Current Herpetology 20(2): 105-133, December 2001 (C) 2001 by The Herpetological Society of Japan

Phylogenetic Relationships of Geoemydine (Reptilia: Bataguridae)

YUICHIROU YASUKAWA,1* REN HIRAYAMA,2 AND TSUTOMU HIKIDA3

1 Tropical Biosphere Research Center , University of the Ryukyus, Nishihara, Okinawa 903-0213, JAPAN 2 Department of Information , Teikyo Heisei University, Uruido 2289, Ichihara, Chiba 290-0193, JAPAN 3 Department of Zoology , Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, JAPAN

Abstract: Monophyly of the batagurid was evalu- ated, and phylogenetic relationships within the subfamily were inferred on the basis of 35 morphological characters. Two approaches, parsimony analysis using the branch and bound algorithm, and neighbor joining clustering of an absolute distance matrix, were used. The results of these analyses yielded phylograms that were almost identical in branching topology, and poorly supported the monophyly of Geoemydinae. This subfamily thus seems to be a metataxon, most likely consisting of the sister group of Batagurinae (Geo- emyda group) and a more primitive stock of Bataguridae ( group). The latter accommodates Mauremys and and its monophyly is not well supported. The former consists of the remaining ten geoemydine genera united by two synapomorphies-absence or reduction of the quadratojugal, and pos- teriorly short-sided anterior neurals. Relationships revealed by our analyses necessitate some changes in the generic classification of Geoemydinae. First of all, Cistoclemmys Gray, 1863 (type : Ci. flavomarginata, often synony- mized to Cuora Gray, 1855 (type species: Cu. amboinensis), is shown to be a valid closely related to Pyxidea and rather than to Cuora (sensu stricto). Fitzinger,1835 (type species: R. punctularia) is shown to be polyphyletic, and Chelopus Cope, 1870 (type species: C. rubida), is resurrected to accommodate R. rubida and R. annulata. The plastral hinge seems to have evolved three times in the Bataguridae-in the - Notochelys clade, Cuora (sensu stricto), and the Cistoclemmys-Pyxidea clade.

Key words: Geoemydinae; Bataguridae; Phylogeny; Geoemyda group; Mau- remys group

INTRODUCTION

* Corresponding author . Tel: + 81-98-895- McDowell (1964) recognized three 8937; Fax: +81-98-895-8966. , Batagurinae, , and E-mail address: [email protected] Testudininae, in the Testudinidae. (Y. Yasukawa) Gaffney and Meylan (1988) raised those 106 Current Herpetol. 20(2) 2001

subfamilies to separate families (- annulata-R. rubida-Geoemyda-Cistoclem- idae, , and Testudinidae, respec- mys-Pyxidea clade being monophyletic tively) of the superfamily , with the family Testudinidae rather than making the Bataguridae the largest and with the other geoemydine genera. In this most divergent family in the hypothesis, the Batagurinae is the sister Testudines. They also divided this family group of the Geoemydinae plus the Testu- into two subfamilies, Batagurinae and dinidae, and the Bataguridae is also para- Geoemydinae (Gaffney and Meylan, 1988). phyletic. The former currently contains 22 species of With respect to batagurine monophyly 12 genera (David, 1994; McCord and and phylogeny within this subfamily, Iverson, 1994) distributed over most of Gaffney and Meylan (1988) approved temperate to tropical Asia, except for the Hirayama's (1984) view because most char- western part (Iverson, 1992). The latter acters Hirayama employed show a low consists of 45 species of 12 genera (David, degree of homoplasy in their cladogram. 1994; McCord et al., 1995, 2000; Fritz et They also shared the view of possible al., 1997; Fritz and Wischuf, 1997; non-monophyly of the Bataguridae with McCord, 1997), occurring in northern Hirayama, but doubted the geoemydine Africa, southern Europe, most parts of phylogeny hypothesized by Hirayama temperate to tropical Asia, central America, because of a very high degree of and northern South America (Iverson, homoplasy postulated therein. Since then, 1992). no comprehensive systematic studies have The first attempt to clarify the phylogeny been carried out for the Bataguridae, of the Bataguridae was made by Hirayama leaving both the geoemydine phylogeny (1984) (as the Batagurinae: see above), and the batagurid monophyly problematic. utilizing cladistic analyses of 37 species Hirayama's (1984) paper was essentially representing all recognized genera mainly an abstract of a much larger work of on the basis of morphological characters. character analysis (not yet published), and Two major groups were recognized, of thus a number of important issues, such as which one, consisting of Batagur, Callagur, detailed descriptions of character states, Chinemys, Geoclemys, Hardella, Hier- were omitted (Gaffney and Meylan, 1988). , Kachuga, , , In addition, a number of taxonomic Ocadia, Orlitia, and , was changes have been made for geoemydine characterized by relatively broad triturating turtles since Hirayama's (1984) analysis surfaces of jaws (=Batagurinae sensu (David, 1994; Obst and Reimann, 1994; Gaffney and Meylan, 1988), whereas the McCord et al., 1995; Yasukawa et al., other, accommodating Cistoclemmys, 1996; Fritz and Wischuf, 1997; Fritz et Cuora, Cyclemys, Geoemyda, , al., 1997; Iverson and McCord, 1997a; Mauremys, , Notochelys, McCord, 1997; Lehr et al., 1998), though a Pyxidea, Rhinoclemmys and Sacalia, was few recent authors suspect some of these characterized by relatively narrow tritu- changes to have been misled by artificial rating surfaces of jaws (=Geoemyd- hybrids and thus are untenable (van Dijk, inae). Hirayama (1984), while defining the 2000; Shi and Parham, 2001; Das, personal broad triturating surfaces as a synapo- communication). These conditions have morph of the Batagurinae, regarded the made it strongly desirable to conduct narrow triturating surfaces in the Geoemy- additional analyses for the geoemydine dinae as a primitive condition of the Bat- phylogeny with detailed methodological aguridae. He thus considered Geoemydinae descriptions and considerations of those as paraphyletic, with the Rhinoclemmys taxonomic changes. We therefore analyzed YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 107 geoemydine phylogeny on the basis of the lacked lateral keels or carapace serration morphological characters of 32 species (i. e., not belong to Ci. g. serrata), but were representing 11 genera (i. e., more than not identified to subspecies level. 70% of extant species representing all There were no essential differences in recognized genera but one). characters examined among Cuora trifasci- ata, Cu. aurocapitata and Cu. pani, MATERIALS AND METHODS although these species differ remarkably from each other in coloration. They are Representatives of the following genera probably very closely related to each other were examined (see appendix for further within Cuora (McCord and Iverson, 1991). details). We thus combined them into a single Cistoclemmys and Cuora. -At present, operational taxonomic unit (OTU) as the the genus Cistoclemmys Gray, 1863 (type Cuora trifasciata species-group. species: Ci. flavomarginata), is usually Cyclemys. -Fritz et al. (1997) described regarded as a junior synonym of Cuora Cyclemys pulchristriata from Vietnam, and Gray, 1856 (type species: Cu. amboinensis) recognized four species, C. dentata, C. (Sites et al., 1984; Ernst and Barbour, oldhamii, C. pulchristriata, and C. tchep- 1989; McCord and Iverson, 1991; Iverson, onensis, for this genus mainly on the basis 1992; David, 1994; Fritz and Obst, 1997; of differences in coloration. On the other Wu et al., 1998). Nevertheless, several hand, Iverson and McCord (1997a), authors regard Cistoclemmys, also accom- analyzing morphometric variation within modating Ci. galbinifrons, as valid (e.g., Cyclemys, described C. atripons from Bour, 1980; Hirayama, 1984; Gaffney and southeastern Thailand and Cambodia, and Meylan, 1988; King and Burke, 1989; Ota, recognized only two species, C. dentata 1991; Zhao and Adler, 1993; Yasukawa and C. atripons, for this genus. As such, and Ota, 1999). We tentatively followed the of Cyclemys is still consider- the latter classification because of differ- ably confused, strongly requiring further ences between the two species of Cisto- verification. We examined 11 specimens of clemmys and the remainder of Cuora sensu Cyclemys, but most of them were skeletal lato (Cuora sensu stricto) (Hirayama, 1984; specimens and thus were not identified to Yasukawa, 1997), and examined the fol- the species level. Detecting no essential lowing species as their representatives- differences in all examined characters, we Cistoclemmys flavomarginata (13 speci- treated all of them as a single sample, mens), Ci. galbinifrons (7), Cuora amboin- Cyclemys, in this analysis. ensis (15), Cu. aurocapitata (4), Cu. Geoemyda. -This is a name-bearing type mccordi (6), Cu. pani (4), Cu. trifasciata genus of the subfamily Geoemydinae. This (5), and Cu. zhoui (4). genus was long regarded as consisting Among the five subspecies of Ci, galbini- solely of Geoemyda spengleri, with two frons, Ci. g. serrata is most divergent, subspecies, G. s. spengleri and G. s. japon- exhibiting a weak pair of lateral keels and ica (e.g., Ernst and Barbour, 1989; Iverson, serration at the posterior margin of the car- 1992). Yasukawa et al. (1992), however, apace (Fritz and Obst, 1997), whereas the elevated both of them to full species, G. other four subspecies are difficult to distin- spengleri and G. japonica, because of their guish from each other (Iverson and remarkable morphological divergences and McCord, 1992a; Obst and Reimann, 1994; prominent geographic isolation from each Lehr et al., 1998). Fritz and Obst (1997) other. Moll et al., (1986), on the basis of suspected full-species status of Ci, g. ser- comparisons with G. spengleri sensu lato, rata. Our specimens of Ci. galbinifrons Heosemys grandis, and H. spinosa, reas- 108 Current Herpetol. 20(2) 2001

signed Heosemys silvatica to Geoemyda. essential differences in any character among We followed this generic arrangement like M. caspica, M. leprosa, and M. rivulata. a few other recent revisions (David, 1994; They were formerly considered as three McCord et al., 1995; Yasukawa and Ota, subspecies of M. caspica, and are most 1999). likely monophyletic (Busack and Ernst, McCord et al. (1995) also assigned their 1980; Ernst and Barbour, 1989; Iverson new species, Geoemyda yuwonoi, and the and McCord, 1994). We thus treat them as two other species, Heosemys depressa and constituting a single OTU, the Mauremys H. leytensis, to Geoemyda. However, leprosa species-group. generic status of the three species remains Melanochelys. -This genus contains two controversial (Fritz and Obst, 1996; Iverson species, M. tricarinata and M. trijuga, of and McCord, 1997b; Yasukawa and Ota, which only M. trijuga (10) was examined. 1999). Recently, McCord et al. (2000) Notochelys. -We examined six specimens assigned G. yuwonoi to a new genus, of N. platynota, the only representative of Leucocephalon, and reassigned G. depressa this monotypic genus. to Heosemys on the basis of analysis of Pyxidea. -This is a monotypic genus mitochondrial DNA variation. Since we consisting solely of P. mouhotii. This did not examine those three species, we species, formerly assigned to Geoemyda tentatively followed McCord et al. (2000). (e. g., McDowell, 1964) or Cyclemys For the genus Geoemyda as defined above, (e. g., Wermuth and Mertens, 1961; Prit- G. japonica (13), G. silvatica (6), and G. chard, 1979), is treated as Pyxidea by spengleri (12) were examined. most recent authors (e.g., Hirayama, Heosemys. -McDowell (1964) assigned 1984; Ernst and Barbour, 1989; Iverson, five species, Heosemys grandis, H. spinosa, 1992; David, 1994). Five specimens were H. depressa, G. leytensis and G. silvatica, examined. to Heosemys, probably on the basis of Rhinoclemmys. -This is the only bat- absence of the quadratojugal (squamosal in agurid genus occurring in central and his terminology). He, however, did not South America, and includes nine spe- directly examine the latter three species, of cies: annulata, areolata, diademata, which H. leytensis and H. silvatica were funerea, melanosterna, nasuta, pulcher- recently reassigned to Geoemyda (see rima, punctularia, and rubida (David above). We examined H. grandis (3) and 1994). Hirayama (1984) suggested the H. spinosa (4). polyphyly of the genus, but all subsequent Leucocephalon. -This genus contains authors have apparently ignored this only one species, Leucocephalon yuwonoi, account (e. g., Ernst and Barbour, 1989; which was formerly assigned to Geoemyda Iverson, 1992; David, 1994). Therefore, we or Heosemys (McCord et al., 2000). We tentatively treated them as composing a sin- have had no chance to examine this spe- gle genus, and examined R. annulata (2), R. cies. areolata (2), R. funerea (3), R. pulcherrima, Mauremys. -This genus currently con- (5), R. punctularia (4), and R. rubida (1). sists of eight species, M. annamensis, M. Sacalia. -Iverson and McCord (1992b) caspica, M. iversoni, M. japonica, M. recognized three species, S. bealei, S. quad- leprosa, M. mutica, M. pritchardi, and riocellata, and S. pseudocellata, for this M. rivulata (Fritz and Wischuf, 1997; genus. They showed some differences in McCord, 1997), of which six, M. annamen- coloration and shell shape, but none in sis (6), M. caspica (1), M. japonica (10), internal morphology. We examined S. M. leprosa (2), M. mutica (11), and M. riv- bealei (2), S. quadriocellata (2), and Sacalia ulata (6), were examined. There were no sp. (2: skeletal specimens not identified at YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 109 the species level). Because no essential employed by McDowell (1964) and differences existed among these specimens Hirayama (1984) were not used in our anal- in any character, we treat them as a single yses, because they showed high intraspe- OTU, Sacalia. cific and/or intrageneric variation in Batagurinae. -To evaluate states of ingroup taxa. These characters include: characters in ingroup taxa and test their the shape of the fissura ethmoidalis, scutel- monophyly, we examined 44 batagurine lation of the forearm, web of the hindlimb specimens of 12 species representing all of digits, and subdivision of the foramen the currently recognized batagurine genera: nervi trigeminalis. See "Characters Exam- Batagur baska (2 specimens), Callagur ined and Distribution of Their States" for borneoensis (3), Chinemys reevesii (10), further details. Geoclemys hamiltonii (2), Hardella thurjii A species-character state matrix (Table 1) (3), Hieremys annandalii (3), Kachuga was analyzed using the Branch and Bound smithii (5), Malayemys subtrijuga (4), algorithm of PAUP (version 3.1.1., Swof- Morenia petersi (2), Ocadia sinensis (4), f ord, 1991) to find all parsimonious Orlitia borneensis (1), and Siebenrockiella networks, which were rooted by the crassicollis (5) (see "Specimens Examined" outgroup species. All characters were set for further details). Das (2001) divided as ordered condition and were equally the genus Kachuga and assigned K. smithii weighted. Three kinds of indices, consis- and other three congeneric species to a tency index (Kluge and Farris, 1969), reten- separate genus, Gray, 1869, tion index (Farris, 1989a), and rescaled usually regarded as a junior synonym or a consistency index (Farris, 1989b), were subgenus of Kachuga (Wermuth and calculated for goodness-of-fit statistics. Mertens, 1977; Moll, 1987). However, we In addition, we applied a neighbor-joining tentatively treated the two genera as a method (Saitou and Nei, 1987) for an single unit, genus Kachuga, because both absolute distance matrix using the genera are possibly valid but probably NEIGHBOR program in PHYLIP (version monophyletic (Hirayama, 1984; Moll, 3.5c, Felsenstein, 1989) following Hikida 1987). In the "Characters Examined and (1993). The network calculated was also Distribution of Their States" section, we rooted by the outgroup species. This refer to these species only by their generic analysis was aimed at evaluating the name. We selected two batagurine genera, validity of the relationships proposed in Orlitia and Siebenrockiella, as the possibly parsimonious trees and to assess the degrees closest outgroup of the Geoemydinae, of differentiation among samples. Since all because these genera exhibited a particular characters were set as ordered condition in similarity, as batagurines, with the latter this study, the absolute distance (diff [y, z]) by having narrow, ridgeless triturating was calculated as cliff (y, z)=|y-z| following surfaces, and lacking the secondary Swofford (1991). The resultant absolute palate. Orlitia and Siebenrockiella, possi- distance matrix is given in Table 2. bly closely related to each other, are likely to be regarded as the most primitive CHARACTERS EXAMINED AND batagurines (McDowell, 1964; Carr and DISTRIBUTION OF THEIR STATES Bickham, 1986; Hirayama, 1984; Gaffney and Meylan, 1988). We examined states of 35 characters as We analyzed 35 characters: 20 for the below for our 26 geoemydine and 12 skull, one for the hyoid apparatus, ten for batagurine samples (Table 1). the shell, one for the pelvic girdle, and Characters Used for the Phylogenetic three for soft parts). Several characters Analysis 110 Current Herpetol. 20(2) 2001

TABLE 1. Species-character state matrix for the subfamily Geoemydinae and its relatives.

1. Frontal exposure onto orbital rim times observed in Siebenrockiella, Rhino- In Chinemys, Hieremys, Malayemys, clemmys pulcherrima, R. funerea, and Morenia, and Orlitia of the batagurines, Cuora amboinensis. In the other species, and Heosemys grandis and Rhinoclemmys the frontal usually reached the orbital rim, punctularia of the geoemydines, the frontal but was sometimes separated by a very was separated from the orbital rim by a weak connection. We coded this character strong connection between the prefrontal as: (0) usually present, without prefrontal- and postorbital. This condition was some- postorbital connection; (1) sometimes YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 111

TABLE 2. Absolute distance matrix for species of the subfamily Geoemydinae and its relatives.

absent, with weak prefrontal-postorbital Cistoclemmys galbinifrons and Geoemyda connection; (2) absent, with strong pre- silvatica had the cranial cavity weakly frontal-postorbital connection. convergent ventrally. We coded this char- acter as: (0) almost parallel; (1) weakly 2. Processus inferior parietalis approximating; (2) approximating. The right and left processus inferior parietalis were almost parallel in most 3. Foramen nervi vidiani batagurids. In Cistoclemmys flavomargin- The foramen nervi vidiani was located ata, Geoemyda japonica, G. spengleri, and anteriorly to the anteroventral part of the Pyxidea, the processus were close to each processus inferior parietalis, and was other ventromedially, and the lower part of bordered by the processus and palatine in the cranial cavity was strongly convergent. most batagurid species. In Geoclemmys, 112 Current Herpetol. 20(2) 2001

Hardella, Morenia, Cistoclemmys, Geo- had no quadratojugal at all. We coded this emyda, and Pyxidea, the foramen was character as: (0) present; (1) absent. located posterolaterally or laterally to the anteroventral part of the processus inferior 7. Contact between quadratojugal and parietalis, bordered by the parietal, pteryg- squamosal oid, and epipterygoid, or sometimes open In batagurines exclusive of Hieremys, the in the pterygoid (2/3 of Geoemyda japonica quadratojugal was firmly connected to the and 3/5 of G. spengleri). We coded this squamosal as well as to the quadrate and character as: (0) bordered by processus the jugal. On the other hand, the quad- inferior parietal and palatine; (1) bordered ratojugal was absent (see above), or if by parietalis, pterygoid, and epiptery- present, did not usually contact the squa- goid; (2) usually on pterygoid. mosal in most geoemydines. In Geoemyda spengleri, Mauremys and Sacalia, however, 4. Medial process of jugal a weak contact was usually present. We In most batagurid species, the medial coded this character as: (0) usually present; process of the jugal was moderately to well (1) absent; (?) no quadratojugal. developed, and its ventromedial tip was expanded. In Cistoclemmys, Geoemyda, 8. Contact between jugal and quad- and Pyxidea, the process was weak and ratojugal had a tapering ventromedial tip. We coded The contact between the jugal and the this character as: (0) moderate to well quadratojugal was present in batagurines developed; (1) weak. except for Hieremys, while it was usually absent in the geoemydines. A weak contact 5. Contact between jugal and pterygoid was present in Geoemyda japonica, G. spen- Most batagurid species had a medial glen, Mauremys, Rhinoclemmys areolata, process of the jugal which was firmly R. funerea, R. pulcherrima, R. punctularia, connected with the pterygoid. In Cisto- and Sacalia. We coded this character as: (0) clemmys flavomarginata, Geoemyda, and present; (1) absent; (?) no quadratojugal. Pyxidea, the connection was usually absent or very weak if present. We coded this 9. Bony beak of upper jaw character as: (0) present; (1) usually absent Most batagurid turtles had a shallow or very weak. notch on the anteroventral margin of the unhooked bony beak composing the upper 6. Quadratojugal jaw. The corresponding portion was Among batagurines, Callagur, Batagur, usually flat, or sometimes only weakly Chinemys, Geoclemmys, Hardella, Malay- notched, with or without making the ante- emys, and Orlitia had a massive quadrato- rior portion hook-shaped as a whole, in jugal, whereas in Kachuga, Morenia, Kachuga, Cuora amboinensis, Cistoclem- Ocadia, and Siebenrockiella the quadra- mys galbinifrons, and Mauremys japonica. tojugal was small to moderate in size. In Cistoclemmys flavomarginata, Cuora On the other hand, most geoemydines exclusive of Cu. amboinensis, Geoemyda, had a much smaller quadratojugal, Pyxidea, Rhinoclemmys annulata, and R. although this element was moderate in rubida, the beak was unnotched and, size in Mauremys and Sacalia, and was slightly but distinctly hooked. We coded exceptionally elongated but laterally this character as: (0) notched and strongly compressed in Geoemyda spen- unhooked; (1) usually flat, but sometimes gleri. Hieremys, Cistoclemmys flavomargi- with a very weak notch or hook; (2) nata, Geoemyda silvatica, and Heosemys unnotched and distinctly hooked. YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 113

10. Ventromedial meeting of maxillae annulata, and R. rubida, the anteromedial at bony beak of upper jaw portion of the upper triturating surface was In most batagurid genera, the tip of the expanded medially and broader than the bony beak of the upper jaw was formed posterior portion, and the posteroventral by the premaxilla, sometimes with very portion of the premaxillae participated in weak participation of the maxilla. In the triturating surface. This character state Geoemyda, the right and left maxillae was usually observed in the batagurines were firmly connected ventromedially except for Orlitia and Siebenrockiella. The with each other, forming the beak. We posterior triturating surface was very coded this character as: (0) absent or narrow in Cistoclemmys, Geoemyda, sometimes present but very weak; (1) Pyxidea, R. annulata and R. rubida, present. whereas it was broad in batagurines. We coded this character as: (0) narrower than 11. Serration of upper labial margin of posterior portion; (1) relatively narrow but maxilla expanding medially; (2) broad and expand- Batagur, Callagur, Hardella, Kachuga, ing medially. Morenia, and Ocadia of the batagurines had moderate to strong serration at the 14. Contact between maxilla and vomer upper labial margin of the maxilla, A firm connection between the maxilla whereas the other batagurines and most and vomer usually existed in most batagurid geoemydines did not have such serration. species. On the other hand, the contact Rhinoclemmys species except for R. was absent in Cuora, Rhinoclemmys rubida had the serration weaker than in areolata, R. funerea, R. pulcherrima, and the above mentioned batagurines. We R. punctularia, and it was very weak or coded this character as: (0) absent; (1) sometimes absent in Cistoclemmys fla- present. vomarginata, Mauremys, and Rhinoclem- mys annulata. We coded this character as: 12. Size and shape of foramen palati- (0) present; (1) very weak, sometimes num posterius absent; (2) absent. The foramen palatinum posterius was small and round in Cistoclemmys, Geo- 15. Position of foramen praepalatinum emyda, and Pyxidea, as well as in the In Cistoclemmys flavomarginata, batagurines. On the other hand, the fora- Cyclemys, Heosemys, Notochelys, Pyx- men was large, and elliptic or oval, about idea, and Sacalia, as well as in all batagur- two times longer than wide, in the other ines, the foramen praepalatinum was geoemydines. We coded this character as: located between the premaxilla and the (0) large, and elliptic or oval; (1) small and vomer. In contrast, the foramen opened round. within the premaxilla in Cuora, Geoemyda japonica, G. spengleri, Mauremys annam- 13. Anteromedial portion of upper trit- ensis, M. mutica, and Rhinoclemmys urating surface except for R. annulata. In the other geo- In Orlitia and Siebenrockiella, and most emydines, the foramen was usually located geoemydines, the anteromedial portion of near the suture between the premaxilla and the upper triturating surface was narrower vomer, or sometimes within the premaxilla. than the posterior portion, and the poster- We coded this character as: (0) between oventral portion of the premaxilla was premaxilla and vomer; (1) sometimes excluded from the surface. In Cistoclem- within premaxilla; (2) within premaxilla. mys, Geoemyda, Pyxidea, Rhinoclemmys 114 Current Herpetol. 20(2) 2001

16. Notch of prefrontal at posterior first. On the other hand, the hyoid was orbital margin weakly ossified (rather cartilaginous), and The prefrontal was usually shallowly the second pair was much shorter than the notched at the posterior orbital margin in first in Cistoclemmys, Geoemyda, Pyxidea, the Cuora trifasciata species-group and Cu. Rhinoclemmys annulata, and R. rubida. mccordi. Such a notch was absent in the We coded this character as: (0) well other species. We coded this character as: ossified with second branchial horns (0) absent; (1) mostly present. slightly shorter than the first; (1) weakly ossified with second branchial horns much 17. Contact between maxilla and pteryg- shorter than the first. oid The maxilla contacted the pterygoid in 21. Plastral hinge most batagurid species. On the other The Plastral hinge was present in Cisto- hand, such a contact was absent in Cisto- clemmys, Cuora, Cyclemys, Notochelys, clemmys flavomarginata, and was very and Pyxidea. They also possessed a well to weak or sometimes absent in Geoemyda. extensively developed musulus testoscapu- We coded this character as: (0) present; (1) laris and m. testoiliacus, which were associ- very weak or sometimes absent; (2) absent. ated with shell closure (Bramble, 1974). Among them, only Cuora amboinensis and 18. Prootic participation in processus Cistoclemmys closed both anterior and trochlearis oticum posterior plastral lobes without gaps Cyclemys, Geoemyda silvatica, Heos- between the carapace and plastron. We emys, Mauremys, Notochelys and Sacalia, did not examine the condition of the as well as batagurines, had the processus muscles in Batagur and Geoclemmys, trochlearis oticum mainly formed by the which lack a plastral hinge. The other prootic. In Cistoclemmys flavomarginata, species lacked a plastral hinge and m. test- Cuora, Geoemyda japonica, Melanochelys, oscapularis and m. testoiliacus were poorly Pyxidea, and Rhinoclemmys, the prootic developed. We coded this character as: (0) exposure was distinctly reduced, with the absent; (1) present but impossible to close processus mainly formed by the quadrate. without gaps; (2) present and possible to In the other geoemydines, the processus close without gaps. was formed by both prootic and quadrate. We coded this character as: (0) mainly by 22. Anal notch of plastron prootic; (1) by both prootic and quadrate; The plastron was distinctly notched (2) mainly by quadrate. posteromedially at the anal scutes in most batagurids. However, the notch was very 19. Quadrate participation in canalis shallow in Cuora mccordi, and absent in cavernosum Cistoclemmys, and Cuora amboinensis. The quadrate participated in the canalis We coded this character as: (0) present; (1) cavernosum in Cuora, while it usually did present but very shallow; (2) absent. not in the remaining batagurids. We coded this character as: (0) absent; (1) usually 23. Serration at posterior margin of present. carapace Hieremys, Siebenrockiella, Cyclemys, 20. Hyoid apparatus Geoemyda japonica, G. spengleri, Heose- In most batagurid turtles, the hyoid was mys, Mauremys japonica, Notochelys, and well ossified, and the second pair of bran- Pyxidea had strong serration at the poste- chial horns was slightly shorter than the rior margin of carapace. In the other YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 115 species, the serration was absent or weakly and Geoemyda. We could not examine the developed only in young individuals. We condition of this character in Batagur and coded this character as: (0) absent, or weak Geoclemmys. We coded this character as: and only in young individuals; (1) strong. (0) present; (1) much reduced or lost.

24. Longitudinal keels on carapace 28. Axillary plastral buttress Chinemys, Geoclemys and Malayemys of In Chinemys, Hieremys, and Morenia of batagurines, and Geoemyda, Melanochelys, the batagurines, and in most geoemydines and Pyxidea of the geoemydines had three having no plastral hinge, the dorsal portion longitudinal rows of strong keels on the of the axillary plastral buttress was carapace with distinct ridges on the bony connected around the portion between the plates. In the other batagurid species, only peripherals and the costals, and the the medial keel was usually developed: the connected portion was distinctly concave. lateral keels were absent or, if present, very In all geoemydines having a plastral hinge weak without distinct ridges on the bony (Cyclemys, Notochelys, Cuora, Cistoclem- plates. We coded this character as: (0) mys, and Pyxidea: see above) the dorsal weak, usually in a single row; (1) promi- portion of the buttress was very short and nent, in three rows. connected to the peripherals, and the connected portion was not or only scarcely 25. Anterior part of neural bones concave. In Mauremys annamensis of the In Hieremys of the batagurines and the geoemydines, and in Geoclemys, Malay- geoemydines exclusive of Mauremys, emys, Orlitia, and Siebenrockiella of the Sacalia, and Notochelys, the anterior batagurines, the dorsal portion of the neurals were usually hexagonal with poste- buttress was long, expanded, and strongly rior sides shorter than anterior sides. In connected to the costals, and the connected the other species, the neurals were also portion was distinctly concave. In the usually hexagonal but the anterior sides other batagurines, the buttress was much were shorter than the posterior sides. We more developed than in M. annamensis, coded this character as: (0) short-sided Malayemys, Orlitia, and Siebenrockiella, anteriorly; (1) short-sided posteriorly. and was very strongly connected to the costals, almost reaching the ribs. We 26. Sutures between lateral sides of coded this character as: (0) very strongly seventh and eighth pleurals connected to costals, almost reaching ribs; In most batagurids, the seventh and (1) strongly connected to costals; (2) eighth pleurals on each side were sepa- connected around the portion between rated by the eighth neural and the ante- peripherals and costals; (3) weakly con- rior suprapygal. In Cistoclemmys, Cuora nected to the peripherals. amboinensis, and Rhinoclemmys rubida, the eighth neural and the anterior suprapy- 29. Inguinal plastral buttress gal were usually absent, and the lateral In Chinemys, Malayemys, Morenia, sides of seventh and eighth pleurals were and Orlitia of the batagurines, and most sutured with each other. We coded this geoemydines, the dorsal portion of character as: (0) absent; (1) present. Inguinal plastral buttress was connected around the suture between the peripher- 27. Cloacal bursae als and the costals, and the connected The cloacal bursae were present in most portion was distinctly concave. In Maur- batagurids, while it was much reduced or emys annamensis, Geoclemys, and Sie- completely lost in Cistoclemmys, Pyxidea, benrockiella, the dorsal portion of the 116 Current Heroetol. 20(2) 2001 buttress was long, expanded, and 32. Radiated markings on plastron strongly connected to the costals, and Radiated markings were observed on the connected portion was distinctly each plastral scute in Cyclemys and Heos- concave. In Hieremys, Cistoclemmys, emys. We coded this character as: (0) Cuora, and Pyxidea, the dorsal portion absent; (1) present. was very short, and the connected por- tion was not concave. In the other 33. Entoplastron batagurines, the buttress was strongly In taxonomy, many authors paid developed, almost touching the rib. We attention to whether the entoplastron was coded this character in the same way as intersected by the humero-pectoral seam Character 28. (e.g., Boulenger, 1889; Smith, 1931; Bour- ret, 1941, Pritchard, 1979; Ernst and 30. Internal choanae Barbour, 1989). Our examination revealed Parsons (1960) examined the structure of that among batagurines, the state of this the choanae for most batagurids. We fol- character was variable, seemingly reflecting lowed his terminology for the structure. the phylogeny (Hirayama, 1984). On the Our examination revealed that Chinemys, other hand, the entoplastron was consis- Hieremys, Ocadia, Orlitia, and Siebenrock- tently intersected by the seam in all the iella of batagurines, and most geoemydines geoemydines but Geoemyda silvatica. had the internal choanae with a flap or a Therefore, the absence or presence of the ridge. Some specimens of Mauremys intersection was not informative for the mutica, Rhinoclemmys pulcherrima, and inference of geoemydine phylogeny. In the Cistoclemmys flavomarginata had the specimens of G. silvatica examined by us, internal choanae with a very weak flap, the entoplastron was intersected by the resembling a ridge. In Mauremys rivulata, seam near the posterior rim (4/5) or over- M. mutica, Melanochelys, and Rhino- lapped by the seam at the posterior rim clemmys pulcherrima, some specimens had (1/5). We thus considered that the absence a flap and others had a ridge. The internal of the intersection represented rather minor choanae had a small papilla in Callagur, individual variation in this species, Hardella, Kachuga, Morenia, 2/4 of although Moll et al. (1986) reported the Notochelys, Cyclemys, Rhinoclemmys absence of intersection of entoplastron by funerea, and R. punctularia. The internal the humero-pectoral seam in this species. choanae lacked a papilla, flap, or ridge in We also examined whether the entoplas- Malayemys and Heosemys. We could not tron was intersected by the gulo-humeral examine this character state in Batagur and seam. In most batagurid species, the Geoclemys. We coded this character as: entoplastron was intersected by this (0) without papilla, flap, or ridge; (1) with seam or barely separated from it (1/10 flap or ridge; (2) usually with papilla. of M. mutica and 2/9 of Sacalia), but in Geoemyda the entoplastron was in a 31. Skin of posterior head separate location posterior to the gulo- The skin of the posterior head was humeral seam. We coded the character as: smooth in most geoemydines, but subdi- (0) entoplastron mostly intersected by gulo- vided into small scales in Cyclemys, Geo- humeral seam; (1) entoplastron separated emyda silvatica, Heosemys, Melanochelys, from gulo-humeral seam. Notochelys, and Pyxidea of the geoemy- dines, as well as in the batagurines. We 34. Antero-dorsal portion of iliac blade coded this character as: (0) subdivided into In most batagurids, the antero-dorsal small scales; (1) smooth. portion of the iliac blade was about as YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 117 long as its postero-dorsal portion (Fig. 1b, Three equally parsimonious trees (tree e-j). Only in Cistoclemmys, the antero- length=107; consistency index (CI)=0.449; dorsal portion was about two times longer retention index=0.762; rescaled consistency than the postero-dorsal portion (Fig. 1c, d). index=0.342) were detected. Mauremys We coded this character as: (0) as long as japonica, the M. leprosa species-group, its postero-dorsal portion; (1) two times and the M. annamensis-M. mutica clade longer than its postero-dorsal portion. were trifurcated in all trees. These trees differed from each other in topology 35. Small foramen on ventral maxilla involving Rhinoclemmys annulata, R. Rhinoclemmys funerea and R. punctu- rubida, and the Geoemyda-Cistoclemmys- laria usually had a very small foramen on Pyxidea clade. In the strict consensus tree the ventromedial portion of the maxilla. (Fig. 3), therefore, their relationships were Such a foramen was absent in the remaining also expressed as trifurcated. batagurids. We coded this character as: (0) The consistency index for these data was absent; (1) usually present. low (CI=0.449). However, this index is known to be highly negatively correlated RESULTS with numbers of OTUs included in an anal- ysis (Sanderson and Donoghue, 1989; Klas- All characters examined were informative sen et al., 1991). Our analysis involved a for the inference of relationships among relatively large number of OTUs (28), and the 26 geoemydine OTUs. the value, while being similar to that in a

FIG. 1. Medial views of pelvic girdles. 118 Current Heroetol. 20(2) 2001

FIG. 3. Strict consensus cladogram of the subfamily Geoemydinae and its relatives. previous empirical study dealing with a Supposing that all character states in the comparable number of OTUs (0.451 for outgroup represent primitive conditions, 28 taxa: Sanderson and Donoghue, 1989), the monophyly of Geoemydinae (Stem A) was much greater than that expected for was supported by three character states: random data (0.131 for 28 taxa; Klassen et character 1 (state 0), presence of frontal al., 1991). Thus, we consider the mono- exposure on orbital rim; 12 (0), large phyletic groups indicated in our results foramen palatinum posterius with elliptic significantly realistic. or oval shape; and 28 (2), axillary plastral At the root of the Geoemydinae (Stem buttress connected around the portion A), characters 1-27 and 31-35 were in the between peripherals and costals. "0" state , whereas the characters 28-30 One synapomorphic character state, 31 were "1". We regarded these character (1) (smooth skin of posterior head), sup- states as ancestral within the Geoemydinae. ported the Mauremys-Sacalia clade (Stem Putative changes of character states in each B), which we henceforth call the Mauremys major stem are given in Table 3. group. YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 119

TABLE 3. Putative changes of character states in major stems of Geoemydinae. Letters denoting stems refer to those in Fig. 3.

The genus Mauremys (Stem C) was supported by two character states: 14 (1), maxilla sometimes contacting vomer; and 15 (1), foramen praepalatinum sometimes opening within premaxilla. Autapomorphy of Sacalia was not detected. The M. annamensis-M. mutica Glade (Stem D) was supported by 15 (2), foramen praepalati- num opening within premaxilla. Stem E, henceforth referred to as the Geoemyda group, was supported by three synapomorphic character states: 7 (1), absence of the squamosal-quadratojugal contact; 8 (1), absence of the jugal-quadra- toj ugal contact; and 25 (1), anterior neurals with shorter posterior sides. The former two character states are seemingly associated with the reduction of the quadratojugal. The state of the third char- acter was constant within the group except for Notochelys. The Notochelys-Cyclemys-Heosemys clade (Stem F) was supported by two synapomorphic character states: 23 (1), strong serration at posterior margin of carapace; and 32 (1), presence of radiated 120 Current Herpetol. 20(2) 2001 markings on plastron. The Notochelys- both plastral hinge and gap between closed Cyclemys clade (Stem G) was supported by shells; 28 (3), very weak axillary plastral three synapomorphic character states: 21 buttress; and 29 (3), very weak inguinal (1), plastral hinge not closing without gap; plastral buttress. The Cuora trifasciata 28 (3), very weak axillary plastral buttress; species group-Cu. mccordi Glade (Stem N) and 30 (2), internal choanae usually with a was supported by a unique character state: small papilla. Monophyly of Heosemys 16 (1), prefrontal notched at posterior (Stem H) was supported by two synapo- orbital margin. morphic character states: 6 (1), loss of Although there were no synapomorphies quadratojugal; and 30 (0), internal choanae uniting the Rhinoclemmys B, the Rhino- without ridge, flap, or papillae. The latter clemmys B-Cistoclemmys-Pyxidea-Geo- was unique among the geoemydines. emyda clade (Stem O) was supported by Heosemys was the sister group of the three character states: 9 (2), unnotched and Notochelys-Cyclemys clade. hooked upper jaw beak; 13 (1), anterome- Stem I was supported by two synapo- dial portion of triturating surface expanding morphic character states: 15 (1), foramen medially; and 20 (1), reduction of hyoid praepalatinum sometimes opening within apparatus. premaxilla; and 18 (2), processus trochle- Monophyly of the Cistoclemmys-Pyxidea- aris oticum mainly formed by the Geoemyda clade (Stem P) was supported quadrate. Stem J was supported by two by seven synapomorphic character states, synapomorphic character states, 9 (1: 3 (1), foramen nervi vidiani located usually flat jaw beak) and 31 (1: smooth anteroventrally or laterally to anteroventral posterior head skin), whereas M. trijuga part of processus inferior parietalis; 4 (1), had one autoapomorphic character state, medial process of jugal with small tip; 27 24 (1: three prominent keels). (1), strong reduction or loss of cloacal Rhinoclemmys was shown to be poly- bursae; 2 (2), strong ventral convergence of phyletic, consisting of two groups, Rhino- cranial cavity; 5 (1), usual absence of the clemmys A (areolata, funerea, punctularia jugal-pterygoid contact; 12 (1), small and and pulcherrima) and Rhinoclemmys B round foramen palatinum posterius; and 24 (annulata and rubida). The Rhinoclemmys (1), three prominent keels on carapace. A-Cuora clade (Stem K) was supported by The former three were unique among geo- three character states: 11 (1), very weak emydines. The Cistoclemmys-Pyxidea clade serration of upper labial margin; 14 (2), (Stem Q) was monophyletic on the basis of absence of the maxilla-vomer contact; and four character states: 15 (0), foramen prae- 15 (2), location of foramen praepalatinum palatinum located between premaxilla and within premaxilla. Within this clade, vomer; 21 (1), presence of plastral hinge; monophyly of the Rhinoclemmys A (Stem 28 (3), very weak axillary plastral buttress; L) was supported by two character states, 8 and 29 (3), very weak inguinal plastral (0: the jugal-quadratojugal contact) and 9 buttress. Validity of Cistoclemmys (Stem (0: notched and hooked beak of upper R) was supported by five synapomorphic jaw), whereas the R. funerea-R. punctu- character states: 21 (2), plastral hinge laria clade was supported by a unique closing shell without gap; 22 (2), absence synapomorphic character state 35 (1: usual of anal notch of plastron; 24 (0), usual presence of small foramen on ventral absence of lateral keels on carapace; 26 (1), maxilla). Cuora (Stem M) was further presence of sutures between lateral sides of supported by four synapomorphic character seventh and eighth pleurals; and 34 (1), states: 19 (1), quadrate participation into very long anterodorsal portion of iliac canalis cavernosum; 21 (1), presence of blade (unique in geoemydines, Fig. 1c, d). YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 121

FIG. 4. Phylogram by neighbor-joining method of the subfamily Geoemydinae and its relatives.

Pyxidea had two autoapomorphic charac- phylogram is largely identical in topology ter states: 23 (1), strong serration of poste- with the strict consensus tree from the rior carapace margin; and 31 (1), posterior parsimony analysis (Fig. 3), and thus is head skin subdivided into small scales. interpreted as supporting the validity of the Monophyly of Geoemyda (Stem S) was latter. One of the differences between the supported by five character states: 10 (1), strict consensus tree and the NJ phylogram ventromedial meeting of maxillae; 33 (1), lay in the relationships within the Rhino- entoplastron separated from gulo-humeral clemmys A. In the latter tree, R. pulcher- seam; 8 (0), presence of the jugal-quadra- rima was united not with the R. funerea-R. tojugal contact; 17 (1), very weak maxilla- punctularia group but with R. areolata. pterygoid contact; and 18 (1) processus Another difference was in the relationships trochlearis oticum formed with prootic and among R. annulata, R. rubida, and the quadrate. The former two character states Cistoclemmys-Pyxidea-Geoemyda group. were unique in the Bataguridae. In addi- In the NJ phylogram, R. annulata and R. tion, the G. spengleri-G. japonica clade rubida were united, collectively constituting (Stem T) was supported by three synapo- the first outgroup to the Cistoclemmys- morphic character states: 3 (2), foramen Pyxidea-Geoemyda group. nervi vidiani sometimes located on ptery- goid; 15 (2), foramen praepalatinum open- DISCUSSION ing within premaxilla; and 23 (1), strong serration at posterior carapace margin. Monophyly of the Bataguridae The first was unique within the Batagur- Based on the analyses of morphological idae. and mithochondrial DNA characters for The phylogram, obtained by the neighbor- major testudine taxa including one batagur- joining (NJ) method using an absolute ine, one geoemydine, three emydid, and distance matrix is shown in Fig. 4. This one testudinid genera, Shaffer et al. (1997) 122 Current Herpetol. 20(2) 2001 YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 123

argued that both Bataguridae and Emy- Monophyly of the Geoemydinae didae are probably monophyletic, and that McDowell (1964) referred to two charac- the latter is the sister group of a clade ter states as discriminating Geoemydinae consisting of the former and the Testu- from Batagurinae-narrow and flat tritu- dinidae. Recently we also found states rating surfaces, and reduction or absence of two morphological characters (i. e., of the quadratojugal. The former probably well developed foramina of axillary and represents a primitive condition of the inguinal musk ducts with penetration into Testudinoidea (Hirayama, 1984; Gaffney the breast chamber, and anterolaterally and Meylan, 1988). In this study, the latter flared iliac blade [Fig. 2]) that seemingly condition is observed in the Geoemyda lend further support to the monophyly group exclusive of G. spengleri. Because of the Bataguridae (Yasukawa, 1997; the Mauremys group had a relatively large Hirayama, Yasukawa and Aoki, unpub- quadratojugal as in Kachuga, Ocadia, lished data), because they occur in most Odsrlitia, and Siebenrockiella, it seems to batagurid turtles but not in the Emydidae be more appropriate to consider the reduc- or Testudinidae. In our examinations, the tion or absence of the quadratojugal as a former character state occurred in all characteristic of the Geoemyda group batagurids exclusive of Morenia. Musk rather than as a synapomorph of the Geo- ducts and indistinct musk foramina also emydinae. exist in some non-testudinoid families of Hirayama (1984) considered that the turtles, such as the , Chelydae, subdivision of the foramen nervi trigemina- , and Platysternidae, and their lis, observed in more than 40% of the spec- occurrence is thus likely to represent primi- imens examined for each geoemydine tive conditions in the Testudines (Gaffney species, is a synapomorph of the Geoemyd- and Meylan, 1988). However, develop- inae besides the loss or reduction of the ment of musk duct foramina to the point quadratojugal. However, Gaffney and of reaching the breast chamber is a condi- Meylan (1988) were concerned about such tion unique to the Bataguridae (Hirayama, an inconsistent occurrence of the former Yasukawa and Aoki, unpublished data). character state, and argued that its use at On the other hand, an anterolaterally this level was dubious. Our additional flared iliac blade was observed in all observations also confirmed the remarkable batagurid, but not in other turtles includ- variability of this character at the intraspe- ing those belonging to non-testudinoid cific level (Yasukawa, unpublished obser- families. We excluded these characters vation). Therefore, we did not include this from our analyses, since these are obvi- character in our phylogenetic analyses. ously not informative for the inference of Although Gaffney and Meylan (1988) relationships among the geoemydines. regarded the loss or reduction of the However, the states of these characters are quadratojugal as "relatively consistent" in possibly synapomorphs of the Bataguridae. geoemydines, this character state turned Thus, in the sections below we take an a out to be confined to the Geoemyda group priori assumption of the batagurid mono- alone (see above). phyly. Our analyses yielded states of three char- acters as possibly supporting monophyly of

FIG. 2. Dorsal views of pelvic girdles. 124 Current Herpetol. 20(2) 2001

TABLE 4. Classifications of geoemydine turtles of the family Bataguridae, derived from results of this study. "*": species not examined; "?": species of dubious generic allocation .

the Geoemydinae: 1 (0), presence of frontal exposure on orbital rim; 12 (0), large and elongated foramen palatinum posterius; and 28 (2), axillary plastral buttress connected around the portion between peripherals and costals. Of these, however, the first and the third were also observed in various species of the Batagurinae (Table 1), Emydidae, and Testudinidae (Gaffney, 1979; Yasukawa and Hirayama, unpub- lished data), and thus seemingly represent primitive conditions of the Testudinoidea. The second was observed in all geoemyd- ines except for the Cistoclemmys-Pyxidea- Geoemyda Glade and thus may be a syn- apomorph of the subfamily. However, we failed to determine whether this state repre- sents a derived or primitive condition, YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 125 because the character is highly variable states exhibited by Mauremys and Sacalia within the Testudinoidea (Gaffney and seem to mostly represent primitive condi- Meylan, 1988; Yasukawa, unpublished tions of the Geoemydinae (Table 1), or of data), and is also seen in Mesozoic prim- the whole family Bataguridae (Hirayama, itive testudinoids such as Mongolemys 1984). Thus, the Mauremys group may be (Hirayama, unpublished data). referred to as the primitive stock of this There are no synapomorphs clearly family. supporting the monophyly of Geoemydinae. Monophyly of the genus Mauremys was The character states shared by most supported by two character states, 14 (1) members of this subfamily largely represent and 15 (1), while Sacalia exhibited no primitive conditions in the Bataguridae autapomorphs in our parsimony analyses. (Hirayama, 1984; Table 1). This may McDowell (1964) suggested that Sacalia is suggest the paraphyletic nature of the discriminated from Mauremys in four char- Geoemydinae as an assemblage of primitive acter states. Of these, however, only one stocks of the Bataguridae. Nevertheless, (a few very large scutes without intervening because we could also detect no synapo- granules on forearms) is a possible synapo- morphs supporting sister group relationship morph of the Sacalia species. Although we with the Batagurinae of any particular did not employ scutellation of the forearm clade within the Geoemydinae, this problem in the phylogenetic analyses due to its remains to be readdressed on the basis of extensive intrageneric variation in the additional data. In consideration of taxo- Geoemyda group, we confirmed the unique nomic stability, we propose to temporarily state mentioned above in this character in retain the recognition of the subfamily Sacalia. Considering it as an autapomorph Geoemydinae as a "metataxon", for which of Sacalia, we regard both Mauremys and no character evidence supports or negates Sacalia as valid. the monophyly (Gauthier et al., 1988). Our analyses revealed that the Geoemyd- Geoemyda group inae consists of the Mauremys and the In our results, the monophyly of the Geoemyda groups. In the following sub- Geoemyda group is supported by three sections, we discuss the phylogeny within character states, absence of the squamosal- each of these groups. We also propose quadratojugal contact (7 [1]), absence of several modifications to the classification the jugal-quadratoj ugal contact (8 [1]), by David (1994) (Table 4) on the basis of and anterior neurals with shorter posterior evolutionary relationships revealed in this sides (25 [1]). Of these, 25 (1) was constant study. within the Geoemyda group exclusive of Notochelys, in which the anterior neurals Mauremys group had shorter anterior sides like those in the The results of this study may support the Mauremys group and most batagurines. monophyly of the Mauremys group as Hirayama (1984) depicted a sister group consisting of Mauremys and Sacalia with relationship between Sacalia and Notochelys one synapomorph (31 [1], smooth skin of as supported by jugal-parietal contact, and the posterior head). Even so, however, this distinctly widened first vertebral which character state was also observed in Stem J sometimes reaches the second marginal. except for Geoemyda silvatica and Pyxidea. The present study revealed that the jugal is This suggests that 31 (1) has evolved more close to the parietal but separated from it than one time in the Geoemydinae, and in Notochelys, while the two make weak reduces it value as evidence for monophyly contact in Sacalia. The first vertebral was of the Mauremys group. Other character widened and usually reached the second 126 Current Herpetol. 20(2) 2001 marginal in Mauremys japonica, the M. we did find a number of remarkable differ- leprosa species-group, Sacalia, and some- ences among Cistoclemmys, Cuora, and times in Notochelys, M. annamensis, M. Pyxidea. We thus consider the three genera mutica, the Cuora trifasciata species- to be valid. group, Cu. zhoui, and Melanochelys. The results of this study confirmed the Thus, it is obviously inadequate to use polyphyly of Rhinoclemmys as proposed these characters to unite Sacalia and by Hirayama (1984), because they divided Notochelys. In the present analyses, the Rhinoclemmys into two groups belonging Notochelys-Cyclemys Glade is supported by to different Glades. Of these, Rhinoclem- three synapomorphic character states, and mys A consisted of two aquatic species, we thus consider this relationship to be far punctularia and funerea, and two terres- more likely than the Notochelys-Sacalia trial species, areolata and pulcherrima. monophyly. Rhinoclemmys B, on the other hand, Except for the position of Notochelys consisted of two terrestrial species with (see above), our results largely support unnotched, hooked beaks, annulata and Hirayama's (1984) hypothesis in that they rubida, that were not united into a mono- also suggest: polyphyly of Cuora (sensu phyletic group by the parsimonious analy- lato) and validity of Cistoclemmys; close sis, but did constitute a single unit in the relationships among Cistoclemmys, Pyxidea NJ phylogram. We thus discuss the and Geoemyda; and polyphyly of Rhino- relationship of these two Rhinoclemmys B clemmys. Sites et al. (1984), on the basis species below. of the analysis of allozymic variation Based exclusively on the coloration and among 22 batagurid species, suggested that the relative depth of the shell, Ernst (1978) Cuora and Cistoclemmys are most closely demonstrated that Rhinoclemmys (as Cal- related to each other. This has been one of lopsis) can be divided into three groups, the major reasons to regard Cistoclemmys the pulcherrima-rubida group, punctularia as a junior synonym of Cuora (e.g., Ernst group (as consisting of R. punctularia, R. and Barbour, 1989; McCord and Iverson, annulata, R. diademata, R. funerea, R. 1991; Iverson, 1992). However, Sites et al. melanosterna, and R. nasuta), and R. (1984) examined very small numbers of areolata. This grouping substantially individuals for most species. The number contradicts our results, especially in the of loci examined in that study was also positions of R. annulata, R. pulcherrima, very small. Our results showed that Cisto- and R. rubida. Considering the remarkable clemmys is related more closely to Pyxidea intersubspecific variation in those characters and Geoemyda than to Cuora, although in both R. pulcherrima and R. rubida Cistoclemmys, Pyxidea, and Geoemyda are (Pritchard and Trebbau, 1984; Ernst and well differentiated from each other (Fig. 4). Barbour, 1989), the grouping proposed by Based on the external comparisons of Ernst (1978) is dubious. subspecies of Cistoclemmys galbinifrons Sites et al. (1981), in the assessment of and Pyxidea, Fritz and Obst (1997) con- allozymic variation among R. areolata, R. cluded that Ci. g. serrata is a distinct funerea, R. pulcherrima, R. punctularia, species which narrows the gap between and R. rubida, suggested close affinity Cuora (sensu lato) and Pyxidea. They among R. areolata, R. punctularia, and R. doubted the validity of both Cistoclemmys funerea, and distinct differentiation among and Pyxidea. However, the number of the areolata punctularia f unerea assem- specimens of Ci. g. serrata examined by blage, R. pulcherrima, and R. rubida. Our them was very small. Moreover, they did results are in accord with those of Sites et not examine internal characters, in which al. (1981) in showing close relationship YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 127 among R. punctularia, R. funerea, and R. no distinct differences in the degree of areolata, but contradict the latter in development of m. testoscapularis between showing the sole divergence of R. rubida. Heosemys and other "non-box" geoemyd- Because the results of Sites et al. (1981) ines (character 21). Additionally, we did were derived from analyses of a relatively not find any character to support mono- small number of loci for very small sam- phyly of the Heosemys group, or of an ples, we consider that our results more assemblage of Cistoclemmys, Cuora, successfully reflect the phylogeny of Rhino- Cyclemys, and Pyxidea. We thus consider clemmys. Bramble's (1974) grouping as unnatural. The type species of Rhinoclemmys Fitz- Bramble (1974) also suggested that plas- inger, 1835, is dorsata Schoepff, tral kinesis of Notochelys arose apart from 1801, which is a junior synonym of R. the Heosemys group. But he hardly punctularia (see Iverson, 1992). Therefore, mentioned the difference in shell-closing punctularia and its relatives (areolata, mechanism between Notochelys and the funerea, and pulcherrima) should be other batagurid box turtles. In adult living retained under Rhinoclemmys, while rubida specimens, both anterior and posterior (type species of Chelopus) and annulata plastral lobes of Notochelys are as movable should be moved to the resurrected genus as those of Cyclemys but less movable Chelopus Cope, 1870, which was formerly than those of Cistoclemmys, Cuora, and regarded as a junior synonym of Rhino- Pyxidea (Yasukawa, unpublished data). clemmys (see Wermuth and Mertens, Both Cyclemys and Notochelys have no 1977). hinge in juveniles, while the other three genera have a weak hinge even in juveniles Parallel evolution of plastral hinge (Yasukawa, unpublished data). In contrast, Within the Bataguridae, a kinetic plastron the very long anterodorsal portion of the equipped with a hinge between the pectoral iliac blade, a condition unique to the and abdominal was observed in five batagurids (character state 34 [1]: Fig. 1c, geoemydine genera, Cistoclemmys, Cuora, d), was observed only in Cistoclemmys. Cyclemys, Notochelys, and Pyxidea. Bram- The shape of the iliac blade appears to be ble (1974) demonstrated distinct differences associated with the closure of the posterior in shell-closing mechanism between emydid plastral lobe (Bramble, 1974). We thus box turtles and batagurid box turtles. He suspect that the most prominent difference argued that in the Geoemydinae the in shell-closing mechanism exists between plastral hinge had evolved two times, in the Cistoclemmys and the other geoemydine common ancestor of Cistoclemmys, Cuora, box turtles, not between Notochelys and Cyclemys, and Pyxidea, and in Notochelys. the others. In the Geoemydinae, the plas- In addition, he noted that Heosemys had a tral hinge seems to have actually evolved more developed musclus testoscapularis three times-in the Notochelys-Cyclemys (a muscle element associated with the Glade (Stem G), Cuora (Stem M), and the closure of the anterior plastral lobe in the Pyxidea-Cistoclemmys Glade (Stem Q). batagurid box turtles: Bramble, 1974) than In adult females of some batagurids the other geoemydines lacking a plastral lacking the plastral hinge, the bony connec- hinge. He thus suspected the derivation tion between the hypoplastron and carapace of Cistoclemmys, Cuora, Cyclemys, and is replaced by a ligamentous connection. Pyxidea from a common Heosemys-like Such plastral kinesis, sometimes called ancestor, and, on the basis of this consider- sexually dimorphic plastral kinesis, is prob- ation, united the five genera in the Heose- ably an adaptation for the passage of rela- mys group. However, our study showed tively large eggs which otherwise could not 128 Current Herpetol. 20(2) 2001 pass through the posterior opening between The NJ phylogram, while supporting the carapace and plastron (Waagen, 1984; aurocapitata-pani-trifasciata-mccordi-zhoui Moll, 1985). Sexually dimorphic plastral assemblage, showed that Cu. amboinensis, kinesis is present in Geoemyda japonica, the type species of the genus, is highly G. spengleri (see Yasukawa et al. [1992]), differentiated from the other congeners. G. silvatica (see Moll et al. [1986]), Heos- Such a relationship was also supported by emys grandis (Yasukawa unpublished data), the cladogram as well. However, we found H. spinosa (see Moll [1985]), Melanochelys no character states definitely uniting the tricarinata (see Waagen [1984]), and Rhino- five species. Therefore, we do not further clemmys spp. (see Pritchard and Trebbau divide this genus. [1984]). Therefore, all members of the Of the currently recognized species of Geoemyda group exclusive of Melanochelys Cuora (sensu lato), Cu. yunnanensis from trijuga actually show plastral kinesis at least Yunnan, China, was not examined in our in adult females. Considering its sporadic study. Examining all syntypes (=all avail- emergences on the phylogenetic tree of able specimens since the description by the Geoemyda group, it is probable that Boulenger [1906]) of this rare species, sexually dimorphic plastral kinesis repre- Ernst (1988) demonstrated a close rela- sents a primitive condition in this group. tionship of Cu. yunnanensis with Cu. tri- fasciata. McCord and Iverson (1991) also Taxonomic notes suggested a close affinity of Cu. yunnan- Based on the strong plastral buttresses ensis with Cu. trifasciata, as well as with in Mauremys annamensis, Savage (1953) Cu. pani and Cu. aurocapitata on the revalidated the monotypic genus Annam- basis of morphometric comparisons emys Bourret 1939, for this species. He among all extant Cuora (sensu lato). also suggested its close affinity to several Therefore, we tentatively retain this spe- batagurines with strong plastral buttresses, cies in the genus Cuora (sensu stricto). such as Batagur, Callagur, Hardella, and Geoemyda silvatica is an enigmatic Kachuga. On the other hand, Iverson and species endemic to the Western Ghats of McCord (1994), in the analyses of morpho- southwestern India (Das, 1991; 2001). Its metric variation in the East Asian Maur- generic allocation has been in dispute. emys, regarded annamensis as the closest Some authors assigned this species to relative of M. mutica, and synonymized Geoemyda, while others assigned it to Annamemys with Mauremys again. The Heosemys (see the Geoemyda subsection present study showed that the buttress of in "Materials and Methods"). Such a M. annamensis is stronger than that of taxonomic confusion is probably attribut- other geoemydines, but much less devel- able to the paucity of studies directly oped than that of the above-mentioned comparing specimens of G. japonica, G. batagurines (see comments for characters silvatica, and G. spengleri. We examined 28 and 29). Except for the buttress, this representatives of all these species in species shared most character states with detail, and our results strongly suggest the M. mutica, supporting Iverson and monophyly of the three species and the McCord's (1994) account (Table 1). validity of the G. japonica-G. spengleri Assignment of this species to the genus clade. Mauremys was also supported by a recent Of the currently recognized species of mithochondrial DNA study, which, how- Rhinoclemmys (sensu lato) R. melanosterna, ever, suggested the closest affinity of M. R. diademata, and R. nasuta were not annamensis with M. iversoni rather than examined. They were once treated as with M. mutica (Honda et al., in press). subspecies of R. punctularia, and then YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 129 were elevated to full species (Pritchard, Aoki, D. Auth, Y. Chigira, R. I. Crombie, 1979; Pritchard and Trebbau, 1984; R. W. Heyer, A. H. Wynn, G. R. Zug, David, 1994). Like R. punctularia and R. H. Endo, S. -I. Ueno, K. Hatooka and Y. funerea, all of the three species are Shibata for allowing us to examine speci- aquatic and have a shallowly notched mens in their cares, and to R. Aoki, A. upper beak (Ernst and Barbour, 1989). In Araya, C. Blanc, T. Hayashi, S. Ikehara, addition, a small foramen on the ventral A. Kamata, S. Kato, T. Kuboya, N. maxilla, which is suggested to be a unique Kamezaki, A. Kozu, M. Matsui, A. synapomorphy of the R. punctularia-R. Mori, H. Ota, S. Sengoku, S. Tanabe funerea clade (35 [1]), was recognized in and M. Tasumi for providing precious juvenile specimens of R. melanosterna specimens. R. Aoki, H. Ota, S. Sengoku, and R. diademata (Hirayama, unpub- M. Matsui and M. Tasumi provided lished data). We thus tentatively retain pertinent literature. We are also much the three species in the genus Rhinoclemmys indebted to I. Das, A. Mori, H. Ota and (sensu stricto). M. Tasumi for critical reading of the manuscript. This study was partially Direction of future studies on the supported by a grant from Fujiwara geoemydine phylogeny Natural History Foundation (to Y. In this study, we attempted as compre- Yasukawa). hensive an analysis as ever made for the phylogeny of the highly diversified subfam- ily Geoemydinae by best use of morpholog- LITERATURE CITED ical information. As a result, we established a best- BOULENGER, G. A. 1889. Catalogue of fitting phylogenetic hypothesis at this the Chelonians, Rhynchocephalians, and level (Figs. 3 and 4), and accordingly Crocodiles in the British Museum (Natural suggested some changes in the classifica- History). Taylor and Francis, London. tion of this subfamily (see above). Nev- 311 p. ertheless, our analysis failed to resolve a BOULENGER, G. A. 1906. Descriptions of new number of ingroup relationships. More- from Yunnan. Ann. Mag. Natur. over, our results, as well as our pre- Hist. (7)17: 567-568. sumption of the batagurine monophyly, BOUR, R. 1980. 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The status of turtle of China, Contribution to Herpetology, 10. in Asia Turtles. p. 15-23. In: P. P. van Society for the Study of Amphibians and Dijk, B. L. Stuart, and A. G. J. Rhodin Reptiles, Ohio. 522 p. (eds.). Asian Turtle Trade: Proceedings of Workshop on Conservation and Trade of APPENDIX Freshwater Turtles and Tortoises in Asia, Chelonian Research Monographs, 2. Specimens Examined. -Catalogue num- Chelonian Research Foundation, Massa- bers of specimens deposited in chusetts. collection of Kyoto University Museum, YASUKAWA ET AL. -PHYLOGENY OF GEOEMYDINE TURTLES 133 turtles collections of Madras Crocodile caspica (1), RH666; M. japonica (10) Bank Trust, Biological collections of KUZ R19575-19580, 36712-36715; M. Teikyo Heisei University, and private leprosa (2) RH453-454; M. mutica kami collections of Ren Hirayama and P. C. H. (5) KUZ R19524-19525, 19541, 19543- Pritchard are preceded by KUZ R, MCBT, 19545; M. m. mutica (6) KUZ R19509- THUb, RH and PCHP, respectively. 19512, RH54, 194; M. rivulata rivulata (6) The other acronyms are those suggested KUZ R36716, 36761, RH541-542, 888, by Leviton et al. (1985). 929-930; Melanochelys trijuga (10) KUZ Geoemydinae: Chelopus annulatus (2 R36718-36720, four unnumbered speci- specimens) RH861-862; C. rubidus (1) mens of MCBT, RH238, 549, 557; RH331; Cistoclemmys flavomarginata fla- Notochelys platynota (6) KUZ R36721- vomarginata (9) NSMT02087-02089, one 367222, RH858, 869, 961-962; Pyxidea unnumbered specimen of OMNH-R, mouhotii (5) KUZ R36723, RH240, 246- KUZ R19561, 36752, RH; 53, 62-63, 430; 247, 269, 273; Rhinoclemmys areolata (2) Ci. f. evelynae (4) NSMT02090-02092, RH654-656; R. funerea (3) 978, 985-986; KUZ R36746; Ci. galbinifrons (7) KUZ R. pulcherrima mani (5) KUZ R36724, R36745, 36757, 36763, RH736-737, 750- THUb17-19, RH835, 836; R. punctularia 751; Cuora amboinensis amboinensis (2) (4) 239, 248, 457, 510; Sacalia bealei (2) KUZ R36704-36705; Cu. a. kamaroma RH525-526; S. quadriocellata (2) KUZ (13) KUZ R19583-19584, 36763, RH34-36, R36725, RH709; Sacalia sp. (2: skeletal 519-520, 596-600, Cu. aurocapitata (4) specimens not identified at the species KUZ R36710, THUb13-14, RH915; Cu. level) RH308, 524. Btagurinae: Batagur mccordi (6) THUb21-22, RH935-936, 950, baska (2) one unnumbered specimen of 981; Cu. pani (4) RH901-903, 987; Cu. tri- MCBT, RH 1002; Callagur borneoensis (3) fasciata (5) KUZ R 36709, RH219-222; KUZ R19560, RH287, THUb10; Chinemys Cu. zhoui (4) KUZ R36706, 36711, RH868, reevesii (10) KUZ R 19591-19593, 36692- 1001; Cyclemys spp. (11) KUZ R 36609, 36698; Geoclemys hamiltonii (2) one 36707-36708, RH134-139, 550-551; Geoe- unnumbered specimen of MCBT, RH920; myda japonica (13) NSMT02083-02086, Hardella thurjii thurjii (3) KUZ R 36699, OMNH-R333-3334, two unnumbered spec- 36701, THUb35; Hieremys annandalii (3) imens of OPM, KUZ R36720-36721; RH236, 587, 928; Kachuga smithii smithii RH480-482; G. silvatica (6) FMNH52515, (5) KUZ R 36759-36760, RH281, THUb49, four unnumbered specimens of MCBT, 52, Malayemys subtrijuga (4) RH140-143, PCHP2725; G. spengleri (12) two unnum- Morenia petersi (2) KUZ R 36702-36703, bered specimens of NSMT, USNM84992, Ocadia sinensis (4) RH201-202, 340-341; KUZ R9994, 12630, 19399-19401, Orlitia borneensis (1) RH871; Siebenrock- THUb15, RH847, 856, 857; Heosemys gran- iella crassicollis (5) KUZ R36762; RH116, dis (3) KUZ R19581, 36718, RH581; H. 706-708. spinosa (4) KUZ R36757, RH185-186, 266-267; Mauremys annamensis (6) KUZ R36700, RH 936-939, THUb 9; M. Accepted: 9 November 2001