LE Templeton 0704913 Msc Project

8/13/2019 LE Templeton 0704913 Msc Project

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Molecular Phylogenetics and Phylogeographyof Neotropical Salamanders

(Genus: Bolitoglossa ).

Liam Templeton (0704913)

The University of Glasgow

August 2013


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Abstract

Plethodontid salamanders are among the most species rich families of the order Caudata .

Further, the majority of species within this family belong to the supergenus Bolitoglossa .

While members of this genus are found in abundance throughout tropical Central America,

comparatively fewer species are known to occur in South America. This observation is

somewhat paradoxical due to the greater landmass and the presence of mega-diverse habitats

as exhibited by South America, and has led to the formation of two major hypotheses: either

that the observed lack of species richness in South America is a result of the recent arrival of

bolitoglossine salamanders via the Panamanian land-bridge (estimated to have formed 3-4

million years ago); or that the real extent of species diversity is obscured by the presence of

cryptic species. Previous attempts to quantify the distribution of genetic diversity as exhibited

by members of this genus in Ecuador have revealed previously unsuspected levels of cryptic

species diversity throughout the upper Amazon. Similarly, divergence time estimates have

indicated an arrival of salamanders in South America that far precedes the completion of the

contemporary land connection. The availability of molecular data for these species is at

present largely restricted to the Amazon. In light of this, the purpose of this study is to

investigate the distribution of genetic diversity of these salamanders in previously

underrepresented coastal and Andean regions of Ecuador in an attempt to better resolve the

existing estimates of both species richness and time of colonisation in this area. Sequence

data from four genes (two nuclear, two mitochondrial) are used to infer phylogenetic

relationships of coastal and Andean specimens of bolitoglossine salamander. Additionally, a

time-calibrated phylogeny is implemented to estimate the time of arrival of salamanders into

South America.


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Keywords Phylogenetics; Phylogeography; Paleogeography; Herpetology; Plethodontidae;

Bolitoglossa; Neotropics; Ecuador; Species diversity; Time calibration; Central America;

South America; Isthmus of Panama; Andes.

Cover - The cover image is a mosaic of four composite images, each made up of the first 100

search results for four of the keywords associated with this project as reported by the image-

hosting site Flikr.

Introduction

Plethodontids, or lungless salamanders (so called due to their ability to breathe through their

skin), are by far the largest and most speciose extant family in the order Caudata

(AmphibiaWeb, 2013); harbouring in the region of 376 species which are known to occupy a

broad range of ecologically diverse niches and habitats. As such, they have been recognised

for their potentially important role in monitoring biodiversity and indicating ecosystem

integrity (Welse & Droege, 2001). The group is currently understood to have originated in

the Appalachian Mountain range of what is now the eastern United States. It is broadly

recognised that periods of orogenesis, and the subsequent creation of stable elevational

gradients, are responsible for the origin and maintenance of species diversity exhibited

among this group (Wake, 1966). This adaptation to lunglessness is thought to have been

primarily derived as a consequence of paedomorphosis (i.e. neoteny) (Ruben & Beucot,

1989), and is also considered to have had a significant influence on the diversification of

species within this group (Lombard & Wake, 1986). Perhaps most notably is the development

of highly specialised hyobranchial apparatus, which have allowed for the development of

novel and efficient methods by which to capture prey (Wake & Elias, 1983).


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The hypothesised northern origin of Caudates and the effects of niche conservatism in their

early diversification have meant that the majority of higher taxonomic units within the order

Caudata currently reside in northern temperate regions (Kozak & Weins, 2010). The largest

and most specious genus of this family, i.e. Bolitoglossa (Wake & Lynch, 1976), is observed

to occur mainly in neo-tropical regions of Central America. This perhaps indicates a recent

expansion of populations into this area followed by a rapid succession of speciation events

throughout this region. Biological phenomena including niche expansion and miniaturisation

are thought to represent key evolutionary processes that have allowed for the seemingly

explosive rate diversification exhibited by many other species belonging to the subfamily

Bolitoglossinae (Hanken & Wake, 1993). Despite the massive level of species diversity

within this group, many species are observed to exhibit extensive, by which species share a

large number of homogeneous morphological features (Wake, 1991).

Principally, an extensive webbing of digits has been widely observed among species of this

group. This feature in particular is acknowledged to be a common indication of an ancestral

adaptation to arboreal life and is considered to have evolved convergently a number of times

within the group (Wake & Lynch, 1976). Numerous colonisation events of diverse habitats

and the repeated independent evolution of similarly derived traits may be partly responsible

for the wealth of species diversity exhibited by this group (Wake, 1987). Further, the

complex geological history of Mesoamerica and the associated biogeographic processes are

likely to have further influenced the rate and extent to which species diversification has taken

place among Central American clades (Rovito et al., 2012).


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Despite the massive diversity of the genus Bolitoglossa as observed in Central America,

comparatively few species are known to inhabit in South America. This difference in

observed diversity of this genus between the two continents is so grand, in fact, that only 28

of the 128 described species that make up the genus Bolitoglossa are known to occur in South

America (Amphibiaweb, 2013); all of which are now understood to belong to the subgenus

Eladinea (Parra-Olea et al., 2004). The only other representative taxa from this group known

to occur in South America, aside from the few members of the subgenus Eladinea, include

only two species from the genus Oedipina (Garcia-Paris & Wake, 2000); all of which belong

to the supergenus Bolitoglossa . This observation is at its surface somewhat paradoxical;

being that the sheer land mass of South America is far greater than that of Central America,

and so might be expected to accommodate a greater number of species. Further, South

America is renowned for its mega diverse habitats and high levels of endemism, such as

those exhibited by the Amazon and Atlantic forests, and hence one might expect a similar

level of diversity to be exhibited among species of salamander.

Of the few species that do occur in South America and which are currently recognised by

science, most of them are observed to exhibit massive distributions across vast areas of dense

tropical forest; the majority of which is likely to be largely unexplored by science. This

observation is not consistent with the comparatively small range sizes as exhibited by most

other Neotropical Salamanders (Rovito et al., 2012). As such, it is possible that species

diversity has been broadly underestimated, and that the inferred distributions reported for

these species are in fact extrapolations of encounters made in the few areas that have been

intensively surveyed. Additionally, much of these encounters are prone to misidentification

due to the cryptic nature of anatomical features exhibited by those animals; features such as

subtle colouration, reduced dentition, and extensive webbing of digits (Crump, 1977); the


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likes of which would typically be greatly informative for the purpose of species

identification. Such characteristics might also be considered to be the cause of much of the

difficulties faced when attempting to classify these animals using morphological features

alone.

This phenomenon, by which species diversity in salamanders is seemingly underrepresented

by current understanding of their systematics, has generally led to the development of two

major hypotheses which can be expressed in terms of the following: either the lack of species

diversity observed in South America is the result of a relatively recent arrival of species via a

land bridge connecting Central and South American (i.e. the Isthmus of Panama) (Wake,

2005); or that the true extent of species richness among South American species groups has

been underestimated by current taxonomic understanding through the presence of cryptic

species diversity (Elmer et al., 2013). In the former, it is speculated that species arriving in

South America from Central America via the Panamanian land bridge would have limited

time to diversify; hence the apparent lack of species diversity that has been previously noted.

In the latter, it is presumed that similarly derived morphological features and convergences of

character evolution have obscured the true level of species diversity exhibited by South

American salamanders, as represented by traditional methods of taxonomic classification.

Further consideration of the presence of deep divergences between Central and South

American clades of salamanders has led to the development of additional hypotheses

(Hanken & Wake, 1982), by which southern Central American populations may have

undergone some form of diversification prior to the formation of the Panamanian land bridge,

and subsequently dispersed into South America following its completion. The alternative

being that species of salamander were able to disperse into South America much earlier than


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was previously thought, and the observed level of divergence exhibited by existing

populations in these areas is the result of subsequent diversification that would have occurred

therein. In this case ancestral populations would have arrived via some form of preexisting

land connection; the likes of which is not yet widely acknowledged to have existed.

The low vagility and generally limited ability to disperse as exhibited by these animals

suggests that it is unlikely that they would have been able to traverse a marine obstacle by

any means other than via some form of land bridge. This suggests that the existing evidence

of a divergence time between Central and South American salamanders predating the

formation of the contemporary land bridge would also indicate the presence of a more ancient

land bridge facilitating the exchange of biodiversity. Further, this limited capacity to disperse

across broad geographic ranges and obstacles has also meant that these animals are ideal

candidates for the exploration of biogeographic processes involved in the origin and

maintenance of biological diversity. Such consideration of the observed distribution of

genetic diversity in these taxa is therefore informative when making inference as to

palaeographic events, which have been otherwise obscured by subsequent geological

processes.

Presently accepted dates for the formation of the contemporary land bridge connecting

Central and South America indicate that it was formed in the region of 4 million years ago

(mya). The exchange in biodiversity that would have followed has since been dubbed the

Great American Biotic Interchange (GABI), and is acknowledged to be a major

biogeographic event in Earth history having greatly influenced the presently observed

distribution of biodiversity throughout the Americas and beyond (Simpsons, 1940). This

event has been well documented in the paleobiology of many mammalian groups (Marshall,

1988; Webb, 1991); offering a seemingly comprehensive understanding of the timings of the


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historical exchange in biodiversity. However, the antiquity and nature of the formation of the

contemporary land bridge has in recent years been the topic of some debate. Novel evidence

has shown that a land connection may have existed much earlier than is currently

acknowledged, which may have facilitated the exchange of terrestrial biodiversity as early as

23-25 mya (Farris et al., 2011; Montes et al., 2012). As it is currently understood, this prior

event may not represent the presence of a fully formed land bridge connecting Central and

South America; rather it is characterized by a narrowing of the seaway between the two

continents and the presence of a shifting island complex.

The arrival of salamanders into South America has been tested numerous times in

concordance with the existing estimates for the formation of the Isthmus of Panama. The

exact nature of this colonization, however, remains unclear. Early estimates suggest a

colonization date sometime during the late Miocene to early Pliocene period (Dunn, 1926).

Further consideration (Brame & Wake, 1963) led to the proposal that ancestral populations

underwent multiple migrations between Central and South America throughout the Pliocene

(~52.5 mya). An analysis of allozyme distance indicates that Central and South American

species diverged upward of 18 mya (Hanken & Wake, 1982), and more recent analyses based

on mitochondrial sequence data confirm reinforce the idea that salamanders were present in

South American prior to the completion of the contemporary land bridge (Parra-Olea et al.,

2004; Wiens et al., 2007).

Most recently, Elmer et al (2013) used divergence time estimates and ancestral area

reconstruction of Bolitoglossan salamanders from Ecuadorian Amazon and Andean regions

to establish the earliest possible age of an endemic South American clade of Plethodontid

salamander and infer a minimum time for colonisation of South America by this group. From


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this, it is reported that the resulting estimates indicate an arrival of salamanders into South

American some time during the early Miocene (23 - 15 MYA). Further, the evolutionary

relationships of salamanders from these regions were also considered for the purpose of

exploring the extent to which diversification has taken place among Ecuadorian populations

of salamander. In doing so it was revealed that South American clades of salamander contain

numerous deep divergences and a level of genetic diversity that was previously unsuspected.

Sampling locations for this study, however, were focussed in upper Amazonian and eastern

Andean regions of Ecuador; and hence there are limitations as to the inferences than can be

made from these results with regards to the distribution of genetic diversity throughout

further regions of South America.

Here, the distribution of genetic diversity as exhibited by South American species of

salamander is considered in areas of Coastal and Western Andean Ecuador; the likes of

which have never before been studied at a molecular level. DNA Sequence information is

generated from multiple genes (two nuclear, two mitochondrial) in an attempt to assemble a

well resolved and informative phylogenetic reconstruction. These data are considered in such

a way so that inference can be made as to the origin and nature of subsequent diversification

of the included taxa. Additionally, new data are combined with existing sets of sequence

information to further explore patterns of diversification and the distribution of genetic

diversity on a national scale throughout areas of Ecuadorian Amazon. Finally, the observed

extent of molecular divergence between Central and South American clades of salamander is

time calibrated using estimated dates for the origin of the subfamily Bolitoglossinae in an

attempt to reevaluate the currently accepted dates for the formation of the Isthmus of Panama.


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Aims

The aims of this study are as follows:

- To investigate the origin and distribution of genetic diversity among populations of

bolitoglossine salamander inhabiting coastal and western Andean regions of Ecuador; either

supporting a relatively recent arrival of individuals into these areas from Central American

populations; or as a result of vicariance following periods of large scale Andean orogeny, in

which case indicating a much deeper divergence between populations.

- To assess the extent to which species richness has been previously underestimated by way

of the identification of cryptic species diversity as represented in the genetic diversity

exhibited amount individuals, and explore the geographical factors that may have influenced

the observed distribution of genetic diversity such as the presence of contemporary land

barriers and major events in geological history.

- The reevaluate the dates from when species are expected to have dispersed into South

America to reinforce that which has previously been demonstrated in published examples;

whereby estimates of divergence between Central and South American Salamanders have

indicated an inter-continental exchange of biodiversity which predates the currently accepted

times for the formation of the Panamanian land-bridge.


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Methods

DNA Extraction and Amplification

Tissue samples from 20 individuals collected from Western Andean and coastal regions of

Ecuador were requested from the collections held at the Museo de Zoologia of the Pontifica

Universidad Catolica de Ecuador. Samples were selected on the basis of the location from

which it was collected. Genomic DNA was extracted from tissue using Qiagen DNeasy

Blood & Tissue Kit, following the manufacturers instructions. The quantity and quality of

DNA extraction products were assessed using a nanodrop spectrophotometer, the results from

which were not used due to several suspected spurious readings. Extractions were instead

visualised by gel electrophoresis to assess the quantity and quality of the DNA held therein.

Samples were loaded in a 2% agarose gel using ethidium bromide as a staining agent and run

at 100 volts and 400 milliamps for approximately 60 minutes before being imaged in a

BioRad Gel Doc XR system.

Once extractions products were observed to have yielded a suitable concentration of DNA,

the following genes were amplified by way of the polymerase chain reaction (PCR) using a

MJ Resarch PTC-220 DNA Engine Dyad Peltier Thermal Cycler: recombination activating

gene 1 (RAG1); Pro-opiomelanocortin (POMC); cytochrome b (CYTB); and 16S ribosomal

RNA (16S). RAG1 was amplified using the primer set RAG1BolitoF & RAG1BolitoR as

described by Elmer et al. (2013). POMC was amplified using the primer set

POMC_DRV_F1 & POMC DRV_R1 as described by Vietes et al. (2007). CYTB was

amplified using the primer set MVZ15L & MVZ16H, as described by Moritz et al. (1992).

16S was amplified using the primer set 16Sc & 16Sd, as described by Evans et al. (2003).


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Protocol used for amplification were as reported in the publication in which the primer was

described, or via a personal communication. The concentration of reagents used for reactions

were as follows:

Reagent Concentration Volume per reaction Concentration per reaction

H2O n/a 26.8ul n/a

dNTPs 10mM 4ul 1mM

Buffer 10X 4ul 1X

MgCl2 25mM 2ul 2.5mM

Forward Primer 10uM 0.4ul 0.1uM

Reverse Primer 10uM 0.4ul 0.1uM

Taq Polymerase 10U/ul 0.4ul 0.1U/ul

Template DNA n/a 2ul n/a

PCR products were visualised by gel electrophoresis using the same protocol as outlined

above. Products for which genes were observed to amplify successfully were purified using a

Machery-Nagel Gel and PCR Clean-up kit, following the manufacturers protocol to remove

excess reagents for sequencing. Clean products were visualised by gel electrophoresis to

determine the concentration of DNA present. Samples were diluted in water to achieve

optimal concentration and volume (30ul at 20ng/ul) of DNA before being sequenced in both

directions by a third-party company (DNA sequencing and services, Dundee). The results

from which were viewed as trace files using the chromatogram viewer 4Peaks (Griekspoor &

Groothuis, 2006) to assess the quality of the sequence.


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Sequence Assembly and Alignment

Sequences were subjected to a BLAST search on the nucleotide database hosted by the

National Centre for Biotechnology Information (NCBI) to confirm the identity of the

sequence as bolitoglossine. By this process, it was discovered that the DNA sequences

yielded for all genes from one sample (Museum no.: QCAZ-A 45262) were consistently and

significantly different from those yielded from other samples. Further, this specimen was not

successfully amplified for the gene RAG1; the primers for which are specifically designed for

bolitoglossine salamanders. As such, it was acknowledged that the sequences of this sample

more closely resembled those of a tropical species of frog (best hit = Hypsiboas pellucens ,

99% identity) and likely did not belong to a salamander as indicated by the museum

collection information provided. It is not clear, however, if this anomaly was a result of a lab

contamination during either the extraction or amplification process, or if instead it was a

result of a mislabeling error on the part of the museum collection from which the sample was

sourced.

Forward and reverse sequences were assembled in Geneious v.5.4 (Drummond et al., 2011)

to form consensus sequences. Sequences were aligned using Clustal Omega (Goujon et al.,

2010) as hosted by the European Bioinformatics Institute (EBI) as part of the European

Molecular Biology Laboratory (EMBL) using the default parameters. Alignments were edited

in MEGA in an attempt to correct any hyper-variable regions, and exported in a NEXUS

format. Additional alignments were generated using datasets as described in previous

publications by Elmer et al (2013) and Parra-Olea et al. (2004); one including additional

Ecuadorian and Amazonian samples for use when investigating the distribution of genetic

diversity on a national (see table of specimens in appendix 2), and the other including


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representative samples of Bolitoglossa species from Central America, for use when

investigating molecular diversification on an inter-continental scale (see table of specimens in

appendix 3). These data were combined with the newly generated sequence for the genes cytb

and rag1 only, being that homologous sequence data for the remaining two genes was largely

unavailable for the existing data sets.

Phylogenetic Inference

The best evolutionary & site heterogeneity model and partitioning scheme of the sequence

matrix was determined using PartitionFinder (Lanfaer et al., 2012). The reading frames of

protein coding sequences were determined by comparing them to homologous sequences and

amino acid translations on GenBank. The ribosomal rRNA subunit 16S was left as a single

data block, as it is understood to contain several non-coding loop and stem regions,

which may independently be subject to variable rates of substitution. It was acknowledged

that it might be possible to identify these respective regions and partition them thusly,

however this was advised against via a personal communication (Lanfear, 2013) on account

of it being a relatively time intensive task for all that it would benefit the outcome of the best

partitioning scheme. The program was run a number of times using different model selection

models (AIC, AICc, BIC), favouring the run that produced the simplest partitioning scheme

(i.e. the fewest number of data partitions).

Phylogenetic relationships of the samples for which new sequence data was generated were

inferred by implementing bayesian methods of phylogenetic reconstruction in BEAST v1.7.5

using the species tree ancestral reconstruction function (*BEAST) as described by Heled &

Drummond (2010). Sequences from a Colombian voucher specimen of Bolitoglossa sima,


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and two Central American Bolitoglossa voucher specimen (Bolitoglossa cerroensis [Costa

Rica], Bolitoglossa biseriata [Panama]) were grouped in used as out-groups for the purpose

of rooting the trees (see table of specimens in appendix 1). Each taxon was allocated an

independent species trait in order to allow inference as to the identity of previously

unidentified specimen based on the observed topology of the tree. The parameters under sites

were informed by the best partitioning scheme as reported by PartitionFinder. All sites were

set with a lognormal uncorrelated relaxed clock with a fixed mean of 1.0 across all genes.

The Monte Carlo Marakov chain (MCMC) was set at 100,000,000 generations, sampling

every 10,000 generations. The resulting log file was viewed in Tracer v1.5 (Rambaut &

Drummond, 2003) to ensure sufficient effective samples size (ESS) and likelihood scores for

the tree and coalescent model. TreeAnnotator v1.7.5 (Rambaut & Drummond, 2002) was

then used to generate maximum clade credibility (MCC) tree from a sample of 10,000 trees,

using a burnin of 10% and a posterior probability limit of 0.0. The resulting trees were

visualized and processed using FigTree (Rambaut & Drummond, 2006). This method was

repeated using data sets for each individual gene to test for congruence among gene trees.

Time-calibration

In order to explore the distribution of genetic diversity on an Ecuadorian scale, including that

which is represented by new sequence data generated from Andean and coastal specimen,

subsequent trees were inferred using the combined data set including the Amazonian data

produced by Elmer et al. (2013). The best partitioning scheme for this data set was

determined using PartitionFinder, and phylogenetic reconstruction was inferred using

BEAST following the same protocol as outlined above. Further, a time calibrated phylogeny

was created in BEAST using the combined data set which included both the Amazonian and


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Central American data sets. The rate of molecular change along branch lengths was

calibrated using a normally distributed basal calibration prior for the divergence of

Bolitoglossinae and Plethodontinae, as indicated by previous research (Vietes, Min, & Wake,

2007; Zhang & Wake, 2009; Pyron & Wiens, 2011). For this purpose, similar to that which

was implemented by Elmer et al. (2013), a prior date of 75 million years with a standard

deviation of 5 million years was used.

Genetic Distance

Inter-individual genetic distances were generated for all new sequences using MEGA 5

(Tamura et al., 2011). Values for genetic distance between mitochondrial genes were

generated using a Kimura two-parameter (K2P) corrected correlation to compensate for the

elevated substitution rate of mitochondrial genes. Genetic distance for nuclear genes was

calculated as an uncorrected p-distance due to their slower evolutionary rate. Individuals

were then grouped as was indicated by the phylogenetic trees generated in BEAST to

calculate between and within group genetic distance for the clades recognised in these trees.

These data were also combined with the Amazonian data produced by Elmer et al. (2013) for

the genes CYTB and RAG1 and grouped as was indicated by the findings in this paper to

inform as to how well these clades are supported with regards to inferred genetic difference.

Trees were combined with locational data for the representative taxa residing at the node tips

in a KML file using a web-based application that was created by Professor Rod Page. This

format allowed for trees to be projected onto an interactive three-dimensional rendering of

the Earths surface (i.e. Google Earth) for the purpose of exploring contemporary landscape

features in three-dimensional space. Similarly, the same locational data were plotted on to a


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two-dimensional rendering of the Earths surface (i.e. Google Maps) using the experimental

online application Google Fusion Tables. Individuals grouped by clade as informed by the

resulting trees, and markers indicating the sampling location of that individual were coloured

accordingly. This allowed for further investigation of geographical features that may have

influenced the observed distribution of genetic diversity on a more local scale.

Geographic Range Expansion

In order to infer the geographic range evolution and dispersal of ancestral species of the

genus Bolitoglossa resulting in their migration into of South America, a Dispersal,

Extinction, Cladogenesis (DEC) model was attempted using the program lagrange and the

methods described by Ree & Smith (2008). This analysis would make use of the time

calibrated inter-continental phylogenetic tree compiled in a matrix of the individuals included

in this tree formatted in such a way so as to indicate their region of origin; either Central

American, South American, or both. However, it was discovered that the existing code

provided for this analysis is incompatible with the latest version of Python, and as such the

analysis could not be completed as intended.

Results

A full table including sampling locations from where the specimen used in this study were

collected can be viewed as using Google Fusion Tables at the following link -

www.google.com/fusiontables/DataSourcedocid=15ovOhHOYEcIMRWXbDR1c9wcyqzij2

BHe1z8y b3E


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By selecting the Map of Latitude tab the sampling locations for each specimen can be

viewed as projected onto Google Maps, allowing for various features to be explored at

different levels of zoom and using different map renderings. Of the specimens represented in

this table, included are all coastal and western Andean specimens for whom sequence data

was generated for the purpose of this study, as well as upper Amazonian specimen used in a

previous study by Elmer et al. (2013). Additionally, three non-Ecuadorian specimens that

were used for the purpose of out-grouping are represented.

Genetic Distance

The inter-individual distance results (Tables 1 4) are variable between different taxa and

different genes exhibit different rates of molecular change. The most highly divergent

sequences appear to be cytochrome b (cytb) (Table 1), with a maximum difference in

sequence identity of 17.1% between individuals QCAZ-A 22346 ( Bolitoglossa sima ,

Esmeraldas) and QCAZ-A 39984 ( B. sp ., Carchi). These individuals are acknowledged to

have been collected in the north of Ecuador; however, the latter from the Andean region (c.f.

coastal). With this considered there is likely a prominent difference in altitude between their

respective sampling sites.

Conversely, two pairs individuals report a genetic distance of zero for this gene, indicating

high level of genetic similarity or even that the two sequences are identical. This was

observed between specimen numbers QCAZ-A 51911 ( B. palmata , Tungurahua) and QCAZ-

A 52616 ( B. peruviana - Morona), and similarly QCAZ-A 52454 ( B. sp . - Tungurahua) and

QCAZ-A 52459 ( B. sp. - Tungurahua). The latter two samples were collected from the same

sampling site. The former, however, are reported to have been collected from sites with a


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considerable amount of geographic distance between them. High levels of sequence

homogeneity are also observed between those two pairs, although they are not observed to be

entirely similar as they are to one another.

Considerably more specimens are observed to exhibit high levels of genetic similarity and

complete sequence homogeneity for the nuclear recombination-activating gene (rag1) (Table

2). These patterns again typically relate to the geographic distribution of the specimens in

question; whereby specimens with neighbouring sample sites are generally observed to

exhibit lower inter-individual genetic distances.

Further consideration of these observed patterns reveals that individuals represented in table

columns 1 5 (QCAZ-A 22176, B. sp .; 22346, B. sima ; 27752, B. sp. ; 31532, B. sima -

Esmeraldas, and 32135, B. sp. - Imbabura) 8 (QCAZ-A 40817, B. sp. - Imbabura), and 14

(QCAZ-A 51867, B. sp. - Esmeraldas) that exhibit evolutionary distances of effectively zero

where all collected from coastal lowland locations. Similarly, those that exhibited high levels

of homogeneity for cytochrome b are also shown to exhibit little or no sequence divergence;

either between or within pairs, as outlined above.

Similar patterns of sequence divergence are observed when considering the inter-individual

estimates of un-corrected evolutionary distance for all coastal and western Andean specimen

(Table 3); whereby specimen collected from sites which are closely associated geographically

exhibit high degrees of sequence similarity. This extent to which this effect is observed for

this gene, however, is not as dramatic as that which is observed for RAG1. Further, while

geographical distance appears to offer some explanation for a lack of sequence divergence at

a region level, it does not seem to be entirely supported at a local level; as some individuals


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which are reported to have been collected from geographically associated sites (e.g. QCAZ-A

22346, B. sima - Esmeraldas & QCAZ-A 31532, B. sima - Esmeraldas) are reported to be

genetically more closely related to individuals from more distant sites.

Comparatively fewer specimens are observed to exhibit genetic distance scores of zero for

mitochondrial gene 16S (Table 4). Those that do exhibit complete sequence identity are also

closely associated by their respective geography, and individuals are reported to have been

collected from the same sampling location. Such observations are made for specimens

QCAZ-A 22176 ( B. sp. Esmeraldas) & QCAZ-A 51867 ( B. sp. Esmeraldas), both of

which are reported to have been collected from the same site, though almost 10 years apart.

Similarly, specimen pairs QCAZ-A 52454 & QCAZ-A 52459 ( B. sp. Tungurahua) and

QCAZ-A 51911 ( B. palmata - Tungurahua) & QCAZ-A 52616 ( B. peruviana Morona), are

also observed to exhibit near complete sequence identity as was reported for other genes.

All identified clades are observed to exhibit high rates of molecular divergence when

considering between-group genetic distances for cytb (Table 5). Values range from a

minimum of 9.2% (Upper Agua Rico vs. Altamazonica, standard deviation [] = 1.7%) to

18.4% (Upper Equatoriana vs. Tungurahua, = 2.9%; Upper Ecuatoriana vs. Upper Agua

Rico, = 2.7%). Values for within group mean distance are also variable, ranging from 0%

(Upper Ecuatoriana, = 0%; Upper Agua Rico, = 0%) to 8.6% (Zamora, 1.4%).

Between and within group un-corrected mean values of evolutionary distance for rag 1 (Table

6) are again observed to be considerably lower in the extent to which genes have diverged

among clades. Further, clades Upper Napo and Zamora are shown to have a genetic distance

of 0% ( 0%). The greatest mean between-group distance is between Pichincha and Upper


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Agua Rico, with a mean value of 2.6% ( 0.6%). Similarly, mean within group genetic

distances are also comparably lower.

Phylogenetic Inference

The results of the best substitution model, site heterogeneity and partitioning scheme for the

data matrix used for inferring phylogenetic relationships of coastal and andean specimen

(Table 7) indicate that codon positions 1 and 2 for RAG1 and POMC should be grouped as a

single data partition, which is best modeled by a Hasegawa, Kishino and Yano (HKY)

substitution model with invariant sites (+I). Codon position 3 for RAG1 and POMC are also

grouped as a single data partition, and are best modeled by a HKY substitution with a gamma

distributed rate variation among sites (+G). Codon position 1 of cytb is partitioned in a data

block with 16S, and is best modeled by a generalised time reversible model with gamma

distributed rates (GTR+G). Codon positions 2 and 3 for cyt b are grouped as separate data

blocks, where position 2 is best modeled by a HKY substitution model and position 3 a

Tamura-Nei model with gamma distributed rates (TrN+G).

The best substitution model, site heterogeneity and partitioning scheme for the matrix

containing sequence data for all Ecuadorian specimens (Table 8) shows that RAG1 has the

same partitioning scheme as with the previous coastal and western andean data set, while

CYTB is again separated into three separated data blocks for each codon position and are

subject to HKY+G, HKY+I, and TrN+G models respectively. For the data set that includes

all Central and South American specimen (Table 9) codon positions 1 and 2 of RAG1 are

partitioned as a single data block subject to a GTR+I+G model, whereas codon position 3 is


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subject to GTR+G. All three codon positions for CYTB are partitioned as separate data

blocks and subject to models GTR+I+G, HKI+I+G, and TrN+G respectively.

The resulting phylogeny from the species tree analysis using the concatenated data matrix

that includes all genes for all coastal and western Andean individuals (Figure 1) shows those

individuals included as out-groups (MVZ 232943, B. biseriata Panama; MVZ-S 2057, B.

sima - Colombia) to form a monophyletic group (pp = 1) with specimens collected from

coastal regions (QCAZ-A 22176, 22346, 27752, 31532, 32135, 40817, and 51867). Further,

these coastal specimens are also observed to form a sub-clade themselves (pp = 1);

henceforth referred to as B. sima Esmeraldas. The remaining taxa in this analysis are also

observed to group monophyletically with each other (pp =1), while each forming their own

sub-clades therein.

Within the Andean clade, specimens QCAZ-A 39984 ( B. sp. Carchi), QCAZ-A 32300 ( B.

sp. Imbabura), and QCAZ-A 45254 ( B. sp . Pichincha) are observed to form an

independent sub-clade (pp = 1) (Figure 1). All of these specimens are reported to have been

collected from various locations across the western ridge of the Andean range. Additionally,

when considering their geographic distribution, each individual is more closely associated

with members of the coastal clade B. sima Esmeraldas than they are to one another.

Additional groupings (pp = 1) are made (Figure 1) between specimens QCAZ-A 52454 ( B.

sp. Tungurahua), QCAZ-A 52459 ( B. sp. Tungurahua), QCAZ-A 51911 ( B. palmata

Tungurahua), and QCAZ-A 52616 ( B. peruviana Morona), in which QCAZ-A 52454 &

52459 and QCAZ-A 51911 & 52616 are observed to pair with each other respectively. These

individuals are reported to have mostly collected from the Tungurahua region in the central


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Andes with the obvious exception of QCAZ-A 52616, whose collection site is in the Morona

province bordering the upper Amazon.

Specimens QCAZ-A 41724 ( B. sp. Zamora), QCAZ-A 42383 ( B. sp. Zamora), QCAZ-A

42467 ( B. peruviana Morona), and QCAZ-A 48195 ( B. sp. Morona) are also observed to

form a monophyletic group (pp = 1). All of these specimens are reported to have been

collected from localities along the eastern ridge of the Andean range. QCAZ-A 51934 ( B.

palmata , Tungurahua) does not appear to belong to any of the other groupings (Figure 1)

from this analysis, despite being identified as B. palmata. In this analysis, it is closely

associated the sub-clade containing the only other recorded sample of B. palmata (QCAZ-A

51911, Tungurahua) used in this study. It is also recognized to have been collected from the

same site as QCAZ-A 51911.

These inferences, as to the groupings of individuals from this analysis described above, can

be observed in relation to their geographic distribution using the map feature included in the

Google Fusion table at the following link:

https://www.google.com/fusiontables/DataSource?docid=10_uig265NTWuqDfe3q48KDajC2

iI6yecF7gtGjk

The colours displayed the map found via the above link are intended to represent the different

sub-clades identified above. The colour scheme loosely matches that used in Figures 1 7,

and groupings can be defined as follows: Green = Coastal; Red = Western Andean; Blue =

Central Andean/Upper Amazonian; Purple = Eastern Andean.

Similarly, a KML tree file viewable in Google Earth is included as a supplementary file.


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Additional analyses (Figures 2 - 7), by which phylogenetic inferences were made using

individual genes and alternate gene pairings (i.e. mitochondrial vs. nuclear DNA trees), show

that there is some incongruence with regards to the inferred relatedness and grouping of

individuals. For example, the resulting topology of the mitochondrial tree (Figure 2) is shown

to be mostly congruent with that as reported by the species tree (Figure 1), other than the fact

that QCAZ-A 51934 ( B. palmata Tungurahua) is now observed to group more closely with

the Eastern Andean sub-clade; whereas it was before shown to more closely resemble the

Central Andean sub-clade. Additionally, rates of molecular change are observed to be greater

than that represented in the previous tree. These patterns are observed to be consistent among

individual gene trees for both mitochondrial genes (Figures 4 5) used in this analysis.

Further, when considering the results of the nuclear DNA tree (Figure 2), most of the major

sub-clades identified in the previous tree appear to be represented. Some incongruence is

observed with regards to the Eastern Andean sub-clade, which was previously observed to

form a monophyletic group. Now it appears as though members of this group have paired

separately with QCAZ-A 51934 ( B. palmata Tungurahua) and the Central Andean sub-

clade. Here, as before, the observed rate of molecular change is observed to differ from the

previous estimate; though this time appears to be much slower. This topology is consistent

with that observed with individual trees (Figures 6 7) for both genes used in this analysis.

The individual gene tree for POMC (Figure 6) is unrooted due to the absence of sequence

data for this gene for those specimens previously used as out-groups.

Estimates of phylogenetic relationships made using sequence data for all Ecuadorian

specimens (Figures 8 - 10) show that none of the new specimens sequenced for the purpose

of this study group with any of the existing sub-clades as described by Elmer et al. (2013).


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One specimen ( B. sp ., QCAZPaul_ECSanFran) that was previously an outlier in the analysis

by Elmer et al. (2013) is shown to group (pp = 0.97) with members of the Eastern Andean

sub-clade (Figure 8). Further, the members of the Eastern Andean sub-clade as identified in

the analyses outlined above are more highly diverged and can be considered to consist of two

separate sub-clades. Namely, these can be described as B. peruviana Morona (QCAZ- A

42467 & QCAZ- A 48195), and B. sp. Zamora (QCAZ-A 41724 & 42383); the latter of

which QCAZPaul_ECSanFran groups with.

These patterns, as reported by the species tree analysis of these taxa (Figure 8), are also well

supported in the single gene tree for cytb (Figure 9), though some incongruence is noticeable

with regards to the observed relatedness between sub-clades. Specifically, the Central

Andean and Eastern Andean sub-clades are here observed to group among the upper

Amazonian clades described by Elmer et al. (2013), whereas the results from the species tree

analysis for these specimens (Figure 10) shows upper Amazonian sub-clades to form a

monophyletic group independent of the coastal and Andean groups. Further incongruences

are observed for the analysis for which only sequence data for rag 1 was used (Figure 10).

Time Calibration

The time of most recent common ancestor (TMRCA) for the group Bolitoglossinae , as

estimated by way of the time calibrated phylogeny (Figure 11) is reported to be around 44

million years before present (mya) (Upper 95% Highest Posterior Density [HPD] = 52.554,

Lower 95% HPD = 3.892). The split between Central and South American species is dated at

approximately 13.5 mya. Additionally, South American clades inhabiting coastal and western

Andean regions of Ecuador are observed to have diverged from all other major Andean and


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Amazonian groups nearly 10 mya. 95% HPD are not given for the latter two dates, as these

groups were not made explicit in the *BEAST input file.

Discussion

Genetic distance

The variability observed among the amount of sequence divergence reported by analysis of

different gene sequences can generally be accounted for by the nature of those genes.

Mitochondrial genes are observed to report consistently greater amounts of sequence

divergence due to the fact that they are not subject to the same selective pressures as nuclear

genes, and as such the sequences that account for these genes are less highly conserved than

those which code for nuclear genes. This is acknowledged to be in part due to their

functionality, or lack thereof. For example, 16s is understood to be mostly non-coding and

does not perform an important function in the cell. This apparent defunct nature of the gene

then allows it to accrue a large number of silent mutations without disrupting influences the

reproductive fitness of that organism. Genes like this are therefore highly informative when

considering the relatedness of organisms and the geographic distribution of genetic diversity

on a local scale (Nicholas et al., 2012).

Some specimens, who are acknowledged to have been collected from sites that are

geographically closely associated, are observed to exhibit greater genetic divergence between

each other than that which they are observed to share with seemingly less closely associated

specimens. In cases such as this it might be presumed that there are some other factors

influencing the diversification of individuals other than simply genetic isolation by distance.


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Altitude has previously been acknowledged to have a profound influence on the isolation and

subsequent divergence of species within this genus (Wiens et al., 2007). Further, the effects

of altitude as a mechanism for genetic isolation are observed to be far greater in tropical

regions than that which has been observed in temperate regions (Kozak & Wiens, 2007). This

is thought to be largely due to the restricted tolerance of variable environmental factors,

which may have evolved in tropical organisms in response to the absence of seasonal

fluctuations in temperature (Janzen, 1967). While the effect of altitude on genetic distance is

not explicitly tested here it may be worth investigating for future studies.

Incongruence among trees

While some incongruence can be observed between these trees it is believed that the topology

as inferred from the mitochondrial DNA tree (figure 2) is most accurate, being that it is

shown to exhibit the greatest overall posterior probability node support scores. The topology

observed in this tree is mostly consistent with the species tree (figure 1) that incorporates

sequence information from all genes sampled for the purpose of this investigation, with the

exception of one individual (QCAZ-A 51934). Additionally, while individuals are observed

to group similarly between these trees there is a considerable difference in the rate of

molecular change and branch lengths as observed in those based on either mitochondrial or

nuclear DNA. This is perhaps understandable, given that nuclear DNA is more highly

conserved due to selective pressure for its correct function as was alluded to previously. With

this consideration, it is accepted that the species tree (Figure 1) be considered the most

reliable representation of the relatedness of species in terms of both the observed topology

and rate of divergence, as it is informed by sequence of all genes.

In spite of this apparent incongruence between mitochondrial and nuclear gene trees, most of


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the identified sub clades appear to be well supported across all trees. This, however, does not

appear to be the case for the sub-clade B. peruviana ; whereby the sister taxa that were

assumed to make up this group based on the species trees and mitochondrial tree are observed

to group more closely with neighbouring sub-clades when considering the relationships of

these taxa as inferred from nuclear genes. Further, these branching patterns are also well

supported, based on their posterior node scores. This observed incongruence may then be

indicative of an extent of divergence between these sister taxa that would be otherwise

overlooked by consideration of the species tree or mitochondrial gene trees alone; suggesting

that the sister taxa represented therein are in fact representatives of two separate sub-clades.

This notion is further supported by the observed patterns of relatedness among all Ecuadorian

specimen as represented in figures 8-10.

Inference of species identity

Being that the majority of specimens included for the purpose of these analyses were not

identified to species level, some inference can be made as to their identity based on the

observed patterns of relatedness represented in the resulting phylogenetic trees. For instance,

of those specimen acknowledged to have been collected from coastal lowland regions of

Ecuador, only one individual (QCAZ-A 22346) is identified to species level ( Bolitoglossa

sima - Esmeraldas). The remaining taxa in this group might then also be assumed to belong to

this species. Mean within group p-distances for this group, however, are reported to be high

(6.6%, = 1.1% for cytb). This might indicate the presence of further genetic diversity within

this group that is not clearly represented in these trees.

Further, while this coastal sub-clade is observed to group closely to the southern Central


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American and South American out-group specimen included in this tree (MVZ 232943, B.

biseriata Panama, and MVZ S 2057 B. sima - Colombia), members of this group are not

observed to group explicitly with the Colombian conspecific specimen. This might represent

some sort of diversification undergone by Ecuadorian representatives of this species and may

represent a subspecies separate to those found in Colombia. However, further consideration

as to the methods by which this inference was made reveals that this specimen was

constrained as monophyletic with the Panamanian out-group specimen (MVZ 232943, B.

biseriata ) and so the resulting patterns of relatedness from these analyses may not be accurate

with regards to the Colombian B. sima (MVZ S 2057) and its Ecuadorian conspecifics. An

amended analysis, for which this taxon was not constrained, was performed; the results of

which are included as an additional file.

None of the specimens that make up the Western Andean clade were described to a species

level, and as such little inference can be made as to their identity. Further, when considered

among the upper Amazonian specimens included for the Ecuadorian analysis, this group was

not observed to closely resemble any of the sub-clades as identified by Elmer et al . (2013).

Of those specimens observed to form the Central Andean clade described in the results, only

two of the specimens included are identified to a species level; neither of which are observed

to be conspecific (QCAZ-A 51911, B. palamata Tungurahua, and QCAZ-A 52616, B.

peruviana Morona) These two individuals are also observed to pair within this group,

despite apparently not belonging to the same species. This observation, along with

consideration of reported genetic distance and sampling locality of these individuals, along

with another individuals (QCAZ-A 51934, B. palmata, Tungurahua), have led to questioning

as to whether these samples had been confused at some point during the process of this study.

Only one of the individuals belonging to the Eastern Andean sub-clade was identified to a


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species level (QCAZ-A 42467, B. peruviana Morona), and as such all other individuals

within this group are assumed to share the same identity. Further consideration based on the

analysis for all Ecuadorian specimens, in addition to the observed incongruence of gene trees

as outline above, might indicate that this preliminary grouping might in fact represent two

geographically separate sub-clades; namely B. sp. Zamora (QCAZ-A 41724 & 42383) and B.

peruviana Morona (QCAZ-A 42467 & 48195). Neither of these groups is observed to group

with any of the major sub-clades as described by Elmer et al. (2013) in the Ecuadorian

analysis. B. sp. Zamora is shown to group with specimen QCAZ-Paul_EC_SanFran ( B. sp );

however, this observation reveals little as to the identity of the members of the group.

Additionally, Elmer et al. (2013) conclude that B. peruviana should be considered endemic to

Peru. Hence, that which has been identified as B. peruviana Morona should perhaps not be

considered to belong to this species either.

Potential miss-identity of specimens

From the relatedness of taxa as indicated by the phylogenetic trees and direct measures of

genetic distance generated for each gene, samples QCAZ-A 51911 and QCAZ-A 52616

appear to be almost identical. This could perhaps be construed as being unusual, since

QCAZ-A 52616 was reportedly collected from a location in the Morona Santiago region,

some distance to the South East of the site from which QCAZ-A 51911 was reportedly

collected. Additionally, sample QCAZ-A 51934, which had previously been identified as the

same species as QCAZ-A 51911 (i.e. Bolitoglossa palmata ) and having been collected from

the same location in the Tungurahua province, is observed to be grouped, albeit

inconsistently, with the Bolitoglossa peruviana clade in the Morona Santiago province to the

South East.


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This anomaly immediately draws question as to whether these two samples (QCAZ-A 51934

& QCAZ-A 52616) had been confused at some point throughout the process of this project.

Similar patterns of relatedness and distance are observed across all genes. Similarly, gene

sequences and identities are noted to be consistent at each stage of the process (i.e. from

chromatogram, to consensus, to alignment, to nexus etc). With this in mind it could then be

reasoned that any confusion of the two samples would have most likely occurred either as a

result of an error during the DNA extraction process or a labeling error on the part of the

Museum; as it is unlikely that the two samples be constantly misidentified throughout the

process of generating and analysing sequence data. In light of this, it is unclear whether the

observed patterns of the distribution of genetic diversity as inferred by the placement of these

two individuals can be trusted.

If this observation were in fact to represent a real biological anomaly, that would then

indicate that QCAZ-A 51934 is not accurately described as belonging to the species B.

palmata , as is indicated by the collection data; and instead would perhaps be more accurately

placed alongside B. peruviana . Further, in the case that the species identification be taken as

accurate, that would then imply that B. palmata has a geographical range that far exceeds the

Andean distribution which is currently accepted (as indicated by the IUCN redlist); one that

extends far into lowland Amazon rainforest while exhibiting little or no genetic

differentiation between these populations. Both of these scenarios are extremely unlikely, and

as such it is reasonable to assume that the observed differences with regards to these isolated

examples are not as a result of a genuine biogeographic phenomenon; nor are they a likely

reason for any kind of taxonomic reform, but instead are most probably explained by some

kind of lab-based or administrative error as described previously.


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event occurred in the region of 10 million years before present (mya). This date is consistent

with proposed dates for major periods of orogeny in the northern extent of the Andean range

(Gregory-Wodzicki, 2000), the likes of which may have influenced the observed pattern of

diversification represented here. Similarly, since this observed divergence is reported to have

occurred prior to or during these periods of orogenesis it may then have facilitated a

migration across montane habitats that would otherwise be difficult for animals that exhibit

low vagility and a limited capacity for dispersal, such as those in question (Garcia-Paris et al.,

2000).

This date is also commonly associated with large-scale marine incursion in the upper

Amazon basin, which is understood to have occurred as a result of eustatic changes in sea

level following periods of deglaciation (Lovejoy et al., 1998). Such an event would have no

doubt created a massive geographical barrier over which these taxa would have been unable

to disperse, and so influencing patterns of species divergence through vicariance and other

similar divergent processes. The subsequent flooding of lowland tropical habitat throughout

the upper Amazon might also have influenced many animals to take refuge in Andean upland

and montane habitats, thus resulting in the observed distribution of diversity as represented in

these analyses (Santos et al., 2009). Any subsequent re-colonisation of these areas, from

either Andean or lower Amazonian populations, would have occurred after the marine

incursion had retreated.


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Time of Colonisation

Time estimates as reported by the chronogram analysis report a colonisation of South

America by ancestral salamander populations that is considerably later than that which was

reported by Elmer et al. (2013). This estimate, however, still predates that which is proposed

for the completion of the contemporary land bridge connecting Central and South America

(i.e. the Isthmus of Panama) by almost 10 million years. This would mean that the earliest

dispersal of Salamanders into South America occurred sometime during the middle Miocene.

The observed later dates as reported by this analysis may be due to the inclusion of more

specimens collected from areas that may have been inhabited much earlier by members of

this genus, therefore improving the resolution of the estimate conducted by Elmer et al.

(2013). The highest posterior density (HPD) values for this analysis described here, however,

are poorly supported; and as such the results from which are likely unreliable.

Further, despite specifying a prior date for the separation between subfamilies

Bolitoglossinae and Plethodintinae of 75 mya ( 6 my) on which the tree was calibrated, the

resulting chronogram estimates this split to have occurred approximately 44 mya. This seems

to draw further question as to the reliability of these estimates. It is acknowledged that root

ages can be manually calibrated in FigTree (Rambaut & Drummond, 2013); however, it is

not clear how robust the resulting time estimates are. The poorly supported values for this

estimate are understood to indicate a lack of convergence of parameters during the Bayesian

sampling process, and would likely benefit from an increased chain-length or sampling

frequency. This would have been difficult to accommodate given the time and computational

constraints of this project. At any rate, it would appear as though this particular analysis

should be repeated if a more reliable time estimate is to be realised.


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If these estimates are considered to be accurate, then the inferred date for the colonisation of

South America by Bolitoglossine salamanders strongly supports the existence of a land

connection between Central and South America that predates the contemporary land bridge.

This finding contributes to a growing number of studies that report evidence to support an

ancient land connection that would facilitate the migration and effective colonisation of

terrestrial organisms between Central and South America. Pinto-Sanchez et al. (2012) report

a similar phenomenon in the frog genus Pristimantis , by which species are inferred to have

undergone multiple colonisation events between Central and South America; many of which

predate the formation of the Isthmus of Panama.

This event has also been hypothesised to have a profound biogeographical effect on the

observed distribution of marine organisms (Bermingham et al., 1997); whereby the closure of

the Isthmus of Panama would have restricted the gene flow between populations inhabiting

the Atlantic and Pacific Oceans. Estimates of the time since divergence of marine organisms

on either side of the Isthmus of Panama generally support a more recent closure of the land

connection (Rocha et al., 2008; Tavera et al., 2012). However, the nature of the earlier land

connection as described previously (Farris et al., 2011; Montes et al., 2012) is such that it

could have facilitated the effective migration of terrestrial individuals via a series of shifting

islands while still allowing dispersal among marine organisms.


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Conclusion

The findings as reported by the methods outlined in this study have shed light on the apparent

distribution of both species and genetic diversity as exhibited by Bolitoglossine salamanders

inhabiting coastal and Andean regions of Ecuador; the nature of which has been previously

underrepresented by any other form of molecular study in these areas. Further, these findings

have informed to some extent the identity of specimens that have not been otherwise

identified to a species level largely due to the cryptic nature. The patterns revealed in the

observed relatedness of these specimens may then be representative of species relationships

beyond that which was considered here, as vast areas of tropical forest and montane habitat

remain unexplored and little molecular data is available for specimen collected those areas

that have been. Of those groups that were identified to inhabit the areas sampled for the

purpose of this study, few were observed to group with existing species groups as described

in previous studies, perhaps representing a level of species diversity that has been otherwise

previously misrepresented. Similarly, those specimens identified as belonging to the species

B. peruviana can be considered to represent at least two independent sub-species of their

own. By the inclusion of specimen from areas that were colonised earlier (i.e. the coast), the

revised time estimates for the colonisation of South America by members of this genus may

represent a better resolution than those that have been previously reported. These new

estimates, however, may be considered erroneous due to the nature of the results as reported

by *BEAST. Regardless, these estimates suggest that the earliest possible date for the

migration of Salamanders into South America is consistently earlier than the accepted timing

for the formation of the contemporary land bridge connecting Central and South America (i.e.

The Isthmus of Panama).


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