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Molecular Phylogenetics and Phylogeography of Neotropical Salamanders (Genus: Bolitoglossa).

Liam Templeton (0704913) The University of Glasgow August 2013



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.


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 imagehosting site Flikr.


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).


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).


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


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


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


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 (~5–2.5 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


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.



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.



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 manufacturer’s 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).


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 H2O dNTPs Buffer MgCl2 Forward Primer Reverse Primer Taq Polymerase Template DNA

Concentration n/a 10mM 10X 25mM 10uM 10uM 10U/ul n/a

Volume per reaction 26.8ul 4ul 4ul 2ul 0.4ul 0.4ul 0.4ul 2ul

Concentration per reaction n/a 1mM 1X 2.5mM 0.1uM 0.1uM 0.1U/ul 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 manufacturer’s 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.


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


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,


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.


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


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 Earth’s 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


two-dimensional rendering of the Earth’s 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.


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 BHe1z8y b3E


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 QCAZA 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


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


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


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


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


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: 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.


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 subclade. 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).


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


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.


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.


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


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


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


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.


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.


An amended map of the distribution of sub-clades and their respective individuals can be found at the following link. Similarly, an amended KML tree can also be found as an additional file along with this manuscript: koW1vaLQUDnHORQ


Based on the phylogenetic analyses outlined here, it is possible to identify at least four clearly defined and well-supported groups represent within Coastal and Andean populations of Bolittoglossine salamanders. Further consideration of the patterns observed in these trees reveals that the identified groups are mostly geographically isolated. Those who are acknowledged to have been collected from coastal regions of Ecuador (Esmeraldas) are shown to form a sub-clade of specimen, provisionally referred to as Bolitoglossa sima Esmeraldas, Also included in this group are two specimen reported to have been collected from a neighbouring Andean province (Imbabura). The fact that this sub-clade also groups monophyletically with specimen included for the purpose of out-grouping is perhaps indicative of a migration of diversity from a Northern ancestral range along the western coast of South America, as opposed to via the upper Amazon basin.

Additionally, the monophyletic clade containing both the coastal Ecuadorian specimen and out-groups is observed to be separate from all Andean and Amazonian lowland sub-clades. This may then represent a traverse migration of ancestral populations across the Andean range, as opposed to around. Time calibrated estimates for this separation indicate that this


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.


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.


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.



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).



This project was partially funded by the Blodwen Lloyd Binns Bequest Fund as awarded by the Glasgow Natural History Society. An additional award was received from the British Herpetological Society as part of their Student Grant Scheme. I would hereby like to express my gratitude toward both of these organisations and their generous financial support. I would also like to give thanks to the following people: Dr. Barbara Mable for her role as course coordinator and in continually assessing my written work to the highest standards; Dr. Kathryn Elmer for acting as project supervisor throughout this process and offering a wealth of insight and encouragement; Ms. Aileen Adam for her instruction and supervision of correct laboratory protocol and etiquette; and finally fellow masters student Ms. Emily Waddell for her comradeship throughout this lengthy process. This thesis is dedicated to my supportive parents, Samuel and Avril Templeton, and the memory of my aunt Kate McKinnon, who sadly passed away at the beginning of last semester.


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