Molecular Phylogenetics and Evolution 99 (2016) 7688

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Genetic divergence in the common bush-tanager Chlorospingus ophthalmicus

(Aves: Emberizidae) throughout Mexican cloud forests: The role of geography,
ecology and Pleistocene climatic fluctuations
Denisse Maldonado-Snchez, Carla Gutirrez-Rodrguez , Juan Francisco Ornelas
Departamento de Biologa Evolutiva, Instituto de Ecologa, AC, Xalapa, Veracruz 91070, Mexico

articleinfo
Article history:
Received 12 August 2015
Revised 23 February 2016
Accepted 13 March 2016
Available online 14 March
2016

Keywords:
Cloud forests
Gene flow
Morphotectonic provinces
Pleistocene glaciations
Population divergence
Subspecies

abstract
By integrating mitochondrial DNA (mtDNA), microsatellites and ecological niche modelling (ENM),
we investigated the phylogeography of Mexican populations of the common bush-tanager
Chlorospingus ophthalmicus to examine the relative role of geographical and ecological features, as
well as Pleistocene climatic oscillations in driving the diversification. We sequenced mtDNA of
individuals collected throughout the species range in Mexico and genotyped them at seven
microsatellite loci. Phylogeographic, population genetics and coalescent methods were used to assess
patterns of genetic structure, gene flow and demographic history. ENM was used to infer contractions
and expansions at different time periods as well as differences in climatic conditions among lineages.
The retrieved mitochondrial and microsatellite groups correspond with the fragmented cloud forest
distribution in mountain ranges and morphotectonic provinces. Differing climatic conditions between
mountain ranges were detected, and palaeodistribution modelling as well as demographic history
analyses, indicated recent population expansions throughout the Sierra Madre Oriental (SMO). The
marked genetic structure of C. ophthalmicus was promoted by the presence of ecological and
geographical barriers that restricted the movement of individuals among mountain ranges. The SMO
was mainly affected by Pleistocene climatic oscillations, with the moist forests model best fitting the
displayed genetic patterns of populations in this mountain range.

1. Introduction
Climate and topography have shaped, separately or together,
the phylogeographical patterns of current populations. Several
studies have shown that historical climatic fluctuations during
the Quaternary led to expansion/contraction cycles of
populations during the glacial and interglacial periods,
influencing the patterns of genetic diversity of different species
(e.g., Soltis et al., 1997; Taberlet et al.,
1998; Hewitt, 1999, 2000). Similarly, topographically complex
landscapes have shaped the genetic architecture of populations
by creating patchiness or corridors in the distribution of optimal
habitat, promoting genetic divergence or gene flow (Chves et
al., 2007; Richardson, 2012; Valderrama et al., 2014).
The distribution of several species in Mesoamerica has been
historically fragmented as a result of geological and climatic

2016 Elsevier Inc. All rights reserved.

events (Navarro-Sigenza et al., 2008; Valderrama et al., 2014).

In particular, species inhabiting cloud forests that are
allopatrically distributed on mountain ranges can be affected by
the restricted mobility of individuals and thus limited gene flow
between isolated populations (Garca-Moreno et al., 2004;
Puebla-Olivares et al., 2008; Barrera-Guzmn et al., 2012;
Ornelas and Gonzlez, 2014). Pleistocene climate oscillations
are also known to influence the distribution of Mesoamerican

(Colinvaux et al., 1996), that can affect the distribution of

Neotropical cloud forest species (Ramrez-Barahona and

cloud forest species by promoting expansion, contraction and

divergence of populations (Gutir rez-Rodrguez et al., 2011;
Ornelas and Gonzlez, 2014). Genetic and palaeoecological data
have suggested that montane species descended towards the
lowlands to survive the cooling periods (Colinvaux et al., 2000;
Jaramillo-Correa et al., 2008), resulting in population
expansions and gene flow. However, as the result of the
substantial changes in precipitation during the glacial periods
extrapolated from the Pleistocene refugia hypothesis for lowland
tropical forests (Haffer, 1969; Toledo, 1982), is based on the
suggestion that colder periods were characterised by increased
humidity while warmer periods were dry. This model posits that
cloud forests were compressed into refugia by the opposing
effects of aridity and cooling during the Last Glacial Maximum
(LGM) at higher elevations. These refugia were located at midelevations in mountain regions with stable temperature and
humidity conditions (Haffer, 1969; Toledo, 1982). Therefore,

Eguiarte, 2013) ,
patterns of diversification could be more complex.
According to Ramrez-Barahona and Eguiarte (2013), two
general models explain the glacial and postglacial dynamics of
Neotropical cloud forest species. The dry refugia model,

populations at high altitudes migrated downslope contracting

into distinct refugia at midelevations, and then re-expanded their
distributional ranges to higher elevations when warmer and
more humid conditions were present (Colinvaux et al., 2000).
Alternatively, for the moist forests model is proposed that cloud

Corresponding author at: Departamento de Biologa Evolutiva, Instituto de

Ecologa, AC, Carretera antigua a Coatepec 351, El Haya, Xalapa, Veracruz
91070, Mexico.
E-mail address: carla.gutierrez@inecol.mx (C. Gutirrez-Rodrguez).
http://dx.doi.org/10.1016/j.ympev.2016.03.014
1055-7903/ 2016 Elsevier Inc. All rights reserved.

D. Maldonado-Snchez et al. / Molecular Phylogenetics and Evolution 99 (2016) 7688

forests were not strongly affected by changes in precipitation.

Consequently, during glacial periods, populations migrated
down-slope resulting in range expansions and connectivity
among populations, and during the warmer interglacial periods,
populations migrated upslope to higher altitudes resulting in
their fragmentation and isolation (Ramrez-Barahona and
Eguiarte, 2013).
Here, we assess the relative role that geographical and
ecological features in Mexican cloud forests as well as
Pleistocene climate fluctuations, played on the
phylogeographic patterns of the common bush-tanager
Chlorospingus ophthalmicus [C. flavopectus; Chesser et al.,
2013], a sedentary species strongly associated to cloud
forests (Peterson et al., 1992). Cloud forests of eastern
Mexico are located along the Sierra Madre Oriental, Sierra
de Los Tuxtlas, Sierra Madre del Sur, sierras in the northern
edge of the Meseta Central and Pacific slope of Chiapas (Fig.
1). Along the Sierra Madre Oriental, cloud forests are further
subdivided into northern, central, and southern areas based
on morphotectonics, soil properties and floristic composition
(Ferrusqua-Villafranca, 1993; Len Paniagua and Morrone,
2009). Previous work has suggested that the geographic
isolation of C. ophthalmicus populations on different
mountain ranges has lead to genetic, morphological, and
acoustic divergence, supporting the recognition of five
subspecies: Chlorospingus ophthalmicus ophthalmicus, C.
ophthalmicus albifrons, C. ophthalmicus dwighti, C.
ophthalmicus postocularis, and C. ophthalmicus wetmorei
(Garca-Moreno et al., 2004; SnchezGonzlez et al., 2007;
Bonaccorso et al., 2008; Weir et al., 2008; Sosa-Lpez et al.,
2013). However, previous genetic evidence is based solely on
mtDNA coding genes, making the addition of independent
markers necessary to determine if the same patterns of
divergence among subspecies are recovered.
By integrating mtDNA and microsatellites markers as
well as ecological niche modelling, we aim to test the effects
of geography, ecology and Pleistocene climatic oscillations
on the genetic variation of C. ophthalmicus, and to associate
the observed genetic patterns with the dry refugia and/or
moist forests models. If geography constitutes a more
important driving force than Pleistocene climatic oscillations,
we expect deep genetic divergence between populations from
different mountain ranges due to longer history of geographic
isolation with no signals of expansion. Alternatively, lower
levels of genetic differentiation between populations will
suggest that Pleistocene climatic oscillations played a more
important role in shaping the observed genetic patterns. The
dry refugia model will be favoured if populations from
different refugia (mountain range) were characterised by
marked genetic differentiation, limited gene flow, loss of
genetic diversity, and demographic expansions during
Pleistocene climate cycles. On the other hand, shallow
genetic differentiation with no geographic structuring of the
resulting lineages, higher levels of genetic diversity and gene
flow during glacial periods in the lowlands as well as little to
no demographic expansions, are expected in the moist forests
model.
2. Materials and methods
2.1. Sample collection
We sampled 176 individuals from 35 localities covering the
geographical range of the species in Mexico (Table 1, Fig. 1).
Sampling localities were chosen based on the fragmented cloud
forest distribution in five mountain ranges and in three
morphotectonic provinces within the Sierra Madre Oriental
(FerrusquaVillafranca, 1993; Fig. 1). We sampled twenty-six
populations along the northern (nSMO), central (cSMO) and the

77

southern parts (sSMO or Nudo de Zempoaltepetl) of the Sierra

Madre Oriental (SMO), one site in Sierra de Los Tuxtlas (ST),
two in Sierra Madre del Sur (SMS), three in the Pacific slope of
Chiapas (pCHIS), and three in central Chiapas (cCHIS) (Table
1, Fig. 1). We captured birds in mist nets and collected two tail
rectrices from each individual for genetic analyses before
releasing them.
2.2. Mitochondrial DNA sequencing and
microsatellite genotyping
We extracted the DNA of C. ophthalmicus individuals and
amplified the first domain of the control region of 176
individuals using PCR; resulting amplicons were purified and
sequenced in both directions. Samples from 181 individuals
were genotyped at seven microsatellite loci by PCR. For details
in laboratory procedures see Supplementary material: Materials
and Methods.
2.3. Relationships among mitochondrial haplotypes
and divergence time estimates
We generated a statistical parsimony haplotype network
using TCS 1.21 (Clement et al., 2000) and the control region
sequence data to infer haplotype relationships. Phylogenetic
relationships and divergence times were assessed using
Bayesian inference and the control region sequences in BEAST
1.5.1, with details given in Supplementary material: Materials
and Methods. To have a more robust haplotype network and
phylogenetic reconstruction and to increase the certainty on our
divergence time estimates, we also performed these analyses by
concatenating the control region sequences to a previously
published fragment consisting of either portions or complete
sequences of protein coding genes/ enzymes (COII, tRNA-lys,
ATPase 6 and 8), from individuals collected in the same
localities as our samples (Supplementary material Table S1). We
included GenBank sequences (Supplementary material Table
S2) of different emberizid species as outgroups to C.
ophthalmicus in the BEAST analyses, according to Barker et al.
(2013), as described in Supplementary material: Materials and
Methods.
2.4. Genetic diversity
We calculated genetic diversity of mitochondrial and
microsatellite data for each collection site, mountain range and
morphotectonic province. All population level analyses using
mitochondrial sequences were only performed on the control
region (n = 176 sequences) because sample size of the combined
dataset (control region plus coding genes) is considerably
smaller (n = 44 sequences). We calculated genetic (h) and
nucleotide (p) diversity in ARLEQUIN 3.01 (Excoffier et al.,
2005).
For microsatellites, we calculated the mean number of alleles
per locus, allelic richness, observed (H O) and expected (HE)
heterozygosities, and the inbreeding coefficient (FIS) combining
all loci in FSTAT 2.93 (Goudet, 2001). Deviations from Hardy
Weinberg equilibrium (HWE) and genotypic linkage
disequilibrium were assessed in GENEPOP on the web 4.0.10
(Raymond

D. Maldonado-Snchez et al. / Molecular Phylogenetics and Evolution 99 (2016) 7688

78

Fig. 1. Geographic distribution of cloud forest and sampling localities (red points) of Chlorospingus ophthalmicus in Mexico. Ovals delimited with dashed lines represent the subspecies distribution
along mountain ranges (Sierra Madre Oriental-C. ophthalmicus ophthalmicus, Sierra de los Tuxtlas-C. ophthalmicus wetmorei, Sierra Madre del Sur-C. ophthalmicus albifrons, Sierra del Sur de
Chiapas-C. ophthalmicus postocularis and Sierra del Norte de Chiapas-C. ophthalmicus dwighti) and ovals in solid lines delimit morphotectonic provinces within the Sierra Madre Oriental according
to Ferrusqua-Villafranca (1993). Refer to Table 1 for code designations.

and Rousset, 1995). We further investigated deviations from

HWE with MICROCHECKER 2.2.3 (van Oosterhout et al.,
2004). Multiple comparisons were corrected with Bonferroni
tests.

2.5. Population genetic differentiation

To determine the distribution of mitochondrial genetic
variation, we carried out three analyses of molecular variance
(AMOVA) in ARLEQUIN with 16,000 permutations. We
performed the AMOVAs treating locations as a single group, and
grouping into five subspecies/mountain ranges or seven groups
considering four mountain ranges and the three SMO
morphotectonic provinces.
We further assessed population genetic differentiation
through pairwise FST comparisons between mountain ranges and
provinces with 1,000 permutations, using ARLEQUIN for
control region and FSTAT for microsatellites. Due to differences
in the effective population size of mtDNA and microsatellites,
discordant pairwise FST values are expected. Additionally, FST
values from microsatellites are expected to be small because of
their high levels of polymorphism (Hedrick, 1999). To address
these issues we calculated pairwise Josts D (Jost, 2008), an
unbiased
estimator
of
differentiation
in
SPADE
(http://chao.stat.nthu.edu.tw/) for both markers, using 1000
bootstrap replicates. Multiple comparisons were adjusted by
Bonferroni tests.
We also inferred population genetic structure based on
microsatellites using a Bayesian clustering analysis in
STRUCTURE
2.1 (Pritchard et al., 2000). We used the admixture model,
with correlated allele frequencies and the LOCPRIOR
function to allow STRUCTURE to use sampling locality
information as a prior. Ten independent chains were run for

79

D. Maldonado-Snchez et al. /Molecular Phylogenetics and Evolution 99 (2016) 76 88

each K, from K = 1 to K = 10. The length of the burn-in was

500,000 and the number of MCMC replications after burn-in
was 1,000,000. To determine the most likely value of groups,
we calculated the DK statistic (Evanno et al., 2005).
Subsequent analyses in STRUCTURE were conducted in
each of the genetic clusters depicted by the previous analysis
to detect additional substructure. For these analyses we ran

separate runs had similar posterior distributions and ESS for

each parameter was at least 50. We report the averages of the
parameter estimates across the four runs and the 90% highest
posterior densities ( HPD ) intervals of each parameter. To
calibrate time estimates, we used the nucleotide substitution rate
of 2.6% (see Supplementary material: Materials and Methods)
and employed a generation time of one year to convert

Table 1
Geographic information of Chlorospingus ophthalmicus collection sites in Mexico including subspecies, geographic distribution in mountain ranges and morphotectonic provinces, and number of
samples (N) analysed with control region (mtDNA) and microsatellites ( SSR ).
Sampling
Mountain
Subspecies
Morphotectonic
State
Location (abbreviation)
N (mtDNA/SSR)
Latitude
Longitude
Altitude ( m )
locality ID
range
province
N
W
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35

SMO

ophthalmicus

nSMO

Hidalgo

Tlanchinol (Tlan)

9/9

21101100

983803500

1413

SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
SMO
ST
SMS
SMS
pCHIS
pCHIS
pCHIS
cCHIS
cCHIS
cCHIS

ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
ophthalmicus
wetmorei
albifrons
albifrons
postocularis
postocularis
postocularis
dwighti
dwighti
dwighti

nSMO
nSMO
nSMO
nSMO
nSMO
nSMO
nSMO
nSMO
cSMO
cSMO
cSMO
cSMO
cSMO
cSMO
cSMO
cSMO
cSMO
cSMO
sSMO
sSMO
sSMO
sSMO
sSMO
sSMO
sSMO
ST
SMS
SMS
pCHIS
pCHIS
pCHIS
cCHIS
cCHIS
cCHIS

Hidalgo
Hidalgo
Hidalgo
Hidalgo
Quertaro
Quertaro
Quertaro
Veracruz
Veracruz
Veracruz
Veracruz
Veracruz
Veracruz
Puebla
Puebla
Puebla
Puebla
Puebla
Oaxaca
Oaxaca
Oaxaca
Oaxaca
Oaxaca
Oaxaca
Oaxaca
Veracruz
Oaxaca
Guerrero
Chiapas
Chiapas
Chiapas
Chiapas
Chiapas
Chiapas

El Potrero (Ptr)
La Majonera (Maj)
Cerro Jarros (Cj)
El Coyol (Cy)
Tres Lagunas (Tlg)
Santa Ins (Si)
El Pemoche (Pmc)
Zacualpan (Zac)
Coapexpan (Coap)
El Riscal (Ris)
Inecol (Ie)
Xico (Xico)
La Ordua (Ord)
La Galera (Gale)
Jonotla (Jon)
Cuetzalan (Cuit)
San Andrs (Sna)
Lagunillas (Lagu)
San Martn Caballero (Smc)
Puente Fierro (Pf)
Pea Verde (Pv)
Ejido Clemencia (Ec)
San Juan Yagila (Yag)
Puerto Soledad (Ps)
La Esperanza (Esp)
Barrio Lerdo (Bl)
Reyes Llano Grande (Llg)
Carrizal de Bravo (Cb)
Volcn Tacan (Vt)
El Triunfo (Tri)
Nueva Colombia (Nc)
Monte Bello (Mb)
Jitotol (Jit)
Pueblo Nuevo (Pn)

8/8
3/3
2/1/1
1/1
2/2
1/1
7/7
9/9
9/9
9/7
4/3
10/9
5/5
2/2
2/2
1/1
3/4
1/1
1/5/5
3/3
11/9
15/15
7/7
15/14
1/1
5/5
2/8
3/4
1/1
9/14
2/3
7/8

20190100
203801700
2100000
2140500
211603500
211003900
211303400
20280100
193102200
192804700
193004700
192403700
192705000
195901000
20104800
20201700
20003600
201301000
18604100
181001200
175004200
181502400
172901200
180504300
173901600
183101300
17103000
17160100
15705300
15430300
154203700
160700000
17301000
171005900

981301700
983403000
99705900
985903500
99703000
99703400
99603400
981805200
965602800
965905100
965602800
965903700
965601300
973603500
973702300
973103500
975001700
975700000
963802400
965004700
964402400
964401800
962202300
965100500
962001300
95903400
974704200
994305900
92603600
92440140
924401500
914300500
925101500
92405900

1967
1880
1836
664
1955
1294
1365
1572
1457
1557
1341
1257
1199
950
914
799
1349
1051
1423
1370
1614
357
2112
2384
2902
1102
1675
1126
3992
1642
1755
569
1575
482

10 chains for each K, from K = 1 to K = 6, as described

above.

2.6. Migration rate estimates

To determine whether divergence occurred in complete
isolation or with gene flow, we used the isolation-withmigration model implemented in IMa (Hey and Nielsen,
2007) on the control region dataset. Because IMa is only
appropriate when population divergence was recent, we only
performed this test to compare the recently diverged genetic
groups of the SMO detected by pairwise FST comparisons
(nSMO vs. cSMO, cSMO vs. sSMO) and by phylogenetic
and AMOVA analyses (nSMO + cSMO vs. sSMO). We ran
the MCMC simulation using Hasegawa-Kishino-Yano model
(HKY) and performed various preliminary analyses to
determine the appropriate prior distributions. To ensure
consistency of the results, the final analyses were repeated
four times using identical
conditions but different starting points with a burn-in period of
50,000,000, and allowing the program to run for 49,999,951 and
4,999,995 steps in chain following burn-in for the nSMO-cSMO
and cSMO-sSMO comparisons, respectively. For the nSMO +
cSMO-sSMO comparison we ran the program for 49,999,951
steps in chain following burn-in. We considered that the
analyses had converged upon a stationary distribution if

migration rates to effective number of migrants per generation.

We estimated migration rates among control region
genetic groups, using this dataset and a maximum-likelihood
coalescent approach in MIGRATE 3.2.16 (Beerli and
Felsenstein, 2001). The first genealogy was started with a
random tree, and the initial theta and migration rate
parameters were obtained from the FST calculations. Five long
(2,000,000 genealogies sampled) and 10 short chains (10,000
genealogies sampled) were run with a burn-in of 10,000. We
also calculated migration rates among microsatellites genetic
groups using the same parameters as for the control region
analysis except that we ran three short chains (7500
genealogies sampled) with 5,000 genealogies as burn-in.
2.7. Demographic history
We tested for demographic expansions on the control
region groups using the Fus Fs neutrality test and assessed
changes in their effective population size (Ne) through time
with Bayesian skyline plots (Drummond et al., 2005) as
implemented in BEAST. We used the substitution model
HKY + G, an uncorrelated lognormal relaxed clock and a
stepwise tree prior with 10 starting groups. Two independent
runs of 10,000,000 generations were performed with trees
and parameters retained every 1000 generations. To confirm
adequate effective sample sizes (EES > 200) for each

D. Maldonado-Snchez et al. / Molecular Phylogenetics and Evolution 99 (2016) 7688

estimated parameter, we visualised the results in Tracer. We

scaled the time axis using the mutation rate of 2.6 %.
2.8. Ecological niche modelling and environmental
variation among genetic groups
To explore whether suitable habitat for C. ophthalmicus has
gone through expansions and contractions at different time
periods, we modelled the potential distribution of C.
ophthalmicus under current conditions, during the Last Glacial
Maximum (LGM, 21 ,000 18,000 BP) and during the Last
Interglacial (LIG, 140,000 120,000 BP). Details of ecological
niche modelling are given in Supplementary material: Materials
and Methods.
We assessed whether environmental conditions differ among
genetic groups by performing climatic niche comparisons
among control region lineages by modelling their habitat
suitability separately, as described in the Supplementary
material: Materials and Methods. We used 22 (cSMO), 9
(nSMO), 36 (sSMO), 15 (ST), 13 (SMS), 46 (cCHIS) and 36
(pCHIS) unique occurrence records and the 10 uncorrelated
climatic variables from the previous analyses. For genetic
groups (nSMO, ST, SMS) with few occurrence records, we
performed a Jackknife model testing to assess model
performance, using the minimum training presence (MTP) as the
decision threshold value for each model, and calculated a Pvalue for each lineage dataset using the script made by Pearson
et al. (2007). We carried out a PCA and used the resulting factor
loadings of the three PCs explaining most of the variation, as
dependent variables in ANOVAs, to determine whether the
bioclimatic variables significantly differed among lineages
(fixed factors). We used Tukeys HSD post-hoc tests to
determine which pairwise comparisons were significantly
different. These analyses were performed in SPSS 21.0.

80

occurred 1.33 Ma and divergence between these and cCHIS

occurred c. 2.77 Ma.
3.2. Genetic diversity
Estimates of intra-population genetic diversity for
mtDNA and microsatellites are summarised in Table 2.
Haplotype diversity (h) was moderate to high for sequences
grouped by mountain range (0.650.98), except SMS (0.33);
and high when grouping by morphotectonic province (0.92
0.95). Nucleotide diversity (p) ranged from 0.004 (SMS) to
0.033 (SMO) across mountain ranges and between 0.012
(sSMO) and 0.019 (nSMO) throughout provinces. The mean
number of alleles, combining all microsatellite loci, across
mountain ranges and provinces ranged from 4.86 (SMS) to
14.42 (SMO) and from 10.14 (nSMO) to 11.86 ( sSMO),
respectively. Allelic richness varied between 3.98 (ST) and
5.35 (pCHIS) across mountain ranges and from 4.94 to 5.21
throughout provinces. Significant deviations from HWE
were detected in all groups of populations, except in SMS
(Table 2). FIS values were positive and significant, suggesting
heterozygote deficiencies. On the other hand, only four of the
collection sites (Ps, Bl, Mb, and Pn) deviated from HWE.
Further analysis of these sites in MICROCHECKER,
revealed that HE and HO differed significantly at some loci
(locus Asl 18 in population Ps, Aca 17 in Mb, Aca 21 in Pn,
and Asl 18 and Aca 01 in Bl). Because deviations from HWE
were not locus specific, as expected when null alleles are
present, we did not attribute the overall deficits to be the
result of null alleles. Deviations from HWE are more likely
the result of the Wahlund effect, due to pooling samples from
different populations. Linkage disequilibrium tests for a total
of 714 comparisons locus/collection site resulted in only two
significant comparisons after Bonferroni corrections.

3.3. Population genetic differentiation

3. Results
3.1. Relationships among mitochondrial haplotypes
and divergence time estimates
Haplotype relationships recovered by the haplotype network
were virtually the same when using the control region or the
combined dataset (control region plus coding genes); only the
results of the former are shown. Five unconnected haplotype
groups, following a strong geographical pattern, were detected
(Fig. 2). The first haplogroup (H1H48, H61H79) is found
exclusively along the SMO, with haplotype sharing between
populations from the cSMO and those from the nSMO, and
sSMO regions. The remaining four haplogroups (ST: H20H25;
SMS: H80 and H81; cCHIS: H50 H54, H57; pCHIS: H49,
H55, H56, H58H60) were formed by haplotypes private to
individuals from each of the mountain ranges
(Fig. 2).
Phylogenetic analyses and divergence time estimates of the
control region and the combined dataset produced nearly
identical results, with wider divergence time intervals for the
control region analysis. We only show the reconstruction and
divergence times for the combined dataset (Fig. 3). Six wellsupported (PP > 0.9) lineages (nSMO + cSMO, sSMO, ST,
SMS, cCHIS, pCHIS) were recovered (Fig. 3). A first split
between pCHIS and the other lineages occurred 4.25 million
years ago (Ma). The ST and SMS lineages diverged from each
other 2.82 Ma and these two lineages diverged from SMO and
cCHIS 3.71 Ma; however, nodes connecting these lineages were
not supported. The split between nSMO + cSMO and sSMO

The AMOVA, in which we treated all locations as a

single group, showed that 80.9% of the genetic variation was
explained by differences among populations and 19.1% by
differences within populations (Table 3). The AMOVA
grouping locations by mountain ranges and morphotectonic
provinces within the SMO found that 73.7% of the variation
was among groups, 23.6% within populations, and
2.8% among populations within groups. When grouping
locations

81

D. Maldonado-Snchez et al. /Molecular Phylogenetics and Evolution 99 (2016) 76 88

Fig. 2. Control region haplotype network and geographic distribution of Chlorospingus ophthalmicus haplotypes throughout Mexican cloud forest. Each circle in
the map represents a collection site with the segments within the circles corresponding to the frequency of a haplotype found in that site. In the haplotype
network, the size of the circle is proportional to haplotype frequency, white small circles represent no sampled haplotypes, and lines mutational changes between
haplotypes.
Fig. 3. Dated Bayesian tree based on the combined dataset (control region plus coding genes) using a relaxed molecular clock as implemented in BEAST and
mutation rates of 2.6% and 2.13% sequence divergence per million years for control region and coding genes, respectively. Colour bars on the side of the tree
represent genetic groups. Values above branches are Bayesian posterior probabilities and those below are divergence times in millions of years (Ma). Purple lines
indicate 95% highest posterior density ( HPD ) intervals in Ma. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

by mountain ranges, the AMOVA showed that 77% of the

variation was explained by differences among groups, 11.9%
by differences within groups and 11% by differences within
populations (Table 3).
Most pairwise FST comparisons calculated for both
markers were lower than Josts D values (Table 4). When
groups of populations corresponding to mountain ranges and
morphotectonic provinces were compared, all FST and Josts
D values were high for the control region. Although pairwise
comparison values (of both statistics) for microsatellites were
smaller than those displayed by the control region, high
levels of genetic differentiation were also detected with these
markers (Table 4).
The assignment tests including all data suggest the
presence of two to four genetic groups (Fig. 4ac). However,
DK supported only two groups: Cluster 1 formed by nSMO,
cSMO, sSMO and cCHIS and Cluster 2 by ST, SMS and
pCHIS (Fig. 4a). Subsequent analyses of these two clusters
as separate datasets detected additional substructure (Fig. 4d
e). The analysis of Cluster 2 depicted three groups: ST, SMS
and pCHIS when K = 3, DK supported the presence of three
groups (Fig. 4d). The analysis of Cluster 1 yielded two
groups: nSMO + cSMO + sSMO and cCHIS when K = 2
(Fig. 4d), and three groups: nSMO, sSMO and cCHIS, with
cSMO being an admixture between nSMO and sSMO
individuals, when K = 3 (Fig. 4e); DK supported the
presence of three groups. Further analysis of groups sSMO
and cCHIS depicted two additional clusters: sSMO and Mb
(a site located in cCHIS), with cCHIS being an admixture
between sSMO and Mb individuals, when K = 2 (Fig. 4f);
DK supported two groups.
3.4. Migration rate estimates

Based on the mutation rate of 0.013 s/s/l/Myr, IMa estimated

the population migration rate, since the separation of nSMO and
cSMO to be low 0.000126 (HPD 90%, 00.415) in the direction
north-to-centre, and high 23.3 (HPD 90%, 5.06106) in the
opposite direction. Population migration rates between cSMO
and sSMO were 2.27 (HPD 90%, 0.2719.31) and 0.83 (HPD
90%, 0.0042.79) in the direction centre-to-south and south-tocentre, respectively. For the nSMO + cSMO-sSMO comparison,
population migration rates were 1.88 (HPD 90%, 0.157.41)
from nSMO + cSMO to sSMO and 0.933 (HPD 90%, 0.005
8.26) in the opposite direction.
Number of migrants per generation (Nm) between control
region groups, estimated in MIGRATE, ranged between 6.5 8
( from all genetic groups to ST) and 8.76 (from cSMO to
nSMO). Migration rates were significantly greater than one only
from sSMO to cSMO (Nm = 1.28), from cSMO to SMS (Nm =
1.6) and from cSMO to nSMO
(Nm = 8.76). Between microsatellites groups N m ranged between
0.96 from pCHIS to nSMO and from nSMO to ST and 2.70
from cSMO to nSMO, and from sSMO + cCHIS to cSMO.
Migration rates between most microsatellite groups were
significantly greater than one.
3.5. Demographic history
Fus Fs values were negative and significant only for SMO
(24.723, P < 0.001) and for the provinces within this mountain

D. Maldonado-Snchez et al. / Molecular Phylogenetics and Evolution 99 (2016) 7688

82

Table 2
Control region and microsatellite diversity of Chlorospingus ophthalmicus in Mexico. We include the number of analysed individuals (N) for both markers. Diversity indices include for mtDNA:
number of haplotypes (Nh), segregating sites (S), haplotype (h) and nucleotide ( p) diversity with their corresponding standard deviations (sd); and for microsatellites: average number of alleles per
locus (A), allelic richness (A R), expected heterozygosity (H E), observed heterozygosity (H O), and inbreeding coefficient F IS. FIS values in bold indicate significant deviations from HardyWeinberg
equilibrium
after
Bonferroni correction
a dash indicates
either thatand
theEvolution
calculation
not76be performed
due to sample size or that samples did not amplify. Refer to Table 1 for site abbreviation.
83
D. Maldonado-Snchez
et and
al. /Molecular
Phylogenetics
99could
(2016)
88
Controlregion

Microsatellites

Nh

h sd

p sd

AR

HE

HO

FIS

6
7
2
2
1
2
1
1
7
5
6
7
4
6
5
2
2
1
3
1
1
4
2
9
6
4
7
1
1
1
3
2
1
2

13
13
8
9
0
7
0
0
12
13
10
12
17
13
12
7
2
0
5
0
0
7
2
14
5
7
27
0
0
0
2
7
0
6
9

0.89 0.09
0.96 0.08
0.67 0.31
1.00 0.50
1.00 0.00
1.00 0.50
1.00 0.00
1.00 0.00
1.00 0.08
0.86 0.09
0.92 0.07
0.94 0.07
1.00 0.18
0.84 0.10
1.00 0.13
1.00 0.50
1.00 0.50 1.00
0.00
1.00 0.27
1.00 0.00
1.00 0.00
0.90 0.16
0.67 0.31
0.95 0.07
0.85 0.06
0.81 0.13
0.86 0.06
1.00 0.00
0.00 0.00
0.00 0.00
0.42 0.19
0.67 0.31
1.00 0.00
1.00 0.50
0.86 0.14

0.022 0.013
0.018 0.011
0.021 0.017
0.037 0.039
0.000 0.000
0.027 0.029
0.000 0.000
0.000 0.000
0.016 0.010
0.015 0.009
0.011 0.007
0.016 0.009
0.042 0.028
0.016 0.0096
0.018 0.012
0.027 0.029
0.007 0.008 0.000
0.000
0.012 0.010
0.000 0.000
0.000 0.000
0.011 0.008
0.005 0.0045
0.018 0.010
0.006 0.004
0.011 0.007
0.029 0.016
0.000 0.000
0.000 0.000
0.000 0.000
0.001 0.002
0.018 0.015
0.000 0.000
0.023 0.024
0.011 0.008

9
8
3

1
2
1
1
7
9
9
7
3
9
5
2
2
1
4
1

5
3
9
15
7
14
1
5
8
14
4
1
3
8

6.57
6.71
2.71

1.67
2.29
1.57
1.57
4.86
5.71
5.29
5.29
3.71
5.29
5.00
2.43
2.71
1.80
3.86
1.83

4.00
2.43
7.29
7.57
6.00
5.29
1.67
4.29
4.71
7.86
4.14
1.50
3.29
5.14

1.77
1.82
1.60

1.55

1.71
1.71
1.74
1.80
1.78
1.77
1.80
1.71
1.64

1.79

1.76
1.50
1.78
1.77
1.77
3.98

1.68
1.63
1.76
1.71

1.73
1.66

0.69
0.73
0.70

0.55

0.65
0.69
0.68
0.70
0.78
0.74
0.72
0.67
0.64

0.79

0.67
0.48
0.73
0.71
0.71
0.09

0.65
0.61
0.70
0.70

0.69
0.59

0.57
0.59
0.61

0.37

0.55
0.57
0.59
0.59
0.76
0.68
0.51
0.42
0.64

0.81

0.54
0.48
0.63
0.52
0.57
0.09

0.49
0.48
0.57
0.57

0.69
0.41

0.20
0.20
0.15

0.44

0.17
0.18
0.14
0.20
0.03
0.09
0.35
0.47
0.15

0.03

0.22
0
0.14
0.28
0.24
0.35

0.28
0.21
0.21
0.03

0.23
0.29

Mountain range/subspecies
SMO
131
SMS
6
ST
15
pCHIS 15
cCHIS 9

60
2
7
6
6

40
3
27
10
12

0.98 0.004
0.33 0.22
0.86 0.057
0.65 0.13
0.89 0.09

0.033 0.017
0.004 0.003
0.029 0.016
0.010 0.006
0.014 0.009

123
6
14
27
11

14.42
4.86
5.29
10.71
6.43

4.58
4.65
3.98
5.35
4.68

0.55
0.65
0.09
0.71
0.61

0.46
0.50
0.09
0.56
0.44

0.19
0.25
0.35
0.23
0.26

Morphotectonic province
nSMO 34
cSMO
54
sSMO
43

19
26
20

18
31
17

0.93 0.03
0.95 0.014
0.92 0.03

0.019 0.011
0.018 0.010
0.012 0.007

32
51
40

10.14
11.57
11.86

4.94
5.00
5.21

0.68
0.72
0.72

0.55
0.61
0.56

0.19
0.15
0.23

N
Collection site
Tlan
Ptr
Maj
Cj
Cy
Si
Pmc
Tlg
Zac
Coap
Ris
Ie
Xico
Ord
Gale
Jon
Cuit
Sna
Lag
Smc
Pf
Pv
Ec
Yag
Ps
Esp
Bl
Llg
Cb
Vt
Mb
Tri
Nc
Jit
Pn

9
8
3
2
1
2
1
1
7
9
9
9
4
10
5
2
2
1
3
1
1
5
3
11
15
7
15
1
5
2
9
3
1
2
7

range (nSMO = 6.89, P < 0.01; cSMO = 6.89, P < 0.01; sSMO
= 6.89, P < 0.01), suggesting demographic expansion along the
SMO. Similarly, Bayesian skyline plots detected an increase in
the effective population size between c. 200,00025,000 years
ago for SMO, its morphotectonic provinces, and pCHIS. The
rest of the genetic groups showed stasis in their effective
population size through time (Supplementary material Fig. S1).
3.6. Ecological niche modelling and environmental variation
among genetic groups
Current, LGM (CCSM, MIROC) and LIG models had high
values (0.94) for the AUC test and the AUC ratio was 1.59 (P <
0.001), indicating high success rate of model performance.
Using the 10 percentile training presence as the logistic
threshold value, a 9.9% omission rate was obtained (Fig. 5).
Under current conditions, the model closely matched the known
distribution for
C. ophthalmicus in the different mountain ranges. The model
also predicted suitable conditions for the species in some
areas of the Sierra Madre Occidental and the Trans-Mexican
Volcanic Belt in Mexico, and from Belize to Panama in
Central America. Although, the identification of additional
areas outside the species range could indicate an overprediction of the model, it could also reflect the presence of
cloud forest fragments with suitable habitat for
Chlorospingus (Fig. 5). Both LGM scenarios predicted a

Table 3
Results of the analyses of molecular variance (AMOVAs) on control region sequences: (a) without a priori grouping samples, (b) five groups defined by subspecies in different mountain ranges, (c)
seven groups defined by mountain range plus the three morphotectonic provinces within SMO.
Source of variation
d.f.
Sum of squares
Variance components
Variation (%)
Fixation indices
D. Maldonado-Snchez et al. / Molecular Phylogenetics and Evolution 99 (2016) 7688
84
Control region
(a) Without groups
Among populations
Within populations

34
141

1766.897
335.483

10.04863
2.37931

80.86
19.14

FST: 0.809***

(b) Mountain range/subspecies

Among groups
Among populations within groups
Within populations

4
30
141

1343.050
423.847
335.483

16.64081
2.58309
2.37931

77.03
11.96
11.01

FCT: 0.770***
FST: 0.890***
FSC: 0.521***

(c) Mountain range + SMO provinces

Among groups
Among populations within groups
Within populations

6
28
141

825.157
78.132
260.665

5.77688
0.21849
1.84869

73.65
2.79
23.57

FCT: 0.736***
FST: 0.764***
FSC: 0.106***

***

P < 0.001.

Table 4
control
region
FST and Jost0s D pairwise comparisons based on ranges. and
intervals.microsatellites between: SMO morphotectonic
In parenthesis are the 95% confidence
Abbreviations
correspond to those in
Table 1.
Comparison
Control region FST
Control region Jost0s D
SMO provinces

SMO provinces vs.

Mountain ranges

Mountain ranges

provinces,

SMO

provinces and mountain

ranges, and mountain
Microsatellites FST
Microsatellites Jost0s D

nSMO vs. cSMO

0.047

0.862 (0.650.96)

0.17

0.032 (0.000.11)

nSMO vs. sSMO

cSMO vs. sSMO
nSMO vs. ST
nSMO vs. SMS
nSMO vs. pCHIS
nSMO vs. cCHIS
cSMO vs. ST
cSMO vs. SMS
cSMO vs. pCHIS
cSMO vs. cCHIS
sSMO vs. ST
sSMO vs. SMS
sSMO vs. pCHIS
sSMO vs. cCHIS
SMO vs. ST
SMO vs. SMS
SMO vs. pCHIS
SMO vs. cCHIS
ST vs. SMS
ST vs. pCHIS
ST vs. cCHIS
SMS vs. pCHIS
SMS vs. cCHIS
pCHIS vs. cCHIS

0.720
0.696
0.864
0.902
0.917
0.838
0.863
0.892
0.908
0.826
0.870
0.916
0.920
0.845
0.784
0.801
0.830
0.694
0.755
0.814
0.566
0.920
0.904
0.855

1.00 (1.001.00)
0.968 (0.890.99)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)
1.00 (1.001.00)

0.045
0.010
0.188
0.151
0.098
0.058
0.168
0.120
0.077
0.049
0.151
0.115
0.076
0.032
0.156
0.116
0.075
0.037
0.217
0.160
0.170
0.121
0.142
0.077

0.117 (0.040.20)
0.008 (0.000.06)
0.439 (0.310.58)
0.389 (0.220.56)
0.309 (0.210.40)
0.112 (0.000.24)
0.428 (0.320.56)
0.345 (0.200.51)
0.268 (0.190.36)
0.105 (0.0020.21)
0.390 (0.250.52)
0.353 (0.190.53)
0.303 (0.220.39)
0.079 (0.000.19)
0.412 (0.310.54)
0.350 (0.200.51)
0.279 (0.210.35)
0.086 (0.000.17)
0.430 (0.220.62)
0.400 (0.280.53)
0.322 (0.150.49)
0.333 (0.140.50)
0.320 (0.120.51)
0.238 (0.100.37)

Significant FST values comparisons after Bonferroni correction are indicated in bold ( a0 = 0.01) for SMO morphotectonic provinces comparisons, a0 = 0.004 for SMO provinces vs. mountain range
comparisons and a0 = 0.005 for mountain range comparisons. Comparisons that were significant before Bonferroni corrections ( a = 0.05) are indicated

wider distribution than currently observed, with contact areas

between some mountain ranges (SMO, ST, cCHIS and
pCHIS) and between all morphotectonic provinces (Fig.
5a,b), indicating that cloud forest was displaced downslope.
The distribution predicted by MIROC was more restricted
than that predicted by CCSM and more similar to the model
under current conditions (Fig. 5a,b), which suggests that this
species had a relatively stable distribution during the last c.
140,000 years BP. Compared to the LGM and the present, the
LIG model yielded a more restricted distribution, suggesting
a

the other two lineages it was not significant (SMS: MTP =

0.130, P = 0.084; nSMO: MTP = 0.514, P = 0.45).
Two principal components explained 66% of the total
variance (PC1 = 45.5%, PC2 = 20.5%); the factor loadings
are provided in Supplementary material Table S3. ANOVAs
of the factor loadings of PC1 and PC2 found significant
variation among genetic groups (Supplementary material
Table S3). The significance values of post-hoc pairwise
comparisons among lineages are in Supplementary material
Table S4.

with a star.

4. Discussion
reduction of suitable habitat for the species during this time
period in Mexico but not in Central America, where cloud
forests had a wider distribution (Fig. 5).
ENMs for most control region groups predicted optimal
climatic conditions within each of their geographic
distributions. All groups had high AUC values (>0.90), and
AUC ratios of 1.75, P < 0.001 (cSMO), 1.78, P < 0.001
(sSMO), 1.43, P < 0.001 (cCHIS) and 1.48 P < 0.001
(pCHIS), indicating a high success rate of model
performance. However, success rate in the jackknife test was
only significant for ST (MTP = 0.410, P < 0.001), while for

4.1. Patterns of genetic differentiation among mountain ranges

and morphotectonic provinces
For the most part, phylogeographic patterns of mitochondrial
and microsatellites data were concordant and suggest that C.
ophthalmicus is constituted by different lineages each one
restricted to a mountain range. These results support the
existence of the previously proposed subspecies distributed
along five mountain ranges: C. o. ophthalmicus (Sierra Madre
Oriental), C. o. albifrons ( Sierra Madre del Sur), C. o. dwighti

85

D. Maldonado-Snchez et al. /Molecular Phylogenetics and Evolution 99 (2016) 76 88

Fig. 4. Assignment of Chlorospingus ophthalmicus individuals to genetic groups (K) based on microsatellite data. Probability of all 181 analysed individuals to belong to an optimal number of
groups (a) K = 2, (b) K = 3, (c) K = 4. Probability of belonging to a group when analysing the resulting clusters separately: (d) ST, SMS, pCHIS (substructure of Cluster 2, when K = 3) and nSMO +
cSMO + sSMO and cCHIS (substructure of Cluster 1, when K = 2); (e) nSMO, sSMO, cCHIS (substructure of Cluster 1, when K = 3); (f) sSMO and Mb (substructure of groups sSMO and cCHIS,
when K = 2). Different colours indicate the proportion of membership to a cluster, and membership to a mountain range, morphotectonic province or collection site is indicated at the bottom of the
graph. Colour lines at the bottom represent control region genetic groups.

(central Chiapas), C. o. postocularis (Pacific slope of Chiapas)

and C. o. wetmorei (Sierra de Los Tuxtlas),
(Garca-Moreno et al., 2004; Bonaccorso et al., 2008; Weir et
al., 2008). Remarkably, several tests based on both
mitochondria and microsatellites suggested further genetic
subdivision within C. o. ophthalmicus (nSMO + cSMO and
sSMO). Although previous phylogeographical studies have
shown similar patterns of population differentiation among
mountain ranges in other bird species (e.g. McCormack et al.,
2008; Navarro-Sigenza et al., 2008) including those on C.
ophthalmicus, genetic differentiation along the SMO was
unnoticed.
Regardless of the genetic differentiation detected within the
SMO, haplotype sharing was apparent between adjacent
morphotectonic provinces (cSMO and nSMO, cSMO and
sSMO). Patterns of shared haplotypes between these lineages
could be the result of a combination of retention of ancestral
polymorphisms with incomplete lineage sorting and gene flow,
two processes that are not mutually exclusive (Hey, 2006).
Coalescent analyses using the isolation-with-migration model
supported that divergence within the SMO provinces occurred
with gene flow, which is not an uncommon process in other bird
taxa (Mil et al., 2009 ; Rodrguez-Gmez et al., 2013).

MIGRATE results for both markers also supported that gene

flow is an important evolutionary force for C. ophthalmicus
inhabiting the SMO, since cSMO interchanged migrants with
both sSMO and nSMO. Gene flow between SMO provinces was
also supported by the STRUCTURE analyses that revealed
admixture between cSMO individuals and those from the two
adjacent provinces. Migration could have been promoted

(a)

(b)

D. Maldonado-Snchez et al. / Molecular Phylogenetics and Evolution 99 (2016) 7688

86

(c)

unsuitable

suitability threshold

(d)

high suitability

Fig. 5. Habitat suitability of Chlorospingus ophthalmicus generated by MAXENT for the LGM under (a) CCSM and (b) MIROC scenarios, the (c) LIG and the (d) present. Colours indicate whether
the habitat is suitable or unsuitable for the species when applying a 10-percentile training presence threshold value of 0.236. In grey are indicated unsuitable areas, in dark blue are the predicted areas
with ideal climatic conditions and in light blue the most suitable areas for the species, with probability values higher than 0.75. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

by the seemly continuity of suitable conditions for cloud forests

during the Pleistocene, as suggested by the LIG and LGM
projections, allowing contact and genetic exchange among
individuals of contiguous SMO provinces.
Interestingly, assignment tests detected finer-scale
subdivision in cCHIS, with individuals from the Mb locality
forming a distinct cluster (Fig. 4f). Such differentiation could be
the result of the geographic location of Mb, which is the most
isolated site of cCHIS and at the lowest elevation. This may
limit dispersal of individuals, allowing the fixation of some
alleles in Mb as previously reported for morphological traits in
other bird species (McCormack and Smith, 2008; Mil et al.,
2009). Additionally, mtDNA indicated that although Mb is
located in cCHIS, it is more closely related to pCHIS localities.
This could be the result of retention of ancestral polymorphisms
and/or historic gene flow among Mb, pCHIs and Guatemala.
The presence of a central depression lacking cloud forest habitat
(see below) separating cCHIS from pCHIS, makes less unlikely
that connectivity and historic gene flow occurred between these
mountain ranges. However, the continuity of cloud forests from
southern Chiapas to Guatemala, shown by the ENMs, might
have facilitated gene flow, resulting in the observed genetic
relationships. Studies that include samples from Central
America, particularly from Guatemala are crucial to have a
better understanding of the patterns of gene flow and genetic
differentiation among Chiapas cloud forests.
One discrepancy between control region and
microsatellites was that the FST value between sSMO and
cCHIS was not significant (after the Bonferroni correction)
with nuclear markers, and assignment tests detected
admixture between them. This suggests recent dispersal
across the Isthmus of Tehuantepec with subsequent genetic
interchange between sSMO and cCHIS individuals as
indicated by the significant migration rates showed by the
microsatellites. The presence of suitable habitat for C.
ophthalmicus at the isthmus, suggested by the ENM for the
present, could have allowed dispersal after a long period of
geographic isolation during the Pleistocene. The Isthmus of
Tehuantepec has not always functioned as a barrier to gene
flow, with different taxa showing shallow or lack
differentiation between populations on either side of the

87

D. Maldonado-Snchez et al. /Molecular Phylogenetics and Evolution 99 (2016) 76 88

isthmus (Ornelas et al., 2010, 2013; Rodrguez-Gmez et al.,

2013).
The genetic differences identified for C. ophthalmicus
coincide with morphological variation previously reported for
the species (Snchez-Gonzlez et al., 2007). Individuals from
different mountain ranges differ in body size and plumage
coloration constituting discrete morphological groups, with a
clinal variation in body size throughout the SMO (SnchezGonzlez et al., 2007). Furthermore, differences in song
structure have also been detected among these same populations
(Sosa-Lpez et al., 2013), supporting the hypothesis that
reproductive isolation has led to different evolutionary pathways
in C. ophthalmicus, as suggested for other montane birds
(McCormack et al., 2008; Gonzlez et al., 2011; Valderrama et
al., 2014).
4.2. Effect of geography and ecology on the patterns of genetic
structure
The deep genetic differentiation detected in C. ophthalmicus
reflects long-term isolation that is clearly associated with the
historical fragmentation of Mexican cloud forests (Ornelas et al.,
2013). The divergence time of C. ophthalmicus lineages dates
from 4.25 to 1.33 Ma, suggesting that these genetic groups
separated during the Pliocene and again in the Pleistocene. The
split time estimated between ST and SMS (2.82 Ma) roughly
matches the time of occurrence of a second eruptive event that
took place during the late Miocene to early Pliocene (7.53 Ma).
This volcanism event has been suggested to have geographically
isolated the Sierra de Los Tuxtlas, in concert with the onset of
the TransMexican Volcanic Belt that occurred during the midMiocene and with the initial volcanic activity at Los Tuxtlas
(Jennette et al., 2003). Similarly, the genetic subdivision of
SMO into two groups (nSMO + cSMO and sSMO) appears to be
a consequence of the late Pliocene volcanic activity and the
movement of major faults active over the past 65 Myr that lead
to the separation of the Nudo de Zempoaltepetl and Sierra de
Jurez (both in the sSMO) from the rest of the SMO (CentenoGarca, 2004). This hypothesis is supported by a
biogeographical analysis of different vertebrates (including
Chlorospingus), suggesting that these two areas constitute a
distinct biogeographic province that resulted from vicariate
events (Len Paniagua and Morrone, 2009).
Although the history of volcanic and tectonic episodes that
characterises the region contributed to some extent to their
isolation, most of the divergence times estimated for C.
ophthalmicus and for other cloud forest taxa (i.e. Ornelas et al.,
2013; Ornelas and Gonzlez, 2014), suggest that only more
ancient divergences were influenced by such events. Therefore,
the observed pattern of genetic differentiation may have resulted
from the isolation of populations due to differing environmental
conditions among mountain ranges and/or the lack of suitable
habitat. Specific habitat requirements may function as barriers to
gene flow, resulting in a pattern of structure due to genetic drift
and/or to diversifying selection (Weir, 2009). This is likely to be
the case for the genetic subdivision observed throughout SMO,
since physiographical differences between these provinces may
have resulted in changes in soil properties of each region,
affecting the chemical and ecological boundaries between them
and thus influencing the floristic composition (FerrusquaVillafranca, 1993).
The fragmented distribution of cloud forest, suggested by the
ENMs, at low elevations, in which Ro Verde and Ro Balsas
(i.e. <300 m) are located, is likely to have promoted the genetic
differentiation between SMS and the other mountain ranges by
acting as a barrier to gene flow, as reported for other birds
associated to cloud forests (Puebla-Olivares et al., 2008).
Similarly, the presence of an arid central depression between the

highlands of Chiapas (pCHIS and cCHIS), in concert with

differences in the climatic conditions between these two
provinces, could be drivers of divergence for a sedentary, cloud
forest-adapted bird species with limited altitudinal migration
such as C. ophthalmicus (Winker et al., 1997).

4.3. Effects of Pleistocene climatic oscillations on the genetic

patterns
It is widely accepted that the altitudinal and latitudinal
distribution of Neotropical cloud forests has been affected by
Pleistocene fluctuations in temperature and humidity (Hugall et
al., 2002; Mil et al., 2000; Ornelas et al., 2013). However, it
less clear whether the genetic patterns of the species are best
explained by the dry refugia model or the moist forests model
for cloud forests (Ramrez-Barahona and Eguiarte, 2013; but see
Ornelas and Gonzlez, 2014; Valderrama et al., 2014). The
genetic patterns shown by C. ophthalmicus throughout the
SMO, including shallow genetic differentiation between
morphotectonic provinces as well as gene flow between them
and high genetic diversity, appear to better fit the moist forests
model. Habitat connectivity throughout the SMO during the
LGM, as indicated by the ENM, plausibly facilitated the
movement of C. ophthalmicus between provinces, allowing
genetic exchange, resulting in higher genetic diversity. These
cloud forest corridors along SMO provinces could account for
the observed patterns of population expansion, further
supporting the moist forests model.
On the other hand, most ENMs indicated that suitable
habitat for C. ophthalmicus is restricted to mountain ranges
(except the CCSM scenario). Despite the possibility of a
slight connection between mountain ranges, differing
climatic conditions of the highlands presumably inhibited
migration between them. The limited cloud forest
connectivity, the differing environmental conditions since the
Quaternary and the time of divergence between lineages
distributed in different mountain ranges (some of them predating the Pleistocene), suggest that genetic differentiation
occurred in allopatry due to long-term geographic isolation.
Although the genetic patterns exhibited by populations
distributed in different mountain ranges match to some extent
those predicted by the dry refugia model, most lineages
showed population stasis, contrary to what the model would
predict. Therefore, it is more likely that the deep allopatric
differentiation exhibited by C. opthalmicus resulted from
geographic isolation that persisted through glacial and
interglacial cycles due to a combination of lack of suitable
habitat and differing environmental conditions between
mountain ranges.
4.4. Taxonomic implications
Previous studies based on coding mtDNA fragments and
morphological traits have suggested that the C. ophthalmicus
species complex is composed of several lineages, each one
restricted to a different mountain range (Garca-Moreno et
al., 2004; SnchezGonzlez et al., 2007; Bonaccorso et al.,
2008): C. ophthalmicusSierra Madre Oriental, C. wetmoreiSierra de Los Tuxtlas, C. albifrons-Sierra Madre del Sur, C.
dwighti-central highlands of Chiapas, and C. postocularisPacific slope of Chiapas. Our investigation based on mtDNA
and microsatellites analyses supports these previous studies,
proving further evidence that each of these lineages has
evolved independently and need to be treated as separate
species according to de Queiroz (2007). Although gene flow
was detected between morphotectonic provinces of the Sierra
Madre Oriental, our results suggest that C. ophthalmicus
from these provinces are also genetically differentiated.

D. Maldonado-Snchez et al. / Molecular Phylogenetics and Evolution 99 (2016) 7688

Detailed morphological and behavioural studies of

individuals from populations along the SMO are necessary to
further investigate whether the detected genetic
differentiation is concordant with patterns of variation on
phenotypic traits.
4.5. Conclusions
The marked genetic divergence of Chlorospingus among
Mexican mountain ranges indicates that geographic isolation
constituted the main evolutionary force driving the
differentiation, which persisted through Pleistocene climatic
oscillations. The shallower genetic differentiation between
morphotectonic provinces within SMO suggests that
Pleistocene climatic oscillations, combined with the presence
of suitable habitat for C. ophthalmicus, had a major impact
on the populations in this mountain range, with the moist
forests model best fitting the displayed genetic patterns.
Acknowledgments
We thank Cristina Brcenas, Jaime Camacho, Sara
Covarrubias, Luis Garca, Clementina Gonzlez, Flor
Rodrguez, Octavio Rojas, Eduardo Ruiz and Antonio
Vasquez for field and laboratory assistance, Adolfo Navarro
Sigenza and Blanca Hernndez from the Museo de Zoologa
de la Facultad de Ciencias, UNAM for donation of tissue
samples, Octavio Rojas for his help with the ENM analyses.
Clementina Gonzlez, Edward Louis Braun and one
anonymous referee provided valuable comments on previous
versions of the manuscript. Permission to conduct fieldwork
was granted by the Mexican government (INE, SEMARNAT,
SGPA/DGVS/02038/07,
SEMARNAT,
SGPA/DGVS/01568/08,
SEMARNAT,
SGPA/
DGVS/02517/09). This work was supported by research
grant (61710) from the Consejo Nacional de Ciencia y
Tecnologa, Mxico (CONACyT, Mxico) to J.F.O, research
funds from the Departamento de Biologa Evolutiva, Instituto
de Ecologa, A.C. (INECOL) granted to C.G-R. (20012-11080) and to J.F.O. (20030/10563), and from the Direccin
General, INECOL. DM-S was supported by undergraduate
(project CB-2005-50060) and Masters (233755) scholarships
from CONACyT.
Appendix A. Supplementary material
Supplementary data associated with this article can be
found,
in
the
online
version,
at
http://dx.doi.org/10.1016/j.ympev.2016.03. 014.
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