ORIGINALARTICLE

Isolation in habitat refugia promotesrapid diversification in a montane tropicalsalamander

Gabriela Parra-Olea1*, Juan Carlos Windfield1, Guillermo Velo-Anton2� and

Kelly R. Zamudio2

INTRODUCTION

Differentiation of montane lineages is of particular interest to

evolutionary biologists because the patchy distribution of

appropriate habitat combined with the geographical isolation

of populations on mountain ‘islands’ may promote diversifi-

cation and increase the rate of speciation for montane-adapted

taxa (Fjeldsa & Lovett, 1997; Jetz et al., 2004). Montane species

1Departamento de Zoologıa, Instituto de

Biologıa, Universidad Nacional Autonoma de

Mexico, Distrito Federal 04510, Mexico,2Department of Ecology and Evolutionary

Biology, Cornell University, Ithaca, NY 14853,

USA

*Correspondence: Gabriela Parra-Olea,

Departamento de Zoologıa, Instituto de

Biologıa, Universidad Nacional Autonoma de

Mexico, Distrito Federal 04510, Mexico.

E-mail: gparra@ibiologia.unam.mx�Present address: CIBIO – Centro de

Investigacao em Biodiversidade e Recursos

Geneticos da Universidade do Porto, Instituto

de Ciencias Agrarias de Vairao, R. Padre

Armando Quintas, 4485-661 Vairao, Portugal.

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

divergence in the Trans-Volcanic Belt of Mexico.

Location The Trans-Volcanic Belt (TVB) of central Mexico, including the states

of Mexico, Morelos, Puebla, Tlaxcala and Veracruz.

Methods We sequenced the cytochrome b mitochondrial DNA gene for 281

individuals from 26 populations and nine mountain ranges in the TVB, and used

Bayesian phylogenetic reconstruction and Markov chain Monte Carlo 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 despite their recent volcanic origin. Populations of P. leprosa

show a pattern of refugial populations in the north-eastern and eastern limits of

the species’ distribution, and genetic evidence of rapid population expansion in

mountain ranges of the central TVB. The oldest divergences among populations

date to c. 3.8 Ma, and the most recent divergences in the central TVB are

Pleistocene in age (c. 0.7 Ma).

Main conclusions Given the timing and order of population diversification in

P. leprosa, we conclude that early isolation in multiple habitat refuges in north-

eastern and eastern mountain ranges played an important role in structuring

population diversity in the TVB, followed by population expansion and genetic

divergence of the central range populations. The dynamic climatic and volcanic

changes in this landscape generally coincide with the history of intra-specific

diversification in P. leprosa. 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, Mexico, montane speciation, phylogeography,

Pleistocene, Plethodontidae, Pseudoeurycea leprosa, Trans-Volcanic Belt,

volcanism.

Journal of Biogeography (J. Biogeogr.) (2012) 39, 353–370

ª 2011 Blackwell Publishing Ltd http://wileyonlinelibrary.com/journal/jbi 353doi:10.1111/j.1365-2699.2011.02593.x

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 are particularly susceptible to climate change (Hughes,

2003; Galbreath et al., 2009). Many cold-adapted montane

organisms experienced range expansion and increased gene

flow during glacial periods and range contractions during

warmer interglacials (Hewitt, 2004; DeChaine & Martin, 2006;

Galbreath et al., 2009). Their low physiological tolerance to

higher temperatures implies that populations isolated on

different mountain ranges should carry signatures of historical

changes associated with either glacial or interglacial periods,

even if the periods of climatic instability were relatively recent

(Knowles, 2000; DeChaine & Martin, 2006).

In the tropics, where the environmental gradient from

lowlands to mountains is most pronounced (Janzen, 1967),

montane regions are important historical centres of diversity

and endemism, even more so than are tropical lowlands

(Smith et al., 2007). Thus, understanding the microevolution-

ary processes promoting diversification, and specifically how

isolation and genetic drift interact to enhance speciation and

diversification in tropical montane species, will provide a

framework for understanding broader patterns of biodiversity

distribution in these habitats (Kozak & Wiens, 2006, 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 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 Pseudoe-

urycea leprosa (Cope, 1869), a tropical salamander endemic to

the Trans-Volcanic Belt (TVB) of central Mexico, where it is

restricted to pine (Pinus), pine–oak (Quercus) and fir (Abies)

forests at elevations between 2000 and 3500 m. The TVB is a

young (Ferrari & Rosas-Elguera, 1999; Ferrari et al., 1999)

volcanic mountain chain that has been modified by volcanism

and climatic cycles through the Plio-Pleistocene, resulting in

large elevational shifts in pine forests and changing patterns of

connectivity among mountain ranges in the system (Brown,

1985; Graham, 1993; McDonald, 1993; Lozano-Garcıa et al.,

2005). The TVB is one of the areas of highest vertebrate

biodiversity in Mexico, second only to the Sierra Madre del Sur

(Luna et al., 2007). These dynamic highlands of Mexico have

been a centre for the 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 that have

contributed to the high rate of local population divergence and

speciation.

In this study we compare the diversity of cytochrome b (cyt

b) mitochondrial gene sequences among populations of

P. leprosa and infer the historical population changes that

contributed to differentiation within this species complex.

Despite its high diversity, the TVB was formed only recently,

beginning in the middle Miocene (Ferrari & Rosas-Elguera,

1999); thus, the 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 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: (1) whether population structure

among independent mountain ranges shows genetic signatures

of isolation, migration and/or drift; (2) whether populations in

the TVB evolved from a single or a few source populations; (3)

whether historical migration patterns from ancestral popula-

tions were directional; and (4) whether the timing of isolation

is concordant with the volcanic origin of the TVB mountains

or with changes in habitat distribution that occurred during

Plio-Pleistocene climatic cycles. We compare our results with

patterns inferred from other plants and animals that are

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 have a severe impact on

the future distribution of appropriate habitat for this and other

species endemic to the TVB (Parra-Olea et al., 2005b). We

discuss the importance of understanding the current geo-

graphical distribution of genetic diversity and the history of

differentiated lineages for the conservation of high-elevation

species in this region.

Geographical context: the TVB

The TVB is a continental magmatic arc composed of nearly

8000 volcanic structures and a few intrusive bodies. It stretches

approximately 1200 km from the Gulf of California to the Gulf

of Mexico (Garcıa-Palomo et al., 2000; Gomez-Tuena et al.,

2007) and defines the southern limit of the uplifted Mexican

Plateau (Mesa Central) (Domınguez-Domınguez & Perez-

Ponce de Leon, 2009). The TVB consists of three distinct

segments, each with its own tectonic, volcanic and geomor-

phological characteristics (Pasquare et al., 1988): the western

section occurs from the Pacific coast to the Colima graben and

includes the Volcan de Colima; the central section extends

from the Michoacan volcanic zone towards either the Valley of

Mexico and the Sierra Nevada (Popocatepetl–Iztaccihuatl)

(Nixon et al., 1987) or the Queretaro–Taxco lineament

(Pasquare et al., 1988) and includes the Nevado de Toluca,

Sierra de las Cruces and Sierra Nevada; finally, the eastern

section extends towards the Gulf of Mexico (Osete et al., 2000)

and includes La Malinche, Pico de Orizaba and Cofre de Perote

(Castillo-Rodrıguez et al., 2007). The range of P. leprosa

encompasses only the central and eastern geomorphological

units of the TVB (Fig. 1).

Isotopic analyses show that the origin of the TVB as a

distinctive geological province dates to the middle/late Mio-

cene (19–10 Ma) and resulted from the progressive counter-

clockwise rotation of the magmatic arc of the Sierra Madre

Oriental (Tamayo & West, 1964; Ferrari et al., 1999; Gomez-

Tuena et al., 2007). The TVB is dominated by geologically

young andesitic stratovolcanoes, some of which form short

G. Parra-Olea et al.

354 Journal of Biogeography 39, 353–370ª 2011 Blackwell Publishing Ltd

north–south volcanic chains that are younger towards the

south. Examples include the Sierra de las Cruces (La Bufa–

Ajusco–Zempoala), Sierra Nevada (Telapon–Iztaccihuatl–

Popocatepetl) and Cofre de Perote–Las Cumbres-Pico de

Orizaba (Fig. 1). Researchers divide the geological evolution of

the TVB into four main episodes: (1) middle to late Miocene

arc (19–10 Ma), (2) late Miocene episode (11–6 Ma), (3) latest

Miocene and early Pliocene volcanism (7.5–3 Ma), and (4) late

Pliocene to Quaternary arc (3.5 Ma–Holocene) (Gomez-

Tuena et al., 2007).

All of the TVB volcanoes are relatively young; their

maximum age is estimated to be 3.7 Ma (Ferrari, 2000; Ferrari

et al., 2000). The oldest cone construction began in the Sierra

de las Cruces (Osete et al., 2000; Garcıa-Palomo et al., 2008), a

volcanic chain between the Mexico and Toluca basins that

consists of eight stratovolcanoes, including Ajusco and Zem-

poala, which were formed in a north-north-west–south-south-

east migration of volcanic activity between 3.6 and 1.8 Ma

(K–Ar dates; Osete et al., 2000). Cone construction in the

Nevado de Toluca began about 2.6 Ma (K–Ar dates; Garcıa-

Palomo et al., 2002). Cone growth in the Sierra Nevada range

began in the late Pleistocene and Holocene, approximately

1.0 Ma (Nixon, 1989). The Iztaccihuatl volcano began to form

about 900,000 years ago (K–Ar; Nixon, 1989). Given the

north–south migration of volcanic activity presented by these

volcanic chains, the Popocatepetl volcano is estimated to be

younger than Iztaccihuatl, but the age of the oldest lava has not

yet been determined (Schaaf et al., 2005). The cones and

highlands in the eastern TVB are La Malinche volcano and the

north–south-trending Cofre de Perote–Las Cumbres–Pico de

Orizaba mountain range. Cofre de Perote lies at the northern

end of the Pico de Orizaba–Cofre de Perote volcanic range,

which is the easternmost volcanic chain of the TVB, separating

the coastal plains from the Altiplano. The early construction of

Cofre de Perote has been dated by K–Ar to about 1.3 Ma

(Carrasco-Nunez & Nelson, 1998; Dıaz-Castellon et al., 2008).

For Pico de Orizaba, the estimated date is 600–300 ka

(Rossotti et al., 2006). La Malinche is the youngest of all of

the TVB volcanoes; the oldest deposits of the Malinche stage

have been dated to 45 ka (Castro-Govea & Claus, 2007).

The distribution and persistence of montane habitat within

the TVB has had a dynamic history due to the Pleistocene and

Holocene glacial cycles (Heine, 1988; Vazquez-Selem & Heine,

2004). Five periods of glacial advances are identified for the

volcanoes in the central TVB: the most extensively recorded

Nexcoalango advance occurred at 195 ka, and reached 3000 m.

The local late Pleistocene glacial maxima occurred in four

pulses. The first pulse (Hueyatlaco 1 advance) peaked at 20–

17.5 ka, the second (Hueyatlaco 2) peaked at 17–14 ka. During

these times the glaciers reached 3400–3500 m. The final two

glacial advances peaked at 12 ka, reaching c. 3800 m (Milpulco

1), and 8.3–7.0 ka (Milpulco 2), reaching 4000 m (Vazquez-

Selem & Heine, 2004). Thus, although the most recent glacial

advances certainly affected the distribution of habitats in the

TVB, they represent only a small portion of the historical

changes that have occurred in this landscape. The impact of

these glaciations on TVB taxa was probably not as dramatic as

that observed at higher latitudes in North America; however,

temperatures decreased by 5–9 �C and the lowest extensions of

pine–oak forest shifted downward by 730–930 m during the

0-10001000-15001500-20002000-25002500-35003500-40004000-45004500-50005000-5500

Elevation (m)

Nevado de Toluca

Sierra de las Cruces

Tlaxco

Malinche

Tres Mogotes

Orizaba

Tlatlauquitepec

Popocatepetl Iztaccihuatl(Sierra Nevada)

Perote

TVB

12

3

45

6

78

9

1011

12

13

14

16

15

17 18 1920 21

22

24

25

23

26

Kilometres0 50 100

Figure 1 Distribution of the 26 populations of Pseudoeurycea leprosa sampled across the Trans-Volcanic Belt (TVB) of Mexico. Ovals group

the localities by mountain ranges. Locality symbols represent the regional clades to which samples from each population belong: trian-

gles = North-eastern haplotypes (NE I, NE II and NE III), diamond = Tres Mogotes, white circles = Central TVB haplotypes, and black

circles = South-east clade. Population 25 (Xometla) is represented by two circles because this population includes haplotypes belonging to

both the Central TVB clade and the South-east clade.

Montane diversification in the tropics

Journal of Biogeography 39, 353–370 355ª 2011 Blackwell Publishing Ltd

late Pleistocene advances (Vazquez-Selem & Heine, 2004), and

the timberline also shifted to 700–900 m below its present

position (Brown, 1985; Graham, 1993; McDonald, 1993;

Lozano-Garcıa et al., 2005).

MATERIALS AND METHODS

Population sampling

We collected tissue samples of P. leprosa from throughout its

distribution along the TVB. We sampled a total of 281

individuals from 26 populations, with a range of 1–30

individuals per site (Fig. 1 and Appendix S1 in Supporting

Information). The sampled populations were distributed

across the following nine mountain ranges (ordered roughly

from west to east): Nevado de Toluca, Sierra de las Cruces,

Iztaccihuatl–Popocatepetl, Malinche, Tlaxco, Tlatlauquitepec,

Cofre de Perote, Orizaba and Tres Mogotes (Fig. 1, Appen-

dix S1). Plethodontids are cryptic salamanders, and P. leprosa

is no exception; thus, the spatial sampling we obtained for this

endemic species represents one of the broadest samplings to

date for population genetic studies in this lineage. We

selected Pseudoeurycea lynchi and Pseudoeurycea lineola as

successively more distant outgroups based on published

higher-level relationships within the Bolitoglossini (Wiens

et al., 2007).

Amplification and sequencing of mitochondrial DNA

(mtDNA)

Total genomic DNA was extracted from ethanol-preserved

tissues (muscle or liver) with DNeasy Tissue Kits (Qiagen Inc.,

Valencia, CA, USA). We used polymerase chain reaction

(PCR) to amplify 675 bp of the mitochondrial cytochrome b

gene (cyt b) using the primers MVZ15 and MVZ16 (Moritz

et al., 1992). The PCR amplifications were performed in a total

volume of 25 lL containing 1 lL DNA template (c. 100 ng

lL)1), 1 U Taq polymerase (Applied Biosystems, Foster City,

CA, USA), 1· PCR buffer with 1.5 mm MgCl2, 0.4 mm

deoxynucleotide triphosphates (dNTPs), and 0.5 lm forward

and reverse primers. The PCR conditions consisted of 35 cycles

with a denaturing temperature of 94 �C (1 min), annealing at

50 �C (1 min) and extension at 72 �C (1 min). Successful

amplicons were purified with shrimp alkaline phosphatase

(1 U) and exonuclease I (10 U) to remove unincorporated

dNTPs and primers. The fragments were sequenced in both

directions using the original amplification primers and BigDye

termination sequencing chemistry (Applied Biosystems). The

sequencing reactions were performed in a total volume of 5 lL

containing 1 lL cleaned PCR product, 0.24 lm primer, 1 lL

Big Dye Ready Reaction Mix, and 1· Sequencing buffer. The

cycle-sequencing products were column-purified with Sepha-

dex G-50 (GE Healthcare, Amersham, Buckinghamshire, UK)

and run on an ABI PRISM 3100 DNA Analyzer (Applied

Biosystems). We checked the resulting electropherograms by

eye before constructing contiguous sequences for each indi-

vidual using Sequencher 4.7 (Gene Codes Corp., Ann Arbor,

MI, USA).

Phylogeographical analyses

We aligned the sequences and identified unique haplotypes for

phylogenetic analyses. We used the program jModelTest

0.1.1 (Posada, 2008) and Akaike information criterion (AIC)

scores to select the appropriate model with which to infer a

population-level phylogeny using Bayesian inference (BI). The

Bayesian analysis, implemented in MrBayes 3.1 (Huelsenbeck

& Ronquist, 2001), consisted of 10 chains sampled every

10,000 generations for 10 million generations. We used two

methods to verify convergence and determine an adequate

burn-in: we examined a plot of the likelihood scores of the

heated chain and checked the stationarity of the chains using

the software Tracer 1.4 (Rambaut & Drummond, 2007). We

discarded a total of 250 trees as burn-in; using the remaining

trees, we estimated the 50% majority-rule consensus topology

and posterior probabilities for each node. We also constructed

two haplotype networks using: (1) a Neighbor-Net algorithm

implemented in SplitsTree 4.6 (Huson & Bryant, 2006),

based on uncorrected patristic distances and a bootstrap

analysis with 1000 replicates, and (2) statistical parsimony

(Templeton et al., 1992) implemented in tcs 1.13 (Clement

et al., 2000). The reticulation and multiple branches shown in

the SplitsTree network allow visualization of conflicting and

ambiguous signals in the data set (Huson & Bryant, 2006).

Genetic diversity and historical demographic analyses

We used Arlequin 3.01 (Excoffier et al., 2005) to estimate the

haplotype diversity (h) and nucleotide diversity (p) (Nei, 1987)

for each clade and mountain range. We characterized the

genetic differentiation among mountain ranges by estimating

DXY (Nei, 1987), the average number of nucleotide substitu-

tions per site between pairs of mountain ranges, in DnaSP 5

(Rozas et al., 2003), and PiXY, the average number of pairwise

differences among haplotypes from different mountain ranges,

in Arlequin 3.01.

We used mismatch analyses of sequences within each clade

to infer historical demographic changes in P. leprosa (Schnei-

der & Excoffier, 1999). Mismatch analysis compares the

frequency distribution of pairwise differences among haplo-

types with that expected under a model of population

expansion. A significant difference between the observed and

expected distributions is tested using a bootstrap approach

(20,000 replicates). The frequency distribution is predicted to

be unimodal for lineages that have undergone recent popu-

lation expansions and multimodal for lineages whose popu-

lations are either subdivided or in equilibrium. We compared

the sum of squared deviations (SSD) between the observed and

expected mismatches 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 each clade and mountain range, we calculated Tajima’s D

G. Parra-Olea et al.

356 Journal of Biogeography 39, 353–370ª 2011 Blackwell Publishing Ltd

(Tajima, 1989) and Fu’s FS (Fu, 1997) as tests of selective

neutrality. Historical population growth predicts significantly

negative values of D and FS, which we tested with 10,000

bootstrap replicates. All three tests for population expansion

were performed in Arlequin 3.01.

To infer the ages of particular haplotype groups in the

P. leprosa topology and to reconstruct demographic changes

over the history of each major lineage, we used the coalescent-

based methods applied in the program beast 1.4.7 (Drum-

mond & Rambaut, 2007). This approach allows for inferences

of population fluctuations over time by estimating the

posterior distribution of the effective population size at

intervals along a phylogeny (Drummond et al., 2005; Drum-

mond & Rambaut, 2007). We estimated demographic changes

for all P. leprosa populations combined, as well as for

populations in each clade. The times to the most recent

common ancestor (TMRCA) for clades of highest support

were obtained using Bayesian Markov chain Monte Carlo

(MCMC) searches. We used a GTR+I+G model of evolution

and implemented an uncorrelated lognormal relaxed molec-

ular-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 the mutation rate of cyt b to

reflect a priori the uncertainty in this parameter (Mueller,

2006; Martınez-Solano & Lawson, 2009). We implemented a

series of coalescent models (Bayesian skyline, Yule and

expansion) to assess any biases model selection might generate

in time estimates. For each analysis, we performed two

independent runs of 30 million generations, sampling every

1000th generation and removing 10% of the initial samples as

burn-in. We combined runs and determined the stationarity of

the posterior distributions for all model 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) and pro-

portion of invariant sites uniform (0,1). The scale operators

were adjusted as suggested by the program.

Finally, we used a coalescence model in Lamarc 2.0

(Kuhner, 2006) to estimate Q (for mtDNA Q = 2Nel, where

Ne is the effective number of individuals and l is the mutation

rate) and migration rates (M) for P. leprosa populations. To

optimize the parameter estimates, we selected 11 representative

populations from the mountain systems, excluding localities

with fewer than four individuals (Tlaxco and Tres Mogotes)

and randomly reduced the sample sizes of populations to 15 to

increase run efficiency (Kuhner, 2006). Default values were

used for the effective population size and migration param-

eters. We performed Bayesian analyses with one long chain, a

burn-in of 1 · 106, and a run of 1 · 106 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 the

maximum probable estimate (MPE) for each parameter.

Parameter convergence was verified by stationarity in param-

eter trends over the length of the chains and the effective

sample sizes (ESS) obtained for each Q and migration rate

using Tracer v1.4. We interpreted ESS values greater than 300

as indicating that the sampled trees were not correlated and

thus represented independent samples.

Isolation by distance and least-cost paths in the TVB

We performed a least-cost path analysis (LCPA) using the

Spatial Analyst extension of ArcGIS 9.2 (ESRI, 2006, Redlands,

CA, USA) and a 1-km resolution digital topographic raster

from the Instituto Nacional de Estadıstica y Geografıa (INEGI,

Mexico), to determine the potential influence of elevation on

gene flow and the 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 sampled in each mountain range as path endpoints

between each pair of mountains. To infer an appropriate cost

for movement among mountain ranges, we reclassified the

elevation layer based on the current elevational distribution of

the species (2500–3500 m a.s.l.) and assumed a higher cost for

movement 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 identified the path resulting in the lowest cost to

reach a given location from a specified source population. We

assumed the following elevation ranges and corresponding

movement costs (in parentheses) through each cell: 0–1000 m

(5); 1000–1500 m (4); 1500–2000 m (3); 2000–2500 m (2);

2500–3500 m (1); 3500–4000 m (2); 4000–4500 m (3); 4500–

5000 m (4); and 5000–5469 m (5). We also calculated the

straight Euclidean distances (ED) between populations and two

geographical distances based on the LCPA identified: (1)

Euclidean distances along every least-cost path (ED-CP); and

(2) the cost of moving along each path (cost path, CP).

To examine isolation by distance (IBD), we used Mantel

tests between the genetic distances (DXY and PiXY) and the

three geographical/cost–distance matrices that were calculated

among mountain ranges. We also used partial Mantel tests to

examine the correlations between genetic distances (DXY and

PiXY) and least-cost distances (ED-CP) while controlling for

the effect of Euclidean distances (ED). These analyses were

performed with 10,000 replications using the program zt

(Bonnet & Van de Peer, 2002).

RESULTS

Diversity of mtDNA

The cyt b 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, among

which 139 sites were variable and 75 were parsimony-

informative. The maximum likelihood (ML)-estimated tran-

sition/transversion ratio was 3.2, with mean nucleotide

frequencies of 27.7% A, 21.5% C, 15.2% G and 35.5% T.

Montane diversification in the tropics

Journal of Biogeography 39, 353–370 357ª 2011 Blackwell Publishing Ltd

The TrN + I + G model of evolution was selected by

jModelTest and used for subsequent Bayesian analyses. All

sequences have been deposited in GenBank (accession num-

bers GQ468424–GQ468493).

Phylogeographical analyses

Both Bayesian analyses (in beast and MrBayes) recovered a

monophyletic P. leprosa with some structured regional groups

of haplotypes (Fig. 2). Three well-supported ‘North-east’ (NE)

clades diverged relatively early in the history of this species; the

NE I clade includes a haplotype from Tlaxco (43) and three of

the four haplotypes from Tlatlauquitepec (45–47); the NE II

clade includes haplotypes from Vigas (50–55) and the

remaining haplotype from Tlatlauquitepec (44); NE III clade

unites the remaining samples from Vigas (48, 56–61) with

those from Teziutlan (48) and Gonzalez Ortega (62–63).

North-east clades I and II are strongly supported with high

posterior probabilities in both beast and MrBayes (nodes 3

and 4; Fig. 2). North-east clade III is recovered with high

support in beast (node 2; Fig. 2) but not recovered in the

MrBayes analysis. The three clades are not geographically

independent, a pattern that may be the result of either

secondary contact between these regions or incomplete lineage

sorting since the time of their isolation. Our phylogeny

provides no resolution for the earliest divergences among these

lineages: the relationships among the three NE clades and the

two samples from Tres Mogotes are unclear, and the nodes at

the base of the tree are unsupported in both analyses. The

topology also infers a South-east (SE) clade (node 6) that

includes all samples from the Pico de Orizaba mountain range

(Xometla, Texmola and Texmalaquilla), and a large Central

TVB clade (node 7) that includes all samples from the Nevado

de Toluca, Sierra de las Cruces, Sierra Nevada and Malinche.

The Neighbor-Net network (Fig. 3) provides a graphical

representation of haplotype groups. Reticulation indicates

alternative mutational pathways (i.e. homoplasy) that occur

mostly inside each group. Our network recovered the six major

groups found in our Bayesian tree in a single network: (1)

Central, (2) SE, (3) NE I, (4) NE II, (5) NE III, and (6) Tres

Mogotes. Using a statistical-parsimony 95% CI, tcs grouped

the 70 P. leprosa sequences into two haplotype networks and

one single haplotype (70, from Tres Mogotes) that was

independent of both networks (Fig. 4). The main network

includes 65 of the haplotypes with a connection limit of 11 bp

and contains the Central, SE and NE II clades as well as the

north-eastern basal haplotypes that were recovered in the

Bayesian tree. A common haplotype (11) is found in the

central mountains (Popocatepetl–Iztaccihuatl and Malinche)

and a south-eastern population (Xometla); additional haplo-

types from these same populations and from the western

mountains (Nevado de Toluca and Sierra de las Cruces) are

separated from this common haplotype by only a few

mutational steps. Higher divergences are found among the

Eastern and South-eastern haplotypes in the network, with 5

(Texmalaquilla, Texmola and Xometla), 10 (Vigas), 14 (Gon-

zalez Ortega) and 23 mutational steps (Tlatlauquitepec)

separating those samples from the Central TVB haplotypes.

The most diverse population is Vigas, with 15 haplotypes

separated by a maximum of 41 mutational steps; the most

closely related haplotypes are from Eastern populations

(Xometla, Tlatlauquitepec). A second network groups the NE

I clade haplotypes with the single haplotype from Tlaxco,

which is separated by six mutational steps.

Genetic diversity and historical demographic analyses

The haplotype diversity (h) and nucleotide diversity (p) for

each clade and mountain range are summarized in Table 1. All

0.01.02.03.04.0

16

47

56

68

17

63

19

45

38

15

59

3

66

42

18

69

5

65

28

13

67

36

43

27

39

33

9

61

10

57

30

41

24

32

44

35

53

7

25

58

21

46

40

50

20

4

23

62

49

11

52

14

1

34

48

64

60

51

31

2

54

22

26

37

12

6

70

8

55

Tres Mogotes

NE Clade III

NE Clade I

NE Clade II

SE Clade

5.06.07.0

29

Central TVB Clade

1.0/1.0

1.0/-

1.0/1.0

1.0/1.0

1.0/1.0

1.0/0.5

1.0/0.6

1

6

2

3

4

5

7

Ma

Figure 2 Ultrametric tree with divergence time estimates from

beast analyses for Pseudoeurycea leprosa haplotypes from 26

populations sampled throughout the Trans-Volcanic Belt (TVB) of

Mexico. Numbers within boxes above branches correspond to

major nodes shown in Table 3. Numbers below branches are

posterior probabilities from Bayesian inference of beast and

MrBayes analyses, respectively. Tip numbers refer to haplotype

numbers in Appendix S1. Older, refugial clades are demarcated by

black bars and symbols, and clades showing genetic signatures of

recent expansion are highlighted in white; symbol shapes for each

clade are concordant with those in Fig. 1. Haplotype 11 in the

Central TVB clade was also collected in the South-east (SE) clade

(identified by the circle after the haplotype name). Shaded hori-

zontal bars correspond to estimates of diversification times (and

confidence intervals) inferred from beast analyses for individual

nodes (Table 3). The scale bar denotes millions of years ago.

G. Parra-Olea et al.

358 Journal of Biogeography 39, 353–370ª 2011 Blackwell Publishing Ltd

clades and mountain systems had low nucleotide diversity

(0.002–0.018) and high haplotype diversity (0.464–0.954).

Genetic distances among mountains ranged from 1.638 to

21.161 and from 0.003 to 0.029 for PiXY and DXY, respectively

(Table 2). The populations from the Central TVB clade were

more closely related to each other and were more genetically

differentiated from north-eastern populations (Perote and

Tlatlauquitepec mountain ranges) than from SE clade popu-

lations (Orizaba mountain ranges; Table 2).

We created mismatch distribution plots for all clades

inferred in our topology, with the exception of NE Clade I,

which includes insufficient haplotypes for analysis (Appen-

dix S1). None of the mismatch analyses (Fig. 5) were signif-

icant; therefore, we could not reject the null hypothesis of

historical population expansion for any of the lineages within

this species (SE clade: SSD = 0.020; P = 0.48, raggedness

index = 0.041; P = 0.73; NE II clade: SSD = 0.027; P = 0.40,

raggedness index = 0.075; P = 0.45; Central–West clade:

SSD = 0.013, P = 0.12; raggedness index = 0.051, P = 0.14;

NE III clade: SSD = 0.010; P = 0.35, raggedness index = 0.054;

P = 0.44). In contrast, both Tajima’s D and Fu’s FS show

evidence for historical population expansion in the Central

TVB clade only (Table 1). Recalculating these tests for the

haplotypes partitioned by the mountain range of origin shows

that this signature of expansion is most evident for Popocate-

petl–Iztaccihuatl and for Malinche (Table 1).

Bayesian skyline plots (BSPs) depict similar scenarios for all

P. leprosa clades, with a low but constant increase in size from

c. 250,000 years ago to the present (Fig. 5a,b). The BSP for all

P. leprosa populations combined shows a decrease in size

approximately 250,000 years ago followed by a rapid expan-

sion. Estimates of TMRCA were highly concordant among

runs (Table 3), independent of the coalescent model applied.

The estimated TMRCA for all P. leprosa was 3.25–3.80 Ma over

all coalescent models. An ancient split occurred between

populations from the NE and SE (clade 4 in Table 3) at about

3.2–3.7 Ma, whereas the Central TVB clade has a TMRCA of

only 0.79–0.83 Ma.

Repeated Lamarc runs resulted in similar posterior prob-

ability distributions and high ESS values for all Q values and

most migration rates. Populations vary in Q (Table 4), ranging

from the lowest value in Ajusco (MPE < 0.001) to the highest

value in Vigas (MPE = 0.011), with a trend of decreasing

population sizes from eastern to western populations. How-

ever, the Central clade populations of Llano Grande and

Atzompa (both in the Popocatepetl–Iztaccihuatl mountain

range) show the highest MPE Q values in the Central TVB, and

their confidence intervals overlap with the largest population

sizes found in eastern populations (Table 4).

The Lamarc migration-rate estimates (M) are scaled to the

mutation rate (m/l). The most probable estimates (MPEs) of

immigration obtained from Lamarc suggest asymmetrical

immigration among mountain ranges (Table 4, Fig. 6).

Among the NE clade populations, gene flow has occurred

principally from Vigas to other populations, with the highest

estimated value from Vigas into Gonzalez Ortega (both in the

Perote mountain range). Migration rates are higher from the

NE II clade (Perote) into populations of the SE clade (Orizaba)

and from the SE clade into the central mountain range of

Malinche, suggesting an expansion from the north-east to the

central TVB via the south-eastern mountain range (Fig. 6).

Indeed, the estimated migration rates from NE clade

0.00100.0010

1826

17

7

1

5

40

11

131415

19

32 1035

4 6

31

3436 41

2743

242523

21

20

2229

30

42

333836

37

289

3281612

4967

69

6564

6668

57 58

6263

61

6059

4856

5051

52

555453

44

70

43

45 4647

70

90

95

96

100

88

Central TVB Clade

SE Clade

NE Clade II

NE Clade III

NE Clade I

Tres Mogotes

Figure 3 Neighbor-Net network for 70 mitochondrial DNA (cytochrome b) haplotypes of Pseudoeurycea leprosa from 26 populations

sampled throughout the Trans-Volcanic Belt (TVB) of Mexico. Circles delimit haplotypes belonging to groups identified in Bayesian

phylogenetic analysis. Bootstrap values for each major clade are enclosed in boxes. Circles represent haplotypes from the Central TVB

mountain ranges and the South-east (SE) clade; triangles represent haplotypes from North-east (NE) clades, and the single diamond

corresponds to the haplotype obtained in the isolated population of Tres Mogotes. Different shaded/stippled symbols group haplotypes by

mountain chains and correspond to the legend in Fig. 4. The bar represents the network scale based on uncorrected patristic distances

among haplotypes.

Montane diversification in the tropics

Journal of Biogeography 39, 353–370 359ª 2011 Blackwell Publishing Ltd

populations to Central TVB populations are low, and the

lowest immigration rates in the entire range of P. leprosa are

into the north-eastern mountain ranges. Within the Central

TVB clade, we found higher migration rates from Malinche to

central and northern Popocatepetl–Iztaccihuatl than to the

southern end of that mountain range, and the Sierra de las

Cruces receives more migrants from the Nevado de Toluca and

Popocatepetl–Iztaccihuatl than vice versa. One caveat is that,

although the MPEs are distinct, all of the immigration rate

95% CIs overlap; thus, these estimates should be interpreted

with caution (Table 4).

Isolation by distance and least-cost paths in the TVB

The genetic and geographical distances between pairs of

mountain ranges are summarized in Table 2 and Appendix S2,

respectively. The Mantel tests revealed no significant isolation

by distance; that is, no significant correlation was found

between any of the three measures of geographical distance

and either 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 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 signif-

icant isolation by distance based on significant correlations

between geographical 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

Mechanisms of diversification in tropical montane

taxa

Many montane regions are hotspots for biodiversity (Rahbek

& Graves, 2001; McCain, 2005), a pattern that has been

attributed to the fact that mountains can act both as cradles for

diversity (by promoting isolation and speciation) and as

museums (by favouring the long-term persistence of lineages)

(Chown & Gaston, 2000; Kozak & Wiens, 2006). Factors that

contribute to the high diversity of montane biotas include

Perote

Nevado de Toluca

Sierra de las Cruces

Popocatepetl-Iztaccihuatl

Malinche

Orizaba

Tlaxco

Tlatlauquitepec

Tres Mogotes

11

N = 1

Figure 4 tcs haplotype network of 70

Pseudoeurycea leprosa haplotypes from 26

populations sampled throughout the Trans-

Volcanic Belt (TVB) of Mexico. Haplotypes

are connected assuming a 95% parsimony

threshold. The size of each haplotype symbol

is proportional to its frequency and black

dots represent mutational steps separating

observed haplotypes. Circles represent

haplotypes from the Central TVB mountain

ranges and the South-east clade; triangles

represent haplotypes from North-east clades,

and the single diamond corresponds to the

haplotype obtained in the isolated population

of Tres Mogotes. Different shaded symbols

group haplotypes by mountain ranges.

G. Parra-Olea et al.

360 Journal of Biogeography 39, 353–370ª 2011 Blackwell Publishing Ltd

topographic complexity, which results in high habitat heter-

ogeneity and environmental diversity (Jetz et al., 2004), and

the fact that mountains can harbour more climatically stable

refugia than lowlands. We now have a good understanding of

the macroevolutionary patterns of species diversity in moun-

tain landscapes (Rahbek, 1995; Rahbek & Graves, 2001) and

some climatic and ecological correlates of this diversity

(Francis & Currie, 2003; Hawkins et al., 2003; Oomen &

Shanker, 2005; McCain, 2007), but few studies have examined

in detail the timing and relative importance of the evolutionary

processes that lead to population splitting and promote lineage

survival in these same landscapes (Cardillo et al., 2005;

Tennessen & Zamudio, 2008). Examining the evolutionary

underpinnings of 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; Ricklefs,

2006; Stevens, 2006; Wiens et al., 2006), and the same pattern

is expected in species belonging to montane assemblages

(Fjeldsa & Lovett, 1997; Ghalambor et al., 2006). The Mexican

highlands are a biodiversity hotspot in North America, with

high degrees of endemism (Ramamoorthy et al., 1993) in

plants (Nixon, 1993), insects (Morrone, 2005), mammals

(Mena & Vazquez-Domınguez, 2005), birds (McCormack

et al., 2008) and reptiles and amphibians (Flores-Villela &

Canseco-Marquez, 2007; Flores-Villela et al., 2010; Bryson

et al., 2011).

Based on our data, we can identify three major events in the

history and evolution of P. leprosa that are temporally

correlated with the geological activity of specific regions of

the TVB: (1) the evolution of P. leprosa in the north-eastern

section of the TVB; (2) the subsequent population isolation

and fragmentation of the north-eastern and south-eastern

populations and the Central clade; and (3) the more recent

westward range expansion of the Central TVB clade.

The evolution of P. leprosa in the north-eastern section of the

TVB

Several lines of evidence indicate that P. leprosa evolved in the

north-eastern section of the TVB in the area of Cofre de

Perote, Veracruz. The presence of haplotypes exclusive to

Cofre de Perote and the fact that north-eastern haplotypes

Table 1 Measures of genetic diversity (± SD), Tajima’s D and Fu’s FS statistics for 26 populations of Pseudoeurycea leprosa from the Trans-

Volcanic Belt of Mexico, estimated by clades and by mountain system.

Localities Nh h (SD) p (SD) D FS

Clades

Central 52 0.952 ± 0.005 0.004 ± 0.002 )1.73* )26.40*

South-east (SE) 9 0.834 ± 0.055 0.004 ± 0.002 )0.34 )1.43

North-east 1 (NE I) 4 0.714 ± 0.180 0.004 ± 0.002 )1.04 0.62

North-east 2 (NE II) 7 0.890 ± 0.074 0.005 ± 0.003 )1.28 )1.15

North-east 3 (NE III) 10 0.851 ± 0.050 0.003 ± 0.002 )0.69 )2.13

Mountain systems

Nevado de Toluca 3 0.4643 ± 0.2000 0.0022 ± 0.0017 )0.01 1.01

Sierra de las Cruces 7 0.8122 ± 0.0372 0.0041 ± 0.0025 )0.28 1.11

Popocatepetl–Iztaccihuatl 34 0.9110 ± 0.0116 0.0037 ± 0.0022 )1.60** )24.91*

Malinche 5 0.8348 ± 0.0365 0.0005 ± 0.0006 )2.00** )9.39*

Orizaba 25 0.8182 ± 0.0586 0.0043 ± 0.0026 )0.13 )0.71

Perote 4 0.9440 ± 0.0186 0.0184 ± 0.0094 0.66 )1.67

Tres Mogotes 1 0.0000 ± 0.0000 0.0000 ± 0.0000 0.00 –

Nh, number of haplotypes; p, nucleotide diversity; h, haplotype diversity; Significant values are given in bold: *P < 0.01, **P < 0.05.

Table 2 Average number of nucleotide substitutions per site, DXY (above the diagonal), and average number of pairwise differences,

PiXY (below the diagonal), between 26 populations of Pseudoeurycea leprosa from mountain ranges of the Trans-Volcanic Belt of central

Mexico. Tlaxco and Tres Mogotes samples were excluded because of low sample sizes for those two mountain ranges.

PiXY/DXY Nevado de Toluca Sierra de las Cruces Popocatepetl–Iztaccihuatl Malinche Orizaba Perote Tlatlauquitepec

Nevado de Toluca – 0.005 0.004 0.006 0.009 0.027 0.028

Sierra de las Cruces 3.638 – 0.004 0.005 0.010 0.026 0.028

Popocatepetl-Iztaccihuatl 4.284 3.770 – 0.003 0.009 0.027 0.028

Malinche 3.042 2.659 1.638 – 0.010 0.029 0.027

Orizaba 9.125 8.428 7.219 6.461 – 0.026 0.028

Perote 19.972 18.572 18.265 17.554 17.408 – 0.029

Tlatlauquitepec 21.161 20.169 19.794 18.862 19.662 18.222 –

Montane diversification in the tropics

Journal of Biogeography 39, 353–370 361ª 2011 Blackwell Publishing Ltd

form three distinct and overlapping subclades indicate that the

eastern mountains may have been occupied by formerly

widespread, contiguous populations that experienced frag-

mentation as a result of geological and climatological events.

We found high within-population sequence divergence in the

north-eastern populations of Vigas and Tlatlauquitepec. Their

high haplotype and nucleotide diversity (Table 1), the early

divergence of those haplotypes in the topology (Fig. 2), their

higher Q values (Table 4) and the sharing of haplotypes among

populations in the eastern and north-eastern mountain ranges

Pseudoeurycea leprosa NE Clade I

0 0.05 0.1 0.15 0.2 0.25 0.30

popu

latio

n si

ze

10

100

1000

1

0 0.25 0.50 0.75 1.00 1.25 1.50

popu

latio

n si

ze

10

100

1000

(a)

(b)

1

all populations

NE Clade II

popu

latio

n si

ze

10

100

1000

1

0 0.10 0.20 0.30 0.40 2 4 6 8 10 12 14pairwise differencestime

2

4

6

8

10

12

0

frequ

ency

14

NE Clade III

popu

latio

n si

ze

100

1000

10

2 4 6 8 10 12 14

20

40

60

0

frequ

ency

80

100

0 0.25 0.50 0.75 1.00 1.25 1.50

Central TVB Clade

0 0.05 0.1 0.15 0.2 0.25 0.30

time

popu

latio

n si

ze

10

100

1000

1

Southeast Clade

popu

latio

n si

ze

100

1000

10

0 0.025 0.05 0.075 0.10 0.125

frequ

ency

1000

2000

3000

4000

5000

6000

7000

2 4 6 8 10 12 14

2 4 6 8 10 12 14

10

20

30

40

50

60

0

frequ

ency

pairwise differences

0.150

Figure 5 Bayesian skyline plots, and pair-

wise mismatch distributions for (a) all pop-

ulations and North-east (NE) clades, and (b)

South-east and Central clades of Pseudoeu-

rycea leprosa uncovered in the Trans-

Volcanic Belt (TVB) of Mexico. The Bayesian

skyline plots show evidence of large increases

in population size for the species as a whole.

For mismatch analyses, the black lines/sym-

bols represent the observed frequency of

pairwise differences among haplotypes, grey

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 expansion;

corresponding results of the goodness-of-fit

and neutrality tests are reported in the text

and in Table 1.

G. Parra-Olea et al.

362 Journal of Biogeography 39, 353–370ª 2011 Blackwell Publishing Ltd

are indicative of older source populations that expanded

throughout the rest of the species’ range. Our TMRCA

estimates indicate that the divergence of the eastern popula-

tions occurred in the Pliocene (3.8 Ma). These timing

estimates correlate with the gap in volcanism that occurred

in the eastern sector of the TVB between the end of the

Miocene and the early Pliocene (c. 5–3 Ma) (Gomez-Tuena

et al., 2007). Volcanism resumed at c. 3.7 Ma to the west of

Mexico City (Mora-Alvarez et al., 1991; Osete et al., 2000) and

the easternmost volcanic chain, Cofre de Perote–Pico de

Orizaba, began to form less than 2 Ma. Our data indicate that

it was during this suspension of volcanism, before the

formation of the Cofre de Perote–Pico de Orizaba mountain

range, that P. leprosa evolved in the easternmost sector of the

TVB. Subsequent fragmentation and isolation of the NE clades

might be correlated with the formation of the volcanoes in this

region.

Population isolation and fragmentation of the north-eastern

populations and the Central TVB clade

High within- and between-population sequence divergence

among haplotypes in the three NE clades indicate early

fragmentation. Geologically, this region is highly dynamic:

several of the volcanoes have been intermittently active since

their formation (Tamayo & West, 1964) and the Cofre de

Perote–La Cumbres–Pico de Orizaba mountain chain has a

history of multiple edifice-collapse events and debris ava-

lanches (Carrasco-Nunez et al., 2006) directed eastward

towards the Gulf of Mexico coast. Moreover, cosmogenic36Cl surface-exposure dating of substrate in four different

valleys of the Cofre de Perote indicate a glacial advance

between 20 and 14 ka followed by recession between 14 and

11.5 ka (Carrasco-Nunez et al., 2010). The Cofre de Perote

volcano is found at the north-eastern limit of the TVB (Fig. 1).

Its structure, geochemistry and volcanic history are signifi-

cantly different from those of the large, predominantly

andesitic stratovolcanoes in other regions of the TVB (Carr-

asco-Nunez et al., 2006; Dıaz-Castellon et al., 2008). Cofre de

Perote reaches 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-Nunez et al., 2010). Moreover, the isolation of

Cofre de Perote from mountain ranges to the west was

probably maintained by the elevational gradient that was

established during the conformation of the eastern end of the

TVB. Combined, these landscape changes may have created

environmental conditions that caused changes in population

sizes and connectivity, thus promoting the divergence of the

NE clades.

Studies of other highly diverse taxa in this region corrob-

orate the conclusion that Cofre de Perote is an important

historical refugium. The plethodontid salamander fauna from

this region is quite 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 the sister taxon of Pseudoeurycea gigantea from

Vigas, with a 4.9% sequence divergence in cyt b and substantial

differences in coloration. Salamanders of the genus Chiro-

pterotriton (Darda, 1994; Parra-Olea, 2003) show the same

pattern of divergence between these two neighbouring regions.

Finally, Cofre de Perote populations of curculionid beetles

(Anducho-Reyes et al., 2008) and pocket gophers (Hafner

et al., 2005) are highly divergent both genetically and mor-

phologically from surrounding populations.

The isolation of the Central clade from the NE clade

occurred during the early Pleistocene (1.3 Ma), coincident

with volcanic activity in the Cuenca de Oriental, the hydro-

logical basin that separates the easternmost Pico de Orizaba–

Cofre de Perote mountain chain from the Malinche volcano to

the west (Fig. 1). The Cuenca de Oriental is a broad, internally

drained inter-montane basin with an average elevation of

2300 m. Volcanism in the Cuenca de Oriental basin has been

active since the late Pleistocene, resulting in the formation of a

series of cinder cones known as xalapaxcos (dry craters) or

axalapaxcos (craters with lakes).

Westward range expansion of the Central TVB clade

Populations from mountain ranges in the central TVB show

very little genetic divergence over a large geographical area, a

Table 3 Divergence time estimates for major nodes and coalescence times for differentiation among haplotypes within mitochondrial

clades of Pseudoeurycea leprosa from the Trans-Volcanic Belt of central Mexico. Mean time estimates and 95% confidence intervals were

inferred using three coalescent models in beast; estimated ages are reported in millions of years ago (Ma). Nodes 1–7 and clade/haplotype

names correspond to those in Fig. 2.

Node Clade name Skyline Expansion Yule

1 All P. leprosa 3.55 (1.21–6.92) 4.68 (1.43–9.96) 2.30 (0.98–4.10)

2 North-east III 0.58 (0.15–1.21) 0.80 (0.19–1.74) 0.57 (0.18–1.09)

3 North-east I 0.88 (0.17–1.87) 1.13 (0.22–2.54) 0.71 (0.63–1.38)

4 North-east II 1.16 (0.29–2.39) 1.47 (0.37–3.29) 0.90 (0.31–1.70)

5 South-east + Central 1.35 (0.40–2.72) 1.76 (0.47–3.82) 1.16 (0.44–2.11)

6 South-east 0.48 (0.12–1.00) 0.65 (0.15–1.47) 0.47 (0.12–0.92)

7 Central 0.73 (0.22–1.46) 1.03 (0.30–2.20) 0.76 (0.30–1.36)

Montane diversification in the tropics

Journal of Biogeography 39, 353–370 363ª 2011 Blackwell Publishing Ltd

Tab

le4

Eff

ecti

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31

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333.

46

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25.7

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74

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40

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118.

35

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67

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8.66

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Xom

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9

(0.0

1–44

8.79

)

112.

08

(0.0

1–71

2.57

)

99.1

1

(0.0

1–57

1.88

)

59.6

4

(0.0

1–63

4.66

)

22.6

5

(0.0

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)

360.

63

(0.0

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9)

402.

13

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43

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8.55

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1–

237.

51)

32.8

9(0

.01–

173.

48)

–

G. Parra-Olea et al.

364 Journal of Biogeography 39, 353–370ª 2011 Blackwell Publishing Ltd

pattern (Figs 3 & 4) typical of populations that have under-

gone geographical expansion (Hewitt, 2004; Cortes-Rodrıguez

et al., 2008). Relatively low values of p and high h for the entire

data set indicate a model of range expansion for the entire

range of P. leprosa, but neutrality tests by region show the

strongest signal of range expansion for the Central TVB clade

populations (Table 1). Rapid population expansion often

results in repeated bottlenecks along the expansion front as

migrants move into newly available habitats and found

populations (Hewitt, 2004). The genetic consequences of these

sequential founding events will be more pronounced when

new habitats are patchily distributed, thus lowering both the

probability of colonization and the number of founders when

colonization does occur (Hewitt, 2004). The stepping-stone

colonization of the mountain ranges in the TVB meets this

requirement, and the low genetic diversity in the westernmost

mountain ranges suggests that genetic drift due to founder

events has significantly reduced the genetic diversity of

populations during range expansion. Founders carrying ances-

tral haplotype 11 colonized the western mountains, and the

small number of mutational steps between that common

haplotype and the others in the newly founded populations

indicates that these novel haplotypes arose in situ during the

relatively short history of independent evolution on each of

the isolated mountain ranges. This historical scenario for the

Pleistocene colonization of the mountains in the central TVB is

corroborated by the estimates of historical migration among

selected pairs of mountain populations. The MPEs of migra-

tion 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 the

posterior distributions corroborate the sequential colonization

of mountain ranges from source populations in the north-

eastern region of the species’ distribution.

Our divergence time estimates for the Central TVB clade are

concordant with the timing of the glacial–interglacial cycles

that characterized the last 0.7 Myr of the Pleistocene and that

are thought to have been the major contributors to biological

diversification during this period (Webb & Bartlein, 1992).

Thus, although Pleistocene climate cycles do not explain the

divergences of relictual populations in the eastern TVB

mountains, the expansion of this species to the already formed

mountains of the central TVB was probably facilitated by the

broad distribution of cooler pine forests during glacial periods.

Diversification and conservation of TVB montane

species

Our phylogeographical results are fully concordant with a

previous study by Lynch et al. (1983) that examined the

relationships among Pseudoeurycea species based on allozymes.

That study included seven populations of P. leprosa along with

samples of Pseudoeurycea longicauda, Pseudoeurycea robertsi and

Pseudoeurycea altamontana and reported high intra-specific

differentiation within P. leprosa. Lynch et al. (1983) found a

‘core group’ of populations with only slight genetic differen-

tiation along the main east–west axis of the TVB. This core

group included populations from the Zempoala, Popocatepetl,

Iztaccihuatl and Malinche volcanic ranges (Nei’s genetic

distance DN = 0.002–0.012), all members of the Central TVB

0-10001000-15001500-20002000-25002500-35003500-40004000-45004500-50005000-5500

Elevation (m)

TVB

12

3

45

6

78

910

11

12

13

14

16

15

17 18 1920 21

22

24

25

23

26

Central Central

NE INE I

NE IIINE III

SE SE

Tres Mogotes Tres MogotesHigh immigration

Low immigration

NE IINE II

Kilometres0 50 100

Figure 6 Historical migration among 26 Pseudoeurycea leprosa populations throughout the Trans-Volcanic Belt of Mexico. Ovals group

populations with haplotypes belonging to different clades, with the exception of the South-east (SE) clade (which includes one haplotype

from Vigas, and shares the common haplotype 11 with the Central Clade). The arrows summarize migration rates among mountain ranges

estimated by Lamarc.

Montane diversification in the tropics

Journal of Biogeography 39, 353–370 365ª 2011 Blackwell Publishing Ltd

clade. In contrast, populations in the eastern portion of the

species’ distribution (Cofre de Perote and Pico de Orizaba)

showed high genetic divergences when compared with each

other and the core populations. The greatest genetic divergence

(DN = 0.539) was found between the northernmost population

(Tlaxco, population 17, Fig. 1) and the south-easternmost

population (San Bernardino, in the vicinity of Tres Mogotes,

population 26, Fig. 1). Genetic divergences between Las Vigas

and Tlaxco and all other populations ranged from 0.240 to 0.437

and from 0.325 to 0.539, respectively. Samples from Xometla

were intermediate, with low genetic divergence values from core

populations (DN = 0.015–0.022) and high genetic divergence

values (DN = 0.167–0.325) from eastern populations. This

population belongs to our SE clade, which is sister to the

Central TVB populations we sampled. The results of Lynch

et al. (1983) indicate that the patterns we recovered based on

mtDNA markers are also represented in patterns of nuclear

diversification. Our more complete sampling from throughout

the range shows evidence of strong divergence among popu-

lations in the eastern mountain ranges, suggesting multiple

isolated refugial populations, and confirms the east–west

direction and recent timing of range expansion for this species.

Our data highlight the potential importance of peripheral

isolation and local adaptation for lineage diversification,

especially in montane-adapted taxa, for which the distribution

of appropriate habitat is naturally discontinuous. Our phylo-

genetic analyses reveal a deep genetic divergence between the

haplotype from the Tres Mogotes population and samples

from all other populations of P. leprosa (Figs 2–4). This

population is located in the extreme south-eastern 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 behaviourally from the remaining

P. leprosa populations. Individuals from the Tres Mogotes

population have persisted at this drier, lower-elevation site by

colonizing a new microhabitat; adults in this population live

within bromeliads during much of the year, in contrast to

those of other P. leprosa populations, which are terrestrial.

The genetic consequences of adaptation to montane habitats

are also evident in the inferred least-cost migration routes

among mountain islands. The effects of the topographic

complexity of the TVB, combined with this species’ low

tolerance for high temperatures, have affected the historical

connectivity and diversification among populations. Neither

measure of pairwise population genetic differentiation corre-

lates independently with Euclidean distance nor with the

distance along least-cost paths, but a partial Mantel test shows

a significant correlation between the 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-cost path

among mountain ranges, that has limited historical dispersal

among these populations once they have become established

on new mountain islands.

Given that montane regions are important centres of

diversification, understanding and maintaining the processes

that lead to variation in species richness are also critical for the

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–Popo-

cateptl, Cumbres del Ajusco, Desierto de los Leones, Nevado

de Toluca, Zoquiapan, Lagunas de Zempoala and La Malin-

che), but our data suggest that these protected areas alone are

not sufficient to preserve most of the genetic diversity found

within this species. In fact, the most diverse populations, which

were historically source populations, are found in the eastern

part of the range and are not currently protected. The north-

eastern highlands of the TVB are threatened by severe

encroachment due to urban and agricultural development

(Garcıa-Romero, 2002; Galicia & Garcıa-Romero, 2007). The

preservation of populations with high genetic diversity is

especially important for preserving adaptive genetic variation

and evolutionary potential in the face of global environmental

change (Spielman et al., 2004; Willi et al., 2007). Genetic drift

can lead to the overall reduction of both neutral and adaptive

genetic variation; therefore, the central TVB populations of

P. leprosa may also show reduced genetic diversity at func-

tional genes that are potentially critical for local adaptation to

changing environments. We know that this species will be

threatened by the projected changes in global temperatures,

which will substantially decrease the distribution of appropri-

ate habitat, especially in the central TVB (Parra-Olea et al.,

2005b). Thus, preservation of the remaining populations in the

eastern TVB is critical because populations in that region have

the strongest chance of persistence under projected climate

change and will retain the highest evolutionary potential for

local adaptation to changing environments (Parra-Olea et al.,

2005b; Isaac, 2009).

ACKNOWLEDGEMENTS

Molecular data were collected in the Evolutionary Genetics

Core Facility and the Cornell Core Laboratories Sequencing

Facility. Analyses benefited from resources of the Computa-

tional Biology Service Unit at Cornell University, a facility

partially funded by Microsoft Corporation. We thank D.B.

Wake, T. Papenfuss, J. Hanken, M. Garcıa-Parıs and E. Recuero

for help with field collections; Luis Canseco, G. Casas-Andreu

and Noemı Matıas for providing tissues; Laura Marquez-

Valdelamar for lab assistance; C.G. Becker for assistance with

GIS analyses; D. Buckley and I. Martınez-Solano for advice on

molecular analyses; and the Zamudio Lab for constructive

comments on earlier versions of the manuscript. G.V.-A. was

supported by a post-doctoral fellowship from the Spanish

Ministerio de Ciencia e Innovacion (ref 2008-0890) and G.P. by

a sabbatical fellowship from UC-MEXUS. Field and laboratory

efforts were partially funded by grants from SEP-CONACyT

(50563) and PAPIIT-UNAM (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 K.R.Z.

G. Parra-Olea et al.

366 Journal of Biogeography 39, 353–370ª 2011 Blackwell Publishing Ltd

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SUPPORTING INFORMATION

Additional supporting information may be found in the online

version of this article:

Appendix S1 Pseudoeurycea leprosa samples included in this

study.

Appendix S2 Geographical measures between pairs of

mountain ranges.

As a service to our authors and readers, this journal provides

supporting information supplied by the authors. Such mate-

rials are peer-reviewed and may be re-organized for online

delivery, but are not copy-edited or typeset. Technical support

issues arising from supporting information (other than

missing files) should be addressed to the authors.

BIOSKETCH

Gabriela Parra-Olea is interested in the historical diversi-

fication and conservation of tropical herpetofauna. She is

currently a faculty member at the Instituto de Biologıa,

Universidad Autonoma de Mexico, and her lab focuses on

population genetics, phylogeography and systematic studies of

reptiles and amphibians endemic to Mexico.

Author contributions: G.P.-O. developed the research ques-

tion; G.P.-O. and K.Z. acquired funding to support fieldwork

and laboratory data collection; J.C.W. completed all field

sampling for the project; J.C.W. and G.V.-A. collected DNA

sequence data; all authors contributed to data analyses;

G.P.-O. and K.Z. led the writing of the paper with important

contributions from others.

Editor: Judith Masters

G. Parra-Olea et al.

370 Journal of Biogeography 39, 353–370ª 2011 Blackwell Publishing Ltd