Nores AND FtEI-o REponrs 113 'lrclttttitttt Cttttsert'ulitttt tttttl Bittlttet'. I 9 O 1998 br Cheloniart Resc'trch Fouttclatiott Molecules, Morphology, and Mud Turtle Phylogenetics (Family Kinosternidae) JonN B. IvnRSoNl tDepartmert of Biologv, Eurlltunt College, Ricltntortcl, Irtcliartct 17 374 U SA I Fa-r: 765-983 - I 197 : E-ntail : joluti @ earlltarn.eclu] Previous phylogenetic analyses of morpholo-eical and protein characters for the mud and musk turtles of the farnily Kinosternidae have resolved many of the hypothetical evo- lutionary relationships arnong its member species (Seidel et al., 1986; Iverson, 199 I ). However, several controversial relationships remained unresolved. This is in part due to the incongruence among the phylogenies produced from mor- phological data alone versus those from only protein data versus those from combined protein and morphological data. Although most of the previously hypothesized species groups were supported by these analyses (Table I ), the monophyly of the Ste mothe rus.._group (carincttLt s, cl ep re s.rrls, rninor, and ocloratus) and the inclusion of leucostontum tn the sco rpioides group (and not with angustiports and clunni) remained ambiguous. This study was originated in order to obtain rnitochondrial DNA sequence data to be submitted to phylogenetic analysis both alone and in combination with the previously published morpholo..eical and protein data. Methocls Blood samples were obtained from all recogn tzedl i v i ng spec ie s of ki no sterni d tuft le s ( Ki nostern i nae and Staurotypinae: Appendix I) except Kinostenton angustiports, K. crectseri.,and K. oa-rctcee,as well as all three subspecies of Kinoste rnon subrubrum (subrubrunt, steinclcrchneri, and ltippoc'repis). DNA isolation and se- quencing was done under the supervision of Brian Bowen at the BEECS Genetic Analysis Core at the University of Florida. MTDNA was isolated using standard phenol/chloro- form methodology (Hillis and Moritz, 1990). Cytochrorne b sequences were amplified via the polymerase chain reaction using the universal primers described by Irwin et al.( l99l). An lS-base "universal" M l3 sequence was added to the 5' end of primers to facilitate automated sequencing. Thennal cycling parameters were: I cycle at94"C (3 min), then 35 cycles at 94"C ( I min), 50"C ( I min), and 72"C ( I min). Standard precautions (including template-free PCRs as nega- tive controls) were used to test for contamination and to insure the accuracy of the reactions (Innes et al., 1990). Streptavidin-coated magnetic beads (Dynabeads M280 streptavidin, Dynal, Sweden) were used to purify PCR products (Mitchell and Merill, 1989). Single stranded tem- plates were generated by denaturing the magnetically-cap- tured double stranded DNA with fresh 0. 15M NaOH and using the released (non-biotinylated) strand as a template for sequencing reactions. Single stranded sequencin-e reactions were conducted with flourescently labeled M l3 primers in a robotic work station (Applied Biosystems model 800); labeled extension products were analyzed with an auto- mated DNA sequencer (Applied Biosystems model 373A). Both the heavy (forward) and light (reverse) strands were seqllenced (at least once) for each individual in order to elirninate ambiguity (except for clunni fforward comple- rnent seqllenced three times] and leucostontunt [forward complement sequenced twice]). Electrophoretograms -qen- erated by the automated sequencer were cross-checked with compllter .-generated sequences to insure accuracy and se- quences were then ali-ened by eye. Any ambiguous base was coded as an unknown. Ali-ened sequences (290 bp; Appendix II) were ana- lyzed cladistically using the maximum parsirnony program PAUP (version 3.1.1; Swofford, 1993) with the composite out._eroup being the Staurotypinae (Stauroh'pus salvinii, S. triporcotus, and Clauclius ongustcttus; after Bramble et al.,, 1984, and Hutchison, l99l . amon-9 others). Analyses were run with unweighted transversions and transitions as well as with tranversions weighted4: I over transitions (the average ratio among the kinosternines); shortest trees were identical under both schemes. Sequence divergence estimates were calculated from the nucleotide sequence differences cor- rected for "multiple hits" by the two-parameter model of Kimura (1980) using PHYLIP 3.5.5. Shortest trees were generated via the heuristic search mode with random addi- tion of taxa and ten replications. Support for nodes was examined via bootstrap with 1000 replications. The cladistic analysis was repeated with the DNA data combined with the 27 unordered morphological characters and the 34 protein electromorph characters (as opposed to Table l. Prior hypotheses concernin-9 the relationships among varions kinosternine turtles. I ) the _qenlls Kirtostenton (seusu srr"icro) is paraphyletic with respect to Srenr otlterus (Seidel et al.. I 986). 2) cctrirtctttts (Zurg. 1966) or ocloratus (Tinkle, 1958) is the most primitive member of the sternotherine groLrp and of the Kinosternini overall. 3 ) carirtcttus, ntinor, and clepres,sus together are monophyletic (Tinkle. 1958). 4) ntirtor and clepressrl.r are sister taxa (Tinkle. I 958: Iverson, I 977: Seidel and Lr-rcchino. 198 I ). 5) buurii and subntbrlun are closely related to each other (Larnb and Lovich. 1990: Lovich and Lamb. 1995) and to the sternotherines (based on protein data from Seidel et al., 1986). 6) lterrerai rs the most primittve Kinostenron (sensu stricto) (e.g., Brarnble et al.. 1984). 7).fluvescen.e is closely related to subrubnurt and baurii (they are ecologic al ly and rnorpho lo-e ic al I y s i mi lar, and nearly parapatric ). 8) artgustiltorts and clurtrti are closely related, if not sister taxa (Le-eler. 1965. Iverson. 1988b). 9) leuc'ostotntun is most closely related to cutgustiltorts and cluruti (Iverson. 1988b. l99l ). and not to the scorpioicles -qroup (but see Iverson. l99l ). l0) sortoriertse and ltirtipe.s are sister taxa (Iverson,, l98l). I I ) the scorpioicles groLlp (crcutunt, olantosae,, chimalhuoca, creoset'i. irfiegrunt, oo.\ocoe. and scorpioicles) is monophyletic (Iverson. I 98 8b. I 99 I ). 12) alarnosoe. clirnullruoco. and oa.rocee are each derived from irttegrun l (Berry. 1978: Berry et al ..1997: Iverson, 1986, 1989). l3) acuttutt and creoseri are sister taxa (lverson, 1988a).