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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: [email protected]�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
vep
op
ula
tio
nsi
zes
(Q)
and
asym
met
ric
mig
rati
on
rate
sin
ferr
edin
La
ma
rc
for
26p
op
ula
tio
ns
of
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ud
oeu
ryce
ale
pros
asa
mp
led
thro
ugh
ou
tth
eT
ran
s-V
olc
anic
Bel
to
f
cen
tral
Mex
ico
.F
or
bo
thp
aram
eter
s,th
em
axim
um
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tim
ate
(MP
E)
isgi
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init
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san
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%co
nfi
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cein
terv
als
(CI)
are
give
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par
enth
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.M
PE
valu
esw
ith
effe
ctiv
esa
mp
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zes
low
erth
an30
0ar
esh
ow
nin
bo
ld.
Ab
ove
the
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al,
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ers
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rto
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rati
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ep
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ns
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edat
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le(i
nb
old
).B
elo
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iago
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um
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em
igra
tio
n
rate
sin
toth
ep
op
ula
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nat
the
left
(in
ital
ics)
.
QM
PE
(95%
CI)
Nev
ado
de
To
luca
Aju
sco
Atz
om
pa
Go
nza
lez
Ort
ega
Lla
no
Gra
nd
e
Mal
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e
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Mal
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asX
om
etla
Nev
ado
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0.00
08
(0.0
00–0
.003
)
–47
4.14
(0.0
2–10
64.6
3)
52.2
7
(0.0
12–3
82.2
8)
3.96
(0.0
1–51
2.95
)
19.3
6
(0.0
1–17
6.66
)
133.
00
(0.0
1–69
8.10
)
126.
02
(0.0
1–72
9.65
)
88.1
4
(0.0
1–57
8.05
)
0.74
(0.0
1–14
1.54
)
0.05
(0.0
1–70
.36)
2.02
(0.0
12–1
96.0
9)
Aju
sco
0.00
00
(0.0
00–0
.001
)
238.
31
(0.0
2–83
7.32
)
–39
.31
(0.0
1–34
7.47
)
16.4
3
(0.0
1–53
6.42
)
32.6
1
(0.0
1–19
6.27
)
114.
07
(0.0
1–65
5.94
)
135.
65
(0.0
1–69
0.81
)
70.9
9
(0.0
1–54
4.39
)
0.95
(0.0
1–13
7.79
)
0.24
(0.0
1–75
.28)
10.8
5
(0.0
1–21
3.70
)
Atz
ompa
0.00
20
(0.0
00–0
.006
)
39.7
8
(0.0
1–52
3.99
)
107.
71
(0.0
1–70
1.63
)
–0.
03
(0.0
1–49
3.38
)
22.6
5
(0.0
1–18
3.19
)
798.
86
(0.0
7–10
36.3
6)
424.
03
(0.0
1–10
74.1
1)
265.
49
(0.0
1–88
5.24
)
0.17
(0.0
1–11
8.30
)
0.19
(0.0
1–55
.97)
25.2
3
(0.0
1–31
8.74
)
Gon
zale
z
Ort
ega
0.00
03
(0.0
00–0
.002
)
30.5
2
(0.0
1–43
2.23
)
102.
36
(0.0
12–6
79.8
4)
11.4
8
(0.0
1–29
0.46
)
–0.
27
(0.0
1–12
9.19
)
78.7
6
(0.0
1–56
8.42
)
64.1
7
(0.0
1–62
8.35
)
28.6
6
(0.0
1–44
8.59
)
15.2
1
(0.0
1–17
6.54
)
44.9
9
(0.0
3–20
5.49
)
11.7
0
(0.0
1–25
4.94
)
Lla
no
Gra
nd
e0.
0041
(0.0
02–0
.012
)
99.1
5
(0.0
1–57
4.62
)
264.
20
(0.0
1–86
7.67
)
80.6
1
(0.0
1–44
0.68
)
3.93
(0.0
1–54
3.13
)
–18
8.25
(0.0
1–80
7.92
)
204.
35
(0.0
1–78
5.50
)
144.
26
(0.0
1–66
4.00
)
2.51
(0.0
1–13
4.40
)
0.66
(0.0
1–52
.45)
24.0
5
(0.0
1–23
4.10
)
Mal
inch
e(E
)0.
0004
(0.0
00–0
.002
)
59.2
5
(0.0
1–53
1.59
)
144.
10
(0.0
1–72
9.34
)
333.
46
(0.6
7–90
3.92
)
3.70
(0.0
1–53
7.59
)
25.7
5
(0.0
1–20
1.89
)
–38
9.20
(0.0
1–10
35.1
5)
234.
74
(0.0
1–86
0.28
)
0.09
(0.0
1–13
1.30
)
0.09
(0.0
1–75
.46)
47.8
2
(0.0
1–33
4.09
)
Mal
inch
e(W
)0.
0003
(0.0
00–0
.002
)
52.0
1
(0.0
1–49
4.81
)
110.
64
(0.0
1–71
8.76
)
124.
66
(0.0
1–62
2.76
)
10.2
0
(0.0
1–53
6.59
)
23.0
1
(0.0
1–17
9.14
)
323.
14
(0.0
1–96
5.82
0)
–20
5.31
(0.0
1–85
8.55
)
0.84
(0.0
1–13
0.60
)
0.46
(0.0
1–62
.88)
42.8
2
(0.0
1–29
5.64
)
Pop
ocat
epet
l0.
0006
(0.0
00–
0.00
2)
83.6
7
(0.0
1–53
7.58
)
153.
55
(0.0
1–74
6.78
)
159.
61
(0.0
1–71
8.06
)
12.7
4
(0.0
1–51
2.56
)
35.1
7
(0.0
1–20
3.22
)
408.
43
(0.0
2–10
51.6
8)
448.
52
(0.0
1–11
03.9
1)
–2.
38
(0.0
1–13
3.38
)
0.71
(0.0
1–60
.47)
28.4
9
(0.0
1–30
8.04
)
Tla
tlau
quit
epec
0.00
349
(0.0
01–
0.01
6)
57.0
2
(0.0
1–47
2.70
)
113.
18
(0.0
1–67
8.25
)
30.3
3
(0.0
1–30
5.24
)
109.
65
(0.0
1–64
0.58
)
0.27
(0.0
1–12
7.88
)
87.0
6
(0.0
1–58
1.27
)
90.2
2
(0.0
1–62
4.99
)
28.2
8
(0.0
1–42
9.74
)
–39
.40
(0.0
2–22
7.49
)
24.7
7
(0.0
1–38
2.09
)
Vig
as0.
0111
4
(0.0
05–
0.02
5)
55.1
9
(0.0
1–51
1.29
)
148.
84
(0.0
1–79
5.57
)
24.5
4
(0.0
1–31
9.48
)
550.
40
(0.0
2–10
47.5
4)
0.86
(0.0
1–12
2.93
)
118.
35
(0.0
1–70
6.66
)
102.
67
(0.0
1–68
6.77
)
47.7
9
(0.0
1–51
9.59
)
100.
38
(0.0
1–52
3.05
)
–11
1.11
(0.0
2–61
8.66
)
Xom
etla
0.00
212
(0.0
01–
0.00
6)
38.3
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
1–15
3.43
)
360.
63
(0.0
1–10
01.6
9)
402.
13
(0.0
1–10
49.4
2)
155.
43
(0.0
1–70
6.81
)
8.55
(0.0
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
REFERENCES
Anducho-Reyes, M.A., Cognato, A.I., Hayes, J.L. & Zuniga, G.
(2008) Phylogeography of the bark beetle Dendroctonus
mexicanus Hopkins (Coleoptera: Curculionidae: Scolytinae).
Molecular Phylogenetics and Evolution, 49, 930–940.
Bonnet, E. & Van de Peer, Y. (2002) ZT: a software tool for
simple and partial Mantel tests. Journal of Statistical Soft-
ware, 7, 1–12.
Brown, B.R. (1985) A summary of late-Quaternary pollen
records from Mexico west of the Isthmus of the Tehuante-
pec. Pollen records of late-Quaternary North American
sediments (ed. by V.M.J. Bryant and R.G. Holloways), pp.
71–94. American Association of Stratigraphic Palynologists
Foundation, Dallas, TX.
Bryson, R.W., Jr, Murphy, R.W., Lathrop, A. & Lazcano-
Villareal, D. (2011) Evolutionary drivers of phylogeo-
graphical diversity in the highlands of Mexico: a case study
of the Crotalus triseriatus species group of montane rattle-
snakes. Journal of Biogeography, 38, 697–710.
Cardillo, M., Orme, C.D.L. & Owens, I.P.F. (2005) Testing for
latitudinal bias in diversification rates: an example using
New World birds. Ecology, 86, 2278–2287.
Carrasco-Nunez, G. & Nelson, S. (1998) Edad y tasa de crec-
imiento del Volcan Cofre de Perote (resumen). Primera
Reunion Nacional de Ciencias de la Tierra, Mexico, D.F.
Carrasco-Nunez, G., Dıaz-Castellon, R., Siebert, L., Hubbard,
B., Sheridan, F. & Rodrıguez, R.R. (2006) Multiple edifice-
collapse events in the Eastern Mexican Volcanic Belt: the
role of sloping substrate and implications for hazard
assessment. Journal of Volcanology and Geothermal Research,
158, 151–176.
Carrasco-Nunez, G., Siebert, L., Dıaz-Castellon, R., Vazquez-
Selem, L. & Capra, L. (2010) Evolution and hazards of a
long-quiescent compound shield-like volcano: Cofre de
Perote, eastern Trans-Mexican Volcanic Belt. Journal of
Volcanology and Geothermal Research, 197, 209–224.
Castillo-Rodrıguez, M., Lopez-Blanco, J. & Palacios, D. (2007)
Multivariate analysis of the location of rock glaciers and the
environmental implications in a tropical volcano: La
Malinche (Central Mexico). Zeitschrift fur Geomorphologie,
51, 39–54.
Castro-Govea, R. & Claus, S. (2007) Late Pleistocene–
Holocene stratigraphy and radiocarbon dating of La
Malinche volcano, central Mexico. Journal of Volcanology
and Geothermal Research, 162, 20–42.
Chown, S.L. & Gaston, K.J. (2000) Areas, cradles and muse-
ums: the latitudinal gradient in species richness. Trends in
Ecology and Evolution, 15, 311–315.
Clement, M., Posada, D. & Crandall, K.A. (2000) TCS: a
computer program to estimate gene genealogies. Molecular
Ecology, 9, 1657–1659.
Cortes-Rodrıguez, N., Hernandez-Banos, B.E., Navarro-
Siguenza, A.G. & Omland, K.E. (2008) Geographic variation
and genetic structure in the streak-backed oriole: low
mitochondrial DNA differentiation reveals recent diver-
gence. The Condor, 110, 729–739.
Darda, D.M. (1994) Allozyme variation and morphological
evolution among Mexican salamanders of the genus Chiro-
pterotriton (Caudata: Plethodontidae). Herpetologica, 50,
164–187.
DeChaine, E.G. & Martin, A.P. (2006) Using coalescent sim-
ulations to test the impact of Quaternary climate cycles on
divergence in an alpine plant-insect association. Evolution,
60, 1004–1013.
Dıaz-Castellon, R., Castillo-Nunez, G. & Alvarez-Manilla, A.
(2008) Mechanical instability quantification of slopes at
Cofre de Perote Volcano, eastern Mexico. Boletın de la
Sociedad Geologica Mexicana, 60, 187–201.
Domınguez-Domınguez, O. & Perez-Ponce de Leon, G. (2009)
La mesa central de Mexico es una provincia biogeografica?
Analisis descriptivo basado en componentes bioticos dul-
ceacuıcolas. Revista Mexicana de Biodiversidad, 80, 835–852.
Drummond, A.J. & Rambaut, A. (2007) BEAST: Bayesian
evolutionary analysis by sampling trees. BMC Evolutionary
Biology, 7, 214.
Drummond, A.J., Rambaut, A., Shapiro, B. & Pybus, O.G.
(2005) Bayesian coalescent inference of past population
dynamics from molecular sequences. Molecular Biology and
Evolution, 22, 1185–1192.
Drummond, A.J., Ho, S.Y.W., Phillips, M.J. & Rambaut, A.
(2006) Relaxed phylogenetics and dating with confidence.
PLoS Biology, 4, 699–710.
Excoffier, L., Laval, G. & Schneider, S. (2005) Arlequin
(version 3.0): an integrated software package for population
genetics data analysis. Evolutionary Bioinformatics Online, 1,
47–50.
Ferrari, L. (2000) Episodes of Cenozoic volcanism and tec-
tonics in central Mexico. Journal of Vertebrate Paleontology,
20, 40A.
Ferrari, L. & Rosas-Elguera, J. (1999) Alkalic (ocean-island
basalt type) and calc-alkalic volcanism in the Mexican vol-
canic belt: a case for plume-related magmatism and prop-
agating rifting at an active margin?: comment and reply.
Geology, 27, 1055–1056.
Ferrari, L., Lopez-Martınez, M., Aguirre-Dıaz, G. & Carrasco-
Nunez, G. (1999) Space-time patterns of Cenozoic arc vol-
canism in central Mexico: from the Sierra Madre Occidental
to the Mexican Volcanic Belt. Geology, 27, 303–306.
Ferrari, L., Conticelli, S., Vaggelli, G., Petrone, C.M. &
Manetti, P. (2000) Late Miocene volcanism and intra-arc
tectonics during the early development of the Trans-Mexi-
can Volcanic Belt. Tectonophysics, 318, 161–185.
Fjeldsa, J. & Lovett, J.C. (1997) Geographical patterns of old
and young species in African forest biota: the significance of
specific montane areas as evolutionary centres. Biodiversity
and Conservation, 6, 325–346.
Flores-Villela, O. & Canseco-Marquez, L. (2007) Riqueza de la
herpetofauna. Biodiversidad de la Faja Volcanica Trans-
mexicana (ed. by I. Luna, J.J. Morrone and D. Espinosa),
Montane diversification in the tropics
Journal of Biogeography 39, 353–370 367ª 2011 Blackwell Publishing Ltd
pp. 407–420. Universidad Nacional Autonoma de Mexico,
Mexico, D.F.
Flores-Villela, O., Canseco-Marquez, L. & Ochoa-Ochoa, L.
(2010) Geographic distribution and conservation of the
herpetofauna of the highlands of central Mexico. Conser-
vation of Mesoamerican amphibians and reptiles (ed. by L.D.
Wilson, J.H. Townsend and J.D. Johnson), pp. 302–321.
Eagle Mountain Publishing Co., Eagle Mountain, UT.
Francis, A.P. & Currie, D.J. (2003) A globally consistent
richness–climate relationship for angiosperms. The Ameri-
can Naturalist, 161, 523–536.
Fu, X.Y. (1997) Statistical tests of neutrality of mutations
against population growth, hitchhiking, and background
selection. Genetics, 147, 915–925.
Galbreath, K.E., Hafner, D.J. & Zamudio, K.R. (2009) When
cold is better: climate-driven elevation shifts yield complex
patterns of diversification and demography in an alpine
specialist (American pika, Ochotona princeps). Evolution, 63,
2848–2863.
Galicia, L. & Garcıa-Romero, A. (2007) Land use and land
cover change in highland temperate forests in the Izta-Popo
National Park, Central Mexico. Mountain Research and
Development, 27, 48–57.
Garcıa-Palomo, A., Macıas, J.L. & Garduno, V.H. (2000)
Miocene to Recent structural evolution of the Nevado de
Toluca volcano region, Central Mexico. Tectonophysics, 318,
281–302.
Garcıa-Palomo, A., Macıas, J.L., Arce, J.L., Capra, L., Garduno,
V.H. & Espındola, J.M. (2002) Geology of Nevado de Toluca
Volcano and surrounding areas, central Mexico. Geological
Society of America Map and Chart Series MCH089. Geo-
logical Society of America, Boulder, CO.
Garcıa-Palomo, A., Zamorano, J.J., Lopez-Miguel, C., Galvan-
Garcıa, A., Carlos-Valerio, V., Ortega, R. & Macıas, J.L.
(2008) El arreglo morfoestructural de la Sierra de Las Cru-
ces, Mexico central. Revista Mexicana de Ciencias Geologicas,
25, 158–178.
Garcıa-Romero, A. (2002) An evaluation of forest deteriora-
tion in the disturbed mountains of western Mexico City.
Mountain Research and Development, 22, 270–277.
Ghalambor, C.K., Huey, R.B., Martin, P.R., Tewksbury, J.J. &
Wang, G. (2006) Are mountain passes higher in the tropics?
Janzen’s hypothesis revisited. Integrative and Comparative
Biology, 46, 5–17.
Gomez-Tuena, A., Orozco-Esquivel, M.T. & Ferrari, L.
(2007) Igneous petrogenesis of the Trans-Mexican Volca-
nic Belt. Geological Society of America Special Paper, 422,
129–181.
Graham, A. (1993) Historical factors and biological diversity in
Mexico. Biological diversity of Mexico: origins and distribution
(ed. by T. Ramamoorthy, R. Bye, A. Lot and J. Fa), pp. 109–
127. Oxford University Press, Oxford.
Hafner, M.S., Light, J.E., Hafner, D.J., Brant, S.V., Spradling,
T.A. & Demastes, J.W. (2005) Cryptic species in the Mexican
pocket gopher Cratogeomys merriami. Journal of Mammal-
ogy, 86, 1095–1108.
Hawkins, B.A., Field, R., Cornell, H.V., Currie, D.J., Guegan,
J.-F., Kaufman, D.M., Kerr, J.T., Mittelbach, G.G., Obe-
rdorff, T., O’Brien, E.M., Porter, E.E. & Turner, J.R.G.
(2003) Energy, water, and broad-scale patterns of species
richness. Ecology, 84, 3105–3117.
Heine, K. (1988) Late Quaternary glacial chronology of the
Mexican volcanoes. Die Geowissenschaften, 7, 197–205.
Hewitt, G.M. (2004) Genetic consequences of climatic oscil-
lations in the Quaternary. Philosophical Transactions of the
Royal Society B: Biological Sciences, 359, 183–195.
Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: Bayesian
inference of phylogenetic trees. Bioinformatics, 17, 754–
755.
Hughes, L. (2003) Climate change and Australia: trends, pro-
jections and research directions. Austral Ecology, 28, 423–
443.
Huson, D. H. & Bryant, D. (2006) Application of phylogenetic
networks in evolutionary studies. Molecular Biology and
Evolution, 23, 254–267.
Isaac, J.L. (2009) Effects of climate change on life history:
implications for extinction risk in mammals. Endangered
Species Research, 7, 115–123.
Janzen, D.H. (1967) Why mountain passes are higher in the
tropics. The American Naturalist, 101, 233–249.
Jetz, W., Rahbek, C. & Colwell, R.K. (2004) The coincidence of
rarity and richness and the potential signature of history in
centres of endemism. Ecology Letters, 7, 1180–1191.
Knowles, L.L. (2000) Tests of Pleistocene speciation in mon-
tane grasshoppers (genus Melanoplus) from the sky islands
of western North America. Evolution, 54, 1337–1348.
Kozak, K.H. & Wiens, J.J. (2006) Does niche conservatism
promote speciation? A case study in North American sala-
manders. Evolution, 60, 2604–2621.
Kozak, K.H. & Wiens, J.J. (2007) Climatic zonation drives
latitudinal variation in speciation mechanisms. Proceedings
of the Royal Society B: Biological Sciences, 274, 2995–3003.
Kuhner, M.K. (2006) LAMARC 2.0: maximum likelihood and
Bayesian estimation of population parameters. Bioinfor-
matics, 22, 768–770.
Lozano-Garcıa, S., Sosa-Najera, S., Sugiura, Y. & Caballero, M.
(2005) 23,000 yr of vegetation history of the Upper Lerma, a
tropical high-altitude basin in central Mexico. Quaternary
Research, 64, 70–82.
Luna, I., Morrone, J. & Espinosa, D. (2007) Biodiversidad de la
faja volcanica transmexicana. Universidad Nacional Auto-
noma de Mexico, Mexico, D. F.
Lynch, J.F., Wake, D.B. & Yang, S.Y. (1983) Genic and mor-
phological differentiation in Mexican Pseudoeurycea (Cau-
data: Plethodontidae) with a description of a new species.
Copeia, 1983, 884–894.
Martınez-Solano, I. & Lawson, R. (2009) Escape to Alcatraz:
evolutionary history of slender salamanders (Batrachoseps)
on the islands of San Francisco Bay. BMC Evolutionary
Biology, 9, 1–14.
McCain, C.M. (2005) Elevational gradients in diversity of small
mammals. Ecology, 86, 366–372.
G. Parra-Olea et al.
368 Journal of Biogeography 39, 353–370ª 2011 Blackwell Publishing Ltd
McCain, C.M. (2007) Area and mammalian elevational
diversity. Ecology, 88, 76–86.
McCormack, J.E., Peterson, A.T., Bonaccorso, E. & Smith, T.B.
(2008) Speciation in the highlands of Mexico: genetic and
phenotypic divergence in the Mexican jay (Aphelocoma ul-
tramarina). Molecular Ecology, 17, 2505–2521.
McDonald, J.A. (1993) Phytogeography and history of the
alpine-subalpine flora of northeastern Mexico. Biological
diversity of Mexico: origins and distribution (ed. by
T. Ramamoorthy, R. Bye, A. Lot and J. Fa), pp. 681–706.
Oxford University Press, Oxford.
Mena, J.L. & Vazquez-Domınguez, E. (2005) Species turnover
on elevational gradients in small rodents. Global Ecology and
Biogeography, 14, 539–547.
Mora-Alvarez, G., Caballero, C., Urrutia-Fucugauchi, J. &
Uchiumi, S. (1991) Southward migration of volcanic activity
in the Sierra de las Cruces basin of Mexico? A preliminary
K–Ar dating and palaeomagnetic study. Geofisica Internac-
ional, 30, 61–70.
Moritz, C., Schneider, C.J. & Wake, D.B. (1992) Evolutionary
relationships within the Ensatina eschscholtzii complex
confirm the ring species interpretation. Systematic Biology,
41, 273–291.
Morrone, J. (2005) Hacia una sintesis biogeografica de Mexico.
Revista Mexicana de Biodiversidad, 76, 207–252.
Mueller, R.L. (2006) Evolutionary rates, divergence rates, and
the performance of mitochondrial genes in Bayesian phy-
logenetic analysis. Systematic Biology, 55, 289–300.
Nei, M. (1987) Molecular evolutionary genetics. Columbia
University Press, New York.
Nixon, G.T. (1989) The geology of Iztaccihuatl volcano and
adjacent areas of the Sierra Nevada and Valley of Mexico.
Geological Society of America Special Paper, 219, 1–58.
Nixon, G.T., Demant, A., Amstrong, R.L. & Harakal, J.E.
(1987) K–Ar and geologic data bearing on the age and
evolution of the Trans-Mexican Volcanic Belt. Geofisica
Internacional, 26, 109–158.
Nixon, K.C. (1993) The genus Quercus in Mexico. Biological
diversity of Mexico: origins and distribution (ed. by T.
Ramamoorthy, R. Bye, A. Lot and J. Fa), pp. 447–458.
Oxford University Press, Oxford.
Oomen, M.A. & Shanker, K. (2005) Elevational species rich-
ness patterns emerge from multiple mechanisms in Hima-
layan woody plants. Ecology, 86, 3039–3047.
Osete, M.L., Ruiz-Martınez, V.C., Caballero, C., Galindo, C.,
Urrutia-Fucugauchi, J. & Harling, D.H. (2000) Southward
migration of continental volcanic activity in the Sierra de
Las Cruces, Mexico: palaeomagnetic and radiometric
evidence. Tectonophysics, 318, 201–215.
Parra-Olea, G. (2003) Phylogenetic relationships of the genus
Chiropterotriton (Caudata: Plethodontidae) based on 16S
ribosomal mtDNA. Canadian Journal of Zoology, 81, 2048–
2060.
Parra-Olea, G., Garcıa-Parıs, M., Papenfuss, T. & Wake, D.B.
(2005a) Systematics of the Pseudoeurycea bellii (Caudata:
Plethodontidae) species complex. Herpetologica, 61, 145–158.
Parra-Olea, G., Martınez-Meyer, E. & Perez Ponce de Leon, G.
(2005b) Forecasting climate change effects on salamander
distribution in the highlands of Central Mexico. Biotropica,
37, 202–208.
Pasquare, G., Garduno, V.H., Tibaldi, A. & Ferrari, L. (1988)
Stress pattern evolution in the central sector of the Mexican
volcanic belt. Tectonophysics, 146, 352–364.
Posada, D. (2008) jModelTest: phylogenetic model averaging.
Molecular Biology and Evolution, 25, 1253–1256.
Rahbek, C. (1995) The elevational gradient of species richness:
a uniform pattern? Ecography, 18, 200–205.
Rahbek, C. & Graves, C.R. (2001) Multiscale assessment of
patterns of avian species richness. Proceedings of the National
Academy of Sciences USA, 98, 4534–4539.
Ramamoorthy, T., Bye, R., Lot, A. & Fa, J. (1993) Biological
diversity of Mexico: origins and distribution. Oxford Univer-
sity Press, Oxford.
Rambaut, A. & Drummond, A.J. (2007) Tracer v1.4. Available
at: http://beast.bio.ed.ac.uk/Tracer.
Ricklefs, R.E. (2006) Evolutionary diversification and the ori-
gin of the diversity–environment relationship. Ecology, 87,
S3–S13.
Rossotti, A., Carrasco-Nunez, G., Rosi, M. & Di Muro, A.
(2006) Eruptive dynamics of the ‘Citlaltepetl Pumice’ at
Citlaltepetl Volcano, eastern Mexico. Journal of Volcanology
and Geothermal Research, 158, 401–429.
Rozas, J., Sanchez-DelBarrio, J.C., Messeguer, X. & Rozas, R.
(2003) DnaSP, DNA polymorphism analyses by coalescent
and other methods. Bioinformatics, 19, 2496–2497.
Schaaf, P., Stimac, J., Siebe, C. & Macıas, J. (2005) Geo-
chemical evidence for mantle origin and crustal processes in
volcanic rocks from Popocatepetl and surrounding mono-
genetic volcanoes, central Mexico. Journal of Petrology, 46,
1243–1282.
Schneider, S. & Excoffier, L. (1999) Estimation of past
demographic parameters from the distribution of pairwise
differences when the mutation rates very among sites:
application to human mitochondrial DNA. Genetics, 152,
1079–1089.
Smith, S.A., de Oca, A.N.M., Reeder, T.W. & Wiens, J.J. (2007)
A phylogenetic perspective on elevational species richness
patterns in Middle American treefrogs: why so few species in
lowland tropical rainforests? Evolution, 61, 1188–1207.
Spielman, D., Brook, B.W. & Frankham, R. (2004) Most spe-
cies are not driven to extinction before genetic factors
impact them. Proceedings of the National Academy of Sciences
USA, 101, 15261–15264.
Stevens, R.D. (2006) Historical processes enhance patterns of
diversity along latitudinal gradients. Proceedings of the Royal
Society B: Biological Sciences, 273, 2283–2289.
Tajima, F. (1989) The effect of change in population size on
DNA polymorphism. Genetics, 123, 597–602.
Tamayo, L.T. & West, R.C. (1964) The hydrology of Middle
America. Handbook of Middle America Indians (ed. by
R. Wauchope), pp. 84–121. University of Texas Press,
Austin, TX.
Montane diversification in the tropics
Journal of Biogeography 39, 353–370 369ª 2011 Blackwell Publishing Ltd
Templeton, A.R., Crandall, K.A. & Sing, C.F. (1992) A cladistic
analysis of phenotypic associations with haplotypes inferred
from restriction endonuclease mapping and DNA sequence
data. III: Cladogram estimation. Genetics, 132, 619–633.
Tennessen, J.A. & Zamudio, K.R. (2008) Genetic differentia-
tion among mountain island populations of the striped
plateau lizard, Sceloporus virgatus (Squamata: Phrynoso-
matidae). Copeia, 2008, 558–564.
Vazquez-Selem, L. & Heine, K. (2004) Late Quaternary
glaciation of Mexico. Quaternary glaciations: extent and
chronology, part III (ed. by J. Ehlers and P.L. Gibbars), pp.
233–242. Elsevier, Amsterdam.
Webb, T., III & Bartlein, P. J. (1992) Global changes during the
last 3 million years: climatic controls and biotic responses.
Annual Review of Ecology and Systematics, 23, 141–173.
Wiens, J.J., Graham, C.H., Moen, D.S., Smith, S.A. & Reeder,
T.W. (2006) Evolutionary and ecological causes of the lati-
tudinal diversity gradient in hylid frogs: treefrog trees
unearth the roots of high tropical diversity. The American
Naturalist, 168, 579–596.
Wiens, J.J., Parra-Olea, G., Garcıa-Parıs, M. & Wake, D.B.
(2007) Phylogenetic history explains elevational biodiversity
patterns in tropical salamanders. Proceedings of the Royal
Society B: Biological Sciences, 274, 919–928.
Willi, Y., Van Buskirk, J., Schmid, B. & Fischer, M. (2007)
Genetic isolation of fragmented populations is exacerbated
by drift and selection. Journal of Evolutionary Biology, 20,
534–542.
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.
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rials are peer-reviewed and may be re-organized for online
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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