Molecular Phylogenetics and Evolution 40 (2006) 517–531
www.elsevier.com/locate/ympev
A molecular phylogeny of tortoises (Testudines: Testudinidae)
based on mitochondrial and nuclear genes
Minh Le a,b,¤, Christopher J. Raxworthy a, William P. McCord c, Lisa Mertz a
a
Department of Herpetology, Division of Vertebrate Zoology, American Museum of Natural History, Central Park West at 79th Street,
New York, NY 10024, USA
b
Department of Ecology, Evolution, and Environmental Biology (E3B), Columbia University, 2960 Broadway, New York, NY 10027, USA
c
East Fishkill Animal Hospital, 455 Route 82, Hopewell Junction, New York 12533, USA
Received 9 October 2005; revised 1 March 2006; accepted 2 March 2006
Available online 5 May 2006
Abstract
Although tortoises of the family Testudinidae represent a familiar and widely distributed group of turtles, their phylogenetic relationships have remained contentious. In this study, we included 32 testudinid species (all genera and subgenera, and all species of Geochelone,
representing 65% of the total familial species diversity), and both mitochondrial (12S rRNA, 16S rRNA, and cytb) and nuclear (Cmos
and Rag2) DNA data with a total of 3387 aligned characters. Using diverse phylogenetic methods (Maximum Parsimony, Maximum
Likelihood, and Bayesian Analysis) congruent support is found for a well-resolved phylogeny. The most basal testudinid lineage includes
a novel sister relationship between Asian Manouria and North American Gopherus. In addition, this phylogeny supports two other major
testudinid clades: Indotestudo + Malacochersus + Testudo; and a diverse clade including Pyxis, Aldabrachelys, Homopus, Chersina, Psammobates, Kinixys, and Geochelone. However, we Wnd Geochelone rampantly polyphyletic, with species distributed in at least four independent clades. Biogeographic analysis based on this phylogeny is consistent with an Asian origin for the family (as supported by the fossil
record), but rejects the long-standing hypothesis of South American tortoises originating in North America. By contrast, and of special
signiWcance, our results support Africa as the ancestral continental area for all testudinids except Manouria and Gopherus. Based on our
systematic Wndings, we also propose modiWcations concerning Testudinidae taxonomy.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Testudinidae; Tortoises; Systematics; Taxonomy; Biogeography; 12S; 16S; cytb; Cmos; Rag2
1. Introduction
The turtle family Testudinidae includes 49 living species of
tortoises in 12 extant genera, all of which are completely terrestrial, and which together represent about 18% of the extant
world turtle diversity (Ernst and Barbour, 1989; Uetz, 2005).
Testudinids are the most broadly distributed non-marine turtle family, occurring on all subpolar continents except Australia, and occupying a wide diversity of habitats varying from
rain forests in Southeast Asia and South America, to deserts
in North America and Africa. Morphologically, tortoises also
*
Corresponding author. Fax: +1 212 769 5031.
E-mail address: [email protected] (M. Le).
1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2006.03.003
show a great diversity of sizes and forms. The largest tortoises,
in the Galápagos (Geochelone nigra), can reach 1.5 m in
length, yet the speckled padloper (Homopus signatus) is
mature at only 10 cm in length. Most genera have highly
domed and rigid carapaces, but hinged-back carapaces occur
in the genus Kinixys, and the genus Malacochersus has a
remarkable Xattened and Xexible carapace for occupying rock
crevices (Crumly, 1984a; Ernst and Barbour, 1989).
Despite previous research, phylogenetic relationships
within the family Testudinidae have remained controversial (Caccone et al., 1999a; Crumly, 1982, 1984a; Gerlach,
2001, 2004; Meylan and Sterrer, 2000; Parham et al.,
2006). One of the perceived problems, voiced by several
authors, concerns the high level of morphological
convergences suspected for some traditionally used
518
M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531
characters (AuVenberg, 1974; Bramble, 1971; Pritchard,
1994). In addition, all testudinid molecular studies to
date have been limited in terms of taxonomic sampling
because they addressed speciWc questions of relationship
within smaller subsets of the family (with the possible
exception of Cunningham’s (2002) study, but this has not
yet become available). These studies included relationships within the genus Gopherus (Lamb and Lydeard,
1994), origin and relationships of Malagasy tortoises
(Caccone et al., 1999a), origin of Galápagos tortoises
(Caccone et al., 1999b), origin of Indian Ocean tortoises
(Austin and Arnold, 2001; Austin et al., 2003; Palkovacs
et al., 2003), relationships within the genus Indotestudo
(Iverson et al., 2001), and relationships within the genus
Testudo (Fritz et al., 2005; Parham et al., 2006; van der
Kuyl et al., 2002).
Nevertheless, the monophyly of the family has been continuously supported by both molecular and morphological
studies; with two groups: Geoemydidae and Emydidae considered sister to testudinids (Crumly, 1984a; GaVney and
Meylan, 1988; Gerlach, 2001; Krenz et al., 2005; Near et al.,
2005; Spinks et al., 2004; van der Kuyl et al., 2002).
Within the Testudinidae, establishing the monophyly of
the genus Geochelone has proven to be especially problematic. As the largest genus of the family, this group currently
includes 10 species, exclusive of Wve species recently
assigned to the genera Manouria and Indotestudo based on
the studies of Crumly (1984a,b) and Hoogmoed and
Crumly (1984), and six extant or recently extinct species
being placed in the genus Aldabrachelys (Dipsochelys) (Austin et al., 2003; Bour, 1982; Gerlach, 2001). AuVenberg
(1974) proposed six subgenera (including Manouria and
Indotestudo) for Geochelone based largely on zoogeographic criteria, however, Crumly (1982) was the Wrst to
argue against their complete use based on his cladistic analysis using 26 cranial characters. This view has also been
corroborated by more recent analyses, which have included
more characters and taxa (Crumly, 1984a; Gerlach, 2001).
Crumly’s (1984a) results also did not support Geochelone
monophyly (Fig. 1). More recent morphological studies
have provided conXicting results: GaVney and Meylan
(1988) and Meylan and Sterrer (2000) found support for a
monophyletic Geochelone, and Gerlach (2001) found
Geochelone to be polyphyletic. In addition, recent molecular studies have shown Malagasy Geochelone paraphyletic
with respect to Pyxis (Caccone et al., 1999a; Palkovacs
et al., 2002), or provided little resolution between Geochelone and other testudinid genera (van der Kuyl et al., 2002).
All of these molecular studies were restricted to mitochondrial genes and only included a subset of Geochelone species.
In terms of their biogeographic history, tortoises oVer an
interesting group for study because of their almost worldwide distribution, including oceanic islands, e.g., the Galápagos, the East Indies, the West Indies, Madagascar, and
the Mascarene Islands. Crumly’s (1984a) study has provided the most detailed discussion of diVerent biogeo-
graphic scenarios of testudinids to date. He suggested true
tortoises originated in North America, based on the occurrence of the oldest known fossil testudinid, Hadrianus
majusculus, from the Early Eocene. Crumly (1984a) also
proposed that tortoises from Europe and Africa were more
closely related to Asian forms than to North American
forms, and that tortoises of the Galápagos, and the Indian
Ocean had rafted to these islands. This latter dispersal scenario has been supported by more recent studies (Austin
and Arnold, 2001; Caccone et al., 1999b; Palkovacs et al.,
2002). However, the origin of the South American tortoises
remains a matter of controversy. Most authors have concluded that tortoises invaded tropical South America from
North or Central America (AuVenberg, 1971; Gerlach,
2001; Simpson, 1943; Williams, 1950); however, both Simpson (1942) and Crumly (1984a) also suggested Africa as a
possible alternative source.
The major objective of our study was to produce a phylogeny of the Testudinidae based on an expanded molecular character dataset, which would be comprehensive at
the generic level, and include all the species available to us
(the majority of species). We ultimately were able to
include species representing all recognized and previously
proposed extant genera and subgenera, representing in
total 65% of all testudinid species. This broad taxonomic
sampling was considered especially important for resolving phylogenetic relationships within the problematic
genus Geochelone, and for this group, we were able to
include all Geochelone species. To provide a robust estimate of phylogeny, we included a broad diversity of
genes, including one mitochondrial protein coding gene
(cytb), two mitochondrial ribosomal genes (12S and 16S),
and two nuclear genes (Cmos and Rag2), the latter of
which have not been previously utilized for resolving relationships between turtles.
2. Materials and methods
2.1. Taxonomic sampling
For our testudinid ingroup, we included 34 taxa (32 species and 2 subspecies) and all genera within the family. We
also included all Geochelone species. For two species:
Indotestudo forstenii and Gopherus agassizii, for which we
were unable to obtain tissue; sequences for these species
(reported by Spinks et al., 2004) were obtained from GenBank. A complete list of all tissues, voucher specimens, and
sequences are provided in Table 1. This molecular phylogenetic analysis includes the most comprehensive taxon sampling achieved to date for testudinids.
For outgroups, we included two species of Geoemydidae
(Rhinoclemmys melanosterna and R. nasuta) and two species of Emydidae (Glyptemys insculpta and Deirochelys
reticularia) based on their reported sister relationships to
testudinids recovered by both morphological and molecular evidence (GaVney and Meylan, 1988; Honda et al., 2002;
Krenz et al., 2005; Near et al., 2005; Spinks et al., 2004).
M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531
519
Fig. 1. Crumly’s (1984a) preferred hypothesis of relationships for the family Testudinidae, based on 57 morphological characters. In contrast to all the
other genera shown here, the genus Geochelone is paraphyletic.
2.2. Molecular data
Both mitochondrial and nuclear DNA were utilized in
this study to resolve relationships at all phylogenetic levels
within the family. Similar approaches have proved successful with other groups exhibiting similar levels of phylogenetic diversity (see Barker et al., 2004; Georges et al., 1999;
Hillis et al., 1996; Pereira et al., 2002; Saint et al., 1998). We
sequenced three regions of the mitochondrial DNA
(mtDNA): the complete cytochrome b sequence, and sections of both the 12S and 16S rRNA genes. For some taxa
(see Table 1), we downloaded cytb and 12S sequences from
GenBank originating from studies by Spinks et al. (2004),
Palkovacs et al. (2002), Caccone et al. (1999a,b), and van
der Kuyl et al. (2002). We also sequenced two nuclear fragments of the Cmos and Rag2 genes. All primers used for
this study (including those developed during the course of
this study) are shown in Table 2.
520
M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531
Table 1
GenBank accession numbers, and associated voucher specimens/tissues that were used in this study
Species names
GenBank
No. (12S)
GenBank
No. (16S)
GenBank
No. (cytb)
GenBank
No. (Cmos)
GenBank
No. (Rag2)
Voucher numbers
for this study
Chersina angulata
Aldabrachelys arnoldii
Aldabrachelys dussumieri
Aldabrachelys hololissa
Geochelone carbonaria
Geochelone chilensis
Geochelone denticulata
Geochelone elegans
Geochelone nigra
Geochelone p. pardalis
Geochelone p. babcocki
Geochelone platynota
Geochelone radiata
Geochelone sulcata
Geochelone yniphora
Gopherus agassiziia
Gopherus polyphemus
Homopus boulengeri
Homopus signatus
Indotestudo elongata
Indotestudo forsteniia
Indotestudo travancorica
Kinixys belliana
Kinixys homeana
Malacochersus tornieri
Manouria emys emys
Manouria emys phayrei
Manouria impressa
Psammobates tentorius
Pyxis arachnoides
Pyxis planicauda
Testudo graeca
Testudo horsWeldii
Testudo kleinmanni
Glyptemys insculpta
Deirochelys reticularia
Rhinoclemmys melanosterna
Rhinoclemmys nasuta
DQ497248
AY081779
DQ497249
AY081783
AB090019
AF175336
DQ497250
AY081785
AF020885
DQ497251
DQ497252
DQ497253
AF020883
AY081787
AF020882
AY434630
AF020879
DQ497254
DQ497255
DQ497256
DQ497269
AY081780
DQ497270
AY081784
AF192926
AF192924
AF192927
AY081786
AF020892
DQ497271
DQ497272
DQ497273
AF020890
AY081788
AF020889
DQ497325
DQ497326
DQ497327
DQ497328
DQ497329
DQ497330
DQ497331
DQ497332
DQ497333
DQ497334
DQ497335
DQ497336
DQ497337
DQ497338
DQ497339
DQ497361
DQ497362
DQ497363
DQ497364
DQ497365
DQ497366
DQ497367
DQ497368
DQ497369
DQ497370
DQ497371
DQ497372
DQ497373
DQ497374
DQ497375
DQ497340
DQ497341
DQ497342
DQ497343
DQ497376
DQ497377
DQ497378
DQ497379
DQ497257
DQ497258
DQ497259
DQ497260
DQ497261
DQ497262
DQ497263
DQ497264
AF020880
AF020881
AF175330
AB090020
AF175332
DQ497265
DQ497266
DQ497267
DQ497268
DQ497277
DQ497278
DQ497279
DQ497280
DQ497281
DQ497282
DQ497283
DQ497284
AF020887
AF020888
DQ497285
DQ497286
DQ497287
DQ497288
DQ497289
DQ497290
DQ497291
DQ497292
DQ497293
DQ497294
DQ497295
DQ497296
DQ497297
DQ497298
DQ497299
DQ497300
DQ497301
DQ497302
DQ497303
DQ497304
DQ497305
DQ497306
AY434562
DQ497307
DQ497308
DQ497309
DQ497310
AY434561
DQ497311
DQ497312
DQ497313
DQ497314
DQ497315
DQ497316
DQ497317
DQ497318
DQ497319
DQ497320
DQ497321
DQ497322
DQ497323
AF258876
AF258877
AY434590
DQ497324
DQ497344
DQ497345
DQ497346
DQ497347
DQ497348
DQ497349
DQ497350
DQ497351
DQ497352
DQ497353
DQ497354
DQ497355
DQ497356
DQ497357
DQ497358
DQ497359
DQ497360
DQ497380
DQ497381
DQ497382
DQ497383
DQ497384
DQ497385
DQ497386
DQ497387
DQ497388
DQ497389
DQ497390
DQ497391
DQ497392
DQ497393
DQ497394
DQ497395
DQ497396
AMCC157819
YU
YU
YU
AMCC157837
AMCC157844
AMCC157838
AMCC106883
FMNH262225
AMCC157839
AMCC157840
AMCC157841
AMCC106882
AMCC111025
AMCC125847
—
FMNH262226
AMCC106886
AMCC125859
AMCC157712
—
AMCC142999
AMCC106880
KU290452
AMCC157829
AMCC157827
AMCC157828
AMCC157716
AMCC157843
RAN34680
AMCC157845
AMCC157846
AMCC144096
AMCC125843
AMCC157833
AMCC157832
AMCC157821
AMCC157826
AF020886
DQ497274
DQ497275
DQ497276
All sequences generated by this study have accession numbers: DQ497248 – DQ497396; YU, Yale University Collection; FMNH, Field Museum of Natural History; AMCC, Ambrose Monell Cryo Collection, American Museum of Natural History (http://research.amnh.org/amcc); RAN, Ronald A. Nussbaum Field Series, University of Michigan, Museum of Zoology; KU, Kansas University Natural History Museum.
a
Genbank sequences only.
DNA was extracted from tissues and blood samples
using the DNeasy kit (Qiagen) following manufacturer’s
instructions for animal tissues. PCR volume for
mitochondrial genes contained 42.2 l (18 l water, 4 l of
buVer, 4 l of 20 mM dNTP, 4 l of 25 mM MgCl2, 4 l of
each primers, 0.2 l of Taq polymerase (Promega), and 4 l
of DNA). PCR conditions for these genes were: 95 °C for
5 min to activate the Taq; with 42 cycles at 95 °C for 30 s,
45 °C for 45 s, 72 °C for 60 s; and a Wnal extension of
6 min. Nuclear DNA was ampliWed by HotStar Taq Mastermix (Qiagen) since this Taq performs well on samples
with low-copy targets and the Taq is highly speciWc. The
PCR volume consists of 21 l (5 l water, 2 l of each
primer, 10 l of Taq mastermix, and 2 l of DNA or
higher depending on the quantity of DNA in the Wnal
extraction solution). PCR conditions for nuclear genes
were the same as above except the Wrst step (95 °C) was
carried out in 15 min, and the annealing temperatures
were 52 and 58 °C for Rag2 and Cmos, respectively.
PCR products were visualized using electrophoresis
through a 2% low melting-point agarose gel (NuSieve GTG,
FMC) stained with ethidium bromide. For reampliWcation
reactions, PCR products were excised from the gel using a
Pasteur pipette, and the gel plug was melted in 300 l sterile
water at 73 °C for 10 min. The resulting gel-puriWed product
was used as a template in 42.2l reampliWcation reactions
with all PCR conditions similar to those used for mitochondrial genes. PCR products were cleaned using PerfectPrep®
PCR Cleanup 96 plate (Eppendorf) and cycle sequenced
using ABI prism big-dye terminator according to manufac-
M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531
521
Table 2
Primers used in this study
Primer
Position
Sequence
Reference
L1091 (12S)
H1478 (12S)
AR (16S)
BR (16S)
CytbG (cytb)
GLUDGE (cytb)
CytbJSi (cytb)
CytbJSr
THR (cytb)
THR-8 (cytb)
CM1 (Cmos)
CM2 (Cmos)
Cmos1 (Cmos)
Cmos3 (Cmos)
F2 (Rag2)
F2-1 (Rag2)
R2-1 (Rag2)
491
947
1959
2561
14368
14358
15011
15030
15593
15585
163
820
163
812
601
590
1312
5⬘-AAAAAGCTTCAAACTGGGATTAGATACCCCACTAT-3⬘
5⬘-TGACTGCAGAGGGTGACGGGCGGTGTGT-3⬘
5⬘-CGCCTGTTTATCAAAAACAT-3⬘
5⬘-CCGGTCTGAACTCAGATCACGT-3⬘
5⬘-AACCATCGTTGTWATCAACTAC-3⬘
5⬘-TGATCTTGAARAACCAYCGTTG-3⬘
5⬘-GGATCAAACAACCCAACAGG-3⬘
5⬘-CCTGTTGGGTTGTTTGATCC-3⬘
5⬘-TCATCTTCGGTTTACAAGAC-3⬘
5⬘-GGTTTACAAGACCAATGCTT-3⬘
5⬘-GCCTGGTGCTCCATCGACTGGGA-3⬘
5⬘-GGGTGATGGCAAAGGAGTAGATGTC-3⬘
5⬘-GCCTGGTGCTCCATCGACTGGGATCA-3⬘
5⬘-GTAGATGTCTGCTTTGGGGGTGA-3⬘
5⬘-CAGGATGGACTTTCTTTCCATGT-3⬘a
5⬘-TTCCAGAGCTTCAGGATGG-3⬘
5⬘-CAGTTGAATAGAAAGGAACCCAAGT-3⬘b
Kocher et al. (1989)
Kocher et al. (1989)
Palumbi et al. (1991)
Palumbi et al. (1991)
Spinks et al. (2004)
Palumbi et al. (1991)
Spinks et al. (2004)
Spinks et al. (2004)
Spinks et al. (2004)
Spinks et al. (2004)
Barker et al. (2002)
Barker et al. (2002)
This study
This study
This study
This study
This study
a
ModiWed from F2R2 (Barker et al., 2004).
ModiWed from R2R1 (Barker et al., 2004); Cmos and Rag2 primer positions corresponding to the positions in chicken genomes with GenBank numbers of M19412 and M58531, respectively; primer positions for mitochondrial genes corresponding to the positions in the complete mitochondrial genome
of Chrysemys picta (Mindell et al., 1999).
b
turer recommendation. Sequences were generated in both
directions on an ABI 3100 Genetic Analyzer.
2.3. Phylogenetic analysis
We aligned sequence data using ClustalX v1.83
(Thompson et al., 1997) with default settings. Data were
analyzed using both parametric and non-parametric methods (see Kolaczkowski and Thornton, 2004): maximum
parsimony (MP) and maximum likelihood (ML) using
PAUP*4.0b10 (SwoVord, 2001) and Bayesian analysis
using MrBayes v3.1 (Huelsenbeck and Ronquist, 2001).
For maximum parsimony analysis, we conducted heuristic analyses with 100 random taxon addition replicates
using the tree-bisection and reconnection (TBR) branch
swapping algorithm in PAUP, with no upper limit set for
the maximum number of trees saved. Bootstrap support
(BP) (Felsenstein, 1985a) was evaluated using 1000 pseudoreplicates and 100 random taxon addition replicates.
Bremer indices (BI) (Bremer, 1994) were determined using
Tree Rot 2c (Soreson, 1999). All characters were equally
weighted and unordered. Gaps in sequence alignments
were treated as a Wfth character state (Giribert and
Wheeler, 1999). Phylogenetic congruence between partitions of the nuclear and mitochondrial datasets was
assessed using the incongruence length diVerence (ILD)
test (Farris et al., 1994). Although the utility of this test as
a measure for data combination has been questioned (e.g.,
see Darlu and Lecointre, 2002; Yoder et al., 2001), we
employed this test to further explore characteristics of
our partitioned data, along with phylogenetic analyses
that we also conducted using these same partitions. These
data were partitioned by genes and DNA organelle
(nuclear or mitochondrial), and also analyzed in their
entirety.
For maximum likelihood analysis the optimal model for
nucleotide evolution was determined using Modeltest V3.7
(Posada and Crandall, 1998). Analyses used a randomly
selected starting tree, and heuristic searches with simple taxon
addition and the TBR branch swapping algorithm. Support
for the likelihood hypothesis was evaluated by bootstrap
analysis with 100 replications and simple taxon addition.
For Bayesian analyses we used the optimal model determined using Modeltest with parameters estimated by MrBayes Version 3.1. Analyses were conducted with a random
starting tree and run for 5 £ 106 generations. Four Markov
chains, one cold and three heated (utilizing default heating
values), were sampled every 1000 generations. Log-likelihood
scores of sample points were plotted against generation time
to detect stationarity of the Markov chains. Trees generated
prior to stationarity were removed from the Wnal analyses
using the burn-in function. Two independent analyses were
started simultaneously. The posterior probability values (PP)
for all clades in the Wnal majority rule consensus tree are
reported. We ran analyses on both combined and partitioned
datasets to examine the robustness of the tree topology
(Brandley et al., 2005; Nylander et al., 2004). In the partitioned analyses, we divided the data into 11 separate partitions, including 12S, 16S, and the other nine based on gene
codon positions (Wrst, second, and third) in cytb, Cmos, and
Rag2. Optimal models of molecular evolution for each partition were selected using Modeltest and then assigned to these
partitions in MrBayes 3.1 using the command APPLYTO.
Model parameters were estimated independently for each
data partition using the UNLINK command.
To test alternative hypotheses of relationships, corresponding tree topologies were compared using the Wilcoxon
signed-ranks test (Felsenstein, 1985b; Templeton, 1983), to
determine if tree length diVerence could have resulted from
chance alone (Larson, 1998). Alternative tree topologies were
522
M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531
Fig. 2. The maximum parsimony tree based on all data combined. This is the strict consensus of two most parsimonious trees, (TL D 4292; CI D 0.39;
RI D 0.50). The combined Wve-gene dataset includes 3387 aligned characters of which 880 are potentially parsimony informative. Numbers above and
below branches are bootstrap (>50%) and Bremer values, respectively. The geographic distribution of each taxon is indicated in the right column.
constructed in MacClade (Maddison and Maddison, 2001)
and then used as constraint trees by importing to PAUP. To
determine biogeographic support for ancestral areas or vicariance events between clades, we utilized Dispersal-Vicariance
Analysis (Ronquist, 1997), as implemented in the program
DIVA 1.1 (Ronquist, 1996). Fully resolved trees based on
MP and ML topologies were imported using distribution
areas (as depicted in Figs. 2 and 4). All optimization settings
used the default, including the maximum number of ancestral
areas that were set as the total number of unit areas.
3. Results
Sequences for all Wve genes were obtained for each of the
32 taxa with tissues. The Wnal matrix consists of 3387
aligned characters: 12S (408 characters), 16S (583 characters), cytb (1140 characters), Cmos (602 characters), and
Rag2 (654 characters). Five taxa have sequences that
include gaps at three loci (cytb, Cmos, and Rag2). For cytb,
Geochelone pardalis babcockii has a single indel that is three
base pairs long at positions 451–453. For Cmos, G. platynota and G. elegans has a single indel that is six base pairs
long corresponding to positions 489–494 of the complete
chicken Cmos sequence (GenBank Accession No. M19412).
For Rag2, Chersina angulata and Kinixys homeana has a
single indel of three base pairs long corresponding to positions 1093–1095 of the partial Pelodiscus sinensis Rag2
sequence (GenBank Accession No. AF369089).
The IDL tests found no signiWcant incongruence
between mitochondrial genes (12S vs 16S, p D 0.07; cytb vs
12S, p D 0.89; cytb vs 16S, p D 0.97), between nuclear genes
(Cmos vs Rag2, p D 0.16), and between the nuclear and
mitochondrial partitions (nuclear DNA vs mtDNA,
p D 0.7). Using MP, we also analyzed the data by gene and
organelle partitions (see Tables 3 and 4). The analysis indicated that Clades 3, 4, and 5 (see Fig. 4) consistently receive
strong support from both mitochondrial and nuclear genes
(Table 3). For the following partitions, the strict consensus
trees include little phylogenetic resolution: 12S, 16S, Cmos,
Rag2, and all the nuclear data. For cytb, although MP
recovered only two equally parsimonious trees, the strict
consensus is poorly resolved concerning deeper level relationships between clades including multiple genera. The
strict consensus tree using all mtDNA was unresolved for
M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531
523
Table 3
MP analyses of data partitions with BP support values for each clade as deWned in Fig. 4
Data
Clade 1 (%)
Clade 2 (%)
Clade 4 (%)
Clade 5 (%)
Clade 6 (%)
Clade 7 (%)
Clade 8 (%)
12S
16S
Cytb
<50
<50
55
63
60
<50
64
95
75
<50
<50
<50
<50
99
<50
<50
<50
<50
<50
<50
<50
<50
<50
<50
79
94
100
81
94
<50
62
72
Cmos
Rag2
<50
<50
<50
<50
70
<50
75
92
62
<50
<50
<50
<50
<50
<50
<50
All Nuclear
<50
55
87
99
72
<50
<50
<50
All MtDNA
Clade 3 (%)
Table 4
Data partitions subject to phylogenetic analyses with maximum parsimony
Data
Total number of
aligned sites
Parsimony
informative characters
12S
16S
cytb
408
583
1140
125
169
475
602
654
All MtDNA
All Nuclear
All data
Cmos
Rag2
MP tree
length
RI
value
CI
value
174
211
616
514
773
2671
0.61
0.58
0.44
0.47
0.44
0.33
117
41
2
66
45
111
90
166
122
0.79
0.85
0.73
0.81
105
5080
2131
1256
769
111
1001
201
3989
293
0.49
0.80
0.37
0.75
3
16
3387
880
1201
4292
0.50
0.39
2
basal nodes of the clade including all the Geochelone
groups—the same clade as recovered in the combined analysis (see Fig. 2). All nodes with BI D 1 in the combined MP
analysis collapsed in this mtDNA tree. Based on the
generally poorly resolved tree topologies recovered using
the partitioned data, and our IDL partition results, we thus
consider the combined data as providing the optimal set
upon which to base phylogenetic inference.
Using the combined dataset (3387 aligned characters,
880 potentially parsimony informative), the MP analysis
recovered two most parsimonious trees with 4292 steps
(CI D 0.39; RI D 0.50). The strict consensus is shown in
Fig. 2. This tree is well resolved with 72% of the nodes
receiving strong support (BP 7 70%) (Hillis and Bull,
1993), and 9% receiving BP support from 65 to 69%. All
nodes in the clades, including Manouria, Gopherus, Testudo,
Indotestudo, and Malacochersus, are well supported, except
for the relationship of Testudo horsWeldii and Malacochersus. We also found that the BP value supporting the sister
relationship between Gopherus and Manouria increases signiWcantly (from 73 to 85% in the MP analysis) by excluding
the species with incomplete data, Gopherus agassizii. Within
the diverse clade, which includes Geochelone, Aldabrachelys,
Kinixys, Homopus, Psammobates, and Pyxis, support was
generally high except for some of the basal relationships.
Incongruence between these two MP trees was conWned to
the genus Aldabrachelys.
Results, including model parameters, of the ML and
Bayesian analyses are shown in Fig. 3 (tree topologies of
both analyses are identical). Similar to the MP analysis, the
ML tree is well resolved with 78% of the nodes receiving
Variable
characters
Number of equally
parsimonious trees
strong support (BP 7 70%). The most weakly supported
nodes are distributed in the clade containing Geochelone.
The topology of the ML cladogram is congruent with the
MP topology for all branches except the following: Malacochersus tornieri sister to either Testudo (BP D 68%, MP) or
Indotestudo (BP D 60%, ML), G. radiata sister to either
Pyxis (BP D 65%, MP) or Geochelone yniphora (BP D 82%,
ML), and the clade including Kinixys + South American
Geochelone (G. carbonaria group) sister to either the clade
including the Malagasy and Indian Ocean species, Chersina, Homopus, Psammobates, and G. pardalis (<50%, MP)
or the clade including G. elegans, G. platynota, and G. sulcata (87%, ML).
In the combined Bayesian analysis (Fig. 3), ¡ln L scores
reached equilibrium after 19,000 generations while the partitioned Bayesian analysis scores reached stationarity after
27,000 generations in both runs. Phylogenetic relationships
supported by the combined and partitioned data are identical to the ML results. Only minor diVerences in the PP values were found between combined and partitioned
Bayesian cladograms: PP values are higher in the partitioned analysis for the following clades: Pyxis and Aldabrachelys (99%), South American testudinids (99%), Kinixys
+ South American testudinids (86%), Testudo (99%), and
A. arnoldi + A. hololissa (74%), and lower for the following:
Chersina + Homopus + Psammobates + Geochelone pardalis
+ Aldabrachelys + Pyxis + Geochelone radiata + G. yniphora
(56%), G. chilensis + G. nigra (93%), and Malacochersus
+ Indotestudo (95%).
All of our analyses, thus, found three well supported
clades of tortoises: (1) Gopherus + Manouria, (2)
524
M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531
Fig. 3. The maximum likelihood (ML) analysis phylogram based on all the data combined, using the GTR+G+I model of molecular evolution (Bayesian
tree topology is identical). Base frequency A D 0.313, C D 0.262, G D 0.193, T D 0.230. ML ¡ln L D 22007.972; rate matrix: A–C D 3.544, A–G D 13.646, A–
T D 3.478, C–G D 0.897, C–T D 42.995, G–T D 1.000; proportion of invariable site (I) D 0.470; gamma distribution shape parameter (G) D 0.553. Total
number of rearrangements tried D 13077, score of best tree found D 21992.392. Numbers above and below branches are ML bootstrap values (>50%) and
Bayesian posterior probabilities, respectively. See Fig. 2 for taxon distribution. ¤The sister relationship between A. arnoldi and A. hololissa, cannot be seen
due to the short branch length.
Indotestudo + Testudo + Malacochersus, and (3) Geochelone
+Pyxis+Aldabrachelys+Homopus+ Chersina + Psammobates
+ Kinixys. It is also evident from these analyses that Geochelone, as currently deWned, is rampantly polyphyletic.
Our phylogenetic results place Geochelone in at least
four independent clades. The Wrst consists of three
species, G. elegans, G. platynota, and G. sulcata (G. elegans
group). The second clade includes all four South American species (G. carbonaria group), which are sister to
Kinixys. G. yniphora and G. radiata from Madagascar
belong to a third clade that also includes Pyxis and
Aldabrachelys. The Wnal clade includes only a single
Geochelone species, G. pardalis, which is sister to the genus
Psammobates.
Biogeographic results are summarized by the area cladogram shown in Fig. 4, which conservatively depicts the
strict congruence between our MP, ML, and Bayesian tree
topologies. The optimized ancestral areas, based on DIVA,
are also shown in Fig. 4. The DIVA ancestral area for the
family Testudinidae is identiWed as including three areas:
Asia, Africa, and North America, and requires a further
seven subsequent dispersals (to South America, Madagascar–Indian Ocean, and Asia). For this three-continent
familial ancestral area, Gopherus represents the North
American lineage, Manouria represents the Asian lineage,
and all other testudinids represent the African lineage. For
the incongruent clades between the MP and ML analyses
(see Figs. 2 and 3), DIVA ancestral areas are optimized as
M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531
525
Fig. 4. Testudinidae area cladogram based on the strict consensus of the tree topologies shown in Figs. 2 and 3. The ancestral area optimizations found by
dispersal-vicariance analysis (DIVA) are shown for each node: Asia (As), Africa (Af), Madagascar and Indian Ocean (MI), North America (NA), and
South America (SA).
follows: Malacochersus + Testudo, Africa; Malacochersus
+ Indotestudo, Africa and Asia; Chersina + Homopus+
Psammobates + Geochelonepardalis + Kinixys + the Geochelone carbonaria group + Aldabrachelys + Pyxis + Geochelone
radiata + G. yniphora, Africa; and the G. elegans group +
Kinixys + the Geochelone carbonaria group, Africa.
4. Discussion
4.1. Phylogeny
Concerning the three major clades of testudinids we Wnd
in this study, the sister group to all other tortoise species
includes Manouria and Gopherus as sister genera (Figs. 2
and 3). This is the Wrst study to sample broadly across the
family and recover this intriguing sister relationship;
although Lamb and Lydeard’s (1994) molecular study also
found weak support for this sister relationship. Other morphological and molecular studies have consistently
reported Manouria to be basal to all other tortoises
(Crumly, 1984a; GaVney and Meylan, 1988; Gerlach, 2001;
Meylan and Sterrer, 2000; Takahashi et al., 2003; Spinks
et al., 2004), with Gopherus usually placed as the next most
basal lineage for the family (Crumly, 1984a; Spinks et al.,
2004). Manouria and Gopherus are distributed in Asia and
North America, respectively, and these two genera show
obvious diVerences in morphological characters, with
Manouria exhibiting several character states considered to
be plesiomorphic, such as the wide anterior edge of the prootic bone (ranging from 80 to 92% of the posterior edge)
and the ventrally and dorsally split supracaudal scute
(Crumly, 1982). This Manouria + Gopherus sister relationship has, to the best of our knowledge, not been previously
considered or discussed, and no synapomorphies have been
proposed for the two genera (GaVney and Meylan, 1988;
Gerlach, 2001; Meylan and Sterrer, 2000; Takahashi et al.,
2003). Winokur and Legler (1975) reported mental glands
in Gopherus, and Ernst and Barbour (1989), also citing
Winokur and Legler, report mental glands in Manouria—
thus suggesting a potentially homologous character. However, our reading of Winokur and Legler’s (1975) study
clearly reveals that for testudinids, these authors only
reported mental glands for Gopherus. In addition, our own
examination of AMNH catalogued material (R-118676 and
R-119966), and living specimens, also failed to Wnd mental
glands in Manouria.
The second major clade found by this study includes
Testudo, Indotestudo, and Malacochersus. A recent molecular study by Parham et al. (2006) based on the complete
mitochondrial genome also recovered this monophyletic
group. Their MP, ML, and Bayesian analyses produced
conXicting results regarding the relationships of Malacochersus tornieri and Testudo horsWeldii. SpeciWcally, their
MP and Bayesian analyses supported the sister relationship
526
M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531
between Malacochersus and Indotestudo, but the ML analysis found Malacochersus sister to all other taxa. The position of T. horsWeldii was unresolved in the MP analysis
(strict consensus tree), but was sister to T. hermanni in the
other analyses. Another study (based on 1124 bp of cytb) by
Fritz et al. (2005) which included almost all forms of the
genus Testudo plus Indotestudo and Malacochersus was also
ambiguous regarding relationships among these three genera. Similarly, we also found conXicting branch support for
relationships within this clade: the MP analysis give moderate support (BP D 68%) for a Malacochersus + Testudo
clade, while the ML and Bayesian results supported a
Malacochersus + Indotestudo
clade
(ML
BP D 60%,
PP D 97%). Interestingly, morphological studies have consistently supported Malacochersus and Testudo as sisters
(Crumly, 1984a; GaVney and Meylan, 1988; Gerlach, 2001).
According to Gerlach (2001), the two genera share the
character of the processus inferior parietalis meeting the
quadrate and partially covering the prootic, while GaVney
and Meylan (1988) found they share four characters: presence of supranasal scales, no contact between the inguinal
and femoral scutes, a viewable ventral tip of the processus
interfenestralis, and the presence of sutures between this
process and the surrounding bones.
Our dataset unambiguously resolves relationships within
the three Indotestudo species. The sister relationship
between I. travancorica and I. elongata is recovered by MP,
ML, and Bayesian analyses with strong support. In previous studies, relationships among these species had
remained uncertain. The morphological study by Hoogmoed and Crumly (1984) found I. travancorica and I. forstenii
to be identical, yet the molecular study by Iverson et al.
(2001) found strong support for the sister relationship
between I. travancorica and I. elongata using MP, but their
ML analysis favored I. travancorica and I. forstenii as sister
taxa. The study by Spinks et al. (2004) also supported this
latter hypothesis.
For the third and largest testudinid clade, we Wnd
support for Wve internal clades: (1) Chersina + Homopus +
Psammobates+ Geochelone pardalis, (2) the Geochelone elegans group, (3) Kinixys, (4) the Geochelone carbonaria group,
and (5) Aldabrachelys + Pyxis+ Geochelone radiata and
G. yniphora. However, relationships between these clades conXict between the MP, ML, and Bayesian analyses, and corresponding branch support is also generally lower (MP
BP< 50%, ML BP < 50–87%, and Bayesian PP 57–100%).
Of special signiWcance (and as suspected by previous
researchers), we Wnd substantial polyphyly for the problematic genus Geochelone. Nevertheless, the phylogenetic
results we present here for Geochelone were not recovered
by previous morphological and molecular studies (Gerlach,
2001; Meylan and Sterrer, 2000; van der Kuyl et al., 2002;
Palkovacs et al., 2002). The strongly supported sister relationships we Wnd between G. pardalis and Psammobates,
between Chersina and Homopus; and the monophyly of
Geochelone elegans group (G. elegans, G. platynota, and G.
sulcata) were not evident in previous morphological stud-
ies; and the molecular study by Palkovacs et al. (2002)
found support for conXicting relationships concerning G.
sulcata and G. elegans between MP, ML, Bayesian, and
neighbor-joining analyses.
Although only weakly supported in terms of branch support (MP and ML BP < 50%, PP 80%), all of our analyses
support the intriguing sister relationship between African
Kinixys and the South American Geochelone carbonaria
group. This hypothesis of relationship is radical: prior morphological and molecular studies placed members of the
Geochelone carbonaria group either sister to Gopherus
(Crumly, 1984a; Gerlach, 2001), or sister to other Geochelone
species: G. elegans, G. pardalis, and G. sulcata (Crumly,
1984a; Meylan and Sterrer, 2000; Palkovacs et al., 2002; van
der Kuyl et al., 2002). Crumly (1984a) did not consider the
South American tortoises to be monophyletic (Fig. 1), but
other researchers have supported monophyly for a sampled
subset of this clade (van der Kuyl et al., 2002), or else otherwise explicitly assumed it (Caccone et al., 1999b; GaVney and
Meylan, 1988; Gerlach, 2001; Meylan and Sterrer, 2000).
The Indian Ocean clade supported by our results:
Aldabrachelys + Pyxis + Geochelone radiata + G. yniphora is
consistent with the molecular results of Austin and Arnold
(2001) and Palkovacs et al. (2002). The latter study, like
ours, found support for a sister relationship between
G. radiata and Pyxis using MP (65% BP), and an alternative sister relationship of G. radiata and G. yniphora using
Bayesian and ML analyses (ML 82% BP, 100% PP). Morphologically, the sister relationship between G. yniphora
and G. radiata has been frequently supported (e.g., Crumly,
1984a; GaVney and Meylan, 1988; Gerlach, 2001; Meylan
and Sterrer, 2000) and Gerlach (2001) reported two synapomorphies: a ventral ridge on the maxilla–premaxilla
suture and keels on the supraoccipital crest. In addition, the
close relationship between Pyxis and G. yniphora + G. radiata was also supported by Gerlach with the synapomorphy
of an indistinct fenestra postotica. By contrast, other studies (e.g., GaVney and Meylan, 1988; Meylan and Sterrer,
2000; Takahashi et al., 2003) have varied widely concerning
the relationship of Pyxis to other testudinid genera. Concerning the Indian Ocean giant tortoises (Aldabrachelys),
for all Wve genes we found almost no variation among the
three forms: A. dussumieri, A. arnoldi, and A. hololissa. Only
two sites showed variation: one for 16S (position 2004) and
the other for Cmos (position 349). These results support the
conclusions of Palkovacs et al. (2003) and Austin et al.
(2003) that all three forms may actually represent a single
species and population originating from Aldabra. However,
these molecular results need to be evaluated by carefully
examining the diagnostic morphological characters used to
describe these species in order to conWrm that these features
do not vary intraspeciWcally.
4.2. IntraspeciWc divergence
The two samples representing diVerent subspecies of
Geochelone pardalis: G. p. pardalis and G. p. babcockii have
M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531
an uncorrected pairwise sequence divergence distance of
5.9% for cytb, the fastest evolving gene in this study with
the highest proportion of variable sites (Table 4). These two
subspecies are also known to exhibit morphological diVerences. The carapace shape of G. p. babcockii is distinctly
convex dorsally, compared to the Xatter G. p. pardalis carapace; and juvenile G. p. babcockii has single carapacial
scute spots, while G. p. pardalis has two (Loveridge and
Williams, 1957). Furthermore, there is a sexually dimorphic
size diVerence: G. p. pardalis males being slightly larger
than females and G. p. babcockii males being much smaller
(Broadley, 1989). G. p. pardalis occurs in western South
Africa and southern Namibia. G. p. babcockii ranges from
Sudan and Ethiopia to Natal and from Cape Province to
southwest Africa and southern Angola (Iverson, 1992;
Loveridge and Williams, 1957). Our preliminary results
suggest that a phylogeographic study of G. pardalis is
expected to reveal high genetic divergence within this species, or species complex.
By contrast, the cytb uncorrected pairwise sequence
divergence distance between the two samples we have of
Manouria e. emys (Burmese tortoise) and M. e. phayrei
(Burmese black tortoise) is just 1.3%. Results showing even
less geographic variation for mtDNA within a testudinid
species have been recently reported for Geochelone radiata
(Leuteritz et al., 2005).
4.3. Biogeographic history
The earliest Testudinidae fossils (not yet described) are
reported to be from the lower Paleocene of Asia (Claude
and Tong, 2004; Hutchison, 1998; Joyce et al., 2004), however older fossils of the putative ancestor (family Lindholmemydidae) for the superfamily Testudinoidea are
known from the upper Cretaceous of Asia (Nessov, 1988;
Sukhanov, 2000). The appearance of testudinids fossils by
the early Eocene in both Europe (Achilemys cassouleti) and
North America (Manouria or Manouria-like fossils and
Hadrianus majusculus), and by the late Eocene in Africa
(Gigantochersina ammon) (Claude and Tong, 2004; Crumly,
1984a; Holroyd and Parham, 2003; Hutchison, 1996, 1998),
indicates that if tortoises Wrst originated in Asia, they must
have soon dispersed to other continents. The abundance of
fossil testudinids also suggests that the fossil record is
unlikely to be substantially underestimating the origin time
for the family. All evidence to date, thus, supports a late
Mesozoic or early Cenozoic origin for the Testudinidae in
Asia, with dispersal to North America, Europe, and Africa
having been achieved by the Eocene. Our DIVA results also
establish the ancestral area for testudinids as including
three continents: Asia, North America, and Africa, and
Wnds Manouria and Gopherus as the extant groups representing the Asian and North American lineages, respectively. Remarkably, all other extant testudinid groups are
associated with the African ancestral area.
The remarkable biographic basal lineage bifurcation
between clades distributed in Asia and North America,
527
which we Wnd here for Manouria and Gopherus, has also
been reported in the family Dibamidae (blind skinks) and
the skink genus Scincella, which are distributed in the
Americas and Asia (Honda et al., 2003; Zug et al., 2001).
These Asian–American divergences may have resulted from
the well-known disruption of Xoral and faunal exchange
across the Bering Strait in the early Eocene; a biogeographic pattern that has been well documented in other
groups (Beard, 2002; Bowen et al., 2002; Maekawa et al.,
2005; Sanmartin et al., 2001 and references therein; TiVney,
1985).
The DIVA ancestral area optimization results provide a
striking conclusion: it is most parsimonious to consider all
testudinids with the exception of Manouria and Gopherus as
having an African ancestral area. The alternative biogeographic scenarios, such as considering the ancestral area for
this large group as Asia or the Palearctic, will require making additional assumptions of dispersal or extinction. Our
results infer the following dispersals from Africa: Indotestudo and the clade including Geochelone platynota + G. elegans dispersing into Asia, Testudo dispersing into Europe,
the clade including Aldabrachelys + Pyxis + Geochelone
radiata + G. yniphora dispersing into the Indian Ocean, and
the G. carbonaria group dispersing into South America.
Africa should therefore be viewed as both a major center of
origin and diversiWcation for testudinid species. Three
clades found by our study are endemic to Africa: Malacochersus, Kinixys, and the diverse clade including
Homopus + Chersina + Psammobates +
Geochelone pardalis, and these groups presumably diversiWed on this continent (the Kinixys belliana population of Madagascar is
typically considered as introduced, see Raselimnanana and
Vences, 2003).
The dispersal of Testudo and Indotestudo from Africa
into Europe and Asia appears to be a novel biogeographic
scenario that has not been previously considered. For
example, Parham et al. (2006) only discuss dispersal events
from a Palearctic center of diversiWcation for Testudo,
Indotestudo, and Malacochersus. In addition, Crumly’s
(1984a) discussion of biogeographic scenarios implied that
African tortoises originated from Asia. However, and by
contrast, the Indian Ocean clade including Aldabrachelys
+ Pyxis + Geochelone radiata + G. yniphora has been previously proposed to have an African origin (Caccone et al.,
1999a; Palkovacs et al., 2002), as supported by our DIVA
ancestral area optimizations (Fig. 4).
However, perhaps the most unusual biogeographic
result concerns the South American + Galápagos tortoises
(Geochelone carbonaria group), which we Wnd are members
of a larger clade otherwise only including species from
Africa, the Indian Ocean, and Asia. To test the previously
proposed North American origin for the Geochelone carbonaria clade, based upon a sister relationship with Gopherus (e.g., Gerlach, 2001) we searched for the shortest
constraint tree that included these clades as sisters. This
constraint tree was 48 steps longer than our most parsimonious hypothesis of relationship, and this step length
528
M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531
diVerence was signiWcant (p < 0.001). By contrast, the consistently recovered sister relationship we found between
African Kinixys and the Geochelone carbonaria clade, and
the distribution of the group sister to this clade, which also
includes Africa, providing strongest support for an African
origin for the South American tortoises (Fig. 4). The DIVA
ancestral area optimizations Wnd this entire clade
(Geochelone + Pyxis + Aldabrachelys + Homopus + Chersina
+ Psammobates + Kinixys) to have an African ancestral
area, thus supporting an early evolutionary history for this
diverse tortoise group in this continent.
The dispersal of tortoises from Africa to South America
might have been aided by westward sea currents, such as
the Equatorial Currents (Fratantoni et al., 2000). These
Xow between the Congo delta and Maranhao in Brazil and
are believed to have been in this direction since the breakup
of Gondwana (Houle, 1999; Parrish, 1993). Several vertebrate animal groups are reported to show a pattern of dispersal from Africa to South America across the Atlantic:
platyrrhine monkeys and caviomorph rodents between 85
and 35 MYA, (Houle, 1999; Huchon and Douzery, 2001;
Mouchaty et al., 2001; Nei et al., 2001; Schrago and Russo,
2003), and Mabuya skinks twice during the last 9 million
years (Carranza and Arnold, 2003). Tortoises are well
known for their ability to Xoat (while extending their heads
above water) and survive without food or freshwater for up
to six months (Caccone et al., 2002), and oceanic dispersal
by testudinid tortoises appears to have occurred many
times. For example: Geochelone to the West Indies (Williams, 1950, 1952), Geochelone to the Galápagos and at
least Wve subsequent dispersals between islands in the archipelago (Caccone et al., 1999b, 2002; Russello et al., 2005),
ancestral Malagasy tortoises to Madagascar from mainland Africa (Caccone et al., 1999a), Aldabrachelys to
Aldabra, Astove, Cosmoledo, Denis, Amirantes, Comoros,
Assumption, and the Granitic Seychelles (Austin et al.,
2003); Cylindraspis (now extinct) to Réunion, Mauritius,
and Rodrigues (Austin and Arnold, 2001); and Hesperotestudo (now extinct) to Bermuda (Meylan and Sterrer, 2000).
4.4. Taxonomic issues
Based on our phylogenetic results, and to reXect monophyletic groupings within the Testudinidae, we propose the
following taxonomic changes:
(1) The name Geochelone (Fitzinger, 1835; type species,
G. elegans) is retained for just three species: G. sulcata,
G. elegans, and G. platynota.
(2) The South American and Galápagos Island species
(Geochelone carbonaria group) are placed in the genus
Chelonoidis (Fitzinger, 1835; type species, C. carbonaria),
which has been previously recognized as a subgenus (Fitzinger, 1856).
(3) Geochelone pardalis is placed in the genus Psammobates (Fitzinger, 1835).
(4) Based on the desirability of maintaining the morphologically distinct and well-supported genera Pyxis and
Aldabrachelys, yet also due to the uncertainty concerning
the relationships of Geochelone yniphora and G. radiata to
these genera, we therefore prefer to conservatively place
both these species in separate monotypic genera. We recommend the use of the available genus name Astrochelys
(Gray, 1873; type species, A. radiata) for G. radiata. However, because there is no available generic name for G. yniphora, we thus propose the following new genus:
Angonoka new genus
Type species. Testudo yniphora Vaillant 1885.
Diagnosis—Testudinid tortoises that have the following
characters: (1) gulars fused to form a single scute; (2) gular
projection thickened and sexually dimorphic in size, projecting well beyond the carapace edge and curved upward
in adult males; (3) highly domed carapace with descending
sides; (4) carapace brown, lacking radiating yellow or black
lines on scutes; (5) no enlarged tubercles on thighs; (6) tail
without an enlarged terminal scale; (7) longitudinal ventral
ridge on the maxilla–premaxilla suture, (8) horizontal keels
on the supraoccipital crest; (9) pectoral scutes narrow and
not contacting the entoplastron.
Content. One species, Angonoka yniphora (Vaillant
1885).
Distribution. Northwest Madagascar.
Etymology. The generic name “Angonoka” is based on
the Malagasy word ‘Angonoka’ which is the local name
for the type species.
5. Conclusions
The broad taxonomic sampling of tortoises we were able
to achieve with this study, coupled with the inclusion of
both mitochondrial and nuclear genes, has resulted in supported tree topologies with both good resolution, and for
many branches, high levels of support. Importantly, these
topologies are also largely congruent, regardless of the
approach taken concerning phylogenetic analysis. We consider this result to represent a substantial advance in our
understanding of testudinid relationships, especially concerning the problematic genus Geochelone, which has been
in a state of substantial taxonomic confusion for the past 30
years. Although our results Wnd Geochelone polyphyletic,
representing at least four independent clades, we anticipate
these Wndings will stimulate new interest within these tortoise groups and strengthen future comparative research.
Despite these systematic advances, further phylogenetic
analysis will be needed to test the relationships we report
here between Indotestudo, Testudo, and Malacochersus, and
the relationships of G. yniphora and G. radiata. The inclusion of additional genes (nuclear and mitochondrial) and
morphological data will likely resolve this uncertainty.
Importantly, a re-examination of the morphological characters will also provide new insights concerning character
evolution for the group, and in addition, aid with the interpretation of the fossil taxa. Unlike most extant reptile families, tortoises have a comparably rich Cenozoic fossil
M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531
record, and thus oVer excellent opportunities for integrating fossil taxa within systematic studies based on both molecules and morphology.
Acknowledgments
M.L. thanks Dr. Eleanor Sterling for the support she has
provided throughout this project. Both JeV Groth and Tom
Near provided valuable help during this study, and we thank
the following individuals for providing valuable tissues: Dr.
George Amato (WCS), Drs. Cheri Asa and Randy Junge
(Saint Louis Zoo), Dr. Sue Lynch (ChaVe Zoo), Dr. Ronald
A. Nussbaum (University of Michigan Museum of Zoology),
Eric Palkovacs, Yale University, Dr. Steve Reichling (Memphis Zoo), Dr. Chris Tabaka (Detroit Zoo), Dr. Harold Voris
(Field Museum of Natural History), and Mr. Truong Quang
Bich, Cuc Phuong National Park, Vietnam. We are also
grateful to Dr. John Iverson for reading and providing helpful comments on the early version of the paper and Drs. Brad
ShaVer and Gisella Caccone and an anonymous reviewer for
suggestions that improved the paper. Margaret Arnold,
David Dickey, and Robert J. Pascocello facilitated the sampling of the specimens at the AMNH. Funding for this project was provided by the National Science Foundation Grant
DEB 99-84496 awarded to C.J.R., the American Museum of
Natural History, the Chelonian Research Foundation, the
Department of Ecology, Evolution, and Environmental Biology, Columbia University, and NASA Grant NAG5-8543 to
the Center for Biodiversity and Conservation at the American Museum of Natural History. We also acknowledge the
generous support provided by the Louis and Dorothy Cullman Program in Molecular Systematic Studies and the
Ambrose Monell Foundation.
Appendix A. Supplementary Data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.ympev.2006.03.003.
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