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doi: 10.1098/rspb.2008.1494
, 893-902276 2009 Proc. R. Soc. B
T.N.C Vidya, Raman Sukumar and Don J Melnick
origins of Asian elephants
Range-wide mtDNA phylogeography yields insights into the
Supplementary data
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Range-wide mtDNA phylogeography yields
insights into the origins of Asian elephants
T. N. C. Vidya
1,2,
*, Raman Sukumar
1
and Don J. Melnick
3
1
Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560 012, India
2
Evolutionary and Organismal Biology Unit, Jawaharlal Nehru Centre for Advanced Scientific Research,
Bangalore 560 064, India
3
Department of Ecology, Evolution and Environmental Biology, Columbia University,
1200 Amsterdam Avenue, New York, NY 10027, USA
Recent phylogeographic studies of the endangered Asian elephant (Elephas maximus) reveal two highly
divergent mitochondrial DNA (mtDNA) lineages, an elucidation of which is central to understanding the
species’s evolution. Previous explanations for the divergent clades include introgression of mtDNA
haplotypes between ancestral species, allopatric divergence of the clades between Sri Lanka or the Sunda
region and the mainland, historical trade of elephants, and retention of divergent lineages due to large
population sizes. However, these studies lacked data from India and Myanmar, which host approximately
70 per cent of all extant Asian elephants. In this paper, we analyse mtDNA sequence data from 534 Asian
elephants across the species’s range to explain the current distribution of the two divergent clades. Based
on phylogenetic reconstructions, estimates of times of origin of clades, probable ancestral areas of origin
inferred from dispersal–vicariance analyses and the available fossil record, we believe both clades
originated from Elephas hysudricus. This probably occurred allopatrically in different glacial refugia, the a
clade in the Myanmar region and the bclade possibly in southern India–Sri Lanka, 1.6–2.1 Myr ago.
Results from nested clade and dispersal–vicariance analyses indicate a subsequent isolation and
independent diversification of the bclade in both Sri Lanka and the Sunda region, followed by northward
expansion of the clade. We also find more recent population expansions in both clades based on mismatch
distributions. We therefore suggest a contraction–expansion scenario during severe climatic oscillations of
the Quaternary, with range expansions from different refugia during warmer interglacials leading to the
varying geographical overlaps of the two mtDNA clades. We also demonstrate that trade in Asian elephants
has not substantially altered the species’s mtDNA population genetic structure.
Keywords: phylogeography; divergent mitochondrial clades; Pleistocene refugia; elephant trade;
Elephas fossils
1. INTRODUCTION
The Asian elephant (Elephas maximus) is endangered, with
a wild population of 41 000–52 000 individuals in 6 per
cent of the range occupied 4000 years ago (Sukumar
2003). It is the sole surviving species of the Proboscidea in
Asia. Studies of its evolutionary history and phylogeo-
graphy are recent enough that their results have not
been integrated into conservation action, although the
flagship role of the elephant for broader conservation in
Asia has been recognized (Duckworth & Hedges 1998;
Sukumar 2003). Fossils and molecular analyses are
valuable in reconstructing evolutionary history, so while
fossil data for the Elephantidae are limited in Asia,
increasing molecular data and new ways of evaluating
them are providing a clearer picture of the species’
phylogeography (Fernando et al.2000,2003;Fleischer
et al. 2001;Vidya et al. 2005).
The largest study of fossil elephantid morphology
indicated that the genus Elephas originated in Africa after
the differentiation of the genus Loxodonta and was present
during the Early Pliocene (Maglio 1973).A recent molecular
study by Rohland et al. (2007) has estimated that Loxodonta
and the Mammuthus–Elephas lineage diverged 7.6 (95%
CI 6.6–8.8) million years ago (Myr ago). The fossil record
alone suggests that this split is more recent (ca 5.5 Myr ago)
(see electronic supplementary material 1). A derivative of the
early Elephas ekorensis–Elephas recki complex colonized
Asia and is thought to have given rise to Elephas planifrons
and Elephashysudricus (Maglio1973). The earliestrecords of
both species were found in the Siwalik Hills in the northern
Indian subcontinent, E. planifrons appearing ca 3.6 Myr ago
and E. hysudricus ca 2.7 Myr ago (see Nanda 2002). Late in
the Early Pleistocene, Elephas namadicus, another derivative
of E. recki, colonized Asia and displaced the earlier Elephas
species across a considerable part of their ranges (Maglio
1973) before disappearing in the Late Pleistocene. However,
E. hysudricus, which was widespread, is considered (based
on dental and cranial evidence) to have given rise to
E. maximus in southern Asia ca 0.25 Myr ago (Maglio
1973) and to Elephas hysudrindicus, a Javan species, ca
0.8–1.0 Myr ago (Maglio 1973;Van den Bergh et al. 1996).
Molecular phylogeographic analyses are often based on
mitochondrial DNA (mtDNA) markers. As mtDNA is
Proc. R. Soc. B (2009) 276, 893–902
doi:10.1098/rspb.2008.1494
Published online 18 November 2008
Electronic supplementary material is available at http://dx.doi.org/10.
1098/rspb.2008.1494 or via http://journals.royalsociety.org.
*Author and address for correspondence: Evolutionary and Orga-
nismal Biology Unit, Jawaharlal Nehru Centre for Advanced Scientific
Research, Bangalore 560 064, India (tncvidya@jncasr.ac.in).
Received 14 October 2008
Accepted 28 October 2008 893 This journal is q2008 The Royal Society
on October 11, 2011rspb.royalsocietypublishing.orgDownloaded from
maternally inherited, stochastic extinctions of mito-
chondrial lineages through the absence of female offspring
are usually the norm unless sufficiently large populations
of females exist. The coexistence of divergent lineages of
mtDNA within a species is therefore rare and requires an
elucidation of the evolutionary and population processes
that led to it (Melnick et al. 1993). The Asian elephant has
two such divergent lineages of mtDNA haplotypes or
clades, the ‘a’ (Fernando et al.2000,2003) or ‘B’ clade
(Hartl et al. 1996;Fleischer et al. 2001) and the ‘b’ or ‘A’
clade (we use the aand bterminology here), with a
sequence divergence of approximately 3 per cent.
Haplotypes from these two clades coexist within popu-
lations, sometimes within small geographical areas
(Fernando et al. 2000;Fleischer et al. 2001), unlike
other mammalian species, in which divergent clades are
usually geographically separate (e.g. Taberlet & Bouvet
1994;Jensen-Seaman & Kidd 2001). Therefore, under-
standing the coexistence and distribution of the two clades
is vital to understanding Asian elephant evolution.
Hypotheses to explain the distribution of the two Asian
elephant clades have invoked introgression of mitochondrial
haplotypes from another species through hybridization,
and/or allopatric divergence, in which mutations accumu-
late and lead to sequence divergenceamong populations that
are geographically separated. More specifically, these
hypotheses include: (i) the introgression of mtDNA from
E. namadicus or an alternative species of Elephas to
E. maximus (Fernando et al. 2000); (ii) allopatric divergence
of populations on the mainlandgiving rise to theaclade and
on Sri Lanka giving rise to the bclade, followed by secondary
contact and admixture ( Fernando et al. 2000); (iii)
introgression of mtDNA from E. hysudrindicus (in the
Sunda region), which gave rise to the bclade, into
E. maximus, which carried the aclade, followed by extensive
trade in elephants bringing the bclade to Sri Lanka and
southern India (Fleischer et al. 2001); and (iv) incomplete
lineage sorting, or the retention of divergent lineages simply
owing to large population size (Fleischer et al. 2001).
Importantly, these hypotheses were based on only four to six
samples from India, which hosts approximately 60 per cent
of the entire Asian elephant population (Sukumar 2003),
and zero tofive samples from Myanmar, which hosts another
10 per cent.
Here, we expand our phylogeographic analysis by
examining mtDNA from 534 Asian elephants across the
species’s range (figure 1), including larger sample sizes
from India (nZ244) and Myanmar (nZ24). The mtDNA
segment analysed (599 base pairs (bp), comprising
the C-terminal of cyt-b, t-RNA
Thr
, t-RNA
Pro
and the
Sri Lanka (82)
Myanmar (24)
Sumatra (34)
Peninsular Malaysia (14)
Borneo (20)
Vietnam (25)
Cambodia (1)
Lao PDR (14)
S India
C India (12)
N India (6)
0.110BP
0.232AE
0.012AF
0.098AG
0.122BO
0.024BN
0.012BM
0.244BL
0.012BK
0.037BJ
0.037BI
0.061BH
Nilgiris (159)
Anamalai-
Periyar (67)
0.196±0.0637
0.030BL
<0.000
0.896BF
0.015BB
0.060BA
Bhutan (13) NE India (63)
Nepal
Bangladesh
China
Thailand
0
1.000BN
0
H,p
1.000AC
0.409±0.1333
0.001±0.0008
0.750BL
0.250BC
0.032BL
0.349AH
0.619AC
0.603±0.0885
0.077BL
0.007±0.0043
0.385AE
0.538AC
0.692±0.0942
0.071BQ
0.005±0.0033
0.500AE
0.286AD
0.143AB
0.200AJ
0.280AK
0.080BO
0.120AD
0.280AB
0.040AA
1.000AB
0
1.000BD
0.014±0.0074
0.823±0.0458
0.042
BW
0.083AF
0.125AH
0.125BQ
0.292BL
0.292BH
0.042AI
0.071BV
0.071BU
0.786BQ
0.071BP
0.147BU
0.441BT
0.176BS
0.176BR
0.059BP
H,p
H,p
H
0.853±0.0190
0.016±0.0083
0.740±0.0522
0.004±0.0025
0.396±0.1588
0.003±0.0021
0.479±0.0435
0.003±0.0020
p
H
p
H
p
H
p
H
p
H
p
H
p
H
p
H
p
H
p
0.813±0.0389
0.006±0.0037
Figure 1. Present Asian elephant distribution (grey) based on Sukumar (2003) and (for India) Vidya et al. (2005), and the
number of individuals sampled (within parentheses), proportions of different haplotypes, and H
ˆand p(expressed as average G
1.96 s.e.) tabled against different populations. Haplotypes beginning with the letter A belong to the aclade and those beginning
with the letter B to the bclade. (See electronic supplementary material 2 for more details about figure 1.)
894 T. N. C. Vidya et al. Asian elephant phylogeography
Proc. R. Soc. B (2009)
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hypervariable left domain of the control region) corre-
sponds to that used by Fernando et al.(2000,2003) and
Fleischer et al. (2001) (minus 76 bp), making it possible to
compare the results across studies. We examined mtDNA
diversity and population differentiation, and then con-
structed phylogenetic trees based on maximum parsimony
(MP), minimum evolution (ME) and Bayesian methods
in order to examine age relationships between haplotypes,
and mismatch distributions to detect historical population
expansions. Finally, we used a nested clade analysis
(NCA) to look for geographical associations of haplotypes
and a dispersal–vicariance analysis to identify areas of
distribution of ancestral haplotypes. Based on our results,
we propose a revised evolutionary hypothesis in which
climatic fluctuations during the Pleistocene were an
important factor shaping Asian elephant phylogeography.
2. MATERIAL AND METHODS
(a)Samples and molecular analysis
We sequenced 365 new individuals and added 169 published
sequences of Fernando et al.(2000,2003). Most (332) new
samples were non-invasively collected fresh dung, mostly
from free-ranging elephants, while the remaining were blood
samples from captive elephants with unambiguous capture
records. Sequences of the African elephant (Loxodonta
afr icana) were obtained from three zoo animals (see
Fernando et al. 2003) and those of the woolly mammoth
(Mammuthus primigenius) from GenBank sequences
NC007596 (Krause et al. 2006) and DQ316067 (Rogaev
et al. 2006). DNA extraction, PCR amplification and
squencing were based on Fernando et al.(2000,2003).
(b)MtDNA diversity and differentiation
We aligned and edited sequences using SEQUENCHER v. 3.1.1
(Gene Codes Corporation 1999). Population structure was
assessed based on locus-by-locus analysis of molecular
variance (AMOVA; Excoffier et al. 1992) and F
ST
(Weir &
Cockerham 1984) values calculated using ARLEQUIN v. 3.1
(Excoffier et al. 2005).
(c)Phylogenetic analyses
Phylogenetic analyses were conducted using MP, ME and
Bayesian approaches, with woolly mammoth and the African
elephant haplotypes as outgroups. MP and ME trees were
constructed using PAUP v. 4 (Swofford 1998), employing a
heuristic search with random addition of sequences and TBR
branch swapping procedure. The Bayesian analysis was
carried out using the HKYCI model, relaxed molecular
clock (uncorrelated lognormal) assumption and normal
priors for HKY kappa (the corrected transition/transversion
ratio), I, and the coalescent population size parameter, in the
program BEAST v. 1.4.6 (Drummond & Rambaut 2006).
Normal priors were also used for divergence times
between the Loxodonta and Mammuthus–Elephas lineages
(meanZ7.6 Myr ago) and between Mammuthus and Elephas
(meanZ6.7 Myr ago) based on Rohland et al.’s (2007)
findings, and between the aand bclades in the Asian
elephant (meanZ1.85 Myr ago; see below). These three
calibration points allowed dating of other internal nodes
(see electronic supplementary material 3 for more details on
phylogenetic analyses).
Mismatch distributions (Rogers & Harpending 1992)
were constructed and similarity between the observed and
simulated mismatches tested using ARLEQUIN v. 3.1. To arrive
at the times of expansion, we calculated a mutation rate for
our 599 bp segment using HKY-corrected pairwise distances
between haplotypes from the software PHYLO_WIN (Galtier
et al. 1996) and a cyt-b-based time calibration. The latter was
carried out with cyt-b-based divergences between the Asian
and African elephants, and between the two Asian elephant
clades as provided by Fleischer et al. (2001), but using
divergence times of 6.6–8.8 Myr between the two species
(Rohland et al. 2007) instead of the previous 5 Myr estimate.
This calculation gave cyt-b-based divergence times between
the two Asian elephant clades of 1.6–2.1 Myr, instead of
1.2 Myr calculated by Fleischer et al. (2001).
(d)Analyses of phylogeography
The NCA was carried out using 530 individuals with
locational data to test the null hypothesis of no geographical
association of haplotypes and, if the null hypothesis was
rejected, to discriminate between the alternative hypotheses
of restricted gene flow, range fragmentation and range
expansion or long-distance colonization ( Templeton 1998).
A haplotype network based on statistical parsimony was
created using TCS v. 1.13 (Clement et al. 2000) and the
network nested into a series of nested clades. Geographical
associations of clades were tested statistically using GEODIS
v. 2.0 (Posada et al. 2000). The inference key of Templeton
(2004) was used to distinguish among alternative hypotheses.
We used relatively small population units/locations (see
electronic supplementary material 4) and land (including
past land bridges) distances (1326 distance measurements)
between locations.
Dispersal–vicariance analysis to reconstruct ancestral area
distributions of the MP and ME phylogenies was carried out
using the program DIVA (Ronquist 1996). In the dispersal–
vicariance analysis of the MP consensus tree, polytomies were
present on individual trees and were not the result of creating
consensus trees. Therefore, polytomies were broken down
into various combinations and reanalysed.
3. RESULTS
(a)Mitochondrial diversity and differentiation
We discovered a total of 33 substitutions (30 transitions, 3
transversions; 31 sites), which resulted in 32 unique
mitochondrial haplotypes, 11 from the aclade and 21
from the bclade, among the 534 individuals sampled
(see electronic supplementary material 5). Analysed as a
single population, Asian elephant haplotype diversity (H
ˆ;
Nei 1987) is (average G1.96 s.e.) 0.871G0.0101, and
nucleotide diversity (p;Nei 1987), 0.0157G0.0080.
Sri Lanka, Myanmar and Vietnam showed the highest
haplotype diversities (figure 1). In addition, Sri Lanka and
Myanmar had the highest nucleotide diversities, while
Vietnam had haplotypes of only the aclade and, therefore,
lower nucleotide diversity (figure 1). Haplotype and
nucleotide diversities in the aclade were 0.801G0.0198
and 0.003G0.0020, respectively, and, in the bclade,
0.781G0.0178 and 0.007G0.0040, respectively. The
average sequence divergence within clades was 0.37 per
cent (range: 0.01–1%) within the aclade and 0.87 per cent
(range: 0.01–1.8%) within the bclade, and the average
sequence divergence between the aand bclades was 3.0 per
cent (range: 2.1–4.0%). Population structure based on
AMOVA showed significant differentiation among regions,
Asian elephant phylogeography T. N. C. Vidya et al. 895
Proc. R. Soc. B (2009)
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among populations within regions and within populations
(see electronic supplementary material 6). Fifty of the fifty-
five pairwise comparisons of F
ST
between populations were
significant after Bonferroni corrections were applied (see
electronic supplementary material 7).
(b)Phylogenetic analyses
MP analysis yielded 5040 equally parsimonious trees (tree
scoreZ125). The majority rule consensus tree is shown in
figure 2. The ME tree (not shown) was similar in topology
to the MP tree with minor rearrangements of the terminal
branches. Log posteriors of the three Bayesian runs
(K1914.28, K1914.21 and K1914.25) indicated conver-
gence of results ( pO0.05 in pairwise tests based on Bayes
factors; see Kass & Raftery 1995). The average log
likelihood of the trees obtained from the three runs
was K1428.72 (95% CI of the highest posterior
densitiesZK1440.84 to 1416.99). A consensus tree of
the 300 003 Bayesian trees (from the three runs) is shown
in figure 3. All three types of tree construction revealed the
distinction between aand bclades with certainty. A
subclade within the bclade, referred to here as the b1
subclade, that consisted of 13 haplotypes—BA, BB, BC,
BF, BH–BO (alphabetically) and BW—was present in the
Bayesian and ME trees and in the MP consensus tree
(figures 2 and 3). Support was poor for this clade in the
MP tree (38%), but this clade consistently appeared in all
the 5040 most parsimonious trees (figure 2), supporting
this structure. This clade was also present in the previous
ME analysis of Fernando et al. (2003). Of the eight bclade
haplotypes that were not part of the b1 subclade,
haplotypes BR, BS, BT and BU formed a monophyletic
group, henceforth referred to as the b2 subclade, in the
Bayesian tree (figure 3). Bootstrap support for this clade
was also lacking in the MP tree (37%), but, again, all the
most parsimonious trees showed this node. The b2
subclade is likely to be the Sumatran subclade of Fleischer
et al. (2001), although we could not confirm this as their
sequences are not publicly available. Haplotype AI was
identified as basal to the aclade in the MP and ME trees,
while no structure was detected within this clade in the
Bayesian tree (figures 2,3). Dating of nodes in the Bayesian
tree indicated that the bclade is older (mean 1.58, 95% CI
1.28–1.86 Myr ago) than the aclade (mean 0.86, 95% CI
0.52–1.21 Myr ago). The b1 and b2 subclades were of
roughly the same age as the aclade (figure 3).
Analysis of mismatch distributions showed that
observed data were explained by the fitted models, with
the combined data of the two clades indicating demo-
graphic stability and the aand bclades indicating
population expansion (see electronic supplementary
material 8). The HKY-corrected distance between clades
was 0.025 and the mutation rate in the mtDNA segment
was estimated to be 1.6 and 1.2 per cent per Myr based on
the mean divergence times, between the Asian and African
elephants, of 6.6 and 8.8 Myr ago, respectively. This was
Laf3
Laf2
Laf1
AJ
AK
AI
AH
AC
AG
AF
AE
AD
AB
AA
BD
BV
BQ
BU
BT
BS
BR
BP
BK
BJ
BI
BL
BH
BN
BO
BW
BM
BC
BB
BA
BF
70
60
100
60
70
83
100
100
67
100
100 100
100
100
100
100
100
100
100
100
68
68
57
57
100
100
100
63
68
70
74
SL
SL
Su
PM, SL or M,
SL or PM, M or Su
PM, M or PM, M or PM or L
Sunda region and/ or SL
*SL, Su,
SL or Su or PM
SL or CV,
NEI or CV
NI or CV, NEI or CV
M or CV
CV
*SL
Mamm1
Mamm2
100
100
100
100
M and/ or SL
and/ or CV
and/ or PM
and/ or Su
Figure 2. Consensus MP tree of Asian elephant haplotypes,
rooted with African elephant (Laf1, Laf2 and Laf3) and
woolly mammoth (Mamm1 and Mamm2) haplotypes as
outgroups. Consensus values are given above the branch and
the bootstrap values (based on 1000 replicates) above 50 per
cent are given below the branch adjoining the corresponding
node. Ancestral areas inferred from dispersal–vicariance
analysis are shown with arrows from the corresponding
nodes. Abbreviations used: B, Borneo; CV, Cambodia–
Vietnam; L, Laos; M, Myanmar; NEI, northeastern India
and Bhutan; NI, northern India; PM, Peninsular Malaysia;
SI, southern India; SL, Sri Lanka; Su, Sumatra. Asterisks
indicate results based on shown as well as alternative
arrangements at those nodes.
BA
BF
BB
BC
BH
BI
BJ
BK
BL
BM
BN
BO
BW
BD
BP
BQ
BV
BR
BS
BT
BU
AA
AB
AC
AD
AE
AF
AG
AH
AI
AJ
Mamm1
Mamm2
Laf1
Laf2
Laf3
0.8 M
y
a
1.0
1.0
1.0
1.0
1.0 0.82
0.78
0.93
0.67
1.0
1.0
0.99
1.0
0.99
0.55
AK
β1
β2
6.68 (6.29–7.07) Myr ago
7.63 (7.24–8.01) Myr ago
1.88 (1.80–1.95) Myr ago
0.86 (0.52–1.21) Myr ago
1.58 (1.28–1.86) Myr ago
0.90 (0.43–1.37) Myr ago
0.95 (0.61–1.31) Myr ago
0.63
α
β
Figure 3. Phylogram from the Bayesian analysis with
posterior probabilities of nodes (greater than 0.5) and mean
and 95% CI of posterior node heights.
896 T. N. C. Vidya et al. Asian elephant phylogeography
Proc. R. Soc. B (2009)
on October 11, 2011rspb.royalsocietypublishing.orgDownloaded from
considerably lower than the rate (3.5%) calculated by
Fleischer et al. (2001), partly owing to the different time
estimate used and partly owing to the smaller HKY-
corrected distance between clades in our larger sample.
Based on the values of t(Z2mtgenerations; mis the
mutation rate over the entire sequence analysed and tis
the time to expansion of a population with initial effective
number of femalesZN
0
to a final size N
1
) from the
mismatch distributions, a generation time of 27 years
estimated (as the average age of reproducing females)
from field data in southern India (C. Arivazhagan,
T. N. C. Vidya & R. Sukumar 2001–2003, unpublished
data) and mutation rates of 1.6 per cent (divergence time
between clades of 1.6 Myr) and 1.2 per cent (divergence
time between clades of 2.1 Myr), respectively the mean
times of expansion of the aclade were approximately
128 000 and 170 000 yr ago and the mean times of
expansion of the bclade were 383 000 and 511 000 yr ago.
N
0
was very low in both clades but confidence limits were
large and upper limits of N
0
were 2190 in the aclade and
4808 in the bclade based on a 1.6 Myr divergence
between clades and 2920 in the aclade and 6410 in the b
clade based on a 2.1 Myr divergence. The values of N
1
(Zq
1
/2m; estimation of mutation parameter q
1
thought to
be biased upwards; Schneider & Excoffier 1999) were
(based on 1.6 and 2.1 Myr divergences, respectively,
between clades) 17 488 and 318 in the aclade, and 15 566
and 20 754 in the bclade. N
0
and N
1
were both
approximately 23 600 (based on a 1.6 Myr divergence)
or 31 500 (based on a 2.1 Myr divergence) based on the
combined data of both clades.
(c)Phylogeographic analyses
Hierarchical nesting of the cladogram generated by
statistical parsimony produced 22 one-step clades, 9
two-step clades, 4 three-step clades, and 1 each of
four-, five- and six-step clades (figure 4). Connections
between the aand bclades did not adhere to the limits of
95 per cent parsimony, and the two clades were treated as
disjointed networks. The distribution of the aand bclades
was clinal as suggested by Fleischer et al. (2001), with the
bclade showing higher frequencies in the southern areas of
the species’ range, and the aclade largely distributed
towards the northern and eastern areas of the range
(figure 1). Nesting level is indicative of the age of
haplotypes in an NCA. Based on the levels of nesting,
clades 2-6 to 2-9 (nested within 3-2 and 3-3, both in turn
nested within 4-1) contained the oldest haplotypes within
the bclade (figure 4). These seven haplotypes in clade 4-1
were distributed largely across Peninsular Malaysia and
Sumatra, but haplotype BP was also fairly common and
widespread in Sri Lanka and BQ was found in Myanmar
and Laos (figure 1; electronic supplementary material 4).
The NCA uncovered strong geographical associations
from 14 out of the 18 clades that could be tested (see
electronic supplementary material 9). Within the aclade,
contiguous range expansion was observed in clades 1-3
and 2-2, and restricted gene flow and isolation by distance
in clade 1-4. Within the bclade, restricted gene flow and
isolation by distance was observed in clades 1-11, 1-12,
2-5 and 4-1, allopatric fragmentation in clade 2-4 and
contiguous range expansion in clade 3-3. Past fragmenta-
tion followed by range expansion was seen in clades 3-4
and 5-1 (see electronic supplementary material 9).
Further analysis of the pattern in clade 5-1, by calculating
the average pairwise distances between haplotypes and
between clades of different levels present in the region
(see Templeton 2001), identified Myanmar as a zone of
secondary contact of haplotypes, following range expan-
sion. The pairwise distance between the geographical
centres of clade 5-1 haplotypes found in Myanmar
(1626 km) was higher than those in the Sunda region
(Peninsular Malaysia and Sumatra; averageZ783 km;
95% CIZ492–1074) and Sri Lanka (averageZ657 km;
95% CIZ376–938), both of which also harbour this
clade. In addition, the average pairwise distances between
geographical centres of clades, which is expected to
decrease with increasing clade level under the scenario
of restricted gene flow (Templeton 2001), remained high
instead up to the clade level that marked the fragmentation
event (average pairwise distance between haplotypes in
Myanmar, 1626 km; between one-step clades, 1645 km;
between two-step clades, 1876 km; between clades 2-5
and 3-3, 2047 km; between clades 2-5 and 4-1, 1776 km).
Similar analysis could not be carried out for clade 3-4
since almost all haplotypes of this clade were confined to
one region each. NCA inference at the level of the entire a
or bclade was inconclusive in the absence of interior-tip
contrast of clades. While aand bclades were analysed
separately thus far in the NCA, when a parsimony network
was constructed adding woolly mammoth haplotypes to
Asian elephant haplotypes in order to examine the
relationship between the two clades, past fragmenta-
tion followed by range expansion was inferred (steps
1-2-11-12-13 followed in inferencekey of Templeton 2004).
Dispersal–vicariance analysis on both MP and ME
phylogenies pointed to Sri Lanka as the ancestral area of
the b1 subclade and Sumatra as the ancestral area of the
b2 subclade. The area occupied by ancestors of the entire
bclade (in both MP and ME trees) could not be
ascertained unambiguously and was identified as the
Sunda region and/or Sri Lanka (figure 2). The ancestral
area of the aclade was pointed out to be Myanmar/
BQ
AD
AB
AC
AJ
AI
AH
AA
AK
AF
AE
AG
BL
BC
BF
BB
BH
BW
BM
BJ
BO BN
BD
BA
BK
BP
BS BT
BU
BR
BV
1-1
1-2
1-3 1-4
1-14
2-1
2-2
2-4
2-5
2-3
2-6
2-7
3-1
3-2
3-4
4-1
6-1
1-22
1-5
1-9
1-8
1-7
1-6
1-13
1-12
1-11
1-10
1-18
1-17
1-16
1-20
1-19
1-21 2-8
2-9
BI
3-3
1-15
5-1
Figure 4. Hierarchical levels of nesting of the haplotype
network generated by statistical parsimony. Each clade is
denoted by two numbers, the first denoting the level of nesting
and the second indicating the number of the clade at that level
of nesting. Each branch between two haplotypes denotes a
single mutation. Dashed lines show non-parsimonious
connections. Empty circles indicate assumed haplotypes.
Asian elephant phylogeography T. N. C. Vidya et al. 897
Proc. R. Soc. B (2009)
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Cambodia–Vietnam based on the MP tree (figure 2) and
Myanmar/northeastern India/Sri Lanka based on the ME
tree. The latter is contingent on the aclade not having
been introduced to Sri Lanka through trade in elephants
(see below).
4. DISCUSSION
(a)Evolutionary history of the aand bclades
The time of divergence, varying complexity and geo-
graphical distributions of the aand bclades suggest
distinct histories. We examine the hypotheses previously
proposed in the context of our results to understand the
phylogeography of the Asian elephant.
(i) Introgression of mtDNA from E. namadicus or an
alternative species of Elephas to E. maximus
Elephas namadicus would seem a logical candidate for the
progenitor of either aor bclade based on its broad
distribution in Asia during the Middle and Late Middle
Pleistocene (ca 0.70–0.15 Myr ago; see electronic supple-
mentar y material 10). However, as pointed out by
Fernando et al. (2000), who first examined this
hypothesis, and Fleischer et al. (2001), the divergence
(including our calculation of 1.6–2.1 Myr) between the a
and bclades is not compatible with the proposed
phylogenetic relationship between E. namadicus and
E. maximus, which are estimated to have diverged over
3.5 Myr ago, based on morphological and stratigraphic
data (Maglio 1973). Fernando et al. (2000) suggested that
introgression of mtDNA could have alternatively occurred
from a different species of Elephas that arose in Asia, but
did not specify the species (see below for an examination
of other species). Unlike Maglio (1973), who suggested
that E. hysudricus gave rise to E. maximus,Aguirre (1969)
suggested a common ancestor of E. hysudricus and
E. maximus in E. planifrons in the Early Pleistocene. If
that were true, it is possible that the two clades originated
in E. hysudricus and E. maximus. However, E. maximus is
considered by others as a recent species and its fossils date
back only to the Late Pleistocene (more recent than
0.25 Myr ago; see electronic supplementary material 10).
(ii) Allopatric divergence of populations on the mainland that
gave rise to the aclade and on Sri Lanka that gave rise to
the bclade, followed by secondary admixture
The overall geographical distribution of the aclade does
suggest a mainland origin and the inferred ancestral areas
of the aclade were largely on the mainland. However,
within the bclade we identified two subclades, b1 and b2,
probably having arisen in Sri Lanka and the Sunda region,
respectively. While one of the interior haplotypes BP
(figure 4) was found in fairly high frequency in Sri Lanka,
others (BP–BV in alphabetical order and BD) were
distributed across the Sunda region and other areas.
Results from the dispersal–vicariance analysis could not
attribute the ancestry of the entire bclade unambiguously
to Sri Lanka or the Sunda region alone. The fossil record,
however, leads us to believe that the bclade may have
arisen in or near Sri Lanka rather than the Sunda region
(see below). The hypothesis of allopatric divergence and
secondary admixture of the two clades appears correct,
with Sri Lanka as an important refugial area during glacial
periods. However, the divergence between clades is not
likely to have occurred between Sri Lanka and India, with
the repeated absence of land bridges between the two
causing the differentiation, because southern India and
central India show only bclade haplotypes. We elaborate
below on a probable mechanism of allopatric divergence
between clades.
(iii) Introgression of mtDNA from E. hysudrindicus
(in the Sunda region), which gave rise to the bclade,
into E. maximus,which already carried the aclade, followed
by extensive trade in elephants bringing the bclade to
Sri Lanka and southern India
As mentioned above, the origin of the entire bclade is not
immediately clear, based on molecular data, but it
probably originated in or near Sri Lanka rather than the
Sunda region, based on fossil data. Even if the bclade did
arise in the Sunda region, the fossil-based time of
divergence between E. hysudrindicus and E. maximus of
0.8–1 Myr ago is lower than our newly calculated
divergence time of 1.6–2.1 Myr ago between the aand b
clades, suggesting that E. hysudrindicus (endemic to Java)
did not give rise to the bclade. In addition, being a recent
species that appeared only in the Late Pleistocene (more
recent than 0.25 Myr ago), E. maximus was presumably
still evolving when the bsubclades were diversifying
independently in Sri Lanka and the Sunda region. Since,
based on fossil evidence, E. maximus evolved from
E. hysudricus and not E. hysudrindicus, and since fossils
of the latter have been recovered only from Java
(see electronic supplementary material 10) and not Sri
Lanka, it is unlikely that the bclade introgressed from
E. hysudrindicus into E. maximus. It is plausible that the a
clade originated in E. maximus (E. hysudricus) as we
discuss below.
The high frequencies of bclade haplotypes in southern
India (100%) and Sri Lanka (66%), and the Sri Lankan
origin of the b1 subclade, are not concordant with long-
distance trade in elephants, dating back to 2000 years at
most, having brought the bclade to these regions.
(iv) Revised hypothesis
Based on our results, fossil data and previous hypotheses,
we present the following hypothesis to explain the
distribution of the two clades of haplotypes. As with
E. namadicus, other Elephas species such as E. planifrons,
E. celebensis and E. platycephalus are also precluded from
being progenitors of either clade owing to fossil-based
divergence times of ca 3.5 Myr between each of them and
E. maximus (Maglio 1973). Similarly, the discordance
between fossil-based E. hysudrindicus–E. maximus diver-
gence and molecular data-based a–bclade divergence
eliminates E. hysudrindicus as a progenitor of either clade
of haplotypes. It therefore appears that both clades had
their origin in E. hysudricus.Elephas hysudricus fossils have
been recorded widely, from the Pinjor horizon of northern
India, the upper Irrawaddies of Myanmar, Kiangsu of
southern China and the Ratnapura fauna of Sri Lanka (see
electronic supplementary material 10). Since E. hysudricus
gave rise to E. hysudrindicus, which was found only on Java
(Maglio 1973), E. hysudricus was presumably found in
the Sunda region also.
Our new calculation of the divergence between the a
and bclades coincides with the beginning of the
Pleistocene. The initiation of the Pleistocene in India
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(as inferred by the magnetostratigraphically dated Olduvai
subchron ca 1.9 Myr ago; Krishnamurthy et al. 1986) was
associated with a cold period (Krishnamurthy et al. 1986;
Singh & Srinivasan 1993) and increasing tectonic
activity in and near the Himalayas in northern India (see
Sahni & Kotlia 1993;Nanda 2002). For ca 2 Myr ago,
E. hysudricus formed part of the ‘Pinjor Fauna’, which
were distributed across the Siwalik Hills (foothills of the
Himalayas) and the upper Irrawaddies of Myanmar
(electronic supplementar y material 10). But from ca
1.9 Myr ago, there was a change in lithostratigraphic
formation in the Siwaliks and the extinction and south-
ward migration into Peninsular India of the Siwalik Pinjor-
associated fauna (Sastry 1997; Nanda 2002,2008). We
think that the Siwalik population of E. hysudricus that
migrated southwards probably gave rise to the bclade and
the population in Myanmar (the inferred ancestral area of
the aclade from the dispersal–vicariance analysis)
extending to southern China, to the aclade.
Thus, the origin of divergent clades from E. hysudricus
probably took place due to the climatic and/or tectonic
changes at the beginning of the Pleistocene, which
resulted in allopatric fragmentation, as supported by the
results of the NCA. Repeated climatic oscillations in the
form of glacial and interglacial periods since the beginning
of the Pleistocene, and the ensuing range changes,
adaptations and reorganization of populations, are
thought to have resulted in large divergences between
populations of various species (Hewitt 2000). Based on
geography and inferred climatic conditions during the
LGM ca 18 000 ya, it is believed that certain relatively
warmer areas including Sumatra and southern Borneo,
northern and eastern Myanmar, Sri Lanka and the
extreme south of India served as glacial refugia during
the Pleistocene (figure 5). Given available data on fossils
and palaeovegetation, it is difficult to identify the exact
region of origin of the bclade. If vegetation during the
beginning of the Pleistocene was similar to that recon-
structed for the LGM (figure 5), the bclade is likely to
have originated in southern India/Sri Lanka. A single fossil
of E. hysudricus thought to date back to the beginning of
the Pleistocene is known from southern India (Prasad &
Daniel 1968). However, if patterns of vegetation were
different from that of the LGM during the period, one
cannot rule out the rest of Peninsular India as refugia
since E. hysudricus fossils of similar ages are known from
various sites in central India and Sri Lanka (electronic
supplementary material 10), and fossil beds older than
1 Myr are very rare in the former and absent in the latter.
Our results from the NCA, showing restricted gene
flow through isolation by distance in clades 2-5 (three out
of the four haplotypes of which are restricted to Sri Lanka)
E. hysudricus ~1.9 Myr ago
β1 subclade
β2 subclade
β clade
α clade
β clade
zone of
contact of
unrelated β
haplotypes
Siwaliks (Himalayan foothills)
thorn forest
open woodland
monsoon forest
semi-evergreen and evergreen forest
Origin of β sub-
clades ~0.9 Myr ago
Figure 5. A simplified schematic of our revised hypothesis to explain the observed distribution of mitochondrial haplotypes.
Arrows of increasing thickness indicate more recent time periods. There were at least two southward migrations, the first ca
1.9 Myr ago resulting in allopatric fragmentation and, consequently, the origin of the aand bclades, and the second ca 0.9 Myr
ago resulting in the origin of b1 and b2 subclades. A subsequent northward expansion gave rise to the zone of contact of
unrelated bclade haplotypes in Myanmar. Palaeovegetation types during the last glacial maximum based on Adams & Faure
(1997) and Gathorne-Hardy et al. (2002) are also shown. Monsoon forests and semi-evergreen and evergreen forests are thought
to have served as Pleistocene glacial refugia.
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and 4-1 (distributed mostly across the Sunda region), and
from the dispersal–vicariance analysis, showing Sri Lanka
and the Sunda region as ancestral areas of the b1 and b2,
subclades, respectively, imply independent diversification
of bclade haplotypes within each of these regions well after
the origin of the bclade. More intriguingly, the secondary
contact of unrelated haplotypes in Myanmar suggests that
these haplotypes did not arise within Myanmar, but
instead resulted from a northward range expansion of b
clade haplotypes from both Sri Lanka and the Sunda
region followed by subsequent admixture in this region.
This may be best explained by the expansion of the bclade
from peninsular India/Sri Lanka to the eastern and
southeastern regions during warm periods, a second south-
ward migration of the clade such that it became isolated in
the southerly areas of Sri Lanka and the Sunda region
(where the b1 and b2 subclades originated), and subsequent
recolonization of the mainland from both regions when
warmer, wetter conditions returned (figure 5).
There were possibly several such north–south move-
ments, as suggested by inferences of range expansion and
past fragmentation followed by the range expansion in our
NCA. The clinal distribution of the two clades thus
appears to be a consequence of possibly inhabiting
different refugia, with range expansions during the warmer
interglacial periods leading to varying distributional
overlaps of the two clades. Two divergent clades of
haplotypes, thought to have expanded from glacial refugia
in southern India and possibly Peninsular Malaysia or
Indochina, have also been reported in the dhole (Cuon
alpinus;Iyengar et al. 2005). It was also found that
Myanmar had haplotypes of both dhole clades, although
the limited sampling precluded testing for a secondary
zone of contact. In an earlier study, two divergent clades of
haplotypes were also found in the rhesus macaque
(Macaca mulatta;Melnick et al. 1993) in Asia, and
Pleistocene glaciations were thought to have created the
separation between the clades around the Brahmaputra
River in northeastern India.
An alternative hypothesis to allopatric fragmentation of
E. hysudricus populations giving rise to the two mtDNA
clades is that of lineage retention within a single
population. We find that the historical effective population
size during the last population bottleneck (less than 50
females in each clade with upper limits of a few thousand
females in each clade, and approximately 23 600 and
31 500 females, based on 1.6 and 2.1 Myr divergences
between clades respectively, if treated as a single
population) is smaller than the 29 800 (based on a
1.6 Myr divergence between clades) or 39 800 (based on
a 2.1 Myr divergence) females that would be required at a
minimum for lineage retention based on applying the
formula of Georgiadis et al. (1994). The plausibility of
several such bottlenecks in the past makes lineage
retention an unlikely explanation for the coexistence of
the aand bclades.
Thus, we also invoke extensive movement to explain
the distribution of the two Asian elephant mtDNA clades,
but unlike Fleischer et al. (2001) we maintain that this
movement was mostly ancient, largely as a response to
climatic conditions, with details of movement possibly
having been additionally influenced by the presence of
heterospecific proboscideans competing for similar niches.
While we cannot detect human-assisted movement of
b-clade haplotypes across Asia, the arrival of the aclade in
Sri Lanka due to trade in elephants cannot be ruled out.
The fairly high frequency of a-clade haplotypes in Sri
Lanka and the presence of a unique haplotype (AG) seem
to support a natural colonization of Sri Lanka. However,
the conspicuous absence of the aclade in central and
southern India, the confinement of the aclade within Sri
Lanka to the southern region, the established historical
trade in elephants between Sri Lanka and Myanmar (see
Sukumar 2003; electronic supplementary material 11)
and the presence of two out of the three Sri Lankan
a-clade haplotypes (AE and AF; the third haplotype AG
differs from AF by a single mutation) in or close to
Myanmar suggest a human-assisted transfer of this clade
to Sri Lanka. Population genetic modelling of peninsular
Indian populations may help in examining whether
elephant captures and reduction in population size due
to historical habitat loss in these regions could have led to
the extinction of the aclade in southern India following a
natural colonization of Sri Lanka by this clade.
This study of phylogeography is unique in sampling
over 1 per cent of the entire population of a large mammal.
We sampled all the major Asian elephant populations with
the exception of those in Thailand. However, D-loop
sequences were available for elephants from Thailand
(GenBank sequences AF317519–AF317535, AF324827–
AF324828, AF368903; submitted by J. Fickel,
D. Kieckfeldt, T. B. Hildebrandt & C. Pitra 2000–2001,
unpublished data) and, although we did not use these
sequences as they were not yet published and were 188 bp
shorter than our other sequences, based on alignment, we
inferred these sequences to be either our haplotypes AB,
AC, AD, AE, AH, BH, BO, BP, BQ and BW, or ones
closely related to them. The presence of these haplotypes
in Thailand is consistent with our proposed phylogeo-
graphic explanation: bclade haplotypes are very similar in
composition to those in Myanmar, and the increased
number of aclade haplotypes geographically close to
Myanmar supports the idea of Myanmar being the
ancestral area of the aclade.
(b)Potential limitations of the study
As with most phylogenetic studies that calibrate dates
based on fossil records, this study also assumes that
divergences based on fossil morphology are reflected in the
gene tree, which may not always be true. Since divergent
clades with coalescence times of over 1 Myr exist in the
Asian elephant, the African elephants (Roca et al. 2005)
and the mammoth (Barnes et al. 2007;Gilbert et al. 2008),
it is possible that extinct species of Elephas also showed
similar unusual patterns. That would lead to larger
uncertainties in the coalescence times of the two clades
and their times of expansions. The molecular dates are
calibrated based on the fossil date for elephantid–
mastodon divergence (Rohland et al. 2007), and therefore
rely on that date being correct (see electronic supple-
mentary material 1).
It has been shown that the NCA produces a large
number of false positives when a panmictic population is
considered (Panchal & Beaumont 2007). If structured
populations show the same results as panmictic popu-
lations, it is possible that some of our inferences in the
NCA are false positives. However, based on simulations in a
panmictic population, the inference of past fragmentation
900 T. N. C. Vidya et al. Asian elephant phylogeography
Proc. R. Soc. B (2009)
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followed by range expansion almost never appears
(Panchal & Beaumont 2007). Therefore, our main
inference of the two clades having arisen due to the past
fragmentation followed by range expansion is not likely to
be a false positive. The test for secondary contact of
unrelated haplotypes in Myanmar also suggests that the
pattern is real. In addition, since the dispersal–vicariance
analysis inferred Sri Lanka and Sumatra as the areas of
origin of the b1 and b2 subclades, respectively, and
Myanmar as an ancestral area of the aclade, and these
areas coincide with Pleistocene glacial refugia, our
inference of a contraction–expansion scenario would be
supported even in the absence of the NCA.
Future work is required to investigate whether the
pattern shown by mtDNA is also shown by nuclear DNA
so that conservation measures are based on results from
multiple genetic markers. A disassociation between
mtDNA and nuclear genes has been observed in the
African elephants (Roca et al. 2005;Roca 2008). In the
Asian elephant, nuclear microsatellite data are available
for India and there is a concordance in population genetic
structure discerned by mtDNA and nuclear DNA
(Vidya et al. 2005).
(c)Population structure
Most of the populations we examined showed distinct
frequencies of various haplotypes and were consequently
significantly differentiated from one another. It is inter-
esting that while Sri Lanka was significantly differentiated
from southern India, it was not significantly differentiated
from central India and Myanmar, which are geographi-
cally farther away. An analysis of population structure
alone would allow this absence of differentiation to be
interpreted entirely as a result of past trade in elephants.
However, F
ST
is an inadequate measure of whether this
movement of matrilines had its origin in ancient
colonization patterns or in historically recent trade in
elephants, and, as explained above, an analysis of
population history supports the former. We therefore
emphasize the need for analyses of population history in
addition to population structure.
Molecular analysis was supported by a USFWS-Asian
Elephant Conservation Fund grant to Prithiviraj Fernando
and D.J.M., a Center for Environmental Research and
Conservation Seed Grant and the Laboratory for Genetic
Investigation and Conservation, Columbia University.
A visiting scholarship was given to T.N.C.V. by Columbia
University. Sequencing from blood-extracted DNA was
carried out by the sequencing facility at the Department of
Biochemistry, Indian Institute of Science. Field sampling was
funded by the Ministry of Environment and Forests, the
Government of India. Samples were collected with research
permissions from the state forest departments of Uttaranchal,
West Bengal, Arunachal Pradesh, Assam, Meghalaya, Orissa,
Jharkhand, Tamil Nadu, Karnataka and Kerala, in India; the
Ministry of Forestry and the Forest Department, Myanmar;
and the Forest Protection Department, Vietnam. We
thank S. Varma, C. Arivazhagan, T. R. Shankar Raman,
G. Dharmarajan and N. Baskaran from the Indian Institute of
Science; G. Polet, WWF Cat Tien National Park Conserva-
tion Project, Vietnam; Y. Htut, Forest Department, Myan-
mar; W. Htun and T. H. Aung , Myanmar Timber
Enterprises; S. Tint and M. Thinn, Ministry of Forestry,
Myanmar; T. V. Thanh, Forest Protection Department,
Vietnam; and N. X. Dang, Institute of Ecology and Biological
Resources, Vietnam, for their help in obtaining samples. Field
assistance was provided by K. Krishna, R. Mohan and many
forest department trackers. We thank Prithiviraj Fernando for
his support and comments on the manuscript, and Avinash
Nanda, Par th Chauhan and Robin Dennell for their
discussion about the fossil records. Prof. W. Hill, three
anonymous referees and a Board Member provided com-
ments that helped to improve the manuscript.
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Correction
Range-wide mtDNA phylogeography yields insights into the origins of Asian elephants
T. N. C. Vidya, Raman Sukumar and Don J. Melnick
Proc. R Soc. B 276, 893– 902 (7 March 2009; Published online 18 November 2008) (doi:10.1098/rspb.2008.1494)
In Vidya et al. [1], the last paragraph on page 896 (continuing to page 897; Results section) provided the rate of
sequence divergence incorrectly as the mutation rate, which was used for the calculation of mean times of expansion
and effective population sizes. We provide the correct values in the two paragraphs below. However, these changes do
not make any difference to the inferences drawn or to the conclusions of the paper.
The last paragraph on page 896 should read as follows:
Analysis of mismatch distributions showed that observed data were explained by the fitted models, with the combined
data of the two clades indicating demographic stability, and the aand bclades indicating population expansion (see
electronic supplementary material, S8). The HKY-corrected distance between clades was 0.025 and the rate of sequence
divergence in the mtDNA segment was estimated to be 1.6 and 1.2 per cent per Myr based on the mean divergence
times, between the Asian and African elephants, of 6.6 and 8.8 Myr ago, respectively. This was considerably lower
than the rate (3.5%) calculated by Fleischer et al.[2], partly owing to the different time estimate used and partly
owing to the smaller HKY-corrected distance between clades in our larger sample. Based on the values of
t
(¼2
m
t
generations;
m
is the mutation rate over the entire sequence analysed and tis the time to expansion of a population
with initial effective number of females ¼N
0
to a final size N
1
) from the mismatch distributions, a generation time of
27 years estimated (as the average age of reproducing females) from field data in southern India (C. Arivazhagan,
T. N. C. Vidya, R. Sukumar 2001– 2003, unpublished data), and rates of sequence divergence of 1.6 per cent (diver-
gence time between clades of 1.6 Myr) and 1.2 per cent (divergence time between clades of 2.1 Myr) per Myr,
respectively, the mean times of expansion of the aclade were approximately 255 700 and 340 900 yr ago and mean
times of expansion of the bclade were 766 100 and 1 021 400 yr ago. N
0
was very low in both clades but confidence
limits were large and upper limits of N
0
were 4380 in the aclade and 9615 in the bclade based on a 1.6 Myr divergence
between clades and 5841 in the aclade and 12 820 in the bclade based on a 2.1 Myr divergence. N
0
and N
1
(¼
u
1
/2
m
;
estimation of mutation parameter
u
1
thought to be biased upwards [3]) were both approximately 47 000 (based on a 1.6
Myr divergence) or 63 000 (based on a 2.1 Myr divergence) based on the combined data of both clades, although the
two clades seemed too different demographically to be combined.
The last but one paragraph on page 900 (Discussion) should read as follows:
An alternative hypothesis to allopatric fragmentation of Elephas hysudricus populations giving rise to the two mtDNA
clades is that of lineage retention within a single population. We find that the historical effective population size
during the last population bottleneck of less than 50 females in each clade with upper limits of a few to several thousand
females in each clade is smaller than the 17 500 females for the aclade or 30 700 females for the bclade that would be
required at a minimum for lineage retention based on applying the formula of Georgiadis et al.[4], using coalescence
times (from the Bayesian tree) of 0.86 and 1.58 Myr for the aand bclades, respectively. The plausibility of several such
bottlenecks in the past make lineage retention an unlikely explanation for the coexistence of the aand bclades.
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