The impact of Quaternary climate oscillations ondivergence times and historical population sizes inThylamys opossums from the Andes
THOMAS C. GIARLA*† and SHARON A. JANSA*†*Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, MN 55108, USA, †J.F. Bell Museum of
Natural History, University of Minnesota, St. Paul, MN 55108, USA
Abstract
Climate oscillations during the Quaternary altered the distributions of terrestrial ani-
mals at a global scale. In mountainous regions, temperature fluctuations may have led
to shifts in range size and population size as species tracked their shifting habitats
upslope or downslope. This creates the potential for both allopatric speciation and
population size fluctuations, as species are either constrained to smaller patches of
habitat at higher elevations or able to expand into broader areas at higher latitudes.
We considered the impact of climate oscillations on three pairs of marsupial species
from the Andes (Thylamys opossums) by inferring divergence times and demographic
changes. We compare four different divergence dating approaches, using anywhere
from one to 26 loci. Each pair comprises a northern (tropical) lineage and a southern
(subtropical to temperate) lineage. We predicted that divergences would have occurred
during the last interglacial (LIG) period approximately 125 000 years ago and that pop-
ulation sizes for northern and southern lineages would either contract or expand,
respectively. Our results suggest that all three north–south pairs diverged in the late
Pleistocene during or slightly after the LIG. The three northern lineages showed no
signs of population expansion, whereas two southern lineages exhibited dramatic,
recent expansions. We attribute the difference in responses between tropical and sub-
tropical lineages to the availability of ‘montane-like’ habitats at lower elevations in
regions at higher latitudes. We conclude that climate oscillations of the late Quaternary
had a powerful impact on the evolutionary history of some of these species, both pro-
moting speciation and leading to significant population size shifts.
Keywords: Andes, Didelphidae, divergence times, population sizes, Quaternary climate oscilla-
tions, Thylamys
Received 29 December 2014; revision received 3 March 2015; accepted 18 March 2015
Introduction
Cyclical climatic changes during the Quaternary altered
the spatial distribution of biomes worldwide, sparking
concomitant evolutionary changes among the organisms
that comprised those biomes (Bennett 1990; Dynesius &
Jansson 2000; Hewitt 2004). The magnitude and trajec-
tory of evolution in the face of shifting climates is
largely dependent on how tightly linked a species is to
its environment (i.e., the extent to which an organism’s
environmental niche is conserved). When a population’s
necessary environmental conditions shift in space—and
if the population does not become extinct—it might
respond by staying in place and coping with the new
environment through adaptive evolution or phenotypic
plasticity. Otherwise, it might track its preferred habitat
into the new area (Holt 1990). The latter scenario is
most likely if a species has a conserved environmental
niche, and mounting evidence suggests that recently
diverged species tend to retain conserved environmental
Correspondence: Thomas C. Giarla, Department of Biological
Sciences and Museum of Natural Science, Louisiana State
University, Baton Rouge, LA 70803, USA. Fax: +1 225 578 3075;
E-mail: [email protected]
© 2015 John Wiley & Sons Ltd
Molecular Ecology (2015) 24, 2495–2506 doi: 10.1111/mec.13173
niche characteristics (Mart�ınez Meyer et al. 2004; Wiens
et al. 2010; Peterson 2011; Quintero & Wiens 2013). If
that is the case, the joint effects of shifting climates and
stable niches can lead to distributional shifts with cas-
cading evolutionary outcomes. The potential outcomes
are especially dramatic for organisms that inhabit
mountainous areas.
Studies examining contemporary climate warming
have shown that some species adapted to live in rela-
tively cold montane habitats are being squeezed into
ever-smaller, patchy ranges at higher elevations, threat-
ening them with extinction (Beever et al. 2003; Parmesan
2006; Galbreath et al. 2009). However, other montane
species are expected to expand their ranges if similar
habitats are available and accessible at higher latitudes
(Guralnick 2007). Differences in the expected distribu-
tional shifts vary by global region. In the tropics, the
potentially cooler temperatures at higher latitudes are
far less accessible than the equivalent temperature
changes available by climbing higher in elevation. As
such, elevational range shifts are much more likely than
shifts in latitude among tropical species experiencing a
warming environment (Colwell et al. 2008). If a montane
species is unable to track its preferred climatic niche to
higher latitudes, the prevailing evolutionary pattern we
expect to observe under warming regimes is one of
demographic decline and population fragmentation.
Furthermore, if these fragmented populations do not go
extinct, population isolation may eventually lead to allo-
patric speciation (Wiens 2004; Wiens & Graham 2005).
As such, the most recent time period for recent montane
speciation (excluding the Earth’s current interglacial)
may be the last interglacial (LIG; 129 800–73 900 years
ago), when global temperatures were considerably war-
mer than today (Rohling et al. 2007).
Researchers have long debated the extent to which
Quaternary climate oscillations affected the timing of
species divergence (Haffer 1974; Fjelds�a 1994; Klicka &
Zink 1997; Avise et al. 1998; Rull 2008, 2011), and there
has been a growing effort to examine their role in shap-
ing species diversity, phylogeographic structure and
demographic shifts in montane systems (Roy 1997; Ko-
zak & Wiens 2006; Ribas et al. 2007; Galbreath et al.
2009, 2010; Shepard & Burbrink 2009). The Andes of
South America are among the most biodiverse moun-
tain ranges in the world (Myers et al. 2000), and numer-
ous studies have implicated Quaternary climate
oscillations in driving this remarkable diversity
(reviewed by Turchetto-Zolet et al. 2013). During the
last glacial maximum (LGM) approximately 26 000–21 000 years before present, temperatures in the tropical
Andes were 5–10 °C cooler than present (Baker et al.
2001; Mourguiart & Ledru 2003) and vegetation com-
munities were shifted downslope relative to their con-
temporary distributions (van der Hammen 1985;
Hooghiemstra et al. 1993). Elevation bands occupy more
surface area at lower elevations in the mountains.
Therefore, at a regional scale, biomes currently
restricted to small areas at high elevations would have
expanded in area when pushed into lower elevations
during previous glacial periods (Urrego et al. 2005).
When climates warmed during interglacial periods,
those same vegetation communities were again forced
farther upslope. This contraction of relatively cool, mon-
tane habitats may have led to a simultaneous reduction
in population size among the terrestrial animal species
that utilize those habitats. In the southern half of the
mountain range, Andean species may have been able to
escape this reduction in available habitat by expanding
into areas with montanelike climates at lower elevations
in the subtropical and temperate zones (e.g. Patagonia
or the lowland Monte desert).
In this study, we investigate the impact of Quaternary
climate oscillations on the evolution and population his-
tory of three pairs of closely related Andean marsupial
lineages in the genus Thylamys. These six lineages have
mostly nonoverlapping distributions extending from
high-elevation habitats in tropical Peru to temperate
Argentina (Fig. 1, Fig. S1, Supporting information; Gia-
rla et al. 2010; Giarla & Jansa 2014; Giarla et al. 2014).
Here, we test two sets of hypotheses concerning the
impact of late-Quaternary climate oscillations on An-
dean Thylamys species: (i) that sister species diverged
due to allopatric speciation during the Quaternary
when the warm LIG forced species into isolated high-
land refugia; and (ii) that montane lineages experienced
population size changes in response to warming and
cooling cycles, modulated by their ability to expand
into more extensive areas at higher latitudes. For the six
Andean lineages studied here, we predict that the more
northerly ‘A’ lineages within each pair would see popu-
lation expansion during cool stages and population
decline during warm stages. In contrast, the southern
‘B’ lineages would see the opposite pattern because
they could expand their ranges during warm stages by
‘escaping’ into montanelike habitats farther south.
Materials and methods
Estimating divergence times
To examine the impact that underlying assumptions
and data types might have on our conclusions, we
employ four analytical approaches for the estimation of
speciation times across cryptic species pairs within Thy-
lamys pallidior, Thylamys sponsorius and Thylamys venu-
stus: (i) a relaxed clock, fossil-calibrated analysis of one
mitochondrial gene; (ii) a relaxed clock, fossil-calibrated,
© 2015 John Wiley & Sons Ltd
2496 T. C. GIARLA and S. A. JANSA
multilocus super-matrix analysis; (iii) a mutation-rate-
scaled, multilocus species tree analysis; and (iv) a series
of three mutation-rate-scaled, multilocus estimates of
divergence times for each cryptic lineage pair under a
two-species coalescent model. The number of loci and
number of individuals included vary depending on the
analysis. Details on sampling strategies and GenBank
accession numbers are provided in Table S1 and
Appendix S1 (Supporting information). Giarla et al.
(2010) identified three mitochondrial haplogroups
within T. venustus (designated A, B, and C), but mul-
tilocus species delimitation tests suggested that haplo-
groups B and C actually represent just one cryptic
species (Giarla et al. 2014). We retain those labels here,
combining B and C into one taxon referred to as
T. venustus B. All of the sequence data used for these
analyses are published: three mitochondrial loci from
Giarla et al. (2010), 14 anonymous nuclear loci and one
X-linked intron from Giarla et al. (2014), and 8 addi-
tional nuclear loci (both exons and introns) from Voss
& Jansa (2009) and Giarla & Jansa (2014). Sequences
from each locus were aligned in GENEIOUS v5.4 (Kearse
et al. 2012) using the MUSCLE algorithm (Edgar 2004)
and adjusted by eye. We assessed nucleotide substitu-
tion models in JMODELTEST v2.1.3 (Darriba et al. 2012) for
each locus and applied the best fitting model to that
subset of the data if it was included in a given analysis.
Fossil-calibrated analysis of mitochondrial sequences. In BEAST
v1.7.5 (Drummond et al. 2012), we inferred an ultrametric,
fossil-calibrated tree of 49 marsupial species, including the
six Andean Thylamys lineages that are the subject of this
ARGENTINA
PARAGUAY
BOLIVIAPERU
CHILE
BRAZIL
Tropic of capricorn
–20º
–25º
–30º
–35º
–15º
–40º
–75º –70º –65º –60º
Pallidior B (10)
Venustus A (7)
Venustus B (20)
Sponsorius A (10)
Sponsorius B (9)
Pallidior A (4)
Elevation (m)0–600601–12001201–19001901–26502651–33003301–37003701–40004001–44004401–48004800–6700
Pallidior A
Pallidior B
Venustus A
Venustus B
Sponsorius A
Sponsorius B
Fig. 1 Map of species distributions for
the six Thylamys lineages included in this
study, with inset species tree from (Gia-
rla et al. 2014). The inset species tree does
not fully sample all Thylamys species;
T. venustus and T. sponsorius are sister
species, but Thylamys pallidior is more
closely related other Thylamys species not
sampled here (Giarla et al. 2010). Shapes
were drawn around collecting localities
for samples sequenced, and the number
of individuals sampled per species is
included in parentheses. Precise locality
information is uploaded to Dryad and
summarized in Table S1 (Supporting
information). Collecting localities used to
draw the shapes are illustrated in Fig. S1
(Supporting information).
© 2015 John Wiley & Sons Ltd
PHYLOGEOGRAPHY OF ANDEAN MARSUPIALS 2497
study, using sequences from the mitochondrial gene cyto-
chrome b (CYTB). We calibrated the tree with two didelp-
hid fossils and one Australian marsupial fossil. These
fossils were used to constrain the minimum age of the split
between Didelphis and Philander (3.3 Ma), the minimum
age of the clade that comprises Monodelphis, Marmosa and
Tlacuatzin (12.1 Ma), and the minimum age of the split
between Dasyuromorphia and Peramelemorphia
(24.7 Ma). Detailed information about the assignment of
fossils to extant clades can be found in Jansa et al. (2014).
The minimum age of the clade containing the fossil was
constrained by a lognormal prior (mean = 1.0) offset by
the estimated age of the fossil. A Yule speciation prior and
a lognormal clock prior were both applied, and all remain-
ing priors were left at their default values. We ran the
MCMC chain for 25 million generations, sampling every
2500 steps. We ensured that effective samples sizes (ESS)
for each estimated parameter exceeded 200 in TRACER v1.6
(Rambaut et al. 2014). After removing the first 10% of trees
as burn-in, we assembled a maximum clade credibility tree
from the post-burn-in sample of trees using TREEANNOTATOR
v1.7.5 (Drummond et al. 2012).
Fossil-calibrated super-matrix analysis. Separate estimates
of divergence dates were extracted from a phylogenetic
tree inferred as part of a previous study examining the
biogeographic history of Thylamys (Giarla & Jansa 2014).
That analysis was performed in BEAST following the
same procedure as described above for the fossil-cali-
brated CYTB analysis, except a super-matrix of 26 con-
catenated loci was used. That super-matrix comprised 3
mitochondrial genes, 22 autosomal loci and 1 X-linked
locus. Details on the BEAST analysis, including the selec-
tion and implementation of partitioning strategies, are
described in Giarla & Jansa (2014).
Mutation-rate-scaled species tree analysis. We conducted a
multilocus species tree analysis of the six focal lineages
using the *BEAST algorithm (Heled & Drummond 2010)
implemented in BEAST. Unlike the first two approaches
described above, this analysis involves only the six focal
Andean lineages and, for each species, includes multi-
ple sequences from 15 nuclear loci and CYTB (Table S1,
Supporting information). Individuals were assigned to
species in *BEAST according to previous species delimita-
tion efforts (Giarla et al. 2010, 2014). We assigned the
appropriate ploidy levels to each gene, and we applied
a Yule species tree prior with a piecewise-linear and
constant root population size model. Rate variation
among lineages was incorporated by applying uncorre-
lated lognormal clock models.
To calibrate the species tree analysis, the clock model
for CYTB was assigned a uniform rate prior ranging
from 0.0172 to 0.0668 substitutions per site per mil-
lion-years (Table S2, Supporting information). This
estimate of the CYTB rate range is based on the 95%
highest posterior density interval (HPDI) estimated for
the CYTB rate for the clade that includes Thylamys and
its sister-genus Lestodelphys from the 26-locus, fossil-cali-
brated super-matrix analysis. All other clock models
received exponential rate priors (mean = 1.0). The
remaining priors were kept at their default values, and
we started two MCMC chains of 125 million genera-
tions, sampling each every 12 500 steps. After assessing
convergence and removing 10% of trees as burn-in, we
combined the two runs in LOGCOMBINER v1.7.5 (Drum-
mond et al. 2012) and assembled a maximum clade
credibility tree from the post-burn-in sample of species
trees using TREEANNOTATOR.
Two-species coalescent models. For the final divergence
time estimation approach, we divided the data set used
for estimating the species tree into three subsets, one
for each pair of cryptic Andean lineages. Each of these
three data sets was analysed independently in BPP v2.2
(Yang & Rannala 2010) with a fixed, two-taxon guide
tree. Priors on effective population sizes (h) and diver-
gence times (s) were assigned diffuse gamma distribu-
tions of Γ(2, 500) and Γ(2, 3000), respectively (Prior
Scheme 1 from Giarla et al. 2014). Interlocus rate hetero-
geneity was allowed under the default Dirichlet prior,
and the heredity scalars for the X-linked locus and
mitochondrial locus were set to their respective values
of 0.75 and 0.25.
We ran the MCMC chain for 2.5 million generations,
sampling every five steps, for a posterior distribution of
500 000 samples. Of these, 50 000 samples were
removed as burn-in, and we assessed chain mixing by
examining ESS values in TRACER. For each pair, the 95%
HPDI BPP estimate for population splitting time (the sparameter) was time-calibrated using the mean substi-
tution rate across the 16 loci used here that were also
used in the 26-locus, fossil-calibrated super-matrix
analysis. To do this, we extracted mean rate estimates
from the maximum clade credibility tree for the clade
that includes Thylamys and its sister-genus Lestodelphys
for each of the 16 loci overlapping with the larger set
from Giarla & Jansa (2014) (Table S2, Supporting infor-
mation).
Modelling demographic fluctuations and rangeexpansions
Extended Bayesian skyline plots. We modelled demo-
graphic changes in each of the six cryptic lineages with
extended Bayesian Skyline plots (EBSPs; Heled &
Drummond 2008) in BEAST. We extracted six separate,
lineage-specific alignments from each of the 16 loci used
© 2015 John Wiley & Sons Ltd
2498 T. C. GIARLA and S. A. JANSA
in the species tree analysis described above. After
dividing the data set into lineage-specific alignments, 13
(of 96) were invariant and excluded from further analy-
sis (Table S3, Supporting information). To set up the six
independent analyses, we applied strict clocks, unlinked
all site models and clock models and assigned the best
fitting available substitution model to each alignment.
For the tree priors, we chose the extended Bayesian sky-
line plot model with a piecewise-linear population size
function and assigned the appropriate ploidy levels to
the X-linked and mitochondrial loci. We assigned expo-
nential priors (mean = 1.0) to all of the ucld.mean
priors, kept all other priors at their default values, and
ran the MCMC chain for 400 million generations (T. pal-
lidior B and T. sponsorius B) or 300 million generations
(all other lineages).
Demographic model comparisons. We compared constant-
size and exponential growth coalescent models for each
lineage using marginal likelihood estimates calculated
via path sampling and stepping stone sampling (PS/
SS), two approaches that have been demonstrated to
provide accurate and reliable marginal likelihood esti-
mates for model comparisons (Baele et al. 2012). Over-
all, we set up 12 analyses in BEAST, one constant-size
coalescent model and one exponential growth coales-
cent model for each of the six lineages (using the same
data sets as for the EBSP analysis). Site models and
clock models were treated the same as for the EBSP
analyses, and either the constant-size or exponential
growth tree prior was chosen. We used the default
Laplace prior on the exponential growth rate, which
allows the MCMC chain to sample both positive and
negative rates. Because PS/SS marginal likelihood esti-
mates can be biased by priors with probability distribu-
tions that do not integrate to 1 (Baele et al. 2013), we
changed the default ‘1/x’ priors for the population size
parameters to lognormal priors (mean = 1.0, SD = 2.0).
The PS/SS calculations rely on well-mixed posterior
distribution to start, so we initialized each marginal
likelihood calculation with 50 million MCMC genera-
tions, sampling every 5000 steps. For all analyses, we
used PS/SS calculations with a chain length of 100 000
generations and 1000 path steps.
Results
Divergence times
Stationarity and high ESS values were attained for all of
the analyses across each of the analytical approaches
used to estimate divergence times (BEAST, *BEAST and
BPP). For the BEAST analysis of CYTB alone, 95% HPD
intervals for divergence estimates between haplogroup
pairs—T. pallidior A vs. B, T. sponsorius A vs. B and
T. venustus A vs. B—extend across a relatively wide
range between approximately 0.25 and 2.0 Ma (Fig. 2,
Fig. S2, Supporting information). The super-matrix
analysis of 26 loci resulted in younger and tighter HPD
intervals for the timing of the three splits, between
approximately 200 000 and 500 000 years ago (Fig. 2,
Fig. S3, Supporting information). Divergence times esti-
mated using the coalescent-based *BEAST and BPP
approaches resulted in congruent, considerably more
Single-Locus: BEAST mtDNA-only
Concatenated: BEAST supermatrix
Multispecies coalescent: *BEAST
Multispecies coalescent: BPP Pal
lidio
rA
vs.
BSp
onso
rius
A v
s. B
Ven
ustu
sA
vs.
B
Last interglacial period(LIG)
0.0 0.5 1.0 1.5 2.0 0.25 0.75 1.25 1.75 2.25
Divergence time (millions of years ago)
Fig. 2 Divergence time estimates for three pairs of montane Thylamys lineages using four different approaches. Bars represent 95%
highest posterior density intervals. Vertical lines within the bars are mean divergence time estimates. A shaded vertical bar illustrates
the duration of the last interglacial (LIG) period (Rohling et al. 2007).
© 2015 John Wiley & Sons Ltd
PHYLOGEOGRAPHY OF ANDEAN MARSUPIALS 2499
recent, and tighter HPD intervals (Fig. 2, Table 1). For
T. pallidior A/B and T. sponsorius A/B, these estimates
overlap with the LIG. For T. venustus A/B, these esti-
mates are more recent still, with HPD intervals for the
timing of the split extending between approximately
20 000 and 75 000 years ago, during the cooling period
between the LIG and the LGM.
Historical demography
Each of the six EBSP runs reached stationarity and
exhibited high ESS values. The x-axes on the EBSPs
were scaled using the mean substitution rate for all loci
in Table S2 (Supporting information). For sister species
not exchanging genes, gene coalescence events are
always older (sometimes considerably so) than the
actual population splitting event that precipitated speci-
ation (Edwards & Beerli 2000), which our coalescent-
based divergence estimates (Fig. 2) attempted to infer.
The long tails on the whole EBSPs (not shown) reflect
the oldest modelled coalescence event across all of the
genes sampled within a species. As such, we illustrate
only the portion of the EBSP that represents a species’
independent coalescent history, separate from its sister
(Fig. 3). For T. pallidior A, the effective population size
gradually increases over the course of its coalescent his-
tory (Fig. 3a). For T. pallidior B, the population size
remains relatively constant until approximately
60 000 years ago, when the population undergoes an
over sixfold increase (Fig. 3b). Thylamys sponsorius A
(Fig. 3c), T. venustus A (Fig. 3e) and T. venustus B
(Fig. 3f) appear to remain approximately the same size
over the course of their demographic history, with
slight declines towards the present. Thylamys sponsorius
B has a complex demographic history, showing a five-
fold population size decline until approximately
40 000 years ago, when it reverses and rapidly expands
to its former population size (Fig. 3d).
Constant-size and exponential growth coalescent tree
priors were compared for each lineage using PS and SS
marginal likelihood estimates (Table 2). In each case,
both estimators gave identical or nearly identical
results. The exponential growth model received a signif-
icantly higher marginal likelihood score as measured by
Bayes Factors (Kass & Raftery 1995) for T. pallidior A
and B, and for T. venustus B. The constant-size model
received a significantly higher marginal likelihood for
T. sponsorius A and T. venustus A, and the constant-size
model could not be rejected for T. sponsorius B. There
was extensive variation in the estimates for the expo-
nential growth rate across runs (Table 2). Thylamys pal-
lidior B, for which the exponential growth model fit the
data best, is estimated to have grown at a rate of 623%
per million-years over its coalescent history, whereas
T. pallidior A and T. venustus B increased at rates of
208% and 143% per million-years, respectively.
Although the exponential growth model did not pro-
vide a significantly better fit to the data than the con-
stant growth model for T. sponsorius B, the overall rate
is negative. However, if the more parameter-rich sky-
line plot model for T. sponsorius B is correct (Fig. 3d), a
simple one-parameter model will not be able to explain
the shift from population decline to population expan-
sion.
Discussion
A major goal of phylogeography is to understand how
ancient earth dynamics shaped the structure of genetic
diversity across landscapes. In montane areas, where
late-Quaternary climate oscillations may have repeat-
edly isolated and reconnected populations, the relation-
ship between population histories and present-day
phylogeographic structure is complex. Late-Quaternary
climate oscillations have been implicated as drivers of
phylogeographic patterns in a number of Andean taxa
(reviewed by Turchetto-Zolet et al. 2013), but the impre-
cision of parameter estimates and lack of statistical tests
have hampered the ability of researchers to tie inferred
evolutionary changes to geological or climatological
events in the earth’s history. The results of this study
offer a significant improvement in the precision of such
estimates among six Andean marsupials.
Divergence during the late pleistocene
Theory indicates that divergence dates estimated from
gene trees (or trees inferred from concatenated alignments
Table 1 Divergence time estimates for three pairs of Andean Thylamys species based on results from *BEAST and BPP analysis of 15
nuclear loci and one mitochondrial gene
pallidior A vs. B sponsorius A vs. B venustus A vs. B
*BEAST BPP *BEAST BPP *BEAST BPP
Mean divergence (years ago) 133 000 105 843 85 500 94 481 50 600 33 584
95% HPD minimum (years ago) 83 000 67 081 56 100 62 985 27 100 19 912
95% HPD maximum (years ago) 192 500 143 624 118 100 126 959 76 300 49 004
© 2015 John Wiley & Sons Ltd
2500 T. C. GIARLA and S. A. JANSA
of multiple loci) will be biased towards older splitting
times relative to those estimated from species trees. This
is because gene tree divergences must predate species tree
divergences in the absence of post-divergence gene flow
(Edwards & Beerli 2000). Despite these known shortcom-
ings, some studies still rely on single-gene and concate-
nated approaches to infer divergence times, including a
recent study that inferred the timing of speciation across
the entire radiation of Thylamys opossums using only a
single mitochondrial marker (Palma et al. 2014). By com-
paring four different divergence dating approaches
(Fig. 2), we illustrate how such analytical decisions can
mislead subsequent evolutionary inferences. Across the
four approaches, results vary in both their mean estimates
and their precision (as measured by 95% HPD intervals).
The fossil-calibrated analyses of CYTB alone offered the
Time (millions of years ago)L
og(p
opul
atio
n Si
ze x
µ)
T. pallidior A T. pallidior B
T. sponsorius A T. sponsorius B
T. venustus A T. venustus B
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2
0.0001
0.001
0.01
0.1
1
0.001
0.01
0.1
1
0.0001
0.001
0.01
0.1
1
0.0001
0.001
0.01
0.1
1
0.0001
0.001
0.01
0.1
1
0.0001
0.001
0.01
0.1
1
0.0001
(a)
(c)
(e)
(b)
(d)
(f)
Fig. 3 Extended Bayesian skyline plots for (a) Thylamys pallidior A, (b) T. pallidior B, (c) Thylamys sponsorius A, (d) Thylamys sponsorius
B, (e) Thylamys venustus A and (f) T. venustus B. The middle lines denote median estimates for population size over time. Lines above
and below the median represent the 95% highest posterior density interval for population size estimates. Vertical dotted lines denote
the onset of the last glacial maximum. The x-axis is timescaled from substitutions/site/million-years to millions of years using the
average substitution rate from Table S2 (Supporting information).
© 2015 John Wiley & Sons Ltd
PHYLOGEOGRAPHY OF ANDEAN MARSUPIALS 2501
least precision and the oldest divergence estimates,
matching theoretical expectations for divergences
estimated from a single-locus (Edwards & Beerli 2000; Ar-
bogast et al. 2002). Divergence time estimates from the
fossil-calibrated super-matrix approach are more precise
than CYTB alone. However, the increased precision is
likely an artifact of concatenation and its simplifying
assumptions (Edwards & Beerli 2000; Kubatko & Degnan
2007). Given the even larger increase in precision we
observed in our coalescent-based divergence time esti-
mates, we rely only on those results as a basis for further
discussion and interpretation. Results from those
approaches place the three splits between ‘A’ and ‘B’
lineages within the last 200 000 years in a narrow
window near the LIG, a time when global temperatures
were at least 2 °C higher than today (Rohling et al. 2007).
The LIG likely represents the most recent time in Earth
history when tropical montane populations were as
fragmented as they have ever been in the past several
million-years.
Our results are broadly concordant with population
divergences seen in other South American species.
Overall, most South American mammalian sister species
(for which data are available) diverged during the Pleis-
tocene, but only two studies pinpoint the divergences
to the period after the onset of the LIG (Turchetto-Zolet
et al. 2013). The paucity of divergence times estimated
to have occurred during the last part of the Pleistocene
might be due to the reliance on gene tree divergences
instead of species tree estimates, which, as discussed
above, tend to overestimate divergence times. In order
to accurately estimate divergence times and compare
them to codistributed taxa, future studies must rely on
methodologies that explicitly incorporate coalescent var-
iation among loci (Edwards et al. 2007; Heled & Drum-
mond 2010; Yang & Rannala 2010) and properly
account for rate variation among lineages (Hope et al.
2014). Otherwise, divergence estimates will be biased
towards older splitting times.
Palma et al. (2014) recently inferred a time tree for
Thylamys using CYTB sequences and two fossil-calibra-
tion points, estimating relatively old divergence times
among species. Their estimates suggested that Thylamys
diverged from its sister-genus Lestodelphys as early as
the Oligocene, with interspecific divergence dates 10
times as old as in our fossil-calibrated super-matrix
analysis (they did not attempt to infer splitting times
for any of the ‘A’ and ‘B’ lineages we study here). The
discrepancy between the results from Palma et al. (2014)
and our multilocus efforts (Fig. 2) is likely driven by
the imprecision inherent to single-locus studies coupled
with imprecise fossil calibrations and inappropriate
prior choices. Palma et al. (2014) calibrated two nodes
using priors with normal distributions, which can lead
to imprecise estimates because it is usually not an
appropriate representation of fossil information (Ho &
Phillips 2009). Moreover, their assignment of two fossil
taxa to crown groups in their tree is unsubstantiated
and does not follow best practices for justifying fossil
calibrations (Parham et al. 2012) because there is no dis-
cussion of how those assignments were made. We
argue that the fossils Palma et al. (2014) used—Thylamys
pinei (Goin et al. 2000) ‘with affinities to T. venustus’ and
Thylamys contrerasi (Deschamps et al. 2012) ‘with affini-
ties to T. pusillus’—should not be linked so tightly to
crown group ages without a more explicit morphologi-
cal study.
Demographic shifts depend on latitude
We predicted that the three northern ‘A’ lineages,
which are presently constrained to tropical montane
Table 2 Marginal likelihood estimates (MLEs) and exponential
growth rates from BEAST analyses using constant-size or expo-
nential change coalescent tree priors. MLEs are based on Step-
ping Stone Sampling (SS) and Path Sampling (PS). Exponential
growth rates are scaled to million-years�1 by assuming a muta-
tion rate of 0.0071 substitutions/site/million-years (Table S2,
Supporting information). 2ln(BF) scores higher than 2 are con-
sidered positive evidence against the null hypothesis (here,
constant size), and scores higher than 20 are considered very
strong evidence against the null hypothesis (Kass & Raftery
1995). The best fitting model (when significant) is marked by
an asterisk
Tree prior MLE (SS) MLE (PS) Growth rate
Thylamys pallidior A
Constant �10944.40 �10944.12 n/a
Exponential* �10941.31 �10941.03 207.96%
2ln(BF) 6.18 6.18
Thylamys sponsorius A
Constant* �14378.64 �14378.27 n/a
Exponential �14383.11 �14382.64 91.87%
2ln(BF) 8.94 8.74
Thylamys venustus A
Constant* �14957.52 �14957.20 n/a
Exponential �14958.55 �14958.23 89.96%
2ln(BF) 2.06 2.06
T. pallidior B
Constant �15730.06 �15729.69 n/a
Exponential* �15723.77 �15723.41 623.03%
2ln(BF) 12.58 12.56
T. sponsorius B
Constant �11999.79 �11999.56 n/a
Exponential �12000.33 �12000.07 �89.53%
2ln(BF) 1.06 1.02
T. venustus B
Constant �17833.98 �17832.79 n/a
Exponential* �17816.29 �17815.11 143.07%
2ln(BF) 35.38 35.36
© 2015 John Wiley & Sons Ltd
2502 T. C. GIARLA and S. A. JANSA
habitats, would have experienced population declines
following post-LGM warming due to compression into
high-elevation habitats. Our results do not match this
prediction. Instead, each ‘A’ lineage had relatively sta-
ble population sizes over the past 200 000 years (Fig. 3),
and a constant-size model could not be rejected for two
of those lineages (T. sponsorius A, and T. venustus A;
Table 2). This unexpected stability suggests that late-
Quaternary climate oscillations had no discernable
effect on population sizes in these tropical Andean lin-
eages. Or, perhaps, the structure of the mountain sys-
tem in the tropical Central Andes is such that ample
habitat space exists at both mid- and high-elevation
areas. In contrast, we predicted that Andean lineages
with ancestral distributions adjacent to subtropical or
temperate areas would have been able to expand their
ranges into those areas during warm stages of the Qua-
ternary (and that such range expansions would trigger
demographic growth detectable in EBSPs). We find evi-
dence for substantial demographic expansion in the
skyline plots for both T. sponsorius B and T. pallidior B,
but not for T. venustus B (Fig. 3). The expansion appar-
ent in the T. pallidior B and T. sponsorius B EBSPs
appears to start before the onset of warming leading up
to the Holocene, but, given the relatively broad HPD
intervals surrounding the demographic functions, it is
impossible to reject our prediction that the expansion
occurred after the LGM. Regardless of the timing, we
suspect that the demographic growth was precipitated
by a southward range expansion into cooler climates at
higher latitudes.
Late-Quaternary range expansions in southern South
America are seen in several other terrestrial taxa,
including amphibians (Nu~nez et al. 2011), squamates
(Olave et al. 2011; Camargo et al. 2013), birds (Masello
et al. 2011) and other mammals (Mar�ın et al. 2007; Hi-
mes et al. 2008; Coss�ıos et al. 2009; Lessa et al. 2010),
suggesting that this pattern might be widespread. How-
ever, phylogeographic data from the open and montane
habitats of central and southern South America are
scarce (Turchetto-Zolet et al. 2013), and future work
should focus on clarifying demographic shifts in
response to climate change in these regions. Accurately
estimating the timing of such demographic shifts is a
challenging but essential part of testing phylogeograph-
ic hypotheses (Hope et al. 2014). The use of relatively
slowly evolving loci might not generate precise enough
estimates for productive investigations into recent evo-
lutionary events on the order of tens of thousands of
years. Phylogenomic-scale data sets incorporating mark-
ers like RAD-seq (Baird et al. 2008) or ultraconserved
elements (Faircloth et al. 2012; Smith et al. 2014) might
be large enough to overcome the sampling error inher-
ent to smaller data sets like ours.
Conclusions
We inferred divergence times and modelled the demo-
graphic histories of six Andean Thylamys lineages, and
our results support extremely recent divergence times
(all within the past 200 000 years) and two noteworthy
shifts in population size (as much as a sixfold expan-
sion in T. pallidior B). Taken as a whole, these results
suggest that late-Quaternary climate oscillations may
have played a role in both stimulating speciation and
(for southern lineages) demographic expansion. Diver-
gence time intervals for two pairs of cryptic lineages
(T. pallidior and T. sponsorius) overlap with the LIG, the
most recent time in Earth’s history when presently
montane species may have been most isolated. Recent
demographic expansions in two lineages with more
southerly contemporary ranges (T. pallidior B and
T. sponsorius B) fit our predictions for extra-tropical
range expansion. None of the three more northerly ‘A’
members of each cryptic pair showed the demographic
decline predicted for species with a more tropical
ancestral distribution, which we expected to have been
forced to contract in size during the post-LGM warm-
ing period. The lack of population size contraction can
be cautiously taken as positive news and suggests that
Central Andean small mammals like Thylamys species
might be more resilient to climate warming than mon-
tane species in other parts of the world (e.g. Rocky
Mountain pikas; Beever et al. 2003). Nonetheless,
although the warming observed since the LGM does
not appear to have reduced the effective population
size for any of the species studied here to date, ongoing
habitat destruction and more extreme warming could
still pose significant extinction risks for these and other
Andean species.
Acknowledgements
This work was supported by grants to T.C.G. from the
National Science Foundation (DEB-1110365), the University of
Minnesota, the Bell Museum of Natural History, the American
Society of Mammalogists and the Society of Systematic Biolo-
gists, and a grant to S.A.J from the National Science Founda-
tion (DEB-0743062). We are grateful to the many collectors and
curators who provided us with tissue and specimen loans as
part of this project. Andrew Simons, Keith Barker, Jake Essels-
tyn, Melissa DeBiasse, Jeremy Brown and three anonymous
reviewers provided helpful comments on earlier drafts of this
work.
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T.C.G. conceived the study and performed the analyses.
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manuscript.
© 2015 John Wiley & Sons Ltd
PHYLOGEOGRAPHY OF ANDEAN MARSUPIALS 2505
Data accessibility
GenBank numbers of the sequences used in this study
are provided in Appendix S1 (Supporting information).
All DNA alignments, BEAST and BPP input files, resulting
tree files and geographic coordinates of collecting locali-
ties for specimens included in this study have been
uploaded to Dryad (doi:10.5061/dryad.n5s02).
Supporting information
Additional supporting information may be found in the online ver-
sion of this article.
Fig. S1 Map of collecting localities for specimens used in this
study, taken from Giarla et al. (2014).
Fig. S2 Ultrametric CYTB tree inferred in BEAST using three
fossil calibrations.
Fig. S3 Ultrametric tree inferred in BEAST using a multilocus
super-matrix and three fossil calibrations.
Table S1 Individuals sequenced for four fifteen nuclear loci as
part of Giarla et al. (2014).
Table S2 Evolutionary rates per locus for Thylamys + Lestodel-
phys.
Table S3 Table of alignments used in the six extended Bayes-
ian skyline plot runs.
Appendix S1 Table of GenBank accession numbers for
sequences used in this study.
© 2015 John Wiley & Sons Ltd
2506 T. C. GIARLA and S. A. JANSA
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