Phylogenetic systematics and historical biogeography of the harlequin frogs (Atelopus ) of Central America based on mitochondrial genes
Juan P. Ramírez1*, Andrew J. Crawford1,2
, César Jaramillo2 , and Roberto Ibáñez2
1 Museo de Historia Natural ANDES and Laboratorio Biom|ics, Departamento de Ciencias Biológicas, Facultad de Ciencias, Universidad de los Andes, Apartado Aéreo 4976, Bogotá, Colombia
2 Smithsonian Tropical Research Institute, Apartado 0843-03092, Panamá, Republic of Panama and Círculo Herpetológico de Panamá, Apartado 0824-00122, Panamá, Republic of Panama.
* Corresponding author. E-mail: [email protected]
Abstract
Populations of amphibians around the world are experiencing severe declines. One of the groups that has experienced the most catastrophic declines are the harlequin frogs of the genus Atelopus
(Anura: Bufonidae). Many species have not been seen for decades, especially in the highlands. The eight Central American species of Atelopus are not an exception to this pattern, as only the five low-elevation species are known to still have surviving populations in the wild, as well as in captivity. However, given that the success of all ex-situ conservation programs depends on having an adequate knowledge of the taxonomy and relationships among species, it is worrying that those aspects of Central American Atelopus are still poorly known. Additionally, insight into the phylogenetic relationships among Central American and South American species of Atelopus could shed light on the recent controversies about the timing of the Great American Biotic Interchange (GABI). In this work, the phylogenetic relationships of seven of the eight known species of Central American Atelopus were inferred based on DNA sequence data of 99 individuals for two mitochondrial genes ( cytb and the 3´ end of COI), for a total of 1353 base pairs. The resulting ancestral area reconstructions suggest that, according to the available data, both a single or a two-time event of colonization of Central America by South American Atelopus are equally probable. Relaxed-clock
analyses of divergence times indicated that Atelopus reached Central America at a minimum age of 4.18 million years ago, in accordance with the newly proposed hypothesis of an older timing of the GABI. These results conform to the recent trend among molecular-based biogeographic studies of not supporting the traditional hypothesis of a recent GABI. In addition, all species of Central American Atelopus were recovered as reciprocally monophyletic, including two previously unrecognized candidate species that need to be incorporated into conservation programs. Future genetic analyses should be undertaken in captive populations of Central American Atelopus in order to look for the existence of other
currently unrecognized species, as well as of unwanted hybridization.
Key words: Historical Biogeography, Phylogenetics, Harlequin frogs, Great American Biotic Interchange, Central America, Isthmus of Panamá.
Introduction
Amphibian species are in the midst of a global conservation crisis. It is estimated that about 43% of amphibians are suffering population declines (Stuart et al., 2004). These declines are probably the result of interactions among several factors of primarily anthropic origin,
including overexploitation, natural catastrophes, habitat destruction, pollution, and the effects of invasive or pathogenic species (Hayes et al., 2010). The most alarming cause of global declines and extinctions of amphibian species is chytridiomycosis, the infection by the chytrid fungus Batrachochytrium dendrobatidis
(Bd). Chytridiomycosis has been identified
as the most important cause of the declines and extinctions suffered by a large proportion of frog species (Skerratt et al., 2007).
Probably the group of amphibians that has suffered the most dramatic population declines and extinctions is the harlequin frogs of the genus Atelopus
(Anura: Bufonidae). The likely
cause of these declines is infection with Bd, as members of Atelopus are very susceptible to chytridiomycosis (DiRenzo et al., 2014). Because of their bright coloration and high local abundance, species of this genus were a prominent element of many Neotropical
communities until about 35 years ago (Guayasamín et al., 2010), but since then most of the species have not been found again in their historic localities (Lötters et al., 2011). Declines of
Atelopus
have been more severe for the species that inhabit higher elevations (above 1000
meters above sea level; La Marca et al., 2005) probably because the lower temperatures present at higher altitudes are associated with higher levels of infectivity and fecundity of Bd (Woodhams et al., 2008). The eight species of Central American Atelopus are not an
exception to this pattern, as the three species restricted to elevations above 1000 m (A. chiriquiensis, A.chirripoensis and A. senex
; Köhler, 2011) are probably extinct (IUCN,
2016), whereas some populations of the remaining five species still remain in the wild (IUCN, 2016).
The success of ex-situ conservation programs depends on having a complete and robust taxonomy, as correct identification of species: 1) allows a more efficient use of resources by avoiding the unnecessary protection of invalid species (Allendorf, Luikart & Aitken, 2012); 2) can prevent neglecting previously unrecognized species in conservation programs
(Daugherty et al., 1990), and 3) helps to avoid the generation of hybrids possibly maladapted to the environments inhabited by their parental species (Frankham et al., 2004). Five species of Central American Atelopus
with remaining wild populations are being reared in captivity
by The Panama Amphibian Rescue and Conservation Project (Cikanek et al., 2014). However, the taxonomy of all Central American Atelopus
taxonomy of most of these species has been evaluated based on characteristics of adult external morphology, of which coloration pattern has been given a special importance, even though it is not always concordant with genetic differentiation in Atelopus
(Noonan &
Gaucher, 2005; Richard & Knowles, 2007). Additionally, the phylogenetic relationships of the species of Atelopus
in Central America are also understudied, as only three species have
been included in published molecular phylogenies of the genus (Lötters et al., 2011; Richard & Knowles, 2007).
Given that Central American Atelopus
seem to be of a South American origin (Lötters et al.,
2011), understanding their phylogenetic relationships and historical biogeography could shed light into the timing of the Great American Biotic Interchange (GABI). The GABI is a phenomenon in which many organisms dispersed between North America to South America and vice versa following the closure of the Isthmus of Panamá (Stehli & Webb, 1985). The timing of this geological event is a controversial topic, as the traditional datation of
approximately 3 million of years ago (MYA) (Keigwin, 1978) has been challenged recently by Bacon et al. (2015) and by Montes et al. (2015), who propose a much older closing of the Isthmus (of 10-6 MYA and 15-13 MYA, respectively). However, recently both of these new proposals have been criticized by O'Dea et al. (2016), as the datasets used on those studies were apparently interpreted erroneously. Even though, there are several molecular studies (reviewed by Cody et al., 2010) that support the older closing of the Isthmus, although most of them lack data of species inhabiting the part of South America closest to Panamá (Pinto et al., 2012). In this work, the phylogenetic relationships of the species of Atelopus
of Central
America are evaluated on the basis of mitochondrial gene sequences in order to determine their taxonomic status, as well as the number and timing of their invasions between Central and South America.
Materials and methods Sampling
The molecular data used in this study consists of DNA sequences of 118 specimens of
Atelopus
, all of them collected or sampled for this publication. The sequences based on
non-collected specimens were obtained from toe-clipsor from skin swabs preserved on 95% ethanol, respectively, whereas those based on collected specimens are based on liver (or leg?) tissue deposited on 95% ethanol. Of these samples, 18 were used as outgroups, as they correspond to species of Atelopus
restricted to South America. More specifically they
correspond to Atelopus
cf. laetissimus, A. spumarius, A, flavescens, A. elegans, A. ignescens,
A. peruensis, A. pulcher, A. epikheistos, A. bomolochos
and two undescribed species from
Cantón Limón and Pachancho, Ecuador. Furthermore, published GenBank sequences of six species of other genera of Bufonidae were also used as outgroups: Bufo gargarizans
minshanicus, Bufo japonicus, Bufo stejnegeri, Duttaphrynus melanostictus, Anaxyrus boreas
and Pseudepidalea viridis
Voucher availability (including museum numbers when applicable), locality data, and GenBank accession numbers for all sequences used in this work are shown on Appendix 1.
Laboratory protocols
Genomic DNA was isolated by using a QIAGEN DNeasy kit. A fragment of 714 base pair (bp) of cytochrome b (cytb
), was amplified with the primers CB1
(5'-CCATCCAACATCTCAGCATGATGAAA-3') and CB3
(5'-GGCGAATAGGAAGTATCATTC-3'). Additionaly, a 639 bp fragment of the 3’ end of the Cytochrome Oxidase I (COI) gene was amplified with the primersCOIf
(5'-CCTGCAGGAGGAGGAGAYCC-3') and COIa
(5'-AGTATAAGCGTCTGGGTAGTC-3'; Palumbi et al. 1991). In order to amplify those fragments of the mitochondrial genome, the PCR protocol of Richard & Knowles (2007) was followed. The obtained PCR products were cleaned by using a QIAquick kit and posteriorly sequenced by means of a X automated sequencer. The obtained DNA
chromatograms were inspected and cleaned by using the software Sequencher 5.0 (Gene Codes Corporation).
The obtained sequences were aligned by eye, as practically no length variations were observed, and checked for the presence of premature codons in the software Mesquite, version 3.10 for Windows (Maddison & Maddison, 2016)
Phylogenetic analyses
Phylogenetic analyses were performed using both Maximum Likelihood (ML) and Maximum Parsimony (MP) criteria. The software PAUP∗, version 4.0a149 for Windows (Swofford, 2000), was used for Heuristic tree searching for MP inference, based on 5000 replicate searches (each from random starting trees) with both the MaxTrees command and the rearrangement limit on each tree set to 100 000. These search conditions were repeated five additional times in order to evaluate the completeness of the first search, retaining the most parsimonious trees obtained per run. In total, 57 equally parsimonious trees were obtained as a result, which only differ slightly among them (only on the branch lengths and/or
phylogenetic positions of few sequences of Atelopus varius
and A. zeteki). One of these
equally parsimonious trees was chosen randomly and it is shown on Figure 1.
The concatenated data matrix was partitioned a priori
both by gene and by codon. The
best-fitting model of all possible combinations of the partitions was identified by means of the software PartitionFinder (Lanfear et al., 2012), under the Bayesian Information Criteria. The results indicated that the best model correspond to a Transversion Model (TVM) with gamma rate heterogeneity and “Invariant” parameters (Yang, 1993). Posteriorly, a total of 100 independent tree searches (distributed in twelve runs of eight independent searches plus one run with only four searches) were run with GARLI version 2.01 (Zwickl, 2006) at the
online platform Cipres (Miller et al., 2010), maintaining the search parameter values as default. The tree with the highest likelihood value is shown in the Supplementary Figure 1, and it has a nearly identical topology in comparison to trees with slightly smaller Likelihood values, indicating that the thoroughness of the search was adequate.
For ML and MP analyses we assessed branch support via 1000 non-parametric bootstrap replicates (Felsenstein 1985). MP bootstrapping was done in PAUP∗, with 30 tree searches performed on each resampled character matrix, and with other search parameters as above. For ML bootstrapping in GARLI, ten sets of 100 replicates of the sequence matrix were run in the online platform Cipres (Miller et al., 2010), again maintaining the parameter values as default. For both MP and ML bootstrap analyses, the obtained bootstrap trees were
summarized as a 50 percent consensus tree (Figure 1, Supplementary Figure 1).
Bayesian phylogenetic analysis was performed with BEAST version 1.8.0 (Drummond et al., 2012), as implemented in the CIPRES portal, and assuming the previously mentioned
partition scheme. Both the posterior distribution of trees and divergence rates were estimated by assuming a relaxed normal clock model of evolution and a calibrated Yule model tree prior (e.g.: a constant speciation rate per lineage, Heled and Drummond, 2012). Values of 0.00957 and 0.0010 for the mean and standard deviation of the normal distribution prior of substitution rates were used. The mean value of that distribution was the
mitochondrial substitution rate of Macey et al. (1998), obtained from several sequences of Eurasian Bufo
, as corrected by Crawford (2003). Additionally, a normal distribution with
mean 0.0010 and standard deviation 0.0001 was used as a prior for the standard deviation of the substitution rate distribution. The values of all other priors were not changed from their default values. 150 million of MCMC generations were run saving one sample tree per 1000 generations, and with the first 15000000 states discarded as burn-in. Stationarity and mixing of the log-likelihood values and parameters estimates of the runs were evaluated by means of the software Tracer version 1.6 (Rambaut and Drummond, 2004). Using Tracer it was also evaluated and confirmed that all evaluated parameters of the post-burnin set of trees had effective sample sizes larger than 200. The obtained set of trees were summarized to the phylogeny shown in the Figure 2 with the software TreeAnnotator 1.5.4. The Bayesian Analysis tree which includes all outgroups is presented in the Supplementary figure 2.
Additionally, a species tree analysis was conducted with the software *Beast (Heled & Drummond, 2010) under the same conditions and parameter values as the MCMC gene tree. For the species tree analysis, the species were defined as the reciprocally monophyletic clades found in the MCMC topology having an uncorrected genetic distance (p-distance) on both the COI and cytb
genes larger than 1.4% to their sister clade. The p-distance between the
sequences of each gene for all samples was calculated with the software Mega7 (Kumar et al., 2016).
For determining the number and direction of invasions between Central and South America, ancestral area reconstructions were conducted with the species tree previously mentioned, by using the Statistical Dispersal and Vicariance Analysis (S-DIVA; Yu, Harris & He, 2015), the Dispersal and Local Extinction and Cladogenesis (DEC; Ree et al., 2008) and the Bayesian inference for discrete areas (BayArea; Landis et al., 2013) biogeographical models, as implemented in the software RASP 3.0 (Reconstruct Ancestral State in Phylogenies, Yu et al., 2015), always under default settings. Species of Atelopus
were coded as being from
Central or South America, each area defined for the purposes of this work as being equivalent to their corresponding geological plates. On the other hand, as none of the included species of other bufonid genera are from the American continent, they were coded as being
“non-American”.
Results
Phylogenetic analyses
The complete constructed alignment consisted of a total of 1353 base pairs from 123
sequences. All the included terminals had sequences of the COI fragment mentioned before, whereas only 5 of the terminals (4% of the total) lack sequences of the cytb
gene. Premature
stop codons were not observed on any of the sequences included in this study. The number and overall proportion of parsimony-informative sites, singletons and invariant sites are summarized on Table 1.
The topologies obtained by the three inference methods (MP, ML and BA) were mostly congruent among them with respect of the ingroup species, only differing in the phylogenetic position of A. senex
and A. chiriquiensis. On the other hand, although the outgroups always
clustered in three well supported clades, as will be shown below, the phylogenetic relationships among them are distinct among the different inference methods. Bootstrap values of the great majority of nodes shared by the MP and ML phylogenies were consistent with their posterior probability values on the Bayesian inference consensus tree. Additionally, the majority of nodes of the three topologies were highly supported (e.g.: presented bootstrap values larger than 75 or posterior probabilities larger than 0.95), with the exception of most internal nodes of A. zeteki
and A. varius.
In all topologies, the genus Atelopus
was recovered as monophyletic. The South American
species of the genus, with the exception of Atelopus elegans
conform three different clades,
all with significant support. These clades are conformed by: a) the species of the Amazonian slopes of the Andes of Perú and Ecuador, and from the Amazonian and Guianan lowlands (Atelopus
sp. “Cantón Limón”, A. pulcher, A. flavescens and A. spumarius), known as the
“Amazonian-Guianan clade” by Lötters et al., (2011), b) species from the high Andes of Ecuador and Peru (Atelopus
and A. peruensis
) , and: c) A. cf. laetissimus from the Sierra Nevada de Santa Marta of
Northwestern Colombia. In both the MP and BI topologies, clade a is the one that diverged in the first place, followed by clade c and by clade b, respectively; whereas in the ML topology the first diverging clade is clade c, then clade a, and finally clade b.
The other species of Atelopus
in this study conform a monophyletic group containing eight
large clades, herein considered as different species. Each one of these clades is assignable to a previously described name, as we included samples from the type localities of all Central American described species of the genus, with the exception of A. chirripoensis
and the
group conformed by the samples from Puerto Obaldía, Panamá, and Capurganá, Colombia. The last mentioned clade is the sister group of all other Central American species. The first group to diverge in the clade of Central American-endemic Atelopus
is conformed by the
species from Southern Panama, A. certus
and A. glyphus, which were recovered as sister
species in all analysis. The next diverging group is either A. senex
(BI), A. senex+A.
chiriquiensis
(MP) or a polytomy formed by A. senex, A. chiriquiensis and a clade conformed
by A. limosus
, A. varius and A. zeteki (ML), although neither of these possibilities has a
significative support. Finally, in the topologies obtained by the three inference methods, A. limosus
is the sister group of a clade conformed by samples identified as A. varius and A.
zeteki
. This clade consists of two sister groups, one found from the extreme southern Costa
Rica to Central Panamá, and the other found only at the east of El Valle del Antón, Central Panamá. Specimens of both of these groups were found sympatrically in Juan Lana, Panamá.
It was found that the genetic distances between the eight clades of Central American Atelopus
were generally much larger than within them (Table 2), with the exception of the clades assigned to the names of A. varius
and A. senex. The last case is especially noteworthy, as for
both genes the sample of the individual with institutional code of MVZ 164816 (from Monteverde, Costa Rica) has genetic distances comparable, and even larger, to those found among the eight clades previously mentioned (Table 2).
Divergence times and ancestral reconstruction
The results of the ancestral area reconstruction done under each of the three biogeographic models were mostly concordant and well-supported (most node reconstructions with probabilities above 95%, Table 3, Supplementary Table 1), and were mapped into the
corresponding nodes of the concatenated Bayesian MCMC phylogeny (Figure 2). According to those results, and to the the calibrated Bayesian tree obtained in this work, it was found that the Most Recent Common Ancestor (MRCA) of the genus Atelopus
existed in the
Oligocene of South America, 28.4851 million of years ago (MYA) (95% Posterior Credibility Interval, CI: 21.3641-36.2952 MYA). Additionally, it was found that the occurrence of either one or two independent events of invasion of species of Atelopus
to Central America from
South America are equally probable. Under the scenario of two Central American invasions, one of them was done by the common ancestor of A.
elegans
from Gorgona Island at 4.7047 MYA (CI: 3.3718-6.2004 MYA), while the other one
was carried out by the ancestor of all other sampled Central American species at 5.5648 MYA (CI: 7.0731-4.1813 MYA), by. On the other hand, a single colonization event could have occurred at 7.167 MYA (CI: 5.4134-9.0478 MYA), with a lineage returning to the Pacific lowlands of South America at an unknown later date. The content of this lineage is limited at the present time to the population of A. elegans
from Gorgona Island only, as there
are not further samples available of other possibly related populations of Chocoan Atelopus
(such as A. spurrelli
, A.longibrachius, A. balios, and the mainland populations of A. elegans).
Discussion
Phylogenetic systematics and biogeography
The phylogenetic relationships within the genus are mostly in accordance to those found by the study of Lotters et al. (2011), also based on mitochondrial DNA sequences, as both studies support the monophyly of Atelopus
and its South American origin. Additionally, both
studies recover the existence of a clade of Amazonian-Guianan species, which is sister to another monophyletic grouping which includes the Andean, Chocoan and Central American species of the genus. However, in contrast with Lotters et al. (2011), in this work we included sequences of at least one species (A.
cf. laetissimus) from the Sierra Nevada de Santa Marta
(SNSM), an isolated mountain range in Northwestern Colombia notorious by its large endemicity. The phylogenetic position of A.
cf. laetissimus is noteworthy, as it is not included
in any of the two previously mentioned clades, but it diverged very early in the history of the genus, being either the first diverging species of Atelopus
, or the sister group of all the clade
formed by the Andean, Chocoan and Central American species. Is interesting to note that a similar pattern is also found in the case of the frogs of the clade Allocentroleniae, in which the earliest diverging group is from the Guianas and Amazonian regions, after which it diverges a species from the SNSM, and then a group which comprises all other known species (Castroviejo-Fisher et al., 2014). However, it is out of the scope of this work to discuss more thoroughly the implications of this common biogeographic pattern.
Regarding the timing and mode of invasion of Atelopus
to Central America, further sampling
of other Chocoan Atelopus
is needed in order to evaluate if there occurred one or two
invasion events, as mentioned before. Additionally, it exists the possibility of the occurrence of even another invasion event of Atelopus
to Central America, as it has been proposed that
the enigmatic A. chirripoensis
is more closely related to the A. ignescens complex than to the
other Central American species of Atelopus
(Savage & Bolaños, 2009). However, this is a
hypothesis very difficult to test, given that A. chirripoensis
has not been found again since
1980, and it is now probably extinct (Savage & Bolaños, 2009). In addition, the great external similarity between this species and those of the A. ignescens
complex can be due not only to
relatedness, but also by convergence, a more parsimonious explanation given that they are both found in very similar páramo
In any case, finding that members of Atelopus
reached Central America as early as 4.2 MYA,
conform to the recent molecular biogeographic studies of not supporting the traditional recent closure of the Panamanian Isthmus and posterior Great American Biotic American
Interchange (e.g: Winston et al., 2016, Pinto et al., 2008). However, it can be argued that these results can also be caused by dispersal prior to the Isthmus formation, a doubtful possibility given that they are based on organisms probably incapable of dispersing over marine barriers (such as army ants or frogs), due to their sensibility to salty water. On the other hand, given that savanna-like environments occurred in Central America during 4-3 MYA (Bacon et al., 2016), it is unlikely that forest animals, such as army ants or frogs of the genus Pristimantis
and Atelopus, could have crossed the Isthmus at the time. Further studies
need to be taken in other groups of terrestrial organisms (such as insects, fossorial reptiles or undergrowth birds) with low-dispersal capabilities in order to confirm the current tendency of not finding evidence of a young GABI. In addition, those studies should also be undertaken on both forest and savanna organisms in order to look for possible additional inconsistencies between their habitat requirements and the prevailing environment found during the time of their invasion to Central America.
Taxonomic and conservation implications
The results obtained in this work are mostly in accordance with the current taxonomic arrangement of the species of Atelopus
. However, there are two Central American clades
whose taxonomic status need further attention. Firstly, the specimens from Puerto Obaldia, Panama and Capurganá, Colombia, could correspond to an undescribed species or to a previously unreported population of the Colombian species A. spurrelli
. However, as
sequences of this last species could not been included in this work, it is currently not possible to distinguish between the two hypotheses previously mentioned. On the other hand, a
specimen from the Monteverde Cloud Forest in Costa Rica (MVZ 164816) is probably a member of an undescribed (and probably extinct) species, given its large genetic distance with respect from all other specimens, including a sympatric individual of its sister species (A. senex
). Further studies based on different lines of evidence (e.g: adult and larvae
morphology, nuclear sequences, differences in calls, among others) need to be done for having a better understanding of the taxonomic status of these candidate species, as well as of the poorly known A. spurrelli.
In addition, we herein also confirmed the results of Richards and Knowles (2007) regarding the distinctiveness of Atelopus zeteki
from A. varius, and we report for the first time the
existence of a locality (Juan Lana, Panama) in which both species are found sympatrically. Finding that
these two species are sympatric in at least one locality opens up the possibility
that they can be hybridizing, a hypothesis that deserves further exploration given their morphological similarity and very recent divergence (of 2.0276 MYA, CI: 1.462-2.678 MYA).
Finding support for the traditional taxonomy of Central American Atelopus
brings hope to the
success of the ex-situ
conservation programs being undertaken with the described species, as
it indicates that resources are being used efficiently in frog conservation, given that all surviving species are being neglected by these programs. Conversely, since each named species appears to be a distinct genetic entity, species ranges are not being underestimated, and no species is being protected unnecessarily. However, this is not the case for the two candidate species been found in this work, as they are not currently part of any ex-situ
or
in-situ
conservation program. Furthermore, finding that the original identification of most
specimens (excepting some specimens of A. varius
and A. zeteki, Appendix 1) is concordant
with the results of the phylogenetic analyses, suggests that cryptic diversity is low among Central American Atelopus
, and that unwanted hybridization resulting from
misidentifications of specimens in captive programs should be rare. At least in the case of A. limosus
this has been shown to be the case (Crawford et al. 2013). Further research should be
done on the additional species currently housed in ex-situ
programs, in order to prevent the
occurrence of genetic erosion by hybridization in captive populations. In addition, sequence data could also be used to determine the identity of captive specimens. Finally, recognizing the taxonomic distinctiveness of the candidate species found herein is urgently needed in order to initiate the corresponding conservation measures to guarantee their long-term survival.
Acknowledgments
Thanks are due to the members of the Biom|ics Lab, specially to S. Herrera, L. Castellanos and P. Montoya, for their comments, corrections and advice about this work. Additionally, JPR also wants to thank V. Romero-Alarcón for the constructive discussions about several theoretical aspects of phylogenetic analyses, and to M. Ramírez-Pinilla for economical support.
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Figures and Tables
Table 1: Number and overall proportion of parsimony-informative sites, singletons and invariant sites for each gene region.
Gene Length of sequence (bp) Invariant sites Singletons Parsimony informativeI sites
Cytb 714 391 25 298
COI 639 397 8 234
Table 2: Uncorrected p-distances for the mitochondrial fragments of COI and cytb (separated by a semicolon) of the species of Atelopus
from Central America. Both
interspecific and intraspecific genetic distances are shown, intraspecific distances being found in the diagonal of the matrix and shaded with gray.
Taxon 1 2 3 4 5 6 7 8 9
1 A. sp. "Capurganá-Puerto Obaldía" 0-0.74; 0-0.86
2 A. certus
4.38-5.17; 7.42-7.98
0-0.63; 0-0.28
3 A. glyphus
4.69-5.01; 7.00-7.42
1.41-2.03;
2.70-3.08 0; 0-0.42
4 A. senex
5.79-6.73; 8.54-9.66 6.73-7.67; 7.70-8.82 6.57-6.88; 7.42-8.40 0.31;0.14;0.01-0.14 5 A. sp.
MVZ
164816 6.42-6.73; 9.94-10.36 4.54-6.89; 9.10 6.10;9.10-9.24 4.69-5.16; 6.58-7.56 0 6 A. chiriquiensis 5.48-5.95; 7.98-8.26 6.42-6.89; 7.25-7.28 6.26-6.41; 7.14-7.28 4.69-5.32;3.92-4.76 4.54-4.69; 5.04 0-0,16; 0-0.28
7 A. limosus
5.63-6.42; 8.54-9.80 5.79-7.04; 7.42-8.40 5.63-6.26; 7.70-8.40 4.54-5.63;5.60-6.86 5.63-5.95; 6.02-6.58 4.85-5.16; 3.36-4.20 0.31-1.25; 0.14-1.12
8 A. varius
3.13-6.73; 8,82-10,64 4.69-7.67; 6.86-7.70 5.00-6.57; 7.42-8,26 4.69-6.73; 4.90-7.84 5.32-6.73; 6.30-7.14 4.07-5.95; 4.06-5.32 3.76-6.42; 3.64-5.04 0-6.73; 0-2.38
9 A. zeteki
6.10-6.73; 9,24-9,80 6.26-7.20; 7.56-7.84 5.79-6.10; 7.84-8.12 5.48-6.10;6.44-6.86 5.63-6.26; 6.58-6.86 4.54-5.01; 4.90-5.46 3.60-4.69; 4.20-5.60 2.66-6.73; 2.80-4.48 0-0.78; 0-0.28
Table 3: Estimated crown ages (in millions of years from the present) and results of the areas reconstructions under three biogeographic models (S-DIVA, DEC, BayArea) for the species of
Atelopus
of Central America, and of selected nodes of other clades of the genus. Ages are
estimated from the results of the concatenated MCMC Bayesian run undertaken on the software Beast. CA=Central America; SA=South America, NA=Non-American.
Node Mean Date (MYA)
95% Posterior Credibility
Interval S-DIVA Reconstruction BayArea Reconstruction DEC Reconstruction
A 2,03 1,46-2,68 CA (100%) CA (100%) CA (100%)
B 2,69 1,96-3,46 CA (100%)
CA (99,94%), CA/SA
(0,06%) CA (100%)
C 3,54 2,68-4,48 CA (100%)
CA (99,48%), CA/SA
(0,52%) CA (100%)
D 3,05 2,19-3,98 CA (100%) CA (100%) CA (100%)
E 3,71 2,78-4,78 CA (100%) CA (100%) CA (100%)
F 5,56 3,18-7,07 CA (100%)
CA (85,77%), CA/SA
(14,23%) CA (100%)
G 1,73 1,11-2,40 CA (100%)
CA (99,77%), CA/SA
(0,23%) CA (100%)
H 7,17 5,41-9,05 SA/CA (100%)
CA (99,94%), CA/SA
(0,06%) CA/SA (100%)
I 4,71 3,37-6,20 SA (100%)
CA/SA (99,98%);
CA/SA/NA (0,02%) CA/SA (100%)
J 12,23 9,93-15,47 SA (100%)
CA/SA (98,77%), CA/SA/NA (1,23%)
A (77,71%), CA/SA (22,29%)
N 16,46 12,28-20,99 SA (100 %)
CA/SA (95,23%),
CA/SA/NA (4,77%) SA (100%)
R 28,49 21,36-36,30 SA (100 %)
SA (99,38%), SA/CA (0,42%), SA/NA
(0,21%) SA (100%)
Supplementary Table 1: Estimated crown ages (in millions of years from the present) and results of the areas reconstructions under three biogeographic models (S-DIVA, DEC,
BayArea) for all nodes of the concatenated MCMC Bayesian run done on the software Beast. CA=Central America; SA=South America, NA=Non-American.
Node
Mean Date (MYA)
95% Posterior Credibility
Interval S-DIVA Reconstruction BayArea Reconstruction DEC Reconstruction
A 2,03 1,46-2,68 CA (100%) CA (100%) CA (100%)
B 2,69 1,96-3,46 CA (100%)
CA (99,94%), CA/SA
(0,06%) CA (100%)
C 3,54 2,68-4,48 CA (100%)
CA (99,48%), CA/SA
(0,52%) CA (100%)
D 3,05 2,19-3,98 CA (100%) CA (100%) CA (100%)
E 3,71 2,78-4,78 CA (100%) CA (100%) CA (100%)
F 5,56 3,18-7,07 CA (100%)
CA (85,77%), CA/SA
(14,23%) CA (100%)
G 1,73 1,11-2,40 CA (100%)
CA (99,77%), CA/SA
H 7,17 5,41-9,05 SA/CA (100%)
CA (99,94%), CA/SA
(0,06%) CA/SA (100%)
I 4,71 3,37-6,20 SA (100%)
CA/SA (99,98%);
CA/SA/NA (0,02%) CA/SA (100%)
J 12,23 9,93-15,47 SA (100%)
CA/SA (98,77%), CA/SA/NA (1,23%)
A (77,71%), CA/SA (22,29%)
K 1,067 0,65-1,52 SA (100 %) SA (100 %) SA (100 %)
L 3,91 2,81-5,10 SA (100 %)
SA (98.98%), CA/SA
(1.02%) SA (100 %)
M 4,79 3,53-6,21 SA (100 %)
SA (96.42%), CA/SA (3.52%), SA/NA
(0.06%) SA (100 %)
N 16,46 12,28-20,98 SA (100 %)
CA/SA (95,23%),
CA/SA/NA (4,77%) SA (100%)
O 4,87 3,49-6,32 - -
-P 5,51 4,10-7,09 SA (100 %)
SA (99.38%), CA/SA (0.42%), SA/NA
(0.21%) SA (100 %)
Q 1,76 1,14-2,43 SA (100 %) SA (100 %) SA (100 %)
R 28,49 21,36-36,30 SA (100 %)
SA (99,38%), SA/CA (0,42%), SA/NA
(0,21%) SA (100%)
S 36,86 27,95-46,51 SA/NA (100%) SA/CA/NA (100%) SA/NA (100%)
T 20,37 15,48-25,86 NA (100%) NA (100%) NA (100%)
U 17,89 13,37-22,38 NA (100%) NA (100%) NA (100%)
V 15,38 11,56-20,45 NA (100%) NA (100%) NA (100%)
W 8,13 5,99-10,41 NA (100%) NA (100%) NA (100%)
Figure 1: One of the 57 equally most parsimonious trees inferred from the dataset of
sequences of two mitochondrial genes (COI and cytb) from the species of Atelopus of Central and South America included in this study. Branch supports are indicated as non-parametric values under Maximum Parsimony and Maximum Likelihood above or below each branches, respectively. Samples names correspond to their museum voucher numbers, or if not
available, to the Genbank accession numbers. Codes after species names indicate their continent of origin: Central America (CA) or South America (SA).
Fortuna, PAN
Cana, Pirré, PAN
Río Farallón, PAN Cerro Tute, PAN
CH 5414, PAN Río Farallón, PAN
Jungurudó, PAN
CH 5417, PAN
Cerro Punta, PAN
Nusagandí, PAN
CH 5613, PAN
CH 5635, PAN
Pequeni, PAN Río María Oeste, PAN
Jordanal, PAN
Santa Rita, PAN Nusagandí, PAN
Fortuna, PAN CH 5636, PAN
Campana, PAN Juan Lana, PAN
Juan Lana, PAN Jordanal, PAN
A. sp. “Pachancho” (KU 217429), PER
CH 5363, PAN
A. spumarius (KZ02), GUY
MVZ 223270, CR
Altos de Piedra, PAN
El Guabal, PAN
A. peruensis (KU 211649), PER
A. epikheistos (KU 211684), PER
Juan Lana, PAN
MVZ 149738, CR
Jungurudó, PAN
A. sp. “Pachancho” (KU 217430), PER
Río Marta, PAN
MVZ 164818, A. aff. senex, CR Cerro Punta, PAN
Pequení, PAN CH 5161, PAN
Jordanal, PAN
Jordanal, PAN MVZ 223277, CR
Jordanal, PAN Río Blanco, PAN
Santa Rita, PAN Cerro Azul, PAN
CH 5412, PAN
A. ignescens (TNHC 62529), ECU Cana, Pirré, PAN
Río Farallón, PAN
A.sp. “Cantón Limón” (QCAZ 14032), ECU
A. ignescens (DMH 90E-45), ECU
Río María Oeste, PAN CR01AvaNewLoc, CR
Jordanal, PAN
A. ignescens (DMH 90E-44), ECU
Peña Blanca, PAN
Cerro Azul, PAN
VCK 247, COL
A. pulcher (KU 211677), PER
Cerro Tute, PAN
MVZ 164816, CR
CH 5614, PAN
Río Colorado, PAN
Jordanal PAN CH 5208, PAN
Río Colorado, PAN
CH 5413, PAN
A. peruensis (KU 211632), PER
Juan Lana, PAN
A.sp. “Cantón Limón” (QCAZ 15098), ECU
Copé, PAN CH 5634, PAN
CH 5207, PAN
A. flavescens(KZ01), GUY
Altos de Piedra, PAN
Juan Lana, PAN CH 5418, PAN Juan Lana, PAN
Juan Lana, PAN Loma Trinidad, PAN Río Marta, PAN Aguas Blancas, PAN
Río Marta, PAN
A. elegans (VCK_25), COL
Campana, PAN
A. pulcher (KU 211676), PER
Pirré, PAN
CH 5637, PAN
VCK 245, COL
Juan Lana, PAN
A. cf. laetissimus (ANDES-A 3664), COL
MVZ 149737, CR
A. elegans (AA01), COL
Río Colorado, PAN
Puerto Piña, PAN
Copé, PAN CH 5164, PAN
Río Farallón, PAN
Jordanal, PAN Río Blanco, PAN
Pirré, PAN
CH 5376, PAN
CH 5548, PAN
MVZ 223279, CR
MVZ 223271, CR
Peña Blanca, PAN Río Marta, PAN
AC3AcgChingazaco PuertoObaldia, PAN
MVZ 203769, CR
Jungurudó, PAN
El Guabal, PAN
CH 5549, PAN
Puerto Obaldia, PAN
A. bomolochos (QCAZ 21123), ECU
100 100 100 56 100 100 96 88 100 100 96 100 100 100 85 72 89 98 78 88 65 99 100 59 79 83 71 100 94 99 86 64 62 92 100 99 98 100 99 65 65 61 63 73 100 57 85 64 78 84 62 75 64 87 89 87 76 84 76 90
Atelopus sp. “Puerto Obaldía-Capurganá”
Atelopus certus Atelopus glyphus Atelopus chiriquiensis Atelopus senex Atelopus limosus Atelopus zeteki Atelopus varius
*
*
*
*
100 100 100 90 100 100 89 98 100 92 73 99 100 76 65 99 95 92 97 92 100 74 100 _ 96 _ 86 82 68 92 95 87 _ 87 78 85 71 92 93 63 78 97 83 99 88 97 99 _ 72 89 99 _ 75 54 89 _ 100 ? ? ?Figure 2: A timetree of Atelopus derived from a relaxed-clock MCMC Bayesian analysis using the software Beast, calibrated using the substitution rates of mitochondrial genes of bufonid frogs of Macey et al., (1998), as corrected by Crawford (2003). Outgroups of genera different from Atelopus are not shown, but their relationships can be seen on the
Supplementary Figure 2. Numbers above branches are the posterior probabilities of adjacent nodes. 95% credibility interval of the age of each node is indicated with blue bars. The green shading indicates an interval between 3.2 to 2.76 millions years ago (MYA) delimited by the end of gene flow among shallow marine animals, and of surface water flowing across the Isthmus, respectively (6). Gray shading correspond to the 95% credibility interval of the oldest node reconstructed as being entirely Central American. The circles on each node correspond to the ancestral area reconstructions under the DEC model and the letters associated to each one of them are as in Table 3.
0 MYA 10 1 1 1 0,93 1 1 1 1 0,98 1 1 1 1 1 1 1 1 1 1 1 1 1 0,50 1 1 0,4 0,92 0,90 1 1 0,14 0,17 1
A. elegans (AA01), COL A. elegans (VCK_25), COL
VCK 245, COLCH 5376, PAN Puerto Obaldia, PAN Puerto Obaldia, PAN VCK 247, COL Puerto Piña, PAN Jungurudó, PAN Jungurudó, PAN Jungurudó, PAN AC3AcgChingazaco Pirré, PAN Pirré, PAN CH 5548, PAN CH 5613, PAN Cana, Pirré, PAN CH 5614, PAN Cana, Pirré, PAN CH 5549, PAN MVZ 203769, CR MVZ 149738, CR MVZ 149737, CR
MVZ 164816, CR
MVZ 164818, A. aff. senex, CR MVZ 223271, CR
Cerro Punta, PAN Cerro Punta, PAN MVZ 223270, CR
1
Santa Rita, PAN Santa Rita, PAN Pequení, PAN Pequení, PAN Nusagandí, PAN Nusagandí, PAN 1 1 1 1 Campana, PAN Campana, PAN Juan Lana, PAN Jordanal, PAN Jordanal, PAN Río María Oeste, PAN CH 5414, PAN Jordanal, PANJordanal, PAN
CH 5413, PAN CH 5412, PAN Jordanal, PAN Jordanal, PAN Juan Lana, PAN Río Farallón, PAN Cerro Azul, PAN
Río Farallón, PANRío María Oeste, PAN Juan Lana, PAN Río Farallón, PAN Río Farallón, PAN Jordanal, PAN
Cerro Azul, PAN Juan Lana, PAN CH 5363, PAN Jordanal, PANCR01AvaNewLoc, CR MVZ 223279, CR MVZ 223277, CR Fortuna, PAN Fortuna, PAN Río Colorado, PAN Copé, PAN CH 5207, PAN CH 5418, PAN CH 5164, PANRío Blanco, PAN CH 5161, PAN CH 5417, PAN CH 5208, PAN Río Colorado, PAN Copé, PAN Peña Blanca, PANRío Colorado, PAN CH 5634, PAN CH 5635, PAN Loma Trinidad, PANPeña Blanca, PAN CH 5637, PAN Cerro Tute, PAN
Cerro Tute, PAN
Río Marta, PAN Aguas Blancas, PANRío Blanco, PAN Río Marta, PAN Río Marta, PANEl Guabal, PAN Altos de Piedra, PAN Altos de Piedra, PAN El Guabal, PAN Juan Lana, PAN Juan Lana, PAN Juan Lana, PAN Juan Lana, PAN Río Marta, PAN CH 5636, PAN
Atelopus varius Atelopus zeteki Atelopus chiriquiensis Atelopus senex Atelopus glyphus Atelopus certus
Atelopus sp. “Puerto Obaldía-Capurganá”
*
*
*
*
*
Atelopus limosus 1 Outgroups (SA) 1 . . B A C D E F G H IFigure 3: Map of Central America showing the localities of the samples used for this work. Each species is indicated with a different colour, as follows: Atelopus chiriquiensis , pale blue; A. senex , gray; A. varius , orange; A. zeteki , yellow; A. limosus , pale blue; A. certus , red;
Supplementary Figure 1: Maximum Likelihood phylogenetic tree inferred from the
sequences of two mitochondrial genes (COI and cytb) from the species of Atelopus of Central and South America included in this study. Numbers above branches represent the
100 100 90 100 100 77 89 98 100 100 92 99 100 73 96 100 76 65 99 88 95 97 92 92 100 74
A. cf. laetissimus (ANDES-A 3664), COL
A.sp. “Cantón Limón” (QCAZ 15098), ECU A.sp. “Cantón Limón” (QCAZ 14032), ECU
A. flavescens(KZ01), GUY A. spumarius (KZ02), GUY A. pulcher (KU 211677), PER A. pulcher (KU 211676), PER
A. peruensis (KU 211632), PER
A. epikheistos (KU 211684), PER
A. peruensis (KU 211649), PER A. ignescens (TNHC 62529), ECU A. ignescens (DMH 90E-45), ECU A. ignescens (DMH 90E-44), ECU
A. elegans (VCK_25), COL
A. sp. “Pachancho” (KU 217429), PER
A. sp. “Pachancho” (KU 217430), PER
A. bomolochos (QCAZ 21123), ECU
A. elegans (AA01), COL VCK 245, COL
CH 5376, PAN
VCK 247, COL Puerto Obaldia, PAN Puerto Obaldia, PAN Puerto Piña, PAN Jungurudó, PAN Jungurudó, PAN Jungurudó, PAN CH 5613, PAN CH 5614, PAN CH 5549, PAN
Pirré, PAN Pirré, PAN
Cana, Pirré, PAN Cana, Pirré, PAN
AC3AcgChingazaco CH 5548, PAN
Cerro Punta, PAN Cerro Punta, PAN MVZ 223271, CR MVZ 223270, CR
MVZ 164818, A. aff. senex, CR MVZ 149738, CR
MVZ 164816, CR MVZ 149737, CR MVZ 203769, CR Santa Rita, PAN Santa Rita, PAN Nusagandí, PAN Pequeni, PAN Nusagandí, PAN Pequení, PAN
Río María Oeste, PAN Campana, PANCampana, PAN Jordanal, PAN
Jordanal, PAN
Jordanal, PAN
Jordanal, PAN
CH 5412, PAN CH 5413, PAN Juan Lana, PANRío Farallón, PAN
Río Farallón, PAN
CH 5414, PAN Jordanal, PAN
Jordanal, PAN
Jordanal, PAN
Juan Lana, PAN Juan Lana, PAN Jordanal, PACH 5363, PANN
Río Farallón, PAN Río Farallón, PAN Cerro Azul, PAN
Cerro Azul, PAN
Río María Oeste, PAN
Juan Lana, PAN Fortuna, PAN Fortuna, PAN CR01AvaNewLoc, CR MVZ 223277, CR MVZ 223279, CR Río Colorado, PANCopé, PAN
CH 5208, PAN CH 5417, PANCH 5161, PAN
Río Blanco, PAN CH 5418, PAN CH 5164, PANCH 5207, PAN Peña Blanca, PAN Río Colorado, PAN Cerro Tute, PAN
Cerro Tute, PAN
Río Marta, PAN Juan Lana, PAN
Juan Lana, PAN
Juan Lana, PAN
Juan Lana, PAN El Guabal, PAN Peña Blanca, PAN
CH 5637, PAN Loma Trinidad, PAN Río Blanco, PAN Río Marta, PAN Río Marta, PAN Río Marta, PAN Aguas Blancas, PANRío Colorado, PAN Copé, PAN
Altos de Piedra, PAN
Altos de Piedra, PAN CH 5635, PAN CH 5634, PAN CH 5636, PANEl Guabal, PAN
Atelopus varius Atelopus zeteki Atelopus limosus Atelopus senex Atelopus chiriquiensis Atelopus glyphus Atelopus certus
Atelopus sp. “Puerto Obaldía-Capurganá”
Supplementary Figure 2: Timetree of Atelopus based on a relaxed-clock MCMC Bayesian Analysis calibrated using the substitution rates of Bufonid frogs of Macey et al., (1998), as corrected by Crawford (2003), and including all used outgroups. The circles on each node correspond to the ancestral area reconstructions under the DEC model and the letters associated to each one of them are as in Table 3 and in the Supplementary Table 3.
0 10 20 30 40 1 1 1 0,93 1 1 1 1 1 0,98 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0,50 1 1 1 0,4 0,92 0,90 0,37 1 1 0,95 1 1 0,14 1 0,17 1 1
A. spumarius (KZ02), GUY
A. flavescens(KZ01), GUY A. pulcher (KU 211676), PER
A. pulcher (KU 211677), PER
A.sp. “Cantón Limón” (QCAZ 14032), ECU
A.sp. “Cantón Limón” (QCAZ 15098), ECU
A. cf. laetissimus (ANDES-A 3664), COL A. ignescens (TNHC 62529), ECU
A. ignescens (DMH 90E-44), ECU
A. ignescens (DMH 90E-45), ECU
A. peruensiA. peruensiss (KU 211632), PER (KU 211649), PER
A. epikheistos (KU 211684), PER
A. bomolochos (QCAZ 21123), ECU
A. sp. “Pachancho” (KU 217429), PER
A. sp. “Pachancho” (KU 217430), PER
A. elegans (AA01), COL
A. elegans (VCK_25), COL
VCK 245, COLCH 5376, PAN Puerto Obaldia, PAN Puerto Obaldia, PAN VCK 247, COL Puerto Piña, PAN Jungurudó, PAN Jungurudó, PAN Jungurudó, PAN AC3AcgChingazaco Pirré, PAN Pirré, PAN CH 5548, PAN CH 5613, PAN Cana, Pirré, PAN CH 5614, PAN Cana, Pirré, PAN CH 5549, PAN MVZ 203769, CR MVZ 149738, CR MVZ 149737, CR
MVZ 164816, CR
MVZ 164818, A. aff. senex, CR MVZ 223271, CR
Cerro Punta, PAN Cerro Punta, PAN MVZ 223270, CR
1
Santa Rita, PAN Santa Rita, PAN Pequení, PAN Pequení, PAN Nusagandí, PAN Nusagandí, PAN 1 1 1 1 Campana, PAN Campana, PAN Juan Lana, PAN Jordanal, PAN Jordanal, PAN Río María Oeste, PAN CH 5414, PAN Jordanal, PANJordanal, PAN
CH 5413, PAN CH 5412, PAN Jordanal, PAN Jordanal, PAN Juan Lana, PANRío Farallón, PAN Cerro Azul, PAN
Río Farallón, PAN Río María Oeste, PAN Juan Lana, PAN Río Farallón, PAN Río Farallón, PAN Jordanal, PAN
Cerro Azul, PAN Juan Lana, PAN CH 5363, PAN Jordanal, PAN CR01AvaNewLoc, CR MVZ 223279, CR MVZ 223277, CR Fortuna, PAN Fortuna, PAN Río Colorado, PAN Copé, PAN CH 5207, PAN CH 5418, PANCH 5164, PAN
Río Blanco, PAN CH 5161, PAN CH 5417, PAN CH 5208, PAN Río Colorado, PAN Copé, PAN Peña Blanca, PANRío Colorado, PAN CH 5634, PAN CH 5635, PAN Loma Trinidad, PANPeña Blanca, PAN CH 5637, PAN Cerro Tute, PANCerro Tute, PAN Río Marta, PAN Aguas Blancas, PANRío Blanco, PAN Río Marta, PAN Río Marta, PAN El Guabal, PAN Altos de Piedra, PAN Altos de Piedra, PAN El Guabal, PAN Juan Lana, PAN Juan Lana, PAN Juan Lana, PAN Juan Lana, PAN Río Marta, PAN CH 5636, PAN
Atelopus varius Atelopus zeteki Atelopus chiriquiensis Atelopus senex Atelopus glyphus Atelopus certus
Atelopus sp. “Puerto Obaldía-Capurganá”
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Atelopus limosus 1 F A A B C D F G H . L M N O P Q R S T U VBufo gargarizans minshanicus Bufo stejnegeri Bufo japonicus Anaxyrus boreas Bufotes viridis Duttaphrynus melanostictus I G E J W K X
Appendix 1
Specimens of Atelopus used for the present study. Museum voucher, locality, latitude,
longitude and GenBank accession numbers are given for all specimens. Museum acronyms
are as follows; ANDES-A = Reptile collection, Museo de Historia Natural ANDES, Bogotá,
Colombia; KU= Kansas University Biodiversity Institute & Natural History Museum,
Lawrence, Kansas; THNC=Texas Natural History Collection, Texas Memorial Museum,
Austin, Texas; QCAZ=Museo de Zoología, Pontificia Universidad Católica del Ecuador,
Quito, Ecuador; MVZ=Museum of Vertebrate Zoology, University of California at Berkeley;
VCK= field number of Vicky Flechas,Universidad de Los Andes, Bogotá, Colombia;
DMH=field number of David M. Hillis, University of Texas at Austin. NA refers to
