Molecular Phylogenetics and Evolution 99 (2016) 7688

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

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

a r t i c l e

i n f o

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

a b s t r a c t
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.
2016 Elsevier Inc. All rights reserved.

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 events

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.

(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 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
(Colinvaux et al., 1996), that can affect the distribution of Neotropical cloud forest species (Ramrez-Barahona and 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

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

Neotropical cloud forest species. The dry refugia model, 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 mid-elevations in mountain
regions with stable temperature and humidity conditions (Haffer,
1969; Toledo, 1982). Therefore, 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 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.

77

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 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 (HO) 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

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

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 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 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-with-migration
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

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

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
locality ID

Mountain
range

Subspecies

Morphotectonic
province

State

Location (abbreviation)

N (mtDNA/SSR)

Latitude
N

Longitude
W

Altitude (m)

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
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
ophthalmicus
wetmorei
albifrons
albifrons
postocularis
postocularis
postocularis
dwighti
dwighti
dwighti

nSMO
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
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

Tlanchinol (Tlan)
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)

9/9
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

2110 1100
20190 100
20380 1700
2100 000
2140 500
21160 3500
21100 3900
21130 3400
20280 100
19310 2200
19280 4700
19300 4700
19240 3700
19270 5000
19590 1000
2010 4800
2020 1700
2000 3600
20130 1000
1860 4100
18100 1200
17500 4200
18150 2400
17290 1200
18050 4300
17390 1600
18310 1300
1710 3000
17160 100
1570 5300
15430 300
15420 3700
16070 0000
1730 1000
17100 5900

98380 3500
98130 1700
98340 3000
9970 5900
98590 3500
9970 3000
9970 3400
9960 3400
98180 5200
96560 2800
96590 5100
96560 2800
96590 3700
96560 1300
97360 3500
97370 2300
97310 3500
97500 1700
97570 0000
96380 2400
96500 4700
96440 2400
96440 1800
96220 2300
96510 0500
96200 1300
9590 3400
97470 4200
99430 5900
9260 3600
92440 140
92440 1500
91430 0500
92510 1500
9240 5900

1413
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

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 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 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 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 P-value for

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

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.

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 well-supported (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
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 (0.920.95). 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
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

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.

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

81

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. 4de). 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-to-centre, 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%, 0.0058.26) in the opposite direction.
Number of migrants per generation (Nm) between control
region groups, estimated in MIGRATE, ranged between 6.58 (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 Nm 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

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

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 (AR), expected heterozygosity (HE), observed heterozygosity (HO), and inbreeding coefficient FIS. FIS values in bold indicate
significant deviations from HardyWeinberg equilibrium after Bonferroni correction and a dash indicates either that the calculation could not be performed due to sample size or
that samples did not amplify. Refer to Table 1 for site abbreviation.
Control region

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
5

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
60
SMS
6
2
ST
15
7
pCHIS
15
6
cCHIS
9
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

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
9
Ptr
8
Maj
3
Cj
2
Cy
1
Si
2
Pmc
1
Tlg
1
Zac
7
Coap
9
Ris
9
Ie
9
Xico
4
Ord
10
Gale
5
Jon
2
Cuit
2
Sna
1
Lag
3
Smc
1
Pf
1
Pv
5
Ec
3
Yag
11
Ps
15
Esp
7
Bl
15
Llg
1
Cb
5
Vt
2
Mb
9
Tri
3
Nc
1
Jit
2
Pn
7

19
26
20

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 over-prediction 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 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

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

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

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
FST and Jost0 s D pairwise comparisons based on control region and microsatellites between: SMO morphotectonic provinces, SMO provinces and mountain ranges, and mountain
ranges. In parenthesis are the 95% confidence intervals. Abbreviations correspond to those in Table 1.
Comparison
SMO provinces

SMO provinces vs.

Mountain ranges

Mountain ranges

nSMO vs. cSMO

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

Control region FST

0.047
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

Control region Jost0 s D

0.862
1.00
0.968
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00

(0.650.96)
(1.001.00)
(0.890.99)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)
(1.001.00)

Microsatellites FST

0.17
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

Microsatellites Jost0 s D
0.032
0.117
0.008
0.439
0.389
0.309
0.112
0.428
0.345
0.268
0.105
0.390
0.353
0.303
0.079
0.412
0.350
0.279
0.086
0.430
0.400
0.322
0.333
0.320
0.238

(0.000.11)
(0.040.20)
(0.000.06)
(0.310.58)
(0.220.56)
(0.210.40)
(0.000.24)
(0.320.56)
(0.200.51)
(0.190.36)
(0.0020.21)
(0.250.52)
(0.190.53)
(0.220.39)
(0.000.19)
(0.310.54)
(0.200.51)
(0.210.35)
(0.000.17)
(0.220.62)
(0.280.53)
(0.150.49)
(0.140.50)
(0.120.51)
(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
with a star.

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 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.

4. Discussion
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 (central Chiapas), C. o. postocularis
(Pacific slope of Chiapas) and C. o. wetmorei (Sierra de Los Tuxtlas),

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

Cluster 1

Cluster 2

(a)

First round: all data analysed (K=2)

(b)

First round: all data analysed (K=3)

(c)

First round: all data analysed (K=4)

(d)

Second round: only

Cluster 2 analysed
(K=3)

Second round: only Cluster 1 analysed (K=2)

(e)

Second round: only Cluster 1 analysed (K=3)

(f)

Third round: only sSMO and cCHIS analysed (K=2)

nSMO

cSMO

sSMO

cCHIS
Mb

ST S
M
S

pCHIS

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.

(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

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

unsuitable

(a)

(b)

(c)

(d)

suitability threshold

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 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 (Snchez-Gonzlez 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

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

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 mid-Miocene 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 (Centeno-Garca, 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 (Ferrusqua-Villafranca, 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
pre-dating 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. wetmorei-Sierra de Los Tuxtlas, C. albifrons-Sierra Madre del Sur, C. dwighti-central highlands of Chiapas,
and C. postocularis-Pacific 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. 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

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

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. (20012-11-080) 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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