Nores AND FtEI-o REponrs 113

were conducted with flourescently labeled M l3 primers in 'lrclttttitttt Cttttsert'ulitttt tttttl Bittlttet'. I 9 robotic work station (Applied Biosystems model 800); O 1998 br Cheloniart Resc'trch Fouttclatiott a labeled extension products were analyzed with an auto- mated DNA sequencer (Applied Biosystems model 373A). Molecules, Morphology, and Mud Both the heavy (forward) and light (reverse) strands Phylogenetics ( ) were seqllenced (at least once) for each individual in to elirninate ambiguity (except for clunni fforward comple- JonN B. IvnRSoNl rnent seqllenced three times] and leucostontunt [forward complement sequenced twice]). Electrophoretograms -qen- tDepartmert of Biologv, Eurlltunt College, erated by the automated sequencer were cross-checked with Ricltntortcl, Irtcliartct 17 374 U SA compllter .-generated sequences to insure accuracy and se- I Fa-r: 765-983 - I 197 : E-ntail : joluti @ earlltarn.eclu] quences were then ali-ened by eye. Any ambiguous base was coded as an unknown. Previous phylogenetic analyses of morpholo-eical and Ali-ened sequences (290 bp; Appendix II) were ana- protein characters for the mud and musk of the farnily lyzed cladistically using the maximum parsirnony program Kinosternidae have resolved many of the hypothetical evo- PAUP (version 3.1.1; Swofford, 1993) with the composite lutionary relationships arnong its member (Seidel et out._eroup being the Staurotypinae (Stauroh'pus salvinii, S. al., 1986; Iverson, 199 I ). However, several controversial triporcotus, and Clauclius ongustcttus; after Bramble et al.,, relationships remained unresolved. This is in part due to the 1984, and Hutchison, l99l . amon-9 others). Analyses were incongruence among the phylogenies produced from mor- run with unweighted transversions and transitions as well as phological data alone versus those from only protein data with tranversions weighted4: I over transitions (the average versus those from combined protein and morphological ratio among the kinosternines); shortest trees were identical data. Although most of the previously hypothesized species under both schemes. Sequence divergence estimates were groups were supported by these analyses (Table I ), the calculated from the nucleotide sequence differences cor- monophyly of the Ste mothe rus.._group (carincttLt s, cl ep re s.rrls, rected for "multiple hits" by the two-parameter model of rninor, and ocloratus) and the inclusion of leucostontum tn Kimura (1980) using PHYLIP 3.5.5. Shortest trees were the sco rpioides group (and not with angustiports and clunni) generated via the heuristic search mode with random addi- remained ambiguous. This study was originated in order to tion of taxa and ten replications. Support for nodes was obtain rnitochondrial DNA sequence data to be submitted to examined via bootstrap with 1000 replications. phylogenetic analysis both alone and in combination with The cladistic analysis was repeated with the DNA data the previously published morpholo..eical and protein data. combined with the 27 unordered morphological characters Methocls Blood samples were obtained from all and the 34 protein electromorph characters (as opposed to 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 Table l. Prior hypotheses concernin-9 the relationships among angustiports, K. crectseri.,and K. oa-rctcee,as well as all three varions kinosternine turtles. subspecies of Kinoste rnon subrubrum (subrubrunt, steinclcrchneri, and ltippoc'repis). DNA isolation and se- I ) the _qenlls Kirtostenton (seusu srr"icro) is paraphyletic with respect to Srenr otlterus (Seidel et al.. I 986). quencing was done under the supervision of Brian Bowen at 2) cctrirtctttts (Zurg. 1966) or ocloratus (Tinkle, 1958) is the most the BEECS Genetic Analysis Core at the University of primitive member of the sternotherine groLrp and of the . MTDNA was isolated using standard phenol/chloro- Kinosternini overall. 3 carirtcttus, ntinor, and clepres,sus together are monophyletic form methodology (Hillis and 1990). ) Moritz, Cytochrorne b (Tinkle. 1958). sequences were amplified via the polymerase chain reaction 4) ntirtor and clepressrl.r are sister taxa (Tinkle. I 958: Iverson, I 977: using the universal primers described by Irwin et al.( l99l). Seidel and Lr-rcchino. 198 I ). 5) buurii and subntbrlun are closely related to each other (Larnb lS-base "universal" M An l3 sequence was added to the 5' and Lovich. 1990: Lovich and Lamb. 1995) and to the end of primers to facilitate automated sequencing. Thennal sternotherines (based on protein data from Seidel et al., 1986). cycling parameters were: I cycle at94"C (3 min), then 35 6) lterrerai rs the most primittve Kinostenron (sensu stricto) (e.g., Brarnble et al.. 1984). cycles ( ( ( at 94"C I min), 50"C I min), and 72"C I min). 7).fluvescen.e is closely related to subrubnurt and baurii (they are Standard precautions (including template-free PCRs as nega- ecologic al ly and rnorpho lo-e ic al I y s i mi lar, and nearly parapatric ). tive controls) were used to test for contamination and to 8) artgustiltorts and clurtrti are closely related, if not sister taxa (Le-eler. 1965. Iverson. 1988b). insure the accuracy the reactions (Innes 1990). of et al., 9) leuc'ostotntun is most closely related to cutgustiltorts and cluruti Streptavidin-coated magnetic beads (Dynabeads M280 (Iverson. 1988b. and not to the scorpioicles (but see l99l ). -qroup streptavidin, Dynal, Sweden) were used to purify PCR Iverson. l99l ). l0) sortoriertse and ltirtipe.s are sister taxa (Iverson,, l98l). products (Mitchell and Merill, 1989). Single stranded tem- I I ) the scorpioicles groLlp (crcutunt, olantosae,, chimalhuoca, plates were generated by denaturing the magnetically-cap- creoset'i. irfiegrunt, oo.\ocoe. and scorpioicles) is monophyletic tured double stranded DNA with fresh 0. 15M NaOH and (Iverson. I 98 8b. I 99 I ). 12) alarnosoe. clirnullruoco. and oa.rocee are each derived from using the released (non-biotinylated) strand as a template for irttegrun l (Berry. 1978: Berry et al ..1997: Iverson, 1986, 1989). sequencing reactions. Single stranded sequencin-e reactions l3) acuttutt and creoseri are sister taxa (lverson, 1988a). tt4 CHEloNlnN CoNSERVATToN AND BroLocv, Volunte 3, Nuntber I - 1998 4 TRI 13 TRI SAL SAL ANG ANG CAR CAR 7 g'- 2 DEP DEP oDo 14 17 MIN MIN oDo 15 ALA 16 INT ALA 13 HIR SCO HER HIR 2 ACU 8SON 5 SON HER 3 CHI ACU INT SCO LEU LEU DUN BAU 3 HIP 11 STE 7 SUB HIP BAU 8 4 SUB STE 15 FLA FLA

Figure l. Single most parsimonious tree (PAUP algorithm) of the Figure 2. Single most parsimonious tree (PAUP algorithm) of the cladistic relationships among kinosternid species (coded by first cladistic relationships among kinosternid species (coded by first three letters of species or subspecies nam see Appendix I) three letters of the species or subspecies name Appendix I) based on the cytochrome b mtDNA sequence data coded in based on 82 mtDNA characters, 34 protein electromorph products., Appendix II. Horizontal node lengths are proportional to branch and 27 morphological characters. Horizontal node lengths are length (number above each branch); numbers below some branches proportional to branch length (number above each branch); num- are 7o support (> 50Vo only) from 1000 bootstrap replications. The bers below some branches are Vc support (> 50Vo only) from 1000 length ts242 steps, and the consistency index is 0.59. The tree was bootstrap replications. The length is 415 steps, and the consistency rooted with the composite outgroup of Clauclins and two species index is 0.53. The tree was rooted with a composite outgroup of of Stauroh-pus. Clauclin s and two species of Stqurott'1ttts. locus characters; see Swofford and Olson, 1990; Mabee and but excluding dunni); and 4) that the scorpioides group Humphries, 1993; and de Queiroz and Lawson, 1994,, for (Table 1) is monophyletic but includes an unnatural hirtipes logic) reported by Iverson (1991). group (hirtipes and sonoriense, as well as herrerai). How- Results the bootstrap values, very few of and Discussion. - Of the 290 cytochrome b ever, as indicated by low nucleotides assayed, l2I (42Vo) were variable among the the relationships are resolved with confidence (only two combined ingroup and outgroup taxa, and 82 of those were values within the exceed 7 47o). In addition, a phylogenetically informative. Among the ingroup taxa l0l strict consensus tree of the shortest tree with trees one and (357o) were variable and 6l of those were informative. Of two steps longer (total -- 295 trees) supported a monophyl- the 101 variable positions, 92 possessed only two nucle- etic Stemotherus, baurii as sister taxon to subrubrum, and otides; of tho se7 4 (80Vo) represented transitions (57 C-T; ll hippocrepis as sister taxon to steindachneri (each with 1007o A-G). Sequence divergence estimates ranged from 0. l7 to of the 295 trees), but these clades and all otherkinosternine taxa 0.27 for staurotypines (Stauroh-pus and Claudius) versus arose from a single unresolved polytomy. Thus, it appears that kinosternines,0.0S to 0. 14 between sternotherines and other neither the protein data alone (Seidel et al., 1986; Iverson, kinosternine turtles, 0.03 to 0.08 among sternotherine spe- l99l ) nor the morphologic data alone (Iverson, 1991) nor the cies, and 0.01 to 0. l8 among other kinosternine turtles. cytochrome b data alone (this paper) are sufficient to resolve The shortest tree generated by the PAUP analysis of the the phylogenetic relationships within the Kinosterninae with cytochrome b nucleotide data (Fig. I ) was completely re- confidence. A total evidence analysis was walranted. solved, and suggested I ) that the group is The phylogenetic analysis of the mtDNA data com- monophyletic; 2) (unexpectedly) that Sternotherus is the bined with the morphological and protein electromorph data sister taxon of dunni; 3) that members of the flavescens sets also generated a single most parsimonious tree (Fig. 2). group ffiavescens, subrubrLtm, and baurii) are the most This tree strongly supported the monophyly of the primitive members of the clade (sensu stricto,, Sternotherus group (based on l4 synapomorphies: 8 mtDNA NorEs AND FtEI-n REponrs 115 tl uniquel, 4 electromorph, and 2 morphological), and within the scorpioicles group as depicted in Fig. 2. The recogn tzed Sternotherus as sister taxon to the analysis of a larger set of characters (particularly including Kinosternon (sensu stricto). Within the Sternotherus group, fast-evolving molecular ones) will be necessary before any odoratil.r was identified as most primitive and sister taxon to resolution of the relationships within this latter group can be the previously recognrzedcarinatus group (Table 1), includ- accepted with confidence. rng carinatus (most primitive) and the sister taxa minor and To test the strength of the support for the shortest depressus (as hypothesized by Tinkle, 1958). combined evidence tree, I generated a507o majority rule tree Within Kinosternon two sister clades were identified. for the best tree along with the79 trees that were one or two in the The first is the flavescens group (Table I ), supported by 7 steps longer (Fig. 3). Each of the major clades evident synapomorphies (3 mtDNA [1 unique], I electromorph, and shortest tree are also resolved in this expanded analysis. In analysis sup- 3 morphologic al 12 uniquel), and in whichflavescens is basal conclusion (compare Table 1), the combined to the sister taxa subrubrum (including steindachneri and ports the monophyly of the genus Sternotherus (and hence hippocrepis) and baurii. Should additional analysis support its resurrection from the synonymy of Kinosternon; see the monophyly of this flavescens group, the genus name Seidel et al., 1986); it indicates that odoratzs is the sister Thyrosternum Agassiz 1857 (type species , Thyrosterm,un taxon to all other Stentotherus;it recognizes a monophyletic pensylvanicum [= K. subrubrumf) is available for this clade; carinatus group (withntinor and depres.sn.T aS sister taxa); it however, that change is not recolilnended here. suggests that the central and eastern kinosternids in the USA The second clade within Kinosternon includes a mono- (sabrubrun't and baurii) are monophyletic (with flavescens phyleti c leucostomunt group (with dunni) as sister taxon to as their sister taxon); and it confirms the monophyly of dunni the combined scorpioides and hirtipes groups. Although the and leucostotnunl, outside of the sco rpioides group. How- latter group was fully resolved in this analysis, minimal ever, it does not support a primitive position for herrerai branch lengths, low bootstrap values, the lack of mtDNA (e.g., Bramble et al., 1984), and it leaves considerable data for two of its probable members (creaseri and oaxacae), ambiguity about the relationships within the combined and biogeographic incompatibilities (e.g ., chimalhuaca on scorpioides and hirtipes species groups. Further analyses the Pacific coast, and herrerai and acutum on the Atlantic) now in progress (J. Parham and J.H. Hutchison, pers. corum.; reduce my confidence in the phylogenetic relationships and D. Starkey and S. Davis, pers. comm.), involving fossils and additional DNA sequence data, respectively, may re- TRI solve the latter relationships. SAL Acknowledgments. M.A. Ewert, S. Guzmdn, M. ANG Klemens, W.P. McCord, P.A. Meylan, P.E. Moler, P. Rosen, CAR M.E. Seidel, R.W. Van Devender, and O. Victoria Castafro DEP provided specimens or tissues sequenced in this study. MIN Although not sequenced for this study, tissues were also oDo provided by C.R. Etchberger, D.R. Jackson, B. Mansell, and J.G. Palis. P. Minx assisted with blood extraction from ALA specimens in the live collection of W.P. McCord. D. HIR Prochaska performed a pilot sequence study that demon- SON strated the potential of the mtDNA for resolving kinosternine HER relationships. The final sequencing would not have been possible without the BEECS Genetic Analysis Core, the JAL University of Florida DNA Sequencing Core, B.W. Bowen, INT E. Almira, A. Garcia, S.E. Encalada, and especially A.M. ACU (Ginger) Clark, all in Gainesville. J. Parham assisted by sco generating the divergence estimates. J.F. Berry, S. Davis, and LEU B. Shaffer provided comments on an early draft of the manu- from the National Science Foundation (DEB DUN script. Support 8005586 and BSR 8404462), Earlham College, the Joseph BAU Moore Museum, and my family is greatly appreciated. SUB HIP Literature Cited

STE Acnssrz, L. 1857. Contributions to the Natural History of the United FLA States of America. Vols. l-2. Boston: Little, Brown and Co., 452pp. BEnny, J.F. 1978. Variation and Systematics in the Kinosternon Figure 3. Majority rule (50Vo) consensus tree of shortest tree (Fig. scorpioides and K. leucostomamcomplexes (Reptilia: Testudines: 2) and79 trees one or two steps longer (415417 steps), illustrating the cladistic relationships among kinosternid turtles based on total Kinosternidae) of Mexico and Central America. Ph.D. Thesis, evidence (see text). University of Utah, Salt Lake City. l 16 CHEloNrnN CoNSERVATToN AND BroLoGv, Volunte 3, Nutttber I - 1998

BERRv, J.F., SEroEL, M.E., AND IvEnsoN. J.8.1997. A new species of Su,op'p'oRp. D.L . 1993. PAUP. Phylogenetic Analysis Using Parsimony. mud turtle (-9enus Kinostenton) from Jalisco and Colirna. Mexico, Version 3.1. Champaign, IL: Illinois Nat. Hist. Suruey, 257 pp. with notes on its natural history. Chelonian Conservation and Swonrono, D.L. AND OLsox, G.J. 1990. Phylogeny reconstruction. In:

Biology 2(3):329-337 . Hillis, D.. and Modtz, C. (Eds.). MolecularSystematics. Sunderland, BnnvBLE, D.M.. HurcHISoN. J.H.. AND Lrclen, J.M. 1984. MA: Sinauer,, pp. 4l l-501. Kinosternid shell kinesis: structure. function and evolution. TrNrLe. D.W. 1958. The systematics and ecology of the Stentotherus Copeia 1984:456-415. curinutus cornplex (Testr.rdinata: ). Tulane Stud. Buru, D.G. 1984. The application of electrophoretic data in system- ZooL 6: I -56. atic str-rdies. Ann. Rev. Ecol. Syst. l5:501-522. ZuG, G.R. 1966. The penial morphology and the relationships of nE QuErRoz, A.., nNo LnwsoN, R. 1994. Phylogenetic relationships of cryptodiran tuftles. Occas. Paper Mus. Zool. Univ. Michigan the garter snakes based on DNA sequence and allozyme variation. 647:I-24. Biol. J. Linn. Soc. 53:209-229. HrLLrs, D., RND Monrrz, C. I 990. Molecular Systematics. Sunderland, {ppnNrnlx I MA: Sinauer. 588 pp. codes and sources of blood. HurcHrsoN, J.H. 1991. Early Eocene Kinosterninae (Reptilia: Three-letter abbreviation Testudines) and their phylogenetic signiticance. J. Vert. Paleo. ANG - Clauclius utgustcttus'. No data: pet trade specimen alive in collection of W.P. McCord. I l:l 45-167 . SAL-srr urro'l1ttt.s sulvinii No data. pet trade specimen alive in collection of W.P. HurcHrsoN, J.H.., AND GnnNasr-n. D.M. 198 I . Hornology of the plastral McCord. scales of the Kinosternidae and related tr-utles. Herpetolo-9ica TRI - Skuruth'pus tt'i2torc'utus'. No data; captive hatched from pet trade specimen 37 (2):73-85. in collection of M.A. Ewerl. CAR-Stenuttlrcnts curhwrrrs: Oklahoma. McCurtain Co., GloverCreek at Hwy INNrs, M.A., GElpnND, D.H., SNrNsrcv, J.J., AND WHrrE. T.J. I 990. PCR 3. NW of Broken Bow: JBI personal collection. Protocols: A Guide to Methods and Applications. San Diego, CA: DEP - Stentotlrcns clepresn u: No data: pet trade specimen alive in collection of Academic Press. W.P. McCord. MIN Stentotltents ninor: Florida. Marion Co.. Rainbow Run: UF (University IRwrN, D.M.. KocHEn. T.D.. AND WrsoN, 1991. Evoh-rtion the - A.C. of of Florida) 80827. cytochrome D gene of mammals. J. Mol. Evol. 32:128-144. ODO - Stenntlrcnts oclortfius: South Carclina. Aiken Co.: personal collection of IvEnsoN, J.B . 1911 . Geographic variation in the musk turtle, M. Klernens. ACU - Kiltostenum ucutturr. No data: pet trade specimen alive in collection of Stemotherus mirrcr. Copeia l97l:502-5ll . W.P. McCord. IvEnsoN, J.B. 198 I . Biosystematics of the Kinostenrcn hirtipes ALA - Kinostentut alunosue: Mexico. Sonom. Presa La Noria. I mi S Alamos species grollp (Testr-rdines: Kinosternidae). Tr"rlane Stud. Zool. Hwy. I mi E road to Cerro Prieto Microondas Station; UF 99877-18. BAU Kirtostenton baurii: Florida. Seminole Co., Oviedo: UF 84965. Bot. 23:I-14. - CHI - Kirtostentott chinrctllutctc'u'. Mexico. Jalisco.2.3 rd. mi S Hwy 200 Rfo IvEnsoN, J.B. 1986. Notes on the natural history of the Oaxaca mud hrrif rcacion brid,se: UF 5 l79l . no ftuther data; specimen in collection of turtle, Kirtostenton oo.rocoe. J. Herpetol. 20:l19-123. DLIN - Kinctstennn tlwui'. Colombia, Olga Victoria Castafro. IvEnsoN. J.B. 1988a. Distribution and status of Creaser's mud turtle FLA - Kinostenumflc:escens'. Nebraska, Garden Co., Gimlet Lalie: UF 99881. (Kirtostenton c'reoseri ). Herpetol. J. l:285 -291. IIER - Kirnstennn lrcrrerai'. Mexico. Veracruz. Xalapa, Las Animas: MVUZ (Museo Zoologica Universidad Veracruzana) 0455. IvEnsoN, J.B. 1988b. Neural bone pattems and the phylogeny of the HIR - Kino,stenlon hinipes: Mexico. Chihuahua, Ojo SE of Galeana; UF turtles of the subfamily Kinosterninae. Milwaukee Public Mus. uncatalogued. Contrib. Biol. Geol. 75:l-12. INT-Ktnostentort integnul: Mexico. Oaxaca, PresaLaNoria.9.0rd. mi. NEjutla, Hwy 175: UF 85004. I v EnsoN,, J . B . I 9 89. Natural history ofthe Alamos mud tu rtle, Kino s te n ton LEU - Kinostenton leucostornton'. Costa Rica. Limon Province, I km S Pr"rerto cil curto s cte ( Kinosternidae ). Southwest. Nat. 34: 134- I 42. Viejo: ASU (Appalachian State Universityl 17430. IvEnsoN. J.B . l99l . Phylogenetic hypotheses for the evolution of SCO - Kirtostentut scorltioitles: No data: pet trade specimen alive in collection of W.P. McCord. modern kinosternine tuftles. Herpetol. Monogr. 5:l -21 . SON - Kinostenrcn sonoriense: Aizona, Madcopa Co., Tempe. canal along Salt Ktuunn, M. 1980. A simple method for estimating evolutionary rate River bottom: specimen alive in collection of P. Rosen. of base substitution through comparative studies of nucleotide SLIB - Kirtostennn subntbntnt .subntbnun: North Carolina, Union Co., Jct SR 1006E and SR 1757N: ASU-WTS 45-92. sequences. J. Mol. Evol. l6: I I l-120. HIP - Kr rto.ste nton subnrbrtun hippocrepis: Florida. Santa Rosa Co., Goose Pond LRtr,ts' T.. RNo LovrcH, J.E,. 1990. Morphometric validation of the N of Munson: UF 80820. (Kirtostenton baurii) in the Carolinas and STE - Kinoste nron s ub ntbnun ste intlachen: Florida, l.ee Co.. no further data; UF 8496 r . . Copeia 1990:61 3-61 8. LecLeR, J.M. 1965. A new species of turtle, genLls Kinostenton, from AppnNorx II Central Amedca. Univ. Kansas hbl. Mus. Natur. Hist. 15:615-625. LovrcH, J.E., AND LRvrs, T. 1995. Morphometric similarity between Cytochrome b sequence data. Abbreviations of taxon names are listed in coded as "9." the turtles Kirtostenron subrubrum hippocrepis and K. buurii. J. Appendix I. Ambi-euous bases are Herpetol. 29:621-624. TRI TTTTGGATCA CTTCTTGGCA TCTGCTTAAT CCTACAAATT ACTACCCGCA MnsEE, P.M.. AND HuH,tpunre,s' J. 1993. Coding polymorphic data: TCTTCTTAGC TATACACTAC TCATCCGACA TATCCCTAGC ATTCTCAACA GTAGCCCACA TTACTCGAGA CGTACAATAC GGCTGACTTA TCCGCAACCT examples from allozymes and ontogeny. Syst. Biol. 42:166- l8 l. ACACGCCAAC GGCCTTTCAA TATTCTTCAT CTGCATTTAT ATGCACATCG MrrcHELL, L.G.. AND MEnnrll, G.R. 1989. Affinity generation of GACGAGGCAT TTACTACGGC TCATACCTCT ATAAAGAAAC TTGAAACACA GGAATTCT|C TACTACTATT AACTATAATA ACTGCATTCA single-stranded DNA for dideoxy sequencing following the poly- SAL TTTTGGATCA CTTCT'IGGCA TCTGCTTAAT CCTACAAATT ACCACCGGCA merase chain reaction. Anal. Biochem. 178:239-242. TCTTCTTAGC TATACACTAC TCATCCGACA TATCCCTAGC ATTCTCAACA GTAGCCCACA TTACTCGAGA CGTACAATAC GGCTGACTTA TCCGCAACCT SernpL, M.E., IvEnsoN, J.8., AND Aor

CAR CT|CGGATCA TTACTCGGCA TCTGCTTAAT CCTGCfuMTC ACTACCGGTA INT CTTCGGATCA TTACTTGGGA CCTGCCTAAC CCTACAAATT ATTACCGGCA TCTTCCTAGC AATACACTAT TCATCTGACA TATCACTAGC ATTCTCATCA TCTTCCTAGC AATACACTAT TCATCTAACA TATCACTAGC ATTCTCATCA GTTACCCATA CAATTCGAGA CGTACAATAC GGCTGACTCA TTCGAAACCT ATCACTCATA CAATTCGAGA CGTACATTAT GGCTGACTCA TCCGAAACCT CCATGCCAAC GGCGCTTCTC TATTCTTTAT ATGTATCTAC ATGCACATTG CCACGCCAAC GGTGCCTCCC TATTTTTTAT ATGTATCTAT ATACACATTG GACGAGGAAT CTACTACGGC TCATACCTGT ACAAAGAAAC TTGAAACACT GACGAGGAAT CTACTACGGA TTATACCTCT ACAAAGAAAC TTGAAACACT GGAATTATAC TATTACTACT AACCATAGCC ACCGCATTCA GGGGTTATAC TATTACTACT AACCATAGCT ACTGCATTCA DEP CTTCGGATCA TTACTCGGCA CCTGCTTAAT CCTACAAATC ACCACCGGCA ACU CTTCGGATCA TTACTTGGCA CCTGCCTAAT CCTACATATT ATTACCGGCA TCTTCCTAGC AATACACTAT TCATCTGACA TATCACTAGC ATTCTCATCA TCTTCCTAGC AATACACTAT TCATCTAACA TATCACTAGC ATTCTCATCA GTCACTCATA CAATTCGAGA CGTACAATAC GGCTGACTCA TTCGAAACCT ATCACCCATA CAATTCGAGA TGTACAATAT GGCTGACTCA TCCGAAACCT CCATGCCAAC GGCGCTTCTC TATTCTTTAT ATGCATCTAT ATGCACATTG CCACGCCAAC GGTGCCTCCC TATTTTTTAT ATGTATCTAT GTACACATTG GACGAGGAAT CTACTACGGC TCATACCTGT ACAAAGAAAC TTGAAACACT GACGAGGAAT CTACTACGGC TCATACTTCT ACAAAAAAAC TTCAAACACT GGAATTATAC TACTACTACT AACCATAGCC ACCGCATTCA GGAATTATAC TACTCCTACT AACCATAGCT ACTGCATICA MIN CTTCGGATCA TTACTCGGCA CCTGTTTAAT CCTACAAATC ATTACTGGTA CHI CTTCGGATCA TTACTTGGCA CCTGCCTAAT CCTACAAATT ATTACTGGCA TCTTCCTAGC AATACACTAC TCATCTGACA TATCATTAGC ATTCTCATCA TCTTCCTAGC AATACACTAT TCATCTGACA TATCACTAGC ATTCTCATCA GTCACCCATA CAATTCGAGA CGTACAATAC GGCTGACTTA TCCGAAACCT ATCACCCATA CAATTCGAGA TGTACAATAC GGCTGACTCA TCCGAAACCT CCATGCCAAC GGCGCTTCCC TATTTTTTAT GTGTATCTAC ATGCATATCG CCACGCCAAC GGTGCCTCCC TATTTTTTAT ATGTATCTAT GTACACATTG GACGAGGAAT CTACTACGGC TCATACCTAT ACAAAGAAAC TTGAAACACC GACGGGGAAT CTACTACGGC TCATACTTCT ACAAAGAAAC TTGAAACACC GGAATTATAC TACTACTACT AACTATAGCT ACCGCATTCA GGAATTATAC TACTCCTATT AACCATAGCT ACTGCATTCA ODO CTTCGGATCA TTACTCGGCA CCTGCTTAAT CCTACAMTC ACCACCGGCA SUB CTTTGGATCA CTACTTGGCA CCTGCT'IAAT CCTACAAATT ACTACCGGTA TCTTCCTAGC AATACACTAT TCATCTGACA TATCACTAGC ATTCTCATCA TCTTCCTAGC AATACACTAT TCATCTCACA TATCATTAGC ATTCTCATCA GTCACCCATA CAATTCGAGA CGTACAATAT GGCTGACTTA TCCGAAACCT ATTACTCATA CAATTCGAGA CGTACAATAC GGCTGACTTA TCCGAAACCT CCATGCCAAT GGCGCTTCCC TATTCTTTAT ATGTATCTAT ATGCACATTG CCATGCCAAC GGTGCTTCCC TATTCTTCAT ATGTATCTAC ATACACATCG GACGAGGAAT CTACTACGGC TCATACCTGT ACAAAGAAAC TTGAAACACT GACGAGGAAT CTACTACGGC TCATATCTTT ACAAAGAAAC TTGAAACACC GGAATTATAC TACTACTACT AACTATAGCC ACCGCATTCA GGAGTTATAC TACTACTACT AATCATAGCT ACTGCAT|CA ALA CTTCGGATCA TTACTTGGCA CCTGCTTAAT ACTACAAATT ATTACCGGCA HIP CTTCGGATCA TTACTTGGCA CCTGCTTAAT CCTACAAATT ACTACCGGTA TCTTCCTAGC AATACACTAT TCATCTGACA TATCACTAGC ATTCCAATCA TCTTCCTAGC AATACACTAT TCATCTGACA TATCATTAGC ATTCTCGTCA ATCACCCATA CAATTCGAGA CGTACAATAC GGCTGACTCA TCCGAAACCT ATCACTCATA CAATTCGAGA CGTACAACAC GGCTGACTTA TCCGAAACCT CCACGCCAAT GGCGCCTCCC TATTTTTTAT ATGTATCTAC ATTCATATTG CCATGCTAAT GGTGCTTCTC TATTTTTCAT ATGTATCTTC ATACACATCG CACGAGGAAT CTACTACGGC TCATACCTCT ACAAAGAAAC TTGAAATACC GACGAGGGAT CTACTACGGC TCATACCTCT ACAAAGAAAC TTGAAACACC GGAATTATAT TACTACTACT AACCATAGCT ACTGCATTTA GGAGTTATAC TACTACTACT AACCATAGCC ACTGCATICA BAU CTTTGGATCA CTACTTGGCA CCTGCTTAAT CCTACAAATT ACTACCGCTA STE CTTCGGATCA TTACTTGGCA CCTGCTTAAT CCTACAAATT ACTACCGGTA TCTTCCTAGC AATACACTAT TCATCTGATA TATCATTAGC ATTCTCATCA TCTTCCTAGC AATACACTAT TCATCCGACA TATCATTAGC ATTCTCATCA ATTACTCATA CAATTCCAGA CGTACAATAC GGCTGACTTA TCCGAAACCT ATCACCCATA CAATTCGAGA CGTACAATAC GGCTGACTTA TCCGAAACCT CCATGCCAAC GGTGCTTCCC TATTCTTCAT ATGTATCTAC ATACACATCG CCATGCTAAT GGTGCTTCTC TATTTTTCAT ATGTATCTAC ATACACATCG GACGAGGAAT CTACTACAAC TCATACCTTT ACAAAGAAAC TTGAAACACC GACGAGGAAT CTACTACGGC TCATACCTCC ACAAAGAAAC TTGAAACACC GGAGTTATAC TACTACTACT AACCATAGCT ACTGCATTCA GGAGTTATAC TACTACTACT AACCATAGCT ACTGCATTTA FLA CTTTGGATCA AIAACTCGGCA CCTGCTTACT CCTACAAACT ACTACCGGCA SCO CTTCGGATCA TTACTTGGCA CCTGCCTAAC CCTACAAATT ATTACCGGCA TCTTCGTAGC AATACACTAC TCATCTGACA TATCATTAGC ATTCTCATCA TCTTCCTAGC AATACACTAT TCATCCGACA TATCACTAGC ATTCTCATCA GTCACCCATA CAATTCGAGA CGTACAATAC GGTTGACTTA TCCGAAACCT ATCACCCATA CAATCCGAGA TGTACATTAT GGCTGACTTA TCCGAAACCT CCACGCCAAT GGGGCTTCCT TATTTTTCAT GTGCATCTAC ATACACATCG CCACGCTAAC GGTGCCTCCT TATTTTT9AT ATGCATATAC GTACACATTC GACGAGGAAT CTACTACGGC TCATACCTCC GCAAAGAAAC TTGAAACACT GACGAGGAAT CTACTACGGC TCATACCTCT ATAAAGAAAC TTGAAACACT GGAGT'IATAC TACTACTACT AACCATAGCC ACTGCATTCA GGAGTTATAC TACTACTACT AACCATAGCT ACTGCATTCA HIR CTTCGGATCA TTACTTGGCA CCTGCCTAAT CCTACAAATT ATTACCGGCA LEU TTTCGGATCA CTACTTGGTA CCTGCC999T CCTACAAATC ACTACCCGTA TCTTCCTAGC AATACACTAT TCATCTGACA TATCACTAGC ATTCTCATCA TCTTCCTAGC AATACACTAC TCATCTGACA TATCATTAGC ATTCTCATCT ATCACCCATA CAATTCGAGA TGTACAATAT GGCTGACTCA TCCGAAACCT ATCACCCATA CAATTCGAGA TGTACAATAC GGCTGACTCA TCCGAAACCT CCACGCCAAT GGTGCCTCAC TATTTTTTAT GTGTATCTAT GTACACATTG CCATGCCAAC GGTGCCTCCC TATTTTTCGC ATGTATCTAC ATACACATCG GACGAGGAAT CTACTACGGC TCATACTTCT ACAAAAACAC TTGAAACACT GACGAGGAAT CTATTACGGA TCGTACTTAT ACAAAGAAAC TTGAAACACC GGAAT'TATAC TACTCCTACT AACCATAGCT ACTGCAT|CA GGAGT'IATAC TACTACTACT TACCATAGCT ACTGCATTTA SON CTTCGGATCA TTACTTGGCA TCTGCCTAAT CCTACAAATT ATTACCGGCA DUN TTT9GGATCA CTACTTCG9A CCTGCgTAAT ACTACAggTC ACTACTGGCA TCTTCCTAGC AATACACTAT TCATCTGACA TATCACTAGC ATTCTCATCA CCTTCCTAGC AATACACTAC TCATCTGATA TATCATTAGC ATTCTCATCC ATTACCCATA CAATTCGAGA TGTACAATAC GGCTGACTCA TCCGAAACCT ATCACTCATA CAATCCGAGA CGTACAATAC GGTTGACTCA TCCGAAACCT ACACGCCAAC GGTGCCTCCA TATTTTTCAT ATGTATCTAT GTACACATTG CCATGCCAAC GGTGCCTCCC TATTCTTCAT ATGTATCTAC ATACACATCG GACGAGGAAT CTACTACGGC TCATACTTCT ACAAAGAAAC TTGAAACACT GACGAGGAAT ATACTACGGA TCATACCTGT ATAAAGAAAC CTGAAACACT GGAATTATAC TACTCCTACT AACCATAGCT ACTGCATTCA GGAAT'|ATAC TACTACTACT CACTATAGCT ACCCCATTCA HER CTTCGGATCA TTACTTGGCA CCTGCCTAAT CCTACAAATT ATTACCGGCA TCTTCCTAGC AATACACTAT TCATCTAACA TATCACTAGC ATTCTCATCA Receivecl: J June 1997 ATCACCCATA CAATTCCAGA TGTACAATAT GGCTGACTCA TCCGAAACCT I CCACGCCAAC GGTGCCTCCC TATTTTTTAT ATGTATCTAT GTACACATTG Revievrecl: 29 March 998 GACGAGGAAT CTACTACGGC TCATACTTCT ACAAAAAAAC TTGAAACACT Revisecl ancl Acceptecl: 28 July 1998 GGAATTATAC TACTCCTACT AACCATAGCT ACTGCATTCA