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Journal of Biogeography
Isolation in habitat refugia promotes rapid diversification in
a montane tropical salamander
Journal of Biogeography
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Original Article
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Parra-Olea, Gabriela; Instituto de Biologia, UNAM, Zoology
Windfield-Perez, Juan Carlos; Instituto de Biologia, UNAM, Zoology
Velo-Anton, Guillermo; Cornell University, Ecology and Evolutionary
Biology
Zamudio, Kelly; Cornell University, Ecology and Evolutionary
Biology
phylogeography, Endemism, glacial refugia, México, plethodontid,
Pleistocene, salamander, Trans-Volcanic Belt, volcanism
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Key Words:
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Parra-Olea et al., Page 1
Original Article
Isolation in habitat refugia promotes rapid diversification in a montane tropical
salamander
Gabriela Parra-Olea1*, Juan Carlos Windfield1, Guillermo Velo-Antón2, and Kelly R. Zamudio2
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Departamento de Zoología, Instituto de Biología, Universidad Nacional Autónoma de México,
Distrito Federal 04510, México
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Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, New York 14853,
USA
*Correspondence: Gabriela Parra-Olea, Departamento de Zoología, Instituto de Biología,
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Universidad Nacional Autónoma de México, Distrito Federal 04510, México. E-mail:
[email protected]
RRH: Montane diversification in the tropics
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LRH: G. Parra-Olea et al.
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Journal of Biogeography
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ABSTRACT
Aim Our goal was to reconstruct the phylogenetic history and historical demography of highly
divergent populations of the endemic plethodontid salamander Pseudoeurycea leprosa, to
elucidate the timing and mechanisms of divergences in the Trans-Volcanic Belt of México.
Location The Trans-Volcanic Belt (TVB) of Central México, including the states of México,
Morelos, Puebla, Tlaxcala and Veracruz.
Methods We sequenced the cytochrome b mitochondrial DNA (mtDNA) gene for 281
individuals from 26 populations and 9 mountain ranges in the TVB, and used Bayesian tree
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reconstruction and MCMC coalescent methods to infer historical demographic parameters and
divergences among populations in each mountain system.
Results We found deep genetic divergences between eastern and central TVB mountain systems
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despite their recent volcanic origin. Populations of P. leprosa show a pattern of refugial
populations in the northeastern and eastern limits of the species’ distribution, and genetic
evidence of rapid population expansion in mountain ranges of the central TVB. The oldest
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divergences among populations date to ~3.8 million of years, and the most recent divergences in
the central TVB are Pleistocene in age (~0.8mya).
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Main conclusions Given the timing and order of population diversification in P. leprosa, we
conclude that early isolation in multiple habitat refuges in northeastern and eastern mountain
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ranges played an important role in structuring population diversity in the TVB, followed by
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population expansion and genetic divergence of the central range populations. We also found
evidence of peripheral isolation of highly divergent forms. The dynamic climatic and volcanic
changes in this landscape generally coincide with the history of intraspecific diversification in P.
leprosa. Combined, climate-driven changes in habitat distribution, in an actively changing
volcanic landscape, have shaped divergences in the TVB and very likely contributed to the high
levels of speciation and endemism in this biodiverse region.
Keywords
Endemism, glacial refugia, México, phylogeography, Pleistocene, plethodontid, salamander,
Trans-Volcanic Belt, volcanism.
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INTRODUCTION
The differentiation of montane species has been of particular interest to evolutionary
biologists, because the patchy distribution of appropriate habitat combined with the geographic
isolation of populations on mountain ‘islands’ will potentially promote diversification and
increase the rate of speciation for montane-adapted taxa (Fjeldså & Lovett, 1997; Jetz et al.,
2004). Montane species are also particularly useful for evaluating historical responses to climate
fluctuations because they are often physiologically adapted to a narrow range of environmental
conditions and thus particularly susceptible to climate change (Brereton et al., 1995; Hughes,
2003; Galbreath et al., 2009). Many cold-adapted montane organisms experienced range
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expansion and increased gene flow during glacial periods (Hewitt, 2004) and range contractions
during warmer interglacials (DeChaine & Martin, 2006; Assefa et al., 2007; Browne & Ferree,
2007). Their low physiological tolerance to higher temperatures implies that populations isolated
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on different mountain ranges should carry the signature of historical changes in connectivity
associated with either glacial or interglacial periods even if the periods of climatic instability
were relatively recent (Knowles, 2000; Smith & Farrell, 2005; DeChaine & Martin, 2006; Assefa
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et al., 2007; Browne & Ferree, 2007; Popp et al., 2008).
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In the tropics, where the environmental gradient from lowland to mountains is most
pronounced (Janzen, 1967), montane regions are important historical centers of diversity and
endemism, even more so than tropical lowlands (Smith et al., 2007). Thus, understanding
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microevolutionary processes promoting diversification, and specifically how isolation and
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genetic drift interact to enhance speciation and diversification in tropical montane species, will
provide a framework for understanding broader patterns of biodiversity distributions in these
habitats (Kozak & Wiens, 2006; Kozak & Wiens, 2007; Smith et al., 2007; Wiens et al., 2007).
Global patterns of species richness across montane and non-montane landscapes are relatively
well-defined (Rahbek, 1995; Jetz et al., 2004; Wiens & Donoghue, 2004; Wiens et al., 2006) but
fine-scale examinations of the processes that engender diversity in mountain species are still
needed. In this study we focus on diversification in a tropical salamander endemic to the TransVolcanic Belt (TVB) of central México, a young (Ferrari et al., 1999; Ferrari & Rosas-Elguera,
1999) volcanic mountain chain that harbors a large percentage of the country’s endemic flora and
fauna (Luna et al., 2007). This mountainous landscape was affected by the climatic cycles of the
Plio-Pleistocene, which resulted in large altitudinal shifts in the pine forests typical of higher
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elevations, and changed patterns of connectivity among mountain ranges in the system (Brown,
1985; Graham, 1993; McDonald, 1993; Lozano-García et al., 2005). The TVB is one of the areas
of highest vertebrate biodiversity in México, second only to the Sierra Madre del Sur (Luna et
al., 2007). These dynamic highlands of México have been a center for diversification of many
plant and animal radiations, including tropical salamanders (Lynch et al., 1983; Luna et al.,
2007) and thus offer an excellent opportunity to study the underlying microevolutionary
processes (Adams et al., 2009) that have contributed to the high rate of local population
divergence and speciation.
We examine population genetic diversification in the plethodontid salamander
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Pseudoeurycea leprosa, a species endemic to the TVB that occurs at elevations between 2000
and 3500 m, and belongs to the diverse Bolitoglossini, the clade that includes most Neotropical
salamanders (Wiens, 2007). We compare diversity of cytochrome b (cytb) mitochondrial gene
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sequences among populations of P. leprosa and infer the historical population changes that
contributed to differentiation in this species complex. Despite its high diversity, the TVB was
formed only recently in the Middle Miocene (Ferrari & Rosas-Elguera, 1999), thus
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diversification of endemic species occurred relatively rapidly. We use this focal taxon to test
hypotheses about how volcanic activity and quaternary glaciations affected the colonization or
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dispersal among mountain ranges in the TVB and to infer the microevolutionary processes that
promoted diversification in this lineage. Specifically, we use our data to investigate whether 1)
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population structure among independent mountain ranges shows the genetic signature of
isolation, migration, and/or drift, 2) whether TVB populations evolved from a single, or few
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source populations, 3) whether historical migration patterns from ancestral populations were
directional, and 4) whether the timing of isolation is concordant with the volcanic origin of TVB
mountains or changes in habitat distribution that occurred during Plio-Pleistocene climatic
cycles. We compare our results to patterns inferred from other plants and animals endemic to this
region, and to an earlier study that examined genetic divergence among a subset of P. leprosa
populations using allozymes (Lynch et al., 1983). Deforestation and anthropogenic climate
change will severely impact the distribution of appropriate habitat for this and other TVB
endemic species in the future (Parra-Olea et al., 2005b); we discuss the importance of
understanding the current geographic distribution of genetic diversity as well as the historical
deployment of lineages for conservation of high-elevation species in this region.
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MATERIALS AND METHODS
Study organism and geographic context
In contrast to temperate zone salamanders, Neotropical salamanders occupy relatively
constant thermal environments, both because tropical climates are less variable (Janzen, 1967)
and because Neotropical salamanders typically occupy sheltered microhabitats (Wake & Lynch,
1976). As a result, tropical salamander populations experience lower variation in body
temperature (Feder, 1976), are generally stenothermal, and thus cannot easily acclimate to large
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temperature fluctuations (Feder, 1978). These characteristics restrict them to small geographic
and elevational ranges (Feder, 1978). Within the genus Pseudoeurycea only two species (P.
leprosa and P. cephalica) have a relatively wide distribution, all others are microendemics or
restricted to single mountain ranges. Pseudoeurycea leprosa is a fully terrestrial species and
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occurs along the length of the TVB, but is restricted to pine, pine-oak and fir forests above 2000
meters.
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The Mexican TVB is a narrow (ca. 160 km) arc of volcanic mountains that stretches 1200
km from the Gulf of California to the Gulf of México (García-Palomo et al., 2000). The
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volcanism that generated this mountain chain began during the late Cretaceous (80 Mya) but the
activity that contributed most to shaping the present mountains dates to the mid-Miocene, and
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reached its greatest intensity during the Plio-Pleistocene (approximately 0.7 Mya) (Tamayo &
West, 1964). This mountain chain defines the southern limit of the uplifted Mexican plateau
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(Mesa Central) (Domínguez-Domínguez & Pérez-Ponce De León, 2009) and consists of three
distinct segments, each with its own tectonic, volcanic, and geomorphological characteristics
(Pasquaré et al., 1988). The westernmost section occurs from the Pacific coast to the Colima
graben and includes the Volcán de Colima; the central section extends from the Michoacán
volcanic zone towards either the Valley of México and Sierra Nevada (Nixon et al., 1987) or
towards the Querétaro-Taxco lineament (Pasquaré et al., 1988) and includes Nevado de Toluca,
Popocatepetl, Iztaccihuatl and Sierra de las Cruces; finally, the eastern section extends toward
the Gulf of México (Osete et al., 2000) and includes La Malinche, Pico de Orizaba and Cofre de
Perote (Castillo-Rodríguez et al., 2007). The range of P. leprosa encompasses only the eastern
and central geomorphological units of the TVB (Fig 1).
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All of the TVB volcanoes are relatively young, with a maximum age estimated at 2.6
Mya (Ferrari, 2000; Ferrari et al., 2000). The oldest cone construction began in Nevado de
Toluca circa 2.6 Mya, whereas in the Popocateptl-Iztaccihuatl range cone growth began in the
late Pleistocene and Holocene, approximately 1.6 Mya (Ferrari et al., 2000; García-Palomo et
al., 2000). The cones and highlands in the eastern TVB date to 1.5-2.0 Mya approximately for
Cofre de Perote (Cantagrel & Robin, 1979). Several of the volcanoes in the TVB have been
intermittently active since their formation in the Pleistocene (Tamayo & West, 1964): Volcán
Colima, in the western mountain range, is currently active and its last major eruption occurred in
1913, Popocateptl last erupted in 1947, and hundreds of smaller volcanoes that compose the
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Michoacán triangle have been active throughout the Quaternary (Johnson & Harrison, 1990).
The distribution and persistence of montane habitat in the TVB has also had a dynamic
history due to the glacial cycles during the Pleistocene and Holocene (Heine, 1988). Available
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data on the most recent events show five periods of glacial advances: one at approximately
20,000 years before present (Nexcoalango) and the other four between 20,000 and 8,300 years
ago (Hueyatlaco I, Hueyatlaco II, Mipulco I and Mipulco II) (Vázquez-Selem & Heine, 2004).
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Climatic cycles began in the early Quaternary and numerous studies of Neotropical ectotherms
have detected the genetic signature of population isolation associated with those earlier events,
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rather than the Last Glacial Maximum (Wüster et al., 2005; Fitzpatrick et al., 2009). Thus,
although the most recent glacial advances certainly affected the distribution of habitat, they are
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representative of a much longer period of changes in landscape due to cyclical climatic change
(Haffer, 1997; Haffer & Prance, 2001). The impact of these glaciations on TVB taxa was
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probably not as dramatic as that observed at higher latitudes (Galbreath et al., 2009) however,
the ice masses on the highest mountains extended altitudinally, dropping the upper tree line 600900 m below its present position (Brown, 1985; Graham, 1993; McDonald, 1993; Lozano-García
et al., 2005). Paleoecological evidence indicates that the climate was cold and moist and
coniferous forests reached their maximum range size with a broader distribution than is present
in México today (Perry, 1991; Millar, 1998; Graham, 1999).
Population Sampling
We collected tissue samples of Pseudoeurycea leprosa from throughout its distribution
along the TVB (Table S1; Fig. 1). We sampled a total of 281 individuals from 26 populations
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with a range of 1-30 individuals per site (Fig. 1, Table 1). The sampled populations were
distributed across the following nine mountain ranges (ordered roughly from west to east):
Nevado de Toluca, Sierra de las Cruces, Iztacchuatl-Popocatepetl, Malinche, Tlaxco,
Tlatlauquitepec, Cofre de Perote, Orizaba, and Tres Mogotes (Table 1, Fig. 1). Plethodontids are
cryptic salamanders, and Pseudoeurycea leprosa is no exception; thus, the large spatial sampling
we obtained for this endemic species represents one of the highest for population genetic studies
in this lineage. We selected Pseudoeurycea lynchi and Pseudoeurycea lineola as successively
more distant outgroups according to published higher-level relationships within the
Bolitoglossini (Wiens et al., 2007).
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MtDNA amplification and sequencing
Total genomic DNA was extracted from ethanol preserved tissues (muscle or liver) with
DNeasy Tissue Kits using manufacturer’s protocols (Qiagen). Polymerase chain reaction (PCR)
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was used to amplify 675 base pairs of the mitochondrial cytochrome b gene (cytb), using the
primers MVZ15 and MVZ16 (Moritz et al., 1992). PCR amplifications were performed in a 25µl
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volume including 1µl of DNA template (~100ng/µl), 1U Taq polymerase (Applied Biosystems),
1× PCR buffer with 1.5 mM MgCl2, 0.4mM dNTPs, and 0.5µM forward and reverse primers.
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PCR reactions consisted of 35 cycles with a denaturing temperature of 94 ºC (1 min), annealing
at 50 ºC (1 min), and extension at 72 ºC (1 min). We electrophoresed the resulting PCR products
with ethidium bromide staining to verify their size. Successful amplicons were purified with
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shrimp alkaline phosphatase (1U) and exonuclease I (10U) to remove non-incorporated dNTPs
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and primers. Fragments were sequenced in both directions using the original amplification
primers and BigDye termination sequencing chemistry. Sequencing reactions were carried out in
a total volume of 5µl with 1µl cleaned PCR product, 0.24µM primer, 1µl Big Dye Terminator
Ready Reaction Mix, and 1X Sequencing buffer (Applied Biosystems). Cycle sequencing
products were column-purified with Sephadex G-50 and run on an ABI PRISM 3100 DNA
Analyzer. We checked electropherograms by eye before constructing contiguous sequences for
each individual using Sequencher version 4.7 (Gene Codes).
Phylogeographic Analyses
We aligned sequences and identified unique haplotypes for phylogenetic analyses in the
program COLLAPSE v1.2 (Posada, 2006). We used the program JMODELTEST v0.1.1 (Posada,
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2008) to select the model of nucleotide substitution that best fit our data. We tested the full
complement of 88 models using the default base frequency and rate variation settings, and ML
optimized base tree for the likelihood calculations (Guindon & Gascuel, 2003). We used AIC
scores to select the appropriate model with which to infer a population-level phylogeny using
fully partitioned Bayesian Inference (BI) implemented in MRBAYES v3.1 (Huelsenbeck &
Ronquist, 2001). The Bayesian analysis consisted of 10 chains sampling every 10,000
generations for 10 million generations. We used two methods to verify convergence and
determine adequate burn-in: we examined a plot of likelihood scores of the heated chain, and
checked the stationarity of chains using the software TRACER v1.4 (Rambaut & Drummond,
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2007). We disregarded a total of 250 trees as burn-in; using the remaining trees, we estimated the
50% majority-rule consensus topology with branch lengths and posterior probabilities for each
node in MRBAYES. Finally, a haplotype network was constructed using statistical parsimony
(Templeton et al., 1992) implemented in TCS v1.13 (Clement et al., 2000).
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Genetic diversity and historical demographic analyses
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We used ARLEQUIN v3.01 (Excoffier et al., 2005) to estimate haplotype diversity (h) and
nucleotide diversity (π) (Tajima, 1983; Nei, 1987) for each population and mountain range. We
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characterized genetic differentiation among mountain ranges by estimating DXY (Nei, 1987), the
average number of nucleotide substitutions per site between pairs of mountain ranges, in DNASP
v5 (Rozas et al., 2003), and PiXY, the average number of pairwise differences among haplotypes
of different mountain ranges, in ARLEQUIN v3.01.
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We used mismatch analyses of sequences within each mountain range to infer historical
demographic changes in Pseudoeurycea leprosa (Schneider & Excoffier, 1999). This analysis
compares the frequency distribution of pairwise differences between haplotypes with that
expected under a model of population expansion. Significant difference between observed and
expected distributions is tested using a bootstrap approach (1,000 replicates). The frequency
distribution is predictably unimodal for lineages that have undergone recent population
expansions and multimodal for lineages whose populations are either subdivided or in
equilibrium. We compared the sum of square deviations (SSD) between the observed and
expected mismatch to test the hypothesis of population expansion (Schneider & Excoffier,
1999); a significant P-value rejects the fit of the data to the expansion model. Additionally, for
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each sampled population, and for each mountain range, we used Tajima’s D (Tajima, 1989) and
Fu’s FS (Fu, 1997) tests of selective neutrality. Historical population growth predicts
significantly negative D and FS values, which we tested with 10,000 bootstrap replicates. All
three tests for population expansion were performed in ARLEQUIN v3.01.
To reconstruct the age of particular groups of haplotypes and changes in demography
over the history of each major lineage, we used the coalescent-based methods applied in the
program BEAST v1.4.7 (Drummond et al., 2005; Drummond & Rambaut, 2007). This approach
allows inferences of population fluctuations over time by estimating the posterior distribution for
effective population size at intervals along a phylogeny (Drummond et al., 2005; Drummond &
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Rambaut, 2007). We estimated changes in demography for all P. leprosa populations combined,
for the populations in the NE I clade, and for populations in the Central TVB clade. Times to
most recent common ancestor (TMRCA) for some clades were obtained using Bayesian Markov
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Monte Carlo searches. We used a GTR+I+G model of evolution and implemented an
uncorrelated lognormal relaxed molecular clock method (Drummond et al., 2006). We used a
normal distribution with a mean of 0.0075 and a standard deviation of 0.0025 as a prior for
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mutation rate in cytb to reflect a priori uncertainty in this parameter (Mueller, 2006; MartínezSolano et al., 2007; Martínez-Solano & Lawson, 2009). We implemented a series of coalescent
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models (Bayesian skyline, exponential, expansion) to assess any bias these models might have
on time estimates. For each analysis, we performed two independent runs of 10-25 million
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generations sampling every 1,000th generation and removing 10% of the initial samples as burnin. We combined runs and determined stationarity of the posterior distributions for all model
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parameters using Tracer 1.4 (Drummond & Rambaut, 2007). We implemented a relaxed
molecular clock with uncorrelated rates among lineages, and the following substitution model
priors: rate parameters uniform (0,500), alpha exponential (1), proportion of invariant sites
uniform (0,1). Scale operators were adjusted as suggested by the program.
Finally, we used a coalescence model in LAMARC 2.0 (Kuhner, 2006) to estimate Θ
(Θ=2Neµ for mtDNA) and migration rates (M) for P. leprosa populations. To optimize
parameter estimations, we selected 11 representative populations from the mountain systems,
excluding localities with less than four individuals (Tlaxco and Tres Mogotes). We followed
suggestions by the authors (Kuhner, 2006) and randomly reduced sample sizes to 15 for those
populations with larger sample sizes, to increase run efficiency. Default values were used for
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effective population size and migration parameters. We performed Bayesian analyses with one
long chain with a burn-in of 1x106 and a run of 1x106 recorded genealogies sampled every 100
steps. We applied a general-time reversal (GTR) model and performed five identical replicate
analyses. LAMARC infers approximate confidence intervals (CIs) around maximum probable
estimates (MPE) for each parameter. Parameter conversion was verified by examining
stationarity in parameter trends over the length of the chains and Effective Sample Sizes (ESS)
obtained for each Θ and migration rates, using TRACER v1.4 2. We interpreted ESS values
greater than 300 as an indication that sampled trees were not correlated and thus represent
independent samples.
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Isolation by distance and least-cost paths in the TVB
We performed a least-cost path analyses (LCPA) using the Spatial Analyst extension in
ARCGIS version 9.2 (ESRI, 2006) to determine the potential influence of elevation on gene flow
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and genetic structure of P. leprosa populations across TVB mountain ranges. We estimated the
least-cost path of migration among mountain ranges, using the centroid of all populations
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sampled in each mountain range as endpoints for paths between pairs of mountains. To infer an
appropriate cost among mountain ranges, we reclassified the elevation layer based on the current
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elevation of the species’ distribution (2500-3500m asl), and assumed higher cost for movements
across terrain at elevations outside that range. A cost raster was created by giving a value to each
cell equal to the cumulative cost of reaching it from the source. From the cost raster, ARCGIS
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identifies the path resulting in the lowest cost to reach location from a specified source
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population. We assumed the following elevation ranges and corresponding movement costs (in
parentheses) through each cell: 0-1000m (5); 1000-1500m (4); 1500-2000m (3); 2000-2500m
(2); 2500-3500m (1); 3500-4000m (2); 4000-4500m (3); 4500-5000m (4); 5000-5469m (5). We
also calculated straight Euclidean distances (ED) between populations and two geographic
distances based on the LCPA identified: 1) Euclidean distances along every least-cost path (EDCP) and 2) the cost of moving along each path (Costpath, CP).
We used Mantel tests to examine Isolation by distance (IBD) between genetic distances
(DXY and PiXY) and the three geographic/cost distance matrices calculated among mountain
ranges. We also used Partial Mantel tests to examine correlations between genetic distances (DXY
and PiXY) and least cost distances (ED-CP), while controlling for the effect of Euclidean
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Parra-Olea et al., Page 11
distances (ED). These analyses were performed with 10,000 replications using the program ZT
(Bonnet & Van De Peer, 2002).
RESULTS
MtDNA diversity
The cytb alignment contained 281 sequences and 675 characters. No insertions or
deletions were found among the sequences, thus alignment was straightforward. We identified 70
unique haplotypes among the samples sequenced, and among those 139 sites were variable and
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75 parsimony-informative. The ML estimated transition/transversion ratio was 3.2, with mean
nucleotide frequencies of 27.7% A, 21.5% C, 15.2% G and 35.5% T. The TrN+I+G model of
evolution was selected by JMODELTEST and used for subsequent Bayesian analyses. All
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sequences have been deposited in GenBank (accession numbers GQ468424-GQ468493).
Phylogeographic Analyses
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The Bayesian analyses recovered a monophyletic P. leprosa with some structured
regional groups of haplotypes. Two well supported “Northeast” (NE) Clades diverged relatively
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early in the history of this species; the NE I clade includes samples from Tlaxco and some but
not all samples from Tlatlauquitepec; the NE II clade includes samples from Vigas and the
remaining samples from Tlatlauquitepec. Thus, despite their strong support, these two clades are
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not geographically independent, a result either of secondary contact and exchange between these
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two regions, or incomplete lineage sorting since the time of their isolation. Our phylogeny did
not provide resolution for the earliest divergences within this group. A basal polytomy includes
other northeastern haplotypes with those from Vigas and Gonzalez Ortega, and the relationship
among those haplotypes, the two NE clades, and the two samples from Tres Mogotes is unclear.
The topology also infers a large Central TVB clade which includs all samples from Nevado de
Toluca, Sierra de las Cruces, Popocatepetl-Iztaccihuatl and Malinche, and a Southeast clade
including all samples in the Pico de Orizaba mountain range (Xometla, Texmola and
Texmalaquilla).
The 70 Pseudoeurycea leprosa sequences were grouped, under the statistical parsimony
95% confidence interval, into two haplotype networks and one haplotype (70, from Tres
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Parra-Olea et al., Page 12
Mogotes) independent of both networks (Fig. 3). The main network groups 65 of the haplotypes
with a connection limit of 11 bp and contains the Central, Southeast, and NE II clades as well as
the northeastern basal haplotypes recovered in the Bayesian tree. A common haplotype (11) is
found in central mountains (Popocatepetl-Itzaccihuatl and Malinche) and a southeastern
population (Xometla); additional haplotypes from these same populations and from western
mountains (Nevado de Toluca and Sierra de las Cruces) are separated from this common
haplotype by only a few mutational steps. Higher divergences were found among Eastern and
Southeastern haplotypes in the network, with five (Texmalaquilla, Texmola and Xometla), 10
(Vigas), 14 (González Ortega) and 23 mutational steps (Tlatlauquitepec) separating those
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samples from Central TVB haplotypes. The most diverse population is Vigas, with 15 haplotypes
separated by a maximum of 41 mutational steps, and with eastern haplotypes as closest related
populations (Xometla, Tlatlauquitepec). A second network groups the NE I clade haplotypes
with the single haplotype from Tlaxco, separated by 6 mutational steps. Finally, the haplotype
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found in the southeasternmost population (70, Tres Mogotes) is isolated from all other
populations in the network.
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Genetic diversity and historical demographic analyses
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Haplotype diversity (h) and nucleotide diversity (π) for each population and mountain
range is summarized in Table 1. The Vigas population showed the highest number of haplotypes,
and highest nucleotide diversity compared to all other populations. Populations from the Central
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TVB had low nucleotide diversity overall, reflecting high sequence similarity. Most mountain
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systems had low nucleotide diversity (0.002-0.018) and high haplotype diversity (0.464-0.944).
Genetic distances among mountains ranged from 1.69-21.16 and 0.003-0.029 for PiXY
and DXY, respectively (Table 2). The populations from the Central TVB clade were more related
to each other, and more genetically differentiated from north-eastern populations (Perote and
Tlatlauquitepec mountain ranges) than from SE Clade populations (Orizaba mountain ranges;
Table 2).
Plots of mismatch distributions (Fig. 4) were multimodal for all P. leprosa populations
combined (SSD=0.025; P = 0.061) and for the NE I clade (SSD=0.062; P = 0.641) but the
Central TVB clade was unimodal (SSD=0.013; P = 0.080). Tajima’s D and Fu’s FS corroborated
evidence for expansion in the Central TVB clade. Fu´s FS test (Table 1) was significant only for
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Calpan (FS = –2.82; P < 0.05) and Nanacamilpa (FS = –2.85; P < 0.01) populations and for
Iztaccihuatl (FS = –24.91; P < 0.05) and Malinche (FS = –9.39; P < 0.01) mountain ranges.
Tajima´s D was significantly negative for Calpan (D = –1.76; P < 0.05) and Rio Frio (D = –1.86;
P < 0.01) populations and for Iztaccihuatl (D = –1.60; P < 0.05) and Malinche (D = –2.00; P <
0.05) mountain ranges.
Bayesian skyline plots (BSP) for all P. leprosa, NE I clade and Central TVB clade
populations are shown in Figure 4. BSPs depict similar scenarios among the NE I clade and the
Central clade, with low but constant increase in size from 200,000 years to the present. In
contrast, BSP for all P. leprosa populations combined shows a decrease in size from 250,000
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followed by a rapid expansion. The estimates of TMRCA were highly concordant among runs
(Table S2), independent of the coalescent model applied. The estimated TMRCA for all P.
leprosa was 3.25-3.80 million years ago over all coalescent models. An old split occurred
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between populations from the NE and SE (clade 4 in Table S2): 3.2-3.7 million years ago,
whereas the Central TVB Clade has a TMRCA of only 0.79-0.83 million years.
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Repeated LAMARC runs resulted in similar posterior probability distributions and high
ESS values for all Θ and most migration rates. Populations vary in Θ (Figure 4 and Table S2),
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ranging from the lowest value in Ajusco (MPE = 0.00001), to the highest value in Vigas (MPE =
0.01114), with a trend of decreasing population sizes from eastern to western populations.
However, the Central clade populations of Llano Grande and Atzompa (both in the Popocatepetl-
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Itzaccihuatl mountain range) showed the highest MPE Θ values for the Central TVB and their
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confidence intervals overlap with the highest population sizes found in eastern populations
(Table S2).
The LAMARC migration rate estimates (M) are scaled to the mutation rate (m/µ). The
most probable estimators (MPE) of immigration obtained from LAMARC suggest an
asymmetrical immigration among mountain ranges (Table 3, Figure 5). Among NE clade
populations, gene flow occurs principally from Vigas to other populations, with the highest
estimated value from Vigas into González Ortega (both in the Perote mountain range). Migration
rates are higher from NE II clade (Perote) into populations of the SE Clade (Orizaba mountain
range), and from SE into the central mountain range of Malinche, suggesting an expansion from
NE to Central TVB via the southeastern mountain range. Indeed, estimated migration from NE to
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Parra-Olea et al., Page 14
Central TVB populations is low and the lowest immigration rates in the entire P. leprosa
distribution are into NE mountain ranges. Within the Central TVB Clade, we found higher
migration from Malinche to central and northern Popocatepetl-Itzaccihuatl than to the southern
end of that mountain range, and Sierra de las Cruces receives more migrants than from Nevado
de Toluca or Popocatepetl-Itzaccihuatl, than vice-versa. One caveat is that although the MPEs
are distinct, all immigration rate 95% confidence intervals overlap, thus estimates should be
interpreted with caution (Table 3).
Isolation by distance and least-cost paths in the TVB
Genetic and geographic distances between pairs of mountain ranges are summarized in
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Table 2 and Table S3, respectively. The Mantel test revealed no significant isolation-by-distance
between all three measures of geographic distance and DXY (ED, Pearson r = 0.13, P = 0.282;
ED-CP, Pearson r = 0.10, P = 0.299; and CP, Pearson r = 0.07, P = 0.334), or PiXY (ED, Pearson
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r = 0.23, P = 0.111; ED-CP, Pearson r = 0.20 P = 0.131; and CP, Pearson r = 0.16, P = 0.162).
However, the partial Mantel test revealed significant isolation-by-distance between geographic
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distance (ED-CP) and both genetic distances (DXY, Pearson r = -0.47, P = 0.04; PiXY, Pearson r =
-0.49, P = 0.03) when Euclidean distances were accounted for.
DISCUSSION
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Many montane regions are hotspots for biodiversity (Myers et al., 2000; Mittermeier et
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al., 2005), a pattern that has been attributed both to the fact that mountains can act as cradles for
diversity by promoting isolation and speciation, but also act as museums, favoring the long term
persistence of lineages (Chown & Gaston, 2000; Kozak & Wiens, 2006; Wiens et al., 2007).
Factors that contribute to high diversity of montane biotas are topographic complexity that result
in high habitat heterogeneity and environmental diversity (Jetz et al., 2004), and the fact that
mountains can harbor climatically stable refugia compared to lowlands. We now have a good
understanding of the macroevolutionary patterns of species diversity in mountain landscapes
(Rahbek, 1995; Rahbek & Graves, 2001) and the climatic and ecological correlates of this
diversity (Francis & Currie, 2003; Hawkins et al., 2003; Oomen & Shanker, 2005; McCain, 2005
2007) but few studies examine in detail the timing and relative importance of evolutionary
processes that lead to population splitting and promote lineage survival in these same landscapes
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Parra-Olea et al., Page 15
(Wiens & Donoghue, 2004; Cardillo et al., 2005; Mittelbach et al., 2007; Tennessen & Zamudio,
2008). Examining the evolutionary underpinnings of other common diversity gradients has
revealed that many patterns are driven by local variation in the rate and timing of lineage
diversification (Cardillo et al., 2005; Stevens, 2006; Wiens et al., 2006; Ricklefs, 2006 ) and the
same pattern is expected to be repeated by individual species belonging to montane assemblages
(Fjeldså & Lovett, 1997; Ghalambor et al., 2006).
The Mexican highlands represent a significant focus of biodiversity in North America with
high degrees of endemism (Ramamoorthy et al., 1993; Bye, 1995) in plants (Nixon, 1993;
Styles, 1993; Turner & Nesom, 1993), insects (Lobo & Halffter, 2000; Morrone & Márquez,
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2003; Morrone, 2005), mammals (Fa & Morales, 2003; Escalante et al., 2005; Mena & VázquezDomínguez, 2005), birds (Escalante et al., 1993; McCormack et al., 2008), reptiles and
amphibians (Flores-Villela & Canseco-Márquez, 2007; Flores-Villela, 2010). Several lines of
evidence indicate that P. leprosa evolved in the northeastern sections of the TVB around the area
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of Cofre de Perote in the state of Veracruz, with subsequent range expansion to the Central TVB.
The presence of haplotypes exclusive to the Cofre de Perote and the fact that northeastern
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haplotypes form two distinct subclades indicate that the eastern mountains may have been
occupied by formerly widespread, contiguous populations which experienced fragmentation
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resulting from geological and climatological events. We found high within-population sequence
divergences for the northeastern populations of Vigas and Tlatlauquitepec, respectively. High
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haplotypic and nucleotidic diversity (Table 2), their basal position of haplotypes in the tree (Fig
1, Table 2), the lack of a signature of demographic expansion, higher Θ values and the sharing of
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haplotypes among populations in eastern and northeastern mountain ranges are indicative of
older source populations for population expansion throughout the rest of the species’ range.
Cofre de Perote volcano is found at the northeastern limit of the TVB (Figure 1). Its
structure, geochemistry, and volcanic history diverge significantly from that of the large
dominantly andesitic stratovolcanoes in other regions of the TVB (Carrasco-Núñez et al., 2007;
Diaz-Castellón et al., 2008). Cofre de Perote is 4282 m at its peak and rises more than 3000 m
above the coastal plain to the east; it formed as a massive low-angle compound shield volcano
that now dwarfs the more typical smaller shield volcanoes of the central and western TVB
(Carrasco-Núñez et al., 2010). The geological age of Cofre de Perote is recent (1.57-2.0 MYr
BP), its isolation from the west and center segments of TVB occurred during the late Pleistocene
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Parra-Olea et al., Page 16
with the formation of the Cuenca Oriental, the hydrological basin that delimits the eastern end of
the TVB. The Cuenca Oriental was reconnected with the México and Apan basins during a
period of intense volcanic activity in the early Holocene (Miller & Smith, 1986). However,
isolation of the Cofre de Perote from mountain ranges to the west was likely maintained due to
the altitudinal gradient established during the conformation of the eastern end of the TVB.
Combined, these landscape changes may have created environmental conditions that caused
population decreases and isolation, thus promoting divergence of the NE clades.
Studies of other highly diverse taxa in this region corroborate the Cofre de Perote as an
important historical refugial area. The plethodontid salamander fauna from this region is quite
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different from that found at lower elevations in the area of Vigas, only 5 km away.
Pseudoeurycea naucampatepetl from Cofre de Perote (Parra-Olea et al., 2005a) is sister taxon to
P. gigantea from Vigas with a 4.9% sequence divergence in cytb and substantial differences in
coloration. Salamanders of the genus Chiropterotriton (Darda, 1994; Parra-Olea, 2003) show the
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same pattern of divergence between these two neighboring regions. Finally, Cofre de Perote
populations of curculionid beetles (Anducho-Reyes et al., 2008) and pocket gophers (Hafner et
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al., 2005) are highly diverged from surrounding populations both genetically and
morphologically.
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In contrast, populations from mountain ranges in the Central TVB show very little
genetic divergence over a large geographic area, a pattern typical or populations resulting from
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geographic expansion (Hewitt, 1996; Grant & Bowen, 1998; Fet et al., 2005; Cortes-Rodríguez
et al., 2008). Relatively low values of π and high h for the entire data set indicate a model of
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range expansion for the entire range of P. leprosa, but neutrality tests and mismatch distributions
by region show the strongest signal of range expansion for the Central TVB clade populations.
Rapid population expansion often results in repeated bottlenecks along the expansion front, as
migrants move into newly available habitats and found populations (Hewitt, 1996). The genetic
consequences of these sequential founding events will be more pronounced when new habitats
are patchily distributed, such that the probability of colonization is low, and when it does occur,
the number of founders is also low (Hewitt, 1996). The stepping-stone colonization of mountains
in the TVB meets this requirement, and the low genetic diversity in the westernmost mountain
islands suggests that genetic drift due to founder’s events have significantly reduced population
genetic diversity during population expansion. Founders carrying ancestral haplotype 11
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colonized the western mountain islands, and the low number of mutational steps among that
common haplotype and the others in those newly founded populations indicate that those novel
haplotypes arose in situ during the relatively short history of independent evolution on each of
the isolated mountain islands. This historical scenario of Pleistocene colonization of mountains
in the Central TVB is corroborated by the estimates of historical migration among selected pairs
of mountain populations. The MPE of migration rates show higher migration from east to west
along the TVB. With a single mtDNA locus, we were not able to infer migration with high
confidence; however, the peaks of posterior distributions corroborate the sequential colonization
of mountain islands from a source populations in the region occupied by NE II clade.
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Our phylogeographic results are fully concordant with a study previously published by
Lynch et al. (1983) that examined relationships among Pseudoeurycea species based on
allozymes. That study included seven populations of P. leprosa along with samples of P.
longicauda, P. robertsi and P. altamontana, and reported high intraspecific levels of
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differentiation within P. leprosa. Lynch et al. (1983) found a ‘core group’ of populations along
the main E-W axis of the TVB that included populations from Zempoala, Popocatepetl,
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Iztaccihuatl and Malinche volcanic ranges, with only slight genetic differentiation among them
(Nei’s genetic distances [DN] = 0.002-0.012). In contrast, populations in the eastern regions of
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the species’ distribution (Cofre de Perote and Pico de Orizaba) showed high genetic divergences
when compared to each other and to the core populations. The greatest genetic divergence (DN =
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0.539) was found between the extreme northern population (Tlaxco, pop 17 Fig. 1) and the
extreme southeastern population San Bernardino (in the vicinity of Tres Mogotes, pop 26 Fig 1).
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Genetic differentiation between Las Vigas and Tlaxco, and all other populations, ranged from
0.240-0.437 and 0.325-0.539, respectively. Their samples from Xometla were intermediate, with
low genetic divergence from the core populations (DN = 0.015-0.022) and large genetic
divergence (DN = 0.167-0.325) from eastern populations. This population belongs to our SE
Clade, which is sister taxon to the Central TVB populations we sampled. The results of the
Lynch et al. (1983) study of allozyme markers indicate that the patterns we recovered based on
mtDNA markers are represented in patterns of nuclear diversification as well. Our more
complete sampling from throughout the range detected high divergences among populations in
the eastern mountain ranges, suggesting multiple isolated refugial populations, and confirmed the
east-west direction and recency of range expansion for this species.
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Parra-Olea et al., Page 18
Our data underscore the importance of peripheral isolation and local adaptation for
lineage diversification, especially in montane-adapted taxa where the distribution of appropriate
habitat is naturally discontinuous. Our phylogenetic analysis revealed deep genetic divergence
between the haplotype from the Tres Mogotes population and samples from other populations
throughout the range, and this haplotype does not cluster with any other sample in the haplotype
network. This population is in the extreme southeastern part of the distribution, and may have
served as a southern refugium. The isolation of this region seems to have caused this population
to diverge both genetically and physiologically from remaining P. leprosa populations.
Individuals from the Tres Mogotes population have persisted at the drier lower elevation site by
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colonizing a new microhabitat; adults in this population live within bromeliads much of the year,
in comparison to other P. leprosa populations that are terrestrial.
The genetic consequence of adaptation to montane habitats is also evident in the results
of least-cost migration routes among mountain islands. The effects of topographic complexity of
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the TVB, combined with this species’ low tolerance for high temperatures, have impacted
historical connectivity and diversification among populations. Both measures of pairwise
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population genetic differentiation do not correlate independently with Euclidean distance and
distance along least cost paths, but a partial Mantel test shows a significant correlation between
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Least-Cost Path distance and genetic differentiation once Euclidean distance has been accounted
for. This pattern indicates that it is not distance alone, but the elevational gradient along the least
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cost path among mountain ranges that has limited historical dispersal among populations, once
they became established on new mountain islands.
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Estimates of divergence times for major clades of P. leprosa populations provide
evidence on whether divergences in this group might be temporally correlated to the formation of
specific regions of the TVB. Our TMRCA estimates under various models of molecular
evolution indicate that divergence of the eastern populations occurred in the Pliocene (3.8 Mya),
while divergence of Central TVB populations diverged in Pleistocene (0.8 Mya). These timing
estimates reject the hypothesis that the formation of the Mexican highlands caused the
diversification of P. leprosa because lineage divergences within the group largely predate the
origin of the volcanic mountain ranges within the TVB. However, uplift of the TVB may have
continued into the Pliocene-Pleistocene (Ferrusquía-Villafranca & González-Guzmán, 2005) and
therefore might have played some role in the more recent differentiation of transvolcanic clades.
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Our divergence timing results for the Central TVB Clade are concordant with the timing of
glacial-interglacial cycles that characterized the last 0.7 Mya of the Pleistocene and that are
thought to have been the major contributor to biological diversification during this period (Webb
& Bartlein, 1992). Thus, although Pleistocene climate cycles do not explain divergences of
relictual populations in the northern and eastern TVB mountains, the expansion of this species to
the already formed mountains of the central TVB was certainly facilitated by the broad
distribution of cooler pine forests during glacial periods.
Given that montane regions are important centers of diversification, understanding and
maintaining the processes that lead to variation in species richness is also critical for
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conservation of montane taxa that are increasingly threatened (Kozak & Wiens, 2006; Smith et
al., 2007; Wiens et al., 2007). Many of the montane habitats of the Central TVB are protected
within National Parks (Parque Nacional Iztaccihuatl-Popocateptl, Cumbres del Ajusco, Desierto
de los Leones, Nevado de Toluca, Zoquiapán, Lagunas de Zempoala, La Malinche) but our data
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suggest that those protected areas alone are not sufficient to preserve most of the population
genetic diversity within this species. In fact, the most diverse, and likely source populations for
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the species in the eastern part of the range are not currently protected, and that area is also
subject to severe encroachment due to urban and agricultural development (García-Romero,
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2002; Galicia & García-Romero, 2007). The preservation of populations with high genetic
diversity is especially important for preserving evolutionary potential in the face of global
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environmental change. Genetic drift leads to the overall reduction of both neutral and adaptive
genetic variation (Frankham, 1996; Spielman et al., 2004; Willi et al., 2007), thus it is possible
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Journal of Biogeography
that central TVB populations of P. leprosa also show reduced genetic diversity at functional
genes that might be critical for local adaptation of populations to changing environments. We
know that this species will be faced with threats from projected changes in global temperatures
that will substantially decrease the distribution of appropriate habitat, especially in the Central
TVB (Parra-Olea et al., 2005b). Thus, preservation of remaining habitats in eastern TVB is
critical because those regions have highest chances of persistence with projected climate change,
and because those populations will retain the highest evolutionary potential for local adaptation
to changing environments (Parra-Olea et al., 2005b; Isaac, 2009).
Journal of Biogeography
Parra-Olea et al., Page 20
ACKNOWLEDGEMENTS
Molecular data for this project were collected in the Evolutionary Genetics Core Facility and the
Cornell Core Laboratories Sequencing Facility. Analyses benefited from resources of the
Computational Biology Service Unit at Cornell University, a facility partially funded by
Microsoft Corporation. We thank D. B. Wake, T. Papenfuss, J. Hanken, M. García-París, and E.
Recuero for help with field collections; Luis Canseco, G. Casas-Andreu and Noemí Matías for
providing tissues; Laura Marquez-Valdelamar for lab assistance; C. G. Becker for assistance
with GIS analyses; D. Buckley and I. Martínez-Solano for advice on molecular analyses; M.
Lopez-Uribe, R. Vega Bernal, and the Zamudio Lab for constructive comments on earlier
r
Fo
versions of the manuscript. During the preparation of this study, Guillermo Velo-Antón was
supported by a postdoctoral fellowship from the Spanish Ministerio de Ciencia e Innovación
(Ref: 2008-0890) and Gabriela Parra by a sabbatical Fellowship from UC-MEXUS. Field and
Pe
laboratory efforts were partially funded by grants from SEP-CONACyT (50563) and PAPIITUNAM (211808) to G. P-O; NSF Tree of Life program (Grant EF-0334939 to D. B. Wake and
M. H. Wake) and an NSF Population Evolutionary Processes Grant (DEB-0343526) to KZ.
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SUPPORTING INFORMATION
Additional supporting information may be found in the online version of this article:
Appendix S1 Collection localities, sample sizes, haplotypes, and voucher specimens for
Pseudoeurycea leprosa samples included in this study.
Appendix S2 Divergence time estimates for major nodes and coalescence times for
differentiation among haplotypes within mitochondrial clades of Pseudoeurycea leprosa.
Appendix S3 Geographic measures between pairs of mountain ranges.
BIOSKETCH
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-Olea is interested in the historical diversification and conservation of tropical herpetofauna. She
is currently a faculty member at the Instituto de Biologia, Universidad Autónoma de México, and
Pe
her lab focuses on population genetics, phylogeography, and systematic studies of reptiles and
amphibians endemic to México.
er
Author contributions: G.P-O. developed the research question; G.P-O and K.Z. acquired funding
to support fieldwork and laboratory data collection; J.C.W. completed all field sampling for the
Re
project; J.C.W. and G.V-A. collected the DNA sequence data; all authors contributed to analyses
of sequence data; G. P-O. and K.Z. led writing of the paper with important contributions from
others.
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FIGURE LEGENDS
Figure 1 Distribution of the 26 sampled populations of Pseudoeurycea leprosa across the TransVolcanic Belt (TVB) of México. Ovals group localities by mountain ranges. Locality symbols
represent the regional clades to which samples from each population belong: triangles =
Northeastern haplotypes (NE I and NE II), diamond = Tres Mogotes, white circles = Central
TVB haplotypes, and black circles = Southeast clade.
Figure 2 Bayesian phylogeny of Pseudoeurycea leprosa haplotypes from 26 populations
throughout the Trans-Volcanic Belt of México. Tip numbers refer to haplotype numbers in the
Appendix S1. Numbers above branches are posterior probabilities from Bayesian Inference.
r
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Older, refugial clades are demarcated by black bars, and clades showing genetic signature of
recent expansion are highlighted in gray; symbols after each clade name are concordant with
those identifying populations in Fig 1. Haplotype 11 in the Central TVB clade was also collected
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in the SE clade (identified by the circle after the haplotype name). Numbers in shaded boxes
correspond to estimates of diversification times (and confidence intervals) inferred from BEAST
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analyses for individual nodes (Appendix S2).
Figure 3 TCS haplotype network of genetic relationships among Pseudoeurycea leprosa
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populations. Haplotypes are connected assuming a 95% threshold. The size of each haplotype
symbol is proportional to its frequency and black dots represent mutational steps separating
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observed haplotypes. Circles represent haplotypes from the Central TVB mountain ranges and
the SE clade; colors group haplotypes by mountain ranges. Triangles represent haplotypes from
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Page 30 of 56
NE clades, and the single diamond corresponds to the haplotype obtained in the isolated
population of Tres Mogotes.
Figure 4 Bayesian skyline plots, and pairwise mismatch distributions for all populations of
Pseudoeurycea leprosa included in the study, and two representative clades (NE I clade and
Central TVB clade). The Bayesian skyline plots show evidence of large increases in population
size for the species as a whole. For mismatch analyses, the black lines/symbols represent the
observed frequency of pairwise differences among haplotypes, gray lines/symbols are the
distribution expected if the population has undergone historical demographic expansion. The
distribution of polymorphism in populations of the Central TVB clade indicates population
Page 31 of 56
Parra-Olea et al., Page 31
expansion; corresponding results of the goodness of fit and neutrality tests are reported in the
text and in Table 2.
Figure 5. Historical migration and timing of diversification in Pseudoeurycea leprosa
populations. Ovals group populations by mountain ranges, and discontinuous lines group the
different clades, with the exception of NE II clade (which includes one haplotype from
Tlatlauquitepec) and the SE clade (which includes one haplotype from Vigas, and the common
haplotype 11). The lower panel summarizes immigration rates among mountain ranges estimated
by LAMARC. Higher and lower immigration rates between populations are identified with arrows
of different colors.
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Table 1 Measures of genetic diversity (+/- SD), Tajima’s D, and Fu’s FS statistics for
Pseudoeurycea leprosa estimated by collection locality and by mountain system. Tlaxco and
Tres Mogotes samples are excluded because of low sample sizes for those two mountain ranges.
Nh
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D
-0.01975
0
0
-0.69681
0
0
0.03217
-1.8689*
0.63863
-1.20064
-1.76429**
-0.30187
-1.05482
-1.31009
-0.93302
-0.8165
-1.23716
0
0.39254
0.90413
1.45884
0
-1.13254
0
0
FS
1.0185
—
—
-0.62348
—
—
-1.41904
-1.58788
0.24158
-2.05648
-2.82924**
-2.8578*
-0.18197
0.76178
-0.00275
0.09021
-0.9218
—
2.81526
1.32573
1.68758
—
2.08813
—
—
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Mountain systems
Nevado de Toluca
Sierra de las Cruces
PopocatepetlIztaccihuatl
Malinche
Orizaba
Perote
Tres Mogotes
π (SD)
0.0022 +/-0.0017
0.0000 +/-0.0000
0.0000 +/-0.0000
0.0006 +/-0.0006
0.0000 +/-0.0000
0.0000 +/-0.0000
0.0058 +/-0.0035
0.0006 +/-0.0006
0.0014 +/-0.0011
0.0012 +/-0.0009
0.0014 +/-0.0010
0.0020 +/-0.0017
0.0003 +/-0.0005
0.0007 +/-0.0007
0.0004 +/-0.0006
0.0005 +/-0.0007
0.0008 +/-0.0008
0.0000 +/-0.0000
0.0051 +/-0.0031
0.0181 +/-0.0093
0.0017 +/-0.0015
0.0000 +/-0.0000
0.0093 +/-0.0058
0.0000 +/-0.0000
0.0000 +/-0.0000
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3
1
1
3
1
1
8
4
4
6
7
5
2
2
2
2
3
1
4
14
2
1
4
1
1
h (SD)
0.4643 +/- 0.2000
0.0000 +/- 0.0000
0.0000 +/- 0.0000
0.4044 +/-0.1304
0.0000 +/- 0.0000
0.0000 +/-0.0000
0.9231 +/-0.0500
0.2215 +/-0.1063
0.7138 +/-0.0358
0.5032 +/-0.0935
0.5988 +/-0.0799
0.9048 +/-0.1033
0.2500 +/-0.1802
0.2500 +/-0.1802
0.3333 +/-0.2152
0.4000 +/-0.2373
0.5238 +/-0.2086
1.0000 +/-0.0000
0.6484 +/-0.1163
0.9011 +/-0.0360
0.6000 +/-0.1753
1.0000 +/-0.0000
0.7143 +/-0.1809
1.0000 +/-0.0000
0.0000 +/-0.0000
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Localities
Nevado de Toluca
Zempoala
Santa Marta
Texcalyacac
Ajusco
Desierto de los Leones
Llano Grande
Rio Frio
Popocateptl
Atzompa
Calpan
Nanacamilpa
Malinche
Malinche
Malinche
Malinche
Texmalaquilla
Texmolae
Xometla
Vigas
González Ortega
Tezuitlán
Tlatlauquitepec
Tlaxco
Tres Mogotes
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3
7
0.4643 +/-0.2000
0.8122 +/-0.0372
0.0022 +/-0.0017
0.0041 +/-0.0025
-0.01975
-0.28474
1.0185
1.11531
34
5
25
4
1
0.9110 +/-0.0116
0.8348 +/-0.0365
0.8182 +/-0.0586
0.9440 +/-0.0186
0.0000 +/-0.0000
0.0037 +/-0.0022
0.0005 +/-0.0006
0.0043 +/-0.0026
0.0184 +/-0.0094
0.0000 +/-0.0000
-1.60172**
-2.00406**
-0.12932
0.66513
0
-24.9135*
-9.39137*
-0.71725
-1.67796
—
Nh, number of haplotypes; π nucleotide diversity; h, haplotype diversity. *P<0.01, ** P<0.05.
Page 33 of 56
Parra-Olea et al., Page 33
Table 2 Average number of nucleotide substitutions per site, DXY (above the diagonal) and
average number of pairwise differences, PiXY (below the diagonal) between mountain ranges.
Tlaxco and Tres Mogotes samples are excluded because of low sample sizes for those two
mountain ranges.
PiXY / DXY
Sierra
de las PopocatepetlCruces Iztaccihuatl Malinche Orizaba
Perote
Tlatlauquitepec
—
0.005
0.004
0.006
0.009
0.027
0.028
3.638
—
0.004
0.005
0.010
0.026
0.028
4.284
3.770
—
0.003
0.009
0.027
0.028
3.042
9.125
19.972
21.161
2.659
8.428
18.572
20.169
1.638
7.219
18.265
19.794
—
6.461
17.554
18.862
0.010
—
17.408
19.662
0.029
0.026
—
18.222
0.027
0.028
0.029
—
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Nevado de Toluca
Sierra de las
Cruces
PopocatepetlIztaccihuatl
Malinche
Orizaba
Perote
Tlatlauquitepec
Nevado
de
Toluca
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1
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Table 3 Effective population sizes (Θ) and asymmetric migration rates inferred in LAMARC for Pseudoeurycea leprosa. MPE (maximum
probable estimate) in italics and 95% CIs are in brackets. In bold, those estimated values with ESS lower than 300. Above the diagonal,
migration rates into all populations shown in bold. Below the diagonal, migration rates into the population shown in italics.
M
Nevado de
Toluca
Ajusco
Atzompa
Gonzalez
Ortega
Llano Grande
Malinche - E
Malinche - W
Popocatepetl
Tlatlauquitepec
Vigas
Xometla
Tlatlauquitepec
Vigas
Xometla
88.14
0.74
0.05
(0.01-729.65)
(0.01-578.05)
(0.01-141.54)
(0.01-70.36)
2.02
(0.012196.09)
114.07
135.65
70.99
0.95
0.24
10.85
(0.01-196.27)
(0.01-655.94)
(0.01-690.81)
(0.01-544.39)
(0.01-137.79)
(0.01-75.28)
(0.01-213.70)
22.65
798.86
424.03
265.49
0.17
0.19
25.23
(0.01-183.19)
rR
(0.07-1036.36)
(0.01-1074.11)
(0.01-885.24)
(0.01-118.30)
(0.01-55.97)
(0.01-318.74)
0.27
78.76
64.17
28.66
15.21
44.99
11.70
(0.01-129.19)
(0.01-568.42)
(0.01-628.35)
(0.01-448.59)
(0.01-176.54)
(0.03-205.49)
(0.01-254.94)
188.25
204.35
144.26
2.51
0.66
24.05
(0.01-543.13)
*
(0.01-807.92)
(0.01-785.50)
(0.01-664.00)
(0.01-134.40)
(0.01-52.45)
(0.01-234.10)
333.46
3.70
25.75
*
389.20
234.74
0.09
0.09
47.82
(0.01-729.34)
(0.67-903.92)
(0.01-537.59)
(0.01-201.89)
(0.01-1035.15)
(0.01-860.28)
(0.01-131.30)
(0.01-75.46)
(0.01-334.09)
110.64
124.66
10.20
23.01
323.14
*
205.31
0.84
0.46
42.82
(0.01-494.81)
(0.01-718.76)
(0.01-622.76)
(0.01-536.59)
(0.01-179.14)
(0.01-965.820)
*
(0.01-858.55)
(0.01-130.60)
(0.01-62.88)
(0.01-295.64)
0.00057
83.67
153.55
159.61
12.74
35.17
408.43
448.52
*
2.38
0.71
28.49
(0.00009-0.002271)
(0.01-537.58)
(0.01-746.78)
(0.01-718.06)
(0.01-512.56)
(0.01-203.22)
(0.02-1051.68)
(0.01-1103.91)
*
(0.01-133.38)
(0.01-60.47)
(0.01-308.04)
0.00349
57.02
113.18
30.33
109.65
0.27
87.06
90.22
28.28
*
39.40
24.77
(0.00083-0.016096)
(0.01-472.70)
(0.01-678.25)
(0.01-305.24)
(0.01-640.58)
(0.01-127.88)
(0.01-581.27)
(0.01-624.99)
(0.01-429.74)
*
(0.02-227.49)
(0.01-382.09)
0.01114
55.19
148.84
24.54
550.40
0.86
118.35
102.67
47.79
100.38
*
111.11
(0.00459-0.025106)
(0.01-511.29)
(0.01-795.57)
(0.01-319.48)
(0.02-1047.54)
(0.01-122.93)
(0.01-706.66)
(0.01-686.77)
(0.01-519.59)
(0.01-523.05)
*
(0.02-618.66)
0.00212
38.39
112.08
99.11
59.64
22.65
360.63
402.13
155.43
8.55
32.89
*
(0.00050-0.005794)
(0.01-448.79)
(0.01-712.57)
(0.01-571.88)
(0.01-634.66)
(0.01-153.43)
(0.01-1001.69)
(0.01-1049.42)
(0.01-706.81)
(0.01-237.51)
(0.01-173.48)
*
Θ MPE
(95% CI)
Nevado de
Toluca
Ajusco
Atzompa
Gonzalez
Ortega
Llano
Grande
Malinche- E
0.00082
*
474.14
52.27
3.96
19.36
133.00
126.02
(0.00017-0.003141)
*
(0.02-1064.63)
(0.012-382.28)
(0.01-512.95)
(0.01-176.66)
(0.01-698.10)
0.00001
238.31
*
39.31
16.43
32.61
(0.00001-0.000556)
(0.02-837.32)
*
(0.01-347.47)
(0.01-536.42)
0.00179
39.78
107.71
*
0.03
(0.00044-0.006185)
(0.01-523.99)
(0.01-701.63)
*
(0.01-493.38)
0.00034
30.52
102.36
11.48
*
(0.00004-0.002554)
(0.01-432.23)
(0.012-679.84)
(0.01-290.46)
*
0.00406
99.15
264.20
80.61
3.93
(0.00168-0.011851)
(0.01-574.62)
(0.01-867.67)
(0.01-440.68)
0.00038
59.25
144.10
(0.00002-0.002465)
(0.01-531.59)
0.00033
52.01
(0.00002-0.001868)
Fo
rP
ee
*
ev
*
Malinche-W Popocatepetl
iew
Page 35 of 56
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Page 37 of 56
Parra-Olea et al., Page 37
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Page 39 of 56
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Journal of Biogeography
Parra-Olea et al., Page 39
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Journal of Biogeography
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Page 40 of 56
Appendix S1 Pseudoeurycea leprosa samples included in this study. For each haplotype identified in our sample, we list the mountain
range, and population of origin, the number of individuals with that haplotype (N), the clade to which it belongs, GPS
coordinates/elevation where those individuals were collected, and the voucher specimens. The population ID numbers correspond to
those in Figure 1. Voucher specimens acronims are as follows: IBH: Coleccion Nacional de Anfibios y Reptiles, Instituto de Biología,
Universidad Nacional Autónoma de México [GP and JCW are unaccessioned specimens from IBH]; MVZ: Museum of Vertebrate
Zoology, University of California, Berkeley; ENCB: Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional; MZFC:
Museo de Zoología de la Facultad de Ciencias, UNAM [AMH, LECHA and ISZ are unaccessioned specimens from MZFC].
Mountain chain
Population
Fo
rP
Clade
Nevado de Toluca Nevado de Toluca
Pop
ID
1
Nevado de Toluca Nevado de Toluca
1
Central
Nevado de Toluca Nevado de Toluca
Sierra de las
Cruces
Sierra de las
Cruces
Sierra de las
Cruces
Sierra de las
Cruces
Sierra de las
Cruces
Sierra de las
Cruces
Sierra de las
Cruces
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
Central
1
Central
Texcalyacac
2
Central
Texcalyacac
2
Texcalyacac
Haplo N
1
6
19 11 37 N
99 50 53 W
Elev
(m)
3310
2
1
19 11 37 N
99 50 53 W
3310
IBH2241
ee
Latitude
Longitude
Voucher
IBH: 22393-22395, 22391, 22310, 22312
3
1
19 11 37 N
3310
IBH22413
13 19 07 14 N
99 30 00 W
3091
Central
5
1
19 07 14 N
99 30 00 W
3091
IBH: 18199, 223119, 18201-18209, 18211,
22318
IBH18200
2
Central
6
rR
99 50 53 W
4
3
19 07 14 N
99 30 00 W
3091
IBH: 18210, 18214, 18212
Desierto de los
Leones
Ajusco
3
Central
7
5
19 16 00 N
99 18 02 W
3300
IBH22428, 22425, 22410, GP795, GP796
4
Central
8
8
19 10 58 N
99 18 00 W
3500
Santa Marta
5
Central
9
3
19 03 41 N
99 19 17 W
3110
IBH: 22459, 22424, 2247, 22418, 22442,
22404, 22439, 22399
IBH: 22438, 22461, 22462
Zempoala
6
Central
10
5
19 03 12 N
99 18 35 W
3200
IBH: 22392, 22419, 22453, 22427, 22458
Nanacamilpa
7
Central
11
1
19 28 49 N
98 35 46 W
2800
JCW051
Nanacamilpa
7
Central
12
1
19 28 49 N
98 35 46 W
2800
JCW053
Nanacamilpa
7
Central
13
1
19 28 49 N
98 35 46 W
2800
JCW058
Nanacamilpa
7
Central
14
2
19 28 49 N
98 35 46 W
2800
JCW054, JCW060
ev
iew
Page 41 of 56
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9
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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30
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33
34
35
36
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Journal of Biogeography
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
Nanacamilpa
7
Central
15
2
19 28 49 N
98 35 46 W
2800
JCW052, JCW061
Río Frío
8
Central
16
1
19 21 58 N
98 41 41 W
3075
IBH22344
Río Frío
8
Central
17
23 19 21 58 N
98 41 41 W
3075
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
Río Frío
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
Popocatepetl-
8
Central
18
1
19 21 58 N
98 41 41 W
3075
IBH: 22451, 22412, 22398, 22445, 22456,
22455, 22444, 22409, 22354, 22360, 22366,
22352, 22363, 22290, 22296, 22289.
JCW386-392. ENCB17783
IBH 22426
8
Central
19
1
19 21 58 N
98 41 41 W
3075
IBH 22446
9
Central
17
3
19 20 20 N
98 43 14 W
3200
IBH 22293, 22283, 22279
Llano Grande
9
Central
Llano Grande
9
Central
Llano Grande
9
Central
ee
Llano Grande
9
Llano Grande
Río Frío
Llano Grande
Fo
rP
20
1
19 20 20 N
98 43 14 W
3200
ENCB17773
21
2
19 20 20 N
98 43 14 W
3200
ENCB17774, ENCB17775
22
2
19 20 20 N
98 43 14 W
3200
Central
23
2
19 20 20 N
98 43 14 W
3200
9
Central
24
1
19 20 20 N
98 43 14 W
3200
ENCB17779,
ENCB17782
ENCB17777
ENCB17780
ENCB17772
Llano Grande
9
Central
25
1
19 20 20 N
98 43 14 W
3200
ENCB17781
Llano Grande
9
Central
26
1
19 20 20 N
98 43 14 W
3200
IBH22276
Atzompa
10
Central
11
25 19 10 50 N
98 33 35 W
3050
Atzompa
10
Central
20
1
19 10 50 N
98 33 35 W
3050
IBH: 22286, 22337, 22320-6, 22330, 22319,
22313-14, 22338-40, 22335, 22331-33,
22389, 22328, 22317, 22315, JCW404
22385
Atzompa
10
Central
27
1
19 10 50 N
98 33 35 W
3050
IBH22431
Atzompa
10
Central
28
5
19 10 50 N
98 33 35 W
3050
IBH: 22433, 22436, 2244, 22435, 22336
Atzompa
10
Central
29
3
19 10 50 N
98 33 35 W
3050
IBH: 22429, 22432, 22430
Atzompa
10
Central
30
1
19 10 50 N
98 33 35 W
3050
IBH22434
rR
ev
iew
Journal of Biogeography
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Page 42 of 56
Iztaccihuatl
PopocatepetlIztaccihuatl
Calpán
11
Central
11
19 19 07 53 N
98 35 30 W
3100
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
Malinche
Calpán
11
Central
31
1
19 07 53 N
98 35 30 W
3100
IBH: 22368, 18226-27, 18217-19, 18221-22
22361, 22358, 22356, 22362, 22367, 22364,
22357, JCW105-108
IBH18220
Calpán
11
Central
32
1
19 07 53 N
98 35 30 W
3100
JCW109
Calpán
Fo
11
Central
33
1
19 07 53 N
98 35 30 W
3100
IBH18224
11
Central
34
1
19 07 53 N
98 35 30 W
3100
JCW104
11
Central
35
8
19 07 53 N
98 35 30 W
3100
Calpán
11
Central
Popocatepetl
12
Central
Popocatepetl
12
Central
Popocatepetl
12
Popocatepetl
Calpán
Calpán
rP
36
1
19 07 53 N
98 35 30 W
3100
IBH: 22386, 22365, 22347, 22346, 22351,
22351, 22359, 22342, 22348
IBH22343
11
1
19 04 22 N
3200
JCW405
36
9
19 04 22 N
98 42 42 W
3200
Central
37
rR
98 42 42 W
7
19 04 22 N
98 42 42 W
3200
12
Central
38
9
19 04 22 N
98 42 42 W
3200
Malinche West
13
Central
11
7
19 15 28 N
98 05 42 W
Malinche West
13
Central
39
1
19 15 28 N
98 05 42 W
Malinche
Malinche North
14
Central
11
4
19 16 37 N
98 02 40 W
Malinche
Malinche North
14
Central
40
1
19 16 37 N
98 02 40 W
Malinche
Malinche South
15
Central
11
5
19 11 14 N
98 01 19 W
Malinche
Malinche South
15
Central
41
1
19 11 14 N
98 01 19 W
Malinche
Malinche East
16
Central
11
7
19 13 48 N
97 58 30 W
Malinche
Malinche East
16
Central
42
1
19 13 48 N
97 58 30 W
29503050
29503050
29503050
29503050
29503050
29503050
29503050
29503050
IBH: 22457, 22454, 22460, 22448, 22387,
22349, 22350, 22355. JCW345.
IBH: 22423, 22437, 22406, 22463, 22288,
22291. JCW406
IBH: 22281-2, 22277, 22284, 22369, 22345,
22370, 22341. JCW343
IBH: 22376, 22383, 22317, 22378, 22375.
JCW39, JCW32
IBH22374
ee
ev
iew
IBH: 22308, 22300, 22306, 22297
IBH22308
IBH: 22377, 22379, 22353, 22396, 22416
IBH22373
IBH: 22303-5, 22298, 22307, 22299, 22301
IBH22302
Page 43 of 56
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22
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Journal of Biogeography
Tlaxco
Tlaxco
17
NE I
43
1
19 41 45 N
98 04 33 W
2880
IBH22417
Tlatlauquitepec
18
NE II
44
1
19 42 47 N
97 32 24 W
2800
ISZ103
18
NE I
45
4
19 42 47 N
97 32 24 W
2800
IBH: 22275, 22271, AMH425, MZFC 20611
19
NE I
46
1
19 42 42 N
97 28 34 W
2800
JCW535 (IBH22267)
19
NE I
47
1
19 42 42 N
97 28 34 W
2800
JCW536 (IBH22256)
Cofre de Perote
Tlautlauquitepec
A
Tlautlauquitepec
A
Tlautlauquitepec
B
Tlautlauquitepec
B
Teziutlán
20
Polytomy
48
1
19 39 21 N
97 15 00 W
Cofre de Perote
Vigas
21
Southeast
49
1
19 37 51 N
97 05 26 W
2985
IBH22334
Cofre de Perote
Vigas
21
Polytomy
50
1
19 37 51 N
97 05 26 W
2985
IBH22272
Cofre de Perote
Vigas
21
NE II
51
1
19 37 51 N
97 05 26 W
2995
IBH22408
Cofre de Perote
Vigas
21
NE II
52
3
19 37 51 N
97 05 26 W
2995
IBH: 22450, 22415, 22384)
Cofre de Perote
Vigas
21
NE II
53
1
19 37 51 N
97 05 26 W
2985
IBH22253
Cofre de Perote
Vigas
21
NE II
54
3
19 37 51 N
97 05 26 W
2995
IBH: 22403, 22411, 2240
Cofre de Perote
Vigas
21
NE II
55
1
19 37 51 N
97 05 26 W
2985
IBH22257
Cofre de Perote
Vigas
21
Polytomy
56
1
19 37 51 N
97 05 26 W
2985
IBH22263
Cofre de Perote
Vigas
21
Polytomy
57
1
19 37 51 N
97 05 26 W
2985
IBH22329
Cofre de Perote
Vigas
21
Polytomy
58
1
19 37 51 N
97 05 26 W
3100
IBH22407
Cofre de Perote
Vigas
21
Polytomy
48
4
19 37 51 N
97 05 26 W
3100
IBH: 22421-22, 22449, 22443
Cofre de Perote
Vigas
21
Polytomy
59
8
19 37 51 N
97 05 26 W
2985
Cofre de Perote
Vigas
21
Polytomy
60
3
19 37 51 N
97 05 26 W
2985
IBH: 22252, 22273, 22258, 22268, 22265,
22262, 22269, 22270
IBH: 22260, 22264, 22254
Cofre de Perote
Vigas
21
Polytomy
61
1
19 37 51 N
97 05 26 W
2985
IBH22255
Cofre de Perote
González Ortega
22
Polytomy
62
3
19 21 2 N
97 14 49 W
3000
IBH22274, 22261, 22251
Cofre de Perote
González Ortega
22
Polytomy
63
2
19 21 2 N
97 14 49 W
3000
IBH: 22266, 22259
Pico de Orizaba
Texmalaquilla
23
Southeast
64
1
18 56 31 N
97 17 24 W
2925
IBH22316
Pico de Orizaba
Texmalaquilla
23
Southeast
65
5
18 56 31 N
97 17 24 W
2925
IBH: 22380, 22381, 22382, 22390, 22327
Pico de Orizaba
Texmalaquilla
23
Southeast
66
1
18 56 31 N
97 17 24 W
2925
IBH22372
Tlatlauquitepec
Tlatlauquitepec
Tlatlauquitepec
Fo
rP
ee
rR
ev
MVZ138396
iew
Journal of Biogeography
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
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Page 44 of 56
Pico de Orizaba
Texmola
24
Southeast
65
1
18 56 08 N
97 14 40 W
2640
MVZ136959
Pico de Orizaba
Xometla
25
Central
11
3
18 58 30 N
97 11 26 W
3100
IBH: 22452, 22401, 2239
Pico de Orizaba
Xometla
25
Southeast
67
2
18 58 30 N
97 11 26 W
3100
IBH22414, 22402
Pico de Orizaba
Xometla
25
Southeast
68
1
18 58 30 N
97 11 26 W
3100
IBH22388
Pico de Orizaba
Xometla
25
Southeast
69
8
18 58 30 N
97 11 26 W
3100
Tres Mogotes
Tres Mogotes
26
Tres
Mogotes
70
2
18 39 28 N
97 29 24 W
2600
22400, 22285, 22280, 22287, 22278, 22292,
GP242, GP262
IBH22295, LECHA3344
Fo
rP
ee
rR
ev
iew
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Journal of Biogeography
Appendix S1 Pseudoeurycea leprosa samples included in this study. For each haplotype identified in our sample, we list the mountain
range, and population of origin, the number of individuals with that haplotype (N), the clade to which it belongs, GPS
coordinates/elevation where those individuals were collected, and the voucher specimens. The population ID numbers correspond to
those in Figure 1. Voucher specimens acronims are as follows: IBH: Coleccion Nacional de Anfibios y Reptiles, Instituto de Biología,
Universidad Nacional Autónoma de México [GP and JCW are unaccessioned specimens from IBH]; MVZ: Museum of Vertebrate
Zoology, University of California, Berkeley; ENCB: Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional; MZFC:
Museo de Zoología de la Facultad de Ciencias, UNAM [AMH, LECHA and ISZ are unaccessioned specimens from MZFC].
Mountain chain
Population
Fo
rP
Clade
Nevado de Toluca Nevado de Toluca
Pop
ID
1
Nevado de Toluca Nevado de Toluca
1
Central
Nevado de Toluca Nevado de Toluca
Sierra de las
Cruces
Sierra de las
Cruces
Sierra de las
Cruces
Sierra de las
Cruces
Sierra de las
Cruces
Sierra de las
Cruces
Sierra de las
Cruces
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
Central
1
Central
Texcalyacac
2
Central
Texcalyacac
2
Texcalyacac
Haplo N
1
6
19 11 37 N
99 50 53 W
Elev
(m)
3310
2
1
19 11 37 N
99 50 53 W
3310
IBH2241
ee
Latitude
Longitude
Voucher
IBH: 22393-22395, 22391, 22310, 22312
3
1
19 11 37 N
3310
IBH22413
13 19 07 14 N
99 30 00 W
3091
Central
5
1
19 07 14 N
99 30 00 W
3091
IBH: 18199, 223119, 18201-18209, 18211,
22318
IBH18200
2
Central
6
rR
99 50 53 W
4
3
19 07 14 N
99 30 00 W
3091
IBH: 18210, 18214, 18212
Desierto de los
Leones
Ajusco
3
Central
7
5
19 16 00 N
99 18 02 W
3300
IBH22428, 22425, 22410, GP795, GP796
4
Central
8
8
19 10 58 N
99 18 00 W
3500
Santa Marta
5
Central
9
3
19 03 41 N
99 19 17 W
3110
IBH: 22459, 22424, 2247, 22418, 22442,
22404, 22439, 22399
IBH: 22438, 22461, 22462
Zempoala
6
Central
10
5
19 03 12 N
99 18 35 W
3200
IBH: 22392, 22419, 22453, 22427, 22458
Nanacamilpa
7
Central
11
1
19 28 49 N
98 35 46 W
2800
JCW051
Nanacamilpa
7
Central
12
1
19 28 49 N
98 35 46 W
2800
JCW053
Nanacamilpa
7
Central
13
1
19 28 49 N
98 35 46 W
2800
JCW058
Nanacamilpa
7
Central
14
2
19 28 49 N
98 35 46 W
2800
JCW054, JCW060
ev
iew
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23
24
25
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PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
Nanacamilpa
7
Central
15
2
19 28 49 N
98 35 46 W
2800
JCW052, JCW061
Río Frío
8
Central
16
1
19 21 58 N
98 41 41 W
3075
IBH22344
Río Frío
8
Central
17
23 19 21 58 N
98 41 41 W
3075
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
Río Frío
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
Popocatepetl-
8
Central
18
1
19 21 58 N
98 41 41 W
3075
IBH: 22451, 22412, 22398, 22445, 22456,
22455, 22444, 22409, 22354, 22360, 22366,
22352, 22363, 22290, 22296, 22289.
JCW386-392. ENCB17783
IBH 22426
8
Central
19
1
19 21 58 N
98 41 41 W
3075
IBH 22446
9
Central
17
3
19 20 20 N
98 43 14 W
3200
IBH 22293, 22283, 22279
Llano Grande
9
Central
Llano Grande
9
Central
Llano Grande
9
Central
ee
Llano Grande
9
Llano Grande
Río Frío
Llano Grande
Fo
rP
20
1
19 20 20 N
98 43 14 W
3200
ENCB17773
21
2
19 20 20 N
98 43 14 W
3200
ENCB17774, ENCB17775
22
2
19 20 20 N
98 43 14 W
3200
Central
23
2
19 20 20 N
98 43 14 W
3200
9
Central
24
1
19 20 20 N
98 43 14 W
3200
ENCB17779,
ENCB17782
ENCB17777
ENCB17780
ENCB17772
Llano Grande
9
Central
25
1
19 20 20 N
98 43 14 W
3200
ENCB17781
Llano Grande
9
Central
26
1
19 20 20 N
98 43 14 W
3200
IBH22276
Atzompa
10
Central
11
25 19 10 50 N
98 33 35 W
3050
Atzompa
10
Central
20
1
19 10 50 N
98 33 35 W
3050
IBH: 22286, 22337, 22320-6, 22330, 22319,
22313-14, 22338-40, 22335, 22331-33,
22389, 22328, 22317, 22315, JCW404
22385
Atzompa
10
Central
27
1
19 10 50 N
98 33 35 W
3050
IBH22431
Atzompa
10
Central
28
5
19 10 50 N
98 33 35 W
3050
IBH: 22433, 22436, 2244, 22435, 22336
Atzompa
10
Central
29
3
19 10 50 N
98 33 35 W
3050
IBH: 22429, 22432, 22430
Atzompa
10
Central
30
1
19 10 50 N
98 33 35 W
3050
IBH22434
rR
ev
iew
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22
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Journal of Biogeography
Iztaccihuatl
PopocatepetlIztaccihuatl
Calpán
11
Central
11
19 19 07 53 N
98 35 30 W
3100
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
PopocatepetlIztaccihuatl
Malinche
Calpán
11
Central
31
1
19 07 53 N
98 35 30 W
3100
IBH: 22368, 18226-27, 18217-19, 18221-22
22361, 22358, 22356, 22362, 22367, 22364,
22357, JCW105-108
IBH18220
Calpán
11
Central
32
1
19 07 53 N
98 35 30 W
3100
JCW109
Calpán
Fo
11
Central
33
1
19 07 53 N
98 35 30 W
3100
IBH18224
11
Central
34
1
19 07 53 N
98 35 30 W
3100
JCW104
11
Central
35
8
19 07 53 N
98 35 30 W
3100
Calpán
11
Central
Popocatepetl
12
Central
Popocatepetl
12
Central
Popocatepetl
12
Popocatepetl
Calpán
Calpán
rP
36
1
19 07 53 N
98 35 30 W
3100
IBH: 22386, 22365, 22347, 22346, 22351,
22351, 22359, 22342, 22348
IBH22343
11
1
19 04 22 N
3200
JCW405
36
9
19 04 22 N
98 42 42 W
3200
Central
37
rR
98 42 42 W
7
19 04 22 N
98 42 42 W
3200
12
Central
38
9
19 04 22 N
98 42 42 W
3200
Malinche West
13
Central
11
7
19 15 28 N
98 05 42 W
Malinche West
13
Central
39
1
19 15 28 N
98 05 42 W
Malinche
Malinche North
14
Central
11
4
19 16 37 N
98 02 40 W
Malinche
Malinche North
14
Central
40
1
19 16 37 N
98 02 40 W
Malinche
Malinche South
15
Central
11
5
19 11 14 N
98 01 19 W
Malinche
Malinche South
15
Central
41
1
19 11 14 N
98 01 19 W
Malinche
Malinche East
16
Central
11
7
19 13 48 N
97 58 30 W
Malinche
Malinche East
16
Central
42
1
19 13 48 N
97 58 30 W
29503050
29503050
29503050
29503050
29503050
29503050
29503050
29503050
IBH: 22457, 22454, 22460, 22448, 22387,
22349, 22350, 22355. JCW345.
IBH: 22423, 22437, 22406, 22463, 22288,
22291. JCW406
IBH: 22281-2, 22277, 22284, 22369, 22345,
22370, 22341. JCW343
IBH: 22376, 22383, 22317, 22378, 22375.
JCW39, JCW32
IBH22374
ee
ev
iew
IBH: 22308, 22300, 22306, 22297
IBH22308
IBH: 22377, 22379, 22353, 22396, 22416
IBH22373
IBH: 22303-5, 22298, 22307, 22299, 22301
IBH22302
Journal of Biogeography
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Page 48 of 56
Tlaxco
Tlaxco
17
NE I
43
1
19 41 45 N
98 04 33 W
2880
IBH22417
Tlatlauquitepec
18
NE II
44
1
19 42 47 N
97 32 24 W
2800
ISZ103
18
NE I
45
4
19 42 47 N
97 32 24 W
2800
IBH: 22275, 22271, AMH425, MZFC 20611
19
NE I
46
1
19 42 42 N
97 28 34 W
2800
JCW535 (IBH22267)
19
NE I
47
1
19 42 42 N
97 28 34 W
2800
JCW536 (IBH22256)
Cofre de Perote
Tlautlauquitepec
A
Tlautlauquitepec
A
Tlautlauquitepec
B
Tlautlauquitepec
B
Teziutlán
20
Polytomy
48
1
19 39 21 N
97 15 00 W
Cofre de Perote
Vigas
21
Southeast
49
1
19 37 51 N
97 05 26 W
2985
IBH22334
Cofre de Perote
Vigas
21
Polytomy
50
1
19 37 51 N
97 05 26 W
2985
IBH22272
Cofre de Perote
Vigas
21
NE II
51
1
19 37 51 N
97 05 26 W
2995
IBH22408
Cofre de Perote
Vigas
21
NE II
52
3
19 37 51 N
97 05 26 W
2995
IBH: 22450, 22415, 22384)
Cofre de Perote
Vigas
21
NE II
53
1
19 37 51 N
97 05 26 W
2985
IBH22253
Cofre de Perote
Vigas
21
NE II
54
3
19 37 51 N
97 05 26 W
2995
IBH: 22403, 22411, 2240
Cofre de Perote
Vigas
21
NE II
55
1
19 37 51 N
97 05 26 W
2985
IBH22257
Cofre de Perote
Vigas
21
Polytomy
56
1
19 37 51 N
97 05 26 W
2985
IBH22263
Cofre de Perote
Vigas
21
Polytomy
57
1
19 37 51 N
97 05 26 W
2985
IBH22329
Cofre de Perote
Vigas
21
Polytomy
58
1
19 37 51 N
97 05 26 W
3100
IBH22407
Cofre de Perote
Vigas
21
Polytomy
48
4
19 37 51 N
97 05 26 W
3100
IBH: 22421-22, 22449, 22443
Cofre de Perote
Vigas
21
Polytomy
59
8
19 37 51 N
97 05 26 W
2985
Cofre de Perote
Vigas
21
Polytomy
60
3
19 37 51 N
97 05 26 W
2985
IBH: 22252, 22273, 22258, 22268, 22265,
22262, 22269, 22270
IBH: 22260, 22264, 22254
Cofre de Perote
Vigas
21
Polytomy
61
1
19 37 51 N
97 05 26 W
2985
IBH22255
Cofre de Perote
González Ortega
22
Polytomy
62
3
19 21 2 N
97 14 49 W
3000
IBH22274, 22261, 22251
Cofre de Perote
González Ortega
22
Polytomy
63
2
19 21 2 N
97 14 49 W
3000
IBH: 22266, 22259
Pico de Orizaba
Texmalaquilla
23
Southeast
64
1
18 56 31 N
97 17 24 W
2925
IBH22316
Pico de Orizaba
Texmalaquilla
23
Southeast
65
5
18 56 31 N
97 17 24 W
2925
IBH: 22380, 22381, 22382, 22390, 22327
Pico de Orizaba
Texmalaquilla
23
Southeast
66
1
18 56 31 N
97 17 24 W
2925
IBH22372
Tlatlauquitepec
Tlatlauquitepec
Tlatlauquitepec
Fo
rP
ee
rR
ev
MVZ138396
iew
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19
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Journal of Biogeography
Pico de Orizaba
Texmola
24
Southeast
65
1
18 56 08 N
97 14 40 W
2640
MVZ136959
Pico de Orizaba
Xometla
25
Central
11
3
18 58 30 N
97 11 26 W
3100
IBH: 22452, 22401, 2239
Pico de Orizaba
Xometla
25
Southeast
67
2
18 58 30 N
97 11 26 W
3100
IBH22414, 22402
Pico de Orizaba
Xometla
25
Southeast
68
1
18 58 30 N
97 11 26 W
3100
IBH22388
Pico de Orizaba
Xometla
25
Southeast
69
8
18 58 30 N
97 11 26 W
3100
Tres Mogotes
Tres Mogotes
26
Tres
Mogotes
70
2
18 39 28 N
97 29 24 W
2600
22400, 22285, 22280, 22287, 22278, 22292,
GP242, GP262
IBH22295, LECHA3344
Fo
rP
ee
rR
ev
iew
Journal of Biogeography
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Page 51 of 56
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Journal of Biogeography
Appendix S2 Divergence time estimates for major nodes and coalescence times for differentiation among haplotypes within
mitochondrial clades of Pseudoeurycea leprosa. Mean time estimate values and 95% confidence intervals were inferred using three
coalescent models in BEAST; estimated ages are reported in millions of years. Nodes 1-9 and clade names correspond to those in
Figure 2.
Clade
1
2
3
4
5
6
7
8
Clade Name
C
C+SE
C+SE+NEII
C+SE+NEI+NEII+Tres
Mogotes
NEII
NEI
SE
All P. leprosa
Fo
C=Central, SE=Southeast, NE=Northeast.
rP
Skyline
0.79 (0.25-1.55)
1.46 (0.43-2.91)
3.29 (1.08-6.42)
ee
Exponential
0.81 (0.31-1.5)
1.30 (0.49-2.59)
2.74 (0.98-5.04)
3.70 (1.3-7.1)
1.21 (0.33-2.46)
0.94 (0.21-2.02)
0.51 (0.14-1.07)
3.80 (1.4-7.38)
rR
3.29 (1.33-5.95)
1.07 (0.33-2.09)
0.85 (0.20-1.71)
0.462 (0.09-0.96)
3.29 (1.33-5.95)
ev
Expansion
0.83 (0.31-1.52)
1.30 (0.49-2.54)
2.77 (0.97-4.8)
3.20 (1.2-5.8)
1.06 (0.34-2.01)
0.86 (0.21-1.69)
0.50 (0.13-1.0)
3.25 (1.3-5.8)
iew
Journal of Biogeography
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Page 52 of 56
Appendix S2 Divergence time estimates for major nodes and coalescence times for differentiation among haplotypes within
mitochondrial clades of Pseudoeurycea leprosa. Mean time estimate values and 95% confidence intervals were inferred using three
coalescent models in BEAST; estimated ages are reported in millions of years. Nodes 1-9 and clade names correspond to those in
Figure 2.
Clade
1
2
3
4
5
6
7
8
Clade Name
C
C+SE
C+SE+NEII
C+SE+NEI+NEII+Tres
Mogotes
NEII
NEI
SE
All P. leprosa
Fo
C=Central, SE=Southeast, NE=Northeast.
rP
Skyline
0.79 (0.25-1.55)
1.46 (0.43-2.91)
3.29 (1.08-6.42)
ee
Exponential
0.81 (0.31-1.5)
1.30 (0.49-2.59)
2.74 (0.98-5.04)
3.70 (1.3-7.1)
1.21 (0.33-2.46)
0.94 (0.21-2.02)
0.51 (0.14-1.07)
3.80 (1.4-7.38)
rR
3.29 (1.33-5.95)
1.07 (0.33-2.09)
0.85 (0.20-1.71)
0.462 (0.09-0.96)
3.29 (1.33-5.95)
ev
Expansion
0.83 (0.31-1.52)
1.30 (0.49-2.54)
2.77 (0.97-4.8)
3.20 (1.2-5.8)
1.06 (0.34-2.01)
0.86 (0.21-1.69)
0.50 (0.13-1.0)
3.25 (1.3-5.8)
iew
Page 53 of 56
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Journal of Biogeography
Appendix S3 Geographic measures between pairs of mountain ranges: A) straight Euclidean
distances (km), B) Euclidean distances along least-cost paths (km), and C) the cumulative cost
along least-cost paths. Tlaxco and Tres Mogotes samples are excluded from the IBD analyses
because of low sample sizes for those two mountain ranges.
Nevad
o de
Toluca
A
Nevado de
Toluca de
Toluca
Orizab
a
Perot
e
57.80
—
96.10
107.49
71.63
—
73.93
89.82
—
48.14
—
—
Nevad
o de
Toluca
Sierra
de las
Cruce
—
71.51
166.14
174.01
130.82
Popocatepetl
-Iztaccihuatl
Malinch
e
Orizab
a
Perot
e
Tlatlauquitepe
c
—
76.69
175.05
176.16
133.43
—
97.80
117.81
73.73
—
71.18
87.78
—
49.75
—
—
55.88
—
126.24
198.11
294.41
297.30
254.50
72.14
144.32
244.77
243.93
202.69
ew
66.11
132.57
224.09
236.66
194.07
vi
119.65
190.15
282.02
294.71
248.89
Re
Nevado de
Toluca
Sierra de las
Cruces
PopocatepetlIztaccihuatl
Malinche
Orizaba
Perote
Tlatlauquitepe
c
Malinch
e
Tlatlauquitepe
c
—
er
B
Popocatepetl
-Iztaccihuatl
Pe
Sierra de las
Cruces
PopocatepetlIztaccihuatl
Malinche
Orizaba
Perote
Tlatlauquitepe
c
Sierra
de las
Cruce
s
r
Fo
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 54 of 56
Page 55 of 56
C
Nevado de
Toluca
Sierra de las
Cruces
PopocatepetlIztaccihuatl
Malinche
Orizaba
Perote
Tlatlauquitepe
c
Nevad
o de
Toluca
Sierra
de las
Cruce
s
Popocatepetl
-Iztaccihuatl
Malinch
e
Orizab
a
Perot
e
Tlatlau
quitepe
c
—
126
286
252
193
—
154
180
112
—
91
148
—
89
—
—
68
—
165
297
456
430
371
111
227
392
355
282
r
Fo
er
Pe
ew
vi
Re
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Journal of Biogeography
Journal of Biogeography
r
Fo
er
Pe
ew
vi
Re
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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