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RES E A R C H A R T I C L E Open Access
The Eurasian invasion: phylogenomic data
reveal multiple Southeast Asian origins for
Indian Dragon Lizards
Jesse L. Grismer
1*
, James A. Schulte II
2
, Alana Alexander
1
, Philipp Wagner
3
, Scott L. Travers
1
, Matt D. Buehler
1
,
Luke J. Welton
1
and Rafe M. Brown
1
Abstract
Background: The Indian Tectonic Plate split from Gondwanaland approximately 120 MYA and set the Indian
subcontinent on a ~ 100 million year collisio n course with Eurasia. Many phylogenetic studies have demonstrated
the Indian subcontinent brought with it an array of endemic faunas that evolved in situ during its journey,
suggesting this isolated subcontinent served as a source of biodiversity subsequent to its collision with Eurasia.
However, recent molecular studies suggest that Eurasia may have served as the faunal source for some of India’s
biodiversity, colonizing the subcontinent through land bridges between India and Eurasia during the early to
middle Eocene (~35–40 MYA). In this study we investigate whether the Draconinae subfamily of the lizar d family
Agamidae is of Eurasian or Indian origin, using a multi locus Sanger dataset and a novel dataset of 4536
ultraconserved nuclear element loci.
Results: Results from our phylogenetic and biogeographic analyses revealed support for two independent
colonizations of India from Eurasian ancestors during the early to late Eocene prior to the subcontinent’s hard
collision with Eurasia.
Conclusion: These results are consistent with other faunal groups and new geologic models that suggest
ephemeral Eocene land bridges may have allowed for dispersal and exchange of floras and faunas between India
and Eurasia during the Eocene.
Keywords: Agamidae, Draconinae, Eocene, Eurasia, India, Faunal exchanges, Landbridges
Background
The collision of the Indian subcontinent (ISC) into
Eurasia caused the formation of some of the world’s
most iconic deserts and mountain ranges, dramatically
changing Asian climates, while simultaneously sculpting
its biodiversity. Much interest has centered on investi-
gating the evolutionary and geological processes that
have influenced the origins and diversification of the
ISC’s unique biotas ([1]; and references therein). Phylo-
genetic studies of birds, dipterocarp trees, terrestrial
gastropods, crabs, freshwater fish, and certain groups of
amphibians, suggests these lineages originated on the
ISC and were a source of biodiversity for regions of Asia
and areas as far west as Africa after the Indian Plate split
off from Gondwanaland [2–7]. However, a suite of
phylogenetic studies across a variety of other taxa sug-
gest an alternative biogeographic hypothesis postulating
Eurasia as the ancestral source of diversity for the ISC.
In these groups Asian lineages dispersed to, and success-
fully colonized, the subcontinent before its hard collision
with Eurasia 25–30 MYA [8–12].
The previous lack of geologic models describing the
fine scale events of the final 50 million years of the ISC’s
collision, left researchers with no mechanistic explan-
ation for the striking differences between these two “ISC
faunal origin ” hypotheses. Fortunately, newer models are
available that take into account continental connections
between the approaching ISC and areas of mainland
* Correspondence: Grismer@ku.edu
1
Department of Ecology and Evolutionary Biology and Biodiversity Institute,
University of Kansas, Dyche Hall, 1345 Jayhawk Blvd., Lawrence, KS
66045-7561, USA
Full list of author information is available at the end of the article
© 2016 Grismer et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Grismer et al. BMC Evolutionary Biology (2016) 16:43
DOI 10.1186/s12862-016-0611-6
Asia prior to the ISC’s collision with Eurasia [13–15].
Acton [13] and Ali and Aitchison [15] hypothesized that
between 34–55 MYA (middle Eocene-late Eocene), India
was connected to Eurasia via land-bridges with Sumatra,
and then along what is now the Thai-Malay Peninsula
and Burma (which would have been one land mass
during this time). Two recent studies have recovered
phylogenetic support for these Eocene land bridges and
hypothesized that these pre-collision continental con-
nections would have allowed for faunal exchanges be-
tween the ISC and Eurasia as the ISC continued
northward [7, 16]. We present data from a diverse radi-
ation of Indian and Southeast Asian lizards that provide
an additional model system, with largershould be large
not "larger" amounts of generic diversity of Indian line-
ages and Asian lineages, to test for phylogenetic supp ort
for these Eoc ene land bridges, which we refer to as the
Eocene Exchange Hypothesis (EEH).
The Draconinae is a subfamily within the lizard family
Agamidae that contains 27 genera and 199 species [17]
comprising approximately 50 % of total Agamid diver-
sity. Members of the Draconinae collectively range
throughout mainland Asia (Indochina), Sundaland,
India, and Sri Lanka (Fig. 1). Draconinae lizards are di-
urnal omnivores exhibiting a range of arboreal and
terrestrial life styles and are some of the dominant mem-
bers of diurnal lizard communities throughout South
and Southeast Asia [18, 19]. To date, only two studies
have investigated the phylogenetic relationships within
the Draconinae. However, both were part of broader sys-
tematic studies on the entire Agamidae family [20, 21].
Moody’s [20] dissertation included 60 extant taxa, was
based on 122 morphological characters, and included
data from 18 fossils. This work was the first study to
hypothesize a Eurasian origin for the Indian draconine
lineages. Macey et al. [21] was the first study to provide
a molecular phylogeny for the Agamidae (including Dra-
coninae), and included an analysis of 72 taxa and one
mitochondrial gene. This analysis demonstrated that
mainland Asian agamids were paraphyletic with respect
to Indian and Sri Lankan lineages. However, multiple
deeper nodes within the Draconinae were characterized
by poor support, resulting in ambiguous relationships
[21]. The authors then used a series of parsimony
methods to suggest that these problema tic areas of the
draconine phylogeny, along with a lack of biogeographic
signal, were likely due to an Indian-Asian faunal ex-
change just after the hard collision, 20–25 MYA. Subse-
quent reviews of Indian-Eurasian collision regarded the
biogeographical interpretations of Macey et al. [21] with
skepticism due to the poorly supported relationships
within the Draconinae ([22]; and references therein).
Since Moody [20] and Macey et al. [21], new Draconinae
genera have been discovered, and previously unsampled
rare genera have been collected, providing additional gen-
etic material for reanalysis of draconine relationships. The
lower per-base cost of next-generation sequencing has also
led to the development of genomic methods extend-
ing the number of genetic markers that have limited
the phylogenetic resolution in previous studies. Here,
we generate a genomic data set of 4536 nuclear loci
derived from ultraconser ved elements (UCEs), along
with traditional Sanger sequencing data , to resolve
the problematic relationships within the Draconinae
reported by Macey e t al. [21]. With the addition of
new ta xa, and genomic sequence -capture data, a na-
lyzed in combination with newly developed geological
models, we are poised to reinterpret the biogeo-
graphic origins of Indian and Southeast A sian draco-
nine lineages. Spe cifically, we tested (1) Moody’s [20]
pre-collision hypothesis ve rsus Macey et al. [21] post-
collision hypothesis for the origins of Indian lineages;
and (2) suggest that a conclusion in favor of M oody’s
[20] pre-collision hypothesis would show phylogenetic
support for the Eocene land bridge connections pro-
posed by Acton [13] and Atchison et al. [14]. We
term this the Eocene Exchange Hypothesis (EEH).
Methods
DNA extraction, Sanger mitochondrial and nuclear DNA
sequence data collection
Genomic DNA was extracted from muscle or liver tissue
samples on loan form La Sierra University, Villanova
University, the California Academy of Sciences, the
Zoologisches Forschungsmuseum Alexander Koenig,
and the Chicago Field Museum. Extractions were pre-
formed using a DNeasy tissue kit (Qiagen, Inc.) and se-
quenced for the mitochondrial and nuclear genes, ND2
(primers from [21]) and RAG-1 (primers from [23]), re-
spectively, using standard PCR and Sanger sequencing pro-
tocols. We edited the sequences and aligned them within
Geneious Pro 5.0.4 (http://www.geneious.com, [24]) and
these new sequence data were combined with existing data
from [21] and [23] (Additional file 1: Table S1). In total, the
dataset included 17 of the 26 draconine genera, including
all but two of the Indian genera (Psammophilus and Cory-
phophylax). H yrdosaurus and Physignathus were not in-
cluded as their phylogenetic affinities are with other agamid
lineages outside of the Draconinae [21]. At least three spe-
cies (or individuals if the genus was monotypic) per genus
were sampled, for a total of 44 individuals. ND2 and RAG-
1 were selected as they are the most frequently sequenced
markers across acrodont lizards and therefore provide max-
imum taxonomic coverage. We used these markers to pre-
liminarily place new genera in a phylogenetic context, and
as a guide tree in our selection of genera for UCE develop-
ment to resolve problematic relationships. No experimental
research was carried out on these animals in this study.
Grismer et al. BMC Evolutionary Biology (2016) 16:43 Page 2 of 11
Ultraconserved elements (UCE) data collection
To resolve the problematic areas in the phylogeny from
the Sanger data (pink nodes : Fig. 2a), we selected 24 in-
dividuals representing 12 genera (underlined taxon
names in Fig. 2) from across four species groups (brown
nodes: Fig. 2a) for ultaconserved element (UCE) enrich-
ment. Sequence-capture data collection followed a modi-
fication of the approach outlined by Faircloth et al. [25].
Briefly, we fragmented genomic DNA with a Covaris S220
ultrasonicator (Covaris , Inc.), and prepared Illumina
libraries using KAPA library preparation kit s (Kapa
Biosystems) and custom sequence tags unique to each
sample [26]. Libraries were pooled into groups of 8
taxa and enriched for 5060 UCE loci (5472 probes).
We amplified enriched pools with a limited-cycle PCR
(18 cycles) and sequenced final libraries on a partial
Ilumina HiSeq 2000 lane. Reads were quality filtered using
the Illumiprocessor [27] wrapper for Trimmomatric [28],
and assembled into contigs using Trinity [29]. Where
alternate alleles differing by less than 5 % sequence diver-
gence (or two nucleotide positions, whatever was greater)
were present in a sample for any given UCE locus, Trinity
retained the allele supported by the largest number of
reads. We used PHYLUCE v. 1.4 (Faircloth et al. [25, 30])
to match contigs to UCE loci and generated two align-
ments in MAFFT [31]: one containing no missing loci
across all individuals (complete) and another containing
data for at least 75 % of taxa per locus (75 % complete),
which returned alignments of 1114 loci and 4536 loci,
respectively.
Phylogenetic and biogeographic analyses
Sanger data
We first used Bayesian analyses with MrBayes 3.2.2 [32] of
the ND2 and RAG-1 datasets independently in the con-
text of the entire Agamidae to ensure that Draconinae was
monophyletic. Once monophyly and lack of conflict be-
tween loci was established, we concatenated the two gene
Fig. 1 Map showing the distribution of Draconin ae and the four biogeographic area (differently-colored borders) used in ancestral
range reconstructions
Grismer et al. BMC Evolutionary Biology (2016) 16:43 Page 3 of 11
partitions for subsequent analyses. We used uniform
priors in MrBayes 3.2.2 and partitioned the dataset by
locus and codon within each locus for just the members
Draconinae sub-family. We then assigned the GTR+ Γ
substitution model for each partition and used three
chains (two hot and one cold), and carried out 100 million
generations, sampled every 10,000 generations. Due to the
risk of substitution saturation, we performed analyses in-
cluding and excluding the third codon position for the
ND2 alignment. Convergence between chains, likelihood
scores, and estimate sample size (ESS) values were evalu-
ated using Tracer 1.6 [33] In order to obtain a reliable root
age for divergence-time estimates within Draconinae, we
expanded our ND2 and RAG-1 datasets to include data
from all acrodont lineages. We analyzed this expanded
dataset using eight acrodont fossils (Additional file 2:
Table S2) within a Bayesian framework in BEAST 2.3 [34]
using the fossilized-birth-death model [35, 36]. The
fossilized-birth-death process provides a model for the
distribution of speciation times, tree topology, and distri-
bution of lineages sampled before the present, and treats
the fossil observations as part of the prior on node time
estimates. We used the root age for the Draconinae result-
ing from this analysis (85 MYA) as a minimum-age cali-
bration for the root of the Draconinae for subsequent
time of divergence estimates within the Draconinae clade.
Sequence-capture data
We performed likelihood analyses in RAxML v.8.1.20
[37] on concatenated datasets for the incomplete (4536
loci) and complete (1114 loci) matrices, using the GTR+ Γ
substitution model, and ran 100 fast bootstrap replicates.
Fig. 2 a Bayesian analysis (in MrBayes) of ND2 and RAG-1 data, with black dots denoting nodes with posterior probabilities above 0.95. Brown
nodes indicate four well-supported species groups (1–4; see text for details) and pink nodes identify poorly supported relationships among these
species groups. Underlined taxon names are genera selected for UCE enrichment. b Multi-species coalescent (“species tree”) from the species tree
estimation using average coalescence times STEAC analysis, using the complete matrix of 1114 UCE loci. Black dots denote nodes with 100
bootstrap support. Brown nodes indicate the four species groups (Group 2 = brown circle; see text for discussion). Blue nodes identify problematic
nodes recovered in Likelihood analysis of the Sanger dataset, resolved with sequence-capture data
Grismer et al. BMC Evolutionary Biology (2016) 16:43 Page 4 of 11
In addition to the concatenated analysis, maximum likeli-
hood gene trees were constructed for each of the UCE loci
included in the complete matrix using Phyluce with
RAxML v.8.1.20 [37], under default settings. Phyluce and
RAxML were also used to generate gene trees for 500
multi-locus bootstraps [38]. Custom R-scripts (R v3.2.0; R
Core Team 2015) and the R library Phybase [39] were
then used to infer the STEAC [40] summary species tree
for the original and bootstrapped data.
Grafted phylogeny and divergence dating
Using 85 MYA as a minimum age limit for the ancestor
of the Draconinae, divergence dates for subclades were
estimated in BEAST 2.3 using the ND2 and RAG-1
datasets with linked clock and tree models. We applied
Birth-Death tree priors and constrained the relationships
to match the results from the analyses of the UCE loci
(blue nodes: Fig. 2b) and let the relationships within
each species group be estimated by the BEAST analyses.
We used a relaxed uncorrelated lognormal clock model
and an exponential prior for the mean rate of each parti-
tion. Default values were used for all other priors, and
the analysis was run for 150 million generations sam-
pling every 12,000 generations, with chain stationarity,
and ESS values were evaluated in Tracer 1.6. The first
25 % of trees were discarded as burn-in and the max-
imum clade credibility tree with median node heights
was summarized using TreeAnnotator 2.3 [34]. We con-
verted our alignments to fasta format using seqmagick
(http://seqmagick.readthedocs.org/en/latest/) . Then, with
the estimate for divergence betwee n Mantheyus and
other draconine species of 85MYA , we estimated the
TMRCA of sub clades based on pairwise Hamming
distances [41] between UCE loci (with a sequence sat-
uration correc tion of 0.95) calculated through fas -
tphylo [42], assuming a naïve strict clock. We carried
out the calculations using a custom R-script [43]. Any
loci where subgroup divergence times exceeded those
of the calibration time were discarde d due to the like-
lihood of incomplete lineage sorting and/or excessive
rate variation. Using the same methods, we then esti-
mated the time to most recent common ancestor
(TMRCA) of the Draco + Ptyctolaemus and spe cies
group 1–4 clades using the estimated age of the Non-
Mantheyus clade. The estimate of the TMRCA of
species group 1–4 was then used to age the split be-
tween Acanthosaura and Pseudocal ates (species group
1), and the ancestor of spe cies groups 2/3/4. The spe-
cies group 2/3/4 TMRCA estimate was then used to
age the split between Salea and Calotes (species
group 2 and 3), and the ancestor of spe cies group 4.
Finally, the estimate for the TMRCA of species group
4 was used to obtain an estimate of the TMRCA of
Certaophora/Ly riocephalus/Cophotis.
Ancestral area reconstructions were performed using
likelihood and Bayesian methods in LAGRANGE within
the program RA SP 3.0 [44], and in RevBayes 10.10 [45]
respectively. Taxa were assigned to their biogeographic
zone (Fig. 1) based on their modern day distributions
and RevBaye s re constructions were visualized using
theonlineresourcePhylowood[46].Traditionally,the
Philippines is not classified as part of Sundaland how-
ever, w e included taxa from this archipelago in the
Sundalan d biogeog raph ic are a because the entire Philippine
agamid fauna is Sundaic in origin.
Results
Sanger mitochondrial and nuclear data phylogenetic
analyses
The B ayesian analyses of the combined Sanger dataset
recovered new relationships that have not been reported
in any previous study (Fig. 2a). Mantheyus was recov-
ered as sister to the remaining Draconinae. The next
lineage to diverge was a well-supported clade containing
Draco, and Ptyctolaemus (Fig. 2a). Lastly, there were
four well-supported species groups (brown nodes :
Fig. 2a). The relationships within each of these species
groups were well supported. However, the relationships
between the species groups were poorly resolved and
characterized by short branches (pink nodes: Fig. 2a). As
the resolution of the relationships between the species
groups is vital for testing hypotheses of Indian or
Eurasian origins, repr esentatives of the taxa from each
of these species groups were included in a phylogenetic
reconstruction from analyses of UCE data.
Sequence-capture data phylogenetic analyses
There were 4536 loci with data for at least 75 % of
the n = 23 individuals included in this study. These loci
had an average length of 644.7 bp (S.D. = 249.7 bp), of
which an average of 10.5 % of sites (S.D. = 20.0 %) were
parsimony informative. The average amount of missing
data per locus was 23.6 % (S.D. = 19.4 %), including both
missing individuals (up to 25 % of individuals at each
locus) and shorter sequence lengths for individuals that
were present (Additional file 3: Table. S3). All analyses of
the sequence-capture data were successful in resolving the
problematic relationships recovered from the Sanger data
(blue nodes; Fig. 2b) and recovered each of the four spe-
cies groups within the Draconinae, with high support
(brown nodes; Fig. 2b), consistent with the results from
the Sanger datasets.
Biogeographic analyses, divergence dating, and
ancestral areas
Both of the methods employed to estimate ancestral
ranges (LAGRANGE and RevBayes analyses) returned
comparable estimates of ancestral areas, however, the
Grismer et al. BMC Evolutionary Biology (2016) 16:43 Page 5 of 11
RevBayes reconstructions were more conservative. Given
the short branch lengths leading to some of the deeper
nodes in our phylogeny, the RevBayes reconstructions
are a better reflection of geology at the times of these
nodes. Therefore only the RevBayes reconstructions are
discussed. The grafted BEAST time-tree (Fig. 3a) was
concordant with the phylogenies derived from the Se-
quence capture data and Sanger data (Fig. 2). The
BEAST time-tree (Fig. 3) indicated the most recent com-
mon ancestor (MRCA) for the Draconinae originated
approximately 92 MYA in mainland Asia ~30 million
years after the ISC broke off Gondwanaland. The MRCA
for Draco and it s relatives most likely originated in
mainland Asia 53 MYA and diverged from the other
mainland Asian and Sundaic lineages around 69 MYA
from a mainland Asian ancestor. The three remaining
species groups appear to have diversified from one an-
other rapidly between 51–59 MYA, most likely from a
mainland Asia ancestor that existed approximately 59
MYA. The Indian endemic Salea (Species group 2) rep-
resents the first invasion of India (D#1: Fig. 3a), having
diverged from a mainland Asian ancestor it shared with
Calotes (Species group 3) approximately 56 MYA
(Fig. 3a). The MRCA for Acanthosaura and Psuedoca-
lotes (species group 1) was estimated at 56 MYA with a
high probability that this ancestor originated in either
mainland Asia or Sundaland (where both genera pres-
ently occur). Within this species group, we recovered
support for a second invasion of India and Sri Lanka,
with the ancestor of Sitana and Otocryptis originating
from a predominantly Sundaic ancestor between 51–27
MYA (D#2: Fig. 3a). Lastly, the MRCA for the Sri Lan-
kan and Sundaland radiations (species group 4) origi-
nated around 51 MYA in Sundaland or Sri Lanka
(Fig. 3a). Within species Group 4, Aphaniotis, Broncho-
cela , and Gonocephalus appear to have diverged from
one another 42 MYA and form the sister line age to the
Sri Lankan genera Lyriocephalus, Cophotis, and Cerato-
phora (Fig. 3a). The Sri Lankan lineages diverged from
one another 28 MYA. We obtained these timing esti-
mates for key divergences and dispersal events using
Sanger data (as they were available for a broader taxo-
nomic sample, including key fossils in comparison with
the UCE data) in BEAST, with the topology constrained
by the results from UCE data. We then crosschecked
these estimates using the minimum divergence time for
Draconinae of 85 MYA, and sequence divergence among
UCE loci between clades of interest. This method is some-
what cruder than the BEAST estimates because it cannot
account for among lineage rate variation. However, the es-
timates obtained using this approach were broadly com-
parable with results or our Bayesian analysis performed in
BEAST (Fig. 4), offering support for our timing of key dra-
conine dispersal events in Southeast Asia.
Discussion
In this study, we utilized unprecedented sampling of the
Draconinae, both in taxonomic diversity and genetic
markers, to give fresh biogeographic insight into the ori-
gins of the Indian and Southeast A sian Draconinae line-
ages. In particular, the thousands of loci generated using
sequence-capture and next-generation sequencing were
successful in reso lving previously problematic relation-
ships within the Draconinae (brown nodes: Fig. 2). Using
the fully resolved UCE phylogeny to constrain the top-
ology of our Sanger dataset, we generated a grafted
Bayesian time tree (Fig. 3a), which supported the hy-
pothesis that there were at least two independent
colonization events of India by Southeast Asian lineages
during the Eocene. These results favor Moody’s [20] pre-
collision hypothesis with the estimated times of the
Eurasian invasions in accordance with the Eocene land
bridges proposed by Acton, [13] and Ali and Aitchison
[14]. These hypothesized land bridges would have con-
nected areas of Eurasia (now Sundaland and the Thai-
Malay peninsula) and the ISC before its collision, and
are the likely conduits for terrestrial faunal exchange
and range expansion in the lineages leading to to-
day’s Indian subcontinent endemics Salea, Sitana,
and Otocr yptis.
The Eocene exchange hypothesis
The first Draconinae invasion into India consisted of a
lineage represented today by the endemic genus Salea,
which descended from a mainland Asian ancestor that
also gave rise to the Indochinese genus Calotes. This
colonization event most likely resulted from an early
Eocene land-bridge connection or an over-water dis-
persal event just prior to the ISC’s conne ction with
Sundaland (Eura sia) 50–55 MYA (Fig. 3b). Given t he
sedentary and arboreal natural histories of extant dra-
conine species , we feel the former hypothesis is more
likely than the latter, although we acknowledge the
possibility of both. We expe ct a broader sampling
within this clade of Southea st Asian, and especially
Indian, species will provide a better e stimate o f the
ancestral area at this node (Salea + Calotes:Fig.3a).
The second dispersal event into India occurred with
the divergence of the Indian and Sri Lankan endemics
Sitana and Otocryptis from an ancestor most likely
found in Sundaland durin g the middle Eocene. This
colonization of the Indian subcontinent most likely
was facilitated via a la nd bridge that connected the
ISC with Sumatra and the Thai-Malay peninsula at 48
MY A. Additionally, the lineage sister to Sitana and Oto-
cryptis, Japalura, and Pseudocalotes, is Phoxophrys (Fig. 3a).
This genus is endemic to the lowland forests of Borneo and
Sumatra—further supporting an India-Sundaland (Eurasia)
connection via Sumatra and the southern portion of the
Grismer et al. BMC Evolutionary Biology (2016) 16:43 Page 6 of 11
a
bc
Fig. 3 (See legend on next page.)
Grismer et al. BMC Evolutionary Biology (2016) 16:43 Page 7 of 11
Thai-Malay Peninsula during the middle Eocene. These in-
dependent colonization events not only support Moody’s
[20] pre-collision biogeographic hypothesis, but also give
additional phylogenetic support for Eocene land bridges
postulated by Acton, [13] andAliandAitchison[14].Our
results contribute to a growing body of literature demon-
strating the possibility of floral and faunal exchange be-
tween India and Eurasia during the Eocene, before the
ISC’s hard collision 20–25 MY A (e.g. freshwater crabs: [7];
rhacophorid tree frogs: Li et al. [16]). Given the ecology of
these organisms, and of the draconine species sampled
here, we feel that it is less likely Eocene faunal exchanges
occurred as the result of over water dispersal events. It is
unclear whether the Eocene land bridges were two separate
spatial/temporal features, versus possibly the same entity,
just changing position as the ISC progressed northward. In
either case, their existence may have provided continental
connections between Southeast Asia and India during the
Fig. 4 Box-and-whisker plots, showing results of our analysis using our UCE_divergence_timing R script (minimum, 25 % quartile, 75 % quartile,
maximum) with a minimum estimate for the age of Draconinae of 85 MYA used to calibrate the ages of the Non-Mantheyus clade. For
subsequent subgroups, the estimated age of the clades were contained within this calibration point. For each group’s divergence timing
estimate, only loci that appeared “clock-like” (ingroup age estimate did not exceed the calibration age) were used. Percentages of loci that were
“clock-like” versus non-“clock-like” (likely affected by rate variation or incomplete lineage sorting), and loci with missing data for outgroups (sister
species of the groups of interest) are shown in pies above box-and-whisker plots (see key). Clades with red arrows show slow-downs relative to
their outgroups i.e. average cumulative branch lengths leading to ingroup taxa from the ingroup/outgroup node are shorter than those leading
to the outgroups (this appears to be correlated with underestimates of divergence times using the naïve strict clock method), clades with green
arrows show rate speed-ups relative to their outgroups i.e. average cumulative branch lengths leading to ingroup taxa are longer than those
leading to the outgroups. Bayesian estimates of divergences times performed in BEAST are shown as small blue diamonds, for comparison
(See figure on previous page.)
Fig. 3 a Time-calibrated Bayesian analysis of ND2 and RAG-1 data, with black dots denoting nodes with posterior probabilities above 0.95,
followed by the estimated divergence time for each node in MYA. Pink circles ident ify nodes where topology w as constrained based on
Likelihood and s pecies tree an alyses of UCE data (Fig. 2B). Brown circ les indicate the four species groups. Biogeographic distributions of
con temporary samples follow area coding depic ted in Fig. 1, with probabilit y of areas a t ances tral nodes from our Bay esian analysis in
RevBayes. Infe rred dispersal events into Indi a are labeled D#1 and D#2, resulting in Indian or Indian/Sri Lankan Salea, Sitana, and Otocryptis.
b Hypothesized position of the ISC and an early Eocene land bridge allowing for the first inferred dispersal event (D#1 in a) from Eurasia into India,
50–55 MYA. c. Hypothesized position of the ISC and a middle-late Eocene land bridge allowing for the second first inferred dispersal event (D#2 in a)
from Eurasia into India between 35–50 MYA (paleomaps modified from Klaus et al. [7])
Grismer et al. BMC Evolutionary Biology (2016) 16:43 Page 8 of 11
Eocene, which could have allowed for terrestrial exchanges
between these areas. These results collectively represent a
broad-scale pattern of faunal exchange between the ISC
and areas of Eurasia before its collision with Asia, at least
partially facilitated by land bridges, which we term the
“Eocene Exchange Hypothesis.” Furthermore, we believe
the reoccurring and somewhat subjective disagreement be-
tween the Indian vs. Asian origins hypotheses [2–12, 16],
have simply identified opposing perspectives of a broad
geographic and temporal conduit of opportunity for faunal
exchange between India and Eurasia. Future studies would
benefit from an attempt to empirically focus on the timing
and direction of faunal exchange between these biogeo-
graphic regions, rather than a prevalence of one scenario
over the other.
Revision of the age of draconinae
Our estimate for the age of Draconinae is significantly
older than those previously published in broad scale
squamate phylogenetic studies (most recently [47]). Our
older estimates are largely due to our consideration of
the acrodont fossils, Mimeosaurus and Priscagama,as
leiolepids rather than stem agamids, following Estes
et al. [48]. These fossils have had a rather turbulent his-
tory of classification, with various studies suggesting
Mimeosaurus was allied with the Chameleonidae [49];
then hypothesized to be located along the branch lead-
ing to Leiolepis and Uromystax [20]; and lastly united
with Priscagama in an extinct subfamily, Priscagaminae
[50], considered to be a stem lineage of Leiolepis and
Uromystax [51].
This confusion has persisted because when Mimeo-
saurus and Pr iscagama were first d escribed, the con -
temporary genera Leiolepis and Uromystax were still
included within the family Agamidae and demonstrated to
be the sister group to the remaining agamids [20] (this re-
lationship has been further confirmed with molecular data
[21, 23, 52, 53]. However, Estes et al. [48] removed Leiole-
pis and Uromystax from the Agamidae and placed them
in their own family (the Leiolepidae), and this taxonomy
has not been followed by subsequent studies. Thus, the
acrodont fossils of Priscagama and Mimeosaurus have
been consistently considered as stem fossils for all aga-
mids and not their sister group, Leiolepis and Uromystax.
We followed the taxonomy of Estes et al. [48] and consid-
ered Mimeosaurus and Priscagama as stem leiolepids and
not stem agamids. It was this placement that lead to our
older estimates of Draconinae origins (85–92 MYA).
However, this estimate is consistent with the ages of new
amber agamid fossils being described out of Indochina
and previous studies on Iguanian lizards ([54]; Bauer et al.,
unpublished data; personal communicat ion with JLG
and PW). We re commend that researchers continue
to follow the taxonomy of [48] with the re cognition
of the Leiolepidae as a distinct family and the place-
ment of priscagamine fossils a s stem to Leiolepis and
Uromystax, as suggested in the original descriptions
of these fossils [20, 50, 51].
Conclusions
The use of additional taxa, sequence-capture data, and
newer geological models—all data not available to previ-
ous studies on Draconinae—resulted in novel and well-
resolved relationships, leading to new biogeographic
insights in this unique subfamily of lizards. Using these
biogeographic insights and a broad comparison with pre-
vious biogeographic literature, we propose the Eocene Ex-
change Hypothesis, and the simple but well supported
assumption that land bridges may have facilitated a broad-
scale pattern of faunal exchange between the ISC and
areas of Eurasia before its collision with Asia during the
Eocene. We expect that with additional sampling of Indian
and mainland Asian species, some factors that may have
biased our biogeographic interpretations within the
Draconinae to (i.e., Indian extinction events), can be eval-
uated. In addition, sampling of additional draconine spe-
cies will allow us to test more fine-scaled hypotheses
concerning dispersal and diversification within this group.
Our phylogenomic analysis add to a growing body of
knowledge addressing the effects of the ISC’s collision on
biogeography and offers new ideas to be tested by future
studies.
Additional files
Additional file 1: Table S1. List of all the species and their associated
localities used in this study. (XLSX 56 kb)
Additional file 2: Table S2. List of all the fossil calibrations, their ages,
and their associated references, used in this study. (DOCX 72 kb)
Additional file 3: Table S3. Per locus metrics for the UCE sequence
capture data. (XLSX 289 kb)
Abbreviations
ISC: Indian subcontinent; EEH: Eocene Exchange Hypothesis; MYA: million
years ago.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JLG, SLT, AA, LJW, and MDB carried out the molecular genetic studies and
participated in the sequence alignment. JLG, JAS, RMB, PW participated in
the design of the study. JLG carried out the analyses and drafted the
manuscript. All authors read and approved the final manuscript.
Acknowledgements
For tissues, we thank Bryan L. Stuart, Aaron Bauer, L. Lee Grismer, Jens
Vindum (CalAcad.) Jimmy McGuire (MVZ), Funding provided by Clarkson
University and NSF through its post-doctoral fellowship program and grants
(DEB-9726064, DEB-9982736, and DEB-0451832). For field assistance we thank
Indraneli Das and for assistance with UCE pipeline we thank Carl H. Oliveros.
Grismer et al. BMC Evolutionary Biology (2016) 16:43 Page 9 of 11
Author details
1
Department of Ecology and Evolutionary Biology and Biodiversity Institute,
University of Kansas, Dyche Hall, 1345 Jayhawk Blvd., Lawrence, KS
66045-7561, USA.
2
Department of Biology, Clarkson University, 8 Clarkson
Avenue, Postdam, NY 13699, USA.
3
Zoologisches Forschungsmuseum
Alexander Koenig Adenauerallee 160, D-53113 Bonn, Germany.
Received: 17 December 2015 Accepted: 8 February 2016
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