow).
Description of the holotype. Body slender, elongate. Head triangular in lateral view
and rounded in dorsal view. Head longer than wide. HL 39.6% of SVL. HW 31.3% of
SVL. Snout acute in lateral view and pointed in dorsal view. SL 42.8% of HL. Nostrils
slightly protuberant, closer to snout than to eyes. Canthus rostralis straight in dorsal
view. Internarial distance smaller than eye diameter. IND about 27.6% of HW. Upper
eyelid covered with tiny tubercles. Eye wide, prominent, about 30.3% of HL.
Tympanum not visible through the skin. Skin around tympanum covered by tiny
granules. Vocal sac not visible. Texture of dorsal skin finely granular. Texture of
dorsolateral skin finely granular. Forelimbs slender. Edges of forelimbs with granules,
in dorsal and ventral view. Upper arms robust. UAL 30.5% of SVL. Small granules
from the outer edge of the mouth to upper arm. HAL 72.4% of UAL. Fingers basally
webbed. Fingers slender, tips unexpanded. Relative length of fingers: I<II<IV<III.
Supernumerary tubercles and accessory palmar tubercles rounded. Palmar tubercle
large and rounded. Subarticular tubercles rounded. Texture of gular region finely
granular. Texture of ventral skin granular. Small granules on venter. Hindlimbs
slender. Edges of thigh to tarsus covered by conical tubercles. THL 51.8% of SVL.
TAL 50.6% of SVL. Tarsus slender. TL 29.8% of SVL. FL 60% of THL. Relative
length of toes: I<II<V<III<V. Inner metatarsal tubercle oval. Outer metatarsal
tubercles small and rounded. Subarticular tubercles rounded. Toes slender and
elongate. Tip of toes not expanded, unwebbed. Cloacal opening slightly above
midlevel of thighs.
Measurement of the holotype (in mm). SVL 12.6; HW 3.9; HL 5.0; SL 2.1; ED 1.2;
IND 1.1; UAL 3.8; HAL 2.7; THL 7.2; TAL 6.9; TL 3.9; FL 4.3.
87
Variation (Fig. 13). The new species presents extensive variation among specimens
(eg. https://bioweb.bio/galeria/FotosEspecimenes/Amazophrynella%20minuta/1).
Sexual dimorphism was observed in SVL, with 11.5–14.7 mm (13.0 ± 0.6 mm, n =
29) in males and 16.1–20.8 mm (18.3 ± 0.9 mm, n = 35) in females. Specimens
(MUBI 7686, MUBI 7698, MUBI 7699, MUBI 7700) from Andoas,Peru, present fewer
tubercles on upper arm. Abundance of granules on ventral surfaces varies in density
(eg. QCAZ 21425, QCAZ 21431, QCAZ 20504, QCAZ 10526, QCAZ 46430). Some
individuals (eg. QCAZ 37761, QCAZ 6095, QCAZ 6105) present one to two large
tubercles on dorsolateral region. Specimens from Pastaza (eg. QCAZ 56342, QCAZ
56354, QCAZ 56361, QCAZ 38599, QCAZ 38679, QCAZ 38722) present greater
abundance of granules on dorsum. Some individuals display different sized blotches
on venter, while in other specimens, blotches are absent (Fig. 13C). In life, belly
coloration varies between yellow to light red. The gular region varies from light red to
red. Thighs, shanks, tarsus and feet vary from light red to red, in dorsal view. Palm
and sole color from light red to orange, in ventral view.
Coloration of the holotype (in life). Head brown, in dorsal view. Dorsum mostly brown.
Flanks reddish brown. Dorsal surfaces of upper arm, arm and hand light brown.
Dorsal surfaces of the thighs, tibia, tarsus and foot light brown. Ventral surfaces of
upper arm light red, arm light brown, palm reddish brown. Gular region reddish
brown. Belly bright red with yellow blotches. Axillar region with yellow granules.
Ventral surfaces of thighs, tarsus and tibia reddish brown, sole reddish brown. Iris
golden and pupil black.
Color in preservative (~70% ethanol, Fig. 14). Almost the same as color in life.
Dorsum became brown. We detected a gradual fading of the red and yellow
coloration of the chest and venter. The blotches on venter became less evident.
Fingers and toes became pale red.
Tadpoles (Fig. 15). Duellman & Lynch (1969) described the tadpole of
Amazophrynella siona sp. nov. as Atelopus minutus based on ten individuals at stage
31 and three at stage 40, from Sarayacu, Province of Pastaza, 400 m a.s.l. The
morphological characteristics described by Duellman & Lynch (1969) are similar to
those observed by us. We analyzed ten tadpoles at stage 30. Body ovoid in dorsal
88
view. Total length 11.0–13.2 mm (11.5 ± 0.84 mm). Body length 3.6–4.8 mm (4.2 ±
0.3 mm); depressed in lateral view. Body height 1.2–1.9 mm (1.5 ± 0.2 mm), body
widest posteriorly. Snout rounded in dorsal and lateral view. Eye diameter 0.3–0.5
mm (0.3 ± 0.1 mm). Eye snout distance 0.9–1.4 mm (1.2 ± 0.14 mm). Nostrils small,
closer to eyes than to tip of snout. Inter nasal distance 0.5–0.75 mm (0.6 ± 0.1 mm).
Inter orbital distance 0.5–0.75 mm (0.6 ± 0.09 mm). Spiracle opening single, sinistral
and conical. Spiracle opening on the posterior third of the body. Centripetal wall
fused with the body wall and longer than the external wall. Upper and lower lips bare,
single row of small blunt teeth, sectorial disc absent. Jaw sheaths finely serrated.
Two upper and three lower rows of teeth. Oral disc weight 0.8–1.1 mm (0.9 ± 0.1
mm). Dorsal fin originating on the tail-body junction, increasing in height throughout
the first third of the tail and decreasing gradually in the posterior two thirds of the tail
to a pointed tip, in lateral view. Ventral fin originating at the posteroventral end of the
body, higher at the first third of the tail, decreasing gradually in height toward tail tip.
Tail length 5.4–8.1 mm (6.8 ± 0.9 mm). Tail height 0.9–1.1 mm (0.9 ± 0.1 mm). Body
and tail rosaceous with small dark pointed flecks on body in fixed specimens. In life,
Duellman & Lynch (1969) reported brown body and spotted tail with black and small
brown flecks on caudal musculature, the entire dorsal fin and posterior third of ventral
fin.
Bioacoustics (Fig. 16). The advertisement call of Amazophrynella siona sp. nov. was
described by Duellman (1978) as the advertisement call of Dendrophryniscus
minutus from Santa Cecilia, Ecuador. We analyzed one call from the Reserva de
Producción Faunistica Cuyabeno, Province of Sucumbíos, Ecuador (QCAZ 18833)
(http://zoologia.puce.edu.ec/Vertebrados/Anfibios). The call was recorded one day
after capture, on February 6, 2002. In our analysis all the call parameters from
Duellman (1978) overlap with the call of the new species. Call trill emitted at irregular
intervals. Note duration 0.03– 0.06 seconds (0.013 ± 0.001 seconds, n = 16). The
fundamental frequency 2000–3240.1 Hz (3000.9 ± 101.79 seconds, n = 16).
Dominant frequency 3647.5–4200 Hz (3757.9 ± 138.1 Hz, n = 16). The number of
pulses 23–28 pulses per note (28.5 ± 5.3 pulses/note, n = 16). Time to peak
amplitude 0.01–0.03 seconds (0.02 ± 0.01 seconds, n = 13). The call has a
downward modulation, reaching its maximum frequency almost at the middle.
89
Distribution and natural history (Fig. 1B). Amazophrynella siona sp. nov. have been
recorded from Ecuador, in Provinces of Orellana, Sucumbíos and Pastaza and Peru
in the Province Andoas, northern Loreto Department. It occurs at elevations ranging
from 200–900 m a.s.l. The species is found in the leaf litter of primary and secondary
forest, terra firme or flooded forest, and swamps. It is active during the day; at night
individuals rest on leaves, usually less than 50 cm above ground. It breeds
throughout the year (Duellman, 1978). This species shows conspicuous sexual
dimorphism, with males being much smaller than females. The amplexus is axillar.
Eggs are pigmented; males call from amidst leaf litter. Duellman & Lynch (1969)
reported that this species deposited its eggs in gelatinous strands 245–285 mm long,
with 245–291 eggs. It can be abundant at some sites (eg., Cuyabeno reserve; SRR
pers. obs.) Given its large distribution range (> 20000 km2) which also includes vast
protected areas and locally abundant populations, we suggest assignment this
species to the Least Concern category.
Etymology. The specific epithet is a noun in apposition and refers to the Siona, a
western Tucanoan indigenous group that inhabits the Colombian and Ecuadorian
Amazon. The Siona inhabit the Cuyabeno Lakes region, an area where
Amazophrynella siona sp. nov. is be abundant. While working in his undergraduate
thesis in the early 1990s, SRR lived with the Siona at Cuyabeno. The Siona chief,
Victoriano Criollo, had an encyclopedic knowledge of the natural history of the
Amazonian forest, superior in extent and detail to that of experienced biologists. His
death, a few years ago, represents one of many instances of irreplaceable loss of
traditional knowledge triggered by cultural change among Amazonian Amerindians.
Amazophrynella xinguensis sp. nov.
urn:lsid:zoobank.org:act:55CD4C19-9A39-4DEB-BA6C-F02F9735BB77
Amazophrynella cf. bokermanni (Vaz–Silva et al. 2015: 208, “Volta grande”, Xingu
River, Pará, Brazil)
Holotype (Fig. 17). INPA–H 35471, adult male, collected at the Sustainable
Development Project (PDS) Virola Jatobá (3°10'06'' S, 51°17'54.2''W), 86 m a.s.l.,
municipality of Anapú, state of Pará, Brazil by E. Hernández and E. Oliveira on
December 06, 2012.
90
Paratypes. Twenty two specimens (males = 4, females = 14, immatures = 4). Brazil:
Pará State: Municipality of Senador José Porfírio: Fazenda Paraíso (2°34'37''S,
51°49'50.3''W), 57 m a.s.l., INPA–H 35482, INPA–H 35493 (adult males), INPA–H
35472 (adult female), E. Hernández and E. Oliveira on December 05, 2012.
Municipality of Anapu: PDS Virola Jatobá, (3°10'06''S, 51°17'54.2''W), 86 m a.s.l.,
INPA–H 35484, INPA–H 35485 (adult males), INPA–H 35473, INPA–H 35474,
INPA–H 35475, INPA–H 35476, INPA–H 35477, INPA–H 35478, INPA–H 35479,
INPA–H 354780, INPA–H 35481, INPA–H 35483, INPA–H 35490, INPA–H 35491,
INPA–H 3592 (adult females), E. Hernández and E. Oliveira on December 06, 2012.
Municipality of Vitória do Xingu, Ramal dos Cocos (3°09'42.1''S, 52°07'41.9''W), 110
m a.s.l., INPA–H 35486, INPA–H 35487, INPA–H 3588, INPA–H 35489 (immatures),
E. Hernández and E. Oliveira on December 04, 2012.
Diagnosis. An Amazophrynella with (1) SVL 17.0–20.0 mm in males, 22.4–26.3 mm
in females; (2) snout pointed in lateral view; (3) upper jaw, in lateral view, protruding
beyond lower jaw; 4) tympanums, vocal sac, parotid gland and cranial crest not
evident; (5) texture of dorsal skin highly granular; (6) abundance of small tubercles
on dorsum, on upper arm and on arms; (7) texture of ventral skin granular; (8) fingers
I and II basally webbed; (9) finger III relative short (HAL/SVL= 0.20–0.22, n = 18);
(10) thumb larger and robust; (11) finger I larger or equal than finger II, FI = 2.1 vs.
FII = 2.1 in adult males, n = 5 and FI = 2.8 mm, vs. FII = 2.9 mm, in adult females, n
= 13; (12) palmar tubercle ovoid; (13) toes slender, basally webbed; in life: (14)
venter greyish; black dots on venter.
Comparison with other species (characteristics of compared species in parentheses).
Amazophrynella xinguensis sp. nov. is more similar to A. bokermanni from which it
can be distinguished by: texture of dorsal skin highly granular (granular); relative size
of fingers: FI ≥ FII mean 2.1 mm, in I vs. 2.1 mm in II in A. xinguensis sp. nov. n = 5
(vs. FI > FII, mean 2.2 mm in FI vs. in 2.0 mm FII in A. bokermanni, n = 7, Fig. 6C vs.
6D); shape of palmar tubercle elliptical (rounded); presence of tubercles on dorsum
(absent); dorsal coloration dark brown (light brown); venter light gray (white); gular
region dark brown (grayish brown). From the other species of Amazophrynella the
new species is easily differentiated by having FI ≥ FII (FI < FII in all the other species,
Fig. 6); its greater SVL of males (KW x2 = 108.6, df = 10, p–value = 0.001, Fig. 5) and
91
its protruding ovoid palmar tubercle (vs. A. teko, A. manaos, A. vote, A. minuta, A.
bokermannni, A. javierbustamantei, A. matses, A. Amazonicola, A. siona sp. nov. A.
teko sp. nov., A. moisesii sp.nov. see Fig. 6).
Description of the holotype. Body robust. Elongate. Head pointed in lateral view and
triangular in dorsal view. Head longer than wide. HL 35.5% of SVL. HW 27.1% of
SVL. Snout acute in lateral view and triangular in dorsal and ventral view. SL 64.0%
of HL. Nostrils slightly protuberant, closer to snout than to eyes. Canthus rostralis
straight in dorsal view. Internarial distance smaller than eye diameter. IND about
20.8% of HW. Upper eyelid covered by small granules. Eye prominent, 30.3% of HL.
Tympanum not visible through the skin. Skin around tympanum covered by tiny
granules. Vocal sac not visible. Texture of dorsal skin highly granular. Rounded small
tubercles on dorsum. Texture of dorsolateral skin granular. Forelimbs thick. Edges of
arms of forelimbs with granules, in dorsal and ventral view. Upper arms robust. UAL
28.5% of SVL. Abundance of small tubercles on upper arm. HAL 68.4% of UAL.
Fingers slender, tips unexpanded. Fingers basally webbed on finger II and finger III.
Relative length of fingers: I≥II<IV<III. Supernumerary tubercles rounded. Palmar
tubercle ovoid. Gular region finely granular. Texture of ventral skin granular. Small
granules in the venter. Hind limbs slender. Edges of thigh to tarsus covered by
conical tubercles. THL 52.2% of SVL. Tibias almost the same length as thighs. TAL
48.9% of SVL. Tarsus slender. TL 29.8% of SVL. FL 60.0% of THL. Relative length
of toes: I<II<III<V<IV. Inner metatarsal tubercle oval. Outer metatarsal tubercles
small and rounded. Subarticular tubercles rounded. Toes slender. Tip of toes not
expanded, basally webbed. Cloacal opening slightly above midlevel of thighs.
Measurement of the holotype (in mm). SVL 18.5, HW 5.0, HL 6.0, SL 3.1, ED 2.1,
IND 1.6; UAL 6.6; HAL 4.1, FI 1.9, FII 1.9, THL 9.7, TAL 9.3, TL 5.7, FL 6.4.
Variation (Fig. 18). Sexual dimorphism was observed in SVL, with 17.7–20.0 mm
(18.9 ± 1.0 mm, n = 5) in males and 22.4–26.3 mm (24.1 ± 1.2 mm, n = 13) in
females. Some individuals (i.e. INPA–H 35473, INPA– H 35477, INPA–H 35475)
present one to two large tubercles on dorsolateral region. The granules on ventral
surfaces are greatly abundant in some individuals (eg. INPA–H 35478, INPA–H
35480, INPA–H 35486). The gular region presents black or brown coloration. Dots on
92
venter display different sizes (small to medium) and abundance (Fig. 18D vs 18A). In
life, ventral surfaces from cream to light gray. Thighs, shanks and tarsus between
cream to white coloration, in ventral view. Palm and sole present different tonalities of
orange, in ventral view.
Coloration of the holotype (in life). Head dark brown, in dorsal view. Dorsum mostly
light brown with brown chevrons. Flanks cream. Dorsal surfaces of upper arm, arm
and hand light brown. Dorsal surfaces of thighs, tibia, tarsus and foot brown. Ventral
surfaces of upper arm, arm and palm cream. Ventral surfaces of thighs, tarsus and
tibia cream, sole black. Gular region cream. Belly cream with tiny black blotches.
White line from the tip of snout to cloaca. Iris golden and pupil black.
Color in preservative (~70% ethanol, Fig. 19). In preservative, the coloration is almost
the same than life. The coloration of the dorsum became dark brown. Gular region
and venter became white. The iris loses its coloration. The fingers and toes became
cream.
Distribution and natural history (Fig. 1B). Amazophrynella xinguensis sp. nov. have
been recorded from State of Pará, Brazil, at three localities: PDS Virola Jatoba,
municipality of Anapú, Fazenda Paraiso, municipality of Senador José Porfirio (right
bank of Xingu River) and Ramal dos Cocos, municipality of Altamira (left bank of
Xingu River), all of them in area of influence of the Belo Monte dam. It occurs in
elevations of 86–106 m a.s.l. This species is found amidst leaf litter. The amplexus is
axillar (Fig. 18C). Reproduction occurs in the rainy season in tiny puddles. Males
were found hidden in the leaf litter. Tadpoles and advertisement call are unknown.
The conservation status of this species remains unknown, but the recent construction
of the Belo Monte hydroelectric complex on the Xingu River represents a threat to
the population status of this species.
Etymology. The specific epithet refers to geographic distribution of the species within
the lower Xingu River basin, Brazil.
Amazophrynella moisesii sp. nov.
urn:lsid:zoobank.org:act:9984F3CB-9416-482D-8F63-5D78C8CDC032
Dendrophryniscus minutus (Bernarde et al. 2011: 120 plate 2, Fig. d)
93
Amazophrynella minuta (Bernarde et al. 2013: 224, 227 plate 7 Fig. c; Miranda et al.
2015: 96)
Holotype (Fig. 20). UFAC–RB 2815 adult male, collected in the Parque Nacional da
Serra do Divisor, Igarapé Ramon (7°27'00"S, 73°45'00"W), 400 m a.s.l., municipality
of Mâncio Lima, Acre, Brazil by Moises Barbosa de Souza on 1 January, 2000.
Paratypes. Thirty eight specimens (males = 18, females = 20, Acre, Brazil: Reserva
Extrativista Alto do Juruá (9°03'00"S, 72°17'00"W), 260 m a.s.l., UFAC–RB 823
(adult male), Moisés B. Souza and Adão J. Cardoso on 26 February 1994, UFAC–
RB 878–879 (adult males), Moisés B. Souza and Paulo Roberto Manzani between
16 and 18 July 1994; UFAC–RB 2606–2611 (adult females), Moisés B. Souza and
M. Nascimento between 7 and 8 March 1998. Parque Nacional da Serra do Divisor:
Igarapé Anil (8°59'00"S, 72°29'00"W), 192 m a.s.l., UFAC–RB 1337–1341 (adult
females), UFAC–RB 1343 (adult female), Moisés B. Souza and William Aiache on 10
November 1994; Zé Luiz lake (8°54'00"S, 72°32'00"W), UFAC-RB 1774–1775 (adult
females), Moisés B. Souza and William Aiache between 9 and 10 November 1996;
Igarapé Ramon (7°27'00"S, 73°45'00"W), 400 m a.s.l., UFAC–RB 1375 (adult
female), Moisés B. Souza and William Aiache between 12 and 13 November 1996,
UFAC–RB 2772–2773 (adult females), UFAC–RB 2816–2817 (adult males), Moisés
B. Souza between 18 and 20 January 2000; Môa River (7°30'00"S, 73°36'00"W), 331
m a.s.l, UFAC–RB 1493 (adult male), Moisés B. Souza and William Aiache between
19 and 20 November 1997, UFAC–RB 2687–2697 (adult males), Moisés B. Souza
on 10 January 2000. Floresta Estadual do Gregório, municipality of Tarauacá
(7°59'00"S, 71°22'36.8"W), 240 m a.s.l., UFAC–RB 5678 (adult female), Moisés B.
Souza and Marilene Vasconcelos between 23 and 26 July 2000; Centrinho do Aluísio
site, municipality of Porto Walter UFAC–RB 6273 (adult male), Paulo Roberto Melo
Sampaio, on 8 January 2014. Municipality of Mâncio Lima, Acre (7°23'10.32"S, 73°
3'31.68"W), MNRJ 91670 (field number PRMS 420) (adult female) Paulo Roberto
Melo Sampaio and Evan M. Twomey on 24 March 2016. Amazonas state:
Municipality of Envira (7º31'16.14"S, 70º1'3.84"W), MNRJ 91669 (field number
PRMS 404) (adult female) Paulo Roberto Melo Sampaio and Evan M. Twomey on 12
March 2016.
94
Diagnosis. An Amazophrynella with (1) SVL 12.2–15.8 mm in males, 16.4–20.9 mm
in females; (2) snout acuminate in lateral view, upper jaw, in lateral view, protruding
beyond lower jaw; (3) snout length protuberant, large for the genus (SL/HL= 0.48–
0.5); (4) cranial crest, vocal slits and nuptial pads absent; (5) small tubercles on
upper arms and posterior area of tympanums; (6) texture of dorsal skin tuberculate;
(7) texture of ventral skin highly granular (8) finger III relative large (HAL/SVL 0.23–
0.25, n = 28); (9) fingers slender, basally webbed; (10) finger I shorter than finger II;
(11) palmar tubercle elliptic; (12) hind limbs relatively large (TAL/SVL 0.51–0.53, n =
28); (13) toes slender basally webbed; in life: (14) venter pale yellow; small irregular
dots on venter.
Comparison with other species (characteristics of compared species in parentheses).
Amazophrynella moisesii sp. nov. is most similar to A. javierbustamantei from which it
can be distinguished by: protruding snout, SL/HL 0.48–0.5, n = 28 (vs. 0.43–0.45, n =
60); snout acuminate, in lateral view (subacuminate); ventral skin highly granular
(coarsely areolate); larger hind limbs, TAL/SVL 0.51–0.53, n = 28 (vs. 0.49–0.51, n =
60); venter bright yellow (pale yellowish orange); small irregular blotches on venter
(tiny rounded points). From the other species of the genus Amazophrynella the new
species is easily differentiated by its large hand, HAL 3.6–5.6 mm (4.62 ± 0.62 mm)
in adult females, 2.5–4.1 mm (3.4 ± 0.52 mm) in adult males (KW x2 = 100.2, df = 10,
p–value = 0.001, Fig. 21); longer SL, adult females 3.4–2.5 mm (3.0 ± 0.2 mm) and
adult males 2.1–3.0 mm (2.6 ± 0.3 mm, KW x2 = 104.3, df = 10, p–value = 0.001, Fig.
22); FI < FII (FI > FII in A. bokermanni, and FI ≥ FII in A. xinguensis sp.nov. - Fig. 6K
vs. 6C and 6K vs. 6D) and venter coloration pale yellow (white, in A. manaos, cream
in A. teko sp. nov., red brown in A. vote and reddish brown in A. siona sp. nov., see
Fig. 7).
Description of the holotype. Body slender, elongate. Head triangular in lateral view
and pointed in dorsal view. Head longer than wide. HL 33.8 % of SVL. HW 30.8% of
SVL. Snout prominent, acuminate in lateral view and pointed in dorsal view. SL
50.9% of HL. Nostrils closer to snout than to eyes. Canthus rostralis straight in dorsal
view. Internarial distance smaller than eye diameter. IND about 30.9% of HW. Upper
eyelid covered by abundant granules on borders. Eye prominent, about 35.7% of HL.
Tympanum not visible through the skin. Skin around tympanum covered by small
95
granules. Vocal sac not visible. Texture of dorsal skin tuberculate. Abundance of
granules on dorsum. Dorsolateral skin granular. Forelimbs slender. Edges of
forelimbs covered by small conical granules, in dorsal and ventral view. Upper arms
slender. UAL 35.2% of SVL. Small conical granules from the outer edge of the mouth
to upper arm. Upper arm covered by abundant medium size granules. Large HAL.
HAL 72.9% of UAL. Fingers basally webbed. Fingers slender, tips unexpanded.
Relative length of fingers: I<II<IV<III. Supernumerary tubercles and accessory palmar
tubercles rounded. Palmar tubercle large and elliptic. Subarticular tubercles rounded.
Texture of gular region tuberculate. Texture of ventral skin highly granular. Small
granules on venter. Hindlimbs slender. Thigh to tarsus covered by conical granules
on borders. THL 54.4% of SVL. Tibias almost the same length as thighs. TAL 53.6%
of SVL. Tarsus slender. TL 33.8% of SVL. FL 74.3% of THL. Relative length of toes:
I<II<V<III<V. Inner metatarsal tubercle rounded. Outer metatarsal tubercles small
and rounded. Subarticular tubercles rounded. Toes slender and elongate. Tip of toes
not expanded, basally webbed. Cloacal opening slightly above middle of thighs.
Measurement of the holotype (in mm). SVL 13.6, HW 4.2, HL 5.1, SL 2.6, ED 1.5,
IND 1.3; UAL 4.8; HAL 3.5, THL 7.4, TAL 7.3, TL 4.5, FL 5.5.
Variation (Fig. 23). Phenotypically, the new species present some variation among
specimens. Sexual dimorphism was observed in SVL, with 12.2–15.8 mm (14.3 ± 1.5
mm, n = 15) in males and 16.4–20.9 mm (18.5 ± 1.6 mm, n =15) in females. Some
specimens present greater abundance of granules on dorsum (eg. UFAC–RB 2690).
Some individuals present greater abundance of small tubercles on dorsolateral
region (eg. UFAC–RB 2611, UFAC–RB 2603, UFAC–RB 2689, UFAC–RB 2692).
Another specimen (UFAC–RB 2610) presents brown chevrons extending from the
head to the vent, in dorsal view. Some individuals (eg. UFAC–RB 829) present a line
on dorsum, extending from the tip of the snout to cloaca. The pale yellow coloration
of ventral surfaces may extend from thighs to the chest or just to the middle of the
venter. In some specimens, the irregular black dots on venter vary in abundance and
size (eg. Fig. 24B vs. 24E). In life and preserved specimens, venter coloration
between pale yellow and yellow. In some individuals, the thighs are abundantly
covered by rounded tiny spots extending to the shank (Fig. 24C vs. 24D).
96
Coloration of the holotype (in life). Head brown, in dorsal view. Dorsum mostly light
brown. Flanks cream with scattered small black dots. Dorsal surfaces of upper arm,
arm and hand light brown. Dorsal surfaces of thighs, tarsus and foot light brown.
Ventral surfaces of upper arm, arm and palm cream. Ventral surfaces of thighs,
tarsus and tibia cream with small black dots. Sole light brown. Fingers cream, in
ventral view. Gular region cream with small dots. Venter pale yellow with small dots.
Iris golden and pupil black.
Color in preservative (~70% ethanol, Fig. 24). Nearly the same as color in life. The
dorsum became light brown. We detected a fading of pale coloration of the chest and
venter became cream. The small irregular dots on venter became less evident. The
hand and foot became cream, in ventral view. The gular region and venter became
cream. The iris loses its coloration.
Distribution and natural history (Fig. 1B). Amazophrynella moisesii sp. nov. have
been recorded from Brasil. State of Acre: municipalities of Cruzeiro do Sul, Mâncio
Lima, Porto Walter and Tarauacá; State of Amazonas: municipality of Envira. Peru:
Department of Huanuco, Panguana, Rio Llullapichis. Due to its abundance and
presence in conservation units of Brazil (Floresta Estadual do Gregório, Reserva
Extrativista do Alto Juruá and Parque Nacional da Serra do Divisor) we recommend
the IUCN Least Concern category.
Etymology. The specific epithet refers to Dr. Moisés Barbosa de Souza, a Brazilian
biologist, professor and friend at the Universidade Federal do Acre (UFAC), to whom
we dedicate this species in recognition of his contributions to herpetological research
and amphibian conservation in the state of Acre, Brazil.
Discussion
To date no study that analyzed a broadly distributed Amazonian taxon
confirmed the existence of just one broadly distributed species (eg., Funk et al.,
2012; Jungfer et al., 2013; Fouquet et al., 2014; Caminer & Ron, 2014; Gehara et al.,
2014; Ferrão et al., 2016). In recent years it has become evident that widespread
species in fact represent species complexes characterized by many deeply divergent
lineages, eg. Adenomera andreae, Dendropsophus minutus, Rhinella margaritifera,
Scinax ruber, Pristimantis ockendeni, Pristimantis fenestratus, Engystomops petersi,
97
Boana fasciata, Physalaemus petersii, Leptodactylus marmoratus and
Osteocephalus taurinus (Fouquet et al., 2007; Padial & Riva, 2009; Angulo &
Icochea, 2010; Funk et al., 2012; Jungfer et al., 2013; Caminer & Ron, 2014;
Fouquet et al., 2014; Gehara et al., 2014; Lourenço et al., 2015). These discoveries
imply that public data deposited in, for example GenBank, Gbif or IUCN are often
flawed and that the numerous metaanalyses (Godinho & Silva, 2018) based on such
data may be imprecise or even inaccurate. As a consequence of not recognizing true
taxonomic diversity of anurans, macroecological studies will fail to recognize actual
patterns of geographic structuring, and ultimately will not contribute to our
understanding of the evolutionary and ecological processes that lead to and are
maintaining this diversity.
Our results suggest that the genus harbors more than twice as many species
as current estimates. In the last several years the systematics and taxonomy of the
genus Amazophrynella has begun to be elucidated (Ávila et al., 2012; Rojas et al.,
2014, 2015, 2016). Resulting from these studies, five new species (A. vote, A.
manaos, A. amazonicola, A. matses and A. javierbustamantei—previously mistaken
for A. minuta) were described. With the description of the four new species in this
study, the total number of nominal species reaches 11 (Fig. 25), representing an
important increase in species diversity of the genus. The number of undescribed
species as a percentage of total is concordant with estimates from previous studies
aiming to elucidate the species diversity of Amazonian frogs (eg. Elmer et al., 2007;
Fouquet et al., 2007; Padial et al., 2012; Ron et al., 2012; Caminer & Ron, 2014;
Gehara et al., 2014; Ferrão et al., 2016). Therefore, our study adds to this growing
body of studies, and confirms the hypothesis that the species diversity within
Amazophrynella is much higher than currently accepted. The four CCS described in
our study present clear differences in diagnostic morphological characters,
divergence at ecological requirements and large genetic distance when compared
with their sister taxa. But it should also be clear that our taxonomic decisions were
conservative, and that numerous putative lineages within Amazophrynella still await
formal description. This conservative approach aims to promote taxonomic stability,
but as a consequence continues, albeit to a lesser degree, to underestimate the true
species diversity of Amazonian anurofauna.
98
A limiting factor of our study was the use of a single molecular marker (16S,
12S and COI mtDNA loci). The potential limitations for species delimitation using
mtDNA have been discussed in literature (eg. Ranalla & Yang, 2003; Yang &
Rannala 2010; Dupuis et al., 2012; Fujita et al., 2012). The use of additional nuclear
markers is generally recommended as the use of these unliked markers has the
potential to improve the accuracy of phylogenetic reconstructions and species
delimitation. In spite of having used only mtDNA loci, our study also provides an
extensive new morphological dataset, bioacoustic data and accurate collecting
locality information which allowed us to associate environmental data with each
specimen. All these additional data support and reinforce the inference based on the
mitochondrial genes.
Our phylogenetic analysis also reveals a striking biogeographic pattern with a
basal eastern and western divergence followed by a northern and southern split
within both eastern/western clades (Fig. 2). Our basal east-west pattern dated to the
Miocene and match similar patterns and divergence times detected in other groups of
frogs (Symula et al., 2003; Noonan & Wray, 2006; Funk et al., 2007; Garda &
Cannatella, 2007; Fouquet et al. 2014). Paleoenvironmental reconstructions of
Amazonian history suggest that there was a large lacustrine region in western
Amazon which began to form at the beginning of the Miocene (~24 Ma) (Hoorn et al.,
2010). This lake and marshland system, known as Lake Pebas, existed in
southwestern Amazonia, and was drained first to the north and then to the east
(Hoorn et al., 2010). Paleoenviromental data suggest marine incursions into western
Amazon during the Miocene, and Noonan & Wray (2006), for example, suggest the
importance of these incursions for the diversification of Amazonian anurofauna. In
general, however, marine incursions remain largely untested as a diversifying force
(Noonan & Wray, 2006; Garda & Cannatella, 2007; Antonelli et al., 2009). In addition,
it is reported that in early Miocene, the Purus arch was still active, and was a
prominent landscape feature in central Amazon (Wesselingh & Salo, 2006;
Figueiredo et al., 2009; Caputo & Soares, 2016) thus this geological formation also
could explain the east-west pattern as well. While other hypotheses, such as
Pleistocene refugia have also been proposed to explain this east-west pattern of
diversity (Pellegrino et al., 2011), the Miocene marine incursions have the best
99
temporal concordance with the basal east-west divergence pattern observed in
Amazophrynella and other Amazonian anuran groups.
The northern and southern split within both the eastern and western clades
occurred in early Miocene (~20.1 Ma) in the eastern Amazonia clades, while the
diversification of the western Amazonian clade commenced in the Middle Miocene
(~16.5 Ma). The beginning of the diversification of these clades appears to be
asynchronous and therefore is unlikely attributable to a single event. The more recent
date of diversification of the western clade is likely to have followed the last marine
incursion, i.e. a colonization of newly available habitat in western Amazon from
eastern Amazon. Independent of the absolute timing these divergence events, the
four subclades are restricted to north and south of the Amazon River, a common
pattern in many vertebrates species groups analyzed at the Amazonia-wide scale
(eg. Kaefer et al., 2012; Ribas et al., 2012; Fouquet et al., 2015; Oliveira et al., 2016).
In the case of Amazophrynella species, ecological characteristics such as small body
side, being a terra firme species and being restricted to reproducing in puddles
(Rojas et al. 2016), clearly evidences these species’ inability to disperse across
rivers. This in turn implies that major Amazonian rivers should limit the distributions of
lineages of Amazophrynella, a pattern observed in our phylogeny. However, the role
of rivers in driving diversification of Neotropical frogs remains controversial (see
Vences and Wake 2007 vs. Lougheed et al., 1999). But it is clear that geological and
climatic changes in the Miocene and Pliocene played an important role in the
diversification of Amazonian vertebrates (Bush, 1994; Glor et al., 2001; Da Silva &
Patton, 1998; Symula et al., 2003; Santos et al., 2009; Kaefer et al., 2012; Fouquet et
al., 2014; Gehara et al., 2014). However, only future process-based studies and
biogeographic hypotheses testing will allowed us to reveal the mechanisms (eg.
dispersion, vicariance, founder event) by which Amazophrynella diversified.
100
Acknowledgments
We would like to thank Ariane Silva, from the herpetological collection of the
Instituto Nacional de Pesquisas da Amazônia (INPA) in Manaus, Brazil, to Fernando
Ayala from the Pontificia Universidad Católica de Ecuador (PUCE) in Quito-Ecuador,
and to O. Aguilar and R. Orellana (MUBI) for providing administrative support and
part of the material for this study. R.R. Rojas thanks Alexander Almeida, Ian Pool
Medina, Richard Naranjito Curto for field support and Mario Nunes for laboratory
assistance.
101
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Table 1. Uncorrected p – distances among mtDNA lineages of Amazophrynella. Molecular distances are based on the 480–bp
fragment of 16S rDNA.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 A. amazonicola
2 A. siona sp. nov. 0.07
3 A. aff. minuta sp1 0.08 0.09
4 A. minuta 0.09 0.09 0.02
5 A. matses 0.09 0.13 0.09 0.09
6 A. aff. matses sp1 0.09 0.13 0.09 0.10 0.02
7 A. javierbustamantei 0.09 0.13 0.08 0.08 0.06 0.06
8 A. moisesii sp. nov. 0.08 0.11 0.08 0.08 0.09 0.09 0.06
9 A. vote 0.12 0.15 0.11 0.11 0.13 0.13 0.11 0.10
10 A. aff. vote sp1 0.12 0.15 0.11 0.11 0.12 0.12 0.12 0.11 0.03
11 A. aff. vote sp2 0.12 0.15 0.11 0.11 0.12 0.12 0.12 0.11 0.04 0.03
12 A. bokermanni 0.12 0.14 0.11 0.11 0.12 0.12 0.11 0.11 0.05 0.05 0.06
13 A. sp2 0.12 0.15 0.10 0.11 0.11 0.11 0.11 0.11 0.07 0.08 0.08 0.07
14 A. sp3 0.11 0.14 0.10 0.10 0.11 0.11 0.12 0.10 0.07 0.07 0.07 0.06 0.04
15 A. xinguensis 0.12 0.15 0.11 0.12 0.13 0.13 0.13 0.11 0.07 0.08 0.08 0.07 0.05 0.06
16 A. manaos 0.13 0.15 0.12 0.13 0.11 0.11 0.12 0.12 0.09 0.09 0.08 0.09 0.09 0.09 0.09
17 A. sp1 0.12 0.15 0.11 0.12 0.11 0.12 0.12 0.13 0.11 0.10 0.09 0.10 0.09 0.10 0.10 0.06
114
115
Table 2. Taxonomic status, congruence and comparison of main diagnostic morphological characters of species identified in
phylogenetic analyses (16S + 12S + COI). Character (-) indicates no data available. CCS= Confirmed Candidate Species;
UCS= Unconfirmed Candidate Species; DCL= Deep Conspecific Lineages; UL= Uncategorized Lineage.
Lineages Status Dorsal skin texture
Ventral skin texture
Head shape
Palmar tubercle
FI vs. FII
Venter coloration
Venter stain
A. manaos CCE Granular Granular Truncate Elliptical I<II White Large blotches
A. teko sp. nov. CCE Highly
granular Highly granular
acute Elliptical I<II Creamy Small blotches
A. sp1 UL Highly
granular Highly granular
acute Elliptical I<II Creamy Small blotches
A. vote CCE Tuberculate Granular Rounded Rounded I<II Reddish-
brown Small dots
A. aff. vote sp1 DCL Tuberculate Granular Rounded Rounded I<II reddish-
brown Small dots
A. aff. vote sp2 DCL Tuberculate Granular Rounded Rounded I<II reddish-
brown Small dots
A. bokermanni CCE Granular Granular Pointed Rounded I>II white Small dots
A. xinguensis sp. nov. CCE Highly
granular Granular Pointed Ovoid I=II Greyish Medium-size dots
A. sp2 UL - - - - - - -
A. sp3 UL - - - - - - -
116
A. matses CCS Spiculate Granular Acute Rounded I<II Yellow Blotches
A. aff. matses sp1 UCS - - - - - - -
A. javierbustamantei CCE Tuberculate Coarsely
areolate Acuminate Rounded I<II Pale
yellow Small dots
A. moisesii sp. nov. CCS Tuberculate Highly
granular Acuminate Elliptical I<II Pale
yellow Tiny points
A. amazonicola CCS Finelly
granular Granular Pointed Rounded I<II Yellow Medium-size blotches
A. siona sp. nov. CCS Finelly
granular Granular Acute Rounded I<II Reddish-
brow Small blotches
A. minuta CCS Highly
granular Granular Pointed Rounded I<II Yellow-
orange Large blotches
A. aff. minuta sp1 DCL Highly
granular Granular Pointed Rounded I<II Yellow-
orange Large blotches
117
Table 3. Descriptive morphometric statistics (in mm) for males of nominal and CCE of Amazophrynella. KW= Kruskal Wallis test, (+)
p-value<0.05.
Variable
A. minuta
(n = 20)
A. matses
(n = 13)
A. javierbustam-antei
(n = 28)
A. moisesii sp. nov (n =15)
A. amazonicola
(n = 15)
A. siona sp. nov. (n = 29)
A. bokerma-nni
(n = 7)
A. xinguensis sp.nov. (n = 5)
A. manaos
(n = 27)
A. teko sp.nov. (n = 13)
A. vote
(n = 14)
KW p-value
SVL 13.5±0.6 12.1±0.6 14.9±0.9 14.3±0.5 14.5±0.7 13.1±0.6 16.3±0.2 18.8±0.9 14.2±0.7 14.8±0.7 13.1±0.7 +
HW 4.2±0.2 3.6±0.2 4.2±0.2 4.3±0.4 4.4±0.3 3.9±0.3 4.8±0.1 5.1±0.2 4.2±0.3 4.5±0.3 4.0±0.7 +
HL 4.9±0.2 4.3±0.3 5.1±0.3 5.4±0.3 5.2±0.3 4.9±2.2 5.7±0.1 6.6±0.2 5.3±0.3 5.3±0.2 4.6±0.3 +
SL 2.3±0.1 2.0±0.3 2.2±0.2 2.6±0.2 2.4±0.2 2.2±0.2 3.0±0.1 3.2±0.1 2.7±0.2 2.5±0.1 2.1±0.2 +
ED 1.4±0.1 1.1±0.1 1.3±0.1 1.6±0.2 1.2±0.1 1.3±0.1 1.7±0.1 2.0±0.1 1.3±0.1 1.5±0.1 1.3± .1 +
IND 1.2±0.1 1.0±0.1 0.9±0.1 1.2±0.1 1.2±0.1 1.1±0.08 1.4±0.1 1.5±0.5 1.1±0.1 1.3±0.1 1.1±0.1 +
UAL 3.8±0.2 3.5±0.4 4.5±0.4 4.8±0.6 4.5±0.3 4.1±0.4 5.4±0.4 6.1±0.5 3.6±0.4 4.8±3.2 3.9±0.5 +
HAL 2.8±0.2 2.7±0.2 3.6±0.4 3.4±0.5 3.2±0.2 2.7±0.2 3.4±0.6 3.7±0.3 2.8±0.6 3.2±0.2 3.0±0.3 +
THL 6.8±0.2 6.2±0.4 7.6±0.7 7.9±0.8 7.7±0.6 7.0±0.4 8.0±0.3 9.5±0.8 6.7±0.3 7.6±0.8 6.5±0.7 +
TAL 6.7±0.3 5.8±0.3 7.6±0.7 7.7±0.9 7.2±0.6 6.6±0.4 7.5±0.3 9.1±0.7 6.9±0.6 7.3±0.5 5.7±0.7 +
TL 4.1±0.2 3.8±0.2 4.7±0.8 5.2±1.2 4.2±0.6 4.1±0.4 4.8±0.4 5.5±0.2 4.6±0.4 4.6±0.4 3.8±1.0 +
FL 4.8±0.4 4.3±0.4 5.7±0.6 5.7± 0.7 5.1±0.4 4.7±0.5 5.6±0.4 6.4±0.2 5.2±0.5 5.5±0.5 4.4±0.6 +
118
Table 4. Descriptive morphometric statistics (in mm) for females of nominal and CCS of Amazophrynella. KW= Kruskal Wallis test,
(+) p-value<0.05.
Variable
A. minuta
(n = 20)
A. matses
(n = 13)
A. javierbustam-antei
(n = 28)
A. moisesii sp. nov (n =15)
A. amazonicola
(n = 15)
A. siona sp. nov. (n = 35)
A. bokerma-nni
(n = 7)
A. xinguensis sp.nov. (n = 13)
A. manaos
(n = 27)
A. teko sp. nov. (n = 17)
A. vote
(n = 14)
KW p-value
SVL 17.4±0.9 17.1±0.7 19.7±1.8 18.5±1.6 18.1±1.1 18.3±0.9 23.4±0.8 24.1±1.2 20.8±2.1 19.2±1.1 16.3±1.6 +
HW 5.1±0.4 4.8±0.4 5.0±0.3 5.1±0.3 5.1±0.4 5.1±0.3 6.4±0.3 6.3±0.3 6.0±0.6 5.4±0.3 4.8±0.4 +
HL 6.0±0.4 5.6±0.3 6.2±0.3 6.4±0.4 6.1±0.4 6.2±0.3 7.9±0.3 7.9±0.3 7.2±0.3 6.5±0.3 5.4±0.4 +
SL 2.7±0.2 2.7±0.3 2.8±0.2 2.9±0.3 1.5±0.2 2.9±0.3 3.6±0.1 3.75±0.2 3.3±0.3 2.9±0.2 2.6±0.3 +
ED 1.7±0.3 1.4±0.2 1.5±0.3 1.9±0.2 1.4±0.1 1.7±0.2 2.2±0.2 2.1±0.1 1.8±0.2 1.8±0.1 1.7±0.2 +
IND 1.4±0.1 1.2±0.2 1.2±0.1 1.4±0.1 1.2±0.1 1.4±0.1 1.6±0.1 1.6±0.1 2.0±0.1 1.5±0.1 1.3±0.1 +
UAL 5.2±0.2 5.2±0.2 6.1±0.6 6.0±0.5 5.5±0.6 5.6±0.4 7.9±0.3 8.0±0.4 5.5±0.3 6.1±0.5 4.9±0.7 +
HAL 3.6±0.3 3.7±0.3 4.6±0.4 4.6±0.5 3.9±0.4 3.9±0.3 4.9±0.2 5.0±0.4 4.4±0.3 4.1±0.3 3.4±0.5 +
THL 8.5±0.9 8.3±0.4 9.6±0.8 9.8±0.4 9.5±0.8 9.4±0.6 11.8±0.7 11.8±0.8 10.2±0.6 9.5±0.5 7.7±0.8 +
TAL 8.4±0.7 8.3±0.4 9.8±0.8 9.6±0.5 9.1±0.7 9.2±0.6 11.0±0.4 11.2±0.6 10.2±0.6 9.4±0.6 7.2±1.0 +
TL 5.4±0.4 5.3±0.4 5.9±0.5 5.7±0.3 5.4±0. 5.7±0.5 6.9±0.4 7.1±0.4 7.1±0.9 5.7±0.4 4.6±0.6 +
FL 6.4±0.7 6.2±0.4 7.2±0.7 7.3±0.7 6.5±0.6 7.0±0.6 8.6±0.5 8.9±0.5 8.1±0.6 7.2±0.62 5.6±0.9 +
119
Table 5. Successful classification in morphological space (males) recovered phylogenetic mt DNA lineages (Eastern and Western
clades). In parenthesis, the percentage of successfully classification. The numbers in the cells represent the numbers of
individuals assigned to each clade by discriminant analyses. UCS and UL were not included.
Lineages
(Eastern clade)
A. manaos (90%)
A. teko sp. nov. (68%)
A. vote (100%)
A. aff. vote sp1 (63%)
A. aff. vote sp2 (0%)
A. bokermanni (50%)
A. xinguensis sp. nov. (80%)
A. manaos 27 0 0 0 0 0 0
A. teko sp. nov. 0 15 0 0 0 1 0
A. vote 0 0 13 0 0 0 0
A. aff. vote sp1 1 1 0 7 2 0 0
A. aff. vote sp2 0 0 0 3 0 0 0
A. bokermanni 1 1 0 0 0 3 1
A. xinguensis sp. nov.
1 0 0 0 0 0 4
Lineages
(Western clade)
A. matses (39%)
A. javierbustamantei (79%)
A. moisesii sp. nov. (31%)
A. amazonicola (85%)
A. siona sp. nov. (59%)
A. minuta (74%)
A. minuta sp1
(0%)
A. matses 5 5 0 1 2 0 0
A. javierbustamantei
1 23 1 0 2 2 0
A. moisesii sp. 0 0 4 0 7 0 2
120
nov.
A. amazonicola 0 1 0 22 2 1 0
A. siona sp. nov. 0 2 2 2 16 5 0
A. minuta 0 0 1 2 23 3
A. minuta aff. sp1 0 0 2 0 0 7 0
121
Table 6. Successful classification in environmental space recovered phylogenetic mt DNA lineages (Eastern and Western clades).
In parentheses, the percentage of successful classifications. The numbers in the cells represent the numbers of individuals
assigned to each clade by discriminant analyses. UCS and UL were not included.
Lineages
(Eastern clade)
A. manaos (77%)
A. teko sp. nov. (90%)
A. vote (80%)
A. aff. vote sp1 (40%)
A. aff. vote sp2 (33%)
A. bokermanni (50%)
A. xinguensis sp. nov. (66%)
A. manaos 7 0 0 0 0 1 0
A. teko sp. nov. 0 10 0 0 0 0 0
A. vote 0 0 4 2 2 0 0
A. aff. vote sp1 1 0 1 2 2 0 0
A. aff. vote sp2 0 0 0 1 1 1 0
A. bokermanni 1 1 0 0 0 2 1
A. xinguensis sp. nov.
0 0 0 0 0 1 2
Lineages
(Western clade)
A. matses (87%)
A. javierbustamantei (100%)
A. moisesii sp. Nov. (62)%)
A. amazonicola (100%)
A. siona sp. nov. (80%)
A. minuta (70%)
A. minuta sp1
(0%)
A. matses 7 0 0 0 0 0 0
A. javierbustamantei 0 6 2 0 0 0 0
A. moisesii sp. nov. 0 0 5 0 0 0 0
A. amazonicola 1 0 0 6 0 0 0
122
A. siona sp. nov. 0 0 0 0 8 2 1
A. minuta 0 0 1 1 7 0
A. aff. minuta sp1 0 0 0 0 1 1 0
123
Figures
Figure 1. Phylogeny and geographic distribution of Amazophrynella. A) Phylogenetic
relationship among nominal and putative species of Amazophrynella based on
Bayesian inference inferred from 1430 aligned sites of the 16S, 12S and COI mtDNA
genes. Numbers in branches represent Bayesian posterior probability. B) Geographic
distribution of Amazophrynella spp. Colors and symbols = occurrence areas for each
clade based on specimens reviewed in collections. Black points = Localities of
genetic collection from specimens. Colors and symbols of clades in the phylogenetic
tree correspond to colors and symbols on the map. Base maps were downloaded as
freely available SRTM maps from https://earthexplorer.usgs.gov/.
124
Figure 2. Time calibrated tree of Amazophrynella with posterior probabilities and
mean age. Blue bars represent 95% HPD.
125
Figure 3. Principal components analyses (PCA) of morphometric and enviromental
variables: Morphometric PCA: A) Eastern clade, B) Western clade. Environmental
PCA: C) Eastern clade, D) Western clade. Symbols and colors represents the clades
recovered by the phylogenetic analyses (Fig.1). UCS and UL were not include.
126
Figure 4. Holotype of Amazophrynella teko sp. nov. (MNHN 2015.136); A) dorsal
view; B) ventral view; C) dorsal view of the head; D) ventral view of the head; E) left
toe; F) left hand. Photos by Rommel R. Rojas.
127
Figure 5. Measurement comparison of SVL between males of nominal species of
Amazophrynella.
128
Figure 6. Comparison of palmar tubercles of nominal species of Amazophrynella. A)
A. teko sp. nov. B) A. siona sp. nov. C) A. xinguensis sp. nov. D) A. bokermanni. E)
A. vote. F) A. amazonicola. G) A. minuta. H) A. matses. I) A. manaos. J) A.
javierbustamantei. K) A. moisesii sp. nov. Elliptical (A, I, J); Rounded (B, E, D, H, F,
G); Ovoid (C). See Table 2. Photos by Rommel R. Rojas.
129
Figure 7. Ventral skin coloration of nominal species of Amazophrynella. A) A. minuta.
B) A. teko sp. nov. C) A. siona sp. nov. D) A. xinguensis sp. nov. E) A. bokermanni.
F) A. vote. G) A. manaos. H) A. amazonicola. I) A. matses. J) A. javierbustamantei,
K) A. moisesii sp. nov Large blotches (A, G); medium size blotches (H); small
blotches (B, I, C); small dots (F, E, J); medium size dots (D); tiny points (K). See
Table 2. Photos by Rommel R. Rojas.
130
Figure 8. Comparison of head profile of nominal species of Amazophrynella in lateral
view. A) A. minuta. B) A. teko sp. nov. C) A. siona sp. nov. D) A. xinguensis sp. nov.
E) A. bokermanni. F) A. vote. G) A. manaos. H) A. amazonicola. I) A matses. J) A.
javierbustamantei. K) A. moisesii sp. nov. Arrow indicates a small protuberance in the
tip of the snout of A. amazonicola. Pointed (A, H, D, E); acute (B, C, I); truncate (G);
rounded (F); acuminate (K, J). See Table 2. Photos by Rommel R. Rojas.
131
Figure 9. Morphological variation in live Amazophrynella teko sp. nov. (unvouchered
specimens). Photos by Antoine Fouquet.
132
Figure 10. Morphological variation of preserved specimens of Amazophrynella teko
sp. nov. Adult males: MHNN 2015.138 (A-B); MHNN 2015.152 (C-D); MHNN
2015.139 (E-F). G-L Adult females: MHNN 2015.141 (G-H); MHNN 2015.143 (I-J);
MHNN 2015.150 (K-L). Photos by Rommel R. Rojas.
133
Figure 11. Oscillogram and spectrogram of the advertisement call of Amazophrynella
teko sp. nov. A) three notes, B) one note.
134
Figure 12. Holotype of Amazophrynella siona sp. nov. (QCAZ 27790); A) dorsal view;
B) ventral view; C) ventral view of head; D) dorsal view of head; E) right hand; F)
right foot. Photos by Rommel R. Rojas.
135
Figure 13. Morphological variation of live Amazophrynella siona sp. nov. QCAZ
51068 (A-B); QCAZ 42988 (C-D); QCAZ 42988 (E-F). Photos by Santiago R. Ron.
136
Figure 14. Morphological variation of preserved specimens of Amazophrynella siona
sp. nov. Adult males: QCAZ 54213 (A-B); QCAZ 11979 (C-D); QCAZ 18826 (E-F).
Adult females: QCAZ 38679 (G-H); QCAZ 6091 (I-J); QCAZ 52434 (K-L). Photos by
Rommel R. Rojas.
137
Figure 15. Tadpole of Amazophrynella siona sp. nov. National Park Yasuni, Ecuador
(QCAZ 24576), stage 30; A) dorsolateral view; B) dorsal view; C) ventral view; D) oral
disc view. Photos by Rommel R. Rojas.
Figure 16. Oscillogram and spectrogram of the advertisement call of Amazophrynella
siona sp. nov. A) three notes, B) one note.
138
Figure 17. Holotype of Amazophrynella xinguensis sp. nov. (INPA-H 35471); A)
dorsal view; B) ventral view; C) ventral view of head; D) dorsal view of head; E) right
hand; F) right foot. Photos by Rommel R. Rojas.
139
Figure 18. Morphological variation of live Amazophrynella xinguensis sp. nov.
(unvouchered specimens). Photos by Emil Hernández-Ruz.
140
Figure 19. Morphological variation of preserved specimens of Amazophrynella
xinguensis sp. nov. Adult males: INPA-H 35482 (A-B), INPA-H 35493 (C-D); INPA-H
35471 (E-F). Adult females: INPA-H 35477 (G-H); INPA-H 35478 (I-J); INPA-H 35479
(K-L). Photos by Rommel R. Rojas.
141
Figure 20. Holotype of Amazophrynella moisesii sp. nov. (UFAC-RB 2815); A) dorsal
view; B) ventral view; C) ventral view of head; D) dorsal view of head; E) right hand;
F) right foot. Photos by Rommel R. Rojas.
142
Figure 21. Measurement comparison of HAL between males of nominal species of
Amazophrynella.
143
Figure 22. Measurement comparison of SL between males of nominal species of
Amazophrynella.
144
Figure 23. Morphological variation in live Amazophrynella moisesii sp. nov.
(unvouchered specimens). Photos by Paulo R. Melo-Sampaio.
145
Figure 24. Morphological variations of preserved specimens of Amazophrynella
moisesii sp. nov. Adult males: UFAC-RB 1698 (A-B); UFAC-RB 2694 (C-D); UFAC-
RB 2815 (E-F). Adult females: UFAC-RB 2608 (G-H); UFAC-RB 2610 (I-J); UFAC-
RB 2607 (K-L). Photos by Rommel R. Rojas.
146
Figure 25. Confirmed candidate species (CCS) of Amazophrynella: A-B) A. minuta
Photo by Rommel R. Rojas; C-D) A. teko sp. nov. Photo by Antoine Fouquet; E-F) A.
siona sp. nov. Photo by Santiago R. Ron; G-H) A. xinguensis sp. nov. Photo by Emil
Hernándes-Ruz; I-J) A. bokermanni Photo by Marcelo Gordo; K-L) A. manaos Photo
by Rommel R. Rojas. M-N) A. amazonicola Photo by Rommel R. Rojas. O-P) A.
matses Photo by Rommel R. Rojas; Q-R) A. javierbustamantei Photo by Juan Carlos
Chapparro; S-T) A. vote Photo by Robson W. Ávila; U-V) A. moisesii sp. nov. Photo
by Paulo R. Melo-Sampaio.
147
CAPITULO III
Description of the advertisement call of four species of Amazophrynella
(Anura:Bufonidae). Rojas, R.R., Carvalho, V., Ávila, R., Kawashita, R., Hrbek, T. &
Gordo, M. (2018). Zootaxa, 4459, 193–196.
148
Description of the advertisement call of four species of Amazophrynella
(Anura:Bufonidae)
ROMMEL R. ROJAS1*, VINICIUS TADEU DE CARVALHO1,2, ROBSON W. ÁVILA2,
RICARDO A. KAWASHITA-RIBEIRO3, TOMAS HRBEK1 & MARCELO GORDO4
1 Laboratório de Genética e Evolução Animal, Departamento de Genética, ICB,
Universidade Federal do Amazonas, Av. Gen. Rodrigo Octávio Jordão Ramos, 6200,
CEP 69077–000, Manaus, AM, Brazil *Corresponding author:
2 Universidade Regional do Cariri (URCA), Campus do Pimenta, Rua Cel. Antônio
Luiz, 1161, Bairro do Pimenta, CEP 63105-100, Crato, CE, Brazil
3 Instituto de Ciências e Tecnologia das Águas, Universidade Federal do Oeste do
Pará, Av. Mendonça Furtado, 2946, CEP 68040-050, Santarém, PA, Brazil.
4 Departamento de Biologia, Instituto de Ciências Biológicas, Universidade Federal
do Amazonas, Av. General Rodrigo Octávio Jordão Ramos, 6200. CEP 69077–000,
Manaus, AM, Brazil
*Corresponding autor: [email protected]
149
Amazophrynella comprises seven small bufonid species with a pan-
Amazonian distribution (Fouquet et al. 2012a, b; Rojas et al. 2016). All species
inhabit the forest leaf litter, breed in seasonal puddles and are diurnally and
nocturnally active (Fouquet et al. 2012b; Rojas et al. 2014; 2015; 2016). Until now
only one nominal species, A. javierbustamantei, and two putative lineages — A. sp.
(Rio Yuyapichis, Peru) and A. aff. minuta (Santa Cecilia, Ecuador) — had their
advertisement calls formally described (Duellman 1978; Schlüter 1981; Rojas et al.
2016). Herein, we described for the first time the advertisement calls from additional
four species of Amazophrynella.
The calls were recorded in uncompressed .wav format, with a Zoom H1 Handy
Recorder (Zoom Corporations, Tokyo, Japan) equipped with an internal microphone,
positioned approximately 1.0–2.5 m from the focal male. All calls were filtered with
Audacity 1.2.2 for Windows (Free Software Foundation Inc., 1991). Praat 4.2.22 for
Windows (Boersma & Weenick, 2006) was used to generate audiospectrograms and
oscillograms at a sampling frequency of 44.0 kHz and 16-bit resolution. Spectral
parameters were analyzed through fast Fourier transformations (FFT) (width 1024
points). We used the Seewave package (Sueur et al. 2008) implemented in the
software R (R Development Core Team, 2008) for figures. Air temperature was
measured immediately after sound recording of each male. Call structures were
visually analyzed in the spectrograms subsequent to which we measured the
following quantitative parameters considered informative in amphibian taxonomy
(Köhler et al. 2017): call duration (s), inter-call interval (s), number of pulses per call,
dominant frequency (Hz), fundamental frequency (Hz), pulse rate (pulses/s) and call
rise time (s) and numbers of harmonics. Call parameter measurements are presented
as range (mean ± standard deviation) throughout the text.
The characterization of the advertisement call of Amazophrynella manaos
Rojas, Carvalho, Gordo, Ávila, Farias & Hrbek, 2014 was based on 20 calls from
three individuals (INPA-H 6983, snout-vent-length (SVL): 13.1 mm; INPA-H 6984,
SVL: 13.6 mm; INPA-H 6987, SVL: 13.9 mm) recorded between 8:30–10:00 h at the
type locality (campus of the Federal University of Amazonas/UFAM , 03°05’37” S,
59°58’26” W), within the city of Manaus, state of Amazonas, Brazil. Air temperature at
the time of recording varied between 26.0 to 27.5°C. The call consisted of a trilled
150
note emitted between regular silent intervals (Fig. 1A). Notes had an upward
amplitude modulation, reaching their maximum intensity near the end of the note.
Note duration ranged between 0.133 and 0.156 s (0.144 ± 0.006 s). Number of
pulses per note ranged from 35 to 51 pulses/note (44.050 ± 5.175 pulses/note). The
inter-note interval was 0.474 to 1.755 s (0.762 ± 0.355 s). The dominant frequency
ranged from 2990.110 to 3427.050 Hz (3338.860 ± 147.153 Hz). The fundamental
frequency ranged from 2617.899 to 3656.613 Hz (3038.247 ± 270.139 Hz). The rise
time ranged from 0.083 to 0.112 s (0.101 ± 0.008 s). Numbers of harmonic varied
from 3 to 5 (4.150 ± 0.587).
The characterization of the advertisement call of Amazophrynella bokermanni
(Izecksohn, 1993) was based on 22 calls from one individual (INPA-H 31861, SVL:
14.2 mm) recorded at 16:00 h around 30 km east from the species type locality
(03°41’20” S, 59°06’19” W), in Juruti, state of Pará, Brazil. Temperature varied from
26°C to 27°C. The call consisted of one trilled note emitted at regular silent intervals
(Fig. 1B). Notes had an upward amplitude modulation increasing in the first half of
the call. Note duration ranged from 0.125 to 0.163 s (0.146 ± 0.011 s). The number of
pulses per note ranged from 26 to 48 pulses/note (40.591 ± 4.767 pulses/note). The
inter-note silent interval ranged from 0.631 to 1.501 s (0.721 ± 0.177 s). The
dominant frequency varied from 3346.143 to 3831.633 Hz (3635.711 ± 159.738 Hz).
The fundamental frequency ranged from 3048.185 to 3410.97 Hz (3195.456 ±
140.472 Hz). Rise time ranged from 0.041 to 0.091 s (0.061 ± 0.015 s). Number of
harmonics varied from 3 to 6 (4.181 ± 0.795).
The characterization of the advertisement call of Amazophrynella vote Ávila,
Carvalho, Gordo, Kawashita-Ribeiro & Morais, 2012 was characterized based on 30
calls from one individual (UFMT 11141, SVL: 15.3 mm) recorded at 10:00 h at the
type locality, at São Nicolau Farm (09°50’43.3” S, 58°13’10.6” W), in the municipality
of Cotriguaçu, state of Mato Grosso, Brazil. Air temperature varied from 26°C to
28°C. The call consisted of one trilled note emitted between regular silent intervals
(Fig. 1C). Notes had an upward amplitude modulation increasing towards the end of
the call. Note duration ranged from 0.098 to 0.150 s (0.127 ± 0.014 s). The number of
pulses per note ranged from 41 to 60 pulses/note (52.410 ± 6.531 pulses/note). The
inter-note silent interval ranged from 0.544 to 1.879 s (0.831 ± 0.389 s). The
151
dominant frequency varied between 3084.211 to 3415.444 Hz (3204.169 ± 75.266
Hz), the fundamental frequency from 3250.921 to 3750.712 Hz (3492.608 ± 155.652
Hz). Rise time ranged from 0.054 to 0.120 s (0.087 ± 0.015 s). Number of harmonics
varied from 3 to 4 (3.133 ± 0.345).
The characterization of the advertisement call of Amazophrynella amazonicola
Rojas, Carvalho, Gordo, Ávila, Farias & Hrbek, 2014 is based on 35 calls from one
individual (MZUNAP 917- SVL: 13.8 mm) recorded at 10:00 h at the type locality
Puerto Almendra (3°49'41''S, 73°22'07''W), department of Loreto, Peru. The
recording temperature varied from 25°C to 26°C. The call consisted of one trill note
issued during irregular intervals (Fig. 1D). Notes had a downward modulation,
reaching maximum frequency almost at the beginning. The duration of the notes was
from 0.013 to 0.025 s (0.018 ± 0.003 s). The number of pulses was from 4 to 12 per
note (8.514 ± 2.331 pulses/note). The inter-note interval was from 0.660 to 2.3240 s
(1.617 ± 0.344 s). The dominant frequency varied from 3054.840 to 3521.22 Hz
(3277.398 ± 93.458 Hz). The fundamental frequency varied from 1404.170 to
2893.010 Hz (2354.207 ± 364.419 Hz), sometimes coinciding with dominant
frequency. The time to peak at maximum frequency was from 0.002 to 0.008 s (0.004
± 0.001 s). Number of harmonics varied from 2 to 3 (2.571 ± 0.502).
Analysis of the advertisement calls suggests a conservative structure of the
calls among species of the genus Amazophrynella. The calls of A. manaos, A.
bokermanni and A. vote are much more similar to each other than to that of A.
amazonicola, the main differences being note duration and number of pulses/note,
fundamental and dominant frequencies. The structure and values of call parameters
of the advertisement call of A. amazonicola are similar to those of A. sp. and A. aff.
minuta from Peru and Ecuador (see Duellman 1978; Schlüter 1981) and A.
javierbustamantei (Rojas et al. 2016), the main differences being the values of the
dominant frequency, note intervals and note duration. The advertisement call of A.
manaos was more similar to that of A. bokermanni in number of pulses per note and
note duration, with the main difference between the two species being the values of
dominant frequency and fundamental frequency. The species A. vote had the
greatest note duration and A. amazonicola had the shortest note duration.
Furthermore, similarities in call structure of the advertisement calls are also reflected
152
in the evolutionary relationships among these species, as suggested by the
phylogenetic hypothesis proposed by Rojas et al. (2016). Bioacoustic data are scarce
for Amazophrynella, but are likely to be important in contributing to the resolution of
the taxonomic status of unconfirmed candidate species (UCS, sensu Vieites et al.
2009) within the framework of integrative taxonomy (Padial et al. 2010).
The senior author thanks Tainara V. Sobroza for help in bioacoustics analyses.
RWA thanks CNPq for his productivity research grant (303622/2015-6). MG thanks
Selvino Neckel-Oliveira for support in Juruti Project.
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155
FIGURE 1. Advertisement call of four species of Amazophrynella. A) A. manaos
(Campus UFAM, INPA-H 6983, 26.0 °C); B) A. bokermanni (Juruti, INPA-H 31861,
27°C); C) A. vote (São Nicolau Farm, UFMT 11141, 27°C); D) A. amazonicola
(Puerto Almendra, MZUNAP 917, 25°C).
156
CAPITULO IV
Redescription of the Amazonian tiny tree toad Amazophrynella minuta (Melin, 1941)
(Anura: Bufonidae) from its type Locality. Rojas, R.R., Fouquet, A., Carvalho, V.,
Ron, S., Chaparro, J., Vogt, R., Ávila, R., Pires, I., Gordo, M. & Hrbek, T. Zootaxa
4482 (3): 511–526.
157
Redescription of the Amazonian tiny tree toad Amazophrynella minuta (Melin,
1941) (Anura: Bufonidae) from its type Locality
Rommel R. Rojas1, Antoine Fouquet2, Vinícius Tadeu De Carvalho1, Santiago Ron3,
Juan Carlos Chaparro4, Richard C. Vogt5, Robson W. Ávila6, Izeni Pires Farias1,
Marcelo Gordo7 & Tomas Hrbek1
1Laboratório de Evolução e Genética Animal (LEGAL), Departamento de Genética,
Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Av. General
Rodrigo Octávio Jordão Ramos, 6200. CEP 69077–000 Manaus, AM, Brazil
2 USR 3456 LEEISA - Laboratoire Ecologie, Evolution et Interactions des Systèmes
Amazoniens, Centre de recherche de Montabo, 275 route de Montabo, BP 70620,
97334 Cayenne, French Guiana.
3 Museo de Zoología, Escuela de Biología, Pontificia Universidad Católica del
Ecuador, Av. 12 de Octubre y Roca, Aptdo. 17–01–2184, Quito, Ecuador.
4 Museo de la biodiversidade-MUBI, Peru.
5 CEQUA, Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da
Amazônia, Av. André Araújo, 2936, Aleixo, CEP 69060-001, Manaus, AM, Brazil.
6 Departamento de Química Biológia, Universidade Regional do Cariri, Campus do
Pimenta, Rua Cel. Antônio Luiz, 1161, Bairro do Pimenta, CEP 63105–100, Crato,
CE, Brazil.
7 Departamento de Biologia, Instituto de Ciências Biológicas, Universidade Federal
do Amazonas, Av. General Rodrigo Octávio Jordão Ramos, 6200. CEP 69077–000
Manaus, AM, Brazil.
*Corresponding autor: [email protected]
158
Abstract
The description of Amazophrynella minuta was published in 1941 by the
Swedish naturalist Douglas Melin based on material from Taracuá (Amazonas state,
Brazil). This description was very brief and based on the morphology of few
specimens with diagnostic characters and color variation not well defined. Moreover,
the type series is currently in poor state of conservation. Consequently, taxonomic
ambiguity surrounds the nominal taxon A. minuta, which hampers the description of
many unnamed congeneric species. Herein, we redescribe A. minuta based on
recently collected specimens from the type locality, we designate a lectotype,
formulate a new diagnosis, provide patterns of morphological variation,
measurements and body proportions
Key words: Amphibians, Brazil, lectotype, systematics, Taracuá, taxonomy
Resumen
La descripción de la especie Amazophrynella minuta fue realizada en 1941
por el naturalista Sueco Douglas Melin en la localidad de Taracuá, estado de
Amazonas, Brasil. La descripción fue muy breve y basada solamente en la
morfología de pocos especímenes. Por ese motivo, los caracteres diagnósticos y el
padrón de coloración no fueron bien definidos en la descripción original, y en la
actualidad la serie tipo se encuentra en malas condiciones de preservación. En este
trabajo, realizamos la redescripción de A. minuta basados en especímenes
recientemente colectados de la localidad tipo, designamos un lectotipo, formulamos
una nueva diagnosis y proporcionamos el patrón de variaciones morfológicas,
medidas morfológicas y proporciones corporales
Resumo
A descrição da espécie Amazophrynella minuta foi publicada no ano 1941
pelo naturalista Sueco Douglas Melin baseado em material proveniente da localidade
de Taracuá, estado do Amazonas, Brasil. A descrição original foi muito breve e
baseada na morfologia de poucos indivíduos. Assim, os caracteres diagnósticos e o
padrão de coloração não foram bem definidos na descrição original, e a serie tipo
encontra-se em condições precárias. Consequentemente há ambiguidade
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taxonômica envolvendo o táxon nominal A. minuta, o que dificulta a descrição de
muitas espécies existentes no gênero Amazophrynella. Neste trabalho, realizamos a
redescrição de A. minuta utilizando espécimes recentemente coletados da localidade
tipo, designamos um lectótipo, formulamos uma nova diagnose morfológica,o padrão
de variação morfológica, medidas morfométricas e proporções corporais.
160
Introduction
The genus Amazophrynella Fouquet, Recoder, Teixeira, Cassimiro, Amaro,
Camacho, Damasceno, Carnaval, Moritz, & Rodrigues 2012a, comprises a group of
small-size bufonids distributed throughout Amazonia (Frost, 2017, Fouquet et al.,
2012b). They inhabit primary forest leaf litter, have diurnal activity and reproduce in
seasonal ponds (Magnusson & Hero, 1991; Ávila et al., 2012; Rojas et al., 2014,
2015). Currently eleven species are described (Rojas et al., 2018)
Douglas Melin described the type species of the genus, Amazophrynella
minuta in 1941, as Atelopus minutus. The description was based on four specimens
collected in 1924 in Taracuá, municipality of São Gabriel da Cachoeira, Amazonas
state, Brazil. The type specimens are deposited in the Göteborgs Naturhistoriska
Museum, Sweden (NHMG 462–465). These specimens are presently in poor state of
conservation, thus, difficult to compare morphologically with other species and
populations of Amazophrynella. In addition, the description is quite brief and lacks
many details in morphological diagnosis, variation, measurements and body
proportions that are perceived today as crucial given the actual diversity of the group.
During the last decade, several studies demonstrated that the genus
Amazophrynella represents a complex of cryptic species (Fouquet et al., 2012b;
Rojas et al., 2016; Rojas et al., 2018) and that many species have been, and still are,
erroneously identified as A. minuta (Fouquet et al., 2012b; Rojas et al., 2016). A
redescription of this species is, therefore necessary to accurately delimit and
describe other species of Amazophrynella.
Preliminary characterization of topotypic A. minuta from the type locality
carried out by Rojas et al. (2014) only provided general morphological characters and
did not describe the morphological variation and body measurements or define the
species’ current distribution. Herein we use extensive morphological, molecular and
bioacoustics data to redescribe A. minuta. We also provide a new combination of
diagnostic characters, and describe morphological variation, reproductive behavior
and geographic occurrence.
Material and methods
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Type locality. The type locality of Amazophrynella minuta is “Taracuá, Rio Uapés,
Brazil” (Melin, 1941) (=Taracuá, 0.10°S, 68.46°W, datum= WGS-84, municipality of
São Gabriel da Cachoeira, Amazonas state, Alto Rio Negro Amerindian Reserve,
Brazil). Topotypical specimens studied herein were collected on August 19 and 20,
2013. See Appendix 1 for examined specimens.
Measurements. The following morphological measurement were taken with a
Mitotuyo digital calliper (0.1 mm precision) with an ocular micrometer in a Zeiss
stereomicroscope: snout-vent length (SVL); head length (HL); head width (HW),
upper eyelid width (UEW); eye diameter (ED); snout length (SL); eye-to-nostril
distance (END); internarinal distance (IND); interorbital distance (IOD); hand length
(HAL); upper arm length (UAL); thigh length (THL); tibial length (TL); tarsal length
(TAL) and foot length (FL) following Kok & Kalamandeen (2008). Sex was determined
by gonadal analysis.
Definition of morphological characters. External morphological nomenclature
follows Kok & Kalamandeen (2008). Main diagnostic characters within
Amazophrynella were defined as follows:
Texture of skin (Figure 1). Most bufonids species are covered with variable–sized
warts (Ford & Cannatela, 1993), defined as, bearing protuberances with keratinized
tip (Kok & Kalamandeen, 2008). We defined texture of skin in Amazophrynella as
follows: tuberculate (Figure 1A), when the species present small sized wart with
conical tips. Granular (Figure 1B), when the species present medium sized warts with
rounded tips. In order to refine these taxonomic character, we considered skin as
“highly granular”, when species present high density of medium sized warts with
rounded tips (Figure 1C) and “finely granular”, when species present low density of
small sized warts with rounded tips (Figure 1D). Spiculate, when the species present
small sized warts with pointed tips (Figure 1E).
Ventral color pattern (Figure 2). We defined ventral color pattern in
Amazophrynella as follows (adapted from Kok & Kalamandeen, 2008): Blotches,
when the species present small to large, irregular light or black markings contrasting
with the background coloration (Figure 2A-D). Dots, when the species present small
or minute, more or less regular light or black markings contrasting with the
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background coloration (Figure 2E-F). Points, when the species present small to
medium-sized roughly round black markings in contrast to the background color
(Figure 2G).
Shape of head. We followed the nomenclature of proposed by Heyer et al. (1990)
page 409.
Shape of palmar and subarticular tubercles (Figure 3). We identified three main
shapes of palmar and subarticular tubercles in species of Amazophrynella, that are
defined as follows: Rounded, when the palmar and subarticular tubercles present a
circular shape, without a distinct point (Figure 3A). Ovoid, when the palmar and
subarticular tubercles present an oval, inverted egg shape, with a tapering or
irregular point (Figure 3B). Elliptical, when the palmar and subarticular tubercles
present an oval shape, with a flattened point (Figure 3C).
Advertisement call. The call was recorded in uncompressed .wav format, with a
Zoom H1 Handy Recorder (Zoom Corporations, Tokyo, Japan) equipped with an
internal microphone, positioned approximately 1.0–2.5 m from the focal male. All
calls were filtered with Audacity 1.2.2 for Windows (Free Software Foundation Inc.,
1991). Praat 4.2.22 for Windows (Boersma & Weenick, 2006) was used to generate
audiospectrograms and oscillograms at a sampling frequency of 44.0 kHz and 16-bit
resolution. Spectral parameters were analyzed through fast Fourier transformations
(FFT) (width 1024 points). Air temperature was measured immediately after each
sound recording. Call structures were visually analyzed in the spectrograms
subsequent to which we measured the following quantitative parameters considered
informative in amphibian taxonomy (Köhler et al., 2017): call duration (s), inter-call
interval (s), number of pulses per call, dominant frequency (Hz), fundamental
frequency (Hz), pulse rate (pulses/s) and call rise time (s) and numbers of harmonics.
Call parameter measurements are presented as range (mean ± standard deviation).
Results
Lectotype designation and redescription justification. Because superficiality in
morphological characters in original description, absent of material other than the
type series and because it is known that the genus Amazophrynella represents a
complex of cryptic species (Fouquet et al., 2012b; Rojas et al., 2018) and many
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similar species may currently be confused by A. minuta, a designation of a lectotype
will aid to taxonomic stability. In addition, that, a redescription will facilitate future
species delimitation within this species complex.
According to the International Code of Zoological Nomenclature (ICZN, 1999)
article 74.1.1: “A lectotype may be designated from syntypes to become the unique
bearer of the name of a nominal species-group taxon and the standard for its
application”. We also follow the recommendations of the article 73C of the ICZN
(2017) to provide data on the lectotype.
Species account
Amazophrynella minuta (Melin, 1941)
Synonymy
Atelopus minutus: Melin (1941: 40, Brazil; Amazonas Taracuá)
Dendrophryniscus minutus: McDiarmid (1971: 39, Brazil, French Guyana, Guyana,
Suriname, Venezuela, Colombia, Ecuador, Peru, Bolivia)
Amazonella minuta: Fouquet et al. (2012b: 832, Brazil, French Guyana, Guyana,
Suriname, Venezuela, Colombia, Ecuador, Peru, Bolivia)
Lectotype. Naturhistoriska Museet, Göteborg, Sweden, NHMG 462, SVL: 13.40 mm,
adult male, collected at Taracuá (0.10°S, 68.46°W) 100 m a.s.l, Amazonas state,
Brazil by Douglas Melin on 1924.
Paralectotypes (Figure 4). NHMG 463, NHMG 465 (adult males), NHMG 464 (adult
female).
Topotypical specimens. INPA–H 32721–34, adult males and INPA-H 32735–40, adult
females collected by Rommel R. Rojas at the same place of lectotype on August 19
and 20, 2013.
Diagnosis. A species of Amazophrynella with: (1) Medium body size for the genus
(see Table 1), adult females 15.0–19.0 mm SVL (n=11), adult males 12.2–14.2 mm
SVL (n=20); (2) snout pointed in lateral view; upper jaw, in lateral view, protruding
beyond lower (3) tympanum, vocal sac, parotid gland and cranial crests
164
inconspicuous; (4) texture of dorsal skin highly granular; (5) abundant small rounded
warts (in part conical) present on dorsal surfaces of forelimbs and hindlimbs; (6)
texture of ventral skin highly granular; (7) fingers slender, basally webbed; (8) finger I
shorter than finger II; (9) finger III relatively long (HAL/SVL 0.2–0.3 mm, n=31); (10)
palmar tubercle elliptical; (11) supernumerary tubercles rounded; (12) long hind limbs
(TAL/SVL 0.4–0.5, n=31); (13) toes slender, basally webbed; (14) toes lacking lateral
fringes ;(15) plantar surfaces of feet bearing one metatarsal tubercle, the inner 2.0x
larger than the outer, outer subconical; supernumerary plantar tubercles rounded;
(16) in life, venter coloration yellow–orange with larger to medium size black blotches
on venter.
Comparisons with other species. Amazophrynella minuta is most similar to A.
amazonicola from which it can be distinguished by (characteristics of compared
species in parentheses) absence of small triangular protrusion at the tip of the snout;
texture of dorsal skin highly granular (granular); palmar tubercle elliptic (rounded).
From A. matses by snout pointed in lateral view; texture of dorsal skin highly granular
(spiculate); venter coloration yellow–orange (pale yellow; Figure 5A vs. 5C). From A.
javierbustamantei by texture of dorsal skin highly granular (tuberculate); palmar
tubercle elliptical (rounded) throat and chest coloration light brown (grayish cream);
large blotches on venter (small points). From A. siona by snout profile pointed
(acute), warts on dorsum (granules) and texture of dorsal skin highly granular (finely
granular). From A. moisesii by snout profile pointed (acuminate), texture of dorsal
skin highly granular (tuberculate), large blotches on venter (tiny points). From A.
bokermanni, A. manaos, A. vote, A. xinguensis and A. manaos the main difference in
the venter coloration yellowish–orange (white in A. manaos, A. bokermanni and A.
teko, red brown in A. vote, brown in A. xinguensis; Figure 5A vs. 5F-J), FI < FII (vs. FI
> FII in A. bokermanni, and FI ≥ FII in A. xinguensis).
Redescription. Body slender, slightly enlarged posteriorly. Sexual dimorphism
observed in SVL, with 12.2–14.7 mm (13.6 ± 0.6 mm, n=20, Table 4) in adult males
and 14.5–19.4 mm (17.5 ± 1.5 mm, n=11) in adult females. Head pointed, longer than
wide: HL 4.6–6.8 mm (5.4 ± 0.5 mm), 36.0% of SVL; HW 3.8–5.8 mm (4.6 ± 0.1 mm),
30.0% of SVL. Snout profile pointed in lateral view; SL 2.1–3.2 mm (2.5 ± 0.3 mm),
46.0% of HL. Canthus rostralis straight and loreal region vertical. Nostrils
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protuberant, closer to snout than to eyes. IND 1.0–1.5 mm (1.2 ± 0.1 mm), 27.0% of
HW. Interorbital and occipital regions flat. Tympanic membrane and tympanic annulus
not apparent. Parotoid glands not visible. Vocal sac not visible in males. Eyes
prominent. Eye diameter 1.2–2.0 mm (1.5 ± 0.2 mm), 29.0% of HL. Eyelid with small
tubercles on borders. Choanae small and circular. Dorsal skin texture highly granular.
Small rounded warts (in part conical) on dorsolateral and lateral surfaces posterior to
eye, on posterior dorsum, and on dorsal surfaces of entire arm. Texture of gular
region, chest and belly highly granular. Forelimbs slender. Forearms robust,
especially in males. UAL 3.5–5.5 mm (4.4 ± 0.7 mm); 29.3% of SVL. Fingers slender.
HAL 2.4–4.5 mm (3.1 ± 0.5 mm); 70.0% of UAL. Edges of forelimbs with scattered
granules, in dorsal and ventral view. Fingers basally webbed. Relative length of
fingers: I < II < IV < III. First finger short, third two times the size of the second, fourth
larger than the first and second. Large elliptical palmar tubercle. Supernumerary
tubercles rounded. Thenar tubercle absent. Tip of fingers unexpanded. Nuptial pad
not evident. Hind limbs slender. Ventral and lateral surfaces of forelimbs granular.
THL 6.3–10.0 mm (7.6 ± 0.1 mm), 50.0% of SVL. TAL 7.4–10.0 mm (7.4 ± 1.0 mm),
49.4% of SVL. TL 3.7–6.7 mm (4.7 ± 0.8 mm); 32.0% of SVL. FL 4.2–8.0 mm (5.5 ±
0.9 mm), 71.1% of THL. Cloacal opening slightly above mid–level of thighs. Grouping
of brownish–yellow granules from the hidden surface of the thighs to the shank.
Basal webbing on foot. Toes lacking lateral fringes. Relative length of toes: I < II < III
< V < IV. First toe very short; second half the size of the third. Elliptical inner
metatarsal tubercle present. Subarticular tubercles visible, more protruding and
swollen in females than males. Foot slender. Tips of toes unexpanded.
Variation. The variation in measurements is presented in Table 1. Specimens from
Japura River (INPA–H 32731, INPA–H 32724, INPA–H 32734) are longer (SL about
1.0 mm) than individuals from the type locality. Some individuals from São Gabriel da
Cachoeira present minor abundance of warts on dorsal surface (i.e. INPA–H32731,
INPA–H 32724 and INPA–H 32734). Mottled warts disseminated on chest and belly
are found in specimens INPA–H32731 (São Gabriel da Cachoeira) and INPA–35496
(Japura River). Some specimens from the type locality (i.e. INPA–H 32728, INPA–H
32735) and from the Japura River (INPA–H 35512), present lower abundance of
warts on arms.
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Coloration in life (Figure 6). Head brown. Dorsum reddish–brown. Flanks light brown.
Dorsal surfaces of the upper arm and arm reddish–brown. Hands and fingers light
brown, in dorsal view. Insertion of the arms present yellow blotches. Dorsal surfaces
of the thighs, tibia, tarsus and feet reddish–brown. Ventral surfaces of upper arm and
arm cream. Palm reddish. Ventral surfaces of thighs mottled cream with small black
blotches. Thighs with a transverse lateral black bar. Posterior region of the thigh and
cloaca yellow, covered by small black blotches. Tarsus with transverse brown bars.
Sole reddish–brown. Gular region and chest grayish–brown. Venter coloration
between yellow to yellow–orange. Venter covered by large to medium size black
blotches. In ventral view, some specimens present yellow blotches at the insertion of
the arms. The specimens INPA–H 32725 (Taracuá) and INPA–H 35494 (Japura
River), display yellow coloration with small black blotches on posterior region of the
thigh near the cloaca. Iris golden and pupil black.
Color in preservative (Figure 7). The color in preservative faded. We noted the
progressive loss of the ventral coloration that became pale yellow. The yellow
coloration on axillary surface disappeared. In ventral view, the color of fingers and
toes lost their reddish intensity and became cream.
Distribution (Figure 8). Amazophrynella minuta is distributed throughout the eastern
border region of the Amazonas state in Brazil, a region also known as “Cabeça do
Cachorro” (“Dog’s head”, English free translation). The species is found in the
following localities: Taracuá (0.10°N, 68.46°W), São Gabriel da Cachoeira (0.16°S,
66.98°W), Cucuí (1.07°N, 66.88°W) and Japura River close to Vila Bittencourt
(1.81°S, 68.98°W), at elevations between 90–105 m a.s.l. In Colombia it is reported
in territories of the Department of Caquetá (0.92°N, 75.67°W) and Vaupés (0.90°S,
69.67°W) (Lynch, 2006) at elevations between 100–200 m a.s.l. (Acosta–Galvis,
2017). In Venezuela (not examined), the species was reported from Raudal de
Danto, Río Cuao (4.53°N, 67.18°W) Amazonas state, at elevation of 105 m a.s.l.
(Rojas–Runjaic et al., 2013).
Ecology. We found all individuals in the morning near a water body. Specimens were
collected in primary “terra firme” forest (“firm land” – non–flooded forest, English free
translation) some in the leaf litter and others among tree roots. Specimens from São
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Gabriel da Cachoeira were collected in a fragment of forest near to the airport; the
individuals were found around a stream with abundant leaf litter. Amplexus is axillar
(Figure 9).
Phylogenetic analysis. The complete phylogenetic relationships of Amazophrynella,
the phylogenetic position of A. minuta and genetic distance between Amazophrynella
spp. were reported in Rojas et al. (2018)- Figure 1 and Table 1).
Advertisement call (Figure 10). The characterization of the advertisement call of
Amazophrynella minuta was based on 14 calls from one individual from São Gabriel
da Cachoeira (INPA-H 32730, SVL: 12.2 mm, recorded between 15:30–16:00 h). Air
temperature at the time of recording varied between 26.0 to 27.0°C. The call
consisted of a trilled note emitted between regular silent intervals. Notes had an
upward amplitude modulation, reaching their maximum intensity near the middle of
the note. Note duration ranged between 0.310 and 0.108 s (0.223 ± 0.068 s).
Number of pulses per note ranged from 18 to 35 pulses/note (26.286 ± 6.557
pulses/note). The inter–note interval was 0.663 to 1.050 s (0.827 ± 0.120 s). The
dominant frequency ranged from 4242.790 to 4562.011 Hz (4419.213 ± 125.642 Hz).
The fundamental frequency ranged from 3201.010 to 3788.520 Hz (3489.211 ±
235.791 Hz). The rise time ranged from 0.045 to 0.083 s (0.059 ± 0.015 s). Number
of harmonics varied from 3 to 5 (4.150 ± 0.587).
Osteology. A detailed review and description of the skeleton of A. minuta were
provided by McDiarmid (1971). In the same publication, McDiarmind (1971) reported
characters that could be tentatively considered as putative genus level
synapomorphies (see Fouquet et al., 2012b).
Discussion
Amazophrynella minuta was described by Melin in 1941 from a few specimens
collected at Taracuá, municipality of São Gabriel da Cachoeira, Amazonas state,
Brazil in 1924. The original description lacked precision in describing morphological
characters such as shape of head, in dorsal and lateral view; texture of dorsal and
ventral skin; formula of fingers and toes; measurements, proportions of body and
details in coloration of the dorsal and ventral surfaces. Since these morphological
characters are important to distinguish and diagnose species of the genus (see
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Izecksohn, 1993; Ávila et al., 2012; Rojas et al., 2014, Fouquet et al., 2012b), herein,
we provided a detailed description of these characters. Furthermore, with the
identification of diagnostic characters and definition of character states of A. minuta,
this work will facilitate the comparison between species of Amazophrynella and
contribute to future descriptions of the remaining unnamed species in the genus
(Rojas et al., 2018).
Morphological traits and phylogenetic relationships suggest that
Amazophrynella minuta is most closely related to A. amazonicola, A. matses and A.
javierbustamantei with which it notably shares coloration and patterns on the belly
(see Figure 5); however, A. minuta differs from these species by snout profile and
texture of dorsal and ventral skin. From the other species of Amazophrynella, A.
minuta differs in SVL, body proportions, size of FI vs. FII; venter coloration, texture of
dorsal skin, type of palmar tubercle and webbing type on fingers (see Comparisons
with other species for details). The morphological crypsis in Amazonian anurans is a
taxonomic problem that hinders descriptions based only on morphological data, but
can and has been overcome in several groups using an integrated approach to
taxonomy (analyses of advertisement calls, morphology, genetics, tadpoles, etc.)
(sensu Padial et al., 2009; Padial et al., 2012; Caminer & Ron 2014)
The description of the advertisement call of A. minuta will contribute to
distinguishing nominal populations from others (i.e. Colombia, Venezuela and Brazil)
in the aim to delimit new species. As expected, the advertisement call of A. minuta is
similar to other species of Amazophrynella (Rojas et al., 2018). As a genus–level
synapomorphic character the advertisement calls of Amazophrynella present a
simple note that varies between 0.0180 to 0.0146 s; a fundamental frequency
between 2354.207 to 3204.169 Hz; a dominant frequency between 3033.86 to
3635.71 Hz; numbers of pluses ranged from 8 to 52.4 and a rise time from 0.004 to
0.107 s. Unfortunately, we could not collect tadpoles of Amazophrynella minuta,
although we made two expeditions to the remote type locality in July 2012 and again
in August 2014. Within the genus, tadpoles are scarce and basic ecology remains
largely unstudied (Rojas et al., 2016). The description of these (tadpoles and
behavior) biological variables in nominal and putative species of Amazophrynella
may provide important data for future species delimitation within the genus.
169
Genetic and morphological data from populations of A. minuta from Colombia
and Venezuela were not analyzed in this study. Given the propensity of hidden
diversity in this group a careful examination of material from these populations is
needed. Future integrative taxonomic analyses will certainly improve our knowledge
of phylogenetic relationships and taxonomy of Amazophrynella.
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Acknowledgements
We thank the people of the community of Taracuá, especially Sr. Maximiliano Correia
Menezes (representative of the Amerindian village of Taracuá and FOIRN), Sr.
Gabriel Correia Menezes, Sr. João Filho Menezes and Sra. Vera Correia for
hospitality and logistic support in Taracuá. We thank Junior Menezes, Max Junior
Menezes, and Bene Menezes for field support. Federação de Organizações
Indígenas do Rio Negro/FOIRN allowed access to Ameridian lands. We thank Göran
Nilson and Anders Larsson (Naturhistoriska Museet, Göteborg) for their
consideration and for sending color photographs of the syntypes of A. minuta. Marcia
Lima de Queiroz and Miss Lana helped in the INPA. Mario Nunez helped with the
molecular analyses. Funding for this work came from CNPq/SISBIOTA Processo No.
563348/2010-0 and SISBIOTA/FAPEAM. Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES) provided financial support and awarded a PhD
fellowship to R. R. R. This work is part of R.R.R.’s PhD Thesis in Genetics,
conservation and evolutionary biology program of INPA. AF has benefited from an
“Investissement d’Avenir” grant managed by the Agence Nationale de la Recherche
(CEBA, ref. ANR-10- LABX-25- 01). RWA thanks CNPq for his productivity research
grant (303622/2015-6). For funding for specimens collection and laboratory work
SRR thanks SENESCYT, Arca de Noe initiative.
171
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(Anura: Bufonidae), for Venezuela. Check List, 9, 1122–1123
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Loreto, Peru. Zootaxa, 3946, 79–103.
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Rojas, R.R., Fouquet, A., Ron, S., Hernandez, E., Melo-Sampaio, P., Chaparro, J.,
Vogt, R., Carvalho, V., Pinheiro, L., Ávila, R., Pires, I., Gordo, M. & Hrbek, T.
(2018). A Pan-Amazonian species delimitation: high species diversity within the
genus Amazophrynella (Anura:Bufonidae). PeerJ, 6, e4941.
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174
Specimens examined.
A. minuta.—BRAZIL: Amazonas State: Uauapés River, Taracuá, INPA–H 32720–23,
INPA–H 32725–26, INPA–H 32728–30, INPA–H 32732, INPA–H 32733, INPA–H
32735–40, NHMG 462, NHMG 463, NHMG 464.; Sao Gabriel da Cachoeira, INPA–
H32731, INPA–H 32724, INPA–H 32734, Japura River, INPA–H 355507–12, INPA–H
35494–98.
A. bokermanni.—BRAZIL: Pará State, Juriti (near 30 km from type locality) INPA–H
31861–65, Amazonas State, Autazes: INPA–35529–30; Pará State: Xingu River INPA
35473–77.
A. vote.—BRAZIL: Mato Grosso State: Cotriguaçu, Fazenda São Nicolau, UFMT–A
11138 (Holotype); UFMT 11136, UFMT 11142, UFMT 11145–50, UFMT 11152–55,
UFMT 4412; Manicoré, Madeira River, INPA–H 12255–56, 12331, 12342–43, 12366–
67 (Paratypes); Parque Estadual do Guariba INPA–H 21558, Novo Aripuanã,
Aripuanã River, INPA–H 12326 (Paratype) INPA–H 35540−50; Estado Amazonas:
Tapauá, Parque Nacional Nascentes do Lago Jari, INPA–H 27412, 27417−19,
27421−23, 27425−26 (Paratypes), Tapauá, Purus river, INPA–H 35551−53,
Manaquiri, INPA–H 35535−39; Estado do Amazonas: Matupiri, INPA–H 31867,
INPA–H 31868, INPA–H 31870–75, INPA–H 31877–80, INPA–H 31882, INPA–H
31883–66.
A. manaos.—BRAZIL: Amazonas State: Campus da Universidade Federal do
Amazonas, INPA–H 31866 (Holotype), INPA–H 6983–64, INPA–H 6987, INPA–H
7797 (paratypes); Presidente Figueiredo, INPA–H 20986, INPA–H 21217, INPA–H
29568–72, INPA–H 30575–77, INPA–H 30572–73 (Paratypes); Reserva Florestal
Adolpho Ducke, INPA–H 21028, INPA–H 21170, INPA–H 21060, INPA–H 31866,
INPA–H 21007–13, INPA–H 20963–INPA-H 20990.
A. matses.—PERU: Department Loreto: Requena, Nuevo Salvador, MZUNAP
921(Holotype), MZUNAP 922–23, MZUNAP 925–27, MZUNAP 934, MZUNAP 936,
MZUNAP 938, MZUNAP 940, MZUNAP 943–44, MZUNAP 948, MZUNAP 952–53,
MZUNAP 955, MZUNAP 958 (paratopotypes); Jenaro Herrera MZUNAP 928–31,
MZUNAP 933, MZUNAP 935, MZUNAP 937, MZUNAP 939, MZUNAP 950, MZUNAP
175
941–42, MZUNAP 946–47, MZUNAP 949 (Paratypes).
A. amazonicola.—PERU: Department Loreto: San Juan Bautista, Puerto Almendra
MZUNAP 901 (Holotype), MZUNAP 906–07, MZUNAP 910–11, MZUNAP 913–17;
MZUNAP 110; MZUNAP 889 (paratopotypes); 58 km of Iquitos–Nauta highway on
Fundo Zamora, MZUNAP 887–88, MZUNAP 900, MZUNAP 902, MZUNAP 886,
MZUNAP 905, MZUNAP 908, MZUNAP 919–20, MZUNAP 924 (Paratypes); Maynas,
Nauta, MZUNAP 909, MZUNAP 918; Fundo UNAP, MZUNAP 242 (Paratype).
A. javierbustamantei.—PERU: Department Madre de Dios: Tambopata, Quebrada
Guacamayo, MHNC 8331(Holotype), MHNC 8316, MHNC 8238, MHNC 8362–63,
MHNC 8245, MHNC 8484, La Pampa, MHNC 1101–04; Nuevo Arequipa, MHNC
8245, MHNC 8331, MHNC 8238, MHNC 8354, MHNC 8484, Rio Tambopata,
MHNSM 9633, MHNSM 9635, MHNSM 9640–42, MHNSM 9644, MHNSM 9646–48;
Manu, Inambari, MHNSM 17993; La Convencion, Camana, MHNSM 2565; Mapi,
MHNC 9939–40. Departamento: Junin: Tambo Poyeni, MHNC 9387; Tsoroja, MHNC
9626, MHNC 9754, MHNC 9756–57, MHNC 9679, MHNC 9680. Department: Cusco:
Urubamba, Urubamba River, MHNC 9626, MHNC 9686–87.
A. moisesii.—BRAZIL: Acre: Parque Nacional da Serra do Divisor: Igarapé Ramon:
UFAC-H 2815 (Holotype), UFAC-H 1375, UFAC-H 2772–2773, UFAC-H 2603, UFAC-
H 2607, UFAC-H 3573–UFAC-H 3575, UFAC-H 2690, UFAC-H 2692, UFAC-H 2815–
2817, UFAC-H 1698; Igarapé Anil: UFAC-H 1337–1343; Zé Luiz lake: UFAC-H 1774–
1775; Môa river: UFAC-H 1493, UFAC-H 2687–2697. Reserva Extrativista Alto do
Juruá: UFAC-H 822–823, UFAC-H 878–879, UFAC-H 2606–2611 Gregório Forest
Reverse: UFAC-H 5678.
A. teko.—BRAZIL: Amazonas state, Trombetas river: INPA-H 35513–35530, Amapa
state: UFPA AA 604. French Guiana: District of Camopi: Alikéné: MNHN 2015.136
(Holotype). District of Saint Laurent du Maroni: Mitaraka layon: MNHN 2015.137,
MNHN 2015.138, MNHN 2015.139, MNHN 2015.140, MNHN 2015.141, MNHN
2015.142, MNHN 2015.143, Pic Coudreau du Sud: MNHN 2015.152, MNHN 2015,
Flat de la Waki: INPA–H 36598. District of Camopi: Mitan: INPA–H 36596, MNHN
2015.144, MNHN 2015.145, MNHN 2015.146, MNHN 2015.147, MNHN 2015.148,
MNHN 2015.149, MNHN 2015.150. District of Saint Georges: Saint Georges: MNHN
176
2015.151, Mémora: MNHN 2015.154, MNHN 2015.155, Saut Maripa: INPA–H 36597,
INPA–H 36610, INPA–H 36599, INPA–H 36601, INPA–H 36600.
A. xinguensis.—BRAZIL: Para state: Sustainable Development Project (PDS) Virola
Jatobá: INPA–H 35471 (Holotype), INPA–H 35484, INPA–H 35485 INPA–H 35473,
INPA–H 35474, INPA–H 35475, INPA–H 35476, INPA–H 35477, INPA–H 35478,
INPA–H 35479, INPA–H 354780, INPA–H 35481, INPA–H 35483, INPA–H 35490,
INPA–H 35491, INPA–H 3592, Fazenda Paraiso: INPA–H 35493, INPA–H 35472,
Ramal dos Cocos: INPA–H 35486, INPA–H 35487, INPA–H 3588, INPA–H 35489.
A. siona.—ECUADOR: Province of Orellana: Yasuni National Park: QCAZ 27790
(Holotype), QCAZ 11981, QCAZ 51068, QCAZ 21425, QCAZ 21431, QCAZ 11973,
QCAZ 11979. Provincia Sucumbios: Reserva de Producción Faunística Cuyabeno:
QCAZ 52433–34, QCAZ 37758–59, QCAZ 37761, QCAZ 6071, QCAZ 6091, QCAZ
6095, QCAZ 6097, QCAZ 6105, QCAZ 6111, QCAZ 6113, QCAZ 6118, QCAZ 6127,
QCAZ 6128, Santa Cecilia, QCAZ 4469, QCAZ 4472, Tarapoa: QCAZ 36331, QCAZ
36336, QCAZ 36338, QCAZ 36357. Provincia de Pastaza: Community of Kurintza:
QCAZ 56342, QCAZ 56354, QCAZ 56361, Villano community, AGIP oil company:
QCAZ 38599, QCAZ 38679, QCAZ 38722, Around Villano community, AGIP oil
company: QCAZ 38642, Community of Kurintza: QCAZ 38809, QCAZ 54213,
Bataburo Lodge: QCAZ 39408, Lorocachi: QCAZ 8902, Lorocachi: QCAZ 56165,
Canelos: QCAZ 52819, QCAZ 52823, QCAZ 17391. Provincia Orellana:
Tambococha: QCAZ 55345, Garzacocha: QCAZ 20504, Yuriti: QCAZ 10526, Kapawi
Lodge: QCAZ 8725, QCAZ 25504, QCAZ 25533 10 km from Puyo: QCAZ 7135.
Provincia Morona Santiago: Pankints: QCAZ 46430. PERU: Department Loreto:
Teniente Lopez: MHNC 7611, MHNC 7685, MHNC 7686, MHNC 7698, MHNC 7699,
MHNC 7700, Jibarito: MHNC 7786, MHNC 7809, MHNC 7814. Shiviyacu: MHNC
14730, near Corrientes River: MHNC 6292.
A. moisesii.—BRAZIL: Acre state: Reserva Extrativista Alto do Juruá: UFAC-RB 823,
UFAC-RB 878–879, Parque Nacional da Serra do Divisor: Igarapé Anil: UFAC-RB
1337–1341, UFAC-RB 1343, Zé Luiz lake: UFAC-RB 1774–1775, Igarapé Ramon:
UFAC-RB 1375, UFAC-RB 2772–2773, UFAC-RB 2816–2817, Môa river: UFAC-RB
1493, UFAC-RB 2687–2697, Gregório forest Reserve: UFAC-RB 5678.
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TABLE 1. Comparisons of measurements (in mm) among males and females of
Amazophrynella minuta (for abbreviations of characters see Materials and Methods).
Character/Sex Male
(n=20)
Female
(n=11)
SVL 13.6 ± 0.6 (12.2–14.7) 17.4 ± 0.9 (14.5–19.4)
HW 4.3 ± 0.2 (3.8–4.8) 5.1 ± 0.4 (4.6–5.9)
HL 5.1 ± 0.3 (4.6–5.8) 6.0 ± 0.4 (5.1–6.9)
SL 2.3 ± 0.2 (2.1–2.6) 2.7 ± 0.2 (2.4–2.9)
ED 1.5 ± 0.2 (1.2–2.0) 1.7 ± 0.3 (1.5–1.9)
IND 1.2 ± 0.1 (1.0–1.4) 1.4 ± 0.1 (1.2–1.5)
UAL 4.0 ± 0.4 (3.5–5.3) 5.2 ± 0.2 (4.6–5.5)
HAL 2.8 ± 0.2 (2.4–3.2) 3.6 ± 0.3 (3.0–4.4)
THL 7.0 ± 0.4 (6.3–8.1) 8.5 ± 0.9 (6.8–10.1)
TAL 6.8 ± 0.4 (6.1–8.1) 8.4 ± 0.7 (7.2–10.0)
TL 4.4 ± 0.6 (3.7–6.7) 5.4 ± 0.4 (4.6–6.0)
FL 4.9 ± 0.4 (4.2–5.8) 6.4 ± 0.7 (5.3–8.2)
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Figure 1. Terminology used in this paper to describe texture of dorsal skin in
Amazophrynella. A) Tuberculate; B) granular; C) highly granular; D) finely granular;
E) Spiculate. See Materials and Methods for character definition.
179
Figure 2. Terminology used in this paper to describe ventral color pattern in
Amazophrynella. A-B) large black blotches over light background; C-D) small black
blotches over light background; E-F) small black dots over light background; G) small
black points over light background. See Materials and Methods for character
definition
180
Figure 3. Terminology used in this paper to describe shape of palmar and
subarticular tubercles in Amazophrynella. A) Rounded; B) oval; C) elliptical. See
Materials and Methods for character definition.
181
Figure 4. Lectotype (A= NHMG 462) and paralectotypes (B= NHMG 465, C= NHMG
463) of Amazophrynella minuta (Melin, 1941) from Taracuá, Rio Uaupés, Amazonas
State, Brazil deposited at the Naturhistoriska Museet, Göteborg, Sweden. Additional
pictures of the types are provided in Figure 3 -page 70 in Ávila et al. (2012). Photos
by: Anders Larson.
182
Figure 5. Amazophrynella spp. A) A. minuta; B) A. bokermanni; C) A. matses; D) A.
javierbustamanteu; E) A. siona; F) A. bokermanni; G) A. manaos, H) A. vote, I) A.
xinguensis, J) A. teko, K) A. moisesii
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Figure 6. Morphological variation in life of Amazophrynella minuta (Melin, 1941) from
type locality. A-F) adult males; G-I) adult females.
184
Figure 7. Morphological variations of preserved specimens of Amazophrynella
minuta (Melin, 1941) from type locality. A-C) adult females; D-F) adult males.
185
Figure 8. Geographic distribution of Amazophrynella minuta (Melin, 1941), star
marks the type locality. Brazil, state of Amazonas: 1) São Gabriel da Cachoeira; 2)
Lago Jabo-Cucui; 3-4) Vila Bittencourt, Japura River. Colombia, Department of
Vaupés: 5) estación biológica Cacaparu; 6) Santander; 7) Vaupés River; 8) Mitu;
Department of Amazonas: 10) Restinga isla mariname; 11) La chorrera; Department
of Caqueta:12) Cabanachagra; 13) Caqueta River. Venezuela; State of Amazonas:
14) Raudal de Danto, Río Cuao.
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Figure 9. Axillary amplexus between specimens of Amazophrynella minuta (Melin,
1941)
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Figure 10. Advertisement call of Amazophrynella minuta (Melin, 1941) from São
Gabriel da Cachoeira, State of Amazonas, Brazil. A) Oscillogram and spectrogram
visualizing three notes; B) Oscillogram and spectrogram visualizing one note.
188
CAPITULO IV
Diversification in Amazonian through the historical biogeography of “terra firme”
tiny tree toads Amazophrynella (Anura: Bufonidae). Rojas, R.R., Fouquet, A.,
Carvalho, V., Ron, S., Ávila, R., Pires, I., Gordo, M. & Hrbek, T. Artigo para ser
submetido na revista Molecular phylogenetics and evolution
189
Diversification in Amazonian through the historical biogeography of “terra
firme” tiny tree toads Amazophrynella (Anura: Bufonidae)
Rommel R. Rojas1, Antoine Fouquet2, Vinícius Tadeu De Carvalho1, Santiago Ron3,
Robson W. Ávila4, Izeni Pires Farias1, Marcelo Gordo5 & Tomas Hrbek1
1Laboratório de Evolução e Genética Animal (LEGAL), Departamento de Genética,
Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Av. General
Rodrigo Octávio Jordão Ramos, 6200. CEP 69077–000 Manaus, AM, Brazil
2 USR 3456 LEEISA - Laboratoire Ecologie, Evolution et Interactions des Systèmes
Amazoniens, Centre de recherche de Montabo, 275 route de Montabo, BP 70620,
97334 Cayenne, French Guiana.
3 Museo de Zoología, Escuela de Biología, Pontificia Universidad Católica del
Ecuador, Av. 12 de Octubre y Roca, Aptdo. 17–01–2184, Quito, Ecuador.
4 Departamento de Química Biológia, Universidade Regional do Cariri, Campus do
Pimenta, Rua Cel. Antônio Luiz, 1161, Bairro do Pimenta, CEP 63105–100, Crato,
CE, Brazil.
5 Departamento de Biologia, Instituto de Ciências Biológicas, Universidade Federal
do Amazonas, Av. General Rodrigo Octávio Jordão Ramos, 6200. CEP 69077–000
Manaus, AM, Brazil.
*Corresponding autor: [email protected]
190
Abstract
Understand the patterns and processes that generated the high species
diversity in Amazonia is essential to develop conservation strategies and provide
information of biodiversity evolution. The frogs genus Amazophrynella comprises
“terra firme” tiny tree toad with an Pan-Amazonian distribution that present and
striking evolutionary pattern. Here, we employed genomic data to reconstruct their
divergence times, ancestral area distributions, dispersal–vicariance events and
temporal pattern of diversification. Discrete and continuous biogeographic inferences
from multiple models. Lineages diversification and areas reconstructions predicted
similar historical biogeography dynamics for Amazophrynella which may suggest that
extant geographical distributions and diversity patterns were influenced strongly by
long-distance dispersal across geographical barriers. In addition, our results suggest
an old connection between Amazonia and Atlantic forest and illustrate how climatic
and landscape modifications preceding the Miocene shaped the initial diversification
of South American Amazonian frogs.
Key words: Anura, biodiversity, biogeography, conservation, diversification, endemic
areas.
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Introduction
The neotropics present the most extraordinary and fascinating biodiversity in
the earth, as such, is a perfect target for research about the origin of biological
diversity (Rull, 2008). Landscape Paleogene reconstructions suggest a continuous
forest connecting the ancient Amazonia and Atlantic forest during Eocene divided by
the origin of drier savanna vegetation comprising by Cerrado, Caatinga and Chaco
(Morley, 2000; Werneck et al., 2012). Phylogenetic evidence also indicates periods of
forest re-connection between both areas (Batalha-Filho et al., 2013; Capurucho et
al., 2018; Costa, 2003; Dal Vechio et al., 2018).However, despite previous studies
that suggest an ancient (Eocene/Miocene) and recent (Pleistocene) divergence
between organism on each side (Fouquet et al., 2012; Pellegrino et al., 2011)
ancestral scenarios reconstructions and biological diversification in early stages of
forest divergence still poorly understood.
The Amazonian present the largest records of flora and fauna in the world
(Jenkins et al., 2013). Geomorphological and climatic fluctuations during Miocene-
Pleistocene has been proposed as important causes of their megadiversity (Antonelli
et al., 2018; Hoorn et al., 2010). Processes as extinction, vicariance and dispersion
and sympatric and allopatric speciation are attributed as the most common
mechanisms in diversification of Amazon taxa (Antonelli et al., 2018; Rull, 2011;
Wiens and Donoghue, 2004). Another evolutionary feature in Amazonian biota is
their not random phylogeographic distribution - endemism areas (Ribas et al., 2012;
Smith et al., 2014).
This striking pattern has been widely studied through lineages diversification of
mammals, birds and lizards (Oliveira et al., 2016; Patton et al., 2000; Ribas et al.,
2012), but historical reconstructions using non-model taxa as amphibians is scarce
(i.e. Noonan and Wray, 2006; Castro-Viejo et al., 2013; Fouquet et al., 2015;
Godinho and Silva 2018). In addition, most of these studies do not explore whether
the observed patterns of biodiversity are concordant with current models of faunal
differentiation in the Amazon basin. Hence, by studying amphibian evolutionary
history, we can gain greater insight into the history of their habitats and of the other
inhabitants of these areas (Castroviejo-Fisher et al., 2014). However, evolutionary
192
history and diversification of Amazonian amphibians is poorly understanding in
neotropics (Castroviejo-Fisher et al., 2014; Fouquet et al., 2007; Gehara et al., 2014;
Godinho and Da Silva, 2018; Santos et al., 2009).
Frogs constitute an excellent group to study species diversification because
their low dispersion abilities compared with birds and mammals (Zeisset and Beebee,
2008), being strong sensitive to environmental and geological modification (Godinho
and Da Silva, 2018), highly depend on habitat quality and have both life stages
(aquatic and terrestrial) (Vences and Wake, 2007). In addition, it is estimated that
almost 40% of species are in danger of extinction (Collins, 2010), and their
populations are progressively decreasing in Andes and Amazonia (Becker et al.,
2016; Catenazzi and von May, 2014). Likewise, only a few frog lineages includes
Amazonian and Atlantic forests counterparts (i.e. Fouquet et al., 2014; Sá et al.,
2018; Thome et al., 2016).
In this study we focus on “terra firme” tiny tree toads of genus Amazophrynella
(Anura: Bufonidae) that constitutes an interesting anuran clade because their
intriguing patterns of phylogenetic evolution (basal east-west followed by a north
south diversification), being distributed by all endemic areas (Rojas et al., 2018) and
being sister clade of endemic species from Atlantic forest (Dendrophryniscus)
(Fouquet et al., 2012a). Amazophrynella comprise seventeen nominal and putative
lineages with a Pan-Amazonian distribution, present small size (snout vent length ≥
26.0 mm) and exhibit high levels of morphological conservation (Rojas et al., 2018).
They are diurnal and crepuscular, found exclusively in leaf litter of primary and
secondary rainforest, their reproduction occurs in small puddles, their eggs are
pigmented and deposited in roots shrubs and under litter (Rojas et al., 2018, 2016,
2015). In summary, Amazophrynella encompasses a taxonomic well resolved group
with a striking phylogenetic and ecological pattern, thus combined with their broad
geographic distribution, make Amazophrynella an ideal species group to study
diversification in Amazonia. But despite this knowledge a comprehensive
biogeographical reconstruction and lineage diversification of genus is still lacking.
Here, using the phylogenetic relationships of Amazophrynella, we explore
lineage diversification and their historical biogeography.
193
Material and methods
Taxonomic and molecular sampling
We used an extensive sampling of the genus Amazophrynella in their entire
Pan-Amazonian distribution with a total of 235 terminals from 35 localities from
seventeen lineages including nominal and putative species from Rojas et al. (2018)
(Supporting Information Appendix S1). Genomic data - Single nucleotide polymorphic
(SNPs) DNA sequence data were obtain from a subset of 23 individuals of
Amazophrynella representing the mtDNA clades (putative and nominal species)
previously delimited by Rojas et al. (2018). We also obtain genomic date from two
individuals of genus Dendrophryniscus and Rhinella margartifera as outgroup.
Molecular procedures
Tissue samples (silver or muscle) were preserved in 100% ethanol and kept at
-20°C for DNA extraction. Standard molecular protocols were used from DNA
extracting to sequencing (Sambrook et al., 1989). Amplification of mitochondrial
genes (12S, 16S and COI) was carried out under the following conditions: 60 s hot
start at 92°C followed by 35 cycles of 92°C (60 sec), 50°C (50 sec) and 72°C (1.5
min). Final volume of the PCR reaction for all genes was 12 μl and contained 4.4 μL
of ddH2O, 1.5 μL of 25 mM MgCl2, 1.25 μL of 10 mM dNTPs (2.5mM each dNTP),
1.25 μL of 10x buffer (75 mM Tris HCl, 50 mM KCl, 20 mM (NH4)2SO4), 1 μL of each
2 μM primer, 0.3 μL of 5 U/μL DNA Taq Polymerase (Fermentas, Lithuania) and 1 μL
of DNA (about 30 ng/μL).
Preparation of the library for genomic data was done according to protocol
developed by Peterson et al. (2012) or ddRADseq libraries. After enrichment PCR
and fragment size selection (400 bp) in the Pippin Prep electrophoresis platform
(Saga Science), all samples were grouped in equimolar quantities and sequenced in
the Genetic Sequencer Generation Ion Torrent PGM, using reagents and sequencing
kits 400pb and chip 318. Voucher specimens for the sampled taxa are listed in
Supporting Information Appendix S1.
The reading of genomic data was processed in the PyRAD (Eaton, 2014) and
used to generate a Matrix of SNPs and concatenated sequences. All alleles used
194
had Least 5X of minimum coverage in each locus and frequency in at least 50% of
the individuals. We obtain a total of 4,152,088 fragments, already filtering the poor
quality sequences. A total of 58% deposition of the library enriched with ion sphere
particles (ISPs) was obtained. PyRAD generated a phylip file with 144,146 bp, which
represents 500 loci with 12070 variable sites and 6077 informative sites of
parsimony.
Phylogenetic methods and molecular clock calibration
We implemented Bayesian phylogenetic analyses in the software package
Beast 2.0 and run a StartBeast analysis (Drummond and Rambaut, 2007). Tree
searches were performed assuming both single models of sequence evolution for
each locus, and Markov chain Monte Carlo (MCMC) searches were made for 10
million generations, sampling every 1000 generations, for a total of 10 000 trees. We
estimated model parameters during runs, and estimated Bayesian posterior
probabilities as the proportion of trees sampled; the trees obtained in the first one
million generations were discarded. The best model of molecular evolution
(GTR+G+I) was estimated in JModelTest (Posada, 2008) and selected using the
Akaike Information Criterion–AIC.
We used the uncorrelated relaxed lognormal clock (Drummond et al., 2006),
with a birth and dead tree prior of speciation. We used divergence time constraints,
applying normal distributions to the prior probabilities of the dates, to the following
nodes a crown age of Amazophrynella + Dendrophryniscus vs. others Bufonidae
52.0–32.0 Ma (mean= 40.2 ± 10.0), Amazophrynella + Dendrophryniscus 49.0–29.0
Ma (mean= 38.1 ± Ma) and eastern vs. western Amazophrynella lineages divergence
obtained from Rojas et al. (2018).
From the MCMC output, we generated the final consensus tree-maximum
clade credibility tree- using Tree Annotator v1.6.2 (part of Beast software package).
For visualization and edition of the consensus maximum clade credibility tree, we
used the program Figtree v.1.3. (Rambaut, 2009).
Analysis of historical biogeography
195
To perform ancestral ranges estimation we excluded non Amazophrynella and
Dendrophryniscus taxa, including only one terminal per area from Starbeast.
Inference of biogeographical history on the dated phylogeny of Amazophrynella we
used the package BioGeography with Bayesian (and likelihood) Evolutionary
Analysis in R Scripts–BioGeoBEARS (Matzke, 2013) using the program R (R
Development Core Team, 2011). We compared six biogeographical models
implemented in BioGeoBEARS to determine their fit to our data. Likelihood values of
the models were compared using Likelihood Ratio Test (LRT), and model–fit was
assessed in BioGeoBEARS by comparing weighted Akaike’s information criterion
scores (Matzke, 2013).
The species distribution areas were assigned based on biogeographic regions
Inambari (In), Guyana (Gu), Napo (Na), Rondonia (Ro), Tapajós (Ta), Xingú (Xi) and
Imeri (Im) and Atlantic Forest (AF) (Olson et al., 2001; Ribas et al., 2012; Smith et al.,
2014). For BioGeoBEARS analysis we used a maximum number of areas (8) per
node unconstrained. This option was selected because the distribution range of the
taxa in past was not detected either in Amazonian, Atlantic forests or both areas and
because the speculative influenced of the geomorphological events in
biogeographical mechanisms (vicariance, dispersion or founded effect), the option no
constraint could be made regarding potential ancestral distributions. Other values
were left at default values (Matzke, 2013).
Phylogeographic analysis
In order to reconstruct the phylogeographic history of Amazophrynella
lineages through time in continuous space (by using location coordinate data for
each lineage), we used the Relaxed Random Walk (RRW), approach proposed by
Lemey et al., (2010). The RRW model involves a Bayesian framework, reconstructing
phylogeographic history on a continuous landscape through the standard Brownian
diffusion process using geographical coordinates, phylogenetic genealogy and
continuous trait (Lemey et al., 2010).
The analysis was performed using the mitochondrial and genomic data. We
used a coalescent prior with constant population size and an uncorrelated log–
normal RRW model as well an GTR+G+I model substitution for each mitochondrial
196
and nuclear marked and length of chain of 107 generations and 10% of cut-off was
used for burn-in. Mixing of parameter sampling, Effective Sample Size (ESS) and
convergence were checked in Tracer 1.5. The resulted tree was summarized with
Tree Annotator 1.7.2. The software SPREAD was used to generate a Keyhole
Markup Language (KML) file which was plotted in a Google Earth map
(http://earth.google.com).
Analysis of lineage diversification
The timing and patterns of diversification in “terra firme” tiny tree toads with
lineage-through-time (LTT) plots. LTT plots depict the cumulative (log) number of
nodes as a function of time. We estimated LTT plots on the basis of calibrated
molecular clock chronograms obtained via bayesian analysis. We also tested
whether significant changes occurred in the diversification rates throughout the
evolutionary history of Amazophrynella by implementing various likelihood-based
statistical methods that simultaneously accommodates undersampling of extant taxa,
rate variation over time, and potential periods of declining diversity (Morlon et al.,
2011). The analyses was made using the software package RPanda (Morlon et al.,
2016) implemented in R (R Development Core Team, 2011).
Results
Phylogenetic and divergence times
The age of the most recent common ancestor (MRCA) of Amazophrynella and
its sister lineage Dendrophryniscus is estimated at c. 34.2 Ma (95% HPD: c. 38.2–
30.5 Ma) in Eocene and middle Oligocene, with high node support (posterior= 1).
Within Amazophrynella the eastern/western divergence is estimated at c. 24.8 Ma
(95% HPD: c. 28.3–21.6 Ma) in late Oligocene and early Miocene. Within the eastern
clade the north and south division is estimated at c. 19.7 Ma (95% HPD: c. 22.6–17.0
Ma) in early Miocene. In western clade, the north vs. south split is estimated at c.
16.5 (95% HPD= 19.0–14.0 Ma) in the middle Miocene (Figure 1). Major
diversification events between lineages within each of the four above clades varied
between 5.0 and 1.0 Ma in late Miocene and Pliocene.
Historical biogeography
197
BioGeo–BEARS analyses of genomic marked were DIVALIKE model including
founder-effect speciation (DIVALIKE+J) as the model that provided the best fit of all
models to our dataset (see in Table 1). The results of the biogeographical model
selected by BioGeo–BEARS are presented in Figure 1.
The biogeographic reconstruction indicates that the MRCA of Amazophrynella
and Dendrophryniscus had a wide distribution range in two areas: Atlantic Forest +
lnambari. Subsequently analysis suggests vicariance between Amazonia vs. Atlantic
Forest. In Amazonian ancestral area were Inambari. Subsequently analysis suggest
a dispersion mechanism from Inambari to Guyana and Imeri. In eastern Amazonia
were detected a dispersion from Guyana to Inambari and from Inambari to Rondonia
and Xingu being Tapajos the last colonized area. In western clade were detected a
dispersion from Imeri to Inambari following by a colonization by dispersion from Imeri
to Napo.
Diversification patterns
The best model to explain Amazophrynella diversification was exponential
variation in speciation with no extinction (Table 2- Figure 2). Phylogeographic
analysis under Brownian motion model (Figure 3) suggested an ancestor of
Amazophrynella and Dendrophryniscus distributed through southern Amazonia +
Cerrado and Atlantic Forest (Figure 3A). This ancestral population remained during
the late Eocene and middle Oligocene c. 44.0–35.0 Ma.
Subsequent scenario showed a widespread ancestral of Amazophrynella
distributed in central Amazonia (Figure. 3B). This ancestral population existing in
middle Oligocene between 35.0–30.0 Ma. The ancestral population of
Amazophrynella split in two demes (eastern and western) around 30.0–20.0 Ma
during late Oligocene and early Miocene (Figure 3C). The two demes expanded and
split in four ancestral population (north and south) that emerged and coexisted in
early Miocene 20.0–15.0 Ma (Figure 3D). Range expansion of four-ancestral
population increase in north-eastern and south-western and Amazonia at 15.0–10.0
Ma (Figure 3E), follow by a north-western and south-eastern range expansion at
10.0–5.0 during late Miocene (Figure 3F). The final expansion of ancestral
populations occurs at 5.0–1.0 Ma during Pliocene (Figure 3G).
198
Discussion
Our results show clear convergence among discrete and continuous
biogeographic inferences from multiple models. Lineages diversification and areas
reconstructions predicted similar historical biogeography dynamics for
Amazophrynella which may suggest that extant geographical distributions and
diversity patterns were influenced strongly by long-distance dispersal across
geographical barriers. The best-fitting model in our diversification analyses are likely
trend of rising diversity with slight variations through time (exponential). Taxon age is
a possible explanation for the observed differences across clades. Thus, because
diversification rates in early adaptive radiation were low (Figure 3) and less niches
were available in initial diversification stages. However, the result should be taken
with caution because the small sample size (n=17 clades) could be a problem
(Freckleton et al., 2002).
Ancestral area reconstruction and diffusion model suggested a connection
from Amazonia to Atlantic forest through south of Cerrado. Hoorn et al. (2010)
suggested that changes in the Amazonian landscape in response to the Andes uplift
could have favored these ancient links. Batalha-Filho et al., (2013). described this
connection during Miocene for birds, but our divergence time estimates were older
(Eocene/Oligocene) presenting a similar pattern with other frogs (i.e De Sá et al.,
2018). Our results contrast with the other two historic connections, along the northern
coast and through current Central Caatinga, called ‘‘young pathway’’ by Batalha-Filho
et al., (2013). Palynological data from Late Pleistocene (10.9–10.5 Ma) of central
Caatinga detected evidences of taxa currently found in Amazonian and Atlantic
Forest, indicating a forest vegetation there along the last glacial cycle (Colinvaux et
al., 2000; Prates et al., 2017).
Our analysis recovers a late Oligocene vicariance between ancestral of
Amazophrynella and Dendrophryniscus. Previously studies suggest climatic
modifications during Eocene and Oligocene that propitiated patches of open
vegetation and changes in floristic composition (Crisci et al., 1991; Jaramillo, 2006,
Grahan, 2010), thus promoting the origin and expansion of a drier savanna
vegetation (Werneck, 2011). These events prevent a long period (44.0-35.0 Ma-
199
Figure 3A) of biotic interchange between Amazonian and Atlantic forest (Batalha-
Filho et al., 2013; Capurucho et al., 2018; Costa, 2003), likely caused ancient
vicariance of forest dwelling amphibians. Generalist species that tolerated dry
environmental conditions could disperse in both wet-forest, as Dendropsophus gr.
minutus, Syncope and Chiasmocleis (Gehara et al., 2014; Sá et al., 2018). However,
Amazophrynella and Dendrophryniscus fail to disperse during forest reconnections
because gallery forest in dry areas must remained unsuitable for ancestral, that
limited their distribution inside areas that presented original or similar niche
conditions that ancestral area, exemplifying as a case of ancestral niche
conservationism.
Initial diversification of Amazophrynella are according with others studies that
pre- date Neogene (Castroviejo-Fisher et al., 2014; Kok et al., 2017; Santos et al.,
2009; Van Bocxlaer et al., 2010). Discrete and continuous models showed a
widespread ancestor in Amazonia, that gradually dispersed from central lowland
Amazonian forests to Guyana and Brazilian Shields, and them diversified during the
last 15.0–10.0 Ma in middle and late Miocene. These diversification scenario follows
the end of marine incursions, high levels of Andean sedimentation and establishment
of modern transcontinental Amazon drainage system (Antonelli et al., 2010; Hoorn et
al., 2010; Rull, 2011). Western Amazonian dispersal routes have most likely occurred
because suitable habitats conditions were available across a wider region after of
marine incursions retractions in the last 10.0–5.0 Ma.
Phylogeographic breaks in eastern vs. western in Amazonia have been widely
detected at different taxonomic levels, including butterflies (Hall and Harvey, 2002);
lizards (Gamble et al., 2008; Glor et al., 2001; Kronauer et al., 2005; Miralles and
Carranza, 2010); frogs (Garda and Cannatella, 2007; Lougheed et al., 1999; Noonan
and Wray, 2006; Symula et al., 2003), birds (Bates et al., 1998); fishes (Farias and
Hrbek, 2008) and mammals (da Silva and Patton, 1993; Steiner and Catzeflis,
2004). Our biogeographic reconstructions suggest an early Miocene (20-15.0 Ma)
east-west divergence. These results are older than previously reported by other
Amazonian taxa as lizards (Gamble et al., 2008; Miralles and Carranza, 2010;
Pellegrino et al., 2011); birds (Eberhard and Bermingham, 2005) and mammals (da
200
Silva and Patton, 1993), but match with time split with other amphibians (i.e. Garda
and Cannatella, 2007; Noonan and Wray, 2006).
Subsequent diversification events in Amazophrynella take place before
Pleistocene dry periods at 5.0-1.0 Mya. Climatic warmer Miocene and the relatively
cooler Pliocene could have favored the high levels of dispersion in lineages. A similar
time estimates diversifications patterns were similar in other species of frogs as
Dendropsophus gr. minutus (Gehara et al., 2014); Rhinella gr. marina (Maciel et al.,
2010); Adenomera gr. andreae (Fouquet et al., 2014); Ranitomeya spp. (Santos et
al., 2009)and Centrolenidae spp. (Castroviejo-Fisher et al., 2014).
Because we coded the presence of taxa in the Amazon region as binary
characters, we ignored distinct distributions within this region. However, is also
apparent that the observed lineages our study are geographically structured, and the
distribution of Amazonian lineages seems to correspond in large part to the proposed
areas of Amazonian endemism (Cracraft 1985; Borges & da Silva 2012). Our
likelihood analysis of discrete biogeographic reconstruction suggested Inambari,
Guyana and Napo as most ancestral areas in Amazonia and a recent colonization in
Tapajos and Imeri. Another striking biogeographic pattern is that one representant of
Inambari (A. aff. vote sp.2) were placed into the Guyana shield clade (Figure 2). This
pattern can be attributed to a long dispersion event (Matzke, 2012); hybridization
and/or the retention of ancestral polymorphic alleles commonly detected in frogs at
genomic scales (Fouquet et al., 2018).
In addition, assuming that extant geographical distributions reflect ancestral
occurrences, it is tempting to suggest that basal east/west diversification in
Amazophrynella took place after populations moved across structural arch (i.e. Purus
arch), or even across marine incursions, and south/north diversification after the
Amazonas river establishment (c. 9.0 Ma -(Hoorn et al., 2017). Future studies should
take into account known areas of endemism for Amazonian amphibians to
investigate the potential impact of historic (i.e. structural arch-Purus arch, marine
incursions, Pleistocene forest fragmentation) and ecological niche evolution in
diversification of Amazonian frogs.
201
Acknowledgements
We thank the people of the community of Taracuá, especially Sr. Maximiliano Correia
Menezes (representative of the Amerindian village of Taracuá and FOIRN), Sr.
Gabriel Correia Menezes, Sr. João Filho Menezes and Sra. Vera Correia for
hospitality and logistic support in Taracuá. We thank Junior Menezes, Max Junior
Menezes, and Bene Menezes for field support. Federação de Organizações
Indígenas do Rio Negro/FOIRN allowed access to Ameridian lands. Marcia Lima de
Queiroz and Miss Lana helped in the INPA. Mario Nunez helped with the molecular
analyses. Funding for this work came from CNPq/SISBIOTA Processo No.
563348/2010-0 and SISBIOTA/FAPEAM. Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES) provided financial support and awarded a PhD
fellowship to R. R. R. This work is part of R.R.R.’s PhD Thesis in Genetics,
conservation and evolutionary biology program of INPA. AF has benefited from an
“Investissement d’Avenir” grant managed by the Agence Nationale de la Recherche
(CEBA, ref. ANR-10- LABX-25- 01). RWA thanks CNPq for his productivity research
grant (303622/2015-6). For funding for specimens collection and laboratory work
SRR thanks SENESCYT, Arca de Noe initiative.
202
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Table 1. Log Likelihood scores for each model of biogeographic dispersal run in
BioGeoBEARS (Matzke, 2013) for genomic marked. Note that the DIVALIKE+ J had
the highest log likelihood score and was therefore chosen as the model that
explained the data best.
Model LnL d e j AIC AIC_wt
DEC -36.38 0.003 0.005 0 76.75 4.8e-05
DEC+J -27.09 1.0e-12 6.3e-10 0.054 60.19 0.19
DIVALIKE -31.85 0.003 1.0e-12 0 67.70 0.0045
DIVALIKE+J -25.68 1.0e-12 1.0e-12 0.049 57.35 0.79
BAYAREALIKE -46.38 0.005 0.081 0 96.75 2.2e-09
BAYAREALIKE+J -29.64 1.0e-07 1.0e-07 0.06 65.28 0.015
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Table 2. Log‐likelihood and AIC values of the six diversification models fitted to the
branching times derived from the Bayesian maximum clade credibility chronogram of
Amazophrynella.
Model LogL AICc AICw
Expanding diversity; constant speciation rate -52.679 107.624 0.416
Expanding diversity; exponential variation in speciation rate
-51.388 107.634 0.484
Expanding diversity; linear variation in speciation rate
-54.253 113.365 0.023
Expanding diversity; constant speciation and extintion rate
-52.665 113.176 0.093
Expanding diversity; exponential variation in speciation rate and constant extinction
-52.388 110.623 0.025
Expanding diversity; linear variation in speciation rate and constant extinction
-52.665 113.176 0.024
213
Figure 1. Graphical representation of the historical biogeography of Amazophrynella.
A) Map delimiting the distribution of each geographic area assigned to each lineage
in the BioGeoBEARS analysis. (B) Bayesian time-calibrate from dd Rad´s genomic
topology. Ages/posterior probability are indicated alongside on the upper and below
of the nodes. The 95% confidence intervals for nodes are highlighted by horizontal
light blue bars. Colored squared indicate the ancestral area and biogeographic
events.
214
Figure 2. Semi-logarithmic lineage-through-time (LTT) plot depicting diversification
patterns of Amazophrynella. LTT plots obtained via penalized likelihood (PL) and
exponential curve of best model of diversification are superimposed.
215
Figure 3. Phylogeographic analysis of Amazophrynella using continuous Relaxed Random
Walks (RRW) based on mt DNA and genomic dataset. Red polygons indicate potential area
of occupancy of the MRCA. (A) Ancestral connection between Amazophrynella and
Dendrophryniscus; (B) ancestral of Amazophrynella in Amazonia; (C-D) east - west
expansion; (E-F) north-south expansion; (G) final diversification in Amazophrynella. Map
were generated using Google Earth (earth.google.com).
216
CONCLUSÕES GERAIS
❖ A diversidade de espécies do gênero Amazophrynella se encontrava subestimada.
Através do uso de diferentes linhas de evidências evolutivas propomos a hipóteses
filogenética de ampla abrangência geográfica para gênero e confirmamos a
existência de onze espécies validas, sendo que outras linhagens ainda não foram
confirmadas. A integração de diversos caracteres evolutivos auxilia em manter uma
taxonomia estável, auxiliar nas decisões taxonômicas e delimitações de espécies. No
entanto, ainda existe uma grande diversidade de espécies escondida dentro do
gênero Amazophrynella.
❖ A história biogeográfica de Amazophrynella indica uma quebra populacional entre
Amazônia e Mata Atlântica no Eoceno, uma divisão basal entre o Leste-Oeste da
Amazônia no Oligoceno e uma quebra norte-sul no Mioceno. Nossas reconstruções
ancestrais sugerem um ancestral amplamente distribuído dividido por mecanismos
de vicariância. A divergência entre Amazônia e Mata atlântica foi provavelmente
influenciado pelo levantamento inicial dos Andes que ocasionaram mudanças
ambientais e períodos secos dentro da antiga floresta continua Sul-Americana. Na
Amazônia, diversos eventos geológicos como a elevação do Arco do Purus e
incursões marinhas poderiam ter ocasionado o padrão leste e oeste, no entanto
nossos tempos de divergência não são completamente sincronizados com as
predições de estas hipóteses. Assim mesmo, a divisão entre o clados do norte e sul
não foi ocasionado pela formação do rio Amazonas e poderia representar uma
pseudo- congruência filogenética. Fatores ecológicos e especialização do nicho
ecológico, assim como forças neutras ocuparam um papel principal na diversificação
e dispersão de Amazophrynella na Amazônia.