Hons Project - Robyn Cuthbertson - Inferring Phylogenies and Distributions of Upper-Andean and Amazonian Frogs From the Genus Pristimantis Using New and Previously Published Sequence

8/13/2019 Hons Project - Robyn Cuthbertson - Inferring Phylogenies and Distributions of Upper-Andean and Amazonian Frogs

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Inferring phylogenies and distributions of upper-Andean and

Amazonian frogs from the genusPristimantis using new and

previously published sequence data.

Pristimantis acuminatus

Name: Robyn Cuthbertson

Matriculation number: 0906976c

Level 4 Honours Project

Supervisor: Dr Kathryn Elmer

Submission date: 22 March 2013


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ContentsAbstract ................................................................................................................................ 2

Introduction .......................................................................................................................... 3

Methods ................................................................................................................................ 6

Data collection .................................................................................................................. 6

Phylogenetic analyses........................................................................................................ 6

Results .................................................................................................................................. 8

Convergence assessment ................................................................................................... 8

Discussion ........................................................................................................................... 13

Phylogenetic relationships ............................................................................................... 13

Model and analyses ......................................................................................................... 17

Convergence on stationarity ............................................................................................ 18

Wider implications .......................................................................................................... 18

Acknowledgements ............................................................................................................. 20

References .......................................................................................................................... 21

Appendix I .......................................................................................................................... 28

Appendix II ........................................................................................................................ 34

Appendix III ....................................................................................................................... 36


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Abstract

The genusPristimantis is the largest of the amphibian genuses with 454 species

currently described and this number is likely to increase with more recently developed

techniques for recognition of cryptic species and the introduction of new molecular

techniques. The deep evolutionary history has been examined and the evolutionary

relationship withEleutherodactylus has been discussed by Heinicke, Duellman and Hedges

(2007), inferring thatPristimantis split from the Eleutherodactylines 37 million years ago,

with rapid speciation 24 million years ago and also with the uplift of the Andes, where

mountain building first appeared around 23 Ma, and the most pronounced peaks followed

between 12 and 4.5 Ma (Hoorn et al., 2010).

My study involved the alignment and phylogenetic analysis of 112 sequences,

followed by the investigation of the distributions of species in a selected clade. This revealed

some relationships between species which support those found by Heinicke, Duellman and

Hedges (2007) and some which differnew evolutionary relationships may be the result of

the introduction of 30 new sequences into the analyses. Wide distribution of some of the

species possibly suggest cryptic species complexes and indicate further studies need to be

done in order to fully delimitate species such asPristimantis ockendeni andPristimantis

chalceus.

The implications for my study and those similar, for example Heinicke, Duellman and

Hedges (2007), on the knowledge of evolutionary history and relationships can have an

impact on the knowledge of the methods of diversification (for example due to topographical

complexities) which in turn can give an indication of the history of the landscape. It can also

affect conservation as subdivision of distributions due to delimitation of species can change

the species conservation requirements.


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Introduction

Frogs of the genusPristimantis (Jimnez de la Espada 1870),the South American rain

frogs, are distributed mainly in Central and South America.They are nocturnal and primarily

arboreal (Kok and Kalamandeen, 2008), and are small in length like most direct developing

frogs (range from 20-50mm) (Heinicke, Duellman and Hedges, 2007).They are also

polychromatic which can cause problems in identification (Kok and Kalamandeen, 2008).

Pristimantisis widely accepted to be the most speciose genus of the Neotropical amphibia,

with an estimated species number of 454 (AmphibiaWeb 2013).This can be validated with

comparison of other genuses in the family Strabomantidae: for example the genus Oreobates

with 21 species listed, and the genusBryophrynewith just eight (AmphibiaWeb, 2013).

With the increasing availability of molecular information it has become of great

interest to researchers to utilise this information in order to infer phylogenies, the overall aim

of which is to discover the topology of the tree and infer evolutionary relationships.

However, the correct phylogenies can be affected by the model chosen to investigate the

evolutionary history (Bollback, 2002).There are a variety of different methods, each with

strengths and weaknesses (Yang and Rannala, 2012).The process of molecular phylogenetics

that is, using DNA sequences to investigate the evolutionary histories and relationships

between species (Hay, Ruvinsky, Hedges, and Maxson, 1995; Darst and Cannatella, 2003;

Fouquet et al., 2007)allows for more precise phylogenetic inference than morphological

classifications can allow.

Species concepts have been discussed recently in terms of reclassifying species, with

changes from the typical biological species concept to phylogenetic and evolutionary
http://amphibiaweb.org/cgi-bin/amphib_query?table=amphib&special=one_record&where-genus=Bryophryne&where-species=abramalagaehttp://amphibiaweb.org/cgi-bin/amphib_query?table=amphib&special=one_record&where-genus=Bryophryne&where-species=abramalagaehttp://amphibiaweb.org/cgi-bin/amphib_query?table=amphib&special=one_record&where-genus=Bryophryne&where-species=abramalagaehttp://amphibiaweb.org/cgi-bin/amphib_query?table=amphib&special=one_record&where-genus=Bryophryne&where-species=abramalagae


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species concepts (Hanken, 1999). South America has many areas of high species diversity

(Myers 2000), with important protected areas such as Manu in Peru and Madidi in Bolivia to

preserve niche biodiversity hotspots (Padial and de la Riva, 2009).There are numerous

hypotheses as to why these areas of diversity exist, such as riverine barriers (Gascon,

Lougheed and Bogart, 1996) and paleogeographic characteristics (Rsnen et al., 1996;

Hughes and Eastwood, 2006).Wang et al., (2009) discuss two causes of speciation: firstly,

where geographic and physical landscape barriers restrict the movement of individuals and so

constrain gene flow between populations, in turn causing genetic drift and/or selection to

result in divergence of phenotype and secondly, where gene flow against immigrant

phenotypes is the result of natural or sexual selection, which causes divergence, and

demonstrates that geographic isolation, although thought to be the primary reason for

diversification and speciation, may not be as fundamental a theory as previously thought.

Generally, it is established that geography has had a major impact on species diversity in

South America, especially in the Andes and Amazonia (Elmer, Dvila and Lougheed, 2007).

Recently Heinicke, Duellman and Hedges (2007) refined the Eleutherodactylines into

three cladesMiddle American, the Caribbean, and finally the South American clade.It is

the later in whichPristimantisis included, and it has been inferred that this genus split from

the rest of the Eleutherodactylines 37 million years ago (Heinicke, Duellman and Hedges,

2007).The range ofPristimantisis central to the Andes, caused by changes in plate tectonics

in the Palaeocene 65-34 million years ago (Hoorn et al., 2010).In terms of geological history

this is very recent and it is apparent thatPristimantisbecame speciose in a short space of

evolutionary time, diversifying rapidly from 24 million years ago (Heinicke, Duellman and

Hedges, 2007).This idea is supported by changes in climate resulting in genetic isolation

resulting in further speciation of the species and the pronounced uplift of the Andes in the last


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124.5 million years (Hoorn et al., 2010).The genus invaded Central America on at least 11

separate occasions, and some of these occurred before the suggested closing of the Isthmus of

Panama (Pinto-Sanchez et al., 2012) around 3 million years ago (Heinicke, Duellman and

Hedges, 2007)for example,Pristimantis ridenswas also established in Central America

before the presence of the land bridge (Wang, Crawford and Bermingham, 2008), again

consistent with the hypothesis of rapid radiation from South America.Arguments for high

diversity of poison frogs in the Amazon basin due to rapid dispersal from neighbouring areas

have also been made (Santos et al., 2009) and so this may also have occurred in Pristimantis.

It is clear that even the current estimations ofPristimantis species numbers may be

lower than the actual number of species present (Fouquet et al, 2007; Lehr, Moravec and

Gagliardi Irrutia,2010) due to unexplored regions, polymorphic species, cryptic species and

species that have extremely similar morphologies.Recent analyses of species in the

Pristimantisgenus include those by Elmer and Cannatella (2008), Siqueira et al., (2008),

Padial and de la Riva (2009), and Arteaga-Navarro and Guayasamin (2011), and with further

work done in the area of phylogenetics and more scientific exploration in the region it is

likely that there will be a continuation of these redefinitions of relationships in the genus.

This study will attempt to determine the phylogenies of the South American clade of

Pristimantis using recently acquired and previously-published sequence data, and

comparisons with other papers will be made in order to infer the likelihood of the accuracy of

results. Using Bayesian analyses, I will closer inspect a single clade in terms of altitude - in

doing so I aim to understand better the patterns of diversity and species richness of the genus

and investigate possible multi-complexes of species.


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Methods

Data collection

This study used data from 121 sequences of 12S ribosomal RNA (12S) and 16S

ribosomal RNA (16S) mitochondrial genes.Of these, 30 were new sequences were provided

by Dr Kathryn Elmer (see Appendix II) and 91 were published sequences were downloaded

from NCBI database (see Appendix I).The South American clade group formed by Heinicke

et al., 2007 (species numbers 162-248) was the source for which accession numbers were

chosen and the outgroup was selected from the same study (see Appendix I).

Phylogenetic analyses

The removal of one outgroup species and the joining of 12S and 16S sequences from

the same species resulted in a total of 112 sequences analysed with a total of 1017 sites.The

sequences were aligned using the default settings of the ClustalW option in Geneious 6.0.6

(Biomatters, 2012), where the cost matrix used was IUB, the gap open cost was 15, and the

gap extend cost was 6.66. MEGA 5.1 was used to extend the flanking regions of the

sequences in order to make them the same length as 12S and 16S sequences have hyper

variable regions which cannot be aligned.Ambiguous regions in the alignment were then

removed using Gblocks 0.91b (Castresana, 2000), in which the default block parameters

were: minimum number of sequences for a conserved position: 9; minimum number of

sequences for a flank position: 14; maximum number of contiguous non-conserved positions:

8; minimum length for a block: 10; allowed gap positions: 0.The resulting alignment was

comprised of 1017 sites.The MrBayes plugin (Hulsenbeck and Ronquist, 2001) in Geneious


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6.0.6 was used to perform Bayesian analyses to reconstruct phylogenies with the GTR + I +

model of evolution (adhering to the methods used by Heinicke, Duellman and Hedges, 2007).

Two parallel runs of the Monte Carlo Markov chain algorithm were conducted with

1,100,000 generations (chain lengths) each.The burn-in length was 160,000 and branch

lengths were unconstrained.The subsampling frequency was 200.

The posterior output was examined to assess the convergence of likelihoods onto the

stationary distribution of the two separate runs.A 50% consensus tree was formed using the

resulting raw tree data (11002 trees) in Geneious 6.0.6 (Biomatters, 2012).The posterior

probability (or clade credibility) values for the branch nodes were examined. Inferred

relationships were examined.

A clade encompassing 32 sequences was chosen for further analysisthis clade was

chosen based on the high proportion of new sequence data and species of interest included.

For this clade, the altitudinal ranges were obtained from AmphibiaWeb (2013), IUCN (2012)

and from the individually collected specimen information.A bar graph was created to

compare these altitudinal rangesto further investigate their biodiversity and possible

relationships.


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Results

Convergence assessment

The parameter estimates included the log likelihood score (LnL), total tree length

(TL), the 4 stationary state frequencies (piT, piA, piG, and piC) and the 6 reversible

substitution rates (rA-T, rA-G, rA-C, rC-G, rC-T, and rG-T), and the shape of the gamma

distribution of rate variation across sites (alpha). The comparison of these parameters from

each of the 2 runs shows that the results of each run were similarasides from the

autocorrelation time which increased fairly substantially. The correlation graph shows a

random distribution, or white noiseof points.The density and relative density graphs for

each run show similar patterns.The trace and sample trace was stochastic around the mean.

These indicate convergence on stationarity in the Bayesian analyses and tree space by

MCMC (see Appendix III).


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Phylogeny assessment

Fig. 1: 50% consensus tree from 11002 raw trees created using the MrBayes plugin in Geneious 6.0.6. (Biomatters 2012)

showing the inferred phylogenies of 111Pristimantis species sequences and 1outgroup sequence. Bayesian posterior

probability values are shown.

Clade

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for

further

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Altitudinal assessment

Fig 2: Comparison of altitudinal distributions of species in the clade encompassing species fromP.orestes toP. diadematus QCAZ18015. For published data, the altitudinal range was taken from IUCN(2012) and AmphibiaWeb (2013), and for new sequence data the altitude where the individual waslocated was taken from the active specimen information sheet provided by K. Elmer.


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Distribution assessment

P.ockendeni: P.simonbolivari:

P.cajamarcensis: P.orestes:


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P.ceuthospilus: P. luteolateralis:

P. chalceus:

Fig 3. Distributions of 7 species ofPristimantis (P. ockendeni, P. ceuthospilus, P. simonbolivari, P.

cajamarcensis, P. chalceus, P. luteolateralis, andP. orestes), from the clade encompassing speciesfromP. orestestoP. diadematus, with altitudinal distributions above 1500m.a.s.l. All images from

IUCN (2012). Species with altitudinal distributions above 1500m.a.s.l were chosen as occurrences atthese altitudes are generally rarer and investigation into their distribution could possibly show multi-

complexes of species.


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Discussion

Phylogenetic relationships

74 of the 99 posterior probability values (75%) in the 50% consensus tree were over

80%, indicating the high likelihood of relationships in the majority of the nodes.However,

there are several incidences where the posterior probability values were less than 70%,

including several of the basal relationships.Unfortunately, because of low support, the deep

relationships of these species cannot be confidently confirmed with this analysis. These deep

relationships are likely to be ancient, and confidence in these phylogenies could mean a better

understanding of the evolutionary histories of the genus overall.

There is one occurance of phylogenetic polytomythese occur where there is a lack

of information, the inability to determine the branching order, or possibly speciation events

that occured at once (Garland and Diaz-Uriarte, 1999).In this case there is phylogenetic

polytomy ofP.nigrovittattus (QCAZ25789).I believe this was due to errors in the analysis

rather than speciation from the rest of the species involved at the same time as the outgroup

Phrynopys bracki.

The new sequencePristimantis altamazonicus QCAZ25382 was grouped separately

from the other 2 newP.altamazonicus and the 1 published sequence ofP.almatazonicus -

instead it was grouped withP.diadematus.This may have been due to discrepancies in

sequence analysis, but as the distributions of the species are similar it is also possible that the

inconsistent sequence ofP. altamazonicus was simply mis-identified and should be namedP.

diadematus. This sequence was also found at a higher altitude than the rest of the species in

theP. diadematus cladethis could indicate that it is in fact a different species altogether,

but further analyses would be needed to investigate this.


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P.sp.QCAZ 25588 and P.sp.QCAZ 25582 are likely to be the same species as there

was high support for a close relationship and they were also found in the same area.The most

closely related species according to my analyses isPristimantis ockendeni.Heinicke,

Duellman and Hedges (2007) analysis found that the closest relative toP.ockendeniwasP.

unistrigatusthis species was found in the similar position in my analyses but was not more

closely related to P.sp.QCAZ 25588 and P.sp.QCAZ 25582 compared toP.ockendeni (see

Fig.4).With the wide distribution and the recognition thatP. ockendeniis yet to be fully

delimited is likely thatP.sp (QCAZ25582 and QCAZ25588) are that of one of the species

that has been or is yet to be delimited fromPristimantis ockendeni.

Fig. 4. Simple representation of the comparison of relationships betweenPristimantisockendeni,Pristimantis unistrigatus and two unidentifiedPristimantis species in my own analysis and the

analysis of Heinicke, Duellman and Hedges (2007).

It is likely that the sequenceP.sp.QCAZ25392 isP.conspicillatus due to the high

posterior probabiliy values for their relationship.The inferred relationships ofP.condor, P.

conspicillatus (QCAZ18007, QCAZ25392) andP.croceoinguinis(QCAZ25544) are similar

to the inferred relationships proposed by Pinto-Snchez (2012) (see Fig.5).They also find

thatP.malkiniis the sister species toP.citrogasterwith relatively high support, but my

Bayesian analysis showedP.malkinito be more closely related toP.lirellus andP.imitatrix,


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with higher support.It is less similar to the relationships proposed by Heinicke, Duellman

and Hedges (2007); they showP.croceoinguinis to be a sister species toP.citrogaster, and

P.condor to be a derived species, whereas my analyses showP.citrogaster andP.

croceoinguinis to be sister species (see Fig.6). With a follow-up study I would consider

removing theP.croceoinguinissequence from the analysis in order to see if the relationships

betweenP.condor, P.conspicillatus and P.citrogasterremained the sameif so we could

conclude that these relationships are more likely to be correct than those proposed by

Heinicke, Duellman and Hedges (2007).

Fig.5.Simple representation of the comparison of relationships between 3 species of Pristimantis inmy own analysis and the analysis of Pinto- Snchez (2012)P conspicillatushere includes the

sequencesP.sp.QCAZ18007, QCAZ25392 and the published sequence ofP.conspicillatus.

Fig.6.Simple representation of the comparison of relationships between 4 species of Pristimantis inmy own analysis and the analysis of Heinicke, Duellman and Hedges (2007)

With further investigation of the species listed in Heinicke, Duellman and Hedges

(2007) asPristimantis sp.AY326002, I found that this sequence is listed as an

Eleutherodactylus species on NCBI.However, this was defined by Darst and Canatella


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(2004) before the analyses done by Heinicke, Duelmann and Hedges (2007), and so I

currently accept the laters definition of the sequence.

P.acuminatusandP.lacrimosuswere not included in the study by Heinicke,

Duellman and Hedges (2007) nor by Pinto-Sanchez et al.,(2007)it is possible that this

study reveals newly discovered relationships, but it would be useful for further

investigations of the relationships between these species and those surrounding them in the

inferred phylogenies as the posterior probability value for splitting ofP.lacrimosuswas

extremely low (52.3) and so this relationship cannot be established with confidence.

Fig.7.Simple representation of the comparison of relationships between 5 species of Pristimantis in

my own analysis and the analysis of Heinicke, Duellman and Hedges (2007).

The 32 species wide clade that was used for altitudinal analyses was chosen as it

encompasses some of the species that have been most recently studied, namelyP.achuar, P.

altamnis, andP.kichwarum (and the previously mentionedP.sp.)and the majority of the

information included in this clade was from the new sequences.

The distribution map forP.ockendenishows a vast range, but this species is

undergoing scrutinisation. The small distributions of the recently delimitedP.kichwarum, P.

altamnis andP.achuarby Elmer and Canatella (2008) indicate that any occurence ofP.

ockendeniwhich has a large distribution should be studied further as the presence of cryptic


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species, each with a smaller seperate distribution, is likely. This may also be true of other

species with wide distributions.

The distribution ofP. chalceus appears to be vast, but of the 25 localities in Ecuador,

20 of them occur below 1000m.a.s.l. (AmphibiaWeb, 2013) and so those at higher altitudes

are probably uncommon. Even so, this wide distribution could indicate that the species name

is an umbrella termfor species that have yet to be delimited. All the sampling was

performed in Ecuador, but for species which appear to have distributions which need to be

further defined sampling further afield would be useful as individuals in those areas may give

an indication of further delimited species.

Model and analyses

Each model available for phylogenetic analyses has its weaknesses.The GTR + I +

model was chosen as it is more parameter rich than models such as the Jukes Cantor model

(Hulsenbeck, Ronquist, Neilson and Bollback, 2001) and has been used in a number of

studies involving phylogenetic relationships (Heinicke, Duellman and Hedges 2007; Santos

et al., 2009; Pinto-Sanchez et al., 2012) and as the comparisons would involve two of these

studies it was beneficial for the same model to be used.It has a relaxed assumption of equal

base frequencies.The I symbol indicates that the base frequencies are stationary, and the

symbol indicates a gamma distribution of rates for sites (Neilson, Lohman and Sullivan,

2001).The MCMC (Markov chain Monte Carlo) algorithm is used by MrBayes to estimate

the posterior probabilities of trees (Hulsenbeck and Ronquist, 2001) and is useful as it takes

phylogenetic uncertainty into account.However, it has been shown that even the widely used

Bayesian methods may not be fully accurate but may be too liberal, especially if the model

used is not complex enough (Suzuki, Glazko and Nei, 2002).With more time, analyses such


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as maximum likelihood and bootstrap probability would be performed to deduce any

discrepancies that may have occurred due to the use of the model and Bayesian methods

chosen.

Convergence on stationarity

The output results show convergence on stationarity when addressing the correlation,

density and trace plots of the runs.The shape of the correlation graph showed no affinity for

increasing or decreasing over time, with a white noise pattern of points, another indication

of convergence. Similarly, initially the shape of the trace and sample trace graphs are

stochastic around the mean, showing that variation of the likelihood around the mean is high,

but then nearing the end of the analysis it varies less so indicating the approach to

stationarity.With analyses run for a longer period of time it is likely that the variation around

the mean would decrease further and give greater confidence in the output.

The standard deviation of the mean was 0.915 and 1.083 for run 1 and run 2 respectivelyit

is expected that during analysis this value should approach 0.Overall, the likelihood of

reaching stationarity in this project was high, but with a repetition of the analysis the program

should be run for longer in order for better confidence in this respect.

Wider implications

As well as the inferrred deep evolutionary history of the genus, assessing the

distribution ofPristimantis is useful in determining which diversification hypotheses is most

likely to be correct - for example, it has been stated that diversification was ancient and due

to tectonic events (with the formation of the Andes) rather than climatic change (Elmer,

Dvila and Lougheed, 2007; Heinicke, Duellman and Hedges, 2007).


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The importance of conservation is amplified by studies similar to my own as it is

difficult to know what real damage anthropogenic practices may have on undiscovered

cryptic species as rapid declines continue (Stuart et al., 2004, Bickford et al., 2007). For

example, the subdivision of wide distribution ranges into multiple smaller regions due to the

occurrence of newly delimited species can intensify the pressure on each new species, or

mean that different conservation practices are needed (Kohler et al., 2005)this may be true

for the example in this study ofPristimantis ockendeni. Globally, 1933 species of amphibians

are listed on the IUCN Red List of Threatened Species as Vulnerable, Endangered or

Critically Endangered (IUCN, 2012) due to known practices such as habitat destruction and

fragmentation, infectious disease, and many enigmatic declines (Stuart et al., 2004). The

continued efforts to revise the taxonomy of thePristimantis genus will enhance the

knowledge of species requirements and the knowledge of the genus overall.


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Acknowledgements

I would like to thank Dr Kathryn Elmer for her continued support throughout my time

working on this project. Her knowledge of the subject was indeed an inspiration and gave me

drive to learn more about the subject. I would also like to thank Roderic Page for his

knowledge of the program Geneious and Roman Biek for the access to the licence for the

same program, and Hans Recknagel for his insight into the program Gblocks and for his

assistance when things were not going smoothly. I would also like to thank Isabel Coombs

for the use of the laptop used for the majority of the preliminary analyses. Thanks also to my

friends and family for putting up with my stress-filled messages and phone calls.

Title page photo credit: Santiago R.Ron, Museo de Zoologa, Universidad Catlica del

Ecuador.


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Appendix I: Table of species names and accession numbers used (Heinicke,

Duellman and Hedges, 2007).

No.

Species Origin

Museum

voucher

Lab tissue

number

Genbank accession no.

12S 16S

162

Pristimantis Heinicke,Duellman andHedges (2007) KU218116 267231 EF493518walkeri

163

Pristimantis Heinicke,Duellman and

Hedges (2007) KU177807 267863 EF493517luteolateralis

164

Pristimantis Heinicke,Duellman andHedges (2007) KU177821 267864 EF493351parvillus

165

Pristimantis Heinicke,Duellman andHedges (2007) KU177638 267865 EF493675chalceus

166


Hedges (2007) KU222023 267253 EF493519ockendeni

167

Pristimantis Heinicke,Duellman andHedges (2007) KU218057 267227 EF493387unistrigatus

168

Pristimantis Heinicke,Duellman andHedges (2007) KU217845 267211 EF493823 EF493663cajamarcensis

169

Pristimantis Heinicke,

Duellman andHedges (2007) KU212216 267198 EF493520ceuthospilus

170

Pristimantis Heinicke,Duellman andHedges (2007) KU212226 267200 EF493521lirellus

171


Hedges (2007) KU215476 267205 EF493824 EF493667imitatrix

172


Hedges (2007) KU217862 267213 EF493669 EF493665croceoinguinis

173

PristimantisHeinicke,

Duellman andHedges (2007) KU215460 267204 EF493670altamazonicus

174


Hedges (2007) KU218257 267249 EF493388orestes

175


Hedges (2007) KU218254 267248 EF493671simonbolivari

176

Pristimantis Heinicke,Duellman andHedges (2007) KU218035 267224 EF493348riveti


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177

Pristimantis Heinicke,Duellman andHedges (2007) KU218096 267228 EF493389versicolor

178


Hedges (2007) KU218025 267222 EF493349

phoxocephalus

179

Pristimantis Heinicke,Duellman andHedges (2007) KU218052 267225 EF493673spinosus

180

Pristimantis Heinicke,Duellman andHedges (2007) KU217863 267214 EF493672cryophilius

181


Hedges (2007) KU219788 267250 EF493674rhodoplichus

182


Hedges (2007) KU219796 267251 EF493377 EF493668wiensi

183

Pristimantis Heinicke,Duellman andHedges (2007) KU212293 267202 EF493825 EF493367petrobardus

184

Pristimantis Heinicke,Duellman andHedges (2007)

MHNSMWED56846 267438 EF493826 E F493664melanogaster

185


Hedges (2007) n/a n/a AM039709 AM039641simonsii

186


Duellman andHedges (2007) KU177637 267866 EF493524appendiculatus

187

Pristimantis Heinicke,Duellman andHedges (2007) KU218028 267223 EF493680pycnodermis

188

Pristimantis Heinicke,Duellman andHedges (2007) KU179090 267867 EF493522dissimulatus

189

Pristimantis Heinicke,Duellman andHedges (2007) KU177658 267868 EF493523calcarulatus

190


Duellman andHedges (2007) KU218021 267221 EF493679orcesi

191

Pristimantis Heinicke,Duellman andHedges (2007) KU218002 267217 EF493676glandulosus

192

Pristimantis Heinicke,Duellman andHedges (2007) KU218015 267218 EF493677inusitatus

193

Pristimantis Heinicke,Duellman andHedges (2007) KU217786 267207 EF493678acerus


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30

194

Pristimantis Heinicke,Duellman andHedges (2007) KU212220 267199 EF493681schultei

195


Hedges (2007) KU291702 171051 EF493351

bromeliaceus

196

Pristimantis Heinicke,Duellman andHedges (2007) KU218147 267246 EF493525subsigillatus

197

Pristimantis Heinicke,Duellman andHedges (2007) KU177812 267869 EF493526nyctophylax

198

Pristimantis Heinicke,Duellman andHedges (2007) No voucher 266036 EF493692shrevei

199


Hedges (2007) BWMC6918 266624 EF493527euphronides

200

Pristimantis Heinicke,Duellman andHedges (2007) No voucher 102308 EF493691rozei

201

Pristimantis Heinicke,Duellman andHedges (2007) KU218109 267230 EF493511gentryi

202


Hedges (2007) KU218013 267229 EF493512truebae

203


Duellman andHedges (2007) KU217871 267215 EF493513curtipes

204

Pristimantis Heinicke,Duellman andHedges (2007) KU177972 267870 EF493689vertebralis

205

Pristimantis Heinicke,Duellman andHedges (2007) KU217836 267210 EF493350buckleyi

206

Pristimantis Heinicke,Duellman andHedges (2007) KU217991 267216 EF493688devillei

207


Duellman andHedges (2007) KU177847 267871 EF493687surdus

208

Pristimantis Heinicke,Duellman andHedges (2007) KU179374 267872 EF493690quinquagesimus

209


n/aKU217998

n/aKU21799

8 AY326003duellmani

210


Hedges (2007) KU177861 267873 EF493514thymalopsoides


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211

Pristimantis Heinicke,Duellman andHedges (2007) KU208508 267439 EF493682ocreatus

212


Hedges (2007) KU218030 267441 EF493683

pyrrhomerus

213

Pristimantis Heinicke,Duellman andHedges (2007) KU218234 267247 EF493515festae

214

Pristimantis Heinicke,Duellman andHedges (2007) KU218227 267437 EF493684leoni

215


Hedges (2007) QCAZ12410 267646 EF493686verecundus

216


Hedges (2007) KU177684 267874 EF493685celator

217

Pristimantis Heinicke,Duellman andHedges (2007) n/a n/a AY326007chloronotus

218

Pristimantis Heinicke,Duellman andHedges (2007) QCAZ16428 267644 EF493516thymelensis

219

Pristimantis Heinicke,Duellman andHedges (2007) n/a n/a AY326005supernatis

220


Duellman andHedges (2007) n/a n/a AY326002sp.

221

Pristimantis Heinicke,Duellman andHedges (2007) USNM33609 101646 EF493699urichi

222

Pristimantis Heinicke,Duellman andHedges (2007) KU218016 267219 EF493698latidiscus

223

Pristimantis Heinicke,Duellman andHedges (2007) QCAZ17101 267635 EF493354colomai

224


Duellman andHedges (2007)

AMNHA12444 267876 EF493697cruentus

225


AMNHA124551 267877 EF493355ridens

226

Pristimantis Heinicke,Duellman andHedges (2007) KU177252 267878 EF493528cremnobates

227

Pristimantis Heinicke,Duellman andHedges (2007) n/a n/a AY326004w-nigrum


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228

Pristimantis Heinicke,Duellman andHedges (2007) KU217830 267209 EF493696actites

229


Hedges (2007) KU222001 267252 EF493695

lanthanites

230

Pristimantis Heinicke,Duellman andHedges (2007) KU177730 267879 EF493693 EF493666crenunguis

231

Pristimantis Heinicke,Duellman andHedges (2007) QCAZ19771 267640 EF493694labiosus

232


Hedges (2007)

MHNSMLR

4341 266049 EF493356sp.

233


Hedges (2007) QCAZ28448 267636 EF493529conspicillatus

234

Pristimantis Heinicke,Duellman andHedges (2007) KU217857 267212 EF493701condor

235

Pristimantis Heinicke,Duellman andHedges (2007) KU212278 267201 EF493700citriogaster

236


Hedges (2007) KU217809 267208 EF493827 EF493660achatinus

237


Duellman andHedges (2007) KU218019 267220 EF493392lymani

238


MHNSM9298 266046 EF493703fenestratus

239

Pristimantis Heinicke,Duellman andHedges (2007) KU291638 171021 EF493702bipunctatus

240


Hedges (2007)

MHNSM100

71 266052 EF493393skydmainos

241


Duellman andHedges (2007) KU215493 267206 EF493353toftae

242

Pristimantis Heinicke,Duellman andHedges (2007) KU173492 267875 EF493706rhabdolaemus

243

Pristimantis Heinicke,Duellman andHedges (2007) n/a n/a AY843586pluvicanorus

244

Pristimantis Heinicke,Duellman andHedges (2007) KU291635 171098 EF493705sagittulus


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245

Pristimantis Heinicke,Duellman andHedges (2007) KU291659 171080 EF493704stictogaster

246


Hedges (2007) KU291627 171070 EF493390

aniptopalmatus

247


MHNSM9267 266050 EF493707peruvianus

248

Pristimantis Heinicke,Duellman andHedges (2007) KU177680 267880 EF493391caprifer

.....

260

Phrynopus Heinicke,Duellman andHedges (2007)

USNM286919 171045 EF493709bracki


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Appendix II: New sequence information provided by Dr Kathryn Elmer

Species nameQCAZnumber

Locality Latitude LongitudeElevation(m.a.s.l)

Pristimantisachuar

25457 Kapawi lodge S 0232'32" W 7651'50" 239

Pristimantis

achuarholotype25463 Kapawi lodge S 0232'32" W 7651'50" 239

Pristimantis

acuminatus16748 EC Yasuni S 040' 36'' W 7623'43'' 230

Pristimantis

acuminatus19664 EC Yasuni S 040' 36'' W 7623'43'' 230

Pristimantis

altamazonicus25382 FMT S 0121'26" W 7751'73" 973

Pristimantis

altamazonicus 25407 La Selva S 0029'89" W 7622'43" 245

Pristimantisaltamazonicus

25535 Kapawi lodge S 0232'32" W 7551'50" 239

Pristimantis

altamnis25808

Comunidad

AsociacinChonta Yacu

N 0006.69 W 7722.46 610

Pristimantis

conspicillatus18007

Estacion

BiologicaJatun Sacha,Napo

S 0104'840" W 7736502 419

Pristimantiscroceoinguinis

18231

Shaime,

Zamora-

ChinchipeS 0418'92" W 7839'84" 907

Pristimantis

croceoinguinis25544 Kapawi lodge S 0232'32" W 7551'50" 239

Pristimantis

diadematus18014

EstacionBiologica

Jatun Sacha,Napo

S 0104'840" W 7736502 419

Pristimantis

diadematus18015

EstacionBiologica

Jatun Sacha,Napo

S 0104'841" W 7736503 420

Pristimantis

diadematus18017

EstacionBiologica

Jatun Sacha,Napo

S 0104'842" W 7736504 421

Pristimantis

kichwarum25579

Estacion


S 0103.740 W 7736879 390

Pristimantis

lacrimosus15982 Azuela S 010'00" W 7739'00" unknown


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Pristimantis

lanthanites25389 La Selva S 0029'89" W 7622'43" 245

Pristimantis

librarius25589 Auca 14 Rd S 0041'85" W 7643'79" 255

Pristimantismalkini

25444

Estacion


S 0104'840" W 7736'502" 419

Pristimantis

martiae25403 La Selva S 0029'89" W 7622'43" 245

Pristimantis

nigrovitattus25789 Lumbaqui N 0006'69" W 7722'46" 610

Pristimantis

ockendeni25576 Cuyabeno S 0000'8" W 7610'4" 230

Pristimantis

ockendeni25768

EstacionBiologica

Jatun Sacha,Napo

S 0104'840" W 7736'502" 419

Pristimantis sp. 18233Shaime,

Zamora-Chinchipe

S 0418'92" W 7839'84" 907

Pristimantis sp. 18235Shaime,

Zamora-Chinchipe

S 0418'93" W 7839'85" 908

Pristimantis sp. 18236Shaime,Zamora-Chinchipe

S 0418'94" W 7839'86" 909

Pristimantis sp. 25392 La Selva S 0029'89" W 7622'43" 245

Pristimantis sp. 25582 Auca 14 Rd S 0041'85" W 7643'79" 255

Pristimantis sp. 25588 Auca 14 Rd S 0041'86" W 7643'80" 256

Pristimantis

variabilis28430 Cuyabeno S 0000'8" W 7610'4" 230


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Appendix III: Output tables and graphs from MrBayes (Hulsenbeck and

Ronquist, 2001)

Table 1.Summary statistics produced from the MrBayes analysis.

Summary statistic LnL_run1 LnL_run2

Mean -17914.636 -17911.164

Standard deviation of mean 0.915 1.083

Median -17914.23 -17911.767

95% HPD lower -17938.889 -17938.827

95% HPD upper -17892.597 -17882.131

Auto-correlation time (ACT) 5380.765 5153.555

Effective sample size (ESS) 173.804 181.467

Fig.S1.Correlation of LnL_run1 and LnL_run2.


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Fig. S2.Comparison of density between LnL_run1 and LnL_run2.

Fig. S3. Comparison of relative density between LnL_run1 and LnL_run2.


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Fig. S4. Trace plot

Fig. S5. Trace plot (sample only)

Documents

Hons Project - Robyn Cuthbertson - Inferring Phylogenies and Distributions of Upper-Andean and Amazonian Frogs From the Genus Pristimantis Using New and Previously Published Sequence