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Spatiotemporal Diversification of the True Frogs (Genus Rana): A Historical Framework for a Widely Studied Group of Model Organisms

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True frogs of the genus Rana are widely used as model organisms in studies of development, genetics, physiology, ecology, behavior, and evolution. Comparative studies among the more than 100 species of Rana rely on an understanding of the evolutionary history and patterns of diversification of the group. We estimate a well-resolved, time-calibrated phylogeny from sequences of six nuclear and three mitochondrial loci sampled from most species of Rana, and use that phylogeny to clarify the group's diversification and global biogeography. Our analyses consistently support an "Out of Asia" pattern with two independent dispersals of Rana from East Asia to North America via Beringian land bridges. The more species-rich lineage of New World Rana appears to have experienced a rapid radiation following its colonization of the New World, especially with its expansion into montane and tropical areas of Mexico, Central America, and South America. In contrast, Old World Rana exhibit different trajectories of diversification; diversification in the Old World began very slowly and later underwent a distinct increase in speciation rate around 29–18 Ma. Net diversification is associated with environmental changes and especially intensive tectonic movements along the Asian margin from the Oligocene to early Miocene. Our phylogeny further suggests that previous classifications were misled by morphological homoplasy and plesiomorphic color patterns, as well as a reliance primarily on mitochondrial genes. We provide a phylogenetic taxonomy based on analyses of multiple nuclear and mitochondrial gene loci.
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DOI:10.1093/sysbio/syw055
Spatiotemporal Diversification of the True Frogs (Genus Rana): A Historical Framework
for a Widely Studied Group of Model Organisms
ZHI-YONG YUAN 1,2,WEI-WEI ZHOU1,XIN CHEN3,NIKOLAY A. POYARKOV,Jr.
4,HONG-MAN CHEN1,
NIAN-HONG JANG-LIAW5,WEN-HAO CHOU6,NICHOLAS J. MATZ KE 7,KOJI IIZUKA8,MI-SOOK MIN9,
SERGIUS L. KUZMIN10,YA-PING ZHANG1,DAVI D C. CANNATELLA11 ,DAV I D M. HILLIS11,,AND JING CHE1
1State Key Laboratory of Genetic Resources and Evolution, and Yunnan Laboratory of Molecular Biology of Domestic Animals,
Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan; 2Kunming College of Life Science, University of Chinese Academy
of Sciences, Kunming 650204, Yunnan; 3Department of Biological Sciences, Dartmouth College, Hanover, HN 03755, USA; 4Department of Vertebrate
Zoology, Biological Faculty, Lomonosov Moscow State University, Leninskiye Gory, GSP-1, Moscow 119991, Russia; 5Animal Department, Taipei Zoo, 30
Xinguang Road, Sec. 2, Taipei 11656, Taiwan; 6Department of Zoology, National Museum of Natural Science, 1st Kuang-Chien Road, Taichung 40453,
Taiwan; 7Division of Ecology, Evolution, and Genetics, Research School of Biology, The Australian National University, ACT 2601, Australia;
8Kanda-Hitotsubashi JH School, 2-16-14 Hitotsubashi, Chiyoda-ku, Tokyo 101-0003, Japan; 9Conservation Genome Resource Bank for Korean Wildlife
(CGRB), Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, 151-742 Seoul, South Korea; 10Institute of
Ecology and Evolution, Russian Academy of Sciences, Moscow 119234, Russia; and 11Department of Integrative Biology, University of Texas at Austin,
Austin, TX 78712, USA
Correspondence to be sent to: Department of Integrative Biology, University of Texas at Austin, Austin, TX 78712, USA or
Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan
E-mail: dhillis@austin.utexas.edu, chej@mail.kiz.ac.cn, catfish@utexas.edu, zhangyp@mail.kiz.ac.cn
The first three authors share equal first authorship.
Received 19 September 2015; reviews returned 11 December 2015; accepted 31 May 2015
Associate Editor: Richard Glor
Abstract.—True frogs of the genus Rana are widely used as model organisms in studies of development, genetics, physiology,
ecology, behavior, and evolution. Comparative studies among the more than 100 species of Rana rely on an understanding of
the evolutionary history and patterns of diversification of the group. We estimate a well-resolved, time-calibrated phylogeny
from sequences of six nuclear and three mitochondrial loci sampled from most species of Rana, and use that phylogeny to
clarify the group’s diversification and global biogeography. Our analyses consistently support an “Out of Asia” pattern with
two independent dispersals of Rana from East Asia to North America via Beringian land bridges. The more species-rich
lineage of New World Rana appears to have experienced a rapid radiation following its colonization of the New World,
especially with its expansion into montane and tropical areas of Mexico, Central America, and South America. In contrast,
Old World Rana exhibit different trajectories of diversification; diversification in the Old World began very slowly and later
underwent a distinct increase in speciation rate around 29–18 Ma. Net diversification is associated with environmental
changes and especially intensive tectonic movements along the Asian margin from the Oligocene to early Miocene. Our
phylogeny further suggests that previous classifications were misled by morphological homoplasy and plesiomorphic color
patterns, as well as a reliance primarily on mitochondrial genes. We provide a phylogenetic taxonomy based on analyses of
multiple nuclear and mitochondrial gene loci. [Amphibians; biogeography; diversification rate; Holarctic; transcontinental
dispersal.]
Biodiversity is distributed heterogeneously across
Earth. Consequently, the determinants of spatial
patterns of diversity are of paramount interest
for biologists (Gaston 2000;Ricklefs 2004). Broadly
distributed, species-rich clades provide an opportunity
to explore the evolutionary processes that drive diversity
across large spatiotemporal scales (e.g., Derryberry et al.
2011;McGuire et al. 2014). Methods combining the
dynamics of diversification (e.g., speciation, extinction)
with biogeographic history allow biologists to test
hypotheses of diversification within and between
regions (Ricklefs 2004;Wiens and Donoghue 2004;
Mittelbach et al. 2007).
If speciation and extinction rates (ignoring dispersal
for the moment) are roughly constant across geographic
areas, then species diversity is influenced by time-
dependent processes (McPeek and Brown 2007).
However, changes in speciation rate (e.g., by adaptive
radiations) or extinction rate (e.g., by climate effects)
can quickly disrupt the correlation between clade age
and species diversity. In many radiations, a pattern
of early, rapid cladogenesis followed by a slowdown
in diversification rate is thought to be related to
ecological constraints (Schluter 2001;Rabosky 2009).
Further, increases in cladogenesis are often associated
with broad-scale environmental changes or geological
processes (e.g., orogenesis).
Diverse clades that are distributed across several
major continental areas provide excellent opportunities
to compare patterns of distribution and diversification
in different regions (Qian and Ricklefs 2000;McGuire
et al. 2014). In this article we investigate the spatial
and temporal patterns of regional and continental-scale
biodiversity in a widely studied clade that is broadly
distributed across Eurasia and the Americas, the true
frogs (genus Rana, sensu AmphibiaWeb 2015).
True frogs are extensively used as model organisms in
studies of development, genetics, physiology, behavior,
ecology, and evolution (see Duellman and Trueb 1986).
The first successful laboratory cloning experiment of
an animal (transfer of a nucleus into an enucleated
egg, resulting in a normal organism) was conducted
1
Systematic Biology Advance Access published July 19, 2016
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2SYSTEMATIC BIOLOGY
in Rana pipiens (Briggs and King 1952), and various
species of Rana continue to be used widely in studies
of physiology and genetics. Many species of Rana are
also commonly studied in the field as well as the
laboratory. One of the most extensive monographs
on the ecology and life history of an amphibian was
based on Rana temporaria (Savage 1962), and studies
of Rana ecology, behavior, conservation, and evolution
have accelerated in recent years (reviewed by Hillis and
Wilcox 2005).
More than 100 described species of Rana
(AmphibiaWeb 2015) range across Europe (12 species)
and Asia (32 species), and from North America to
the northern half of South America (57, plus several
recognized but not yet described species; Hillis and
Wilcox 2005). Species occur in a wide variety of
habitats including tundra, temperate coniferous and
deciduous forests, grasslands, deserts, brackish-water
marshes, freshwater streams and lakes, montane
cascades, semitropical cloud forests, and tropical
rainforests (Hillis and Wilcox 2005). Many species
are of conservation concern, and several species are
recently extinct or threatened with extinction. Despite
the diversity of the genus and its importance in many
biological investigations, no comprehensive analysis of
the spatial patterns and drivers of diversity for Rana has
been published.
The Eurasian species are morphologically
conservative. They possess prominent dorsolateral
folds, a dark temporal mask, and a body that is
countershaded in various shades of brown, leading to
the common English name “brown frogs” (Boulenger
1920;Liu and Hu 1961). This color pattern occurs in
many species in Europe, Asia, and North America,
although several New World species groups show far
greater morphological differentiation. This greater
morphological diversity correlates with their varied
ecologies, physiologies, behaviors, and morphological
structures associated with mating calls and habitat
(Hillis and Wilcox 2005). These differences in patterns
of species diversity and biological divergence make
Rana an excellent group for studying the processes of
biodiversity generation.
Most previous studies of Rana have been restricted
to particular geographic regions, or have used very
limited sampling of global taxa, or have been limited
to analyses of mtDNA sequences. For example, mtDNA
analyses (sometimes with small amounts of nuclear
DNA (nuDNA)) of Rana have included species in Europe
(e.g., Veith et al. 2003), the New World (e.g., Hillis
and Wilcox 2005), the mainland of East Asia (e.g.,
Che et al. 2007a), and the Asian islands (e.g., Tanaka-
Ueno et al. 1998;Matsui 2011). We expand on these
studies by analyzing six nuclear and three mitochondrial
genes for a large majority of the extant species (90
species; Supplementary Table S1, available on Dryad
at http://dx.doi.org/10.5061/dryad.ck1m7) across the
entire distribution in Eurasia and the Americas, and
by estimating divergence times and patterns of net
diversification.
The family Ranidae began to diversify about 57 Ma
(Bossuyt et al. 2006;Wiens et al. 2009), long after the
breakup of Pangaea (>120 Ma, Sanmartín et al. 2001).
Therefore, the global distribution of the family must
have resulted from intercontinental dispersal. Based on
limited sampling of Rana,Bossuyt et al. (2006) suggested
that the ancestor(s) of the American Rana reached the
New World in one or two waves from Eurasia, without
specifying the route of dispersal. Macey et al. (2006)
suggested two alternative hypotheses: two dispersals
from Asia to America via Beringia, or one dispersal from
Asia to America with a second back-dispersal to Asia. We
asked the following questions:
(1) When did the intercontinental dispersal of Rana
occur between Eurasia and the Americas? Were
these dispersals via trans-Atlantic land bridges
(Case 1978) or by trans-Beringian (Pacific) land
bridges (Macey et al. 2006)? Was there only one
dispersal from Eurasia to the Americas, with
a dispersal back to Eurasia, or were there two
distinct dispersal events into the New World?
When did major dispersal events within Eurasia
and the Americas occur (e.g., into Europe, and into
the Neotropics)?
(2) To what extent was dispersal into new areas
accompanied by increased diversification rates?
Were speciation rates highest when a lineage
entered new regions, or were they relatively
constant?
(3) How did geologic events and environmental
changes influence the diversification of Rana in the
Old World (especially the diversity in East Asia)
and the New World?
MATERIALS AND METHODS
Taxon Sampling
Complete sampling of all species in Rana was
a challenge due to the large number of species,
their intercontinental distribution, and rarity or recent
extinction of some species. Our analyses included 82 of
the currently recognized species of Rana as well as eight
undescribed taxa (Supplementary Table S1, available
on Dryad). All major lineages were included (Tanaka
et al. 1996;Veith et al. 2003;Hillis and Wilcox 2005;Che
et al. 2007a;Matsui 2011). Based on other studies, the
relationships of the unsampled species (Supplementary
Table S2, available on Dryad) are uncontroversial and the
species are distributed evenly across the tree. Thus, our
sampling bias is negligible. Four species from Odorrana,
Pelophylax,Hylarana, and Rugosa (Ranidae) were chosen
as outgroup taxa based on Che et al. (2007b). New
sequences from 80 species were analyzed, along with
14 species from GenBank (Veith et al. 2003;Hillis and
Wilcox 2005;Che et al. 2007a) (Supplementary Table S1,
available on Dryad).
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2016 YUAN ET AL.—SPATIOTEMPORAL DIVERSIFICATION OF THE TRUE FROGS 3
DNA Extraction, Amplification, and Sequencing
We extracted DNA from muscle or liver tissues (either
frozen or preserved in 95% ethanol) using standard
phenol-chloroform extraction protocols (Sambrook et al.
1989). We amplified and sequenced three fragments
of mitochondrial DNA (mtDNA) and six nuDNA loci
(Supplementary Table S1, available on Dryad). The
mitochondrial genes included 12–16S (partial sequence
of 12S ribosomal RNA gene, complete sequence of
tRNAVal and partial sequence of 16S ribosomal RNA),
CYTB (cytochrome b) and ND2 (partial sequence of
NADH dehydrogenase subunit 2; complete sequence of
tRNAAla, tRNAAln, tRNATrp ; and partial sequence of
the light strand replication origin). The nuDNA markers
(Supplementary Table S3, available on Dryad) included
RAG1 (partial sequence of recombinase activating 1
protein gene), RAG2 (partial sequence of recombinase
activating 2 protein gene), BDNF (partial sequence
of brain-derived neurotrophic factor gene), SLC8A3
(partial sequence of solute carrier family 8 member
3), TYR (exon 1 of tyrosine precursor gene), and
POMC (partial sequence of pro-opiomelanocortin A
gene). We used standard polymerase chain reactions
(PCRs) in 25-L reactions with the following protocol:
initial denaturation step with 5 min at 95°C, 35 cycles
of 94°C for 1 min, 41–57°C (depending on primers;
Supplementary Table S3, available on Dryad) for 1
min, 72°C for 1 min and a single final extension at
72°C for 10 min. PCR products were purified with
a Gel Extraction Mini Kit (Watson BioTechnologies,
Shanghai). The purified product was used as the
template DNA for cycle-sequencing reactions performed
using BigDye Terminator Cycle Sequencing Kit (v2.0,
Applied Biosystems). Sequencing was conducted on
an ABI PRISM 3730 (Applied Biosystems) automated
DNA sequencer. We sequenced amplified fragments
with PCR primers in both directions and subjected the
raw sequences to BLAST searches in GenBank to verify
the amplifications.
Nucleotide sequences were aligned using ClustalX
v1.81 (Thompson et al. 1997) with default parameters,
and checked for ambiguously aligned regions in MEGA
5.0 (Tamura et al. 2011). Saturation tests for each gene
and gene partitions were done using DAMBE (Xia and
Xie 2001).
Phylogenetic Analyses
Bayesian inference (BI), maximum likelihood
(ML), and maximum parsimony (MP) analyses were
conducted using the nine concatenated gene fragments.
BI analyses were performed in MrBayes v3.1.2 (Ronquist
and Huelsenbeck 2003) using the optimal partitioning
strategy and best-fit nucleotide substitution model for
each region (Supplementary Table S4, available on
Dryad) selected by PartitionFinder v1.1.1 (Lanfear et al.
2012). Four incrementally heated Markov chains (using
the default heating value of 0.1) were run for 10 million
generations each while sampling the chains at intervals
of 1000 generations. Two independent runs were carried
out. We discarded the first 50% of the samples as burn-
in, and log-likelihood scores were tracked to assure
convergence (effective sample size, ESS, values >200).
We assessed topological convergence using AWTY
(Nylander et al. 2008) to visualize the cumulative split
frequencies in the set of posterior trees, as recommended
by Moyle et al. (2012). The average split frequency value
was set to <0.01 to assure convergence. The samples
were summarized as a majority-rule consensus tree and
the frequencies of nodal resolution were interpreted as
Bayesian posterior probabilities (BPPs).
The ML trees were estimated using RAxML v7.0.4
(Stamatakis 2006). We used GTR+as the substitution
model, based on the partitioning strategy selected using
PartitionFinder (Supplementary Table S4, available on
Dryad). Support values were estimated from 1000
nonparametric bootstrap pseudoreplicates.
MP analyses were implemented using PAUP* 4.0b10a
(Swofford 2003). The heuristic MP searches were
executed for 1000 replicates using tree-bisection-
reconnection branch swapping. All characters were
treated as unordered and equally weighted. We
used bootstrap analysis (BP) conducted with 1000
pseudoreplicates to assess nodal support. Gaps were
treated as missing data.
We conducted exploratory BI analyses of a
concatenated mitochondrial data set and a concatenated
nuclear data to assess the separate contributions of
each data type to the tree derived from all concatenated
genes. We also conducted species tree estimation
using the gene-tree-based coalescent methods ASTRAL
(Mirarab et al. 2014) and MP-EST (Liu et al. 2010). We
treated the three fragments of mtDNA as one locus, and
the six nuDNA loci separately. For each of the seven
loci, we generated 100 bootstrap replicates in RAxML
v7.0.4 (Stamatakis 2006), and used the GTR+model of
sequence evolution. MP-EST analyses were conducted
using the STRAW server (Shaw et al. 2013), and ASTRAL
analyses conducted using ASTRAL v4.7.1 (Mirarab et al.
2014).
Divergence Times
Divergence times were estimated using BEAST v1.7.5
(Drummond et al. 2012) using a relaxed uncorrelated
clock (Drummond et al. 2006). The substitution models
and partitions were the same as used in the BI analyses.
A Yule process was chosen for the tree prior. We used
two independent analyses of 40 million generations each
with sampling every 1000 trees. Convergence of the
chains was determined with Tracer v1.5 (Rambaut and
Drummond 2007), with target ESS values more than 200
for all parameters; 25% of samples were discarded as
burn-in.
Nodes 2, 9, 5, and 17 (as shown in Fig. 1) were used
for calibration. Node 2 was calibrated using a previous
age estimate from Bossuyt et al. (2006; Fig. 3, their node
19). A normal prior distribution with mean of 31.2 and
SD of 8.1 Ma yielded 2.5% and 97.5% quantiles of 15.3
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4SYSTEMATIC BIOLOGY
FIGURE 1. The BI tree derived from the combination of six nuDNA and three mtDNA loci. Branches without support symbols were strongly
supported in all three phylogenetic analyses (BI, ML, and MP); we treated bootstrap proportions 70% and BPPs 95% as significantly supported
(). Bootstrap proportions <70% and BPPs <95% were considered weakly supported (). Support values are shown only for branches that were
weakly supported in at least one of the three analyses, in which case support values are shown in order for BI, ML, and MP analyses. Colors
of branches indicate the geographic distribution of extant taxa. Numbers on branches correspond to clades discussed in this study. Numbers
with a black box correspond to the nine subgenera (plus Rana sylvatica) referred to in Supplementary Table S2, available on Dryad. Photographs
depict the morphological variation of Rana across clades 1, 4, 5, 7, 8, 9, 11, 13, 15, 17, and 18. Frog illustrations are used with permission from
David Hillis, Hui Zhao, Todd Pierson, Andreas Noellert, and Richard Sage.
and 47.1 Ma. Three node calibrations were based on
fossil Rana representing the R. pipiens group (node 9),
the Rana catesbeiana group (node 5), and the R. temporaria
group (node 17). The last is based on a single articulated
individual. The first two are represented primarily by
ilia, one of the most commonly preserved elements of
Tertiary fossil frogs. As noted by Parmley et al. (2010), it
is relatively easy to diagnose Rana ilia from other genera
due to the extremely prominent iliac crest. However,
the ilia are less easily distinguished among species
within Rana.
Assignment of the fossils to species groups by
previous authors was based on overall similarity based
on comparisons of several species of Rana. Because
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2016 YUAN ET AL.—SPATIOTEMPORAL DIVERSIFICATION OF THE TRUE FROGS 5
FIGURE 2. Map showing the generalized distribution areas of the extant Rana species and schematic drawings that illustrate possible dispersal
events among the four major Holarctic areas under H0, H1, and H2. Seven major geographic regions (A–G) are indicated using colors. H0 is the
null model, which assumes equal rates of dispersal between any two regions. H1 is the trans-Beringian hypothesis, which is further subdivided
into H1a, with dispersals between East Asia and western North America and eastern North America, in either direction; H1b, two independent
dispersals from East Asia to western North America and eastern North America (Macey et al. 2006); H1c, one dispersal from East Asia to eastern
North America and one back to East Asia from western North America (Macey et al. 2006). H2 is the trans-Atlantic hypothesis: dispersal(s)
occurred between Europe and western North America or eastern North America.
the authors explicitly allocated each fossil to a well-
circumscribed clade of species, we inferred that the fossil
is nested within that group, and used it to calibrate the
most recent common ancestor (MRCA) of that group.
The exception was the R. temporaria group fossil (see
below).
We used a lognormal distribution to sample the prior
for the age of each node. To parameterize the priors we
used the BEAUTI user interface to set the “offset” as the
minimum age of the fossil, the mean at 2.0 (standard
scale), and the standard deviation at 1.0 (natural log).
The mean and standard deviation were chosen so that
the upper 97.5% quantile limit of each calibration age
was 9–10 Ma older than the minimum age. Because no
fossils that can be clearly allocated to Rana are older than
20 Ma, we view these limits as reasonable.
The calibration of node 9 was based on several “Rana
cf. Rana pipiens” individuals from the Early Miocene
Thomas Farm locality in Florida (18 Ma; Holman 1965,
1968). Holman concluded that the fossil ilia were not
distinguishable from some extant species within the
R. pipiens group (although they are from others), so we
infer that the fossil is nested within the R. pipiens clade
(node 9) and thus the clade had begun to diversify at
least 18 Ma. The 2.5% and 97.5% quantiles of the prior
distribution are 18.2 and 26.6 Ma.
Calibration of node 5 was based on Hottell Ranch
Site fossils (15 Ma, Barstovian, Miocene), identified as
Rana near R.“clamitans”(Voorhies et al. 1987), a species
within the R. catesbeiana clade. Therefore, the MRCA of
the R.catesbeiana group was calibrated at a minimum
age of 15 Ma with 2.5% and 97.5% quantiles of 15.2 and
23.6 Ma. Voorhies et al. (1987) also identified “R. pipiens
complex” ilia in their sample, suggesting that the authors
were confident in the assignment of their R.clamitans
fossil.
Calibration of node 17 was based on the single
articulated fossil (R. temporaria group) from
Dietrichsberg, Germany, dated at 19–20 Ma, early
Miocene (Böhme 2001). Because the author did not state
whether the fossil was nested within the R.temporaria
clade, as opposed to being its sister species, we used it to
calibrate the node immediately ancestral to the MRCA
of the European clade; that is, the divergence of Rana
asiatica from the European species of the R.temporaria
group (node 17). We set the minimum age at 19 Ma;
2.5% and 97.5% quantiles are 19.2 and 27.6 Ma.
To test the sensitivity of our time calibration to
particular calibration points, we used a jackknife analysis
(Near et al. 2005). We successively deleted each of
the individual fossil calibration points, repeated the
calibration analyses, and compared the median age
estimates with those ages from the complete analysis
(Supplementary Table S5: Analyses 1–4, available on
Dryad). Removing the R.catesbeiana group fossil yielded
age estimates that were on average 8% younger than the
estimates based on all fossil calibrations (Analysis 1).
Similarly, removing the R. temporaria group fossil
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6SYSTEMATIC BIOLOGY
(Analysis 4) yielded age estimates that on average
were 9% younger than those of Analysis 1. In contrast,
jackknife deletion of the R. pipiens group fossil (Analysis
3) yielded ages that were an average of 4% older than
those of Analysis 1. Despite the modest systematic
differences (older or younger) between the jackknife
estimates and the complete analysis, the 95% credible
intervals of the ages overlapped broadly, and so we
used the calibration based on all fossils for our
biogeographical analyses.
Biogeographic Multimodel Inference and Ancestral
Range Estimation
To estimate the ancestral range for Rana, we defined
seven major geographic regions (Fig. 2): (A) East Asia
(mainland +islands), (B) Central Asia, (C) Europe, (D)
western North America, (E) eastern North America, (F)
Mexican Plateau and surrounding region, and (G) the
Neotropics. We distinguished region B based on its arid
continental climate and distinct anuran fauna compared
with region A, and separated region C from regions A
and B along the Ural Mountains and Caspian Sea, the
area of the ancient Turgai Straits (Tiffney 1985a;Briggs
1995). We first divided the Americas into temperate
and tropical regions, and then further divided North
American temperate regions into eastern NA (E) and
western NA (D) along the Rocky Mountains (Tiffney
1985b;Sanmartín et al. 2001;Donoghue and Smith 2004).
We also defined an impor tant transition area between the
temperate and tropical regions that is centered on the
Mexican Plateau (F), a hotspot of diversity in Rana. This
region includes the montane areas of southern Arizona
and New Mexico, from the Mogollon Rim southward,
as well as the Edwards Plateau in Texas southward.
The southern boundary of this region is defined by
the montane regions of Mexico north of the Isthmus of
Tehuantepec (Fig. 2). The Neotropics (G) lies south of this
boundary, and along the lowlands of both the Atlantic
and Pacific coasts. Areas A–C are collectively referred to
as Old World, and D–G as New World.
We estimated model parameters and ancestral range
probabilities using the DEC and DEC+J models in
the BioGeoBEARS R package (Matzke 2013a;R Core
Development Team 2014). The DEC model describes
the temporal change in the range of a species, and
distinguishes anagenetic change (between nodes) from
cladogenetic change (at a node). It estimates two free
parameters describing anagenesis: d, the rate of dispersal
(range expansion) and e, the rate of extinction (range
contraction) (Ree and Smith 2008). The DEC+J model
includes a third parameter, j, which describes the
relative weight of founder event speciation during
cladogenesis (Matzke 2013b). Under this model, which
resembles founder event speciation, an ancestor in
area A instantaneously “jumps” to area B, leaving one
descendant in A and one in B. An increase in jreduces the
weight of the traditional DEC cladogenesis processes.
We followed the terminology of Matzke (2014)
for cladogenetic events. Vicariance is the splitting
of a species’ range such that the two descendants
have non-overlapping ranges. In sympatric-subset
cladogenesis, the range ABC yields two descendant
ranges that overlap, such as (ABC, A), or (ABC, B).
In sympatric range-copying cladogenesis, the ranges of
each descendant node are identical with their ancestor.
To test the hypotheses of intercontinental dispersal,
we used dispersal matrices (Supplementary Table S6,
available on Dryad) to indicate the probability of
dispersal events between two areas. We first performed
an unconstrained analysis, assuming equal probability
of dispersal among all adjacent areas (Fig. 2), as our null
model (H0) to compare to hypotheses of Trans-Beringian
(H1) and the Trans-Atlantic dispersals (H2).
Under the Trans-Beringian hypothesis (H1), we
considered (a) dispersals only between East Asia and
western NA and eastern NA, in either direction; (b)
two independent dispersals from East Asia to western
NA and eastern NA (Macey et al. 2006); and (c) one
dispersal from East Asia to eastern NA and one back
to East Asia from western NA (Macey et al. 2006). Under
the Trans-Atlantic hypothesis (H2), we tested whether
dispersal(s) occurred between Europe and western NA
or eastern NA.
We assigned a value of 1 for unrestricted dispersal
between two areas and 0.001 for disallowed dispersal
(0.0 causes computational difficulties; Supplementary
Table S6, available on Dryad). For all analyses the
maximum number of areas was set at four. We specified
allowed connections with an area adjacency matrix
(Supplementary Table S7, available on Dryad). The fit of
all models to the data was compared using AIC, and the
combination of connectivity model (dispersal matrices)
and DEC/DEC+J was determined using relative model
weights (Supplementary Table S9, available on Dryad).
The ML estimates for each model were then used to
calculate the ancestral range probabilities at each node of
the tree. Given that all species of Odorrana, the immediate
outgroup of Rana (Che et al. 2007b;Stuart 2008), occur in
East Asia, we fixed the node below Rana to have an East
Asian distribution using the BioGeoBEARS “fixlikes”
option in all analyses.
Diversification Patterns
Diversification analyses used (1) the entire genus Rana,
and (2) subsets of species within a region of interest. We
used the R package ape (Paradis et al. 2004) to generate a
lineage-through-time (LTT) plot based on the maximum
clade credibility tree and 95% credible interval obtained
by random sampling of 1000 trees from the post-burnin
posterior distribution of the BEAST analysis using
Burntrees v.0.1. (http://www.abc.se/~nylander/).
Outgroups were excluded. In addition, we explored
regional diversification of Rana using scripts provided
by Mahler et al. (2010) to visualize the accumulation of
lineages within different regions. These differ from LTT
plots in that they reflect diversity due to both in situ
diversification and independent immigration events
(see McGuire et al. 2014).
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2016 YUAN ET AL.—SPATIOTEMPORAL DIVERSIFICATION OF THE TRUE FROGS 7
We performed four LTT analyses to explore regional
patterns of diversity. First, we compared Old World and
New World species. Second, we compared individual
regions: East Asia, Europe (including central Asia
considering their close relationships), western NA, and
eastern NA. Third, we partitioned the East Asia region
into mainland and islands to examine the contribution
of species from East Asian islands, for example, Japan
and Taiwan. Last, we compared western NA, eastern
NA, the Mexican Plateau, and the Neotropics to identify
which regions contributed the lineage accumulation in
clade 3 (Fig. 1).
Because the LTT plots measure only the accumulation
of species and not speciation/extinction rates, we used
BAMM (Rabosky 2014) and MEDUSA (Alfaro et al. 2009)
to examine diversification rate heterogeneity along the
branches. Analyses were conducted using three groups:
all Rana, Old World Rana (Fig. 1: clades 1 +11 +
14), and New World Rana (using clade 3 only, because
clade 13 represents an independent dispersal into the
New World).
The BAMM analyses used 200 million generations,
sampling every 10,000 generations, and sampling event
data every 10,000 generations. We simulated the prior
distribution on the number of rate shifts. The effect of
unsampled taxa was determined by using a nonrandom
incomplete taxon-sampling correction. We removed the
first 30% of generations as burn-in after checking MCMC
convergence using BAMMtools (Rabosky et al. 2013;
Rabosky 2014). We checked the ESS (target value of 300)
using the CODA package (Plummer et al. 2006). The
final results, including the credible set of rate shifts,
the best shift model, and rate-through-time curves, were
summarized and visualized using BAMMtools.
We also estimated speciation/extinction rates using
MEDUSA, part of the R package geiger (Harmon et al.
2008). The former fits a birth–death model to the
phylogeny (Alfaro et al. 2009) and detects significant
rate shifts along the branches. We ran analyses using
the default AIC threshold. The MEDUSA algorithm
first estimates likelihood and AIC scores with the
simplest birth–death model (two parameters, band d).
Then, it compares the AIC scores of this model with
an incrementally more complex model that includes
five parameters (two birth parameters and two death
parameters owing to one break point and a shift-location
parameter). This process is terminated when further
addition of parameters does not improve the AIC score.
RESULTS
Phylogenetic Analysis
Our final data set included 94 sequences from 12S–
16S; 70 from CYTB;69fromND2;76fromRAG1,RAG2,
BDNF, and SLC8A3; and 77 from POMC and TYR, plus
sequences downloaded from GenBank (Supplementary
Table S1, available on Dryad). Seventy-seven species
included both mtDNA and nuclear sequences, whereas
17 species had GenBank mtDNA sequences only; tissues
were not available for additional analysis. For the 77
taxa with both nuclear and mtDNA data, some samples
were not successfully amplified for all genes, including
four from CYTB; six from ND2; and one from each of
RAG1,BDNF, and SLC8A3. We deposited the 639 new
sequences in GenBank (No. KX269176–KX269814).
The final matrix consisted of 7250 positions, including
1849 parsimony-informative sites (Supplementary
Table S4, available on Dryad). We translated all
protein-coding gene sequences into amino acids to
check for stop codons. In the fragment of 12S–16S
and POMC, regions of ambiguous alignment were
excluded. No indication of a saturation effect was
detected (Supplementary Table S8, available on Dryad).
The preferred substitution models for each partition are
listed in Supplementary Table S4, available on Dryad.
The BI, ML, and MP trees were very similar (Fig. 1and
Supplementary Fig. S1, available on Dryad). The main
conflict between mtDNA and nuclear genes involved
the position of Rana sylvatica and the relationships
among the three major New World groups within clade
6 (Supplementary Fig. S2, available on Dryad). Our
species-tree analyses (Supplementary Fig. S3, available
on Dryad) produced results that were consistent with
our concatenated analyses (Fig. 1), but with somewhat
less resolution. Given the greater resolution of the
concatenated analysis, and its general agreement with
the species-tree analysis, we used the concatenated
analysis for our biogeographic assessment (Figs. 1and
3). Our analyses resolved the following seven major
geographic lineages of Rana with strong support: Rana
weiningensis (clade 1) from southwestern China was
the sister group of the remaining taxa, which together
included a New World clade 3 (eastern North America,
Mexican Plateau, and Neotropics), southwestern China
(clade 11; only Rana shuchinae), western North America
(clade 13), southwestern China and northern Vietnam
(clade 15), Central Asian R.asiatica plus Europe (clade
17), and East Asia (clade 18), including the mainland and
adjacent islands.
We found that species of Old World Rana are
paraphyletic with respect to the two New World clades.
The major well-differentiated, morphologically and
ecologically distinct clades within New World Rana
largely support the traditional subgeneric designations
for the genus (Dubois 1992;Hillis and Wilcox 2005;
Hillis 2007;AmphibiaWeb 2015). The New World
clade 13 consists of the Rana boylii group in western
North America (the subgenus Amerana). The second
New World clade (clade 3) includes the remaining
North and South American taxa, with five strongly
differentiated subclades (Fig. 1): R. sylvatica (clade 4),
which forms the sister group of the remaining species;
the R. catesbeiana group (clade 5; the subgenus Aquarana);
the Rana palmipes group (clade 7; the subgenus Lithobates,
although that subgenus was further subdivided by
Hillis and Wilcox 2005); Rana sierramadrensis plus the
Rana tarahumarae group (clade 8; the subgenus Zweifelia,
although that name was used for the R. tarahumarae
group alone by Hillis and Wilcox 2005); and the R. pipiens
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8SYSTEMATIC BIOLOGY
FIGURE 3. Spatiotemporal reconstruction of the true frogs based on the Trans-Beringia hypothesis (H1b). Results based on the (a) DEC and (b)
DEC+J likelihood methods implemented in BioGeoBEARS are presented. The tree topology, derived from BEAST analyses, is consistent with
the Bayesian tree of Figure 1. Branch lengths are proportional to divergence times. Means and 95% confidence intervals of divergence times are
presented in Supplementary Figure S5, available on Dryad. Colors reflect biogeographic designations (for species at tips) and most probable
states at each node. Gray colors indicate nodes with multiple ancestral states. States at nodes represent the most probable ancestral area before
the instantaneous speciation event, whereas those on stems represent the state of the descendant lineage immediately after speciation. Some
node and stem labels are omitted to reduce clutter; in all cases these are identical to the state at both the ancestral and descendant nodes. The
corresponding pie charts for the proportional likelihoods of ancestral area states at each node and stem are presented in Supplementary Figure
S6, available on Dryad. Abbreviations: y, sympatric range-copying speciation; s, sympatric-subset speciation; v, vicariance; j, jump-dispersal or
founder event speciation. Arrows show the dispersal events.
group (clade 9; the subgenus Pantherana). Although each
of these distinct groups is strongly supported, the higher
relationships among groups have weaker support.
The East Asian taxa (clades 1, 11, 15, and 18)
formed a polyphyletic group. Clade 18 included five
well-supported subclades (Fig. 1and Supplementary
Fig. S4, available on Dryad): clades 19 and 22 included
exclusively the East Asian species from Taiwan and
Japan; 20, 21, and 23 included the remaining insular
species and closely related taxa from the East Asian
mainland. The unresolved relationships among the five
subclades suggest rapid radiation.
Divergence time estimates from the BEAST
chronograms are shown in Figure 3(with details
in Supplementary Fig. S5, available on Dryad). These
are described in concert with the historical biogeography
below.
Historical Biogeography
The DEC+J class of models always conferred a much
higher likelihood on the data than DEC (log-likelihood
differences of 11–16 units; Supplementary Table S9 and
Supplementary Fig. S6, available on Dryad). Within
each class, all submodels that allowed dispersals
across Beringia (H1) were favored over the Trans-
Atlantic dispersal hypothesis (H2) (Supplementary
Table S9, available on Dryad). Among the Beringian
dispersal hypotheses, hypothesis H1b (two independent
dispersal events from East Asia to North America)
received the strongest support. Thus, we used
this model in our evaluation of ancestral range
estimates.
We contrasted the results of the DEC and DEC+J
models (Fig. 3) under hypothesis H1b. DEC (Fig. 3a)
estimated the dispersal rate (r) as 0.0023 and the
extinction rate (e) as 0.0. By definition, the jump-dispersal
rate (j) was 0.0 under this model. We identified nine
dispersal events (horizontal arrows on Fig. 3a). In almost
all cases, these were followed by a vicariance event (v)
that split the ancestral range. We inferred 10 vicariance
and 7 sympatric-subset events (s). The remaining events
we inferred were sympatric range-copying events, in
which the ranges of both descendants were identical to
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2016 YUAN ET AL.—SPATIOTEMPORAL DIVERSIFICATION OF THE TRUE FROGS 9
FIGURE 4. Summary of major range shifts (dispersal and vicariance) of Rana over three time periods (a–c). For details of the range shifts see
Figure 3. (a1) Dispersal to eastern North America within the interval 48–43 Ma, followed by vicariance at 43 Ma. (a2) Dispersal to western North
America 43–34 Ma, followed by vicariance at 34 Ma. (b1) Dispersal to Europe +Central Asia (composite area) during 29–25 Ma (or possibly
earlier; see text). (b2) Europe and Central Asia split at 20 Ma by vicariance. (b3) Dispersal to the Mexican Plateau +Neotropics composite area
during 37–32 Ma; the Neotropics split from its sister-group (eastern North America +Mexican Plateau clade) by vicariance at 32 Ma; divergence
at 31 Ma of the first of several Mexican Plateau clades. (c1) Repeated dispersals among East Asia Islands and the mainland during 20–5 Ma. (c2)
At least two dispersals from the Mexican Plateau to the Neotropics during 13–10 Ma. Taxon names in panels are subgenera.
those of the ancestor (y); most of these are not shown to
avoid visual clutter.
We focus first on the divergences of the earliest-
diverging branches. Under the DEC model (Fig. 3a),
range expansions can be described by a combination
of dispersal followed by vicariance or sympatric-subset
events. Because the reconstruction of areas at some nodes
is not definitive, we report general patterns rather than
enumerating each event.
The MRCA of Rana dated to the Eocene at about
48 Ma (95% credibility interval [CI]: 55–40 Ma). The
first dispersal involved a range expansion through
Beringia and then across high latitudes of North America
into eastern North America about 48–43 Ma (Fig. 4a),
followed by vicariance at 43 Ma resulting in an eastern
North American clade 3 and an Asian clade 10 whose
ancestral range is reconstructed as East Asia (Area A).
The latter then underwent a second range expansion
across Beringia and then south into western North
America 43–34 Ma (Fig. 4a), followed by vicariance (node
12) at 34 Ma, yielding a western North American clade
(13) and a clade of Eurasian species (clade 14). The
Eurasian clade subsequently split into an East Asian
clade and a clade in Europe and Central Asia (Fig. 4b).
The East Asian lineage continued to diversify into a series
of lineages, some of which dispersed into the East Asian
islands (the Japanese islands, Sakhalin, the Kurils, and
Taiwan; Supplementary Fig. S4, available on Dryad, and
Fig. 4c).
In the New World radiation, the first invasion of Rana
from Asia underwent a primary split into two lineages:
R. sylvatica versus the remaining species. Then another
split occurred among the majority of species between
clade 5 (Aquarana; the North American water frogs) and
a clade that expanded into the Mexican Plateau and
the Neotropics (Fig. 4b). Shortly thereafter (~32 Ma),
this latter clade split to form clade 7 (Lithobates;the
Neotropical true frogs), and in turn a second lineage
split at approximately 31 Ma, yielding clade 8 (Zweifelia;
the torrent frogs) on the Mexican Plateau, and the
large clade 9 (Pantherana; the leopard frogs; Fig. 4c).
Within Pantherana, two lineages, R. forreri, and a clade
comprising R. macroglossa, sp. 4, sp. 5, and sp. 6,
diverged from their close relatives at approximately 11
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10 SYSTEMATIC BIOLOGY
FIGURE 5. Rana species accumulation curves as estimated for regions: (a) the Old World (clades 1, 11, and 14; see Fig. 1) and New World
(clades 3 and13); (b) East Asia (mainland +islands, clades 1, 11, 15, and 18), Europe and Central Asia (clade 17), the New World (clades 3 and 13);
(c) Old World, eastern North America, western North America, Mexican Plateau and surrounding region, and Neotropics; (d) New World, East
Asia mainland, East Asia islands, Europe and Central Asia. The y-axis and x-axis show the historical lineage diversity estimates and relative
branching times obtained from the time-calibrated phylogeny, respectively. Pie charts are color-coded to reflect the proportional likelihoods of
alternative ancestral state reconstructions.
Ma to occupy the Neotropics. The remaining species
of Pantherana are found in eastern and northern North
America and the Mexican Plateau. In contrast, clade 13
(Amerana) gave rise to several species beginning at 23 Ma,
but none of the descendants expanded east or south into
other areas. Ambiguities of ancestral range estimation
(Fig. 3) make precise interpretation of the biogeographic
history difficult, but at least four dispersal/vicariance
events occurred between eastern North America and the
Mexican Plateau.
Under the DEC+J model (Fig. 3b), these patterns can
be explained by 14 jump dispersals (jsymbol at the node,
and vertical arrows). The jparameter was estimated
to be 0.0279, and dand ewere 0.0. Under this model,
no vicariance events are inferred. We inferred seven
sympatric range-copying cladogenetic (y) events, but
no sympatric-subset cladogenetic events. Although the
estimated ranges and inferred mechanisms differed, the
locations of range shifts on the tree were similar under
both models.
Diversification Patterns
The standard LTT plot for all Rana depicted a near-
constant accumulation of lineages through time until
about 12 Ma, after which there was a gradual slowdown
(Supplementary Fig. S7, available on Dryad). However,
diversification plots of regions (Fig. 5) indicated several
region-specific patterns.
Generally, the Old World species (Fig. 1, clades 1 +11
+14) show a different trajectory of lineage accumulation
compared with taxa of the New World (Fig. 1, clade 3 +
13) (Fig. 5a). Further regional plotting indicates that East
Asian clades 1 +11 +18 and the larger clade 3 from
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2016 YUAN ET AL.—SPATIOTEMPORAL DIVERSIFICATION OF THE TRUE FROGS 11
FIGURE 6. Temporal dynamics of diversification rate through the evolutionary history of Rana revealed by BAMM analyses.Rate-through-time
curves depict clade-specific net diversification trajectories for (a) all Rana species, (b) the New World clade 3, and (c) the Old World species (clades
1, 11, and 14; Fig. 1). Shading intensity reflects relative probability of a given diversification trajectory, with upper and lower bounds denoting
the 90% Bayesian credible interval on the distribution of rates through time. A period of elevated diversification rate in (c) is highlighted by a
rectangle.
eastern NA, the Mexican plateau, and the Neotropics
are important in explaining the diversity of the Old
World and New World, respectively (Fig. 5b). The Old
World Rana began species accumulation about 48 Ma
ago, slowly at first, until 29 Ma; this was followed by
continuous cladogenesis, and acceleration of the rate of
lineage accumulation (Fig. 5a). Plots of the East Asian
lineages (Fig. 5b,d) indicate that East Asian islands
account for a substantial portion of Old World Rana
diversity, especially given the small size of the islands
relative to the mainland.
Generally, lineage accumulation in the New World
showed a faster rate than in the Old World (Fig. 5a,b),
and especially an increase in the rate of cladogenesis
around 14 Ma. This increase is associated with separate
radiations in eastern North America, the Mexican
Plateau, and the Neotropics, of which the radiation on
the Mexican Plateau had the greatest impact (Fig. 5c).
In contrast, species accumulation in western North
America was relatively slow and constant, resulting in
only eight extant species after the second dispersal from
East Asia (Fig. 5b).
Unlike the regional LTT analyses, BAMM analysis
of all Rana indicated an initial high net diversification
rate that gradually decreased through time (Fig. 6a and
Supplementary Figs. S8a and S9a, available on Dryad).
Analyses further supported strongly heterogeneous
diversification dynamics between the Old World and
New World species (Fig. 6and Supplementary Figs. S8b,c
and S9b,c, available on Dryad). The pattern for the New
World species (clade 3) supports diversity-dependent
speciation, with a pattern of initial high diversification
that gradually decreased through time (Fig. 6b). In
contrast, both BAMM and MEDUSA analyses detected a
more sudden rate shift among the Old World species
(Fig. 6c and Supplementary Figs. S8c, S9c, and S10,
available on Dryad). The net diversification rate from
BAMM shows a hump-shaped curve for the Old World
species, with an increased rate from 29 to 18 Ma (about
0.14 species/Ma; Fig. 6c). The MEDUSA analysis also
suggested that the current diversity of Old World Rana
(clades 1 +11 +14) is best explained by one rate
shift (AICc threshold =2.01; Supplementary Fig. S10,
available on Dryad); the average speciation rate increases
significantly from 0.013 to 0.093 after the split of
R. shuchinae from other Rana at approximately 37 Ma.
DISCUSSION
Phylogeny and Classification of Rana
Our analyses yield a comprehensive and well-resolved
phylogeny for the genus Rana and strongly confirm
its monophyly. Our treatment of Rana is largely
consistent with that used by AmphibiaWeb, except that
R. cangyuanensis is now considered part of Odorrana
(Fei et al. 2012). Previous studies have been hampered
by sampling restricted geographic regions or limited
species groups with limited gene markers, mostly based
on mtDNA analyses (e.g., Tanaka et al. 1996,Tanaka-
Ueno et al. 1998;Veith et al. 2003;Hillis and Wilcox 2005;
Frost et al. 2006;Che et al. 2007a;Matsui 2011). In contrast,
our tree is based on deep taxon sampling and multiple
nuclear and mitochondrial loci. Prior studies did not
recover most of the phylogenetic relationships because
they did not simultaneously consider both Eurasian and
American species.
The two New World clades of Rana (3 and 13 in Fig. 1)
clearly nest within East Asian clades (1, 11, 15, and 18 in
Fig. 1), and thus the simple sister pairs of intercontinental
disjunct Rana lineages (e.g., Macey et al. 2006) cannot be
assumed. This finding for Rana mirrors the conclusions
of Wen (1999), who considered similar intercontinental
distributions of several plant groups, and found that
many taxa traditionally treated as disjunct sister groups
were polyphyletic or paraphyletic. Our study highlights
the importance of thorough sampling for phylogenetic
history constructions.
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12 SYSTEMATIC BIOLOGY
The phylogenetic relationships we recovered do not
support some of the taxonomic assignments suggested
by Feietal.(2012) and Frost (2015) (Supplementary
Table S2, available on Dryad). For example, Pseudorana
from both Feietal.(2012) and Frost (2015) is paraphyletic.
Clade 15 contains Rana johnsi,Rana zhengi, and Rana
sangzhiensis (Fig. 1and Jing Che unpublished data) and
these are not the sister taxa of clade 1 (R. weiningensis).
Morphological homoplasy has led to some of the
taxonomic confusion surrounding the genus Rana. The
presence of grooves on the toe pads of adults in the
species in clades 1, 15, and Rana sauteri (within clade 19),
and an abdominal sucker in the tadpole of R. sauteri,have
led to various taxonomic proposals for these species. For
example, Pseudorana (Fei et al. 1990) and Pseudoamolops
(Jiang et al. 1997) were established as separate genera
but with various compositions of species (Dubois 1992;
Ye et al. 1993;Fei et al. 2012). The recognition of
Pseudoamolops has long been rejected by phylogenetic
studies (e.g., Tanaka-Ueno et al. 1998;Che et al. 2007a,
2007b) and our results corroborate these findings. Frost
et al. (2006) included both Pseudorana and Pseudoamolops
in Rana, and at the same time, recognized clade 3 of
this study as Lithobates based on limited species and
gene sampling. However, that arrangement makes the
remaining Rana paraphyletic. Later, largely based on
the phylogeny of Che et al. (2007b), Frost (2015) revived
the use of Pseudorana and recognized genera Pseudorana,
Lithobates, and Rana as the three genera of “true frogs”.
However, this action still does not result in entirely
monophyletic taxa (Supplementary Table S2, available
on Dryad).
Even if these groups were fixed by transferring
species among these taxa, the major morphological,
ecological, and behavioral differentiation within Rana
does not occur among these poorly differentiated taxa
(Pseudorana,Rana, and Lithobates sensu Frost 2015),
but within them. For example, biologists have a
much greater need for names of well-differentiated
groups including Pantherana(t he leopardfrogs), Zweifelia
(the torrent frogs), Lithobates (the Neotropical true
frogs), and Aquarana (the North American water
frogs), than for a taxon that combines these groups.
Hillis and Wilcox (2005) provided nested, clade-
based names at both levels, but Dubois (2006)
argued that such nested subgeneric names were
incompatible with the rules of the International Code
of Zoological Nomenclature (ICZN). Hillis (2007)
disagreed with Dubois’ interpretation of the ICZN rules,
but nonetheless suggested a compromise classification
that recognized the distinct, nonoverlapping clades
within Rana as subgenera.
Based on our phylogenetic analyses and the lack of
any diagnostic morphological characters for the putative
genera recognized by Feietal.(2012)orFrost et al.
(2006), and the clear monophyly of the larger group,
we retain all these species in the traditional genus Rana
(Supplementary Table S2, available on Dryad). This also
has the desirable outcome of retaining the traditional
binomial species names for these well-studied species,
and thus retaining the connections of these names to
their corresponding extensive literatures.
We support the recognition of the major diverse
lineages within Rana (the groups that show significant
molecular, morphological, ecological, and behavioral
divergences) as subgenera (Supplementary Table S2,
available on Dryad), which largely match the traditional
major distinct species groups long recognized by
numerous authors (Zweifel 1955;Case 1978;Farris et al.
1980,1983;Hillis et al. 1983,1984;Hillis 1988,2007;
Hillis and de Sá 1988;Dubois 1992;Hillis and Wilcox
2005). The subgenera of Rana are Pseudorana (clade 1),
Aquarana (clade 5), Lithobates (clade 7), Zweifelia (clade 8),
Pantherana (clade 9), Liuhurana (clade 11), Amerana (clade
13), and Rana (clade 14) (Fig. 1, Supplementary Table S2,
available on Dryad).
The inferred relationships of R. sylvatica differed in
the trees derived from nuclear versus mitochondrial
genes (Supplementary Fig. S2, available on Dryad), and
different studies and data sets have placed this species
at various places within clades 3 and 10 (Case 1978;
Farris et al. 1980,1983;Post and Uzzell 1981;Hillis and
Davis 1986;Hillis and Wilcox 2005). In our combined
analysis, R. sylvatica forms the sister group of all the
other remaining New World Rana (Fig. 1), which is
consistent with Case (1978) based on immunological
data. However, the support is weak (Fig. 1), because
mtDNA sequences conflict with the signal from the six
nuclear genes regarding the placement of R. sylvatica
(Supplementary Fig. S2a,b, available on Dryad). The
different resolutions for this species may indicate
hybridization or incomplete lineage sorting among the
ancestral lineages of clade 3.
Biogeographic Processes Inferred from DEC and DEC+J
Models
The DEC model most often interprets range evolution
as expansion into an unoccupied area (i.e., dispersal)
(Matzke 2014), followed by splitting of the ancestral
range by vicariance or sympatric-subset cladogenesis
into two daughter species with more restricted ranges. In
total, nine dispersals followed by 10 vicariance speciation
and 7 sympatric-subset events were inferred, for a total
of 16 range changes under DEC compared with 14 under
DEC+J. In some cases, even though DEC and DEC+J
reconstructed the same number of changes in ancestral
range, the interpretation (e.g., dispersal vs. vicariance)
may differ because of differences in the models.
Although the DEC+J model was significantly favored
over DEC under all scenarios, it is worth considering
its suitability for this analysis given that it was
developed to accommodate founder event speciation
(jparameter), which is common in oceanic island
systems (Matzke 2014). Under DEC+J, the ancestral
ranges of all nodes involve a single area (Fig. 3b).
Pie charts of the relative likelihoods of alternative
reconstructions (Supplementary Fig. S6c4, available on
Dryad) showed low likelihoods, except for ancestral
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2016 YUAN ET AL.—SPATIOTEMPORAL DIVERSIFICATION OF THE TRUE FROGS 13
node 17. This suggests that founder event speciation
was more common than vicariance or sympatric-subset
speciation.
In contrast, DEC range reconstructions include 18
nodes with >1 area (not including the “corner” nodes).
These reflect classic dispersal into new regions. The
reconstructions for several nodes are ambiguous, having
alternative possibilities with similar likelihoods.
The DEC+J model typically yields higher overall
likelihood scores and the range estimates are
unambiguous, as expected (Matzke 2013b,2014).
Thus, DEC+J presents a “cleaner” scenario. However,
it is worth considering whether the DEC+J model is
biologically meaningful to dispersal between contiguous
areas within a continent. An improvement in likelihood
score indicates a better fit of data to a particular model
only, which does not mean that the model offers a better
biological explanation for the data. Notwithstanding,
the DEC class of analyses also may be biased depending
on how the distributional data were scored. Matzke
(2014) pointed out that in his simulations DEC+J tended
to be favored in trees with terminal taxa that occupied
a single area, whereas DEC was favored when terminal
taxa occupied multiple areas. Because we assigned
all terminal taxa to a single area, the favoring of
DEC+J is not surprising. Clearly, the choice of terminal
assignment to one or more areas may affect model
selection.
Historical Biogeography
The origin and evolution of Rana has been of long-
standing interest to systematists and biogeographers
(e.g., Case 1978;Veith et al. 2003;Macey et al. 2006).
Our extensive sampling suggests that Rana originated
in the East Asia region, perhaps in southwestern China
(Figs. 1and 3) where three distinct, well-differentiated
groups occur: R. weiningensis (clade 1), R. shuchinae
(clade 11), and the sister species R. zhengi and R. johnsi
(clade 15) (Fig. 1). The former two species have limited
distributions at high elevations on the Yunnan-Guizhou
Plateau (1700–2950 m and 2760–3800 m, respectively).
Species in clade 15 (including R. sangzhiensis) occur in
the lowlands of Southwest China and adjacent northern
Vietnam.
The DEC and DEC+J models both suggest an “Out
of Asia” pattern involving two independent dispersals
from East Asia into the New World (Fig. 4a), and a third
one into Europe and Central Asia (Fig. 4b). This basic
biogeographic pattern was established 48–25 Ma.
Under DEC, a range expansion approximately 48–
43 Ma (Eocene) from East Asia into eastern North
America (across northern North America) was followed
by a vicariance event at 43 Ma. Subsequently, a second
range expansion from East Asia to western North
America (south along the Pacific coast of North America)
occurred approximately 43–34 Ma (Eocene) followed by
vicariance at 34 Ma, which restricted the R. boylii group
(Amerana) to western North America. Under DEC+J,
range evolution would be explained by jump dispersal,
which is “instantaneous” speciation. The first such event
(to eastern North America) occurred at 43 Ma, and the
second (to western North America) at 34 Ma.
Are these models supported by geological and
paleoecological information? During the Eocene (56–
34 Ma), a continuous belt of boreotropical forest
extended over the entire Northern Hemisphere from
Asia through North America across Beringia (Wolfe
1975,1987). This would have allowed considerable trans-
Beringian exchange of terrestrial biota adapted to warm-
temperate climates (Tiffney 1985a). Thus, during the
early branching of Rana, it seems there was no obvious
barrier to dispersal across Beringia. In the mid-Tertiary,
beginning about 35 Ma, mixed deciduous hardwood and
coniferous forests began to dominate in Beringia (Wolfe
1987), which perhaps facilitated the second dispersal of
Rana (clade 13). The end of the Eocene event marked a
dramatic climatic change (Sanmartín et al. 2001)froma
“greenhouse” to an “icehouse” world. From 14–10 Ma
to 3.5 Ma, cold taiga forest covered the Beringia bridge
(Wolfe 1987), rendering additional dispersals of Rana
unlikely. Asia and North America remained joined by
Beringia until the opening of the Bering Strait at 5.5 or
5.3 Ma (Marincovich and Gladenkov 1999;Gladenkov
et al. 2002).
The lack of a dispersal barrier suggests that range
expansion to North America can be explained by DEC
without including the J parameter. Under DEC+J, jump
dispersal is typically associated with an inhospitable
barrier such as ocean separating two continents or
islands (Matzke 2013b).
The third major event, the occupation of Europe and
Central Asia (Fig. 5b) from East Asia, took place from 29
to 25 Ma (under DEC; ambiguity in the reconstruction
might push the 29 Ma limit back to 34 Ma). This event
corresponds with the receding of the Turgai Sea at 30–29
Ma, which likely acted as a barrier for biotic exchanges
between Europe and Asia from the Jurassic to the Eocene
(Briggs 1995;Sanmartín et al. 2001). The ancestor of clade
17, which occupied Europe +Central Asia, experienced
vicariance at 20 Ma. This isolated R. asiatica in Central
Asia from its sister group, the European R. temporaria
group. DEC+J depicts the two range shifts as a jump
dispersal from East Asia to Europe at 25 Ma, followed
by jump dispersal from Europe back to Central Asia
at 20 Ma (Fig. 3). Progressive aridification of Central
Asia beginning about 22 Ma (Guo et al. 2008)may
have contributed to the present disjunction between the
European R.temporaria group, R. asiatica, and clade 18
(the remaining East Asian species). Savage (1973)first
proposed that this aridification event led to the range
disjunction of the amphibian faunas of Europe and East
Asia, and our analyses support this hypothesis for Rana.
Other amphibian groups that may fit this pattern include
Bombina (Zheng et al. 2009), Hyla (Li et al. 2015), and two
clades within Salamandridae (Zhang et al. 2008).
Finally, expansion of Rana into South America
occurred across the Isthmus of Panama. The three strictly
South American species of Rana form a single clade that
originated 16 Ma, and then diversified by 9 Ma (Fig. 3).
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14 SYSTEMATIC BIOLOGY
This finding supports dispersal across the Panamanian
Isthmus in the middle Miocene, which is consistent with
recent geological evidence that supports the formation
of the Panamanian Isthmus at this time (Montes et al.
2015).
Although vicariance appears to have played a
dominant role in speciation, strict geographic barriers do
not appear to have been present in all speciation events.
Heterogeneous landscapes, facilitated by differentiation
in ecological dimensions, may have also driven
speciation. The three New World areas (Fig. 2)are
effectively ecological islands for speciation in Rana;
almost no widespread species range across any two
regions. The Mexican Plateau, as the transition zone
between temperate and tropical regions, provides
unique and restricted habitats that may drive ecological
speciation. This might explain the numerous endemic
species associated with this biodiversity hotspot (e.g.,
Mittermeier et al. 2005). Under DEC+J, dispersals from
eastern NA to the Mexican Plateau and Neotropics
appear to have occurred since the late Eocene,
approximately 36 Ma. In the Neogene, we inferred
frequent dispersals and speciation events (Fig. 3b). This
jump-dispersal scenario for American Rana taxa should
be further tested with other taxa, such as pitvipers and
bunchgrass lizards (Castoe et al. 2009;Bryson et al.
2012).
Trans-Atlantic versus Trans-Beringian Dispersal
Our biogeographic analysis supports two
independent dispersals of Rana from East Asia to North
America via the Beringian land bridges (Fig. 2: H1b).
In contrast, the hypothesis of a trans-Atlantic bridge
connecting North America and Europe, as suggested
by Case (1978) and illustrated in Figure 2(H2), received
little statistical support (Supplementary Table S9,
available on Dryad). The trans-Atlantic dispersal route
does not seem likely based on geological considerations.
According to the ancestral range reconstructions for
model H2 (Supplementary Fig. S6e, available on Dryad),
one jump dispersal (DEC+J) or range expansion (DEC)
from East Asia to Europe would have had to occur
prior to the trans-Atlantic dispersal between Europe
and eastern North America. The dispersal from East
Asia to Europe would have had to occur by 48 Ma,
but the Turgai Sea separated Asia from Europe at that
time. Only after 30–29 Ma (Briggs 1995;Sanmartín
et al. 2001) were extensive biotic exchanges between
Europe and Asia possible. The lack of a sister-group
relationship between the Rana of Europe and North
America also argues strongly against the trans-Atlantic
scenario.
The disjunct distributions of many taxa across the
Holarctic region have attracted considerable attention
of botanists and zoologists (e.g., Wen 1999;Sanmartín
et al. 2001;Donoghue and Smith 2004). Most groups
of temperate forest plants originated and diversified
in eastern Asia, and then dispersed out of Asia across
Beringia, mostly during the last 30 Ma (Donoghue and
Smith 2004). In contrast, no general “Out of Asia” pattern
has been reported for terrestrial animals. However, most
terrestrial animal examples involve insects, birds, and
mammals (Sanmartín et al. 2001). Do amphibians and
reptiles show similar biogeographic patterns to plants,
or to insects, birds, and mammals?
A considerable body of literature exists on the
relationships of amphibians and reptiles that are
endemic to at least two of the following major
Holarctic regions: Europe, East Asia, western North
America, and eastern North America. There have been
28 hypothesized dispersal events of amphibians and
reptiles among these four regions (Table 1; Fig. 7).
Amphibians involved in these dispersal events include
groups of Cryptobranchidae, Salamandridae, Proteidae,
Plethodontidae, Hylidae, and Ranidae (this study),
and reptiles include Elapidae, Viperidae, Natricinae,
Lampropeltini, Scincidae, Alligatoridae, Trionychidae,
Emydidae, Geoemydidae, and Testudinidae (Table 1;
Spinks et al. 2004;Min et al. 2005;Smith et al. 2005;
Le et al. 2006,2014;Macey et al. 2006;Burbrink and
Lawson 2007;Roos et al. 2007;Vieites and Wake 2007;
Le and McCord 2008;Pramuk et al. 2008;Wüster
et al. 2008;Zhang et al. 2008,Zhang and Wake 2009;
Hua et al. 2009;Kelly et al. 2009;Spinks and Shaffer 2009;
Van Bocxlaer et al. 2010;Brandley et al. 2011;Garcia-Porta
et al. 2012;Guo et al. 2012;Pyron et al. 2013;Pyron 2014;
Li et al. 2015). Although Proteidae is also distributed
in North America and Europe, the divergence between
the relevant taxa is older than the breakup of Laurasia
(108 Ma; Pyron 2014), and so Proteidae is not included
in these comparisons. The remainder of the estimated
dispersal times for the amphibians and reptiles ranged
from the Early Eocene to the Middle Miocene (Table 1).
The biogeographic origins of these clades largely fit
the pattern seen among Holarctic plants (Donoghue
and Smith 2004), with strong and repeated support
for trans-Beringian over trans-Atlantic dispersal (Fig. 7).
In contrast, support exists for just three trans-Atlantic
dispersals (Salamandridae and Plethodontidae; Zhang
et al. 2008;Zheng et al. 2009; and Emydidae; Spinks and
Shaffer 2009).
Lineage Diversification in the Old versus New World
The lineages of Old and New World Rana have
different trajectories of diversification (Figs. 5and 6).
Of the two clades that resulted from trans-Beringian
dispersals, Amerana (clade 13, western North America),
does not seem to have greatly influenced New World
diversity. In contrast, clade 3, comprising the species
of eastern North America, the Mexican Plateau, and
the Neotropics rapidly radiated (Figs. 5b and 6b; 0.17
species/Ma), probably owing to the availability of
diverse new ecological opportunities. BAMM analyses
suggest a diversity-dependent process (Fig. 6b; Rabosky
et al. 2007), resulting in saturation of ecological niche
space and a decline in the speciation rate (Walker and
Valentine 1984;Valentine 1985;Rabosky and Lovette
2008).
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2016 YUAN ET AL.—SPATIOTEMPORAL DIVERSIFICATION OF THE TRUE FROGS 15
TABLE 1. Disjunct distributions of Eurasian and North American amphibians and reptiles
Disjunction pattern number, direction of dispersal, and estimated divergence time (Ma, in parentheses)
Taxa A–E A–C A–D C–D C–E D–E A–NA C–NA Reference(s)
Cryptobranchidae 1, A to E
(43) Zhang and Wake (2009);
Pyron (2014)
Ranidae (Rana) 2,AtoE
(43)
17,AtoC
(25)
13,AtoD
(34)
This study
Salamandridae (Modern Eurasia newts) 18, C to A
(27) Zhang et al. (2008); Pyron (2014)
Salamandridae (Primitive Eurasia newts) 19, C to A
(27) Zhang et al. (2008); Pyron (2014)
Salamandridae (Tar icha and Notophthalmus) 27, ? (35) Zhang et al. (2008); Pyron (2014)
Salamandridae (New World salamandrids) 24,Cto
NA (43.5) Zhang et al. (2008); Pyron (2014)
Plethodontidae (Hydromantes) 25,DtoC
(33) Pyron (2014); Vieites and Wake
(2007)
Plethodontidae (Karsenia)12,NAto
A(51) Pyron (2014); Minetal.(2005);
Vieites and Wake (2007)
Plethodontidae (Aneides) 26, ? (40) Pyron (2014);
Vieites and Wake (2007)
Plethodontidae (Plethodon) 28, ? (50) Pyron (2014);
Vieites and Wake (2007)
Hylidae (Hyla) 7,EtoA
(16)
20,AtoC
(27) Li et al. (2015); Pyron (2014)
Scincidae (Plestiodon) 3,AtoE
(24) Brandley et al. (2011)
Scincidae (Scincella) 4,AtoE
(?) Pyron et al. (2013)
Viperidae 11,Ato
NA (24) Wüster et al. (2008)
Elapidae 6, A to E
(25) Kelly et al. (2009)
Colubrinae 22, A to C
(26–25)
10,Ato
NA
(26–25)
Burbrink and Lawson (2007)
Natricinae 21, A to C
(27)
9, A to
NA (27) Guo et al. (2012)
Alligatoridae (Alligator) 8,EtoA
(53–47) Wu et al. (2003);
Roos et al. (2007)
Trionychidae (Rafetus and Apalone) 5,AtoE
(43)
15,AtoC
(21) Le et al. (2014)
Emydidae (Emys)23,EtoC
(17) Spinks and Shaffer (2009)
Geoemydidae (Mauremys) 14,AtoC
(30–18) Spinks et al. (2004);
Le and McCord (2008)
Testudinidae (Testudo) 16,AtoC
(?) Le et al. (2006)
Notes: Twenty-two clades with disjunct distributions are assigned to one of the eight two-area categories. Where the divisions of eastern and western North America are ambiguous, we
combined them as North America (NA). “?” indicates that the time or place of origin is ambiguous. Numbers in parentheses represent the time of divergence (Ma). Numbered events refer
to the disjunctions in Figure 7. Abbreviations: A, East Asia (mainland +islands); C, Europe; D, western North America; E, eastern North America. References refer to the phylogenetic
studies from which data were obtained.
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16 SYSTEMATIC BIOLOGY
FIGURE 7. Inferred ancestral areas and directions of movement
among the four major Holarctic areas for amphibians and reptiles.
Letter designations for the major geographic areas are the same as
in Figure 2 (A: East Asia; C: Europe; D: western North America; E:
eastern North America). Arrows point from the inferred ancestral area
to the newly colonized area. The line without arrowheads between
eastern and western North America indicates three cases in which
the direction of dispersal is ambiguous. The dotted line indicates two
cases of possible (but somewhat ambiguous) trans-Beringian dispersal.
Table 1presents details for the 28 numbered inferred dispersal events.
A distinct increased rate of lineage accumulation
occurred at around 14 Ma (clade 3; Fig. 5b),
corresponding to dispersals among the Mexican Plateau,
eastern North America, and Neotropics (Figs. 3and 4c2),
as well as in situ diversification of Mexican taxa. The rate
increase generally corresponds to the Middle Miocene
formation of the Trans-Mexican Volcanic Belt (Ferrari
et al. 1999;Gómez-Tuena et al. 2007), which has likely
contributed to the diversification of numerous taxa (e.g.,
Bryson et al. 2011, 2012).
In contrast, lineage accumulation of the Old World
Rana (clades 1, 11, and 14) proceeded very slowly, then
underwent a distinct net diversification rate-shift at
around 29–18 Ma (about 1.4 species/Ma; Fig. 6c and
Supplementary Figs. S7c and S8c, available on Dryad).
Closing of the Turgai Strait after 29 Ma (Briggs 1995)
permitted the dispersal of Rana into western Eurasia.
Clade 17 further diversified in the Mediterranean region
around 18 Ma, possibly in association with the onset of
Asian monsoons at the Oligocene/Miocene boundary
(Sun and Wang 2005).
Lineage accumulation of the East Asian clades 19–23
(Fig. 1) occurred over a short period of time (23–20 Ma)
(Fig. 3) in association with island formation (Fig. 5b,d).
From 23 to 15 Ma, rifting and tectonic deformation
associated with block rotations and volcanism resulted
in the substantial fragmentation of the East Asian margin
and formation of islands as a continental sliver (Taira
2001;Itoh et al. 2006). Following the initial opening of
the Sea of Japan in the Late Oligocene (Otofuji et al.
1985;Ingle 1992;Jolivet et al. 1994;Isozaki 1997;Taira
2001), the archipelagos of Japan rotated into their present
configuration about 15 Ma (Taira 2001). The five clades
(19–23; Fig. 1) along the Asian margin formed well
before the origin of the present island system. Thus, our
results support a role for Miocene tectonic events in the
diversification of East Asian Rana.
Our analysis provides a framework for understanding
and interpreting the biology of the well-studied frogs of
the genus Rana throughout Eurasia and the Americas.
Numerous studies of the biology of these frogs can be
facilitated by a better understanding of their phylogeny
and biogeography.
AUTHOR CONTRIBUTIONS
Conceived and designed the study: J.C., D.M.H.,
D.C.C., and Y.P.Z. Collected samples and sequence
data: Z.Y.Y., W.W.Z., N.A.P., H.M.C., N.H.J.L., W.H.C.,
K.I., M.S.M., S.L.K., D.M.H., and J.C. Conducted and
evaluated data analyses: Z.Y.Y., W.W.Z., X.C., N.J.M.,
D.C.C., D.M.H., and J.C. Discussed and drafted the
manuscript: Z.Y.Y., W.W.Z., X.C., N.A.P., N.J.M., Y.P.Z.,
D.C.C., D.M.H., and J.C. All authors have read and
approved the final version of the manuscript.
SUPPLEMENTARY MATERIAL
Data available from the Dryad Digital Repository:
http://dx.doi.org/10.5061/dryad.ck1m7.
FUNDING
This work was supported by the Strategic Priority
Research Program (B) Grant XDB13020200 of the
Chinese Academy of Sciences (CAS) to J.C.; National
Natural Science Foundation of China [31321002 to
Y.P.Z., 31090250 to J.C., and 31401966 to W.W.Z.], the
program of State Key Laboratory of Genetic Resources
and Evolution, Kunming Institute of Zoology, CAS to
N.A.P. [GREKF14-1], and a National Science Foundation
Assembling the Tree of Life grant to DCC and DMH
[AmphibiaTree; DEB 0334952]. J.C. was supported by
the Youth Innovation PromotionAssociation CAS, and to
study abroad in the University of Texas at Austin and the
University of Califor nia at Berkeley by China scholarship
council ([2014]3012) and Chinese Academy of Sciences
([2011]31). D.M.H. was supported for collaborative visits
to the Kunming Institute of Zoology by an Einstein
Professorship from the Chinese Academy of Sciences.
N.A.P. was supported by the Russian Foundation of Basic
Research [RFBR Taiwan 14-04-92000; RFBR 15-29-02771]
and Russian Science Foundation [RSF 14-50-00029].
N.J.M. was supported by a NIMBioS fellowship under
NSF Award [EFJ0832858], and ARC DECRA fellowship
[DE150101773].
ACKNOWLEDGMENTS
We thank Jie-Qiong Jin for assisting with sample
collection and laboratory work, Luke Mahler for the
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2016 YUAN ET AL.—SPATIOTEMPORAL DIVERSIFICATION OF THE TRUE FROGS 17
use of his scripts, Jim McGuire for discussion regarding
regional lineage accumulation, Mariana Vasconcellos
for her help on biogeographic analyses, Shao-Yuan
Wu and Bao-Lin Zhang for their help on species-
tree analyses, Ted Papenfuss for discussions regarding
transcontinental dispersals of amphibians and reptiles,
Robert W. Murphy for suggestions on the manuscript,
and Richard Glor and three anonymous reviewers for
valuable suggestions during manuscript review. Amy
Lathrop and Jingting Liu assisted in figure production.
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