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 relation- ships 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, Psam- mobates, Kinixys, and Geochelone. However, we Wnd Geochelone rampantly polyphyletic, with species distributed in at least four indepen- dent 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 show a great diversity of sizes and forms. The largest tortoises, in the Galápagos (Geochelone nigra), can reach 1.5m in The turtle family Testudinidae includes 49 living species of length, yet the speckled padloper (Homopus signatus) is tortoises in 12 extant genera, all of which are completely ter- mature at only 10cm in length. Most genera have highly restrial, and which together represent about 18% of the extant domed and rigid carapaces, but hinged-back carapaces occur world turtle diversity (Ernst and Barbour, 1989; Uetz, 2005). in the genus Kinixys, and the genus Malacochersus has a Testudinids are the most broadly distributed non-marine tur- remarkable Xattened and Xexible carapace for occupying rock tle family, occurring on all subpolar continents except Austra- crevices (Crumly, 1984a; Ernst and Barbour, 1989). lia, and occupying a wide diversity of habitats varying from Despite previous research, phylogenetic relationships rain forests in Southeast Asia and South America, to deserts within the family Testudinidae have remained controver- in North America and Africa. Morphologically, tortoises also sial (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 * Corresponding author. Fax: +1 212 769 5031. authors, concerns the high level of morphological E-mail address: [email protected] (M. Le). convergences suspected for some traditionally used
1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2006.03.003 518 M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531 characters (AuVenberg, 1974; Bramble, 1971; Pritchard, graphic scenarios of testudinids to date. He suggested true 1994). In addition, all testudinid molecular studies to tortoises originated in North America, based on the occur- date have been limited in terms of taxonomic sampling rence of the oldest known fossil testudinid, Hadrianus because they addressed speciWc questions of relationship majusculus, from the Early Eocene. Crumly (1984a) also within smaller subsets of the family (with the possible proposed that tortoises from Europe and Africa were more exception of Cunningham’s (2002) study, but this has not closely related to Asian forms than to North American yet become available). These studies included relation- forms, and that tortoises of the Galápagos, and the Indian ships within the genus Gopherus (Lamb and Lydeard, Ocean had rafted to these islands. This latter dispersal sce- 1994), origin and relationships of Malagasy tortoises nario has been supported by more recent studies (Austin (Caccone et al., 1999a), origin of Galápagos tortoises and Arnold, 2001; Caccone et al., 1999b; Palkovacs et al., (Caccone et al., 1999b), origin of Indian Ocean tortoises 2002). However, the origin of the South American tortoises (Austin and Arnold, 2001; Austin et al., 2003; Palkovacs remains a matter of controversy. Most authors have con- et al., 2003), relationships within the genus Indotestudo cluded that tortoises invaded tropical South America from (Iverson et al., 2001), and relationships within the genus North or Central America (AuVenberg, 1971; Gerlach, Testudo (Fritz et al., 2005; Parham et al., 2006; van der 2001; Simpson, 1943; Williams, 1950); however, both Simp- Kuyl et al., 2002). son (1942) and Crumly (1984a) also suggested Africa as a Nevertheless, the monophyly of the family has been con- possible alternative source. tinuously supported by both molecular and morphological The major objective of our study was to produce a phy- studies; with two groups: Geoemydidae and Emydidae con- logeny of the Testudinidae based on an expanded molecu- sidered sister to testudinids (Crumly, 1984a; GaVney and lar character dataset, which would be comprehensive at Meylan, 1988; Gerlach, 2001; Krenz et al., 2005; Near et al., the generic level, and include all the species available to us 2005; Spinks et al., 2004; van der Kuyl et al., 2002). (the majority of species). We ultimately were able to Within the Testudinidae, establishing the monophyly of include species representing all recognized and previously the genus Geochelone has proven to be especially problem- proposed extant genera and subgenera, representing in atic. As the largest genus of the family, this group currently total 65% of all testudinid species. This broad taxonomic includes 10 species, exclusive of Wve species recently sampling was considered especially important for resolv- assigned to the genera Manouria and Indotestudo based on ing phylogenetic relationships within the problematic the studies of Crumly (1984a,b) and Hoogmoed and genus Geochelone, and for this group, we were able to Crumly (1984), and six extant or recently extinct species include all Geochelone species. To provide a robust esti- being placed in the genus Aldabrachelys (Dipsochelys) (Aus- mate of phylogeny, we included a broad diversity of tin et al., 2003; Bour, 1982; Gerlach, 2001). AuVenberg genes, including one mitochondrial protein coding gene (1974) proposed six subgenera (including Manouria and (cytb), two mitochondrial ribosomal genes (12S and 16S), Indotestudo) for Geochelone based largely on zoogeo- and two nuclear genes (Cmos and Rag2), the latter of graphic criteria, however, Crumly (1982) was the Wrst to which have not been previously utilized for resolving rela- argue against their complete use based on his cladistic anal- tionships between turtles. ysis using 26 cranial characters. This view has also been corroborated by more recent analyses, which have included 2. Materials and methods more characters and taxa (Crumly, 1984a; Gerlach, 2001). Crumly’s (1984a) results also did not support Geochelone 2.1. Taxonomic sampling monophyly (Fig. 1). More recent morphological studies have provided conXicting results: GaVney and Meylan For our testudinid ingroup, we included 34 taxa (32 spe- (1988) and Meylan and Sterrer (2000) found support for a cies and 2 subspecies) and all genera within the family. We monophyletic Geochelone, and Gerlach (2001) found also included all Geochelone species. For two species: Geochelone to be polyphyletic. In addition, recent molecu- Indotestudo forstenii and Gopherus agassizii, for which we lar studies have shown Malagasy Geochelone paraphyletic were unable to obtain tissue; sequences for these species with respect to Pyxis (Caccone et al., 1999a; Palkovacs (reported by Spinks et al., 2004) were obtained from Gen- et al., 2002), or provided little resolution between Geoche- Bank. A complete list of all tissues, voucher specimens, and lone and other testudinid genera (van der Kuyl et al., 2002). sequences are provided in Table 1. This molecular phyloge- All of these molecular studies were restricted to mitochon- netic analysis includes the most comprehensive taxon sam- drial genes and only included a subset of Geochelone spe- pling achieved to date for testudinids. cies. For outgroups, we included two species of Geoemydidae In terms of their biogeographic history, tortoises oVer an (Rhinoclemmys melanosterna and R. nasuta) and two spe- interesting group for study because of their almost world- cies of Emydidae (Glyptemys insculpta and Deirochelys wide distribution, including oceanic islands, e.g., the Galá- reticularia) based on their reported sister relationships to pagos, the East Indies, the West Indies, Madagascar, and testudinids recovered by both morphological and molecu- the Mascarene Islands. Crumly’s (1984a) study has pro- lar evidence (GaVney and Meylan, 1988; Honda et al., 2002; vided the most detailed discussion of diVerent biogeo- 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 (mtDNA): the complete cytochrome b sequence, and sec- tions of both the 12S and 16S rRNA genes. For some taxa Both mitochondrial and nuclear DNA were utilized in (see Table 1), we downloaded cytb and 12S sequences from this study to resolve relationships at all phylogenetic levels GenBank originating from studies by Spinks et al. (2004), within the family. Similar approaches have proved success- Palkovacs et al. (2002), Caccone et al. (1999a,b), and van ful with other groups exhibiting similar levels of phyloge- der Kuyl et al. (2002). We also sequenced two nuclear frag- netic diversity (see Barker et al., 2004; Georges et al., 1999; ments of the Cmos and Rag2 genes. All primers used for Hillis et al., 1996; Pereira et al., 2002; Saint et al., 1998). We this study (including those developed during the course of sequenced three regions of the mitochondrial DNA 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 GenBank GenBank GenBank GenBank Voucher numbers No. (12S) No. (16S) No. (cytb) No. (Cmos) No. (Rag2) for this study Chersina angulata DQ497248 DQ497269 DQ497292 DQ497325 DQ497361 AMCC157819 Aldabrachelys arnoldii AY081779 AY081780 DQ497293 DQ497326 DQ497362 YU Aldabrachelys dussumieri DQ497249 DQ497270 DQ497294 DQ497327 DQ497363 YU Aldabrachelys hololissa AY081783 AY081784 DQ497295 DQ497328 DQ497364 YU Geochelone carbonaria AB090019 AF192926 DQ497296 DQ497329 DQ497365 AMCC157837 Geochelone chilensis AF175336 AF192924 DQ497297 DQ497330 DQ497366 AMCC157844 Geochelone denticulata DQ497250 AF192927 DQ497298 DQ497331 DQ497367 AMCC157838 Geochelone elegans AY081785 AY081786 DQ497299 DQ497332 DQ497368 AMCC106883 Geochelone nigra AF020885 AF020892 DQ497300 DQ497333 DQ497369 FMNH262225 Geochelone p. pardalis DQ497251 DQ497271 DQ497301 DQ497334 DQ497370 AMCC157839 Geochelone p. babcocki DQ497252 DQ497272 DQ497302 DQ497335 DQ497371 AMCC157840 Geochelone platynota DQ497253 DQ497273 DQ497303 DQ497336 DQ497372 AMCC157841 Geochelone radiata AF020883 AF020890 DQ497304 DQ497337 DQ497373 AMCC106882 Geochelone sulcata AY081787 AY081788 DQ497305 DQ497338 DQ497374 AMCC111025 Geochelone yniphora AF020882 AF020889 DQ497306 DQ497339 DQ497375 AMCC125847 Gopherus agassiziia AY434630 AY434562 — Gopherus polyphemus AF020879 AF020886 DQ497307 DQ497340 DQ497376 FMNH262226 Homopus boulengeri DQ497254 DQ497274 DQ497308 DQ497341 DQ497377 AMCC106886 Homopus signatus DQ497255 DQ497275 DQ497309 DQ497342 DQ497378 AMCC125859 Indotestudo elongata DQ497256 DQ497276 DQ497310 DQ497343 DQ497379 AMCC157712 Indotestudo forsteniia AY434561 — Indotestudo travancorica DQ497257 DQ497277 DQ497311 DQ497344 DQ497380 AMCC142999 Kinixys belliana DQ497258 DQ497278 DQ497312 DQ497345 DQ497381 AMCC106880 Kinixys homeana DQ497259 DQ497279 DQ497313 DQ497346 DQ497382 KU290452 Malacochersus tornieri DQ497260 DQ497280 DQ497314 DQ497347 DQ497383 AMCC157829 Manouria emys emys DQ497261 DQ497281 DQ497315 DQ497348 DQ497384 AMCC157827 Manouria emys phayrei DQ497262 DQ497282 DQ497316 DQ497349 DQ497385 AMCC157828 Manouria impressa DQ497263 DQ497283 DQ497317 DQ497350 DQ497386 AMCC157716 Psammobates tentorius DQ497264 DQ497284 DQ497318 DQ497351 DQ497387 AMCC157843 Pyxis arachnoides AF020880 AF020887 DQ497319 DQ497352 DQ497388 RAN34680 Pyxis planicauda AF020881 AF020888 DQ497320 DQ497353 DQ497389 AMCC157845 Testudo graeca AF175330 DQ497285 DQ497321 DQ497354 DQ497390 AMCC157846 Testudo horsWeldii AB090020 DQ497286 DQ497322 DQ497355 DQ497391 AMCC144096 Testudo kleinmanni AF175332 DQ497287 DQ497323 DQ497356 DQ497392 AMCC125843 Glyptemys insculpta DQ497265 DQ497288 AF258876 DQ497357 DQ497393 AMCC157833 Deirochelys reticularia DQ497266 DQ497289 AF258877 DQ497358 DQ497394 AMCC157832 Rhinoclemmys melanosterna DQ497267 DQ497290 AY434590 DQ497359 DQ497395 AMCC157821 Rhinoclemmys nasuta DQ497268 DQ497291 DQ497324 DQ497360 DQ497396 AMCC157826 All sequences generated by this study have accession numbers: DQ497248 – DQ497396; YU, Yale University Collection; FMNH, Field Museum of Natu- ral History; AMCC, Ambrose Monell Cryo Collection, American Museum of Natural History (http://research.amnh.org/amcc); RAN, Ronald A. Nuss- baum 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 extraction solution). PCR conditions for nuclear genes using the DNeasy kit (Qiagen) following manufacturer’s were the same as above except the Wrst step (95 °C) was instructions for animal tissues. PCR volume for carried out in 15 min, and the annealing temperatures mitochondrial genes contained 42.2 l (18 l water, 4 l of were 52 and 58 °C for Rag2 and Cmos, respectively. V bu er, 4 l of 20 mM dNTP, 4 l of 25 mM MgCl2, 4 l of PCR products were visualized using electrophoresis each primers, 0.2 l of Taq polymerase (Promega), and 4 l through a 2% low melting-point agarose gel (NuSieve GTG, of DNA). PCR conditions for these genes were: 95 °C for FMC) stained with ethidium bromide. For reampliWcation 5 min to activate the Taq; with 42 cycles at 95 °C for 30 s, reactions, PCR products were excised from the gel using a 45 °C for 45 s, 72 °C for 60 s; and a Wnal extension of Pasteur pipette, and the gel plug was melted in 300 l sterile 6 min. Nuclear DNA was ampliWed by HotStar Taq Mas- water at 73 °C for 10 min. The resulting gel-puriWed product termix (Qiagen) since this Taq performs well on samples was used as a template in 42.2l reampliWcation reactions with low-copy targets and the Taq is highly speciWc. The with all PCR conditions similar to those used for mitochon- PCR volume consists of 21 l (5 l water, 2 l of each drial genes. PCR products were cleaned using PerfectPrep® primer, 10 l of Taq mastermix, and 2 l of DNA or PCR Cleanup 96 plate (Eppendorf) and cycle sequenced higher depending on the quantity of DNA in the Wnal 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) 491 5Ј-AAAAAGCTTCAAACTGGGATTAGATACCCCACTAT-3Ј Kocher et al. (1989) H1478 (12S) 947 5Ј-TGACTGCAGAGGGTGACGGGCGGTGTGT-3Ј Kocher et al. (1989) AR (16S) 1959 5Ј-CGCCTGTTTATCAAAAACAT-3Ј Palumbi et al. (1991) BR (16S) 2561 5Ј-CCGGTCTGAACTCAGATCACGT-3Ј Palumbi et al. (1991) CytbG (cytb) 14368 5Ј-AACCATCGTTGTWATCAACTAC-3Ј Spinks et al. (2004) GLUDGE (cytb) 14358 5Ј-TGATCTTGAARAACCAYCGTTG-3Ј Palumbi et al. (1991) CytbJSi (cytb) 15011 5Ј-GGATCAAACAACCCAACAGG-3Ј Spinks et al. (2004) CytbJSr 15030 5Ј-CCTGTTGGGTTGTTTGATCC-3Ј Spinks et al. (2004) THR (cytb) 15593 5Ј-TCATCTTCGGTTTACAAGAC-3Ј Spinks et al. (2004) THR-8 (cytb) 15585 5Ј-GGTTTACAAGACCAATGCTT-3Ј Spinks et al. (2004) CM1 (Cmos) 163 5Ј-GCCTGGTGCTCCATCGACTGGGA-3Ј Barker et al. (2002) CM2 (Cmos) 820 5Ј-GGGTGATGGCAAAGGAGTAGATGTC-3Ј Barker et al. (2002) Cmos1 (Cmos) 163 5Ј-GCCTGGTGCTCCATCGACTGGGATCA-3Ј This study Cmos3 (Cmos) 812 5Ј-GTAGATGTCTGCTTTGGGGGTGA-3Ј This study F2 (Rag2) 601 5Ј-CAGGATGGACTTTCTTTCCATGT-3Јa This study F2-1 (Rag2) 590 5Ј-TTCCAGAGCTTCAGGATGG-3Ј This study R2-1 (Rag2) 1312 5Ј-CAGTTGAATAGAAAGGAACCCAAGT-3Јb This study a ModiWed from F2R2 (Barker et al., 2004). b ModiWed from R2R1 (Barker et al., 2004); Cmos and Rag2 primer positions corresponding to the positions in chicken genomes with GenBank num- bers 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). turer recommendation. Sequences were generated in both For maximum likelihood analysis the optimal model for directions on an ABI 3100 Genetic Analyzer. nucleotide evolution was determined using Modeltest V3.7 (Posada and Crandall, 1998). Analyses used a randomly 2.3. Phylogenetic analysis selected starting tree, and heuristic searches with simple taxon addition and the TBR branch swapping algorithm. Support We aligned sequence data using ClustalX v1.83 for the likelihood hypothesis was evaluated by bootstrap (Thompson et al., 1997) with default settings. Data were analysis with 100 replications and simple taxon addition. analyzed using both parametric and non-parametric meth- For Bayesian analyses we used the optimal model deter- ods (see Kolaczkowski and Thornton, 2004): maximum mined using Modeltest with parameters estimated by MrBa- parsimony (MP) and maximum likelihood (ML) using yes Version 3.1. Analyses were conducted with a random PAUP*4.0b10 (SwoVord, 2001) and Bayesian analysis starting tree and run for 5 £106 generations. Four Markov using MrBayes v3.1 (Huelsenbeck and Ronquist, 2001). chains, one cold and three heated (utilizing default heating For maximum parsimony analysis, we conducted heu- values), were sampled every 1000 generations. Log-likelihood ristic analyses with 100 random taxon addition replicates scores of sample points were plotted against generation time using the tree-bisection and reconnection (TBR) branch to detect stationarity of the Markov chains. Trees generated swapping algorithm in PAUP, with no upper limit set for prior to stationarity were removed from the Wnal analyses the maximum number of trees saved. Bootstrap support using the burn-in function. Two independent analyses were (BP) (Felsenstein, 1985a) was evaluated using 1000 pseu- started simultaneously. The posterior probability values (PP) doreplicates and 100 random taxon addition replicates. for all clades in the Wnal majority rule consensus tree are Bremer indices (BI) (Bremer, 1994) were determined using reported. We ran analyses on both combined and partitioned Tree Rot 2c (Soreson, 1999). All characters were equally datasets to examine the robustness of the tree topology weighted and unordered. Gaps in sequence alignments (Brandley et al., 2005; Nylander et al., 2004). In the parti- were treated as a Wfth character state (Giribert and tioned analyses, we divided the data into 11 separate parti- Wheeler, 1999). Phylogenetic congruence between parti- tions, including 12S, 16S, and the other nine based on gene tions of the nuclear and mitochondrial datasets was codon positions (Wrst, second, and third) in cytb, Cmos, and assessed using the incongruence length diVerence (ILD) Rag2. Optimal models of molecular evolution for each parti- test (Farris et al., 1994). Although the utility of this test as tion were selected using Modeltest and then assigned to these a measure for data combination has been questioned (e.g., partitions in MrBayes 3.1 using the command APPLYTO. see Darlu and Lecointre, 2002; Yoder et al., 2001), we Model parameters were estimated independently for each employed this test to further explore characteristics of data partition using the UNLINK command. our partitioned data, along with phylogenetic analyses To test alternative hypotheses of relationships, corre- that we also conducted using these same partitions. These sponding tree topologies were compared using the Wilcoxon data were partitioned by genes and DNA organelle signed-ranks test (Felsenstein, 1985b; Templeton, 1983), to (nuclear or mitochondrial), and also analyzed in their determine if tree length diVerence could have resulted from entirety. 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) long corresponding to positions 489–494 of the complete and then used as constraint trees by importing to PAUP. To chicken Cmos sequence (GenBank Accession No. M19412). determine biogeographic support for ancestral areas or vicar- For Rag2, Chersina angulata and Kinixys homeana has a iance events between clades, we utilized Dispersal-Vicariance single indel of three base pairs long corresponding to posi- Analysis (Ronquist, 1997), as implemented in the program tions 1093–1095 of the partial Pelodiscus sinensis Rag2 DIVA 1.1 (Ronquist, 1996). Fully resolved trees based on sequence (GenBank Accession No. AF369089). MP and ML topologies were imported using distribution The IDL tests found no signiWcant incongruence areas (as depicted in Figs. 2 and 4). All optimization settings between mitochondrial genes (12S vs 16S, p D 0.07; cytb vs used the default, including the maximum number of ancestral 12S, p D 0.89; cytb vs 16S, p D 0.97), between nuclear genes areas that were set as the total number of unit areas. (Cmos vs Rag2, p D 0.16), and between the nuclear and mitochondrial partitions (nuclear DNA vs mtDNA, 3. Results p D 0.7). Using MP, we also analyzed the data by gene and organelle partitions (see Tables 3 and 4). The analysis indi- Sequences for all Wve genes were obtained for each of the cated that Clades 3, 4, and 5 (see Fig. 4) consistently receive 32 taxa with tissues. The Wnal matrix consists of 3387 strong support from both mitochondrial and nuclear genes aligned characters: 12S (408 characters), 16S (583 charac- (Table 3). For the following partitions, the strict consensus ters), cytb (1140 characters), Cmos (602 characters), and trees include little phylogenetic resolution: 12S, 16S, Cmos, Rag2 (654 characters). Five taxa have sequences that Rag2, and all the nuclear data. For cytb, although MP include gaps at three loci (cytb, Cmos, and Rag2). For cytb, recovered only two equally parsimonious trees, the strict Geochelone pardalis babcockii has a single indel that is three consensus is poorly resolved concerning deeper level rela- base pairs long at positions 451–453. For Cmos, G. platy- tionships between clades including multiple genera. The nota and G. elegans has a single indel that is six base pairs 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 3 (%) Clade 4 (%) Clade 5 (%) Clade 6 (%) Clade 7 (%) Clade 8 (%) 12S <50 63 64 <50 <50 <50 <50 <50 16S <50 60 95 <50 99 <50 <50 <50 Cytb 55 <50 75 <50 <50 <50 <50 <50 All MtDNA 79 94 100 81 94 <50 62 72 Cmos <50 <50 70 75 62 <50 <50 <50 Rag2 <50 <50 <50 92 <50 <50 <50 <50 All Nuclear <50 55 87 99 72 <50 <50 <50
Table 4 Data partitions subject to phylogenetic analyses with maximum parsimony Data Total number of Parsimony Variable MP tree RI CI Number of equally aligned sites informative characters characters length value value parsimonious trees 12S 408 125 174 514 0.61 0.47 117 16S 583 169 211 773 0.58 0.44 41 cytb 1140 475 616 2671 0.44 0.33 2 Cmos 602 66 111 166 0.79 0.73 105 Rag2 654 45 90 122 0.85 0.81 5080 All MtDNA 2131 769 1001 3989 0.49 0.37 3 All Nuclear 1256 111 201 293 0.80 0.75 16 All data 3387 880 1201 4292 0.50 0.39 2 basal nodes of the clade including all the Geochelone strong support (BP 7 70%). The most weakly supported groups—the same clade as recovered in the combined anal- nodes are distributed in the clade containing Geochelone. ysis (see Fig. 2). All nodes with BI D 1 in the combined MP The topology of the ML cladogram is congruent with the analysis collapsed in this mtDNA tree. Based on the MP topology for all branches except the following: Malac- generally poorly resolved tree topologies recovered using ochersus tornieri sister to either Testudo (BP D 68%, MP) or the partitioned data, and our IDL partition results, we thus Indotestudo (BP D 60%, ML), G. radiata sister to either consider the combined data as providing the optimal set Pyxis (BP D 65%, MP) or Geochelone yniphora (BP D 82%, upon which to base phylogenetic inference. ML), and the clade including Kinixys + South American Using the combined dataset (3387 aligned characters, Geochelone (G. carbonaria group) sister to either the clade 880 potentially parsimony informative), the MP analysis including the Malagasy and Indian Ocean species, Cher- recovered two most parsimonious trees with 4292 steps sina, Homopus, Psammobates, and G. pardalis (<50%, MP) (CI D 0.39; RI D 0.50). The strict consensus is shown in or the clade including G. elegans, G. platynota, and G. sul- Fig. 2. This tree is well resolved with 72% of the nodes cata (87%, ML). receiving strong support (BP 7 70%) (Hillis and Bull, In the combined Bayesian analysis (Fig. 3), ¡ln L scores 1993), and 9% receiving BP support from 65 to 69%. All reached equilibrium after 19,000 generations while the par- nodes in the clades, including Manouria, Gopherus, Testudo, titioned Bayesian analysis scores reached stationarity after Indotestudo, and Malacochersus, are well supported, except 27,000 generations in both runs. Phylogenetic relationships for the relationship of Testudo horsWeldii and Malacocher- supported by the combined and partitioned data are identi- sus. We also found that the BP value supporting the sister cal to the ML results. Only minor diVerences in the PP val- relationship between Gopherus and Manouria increases sig- ues were found between combined and partitioned niWcantly (from 73 to 85% in the MP analysis) by excluding Bayesian cladograms: PP values are higher in the parti- the species with incomplete data, Gopherus agassizii. Within tioned analysis for the following clades: Pyxis and Aldabr- the diverse clade, which includes Geochelone, Aldabrachelys, achelys (99%), South American testudinids (99%), Kinixys Kinixys, Homopus, Psammobates, and Pyxis, support was + South American testudinids (86%), Testudo (99%), and generally high except for some of the basal relationships. A. arnoldi + A. hololissa (74%), and lower for the following: Incongruence between these two MP trees was conWned to Chersina + Homopus + Psammobates + Geochelone pardalis the genus Aldabrachelys. + Aldabrachelys + Pyxis + Geochelone radiata + G. yniphora Results, including model parameters, of the ML and (56%), G. chilensis + G. nigra (93%), and Malacochersus Bayesian analyses are shown in Fig. 3 (tree topologies of + Indotestudo (95%). both analyses are identical). Similar to the MP analysis, the All of our analyses, thus, found three well supported ML tree is well resolved with 78% of the nodes receiving 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 Biogeographic results are summarized by the area clado- +Pyxis+Aldabrachelys+Homopus+ Chersina +Psammobates gram shown in Fig. 4, which conservatively depicts the +Kinixys. It is also evident from these analyses that Geoche- strict congruence between our MP, ML, and Bayesian tree lone, as currently deWned, is rampantly polyphyletic. topologies. The optimized ancestral areas, based on DIVA, Our phylogenetic results place Geochelone in at least are also shown in Fig. 4. The DIVA ancestral area for the four independent clades. The Wrst consists of three family Testudinidae is identiWed as including three areas: species, G. elegans, G. platynota, and G. sulcata (G. elegans Asia, Africa, and North America, and requires a further group). The second clade includes all four South Ameri- seven subsequent dispersals (to South America, Madagas- can species (G. carbonaria group), which are sister to car–Indian Ocean, and Asia). For this three-continent Kinixys. G. yniphora and G. radiata from Madagascar familial ancestral area, Gopherus represents the North belong to a third clade that also includes Pyxis and American lineage, Manouria represents the Asian lineage, Aldabrachelys. The Wnal clade includes only a single and all other testudinids represent the African lineage. For Geochelone species, G. pardalis, which is sister to the genus the incongruent clades between the MP and ML analyses Psammobates. (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 Manouria exhibiting several character states considered to + Indotestudo, Africa and Asia; Chersina + Homopus+ be plesiomorphic, such as the wide anterior edge of the pro- Psammobates + Geochelonepardalis + Kinixys +the Geoche- otic bone (ranging from 80 to 92% of the posterior edge) lone carbonaria group + Aldabrachelys + Pyxis + Geochelone and the ventrally and dorsally split supracaudal scute radiata + G. yniphora, Africa; and the G. elegans group + (Crumly, 1982). This Manouria + Gopherus sister relation- Kinixys +the Geochelone carbonaria group, Africa. ship has, to the best of our knowledge, not been previously considered or discussed, and no synapomorphies have been 4. Discussion proposed for the two genera (GaVney and Meylan, 1988; Gerlach, 2001; Meylan and Sterrer, 2000; Takahashi et al., 4.1. Phylogeny 2003). Winokur and Legler (1975) reported mental glands in Gopherus, and Ernst and Barbour (1989), also citing Concerning the three major clades of testudinids we Wnd Winokur and Legler, report mental glands in Manouria— in this study, the sister group to all other tortoise species thus suggesting a potentially homologous character. How- includes Manouria and Gopherus as sister genera (Figs. 2 ever, our reading of Winokur and Legler’s (1975) study and 3). This is the Wrst study to sample broadly across the clearly reveals that for testudinids, these authors only family and recover this intriguing sister relationship; reported mental glands for Gopherus. In addition, our own although Lamb and Lydeard’s (1994) molecular study also examination of AMNH catalogued material (R-118676 and found weak support for this sister relationship. Other mor- R-119966), and living specimens, also failed to Wnd mental phological and molecular studies have consistently glands in Manouria. reported Manouria to be basal to all other tortoises The second major clade found by this study includes (Crumly, 1984a; GaVney and Meylan, 1988; Gerlach, 2001; Testudo, Indotestudo, and Malacochersus. A recent molecu- Meylan and Sterrer, 2000; Takahashi et al., 2003; Spinks lar study by Parham et al. (2006) based on the complete et al., 2004), with Gopherus usually placed as the next most mitochondrial genome also recovered this monophyletic basal lineage for the family (Crumly, 1984a; Spinks et al., group. Their MP, ML, and Bayesian analyses produced 2004). Manouria and Gopherus are distributed in Asia and conXicting results regarding the relationships of Malac- North America, respectively, and these two genera show ochersus tornieri and Testudo horsWeldii. SpeciWcally, their obvious diVerences in morphological characters, with 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 analy- ies; and the molecular study by Palkovacs et al. (2002) sis found Malacochersus sister to all other taxa. The posi- found support for conXicting relationships concerning G. tion of T. horsWeldii was unresolved in the MP analysis sulcata and G. elegans between MP, ML, Bayesian, and (strict consensus tree), but was sister to T. hermanni in the neighbor-joining analyses. other analyses. Another study (based on 1124 bp of cytb) by Although only weakly supported in terms of branch sup- Fritz et al. (2005) which included almost all forms of the port (MP and ML BP < 50%, PP 80%), all of our analyses genus Testudo plus Indotestudo and Malacochersus was also support the intriguing sister relationship between African ambiguous regarding relationships among these three gen- Kinixys and the South American Geochelone carbonaria era. Similarly, we also found conXicting branch support for group. This hypothesis of relationship is radical: prior mor- relationships within this clade: the MP analysis give moder- phological and molecular studies placed members of the ate support (BP D 68%) for a Malacochersus + Testudo Geochelone carbonaria group either sister to Gopherus clade, while the ML and Bayesian results supported a (Crumly, 1984a; Gerlach, 2001), or sister to other Geochelone Malacochersus + Indotestudo clade (ML BP D 60%, species: G. elegans, G. pardalis, and G. sulcata (Crumly, PP D 97%). Interestingly, morphological studies have con- 1984a; Meylan and Sterrer, 2000; Palkovacs et al., 2002; van sistently supported Malacochersus and Testudo as sisters der Kuyl et al., 2002). Crumly (1984a) did not consider the (Crumly, 1984a; GaVney and Meylan, 1988; Gerlach, 2001). South American tortoises to be monophyletic (Fig. 1), but According to Gerlach (2001), the two genera share the other researchers have supported monophyly for a sampled character of the processus inferior parietalis meeting the subset of this clade (van der Kuyl et al., 2002), or else other- quadrate and partially covering the prootic, while GaVney wise explicitly assumed it (Caccone et al., 1999b; GaVney and and Meylan (1988) found they share four characters: pres- Meylan, 1988; Gerlach, 2001; Meylan and Sterrer, 2000). ence of supranasal scales, no contact between the inguinal The Indian Ocean clade supported by our results: and femoral scutes, a viewable ventral tip of the processus Aldabrachelys + Pyxis + Geochelone radiata + G. yniphora is interfenestralis, and the presence of sutures between this consistent with the molecular results of Austin and Arnold process and the surrounding bones. (2001) and Palkovacs et al. (2002). The latter study, like Our dataset unambiguously resolves relationships within ours, found support for a sister relationship between the three Indotestudo species. The sister relationship G. radiata and Pyxis using MP (65% BP), and an alterna- between I. travancorica and I. elongata is recovered by MP, tive sister relationship of G. radiata and G. yniphora using ML, and Bayesian analyses with strong support. In previ- Bayesian and ML analyses (ML 82% BP, 100% PP). Mor- ous studies, relationships among these species had phologically, the sister relationship between G. yniphora remained uncertain. The morphological study by Hoogm- and G. radiata has been frequently supported (e.g., Crumly, oed and Crumly (1984) found I. travancorica and I. forstenii 1984a; GaVney and Meylan, 1988; Gerlach, 2001; Meylan to be identical, yet the molecular study by Iverson et al. and Sterrer, 2000) and Gerlach (2001) reported two syna- (2001) found strong support for the sister relationship pomorphies: a ventral ridge on the maxilla–premaxilla between I. travancorica and I. elongata using MP, but their suture and keels on the supraoccipital crest. In addition, the ML analysis favored I. travancorica and I. forstenii as sister close relationship between Pyxis and G. yniphora + G. radi- taxa. The study by Spinks et al. (2004) also supported this ata was also supported by Gerlach with the synapomorphy latter hypothesis. of an indistinct fenestra postotica. By contrast, other stud- For the third and largest testudinid clade, we Wnd ies (e.g., GaVney and Meylan, 1988; Meylan and Sterrer, support for Wve internal clades: (1) Chersina + Homopus + 2000; Takahashi et al., 2003) have varied widely concerning Psammobates+ Geochelone pardalis, (2) the Geochelone ele- the relationship of Pyxis to other testudinid genera. Con- gans group, (3) Kinixys, (4) the Geochelone carbonaria group, cerning the Indian Ocean giant tortoises (Aldabrachelys), and (5) Aldabrachelys+Pyxis+Geochelone radiata and for all Wve genes we found almost no variation among the G. yniphora. However, relationships between these clades con- three forms: A. dussumieri, A. arnoldi, and A. hololissa. Only Xict between the MP, ML, and Bayesian analyses, and corre- two sites showed variation: one for 16S (position 2004) and sponding branch support is also generally lower (MP the other for Cmos (position 349). These results support the BP< 50%, ML BP< 50–87%, and Bayesian PP 57–100%). conclusions of Palkovacs et al. (2003) and Austin et al. Of special signiWcance (and as suspected by previous (2003) that all three forms may actually represent a single researchers), we Wnd substantial polyphyly for the problem- species and population originating from Aldabra. However, atic genus Geochelone. Nevertheless, the phylogenetic these molecular results need to be evaluated by carefully results we present here for Geochelone were not recovered examining the diagnostic morphological characters used to by previous morphological and molecular studies (Gerlach, describe these species in order to conWrm that these features 2001; Meylan and Sterrer, 2000; van der Kuyl et al., 2002; do not vary intraspeciWcally. Palkovacs et al., 2002). The strongly supported sister rela- tionships we Wnd between G. pardalis and Psammobates, 4.2. IntraspeciWc divergence between Chersina and Homopus; and the monophyly of Geochelone elegans group (G. elegans, G. platynota, and G. The two samples representing diVerent subspecies of sulcata) were not evident in previous morphological stud- Geochelone pardalis: G. p. pardalis and G. p. babcockii have M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531 527 an uncorrected pairwise sequence divergence distance of which we Wnd here for Manouria and Gopherus, has also 5.9% for cytb, the fastest evolving gene in this study with been reported in the family Dibamidae (blind skinks) and the highest proportion of variable sites (Table 4). These two the skink genus Scincella, which are distributed in the subspecies are also known to exhibit morphological diVer- Americas and Asia (Honda et al., 2003; Zug et al., 2001). ences. The carapace shape of G. p. babcockii is distinctly These Asian–American divergences may have resulted from convex dorsally, compared to the Xatter G. p. pardalis cara- the well-known disruption of Xoral and faunal exchange pace; and juvenile G. p. babcockii has single carapacial across the Bering Strait in the early Eocene; a biogeo- scute spots, while G. p. pardalis has two (Loveridge and graphic pattern that has been well documented in other Williams, 1957). Furthermore, there is a sexually dimorphic groups (Beard, 2002; Bowen et al., 2002; Maekawa et al., size diVerence: G. p. pardalis males being slightly larger 2005; Sanmartin et al., 2001 and references therein; TiVney, than females and G. p. babcockii males being much smaller 1985). (Broadley, 1989). G. p. pardalis occurs in western South The DIVA ancestral area optimization results provide a Africa and southern Namibia. G. p. babcockii ranges from striking conclusion: it is most parsimonious to consider all Sudan and Ethiopia to Natal and from Cape Province to testudinids with the exception of Manouria and Gopherus as southwest Africa and southern Angola (Iverson, 1992; having an African ancestral area. The alternative biogeo- Loveridge and Williams, 1957). Our preliminary results graphic scenarios, such as considering the ancestral area for suggest that a phylogeographic study of G. pardalis is this large group as Asia or the Palearctic, will require mak- expected to reveal high genetic divergence within this spe- ing additional assumptions of dispersal or extinction. Our cies, or species complex. results infer the following dispersals from Africa: Indote- By contrast, the cytb uncorrected pairwise sequence studo and the clade including Geochelone platynota + G. ele- divergence distance between the two samples we have of gans dispersing into Asia, Testudo dispersing into Europe, Manouria e. emys (Burmese tortoise) and M. e. phayrei the clade including Aldabrachelys + Pyxis + Geochelone (Burmese black tortoise) is just 1.3%. Results showing even radiata + G. yniphora dispersing into the Indian Ocean, and less geographic variation for mtDNA within a testudinid the G. carbonaria group dispersing into South America. species have been recently reported for Geochelone radiata Africa should therefore be viewed as both a major center of (Leuteritz et al., 2005). origin and diversiWcation for testudinid species. Three clades found by our study are endemic to Africa: Malac- 4.3. Biogeographic history ochersus, Kinixys, and the diverse clade including Homopus+ Chersina + Psammobates + Geochelone par- The earliest Testudinidae fossils (not yet described) are dalis, and these groups presumably diversiWed on this conti- reported to be from the lower Paleocene of Asia (Claude nent (the Kinixys belliana population of Madagascar is and Tong, 2004; Hutchison, 1998; Joyce et al., 2004), how- typically considered as introduced, see Raselimnanana and ever older fossils of the putative ancestor (family Lind- Vences, 2003). holmemydidae) for the superfamily Testudinoidea are The dispersal of Testudo and Indotestudo from Africa known from the upper Cretaceous of Asia (Nessov, 1988; into Europe and Asia appears to be a novel biogeographic Sukhanov, 2000). The appearance of testudinids fossils by scenario that has not been previously considered. For the early Eocene in both Europe (Achilemys cassouleti) and example, Parham et al. (2006) only discuss dispersal events North America (Manouria or Manouria-like fossils and from a Palearctic center of diversiWcation for Testudo, Hadrianus majusculus), and by the late Eocene in Africa Indotestudo, and Malacochersus. In addition, Crumly’s (Gigantochersina ammon) (Claude and Tong, 2004; Crumly, (1984a) discussion of biogeographic scenarios implied that 1984a; Holroyd and Parham, 2003; Hutchison, 1996, 1998), African tortoises originated from Asia. However, and by indicates that if tortoises Wrst originated in Asia, they must contrast, the Indian Ocean clade including Aldabrachelys have soon dispersed to other continents. The abundance of + Pyxis + Geochelone radiata + G. yniphora has been previ- fossil testudinids also suggests that the fossil record is ously proposed to have an African origin (Caccone et al., unlikely to be substantially underestimating the origin time 1999a; Palkovacs et al., 2002), as supported by our DIVA for the family. All evidence to date, thus, supports a late ancestral area optimizations (Fig. 4). Mesozoic or early Cenozoic origin for the Testudinidae in However, perhaps the most unusual biogeographic Asia, with dispersal to North America, Europe, and Africa result concerns the South American + Galápagos tortoises having been achieved by the Eocene. Our DIVA results also (Geochelone carbonaria group), which we Wnd are members establish the ancestral area for testudinids as including of a larger clade otherwise only including species from three continents: Asia, North America, and Africa, and Africa, the Indian Ocean, and Asia. To test the previously Wnds Manouria and Gopherus as the extant groups repre- proposed North American origin for the Geochelone car- senting the Asian and North American lineages, respec- bonaria clade, based upon a sister relationship with Gophe- tively. Remarkably, all other extant testudinid groups are rus (e.g., Gerlach, 2001) we searched for the shortest associated with the African ancestral area. constraint tree that included these clades as sisters. This The remarkable biographic basal lineage bifurcation constraint tree was 48 steps longer than our most parsimo- between clades distributed in Asia and North America, nious 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 con- Aldabrachelys, yet also due to the uncertainty concerning sistently recovered sister relationship we found between the relationships of Geochelone yniphora and G. radiata to African Kinixys and the Geochelone carbonaria clade, and these genera, we therefore prefer to conservatively place the distribution of the group sister to this clade, which also both these species in separate monotypic genera. We rec- includes Africa, providing strongest support for an African ommend the use of the available genus name Astrochelys origin for the South American tortoises (Fig. 4). The DIVA (Gray, 1873; type species, A. radiata) for G. radiata. How- ancestral area optimizations Wnd this entire clade ever, because there is no available generic name for G. yni- (Geochelone + Pyxis + Aldabrachelys + Homopus + Chersina phora, we thus propose the following new genus: + Psammobates + Kinixys) to have an African ancestral Angonoka new genus area, thus supporting an early evolutionary history for this Type species. Testudo yniphora Vaillant 1885. diverse tortoise group in this continent. Diagnosis—Testudinid tortoises that have the following The dispersal of tortoises from Africa to South America characters: (1) gulars fused to form a single scute; (2) gular might have been aided by westward sea currents, such as projection thickened and sexually dimorphic in size, pro- the Equatorial Currents (Fratantoni et al., 2000). These jecting well beyond the carapace edge and curved upward Xow between the Congo delta and Maranhao in Brazil and in adult males; (3) highly domed carapace with descending are believed to have been in this direction since the breakup sides; (4) carapace brown, lacking radiating yellow or black of Gondwana (Houle, 1999; Parrish, 1993). Several verte- lines on scutes; (5) no enlarged tubercles on thighs; (6) tail brate animal groups are reported to show a pattern of dis- without an enlarged terminal scale; (7) longitudinal ventral persal from Africa to South America across the Atlantic: ridge on the maxilla–premaxilla suture, (8) horizontal keels platyrrhine monkeys and caviomorph rodents between 85 on the supraoccipital crest; (9) pectoral scutes narrow and and 35 MYA, (Houle, 1999; Huchon and Douzery, 2001; not contacting the entoplastron. Mouchaty et al., 2001; Nei et al., 2001; Schrago and Russo, 2003), and Mabuya skinks twice during the last 9 million Content. One species, Angonoka yniphora (Vaillant years (Carranza and Arnold, 2003). Tortoises are well 1885). known for their ability to Xoat (while extending their heads Distribution. Northwest Madagascar. above water) and survive without food or freshwater for up Etymology. The generic name “Angonoka” is based on to six months (Caccone et al., 2002), and oceanic dispersal the Malagasy word ‘Angonoka’ which is the local name by testudinid tortoises appears to have occurred many for the type species. times. For example: Geochelone to the West Indies (Wil- liams, 1950, 1952), Geochelone to the Galápagos and at 5. Conclusions least Wve subsequent dispersals between islands in the archi- pelago (Caccone et al., 1999b, 2002; Russello et al., 2005), The broad taxonomic sampling of tortoises we were able ancestral Malagasy tortoises to Madagascar from main- to achieve with this study, coupled with the inclusion of land Africa (Caccone et al., 1999a), Aldabrachelys to both mitochondrial and nuclear genes, has resulted in sup- Aldabra, Astove, Cosmoledo, Denis, Amirantes, Comoros, ported tree topologies with both good resolution, and for Assumption, and the Granitic Seychelles (Austin et al., many branches, high levels of support. Importantly, these 2003); Cylindraspis (now extinct) to Réunion, Mauritius, topologies are also largely congruent, regardless of the and Rodrigues (Austin and Arnold, 2001); and Hesperote- approach taken concerning phylogenetic analysis. We con- studo (now extinct) to Bermuda (Meylan and Sterrer, 2000). sider this result to represent a substantial advance in our understanding of testudinid relationships, especially con- 4.4. Taxonomic issues cerning the problematic genus Geochelone, which has been in a state of substantial taxonomic confusion for the past 30 Based on our phylogenetic results, and to reXect mono- years. Although our results Wnd Geochelone polyphyletic, phyletic groupings within the Testudinidae, we propose the representing at least four independent clades, we anticipate following taxonomic changes: these Wndings will stimulate new interest within these tor- (1) The name Geochelone (Fitzinger, 1835; type species, toise groups and strengthen future comparative research. G. elegans) is retained for just three species: G. sulcata, Despite these systematic advances, further phylogenetic G. elegans, and G. platynota. analysis will be needed to test the relationships we report (2) The South American and Galápagos Island species here between Indotestudo, Testudo, and Malacochersus, and (Geochelone carbonaria group) are placed in the genus the relationships of G. yniphora and G. radiata. The inclu- Chelonoidis (Fitzinger, 1835; type species, C. carbonaria), sion of additional genes (nuclear and mitochondrial) and which has been previously recognized as a subgenus (Fitz- morphological data will likely resolve this uncertainty. inger, 1856). Importantly, a re-examination of the morphological char- (3) Geochelone pardalis is placed in the genus Psammo- acters will also provide new insights concerning character bates (Fitzinger, 1835). evolution for the group, and in addition, aid with the inter- (4) Based on the desirability of maintaining the morpho- pretation of the fossil taxa. Unlike most extant reptile fami- logically distinct and well-supported genera Pyxis and lies, tortoises have a comparably rich Cenozoic fossil M. Le et al. / Molecular Phylogenetics and Evolution 40 (2006) 517–531 529 record, and thus oVer excellent opportunities for integrat- Beard, C., 2002. East of Eden at the Paleocene/Eocene boundary. Science ing fossil taxa within systematic studies based on both mol- 295, 2028–2029. ecules and morphology. Bour, R., 1982. 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Cheri Asa and Randy Junge analyses, partition choice, and the phylogenetic relationships of scincid (Saint Louis Zoo), Dr. Sue Lynch (ChaVe Zoo), Dr. Ronald lizards. Syst. Biol. 54, 373–390. A. Nussbaum (University of Michigan Museum of Zoology), Bremer, K., 1994. Branch support and tree stability. Cladistics 10, 295–304. Broadley, D.G., 1989. Geochelone pardalis. In: Swingland, I.R., Klemens, Eric Palkovacs, Yale University, Dr. Steve Reichling (Mem- M.W. (Eds.), The Conservation Biology of Tortoises. IUCN, Gland, phis Zoo), Dr. Chris Tabaka (Detroit Zoo), Dr. Harold Voris Switzerland, pp. 43–46. (Field Museum of Natural History), and Mr. Truong Quang Caccone, A., Amato, G., Gratry, O.C., Behler, J., Powell, J.R., 1999a. A Bich, Cuc Phuong National Park, Vietnam. We are also molecular phylogeny of four endangered Madagascar tortoises based grateful to Dr. John Iverson for reading and providing help- on mtDNA sequences. Mol. Phylogenet. Evol. 12, 1–9. 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