Herpetologists' League

Phylogeographic Patterns in subrubrum and K. baurii Based on Mitochondrial DNA Restriction Analyses Author(s): DeEtte Walker, Paul E. Moler, Kurt A. Buhlmann, John C. Avise Source: Herpetologica, Vol. 54, No. 2 (Jun., 1998), pp. 174-184 Published by: Herpetologists' League Stable URL: http://www.jstor.org/stable/3893425 Accessed: 25/11/2008 13:21

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http://www.jstor.org 174 HERPETOLOGICA [Vol. 54, No. 2

trial Vertebrates in the Neotropical Realm. Dr. W SHERBROOKE, W C. 1975. Reproductive cycle of a Junk, The Hague, The Netherlands. tropical lizard, Neusticurus ecpleopus Cope. in PARKER, H. W 1935. The lizards of Trinidad. Trop. Peru. Biotropica 7:194-207. Agric. 12:65-70. SILVA, J. M. C. 1995. Avian inventory of the cerrado PETERS, J. A., AND R. DONOSO-BARROS. 1986. Cat- region, South America: implications for biological alogue of the Neotropical Squamata. Part II, Liz- conservation. Bird Cons. Int. 5:291-304. ards and Amphisbaenians (Revised ed.). Smithson- VANZOLINI, P. E. 1961. Bachia: especies brasileirase ian Institution Press, Washington, D.C., U.S.A. conceito generico (Sauria, Teiidae). Pap. Av. Dep. PINTO, M. N. 1994. Cerrado: Ocu- CaracterizaVao, Zool., S. Paulo 14:193-209. pa,Vo e Perspectivas (2nd ed.). Editora Universi- . 1966. dade de Brasilia, Brasilia, Brasil. Sobre o segundo exemplar de Bachia PROJETO RADAMBRASIL. 1982a. Folha SD. 21 Cu- bresslaui (Sauria, Teiidae). Pap. Av. Zool., S. Paulo iaba; Geologia, Geomorfologia, Pedologia, Vegeta- 19: 189-192. Vao e Uso Potencial da Terra. Minist6rio das Minas VITT, L. J. 1982. Sexual dimorphism and reproduc- e Energia, Rio de Janeiro, Brasil. tion in the microteiid lizard, Gymnophthalmus . 1982b. Folha SF. 21 Campo Grande; Geo- multiscutatus. J. Herpetol. 16:325-329. logia, Geomorfologia, Pedologia, VegetaVyo e Uso Potencial da Terra. Ministerio das Minas e Energia, Accepted: 29 June 1997 Rio de Janeiro, Brasil. Associate Editor: Daniel Formanowicz, Jr.

Herpetologica,54(2), 1998, 174-184 ? 1998 by The Herpetologists' League, Inc.

PHYLOGEOGRAPHIC PATTERNS IN KINOSTERNON SUBRUBRUM AND K. BAURII BASED ON MITOCHONDRIAL DNA RESTRICTION ANALYSES

DEETTE WALKER,' PAUL E. MOLER,2 KURT A. BUHLMANN,3 AND JOHN C. AVISE' 'Department of Genetics, University of Georgia, Athens, GA 30602, USA 2Wildlife Research Laboratory, Game and Fresh Water Fish Commission, 4005 South Main Street, Gainesville, FL 32601, USA 3Savannah River Ecology Laboratory, Drawer E, Aiken, SC 29801, USA

ABSTRACT: We used restriction assays of mitochondrial (mt) DNA to estimate phylogeographic variation in two sister taxa of muid in the southeastern United States. Extensive mtDNA variation characterized Kinosternon subrubrum and, to a lesser degree, K. baurii. Each of 26 mtDNA haplotypes from the 83 assayed specimens was localized spatially. Collectively, these mtDNA haplotypes demarcated four major matrilineal assemblages, each with a well defined re- gional distribution: a western group (A) in Missouri and Louisiana, a central group (B) throughout the Gulf coastal states, an eastern group (C) along the Atlantic coastal states north of Florida, and a southern group (D) in peninsular Florida. All assayed samples of K. baurii belonged to the mtDNA C assemblage. The two in Florida are thus highly distinct in mtDNA genotype, but they exhibit minimal mtDNA divergence along the Atlantic coastal states. These findings raise questions concerning the evolutionary history and of these two recognized species. MtDNA phy- logeographic patterns in the baurii/subrubrum complex are remarkably similar to those reported previously for two other southeastern kinosternids, minor and S. odoratus. Key words: Mud turtles; Phylogeography; Gene flow; Population struicture; Southeastern United States; Kinosternon.

MUD turtles (Kinosternon) are semi- Ernst et al., 1994), a habit that may influ- aquatic organisms typically associated with ence patterns of inter-drainage gene flow slow-moving, often ephemeral waters such (Gibbons, 1983) and geographic popula- as shallow bayous, swamps, and ditches. tion structure. Sixteen species of mud tur- These turtles commonly are observed tra- tles are recognized in North, Central, and versing land (Ernst and Barbour, 1989; South America (Ernst et al., 1994), two of June 1998] HERPETOLOGICA 175

***K. b auriit K. s. hippocrepis K. s. subrubrum K. s. steindachneri

FIG. 1.-Map of the southeastern United States showing collection sites for mud specimens (black dots, K. subrubrum; stars, K. baurii). The described range of K. baurii is to the east and south of the heavy line (i.e., the Atlantic coastal plain and all of peninsular Florida).

which (Kinosternon subrubrum and K. er basin. The Florida mud turtle, K. s. baurii) occur in the southeastern United steindachneri, is confined to the Florida States. peninsula. The Mississippi mud turtle, K. Three of K. subrubrum cur- s. hippocrepsis, inhabits primarily western rently are recognized (Conant and Collins, Mississippi, Louisiana, and portions of Ar- 1991; Ernst et al., 1994: Fig. 1). The east- kansas, Oklahoma, and Texas. Intergrada- ern mud turtle, K. s. subrubrum, occurs tion is reported between these subspecies along the Atlantic coast from Long Island, where their ranges adjoin or overlap New York to northern Florida and west (Ernst et al., 1974; Iverson, 1977). The into the lower and central Mississippi Riv- , K. baurii, occurs along 176 HERPETOLOGICA [Vol. 54, No. 2 the Atlantic coast from southern Virginia TABLE 1.-MtDNA haplotypes observed in Kinoster- to the Florida Keys (Ernst et al., 1994; non subrubrum and K baurii. Letters from left to right in the descriptions represent digestion profiles Lamb and Lovich, 1990; Mitchell, 1994). for the restriction enzymes BanI, Bcll, BglI, BglII, Most southern specimens of K. baurii DraII, EcoRI, Hindll, HindIII, KpnI, NciI, NsiI, display pronounced stripes on the cara- PvuII, StuI, and XbaI. pace and head, but these stripes ebb in Haplotype No. of northern specimens, which causes identi- code individuals Description fication difficulties with K. s. subrubrum, K subrl 3 CCCDCCCCCCCCCD a subspecies lacking such markings (Lamb, K subr2 3 CBCDCCCCCCCCCD 1983a,b; Lamb and Lovich, 1990). Prior K subr3 6 CBCDCCCCCDCCCC phylogenetic analyses based on allozymes K subr4 1 BBCDDCCCBDCCCC K subr5 1 CBCDCCCFCDCCCC (Seidel et al., 1986), karyology (Sites et al., K subr6 3 CBCDCCCCCCBCCC 1979), and morphology (Iverson, 1991) K subr7 2 CBCDBCCCCDCCCC suggested that K. baurii and K. subrubrum K subr8 1 CBCDBCCCCDCCDC are closely related sister taxa within the Ki- K subr9 9 BACCFCABBBEDCC nosternidae, but these studies were not K subrlO 2 AACCFCABBBEDCB K subrll 1 BACCFCBBBBEDCC designed to assess geographic variability K subrl2 3 BACCFCABBBEECC within either species. Here we examine K subrl3 1 DACDECGACAFCBC mitochondrial (mt) DNA variation within K subrl4 1 DACDECGADCFCCC and between geographic populations of K. K subrl5 1 DACDECDACCFCCC K subrl6 2 DACEECDACBFCCC baurii and K. subrubrum. K subrl7 2 BACBFCABBFEDCC K subrl8 5 BACCFCABBFEDCC MATERIALS AND METHODS K subrl9 16 CEDCEBEECCHCFC K subr20 1 BACGFCABBBEDCB Samples and Laboratory Procedures K baur2l 4 CBCDCCCCCCCACC We collected 64 specimens of K. subru- K baur22 1 CBCDCCCCCCCCCC brum from 32 locales and 19 specimens of K baur23 10 CBCDCCFCCCCBCC K. baurii from 11 locales (Fig. 1, Appendix K baur24 1 CBCDCCFECCCBCC K baur25 2 CBCDCCICCCCBCC I). The specimens of K. baurii from Florida K baur26 1 CACDCCFCCCCBCC were easily distinguished morphologically from K. subrubrum because they displayed the characteristic stripes on the carapace and head. Specimens of K. baurii from At- gested by 14 restriction enzymes (Table 1) lantic coast drainages had head stripes; following recommendations of the manu- their identification to species by Joseph facturer (Boehringer Mannheim). Frag- Mitchell was based on these morphological ments were radioactively end-labeled us- criteria. In addition, morphological species ing Klenow and 32P-labeled nucleotides, assignments were confirmed by application size-separated by electrophoresis through of the discriminant function analyses de- 1.2-1.5% agarose gels, and visualized by fined in Lamb (1983b), as applied to five autoradiography (Lansman et al., 1981). shell characteristics measured in all speci- The digestion profiles proved informative mens of mud turtles collected along the At- in the sense that they yielded restriction lantic coast from the Carolinas through fragment length polymorphisms (RFLP's) Florida. All specimens are deposited in The whose differences within and between K. University of Georgia Museum of Natural baurii and K. subrubrum provisionally History (UGAMNH 28567-28648) except could be interpreted as restriction site those from Cohoke Mill Creek (Virginia) gains or losses. which were donated to the Smithsonian Museum (USNM numbers 515120,515121, Data Analyses 515124-515127, 515212, and 515213). Each mtDNA digestion profile was as- We extracted mtDNA from heart, liver, signed a letter code (Table 1). These let- and muscle tissues following Lansman et ters were compiled for each individual into al. (1981). Closed-circular mtDNA was di- a composite mtDNA haplotype. From the June 1998] HERPETOLOGICA 177 presence/absence matrixof restrictionsites tween mtDNA genotypes in separate geo- summarizing these haplotypes, sequence graphic regions. divergences (Nei and Li, 1979) and ge- Parsimony networks (Fig. 2; see also notypic and nucleotide diversities (Nei, legend to Fig. 3) and a neighbor-joining 1987) were calculated. tree (Fig. 3) for the mtDNA haplotypes Phenetic relationships among haplo- are based strictly on genotypic considera- types were inferred from the genetic dis- tions and essentially agree in all major fea- tance matrix using the neighbor-joining tures. Four fundamental phylogenetic (N-J) method (Saitou and Nei, 1987) as groups (A-D), each showing a strong geo- implemented in PHYLIP (Felsenstein, graphic orientation (Fig. 4), are evident. 1991) and rooted by the mid-point crite- Group A occurs in the western-most por- rion. We conducted parsimony analyses tion of the range of K. subrubrum; group from the presence/absence matrix of re- B occurs in the central portion of the striction sites using the heuristic search range of K. subrubrum in the Gulf coastal option in PAUP (Swofford, 1990). Statis- states; group C includes K. subrubrum tical support was based on 1000 bootstrap from Atlantic coastal states north of Flor- pseudoreplicates. A parsimony network ida, plus all individuals of K. baurii (in- was hand-generated using observed num- cluding those from the Florida peninsula); bers of restrictionsite differences between and group D consists of all specimens of the mtDNA haplotypes. Outgroup taxa K. subrubrum from the Florida peninsula. were not employed to root the parsimony The mean levels of genetic divergence es- networks, because other assayed species of timated among haplotypes within each as- (Walker et al., 1995, 1997) semblage (0.000, 0.003, 0.006, and 0.008 proved too divergent in most of the for groups A, B, C, and D, respectively) mtDNA digestion profiles to permit secure typically are much smaller than those be- scoring of restriction site changes. tween assemblages (0.071, 0.068, 0.057, 0.054, 0.032, and 0.038, respectively, for RESULTS the paired combinations A-B, A-C, A-D, The mtDNA restriction site differences B-C, B-D, and C-D). between K. baurii and some specimens of Intra-assemblage genetic variation dif- K. subrubrum were minimal, so the data fered considerably among the four genetic were analyzed collectively. In total, the 14 groups. The largest numbers of mtDNA informative restriction enzymes revealed haplotypes (seven and eight, respectively) 26 different mtDNA haplotypes:20 for K. were observed in genetic groups B and C. subrubrum and six for K. baurii (Table 1). However, the highest values for genotypic The mtDNA molecule was approximately diversity (0.918) and nucleotide diversity 16.3 kilobases in length in both species, (0.013) occurred in the Florida peninsula with no evident size differences among in- with turtles representing both the C and dividuals. A mean of 43 restriction sites D groups co-occurring there. per individual was scored, reflecting 451 In peninsular Florida, all specimens as- base pairs of recognition sequence or signed by morphology to K. subrubrum about 2.8% of the mtDNA genome. belonged to mtDNA group D, and all The gel digestion profiles were inter- specimens morphologically referable to K. preted with respect to restriction site baurii belonged to group C. However, changes by methods described in Avise group C (with bootstrap support 99%: Fig. (1994). Fifty-nine of the 81 scored restric- 3) also included all sampled turtles from tion sites were variable (Table 2), and 31 the Atlantic coastal states, regardless of were informativephylogenetically (i.e., not taxonomic status (K. baurii or K. subru- confined to a single individual). For the brum). Within the C group, specimens of pooled collection of samples, estimated K. baurii from Florida differed consistent- genotypic diversity was 0.927 and nucleo- ly from those in Georgia and Virginia by tide diversity was 0.041. Most of this di- at least two restriction site changes (Fig. versity stemmed from large differences be- 2). TABLE 2.-Presence (1) versus absence (0) matrix of mtDNA restriction sites in Kinostemnon subrubrum and K, baurii.

sites HaplotypetRestriction code BanI BclI BglI BglII Drall EcoRI HindIl Hindill KpnI NciI NsiI PvuII StuI XbaI K subrl 1111100 11110 11 00000 11111110 110 111111100000000 11100 1100 10000 1100000 11000 111000 1110 K subr2 1111100 11100 11 00000 11111110 110 111111100000000 11100 1100 100000 1100000 11000 111000 1110 K subr3 1111100 11100 11 00000 11111110 110 111111100000000 11100 1100 11000 1100000 11000 111000 1100 K subr4 1111110 11100 11 00000 01111110 110 111111100000000 11100 1110 11000 1100000 11000 111000 1100 K subr5 1111100 11100 11 00000 11111110 110 111111100000000 01100 1100 11000 1100000 11000 111000 1100 K subr6 1111100 11100 11 00000 11111110 110 111111100000000 11100 1100 10000 1110000 11000 111000 1100 K subr7 1111100 11100 11 00000 11111100 110 111111100000000 11100 1100 11000 1100000 11000 111000 1100 K subr8 1111100 11100 11 00000 11111100 110 111111100000000 11100 1100 11000 1100000 11000 111010 1100 1 K subr9 1111110 11101 11 11000 10100101 110 101011111000000 10100 1110 10110 1101100 10000 111000 1100 > K subrlO 1111111 11101 11 11000 10100101 110 101011111000000 10100 1110 10110 1101100 10000 111000 1101 t K subrll 1111110 11101 11 11000 10100101 110 101011111100000 10100 1110 10110 1101100 10000 111000 1100 O K subrl2 1111110 11101 11 110 00 10100101 110 10101111000000 10 100 1110 10110 1101100 1010 0 111000 1100 t K subrl3 1101100 11101 11 1o0o1o0000000 10100100 110 111111110010000 10101 1100 1101110 11000 111100 1100 O K subrl4 1101100 11101 11 00000 101 00100 110 111111110010000 10101 1101 10000 1101110 11000 111000 1100 2 K subrl5 1101100 11101 11 00000 1010 0100 110 111111110000000 10101 1100 10000oo0 1101110 1100 0 111000 1100 K subrl6 1101100 11101 11 10010 10100100 110 111111110000000 10101 1100 10110 1101110 11000 111000 1100 K subrl7 1111110 11101 11 11100 10100101 110 101011111000000 10100 1110 10111 1101100 10000 111000 1100 K subrl8 1111110 11101 11 11000 10100101 110 10101111000000 10100 1110 10111 1101100 10 000 111000 1100 K subrl9 1111100 11001 10 11000 10100100 1 010101100001110 100 10 1100 10000 1101011 11000 10 0001 1100 K subr20 1111110 11101 11 11001 10100101 110 101011111000000 10100 1110 10110 1101100 10000 111000 1101 K baur2l 1111100 11100 11 00000 11111110 110 111111100000000 11100 1100 10000 1100000 11001 111000 1100 K baur22 1111100 11100 11 00000 11111110 110 111111100000000 11100 11000o0 1000 1100 0 0 0 1100 0 111000 1100 K baur23 1111100 11100 11 00000 11111110 110 110111100000000 11100 1100 10000 1100000 11010 111000 1100 K baur24 1111100 11100 11 00000 11111110 110 110111100000000 01100 1100 10000 1100000 11010 111000 1100 K baur25 1111100 11100 11 000o00 11111110 110 110111100000001 11100 1100 10000 100000 11010 111000 1100 K baur26 1111100 11101 11 00000 11111110 110 110111100000000 11100 1100 10000 1100000 11010 111000 1100 June 1998] HERPETOLOGICA 179

K.subrl

K.subr2 K.baur2l

HI4K.baur22 0.002 K.subr6 A K.subr4 .subr5 group K.subr3 C 9 9 l / ~~~~~~~~~~~~26C K.subr7 K.subr8

K.baur26 K.baur23

K.baur25 K.baur24 17 K.subrI2

18 1 K.subrl 1 13 D 1 00 K.subrl7 K.subr18 group K.subr20 B B Kasubri 0 K.subrg 168 K.subr16 FIG. 2.-Hand-generated parsimony network esti- mating relationships among the composite mtDNA K.subr3 group haplotypes (numbered as in Table 1) scored in the K.subri4 D collections of K. baurii (indicated by asterisks) and K.subrl 5 K. subrubrum. Slashes along branches are inferred K.subrl9 J A character state (restriction site) changes, and those FIG. 3.-Neighbor-joining tree for mtDNA haplo- along branches the four connecting major genotypic types (numbered as in Table 1) in mud turtles. The groups (A-D) represent the minimum numbers of tree is mid-point rooted and branch lengths are such changes between any of these representatives drawn according to numbers of inferred restriction respective groups. site changes. A computer-generated parsimony net- work (not shown, but with consistency index 0.76) essentially agreed in identifying all major mtDNA and levels of DISCUSSION groups, yielded bootstrap support (>65%) that are shown here superimposed on the Genetic Variation and Phylogeographic neighbor-joining tree. Patterns In several respects, the levels and spatial Second, most of the mtDNA haplotypes distributions of mtDNA variation in the observed in Kinosternon were localized Kinosternon subrubrum/baurii complex geographically, usually confined to a single are remarkably similar to those reported site or set of adjacent locales (Fig. 4). The previously for two other species of kino- primary exceptions involved haplotype 19 sternid turtles in the southeastern United in K. subrubrum, which was observed both States: the musk turtle, Sternotherus mi- in southern Missouri and southern Loui- nor (Walker et al., 1995), and the stinkpot, siana, and haplotype 9 in this same species, S. odoratus (Walker et al., 1997). First, which was found in sites from northern mtDNA variation is extensive. The geno- Georgia, Mississippi, Alabama, and the typic diversity value (0.927) observed in panhandle of Florida (Fig. 4). However, the collection of Kinosternon approximates any conclusions about local population values reported within S. minor (0.859) structure in these turtles must remain and S. odoratus (0.899) and the overall nu- tempered given the small numbers of cleotide diversity (0.041) in the assayed specimens assayed per locale. Kinosternon complex surpasses such esti- Third, the numerous mtDNA haplo- mates within either of the species of Ster- types in the species of Kinosternon align notherus examined (0.017 and 0.016, re- phylogenetically into highly distinct groups spectively). that also show a striking macro-geographic 180 HERPETOLOGICA [Vol. 54, No. 2

E FAZ~~~~~~~~~~~~~~~~~~~~21

1 ~~~~~~~~~~~~O

2~~~~~~~~~

2 23 25D(C

FIG. 4.-Geographic distributions of mtDNA haplotypes (numbered as in Table 1) in mud turtles and of the four major mtDNA groups. orientation (Fig. 4). Four genetically dis- baurii), the Atlantic coastal populations tinct assemblages (A-D) were observed in were dramatically divergent in mtDNA the Kinosternon complex, compared to cornposition from those to the west and two in S. minor and 3-4 in S. odoratus. along the upper Gulf coastal states. Fur- The geographic distributions of mtDNA thermore, in both the Kinosternon com- groups A, B, and D (Fig. 4) generally con- plex and in S. odoratus, populations in form well to the described ranges of the peninsular Florida displayed pronounced three conventionally recognized subspe- mtDNA differences from those along the cies within K. subrubrum (Fig. 1), with the Atlantic coast to the north and from those exception that mtDNA group B apparently in all Gulf coastal states to the west. Such does not extend into the Atlantic coastal patterns, in general, complement those states that traditionally are included within observed in several species of freshwater the range of K. s. subrubrum (Figs. 1, 4). fish in the southeasternU.S. (Bermingham The overall distributions of the major and Avise, 1986), as well as in some ter- phylogeographic assemblages were re- restrial vertebrates (reviewed in Avise, markably similar for the species of Kino- 1996). Probably, numerous details in the sternon and Sternotherus. In all cases (S. historicalpatterns of drainageisolation and minor, S. odoratus, and K. subrubrumi coalescence and their influences on gene June 1998] HERPETOLOGICA 181 flow (Bermingham and Avise, 1986; Swift C and D groups as these latter are from et al., 1985) have contributed to these ob- one another. Finally, in contradistinction served genetic patterns in freshwater tur- to morphological patterns mentioned tles. above (Lovich and Lamb, 1995), samples of K. s. hippocrepis (assemblage A) proved Relationships between K. subrubrum and highly divergent in mtDNA composition K. baurii from K. baurii (assemblage C). Thus, with Considerable discussion has centered on respect to matrilineal ancestry, K. subru- the topic of morphological and taxonomic brum appears to be genealogically para- distinction between K. subrubrum and K. phyletic (Neigel and Avise, 1986) in rela- baurii. In an investigation of the subspe- tion to K. baurii (Fig. 3). cific status of a lower Florida Keys popu- One conceivable explanation for the ap- lation of K. baurii, Iverson (1978) con- parent paraphyly is that the current mt- cluded that the highly variable color pat- DNA restriction site data provide grossly terns on the head and carapace were un- inadequate descriptions of matrilineal re- reliable in distinguishing populations of K. lationships within the Kinosternon com- baurii. According to Lamb (1983a,b), plex, perhaps because of scoring difficul- these patterns also cause occasional misi- ties associated with inferences from diges- dentifications with sympatric K. s. subru- tion profiles alone. However, this is un- brum. Lamb (1983a,b), using multivariate likely because we also have sequenced discriminant function analyses of morpho- portions of the mtDNA control region metric characters, concluded that the two from representative samples of K. subru- species in Florida and along the Atlantic brum and K. baurii, and all conclusions Coast could be separated reliably. These about the major mtDNA phylogeographic analyses also demonstrated that the range groups within the complex are supported of K. baurii extends into Georgia and fully (Walker et al., 1998). Alternatively, South Carolina. A broader geographic sur- several competing evolutionary explana- vey by Lamb and Lovich (1990) again in- tions might account for the paraphyletic dicated that the two species were distin- pattern observed. Perhaps K. baurtii and K. guishable along the Atlantic coast, and ex- subrubrum are "good" biological species, tended the described range of K. baurii as is suggested by partial sympatry and the into southeastern Virginia. However, a lat- morphometric differences, but K. baurii is er assessment of samples of K. baurii a recent phylogenetic derivative of K. sub- against the western subspecies (hippocre- rubrum. Consistent with this possibility is pis) of K. subrubrum failed to distinguish that K. baurii may have split recently from these taxa by the same morphometric cri- Atlantic-like populations of K. subrubrum, teria (Lovich and Lamb, 1995). accounting for its overall mtDNA similar- The current mtDNA restriction site data ity to K. subrubrum in Atlantic coast drain- place all assayed samples morphologically ages, and that the species secondarily in- referable to K. baurii in the C matrilineal vaded the Florida peninsula, thus account- group, which extends from southern Vir- ing for its strong mtDNA divergence from ginia to southern peninsular Florida. All K. subrubrum in that area. Such a history individuals of K. subrubrum collected also might account for the carapace and from the Atlantic coastal states north of facial patterns wherein specimens of K. Florida also belong to this mtDNA C baurii tend to be easier to differentiate group. However, all peninsular Florida from K. subrubrum in Florida than along specimens referable by morphology to K. the Atlantic coast. On the other hand, his- subrubrum belong to the sharply differ- torical introgressive hybridization in some entiable mtDNA D assemblage. Further- other plant and taxa is known to more, K. subrubrum across its broader produce occasional gene tree/species tree geographic distribution to the west dis- discordances (Avise, 1994). Perhaps sam- plays at least two other matrilineal assem- ples of K. subrubrum from the Atlantic blages that are at least as distinct from the coastal states are similar in mtDNA com- 182 HERPETOLOGICA [Vol. 54, No. 2 position to samples of K. baurii because Acknowledgmnents.-We thank the following for help with the collections: R. Babb, I. Barak, V. Burke, gender-asymmetric hybridization has M. Case, A. Davis, S. Doody, S. Emms, M. Goodis- moved mtDNA of K. baurii into K. sub- man, M. Hare, C. Hobson, C. Holod, T. Ingstrom, rubrum, or vice versa, in this geographic D. Jansen, T. Johnson, A. Jones, B. Mansell, P. May- area. Further evaluation of possibilities in- ne, J. Mitchell, B. Nelson, G. Ortf, P. Prodohl, F. volving hybridization will require evidence Rose, C. Starlin, D. Stevenson, R. Vandevender, D. Wilson, K. Wood, and personnel from the Avise lab- from nuclear genes. In any event, the oratory and The University of Georgia Golf Course. mtDNA data strongly indicate that any We also thank personnel from the National Fish such hybridization has not genetically Hatcheries of Bo Ginn, Carbon Hill, Tupelo, McKin- merged K. subrubrum and K. baurii in ney Lake, Orangeburg, Warm Springs, and Welaka. All specimens were collected under relevant state peninsular Florida. permits. Work was supported by a National Institutes It is possible that populations of K. bau- of Health training grant to DeEtte Walker, by De- rii from the Atlantic coastal states always partment of Energy contract DE-FC09-96SR18546 have been classified improperly as "K. sub- between the U.S. Department of Energy and The rubrum". If so, a revised for K. sub- University of Georgia's Savannah River Ecology Lab- range oratory, and by a National Science Foundation grant rubrum would include the Florida penin- to John Avise. sula and all relevant Gulf coastal and in- terior states to the west, but would not ex- LITERATURE CITED tend northward along the Atlantic coast which instead is occupied solely by K. bau- AVISE, J. C. 1994. Molecular Markers, Natural His- tory and Evolution. Chapman & Hall, New York, rii. The two species thus overlap only in New York, U.S.A. the Florida peninsula. Arguing against this . 1996. Toward a regional conservation genet- possibility is the morphological separation ics perspective: phylogeography of faunas on the between these two species in Atlantic southeastern United States. Pp. 431-470. In J. C. Avise and J. L. Hamrick (Eds.), Conservation Ge- coastal regions (Lamb 1983a,b; Lamb and netics: Case Histories from Nature. Chapman & Lovich, 1990; current study). Hall, New York, New York, U.S.A. Another taxonomic revision imaginable AVISE, J. C., AND R. M. BALL, JR. 1990. Principles of would be to consider the highly divergent genealogical concordance in species concepts and biological taxonomy. Oxford Surv. Evol. Biol. 7:45- mtDNA groups A, B, and D within K. sub- 67. rubrum to reflect the presence of distinct AVISE, J. C., AND K. WOLLENBERG. 1997. Phyloge- phylogenetic (and, or, biological) species. netics and the origin of species. Proc. Natl. Acad. However, none of these or other such tax- Sci. USA 94:7748-7755. onomic alterations can as yet be recom- BERMINGHAM, E., AND J. C. AVISE. 1986. Molecular zoogeography of freshwater fishes in the south- mended with great certitude, because the eastern United States. Genetics 113:939-965. genetic assays thus far are confined to a CONANT, R., AND J. F. COLLINS. 1991. A Field Guide single "gene" (mtDNA). In principle, to and Amphibians, Eastern and Central "gene trees" can differ from "species North America. Houghton Mifflin, Boston, Mas- sachusetts, U.S.A. trees" for several plausible historical- ERNST, C. H., AND R. W BARBOUR. 1989. Turtles of demographic reasons, such as idiosyncratic the World. Smithsonian Institution Press, Washing- sorting of gene-tree lineages in transitional ton, D.C., U.S.A. populations that are large relative to inter- ERNST, C. H., R. W BARBOUR, E. M. ERNST, AND J. nodal R. BUTLER. 1974. Subspecific variation and inter- times as measured in organismal gradation in Florida Kinosternon subrubrum. 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Road, east of SR 12, Liberty Co., FL (n = 1); Bayou Smithsonian Institution Press, Washington, D.C., Lafourche drainage-Laurel Plantation Road near U.S.A. Thibodaux, LaFourche Parish, LA (n = 1); Bayou NEI, M. 1987. Molecular Evolutionary Genetics. Co- Boeuf near Kraemer, LaFourche Parish, LA (n = 2); lumbia University Press, New York, New York, Bayou L'Ourse drainage-bayou on SR 70, Assump- U.S.A. tion Parish, LA (n = 6); Bayou L'Ourse, Assumption NEI, M., AND W-H. Li. 1979. Mathematical model Parish, LA (n = 2); Coosa (Mobile) River basin- for studying genetic variation in terms of restriction James Floyd State Park, Chattooga Co., GA (n = 3); endonucleases. Proc. Natl. Acad. Sci. USA 76: Withlacoochee River basin-Hwy 19, 10 km. north of 5269-5273. Crystal River, Citrus Co., FL (n = 1); Edisto River NEIGEL, J. E., AND J. C. AVISE. 1986. Phylogenetic basin-Orangeburg National Fish Hatchery ponds, relationships of mitochondrial DNA under various Orangeburg Co., SC (n = 2); Mississippi River ba- demographic models of speciation. Pp. 515-534. In sin-cypress swamp, Big Cane Conservation Area, E. Nevo and S. Karlin (Eds.), Evolutionary Pro- Butler Co., MO (n = 5); Ochlockonee River basin- cesses and Theory. Academic Press, New York, County Road 268 near intersection with County Road New York, U.S.A. 65B, Gadsden Co., FL (n = 1); Apalachicola National PAMILO, P., AND M. NEI. 1988. Relationships be- Forest Rd. 13, Liberty Co., FL (n = 1); Ogeechee tween gene trees and species trees. Mol. Biol. Evol. River basin-ponds on Ft. Stewart Military Base, 5:568-583. Bryan Co., GA (n = 5); Pascagoula River basin- SAITOU, N., AND M. NEI. 1987. The neighbor-joining Lake Ivy, Clarkco State Park, Clark Co., MS (n = 3); method: a new method for reconstructing phylo- Pee Dee River basin-McKinney Lake National Fish genetic trees. Mol. Biol. Evol. 4:406-425. Hatchery ponds, Richmond Co., NC (n = 3); St. SEIDEL, M. E., J. B. IVERSON, AND M. D. ADKINS. Mark's River basin-Hwy 319 north of Tallahassee, 1986. Biochemical comparisons and phylogenetic Leon Co., FL (n = 1); pond south of Tallahassee near relationships in the Kinosternidae (Testudi- junction of Hwys. 319/263 and Hwy 363, Leon Co., nes). Copeia 1986:285-294. FL (n = 2); Savannah River basin-Long Creek, SITES,J. W, JR., J. W BICKHAM,M. W HAIDUK,AND Oglethorpe Co., GA (n = 1); Hwy 22 near Philomath, J. B. IVERSON. 1979. Banded karyotypes of six taxa Oglethorpe Co., GA (n = 1); ditch near Tillman, Jas- of kinostemid turtles. Copeia 1979:692-698. per Co., SC (n = 1); Sopchoppy River basin-Apa- 184 HERPETOLOGICA [Vol. 54, No. 2 lachicola National Forest Rd 13, Wakulla Co., FL (n near Oak Hill, Volusia Co., FL (n = 1); Ogeechee = 1); Tickfaw River basin-swamp on Hwy 15 near River basin-Bo Ginn National Fish Hatchery, Jen- Pontchatook, Tangipohoa Parish, LA (n = 3); Tom- kins Co., GA (n = 1); St. John's River basin-Welaka bigbee (Mobile) River basin-Carbon Hill National National Fish Hatchery, Putnam Co., FL (n = 1); Fish Hatchery ponds, Walker Co., AL (n = 1); Tupelo Hwy 441, Payne's Prairie, Alachua Co., FL (n = 1); 346 Alachua FL = National Fish Hatchery, Lee Co., MS (n = 2); Yellow Co. Rd near Hwy 121, Co., (n 2); 471 south of Tar- River basin-Yellow River Flood Plain, Hwy 90, Oka- Withlacoochee River basin-Hwy rytown, Sumter Co., FL (n = 1); Hwy 19, 10 km. loosa Co., FL (n = 2); Shoal River at Hwy 90, Oka- north of Crystal River, Citrus Co., FL (n = 1); Hwy = loosa Co., FL (n 1); York River basin-Cohoke 50, approximately 4 km west Sumter Co. line, Her- Mill Creek, King William Co., VA (n = 3); Florida nando Co., FL (n = 1); York River basin-Cohoke coastal integrated drainages-Chassahowitzka Wild- Mill Creek, King William Co., VA (n = 4); Florida life Management Area, Hernando Co., FL (n = 2); coastal integrated drainages-Snapper Creek Canal, Florida disjointed drainages-West Palm Beach, Dade Co., FL (n = 4); Florida disjointed drainages- Palm Beach Co., FL (n = 2). Dade/Collier Training Airport, Collier Co., FL (n = K. baurii: Indian River basin-Maytown Road 1).

Herpetologica, 54(2), 1998, 184-206 ? 1998 by The Herpetologists' League, Inc.

THE PHYLOGENETIC POSITION OF THE MEXICAN BLACK- TAILED PITVIPER (SQUAMATA: VIPERIDAE: CROTALINAE)

RONALD L. GUTBERLET, JR. Department of Biology, Box 19498, The University of Texas at Arlington, Arlington, TX 76019-0498, USA

ABSTRACT: Phylogenetic analyses of 52 morphological characters from Agkistrodon contortrix, Atropoides nummifer, Bothriechis bicolor, B. lateralis, B. nigroviridis, B. schlegelii, Bothrops asper, Cerrophidion godmani, Gloydius blomhoffii, Ophryacus undulatus, Porthidium melanurum, P. na- sutum, and P. ophryomegas indicate that the Mexican black-tailed pitviper (Porthidium melanurum) is more closely related to Ophryacus undulatus than it is to its congeners P. nasutum and P. ophryomegas. To achieve a monophyletic classification, P. melanurum is placed in the Ophry- acus. Key words: Crotalinae; Morphology; Ophryacus; Phylogenetic systematics; Porthidium; Porthi- dium melanurum; Viperidae

RECENT investigations into relationships (sensu Burger, in Perez-Higareda et al., among Neotropical pitvipers have led to 1985) is polyphyletic, and new genera the generic recognition of several putative- were proposed to rectify this unnatural ly monophyletic groups. In an unpublished grouping. Werman (1992) erected the ge- doctoral dissertation, Burger (1971) divid- nus Atropoides, the jumping pitvipers, to ed the morphologically diverse and poten- contain three of the 14 species formerly tially polyphyletic genus Bothrops into five included in Porthidium: A. nummifer, A. genera: Bothriechis, Bothriopsis, Bothrops, olmec, and A. picadoi. Campbell and La- Ophryacus, and Porthidium. Subsequent- mar (1992) segregated an additional three ly, Perez-Higareda et al. (1985) published species of Porthidium into Cerrophidion, this taxonomic arrangement. Campbell the montane pitvipers: C. barbouri, C. and Lamar (1989) adopted this classifica- godmani, and C. tzotzilorum. tion but suggested that additional studies These revisions reduced the content of into the relationships and generic limits of Porthidium to eight species. Solorzano these groups, especially Porthidium, were (1994) later increased the number of spe- needed. cies in Porthidium to nine with the de- Further study revealed that Porthidium scription of P. volcanicum. Eight of these