Ribosomal Dna and the Phylogeny of Frogs

Home , Hyloidea, Mesobatrachia, Ranoidea

HerpetologicalMonographs, 7, 1993, 118-131 ? 1993 by The Herpetologists'League, Inc.

RIBOSOMAL DNA AND THE PHYLOGENY OF FROGS

DAVID M. HILLIS, LOREN K. AMMERMAN,1 MICHAEL T. DIXON,' AND RAFAEL 0. DE SA2 Department of Zoology, The University of Texas, Austin, TX 78712, USA

ABSTRACT: Phylogenetic analysis of 1656 aligned sites in the 28S ribosomal RNA gene of frogs supports some of the recently recognized higher groups of anurans but provides counter-support for others. The 28S rDNA data support the monophyly of the recently recognized Pipanura (me- sobatrachians plus neobatrachians), which in turn indicates paraphyly of archaeobatrachians. Me- sobatrachians (pelobatoids plus pipoids), which are either considered paraphyletic or weakly supported as monophyletic in morphological analyses, also receive support as a monophyletic group from the 28S rDNA data. Hyloidea (= Bufonoidea), which is widely recognized but lacks morphological support, receives some molecular support as being monophyletic. However, Ranoidea, which is supported by morphology, is counter-supported by ribosomal DNA. In particular, dendrobatids do not group with ranids (but sometimes group with hyloids). A combined analysis of the molecular data with the morphological data of Duellman and Trueb (1986:Biology of Am- phibians) supports Pipanura, Mesobatrachia, Neobatrachia, and Hyloidea, but shows the ranoids as paraphyletic (with Dendrobatidae related to Hyloidea). The agreement between molecular and morphological data in several regions of the anuran tree indicates an approaching stabilization of traditionally labile higher frog classification. Key words: Anura; Frogs; Phylogeny; Systematics; Ribosomal DNA

THE higher phylogenetic relationships of stabilizing as new data have been of anurans are so poorly resolved that the brought to bear on the problem. major competing hypotheses share little To date, most of the relevant data have common ground. Twenty years ago, the come from morphological analyses of adult major subdivisions within frogs were the and larval frogs (summarized in Duellman subject of considerable debate (e.g., com- and Trueb, 1986; Ford and Cannatella, pare the classifications of Starrett, 1973, to 1993). Contributions from cytogenetics and those of Lynch, 1973). Today, although molecular biology have been compara- some progress toward stabilization of frog tively minor (see Hedges and Maxson, classification has occurred, there still ap- 1993; Hillis, 1991a; Morescalchi, 1973). pears to be little consensus among system- The reasons that frog phylogeny has been atists about relationships among the major such a difficult problem probably include groups of frogs (e.g., compare Hedges and all of the following: Maxson, 1993, to Ford and Cannatella, (1) The major lineages of frogs proba- 1993). Although a few major groups of bly diversified over a relatively short span anuran families are widely recognized, of time in the Mesozoic (Milner, 1988), so some families (such as Dendrobatidae, the frog tree is one of long terminal Sooglossidae, and Pelobatidae) are regu- branches leading back to small internodes. larly shifted back and forth among the This shape of tree is the most difficult type higher categories by the various authori- to reconstruct correctly, and is the most ties. In short, there is no widely accepted likely to lead to misleading or ambiguous classification of anurans because the in- results (see Swofford and Olsen, 1990). ferred phylogenies have shown few signs (2) Most phylogenetic studies of frogs (and especially molecular studies) have tended to include single exemplars to rep- resent major monophyletic groups, which PRESENTADDRESS: Department of Biology, Texas the identified in Wesleyan University, Forth Worth, TX 76105, USA. compounds problem (1) 2 PRESENTADDRESS: Department of Biology, Uni- above. Unlike (1), however, this problem versity of Richmond, Richmond, VA 23173, USA. can be corrected by expanding published 118 1993] HERPETOLOGICAL MONOGRAPHS 1993] HERPETOLOGICAL MONOGRAPHS 119~~~~~~~__119 I

databases to include more taxa. As more tential for estimating higher anuran rela- taxa are added to the analyses, the long, tionships. Although we are aware of the unbroken branches will be divided and need to add additional taxa, our sample of thereby shortened. Hopefully, such ap- species includes enough diversity to test proaches will gradually result in better es- some of the widely recognized (although timates of relationships. poorly supported) anuran groups. While (3) Morphological and cytogenetic previous studies of frog phylogeny have variation in frogs is surprisingly slight varied considerably in their conclusions, compared to other vertebrate groups of a the following higher taxa have been rec- similar age and species diversity. This ognized the most consistently: leaves systematists with relatively few Archaeobatrachia.-This name is ap- morphological or cytogenetic characters plied by different authors to several dif- that are informative about higher frog re- ferent groups of taxa. However, the group lationships, despite the fact that the taxa usually includes Ascaphidae, Bombinato- have been sampled far more intensively ridae, Discoglossidae,Leiopelmatidae, and for morphological and cytogenetic studies the Mesobatrachia (see below) (cf. Can- than for molecular studies. natella, 1985; Duellman, 1975; Hedges and (4) Although there is considerable mo- Maxson, 1993; cf. Laurent, 1979, 1986; lecular variation among major groups of Reig, 1958). When it has been considered frogs, molecular studies of frog relation- to be a monophyletic group, Archaeoba- ships have tended to focus on far too few trachia usually has been viewed as the sis- potentially informative characters to ter taxon of the remaining anurans (e.g., achieve any kind of robust support for or Hedges and Maxson, 1993). However, the against a particular phylogenetic hypoth- monophyly of this group appears highly esis. For instance, Hillis and Davis (1987) doubtful (Cannatella,1985; Ford and Can- examined restriction site and length vari- natella, 1993); in fact, the part of the an- ation in the 28S rRNA gene of 54 species uran tree that shows the strongest resolu- representing 17 families, but were unable tion from previous morphological analyses to make any robustconclusions about high- indicates the paraphyly of Archaeobatra- er frog phylogeny because of the small chia (Hillis, 1991a). number of changes. More recently, Hedg- Pipanura.-The grouping of mesoba- es and Maxson (1993) examined 333 trachians plus neobatrachians has been aligned sites in the mitochondrial 12S recognized by several recent authors (e.g., rRNA gene among 20 frogs, and found no Cannatella, 1985; Duellman and Trueb, nodes that they considered significantly 1986; Ford and Cannatella, 1993; Hillis, supported. A major problem with molec- 1991a; Sokol, 1975, 1977). Ford and Can- ular studies continues to be the tradeoff natella (1993) explicitly defined this group between sampling intensity among taxa and named it Pipanura,although they not- and sampling intensity among sites in the ed that the name Ranoidei had been pro- genome. Examinationof few taxa for many posed for this clade by Sokol (1977). The characters can lead to the problem noted latter name is usually used in a more re- in (2) above, whereas examination of many stricted sense (see Dubois, 1984). Recog- taxa for few characters produces poor res- nition of the Pipanura is obviously in olution. Hopefully, this problem will also conflict with the recognition of Archaeo- be temporary as more complete gene se- batrachia, if the latter group is considered quences accumulate for larger numbers of to include Mesobatrachia(i.e., in the sense taxa. of Duellman, 1975; Hedges and Maxson, Because of these limitations, there are 1993; or Reig, 1958). Among those who no strongly supported phylogenies that re- have recognized the Pipanura as a mono- late most of the families of frogs. The pur- phyletic group, opinion is divided as to pose of this paper was to examine a rela- whether the remaining taxa (discoglos- tively long and evolutionarily conservative soids) form a monophyletic sister group gene in enough frogs to determine its po- (e.g., Duellman and Trueb, 1986; Sokol, 120 HERPETOLOGICAL MONOGRAPHS [No. 7

1975) or are paraphyletic with respect to particular, the families Dendrobatidae and Pipanura (e.g., Cannatella, 1985; Ford and Sooglossidae have been the most problem- Cannatella, 1993; Hillis, 1991a; Lynch, atic (see Ford, 1989; Nussbaum, 1980). 1973). Sooglossids have been placed within the Mesobatrachia.-Mesobatrachia (Can- ranoids (e.g., Duellman, 1975; Griffiths, natella, 1985; Laurent, 1979) or Pipoidei 1959), within the hyloids (see Ford and (Dubois, 1984) has been less consistently Cannatella, 1993), or in the sister group to recognized, and the support of this group hyloids plus ranoids (e.g., Duellman and from morphological data is weak (Ford Trueb, 1986; Lynch, 1973). Dendrobatids and Cannatella, 1993; Hillis, 1991a). Me- have been considered hyloids by many sobatrachia consists of the last common an- (e.g., Laurent, 1979, 1986; Lynch, 1971, cestor of Pipidae, Rhinophrynidae, Pelo- 1973; Noble, 1922, 1931), despite the fact dytidae, Pelobatidae, and Megophryidae that they seem to have a full suite of ranoid (the latter two families are often combined synapomorphies (e.g., Duellman and into one) and all of its descendants (Ford Trueb, 1986; Ford, 1989, in press; Ford and Cannatella, 1993). The monophyly of and Cannatella, 1993; Griffiths, 1963). the pipids and rhinophrynids (usually Ranoidea.-This group of neobatrachi- grouped together with the extinct Palaeo- ans traditionally has been united on the batrachidae as the Pipoidea) is well basis of a firmisternal pectoral girdle [but supported by both adult and larval mor- see Ford and Cannatella (1993) for addi- phology (Cannatella, 1985; Ford and Can- tional synapomorphies]. Firmisterny is thus natella, 1993), although other relationships viewed as the derived condition, with an have been suggested (Maxson and Daugh- arciferal girdle seen as the ancestral con- erty, 1980). The remaining families are dition. Firmisternal girdles are also found often grouped together in the Pelobato- in some pipids, where the condition is idea, but the support for the monophyly widely regarded as convergent. Dendro- of this taxon is not strong (Hillis, 1991a). batids also have firmisternal pectoral gir- Neobatrachia.-This is the most consis- dles, which is part of the evidence used to tently recognized group of frogs, and is place Dendrobatidae in this group (Ford, supported by five morphological synapo- in press). As defined by Ford and Can- morphies (see Ford and Cannatella, 1993). natella (1993), Ranoidea includes "the It is defined by Ford and Cannatella (1993) common ancestor of hyperoliids, rhacoph- as "the most recent common ancestor of orids, ranids, dendrobatids, Hemisus, ar- living hyloids (myobatrachids, leptodac- throleptids, and microhlids, and all of its tylids, bufonids, hylids, centrolenids, pseu- descendants." dids, sooglossids, Heleophryne, brachy- We chose at least two of what are con- cephalids, Rhinoderma, and Allophryne) sidered to be among the most divergent and Ranoidea ... and all of its descen- taxa from each of these groups to have dants." Among recent classifications, only minimal tests of monophyly of the widely that of Starrett (1973), which was based recognized clades of frogs. We examined on Orton's (1953, 1957) tadpole types, has the large subunit, nuclear ribosomal RNA not recognized a monophyletic Neobatra- gene (encoding 28S rRNA) because this chia. gene shows considerable promise for ex- Hyloidea.-This group, for which the amining phylogenetic relationships across junior synonym Bufonoidea was formerly the Mesozoic (Hillis and Dixon, 1991), used (Dubois, 1986), has been widely rec- when the major groups of anurans presum- ognized but is unsupported by morpho- ably diverged. logical synapomorphies (Ford and Can- natella, 1993). Despite the widespread MATERIALS AND METHODS recognition of a primary division in Neo- High-molecular-weight DNA was iso- batrachia between hyloids and ranoids in lated from muscle tissue from Latimeria anuran classifications, there are several chalumnae (Actinistia), Leiopelma ham- families that have been shifted between iltoni (Leiopelmatidae), Spea multiplicata these two groups by various authors. In (Pelobatidae), Rana catesbeiana (Rani- 1993] HERPETOLOGICALMONOGRAPHS 121 dae), Allobates femoralis (Dendrobati- Swofford's (1990) Phylogenetic Analysis dae), Ceratophrys ornata (Leptodactyli- Using Parsimony (PAUP) program, ver- dae), Gastrotheca pseustes (Hylidae), and sion 3.0s. The amniote (Mus) and coel- Nesomantis thomasetti (Sooglossidae) fol- acanth (Latimeria) sequences were treat- lowing the protocol of Hillis and Davis ed as outgroups. All changes among (1986). Each sample of DNA was cleaved character states were weighted equally, and with the restriction enzyme Eco RI and gaps were treated as a fifth character state. ligated into a lambda vector (XgtlO for Regions of the gene that pair during sec- Rana and Gastrotheca, Lambda Zap II ondary structural folding were not weight- [Stratagene] for the others) to produce a ed by one-half as suggested by Wheeler subgenomic library (Hillis et al., 1990). The and Honeycutt (1988) because this over- Rana and Gastrotheca libraries were compensates for non-independence of the screened by filterlift hybridization with a data (Dixon and Hillis, 1993). However, cloned mammalian 28S rRNA gene (see we recognize that equal weighting could Hillis and Davis, 1987); the remaining li- introduce bias resulting from the weak in- braries were screened with the isolated 28S terdependence among paired sites. The rRNA gene of Rana catesbeiana (pE2528). presence of phylogenetic signal in the se- Positive plaques were selected and the in- quences was evaluated by examining the serts were subcloned into the vector skewness of the resulting tree-length dis- pBluescript (Strategene). Subclones were tributions (Hillis, 1991b; Hillis and Huel- verified by restriction digestion, Southern senbeck, 1992). The skewness statistic g, blotting, and sequencing. can be used to evaluate whether or not a Plasmid DNA was purified using the data matrix contains more structure than protocols described by Hillis et al. (1990), is expected from variation that is random denatured in alkali, and sequenced by the with respect to phylogenetic history. We base-specific dideoxynucleotide chain ter- did not use non-parametric bootstrapping mination method (Sanger et al., 1977) us- (Felsenstein, 1985) because interpretation ing modified T7 DNA polymerase (Tabor of bootstrapping results is not straightfor- and Richardson, 1987). Sequencing prim- ward and bootstrap proportions are not ers and their locations are given in Hillis comparable among branches on a tree or et al. (1991) or Hillis and Dixon (1991). among studies (Hillis and Bull, in press). Reaction products were separated on 4- RESULTS 6% polyacrylamide gels and visualized by autoradiography. DNA sequences were We aligned 1656 base-positions across aligned using the alignment subroutines the ten taxa (Fig. 1), of which 336 positions described by Pustell and Kafatos (1982, were variable. Our sequences spanned 1984, 1986). In addition to the taxa listed three sections of the 28S gene: Mus posi- above, we aligned the published 28S rDNA tions 110-425,1132-1789, and 3342-4134. sequences of Xenopus laevis (Pipidae; Parsimony analysis of this data matrix re- Ware et al., 1983; as corrected by Ajuh et sulted in three most parsimonious trees, al., 1991) and Mus musculus (Amniota; which differed only in the placement of Hassouna et al., 1984). Regions of ques- Allobates (Fig. 2). These three trees were tionable alignment were excluded from 375 steps long, with a consistency index of phylogenetic analyses. 0.622 (excluding uninformative charac- To compare the results from the 28S ters). rDNA data to morphology, we re-analyzed The skewness analysis indicated a sig- the data of Duellman and Trueb (1986) nificant amount of phylogenetic signal in for the same families that we examined. the 28S rDNA data matrix (g, = -1.34; P We also combined the molecular and mor- < 0.01). Not surprisingly, the best sup- phological data to evaluate the relative ported internal branch separated the in- strength of phylogenetic support from the group and outgroup taxa (Fig. 2). The two data sets. tree-length distribution of the possible res- All possible tree topologies were evalu- olutions within frogs was also strongly left = ated under the parsimony criterion using skewed (gl -0.57; P < 0.01), indicating 122 HERPETOLOGICAL--- --HERPETOLOGICAL ------MMONOGRAPHS-.- - -- OA [No. 7 ---122 [No...

01120 01140 01160 01180 Mus GGGTCGCGGCTTAGGGGCGGCGCGCAGCGCCGCACCCTTTACACCGCATGCCTTCTGGGTGAGGGGCCGCGG Xen GGGTCGCGGCTTAGGGGCGGGCGGGCCGCCGCGCCCTGCACACCGCATGCCCTCTGGCCTGGGGGGGCCGC Spe GGGTCGCGGCTTANGGGCCGGCGGGCCC-CGCAGCCCTTTACACCGCATTCCCTCTGGCCTGGGGGGGTGAC Ran GGGTCGCGGCTTAGGGGCANGCAGGCCGCCCGCGCCCTTTACACCGCATGCCCTCCGGCCTGGGTGGGCCGC Gas GGGTCGCGGCTTANGGGCGNGCGGGC-----NNNCCCTTTACACCGNATGNCCTCTGGNNNGGGTGGGGCCG Lei GGGTCGCGGCTTAGGGGCGGGCTGGCCG--CGCGCCCTTTACACCGCATGCCCTCTGGCCTGG-TGGGGCCG Nes GGGTCGCGGCTTAGGGGCGGGCGGGC-CGCCCTCTACACCGCATGCCCTCTGGCCTGGGTGGGGCCG Den NNNNNNNNGCTTAGGGGCAGGCGGGCCG-CCGAGCCCTGCACACCGCATGCCCTCTGGCCTGG-TGGGGCCG Cer GGGTCGCGGCTTAGGGGCGAGCGGGCCG-CCGCGCCCTTTACACCGCATGCCCTCTGGCCTGGATGGGGCCG Lat GGGTCGCGGCTTAGGGGCGAGCAGACCG-CCGCGCCCTTTACACCGCATGTCTTCTGGG-TGGAGGGGCC-G

02100 02120 02140 Mus CGAGCACCCCCCGGGTTCAGGAAGACTAGCTCCGGG-TCGGGCACCTGCCACACTCCGGCCATCGCCGGGGG Xen GCCGAGCCCCC-GGGTTCAGGAAGACTAGCTCCGGG-TCGGGCGCCTGCCACAATCCGGCCACCGCCGGGGG Spe TGTGAGCCCTT-GGGTTCAGGAAGACTAGCTCTGGG-TCGGGCGCCTGCCACAATCCGGCCATCGTTGGGNT Ran AGCGAGCCCCCNGGGTTCAGGAAGACTAGCTCCGGG-TCGGGCGCCTGCCACAATCCGGCCATCGCCGGGGG Gas NANCNAGNNNNNGGGTTCAGGAAGACTAGCTCCGGG-TCGCGCGCCTGCCACAATCCGGCCATCGCCGGGGG Lei CAGCGAGCCCCCGGGTTCAGGAAGACTAGCTCCGGG-TCGGGCACCTGCCACAATCCGGCCATCGCCGGGGG Nes CAGCGAGCCCC-GGGTTCAGGAAGACTAGCTCCGGG-TCGGGCGCCTGCCACAATCCGGCCANCCGCCGGGG Den CAGCGAGCCCCCGGGCTCAGGAAGATTATCACCGGGGTCGGGCGCCTGCCACCATCCGGCCATCGCCGGGGG Cer CAGCGAGCCCCCGGGCTCAGGAAGACTAGCTCCGGG-TCGGGCGCCTGCCACAATCCGGCCATCGCCGGGG- Lat CGGCGAGCCCCCGGGTTCAGGAAGACTAACTCCGAA-TCGGGTGCCTGCCACGCTCCGGCCATCGCCGGGGG

02160 02180 03100 03120 Mus CCG-CGCGGCCCGAGCCCAGAAGGGCCTCAGCCCAACGAACCCTTACGTCGGGTTTCGCCCACCATTTGAGG Xen CCG-CGCCGCCCTGGGCCAGAGGAGCCTCAGCCCAACAAACCCTTACGTCGGGTTTCGCCCACCATTTGAGG Spe TNGTCGCCNCCCTGGGCCAGATGTGCCTCAGCCCAACAAACCCTTACGTCGGGTTTCGCCCACCATTTGAGG Ran GCCGCGCCGCCCTGGGCCGGAGGGGCCTCAGCCCAACAAACCCTTACGTCGGGTTTCGCCCACCATTTGAGG Gas CCG-CGCCGCCCTGGGCCAGAGGGGCCTCAGCCCAACAAACCCTTACGTCGGGTTTCGCCCACCATTTGAGG Lei NCG-CGCCGCCCTGGGCCAGAGGAGCCTCAGCCCAACAAACCCTTACGTCGGGTTTCGCCCACCATTTGAGG Nes GCCGCGCCGCCCTGGGCCAGAGGGGCCTCAGCCCAACAAACCCTTACGTCGGGTTTCGCCCACCATTTGAGG Den NCCGCGGCGCCCTNGGCCAGANGGGCCTCAGCCCAACAAACACTTACGTCGGGTTTCTCCCACCATTTGAGG Cer -CCGCGCCGCCCTGGGCCAGAGGGGCCTCAGCCCAACAAACCCTTACGTCGGGTTTCGCCCACCATTTGAGG Lat G--CCCCCGCCCTGGCCCAGAAGAGCCTCAGCCCAACAAACCCTTACGTCGGGTTTCGCCCACCATTTGAGG

03140 03160 03180 Mus TAGATTCCGATTTATGGCCGTGCT-CTGGCTATCAGTTGTTCATGGCATTCCCTTTCAACTTTTCTTGAAAC Xen TAGATTCCGATTTATGGCCGTGCT-CTGGCTATCGCCTGTTCATGGCATTCCCTTTCAACTTTTCTTGAAAC Spe TAGATTCCGATTTATGGCCGTGCT-CTGGCTATCAGCTGTTCATGGCATTTC-GGTCAACTT"TC--GAAAC Ran TAGATTCCGATTTGTGCCCGCGCT-CTGGCTATCGCCTGTTCATGGCATTCCCTTTCAANNNNNNNNNNNNN Gas TAGATTCCGATTTATGGCCGCGCT-CTGGCTATCGCCTGTTCATGGCATTCC-TTTCAACTTTTCTTGAAAC Lei TAGATTCCGATTTATGGCCGTGCT-CTGGCTATCAGCTGTTCATGGCATTCCCTTTCAACTTTTCTTGAAAC Nes TAGATTCCGATTTATGCCCGCGCT-CTNGCTATCGCCTGTTCANGGCATTCCCTTTCAACTTTTCTTGAAAC Den TAGATTCCGATTTATGGCCGCGCT-CTGGCTATCGCCTGTTCATGGCACTCCCTTTCAACTTTTCTTGAAAC Cer TAGATTCCGATTTATGGCCGNGCT-CTGGCTATCGTCTGTTCATGGCATTCC-TTTCAACTTTTCTTGAAAC Lat TAGATTCCGATTTATGGCC-TGCTGCTGGCTATCAGCTGTTCATGGCATTCC-TTTCAAC'"I"TCTTGAAAC

04100 04120 11140 11160 Mus TTCTCTCTCAAGTT-CTCCCGCACTTTGGCAAAACTTTGTGCCTGGTTCCTCAGATTGCGCACGCGCTCA Xen TTCTCTCTCAAGTT-CTCCCGCACTTTGGCAAAACTTTGTGCCTGGTTCCTCAGATTGCGCGCGCGCTCA Spe TTCTCTCTCAAGTTTCTCCCGCACTTTGGCAAAACTTTGTGC-TGGTTCCTCAGATTGCGCGCGCGCTCA Ran NNN NNNNNNNN NNNN NNNNNNNNNNNNNNNNNNNNN Gas TTCTCTCTCAAGTT-CTCCCGCACTTTGGCAAAACTTTGTGCCTGGTTCCTCAGATTGCGCGTCGCGTCA Lei TTCTCTCTCAAGTT-CTCCCGCACTTTGGCAAAACTTTGTGCCTGGTTCCTCAGATTGTGCGCGCGCTCA Nes TTCTCTCTCAAGTT-CTCCCGCACTTTGGCAGNNNN NNN NNNNNNNN Den TTCTCTCTCAAGTT-CTCNCNNNNNNNNNNNNNNNNNNNNNNNNNNTCA Cer TTCTCTCTCAAGTT-CTCCCGCACTTTGGCGAAACTTTGTGCCTGGTTCCTCAGATTGCGCGCGCGCTCA Lat TTCTCTCTCAAGTT-CTCCCGCACTTTGGCAAAACTTTGTGCCTGGTTCCTCAGATTGCGCACGCGCTCA

FIG. 1.-Aligned DNA sequences from Mus (Mus), Xenopus (Xen), Spea (Spe), Rana (Ran), Gastrotheca (Gas), Leiopelma (Lei), Nesomantis (Nes), Allobates (Den), Ceratophrys (Cer), and Latimeria (Lat). Numbers refer to the position in the mouse gene as reported by Hassouna et al. (1984). Sequences enclosed in square brackets were not aligned and were not used in the phylogenetic analysis. 1993]L993] HERPETOL~OGICALHERPETOLOGICAL MONOGRAPHSI[MONOGRAPHS 123

11180 12100 12120 Mus G--TCCCCGAGCAG-GCTTTCGGCGGCACCGCGTTACTTCCACT[TCCCGGGGCGGGCCCCCG] Xen GCCTCCCTGAGACGCGCTTTGGG--ACACCGCGTTACTTCCACT[CCCGGCCCCGCGGGGCC] Spe GCCTCCC-GAGCGTCGCTTTGGG--ACACCGCGTTACTTCCACT[CCCCCCCGCTGGGGCC] Ran GCTCCC-GAGCGTNGTTGGGGNNN N-ACACCGCGTTACTTCCAC I I Gas GCCTCCC-GAGCGTNGCTTGGGG--ACACCGCGTTACTTCCACT[ACCCGGGTCTGGGGCC] Lei GCCTCCCGGAGA---GCTTTGGG--ACACCGCGTTACTTCCACT [CCGCCCCNCGGGGCC] Nes NNNNNNNNJNNNNNNNNNNNNNNNNNN [ I Den NCCTCCNNNNNNGT-GCTTTCGGGCGNNNTNNGTTACTTCCACT[CCCGGCCGNC] Cer GCCTCCC-GAGCGT-GCTTTGGG--ACACCGCGTTACTTCCACT[CCCGGCCCCGCGGGGC] Lat GTCTCCCGTCNNNTCGCTTTCGGG-GTACCGCGTTACTTTCACT[CCGCGCGCCCGGCC]

12140 12160 Mus GGCTCCACCCTAGGG[CTCCGGAGAGGTCAGGCGGCTCCCG] Xen GACTCCACCCTAGGG[CGGCGGGGAGGGAGGCGGGGGGGCCCCCGCCCCCCCCCGCGGCCGCCCGI Spe GACTCCACCCTAGCG[GCGGCGCGCCGCCCGCGTGGTGGCCGGGCAGAGCGGCCGTGGCAGGCCTCCATI Ran NNNNNNNNNNNNNNN[CGGCCGCCCGI Gas GACTCCACCCTAGGG[CCGAGCAAGCCNGGCAGGCGCG] Lei GACTCCACCCTAGGG[CGCGGCGCGCCGCCTG] Nes NL'NNNNNSNNNNNNNNN[ I Den GACTCCACCCTAGGG[CTCCGCGGCTNNTGCCTCCCG) Cer GACTCCACCCTAGCG[CGGCGGGCAGGCGCCAGTNNTGCCGCCCG] Lat GACTCCACCCTAGGG[GTGCGGAGCACGCCCCCCG]

12180 13100 13120 13140 Mus CGTGGTGGCCGGGCAGAGCGGGCGGCGCGGCCCCTCCACCTCGTGCTCGCATGCGCAATCCTGGGCTTTCTA Xen CGTGGTGGCCGGGCAGAGCGGGCGGGGCAGCCCCTCCACCTCGCACTCGCGCGCGCTATCCTGGGCTTTCTA Spe GGTG----- CGGGCAGAGCGG-CGTGGCAGGCCCTCCACCTCGCTCTCGCGCGCGCTATCCTGGGCTTTCTA Ran CGTGGTGGCCGGGCAGAGCGGGCG---GCGGCCCCTCCACCTCGTACTCGCGCGCACCGTCCTGGGCTTTCTA Gas CGTGGTGGCCGGGCAGAGCGG-CGAGGCAGCCCCTCCACCTCGTACTCGCGCGCGCNATCCTGGGCTTTCTA Lei CGTGGTGGCCGGGCAGAGCGGGCGAGGCAGCCCCTCCACCTCGTACTCGCACGCACTATCCTGGGCTTTCTA Nes CGTGGTGGCCGGGCAGAGCGGGCG-GGCGGCCCCTCCACCTCGTACTCGCACGCGCTATCCTGGGCTTTCTA Den CGTGGTGGCCGGGCAGAGCGGGCG-GGCAGCCCCTCCACCTCGTACTCGCGCGCACGATCCTGGGCTTTCTA Cer CGTGGTGGCCGGGCAGAGCGGGCG--GCAGCCCCTCCACCTCGTACTCGCGCGCGCTATCCTGGGCTTTCTA Lat CGTGGTGGTCGGGCAGAGTGGGCGTCGCAGCCCCTCCACCTCGTACTCGCACGCACTATCCTGGGCTTTCTA

13160 13180 14100 Mus CCACTTGATACGAACCCGTCCCGCTTCGGTCTCCTTTGAGACCACCTCCAGGCATCGCCAGGACTGCACGTT Xen CCACTTGATACGGACCCGTCCCGCTTCGGTCTCCTTTGAGACCACCTCCAGGCATCGCCAGGACTGCACGTT Spe CCACTTGATACGGACCCGTC-CGCTTCGGTCTCCTTTGAGACCACCTCCAGGCATCGCCAGGACTGCACGTT Ran CCACTTGATACGGACCCGTCCCGCTTCGGTCTCCTTTGAGACCACCTCCAGGCGTCGCCAGGACTGCACGTT Gas CCACTTGATACGGACCCGTCCNGCTTCGGTCTCCNNTGAGACCACCTCCAGGCGTCGCCAGGANTGCACGTT Lei CCACTTGATACGGACCCGTCCCGCTTCGGTCT TNNNNNNN Nes CCACTTGATACGGACCCGTNNNNNNNNNNNNNNTGAGAC-ACCTCCAGGCGTNNNNAGGANTGCANGTT Den CCACTTGATACGGACCCGTCCCGCTTCGGTCTCCTTTGAGACCACCTCCANNNNNCGCCAGGACTGCACGTT Cer CCACTTGATACGGACCCGTCCCGCTTCGGTCTCCTTTGAGACCACCTCCAGGCGTCGCCAGGACTGCACGTT Lat CCACTTGATACGGACCCGTCCCGCTTCGGTCTCCTTTGAGACCACCTCCANGCATCGCCAGGACTGCACGTT

14120 14140 14160 14180 Mus TAGCCAGCAGGCT-GGACCCATATCCCCGCTTTCTGATTAGCTTGGTAGATCATCGACCAAGGGAGGCTTCA Xen TAGCCAGCAGGCT-GGACCCATATCCCCGCTTTCTGATTAGCTTGGTAGATCATCGACCAAGGGAGGCTTGA Spe TAGCCAGCAGGCT-GGACCCATATCCCCGCTTTCTGATTAGCTTGGTAGATCATCGACCAAGGGAGGCTTCA Ran TAGCCAGCAGGCT-GGACCCATATCCCCGCTTTCTGATTAGCTTGGTAGATCATCGACCAAGGGAGGCTTCA Gas TAGC-AGCAGGGT-GGACCCATATCCCCGNNTTCTGATTAGCTTGGTAGATCATCGACCAAGGGAGGCTTCA Lei TNNNNNNNAGGCT-GGACCCATATCCCCGCTTTCTGATTAGCTTGGTAGATCATCGACCAAGGGAGGCTTCA Nes TAGC-AGCAGGNT-GGNNNCATATCCCCGCTTTCTGATTAGCTTGGTAGATCATCGACAAGGGAAGGNTTCA Den TNNNNNNNAGGCT-GGACCCATATCCCCGCTTTCTGATTAGCTTGGTAGATCATCGACCAAGGGAGGCTTCA Cer TAGCCAGCAGGCTGGACCCATATCCCCGCTTTCTGATTAGCTTGGTAGATCATCGACCAAGGGAGGCTTCA Lat TAGCCAGCAGGCTTGGACCCATATCCCCGCTTTCTGATTAGCTTGGTAGATCATCGACCAAGGGAGGCTTCA

FIG. 1.-Continued. 124 HERPETOLOGICALHERPETO3LOGICAL MONOGRAPHS [No. 7

15100 15120 15140 Mus AAGGGAGTCCTATCGACCGCGAG[AGCGAGGGCTGCATGC] GTCAAAATAGGCCATTTCGCT Xen AAGGGAGTCCTATCGACCGCGAG[CAGGCAGC] GTCAAAATAGGCCATTTCGCT Spe AAGGGAGTCCTATCGACCGCGAG[AGAGGNAGGC] GTCAAAATAGGCCATTTCGCT Ran AAGGGAGTCCTATCGACCGCGAG[AGCCGTGC] -TCAAAATAGGCCATTTCGCT Gas AAGGGAGTCCTATGCACCGCGAG[TGAGTTACANAAGTCGCNNC] GTCAAAATAGGCCATTTCGCT Lei AAGGGAGTCCTATCGACCGCGAG[TCCGGT] GTCAAAATAGGCCATTTCGCT Nes AAGGGAGTCCTATCGACNNAGCC[NCNCTCCT] GTCAAAATAGGCCATTTCGCT Den AAGGGAGTCCTATCGACCGCGAG[CAGCGTTGGGCTCTTTGGGAGCTC] GTCAAAATAGACCATTTCGCT Cer AAGGGAGTCCTATCGACCGCGAG[TGGGGGCTTC] GTCAAAATAGGCCATTTCGCT Lat AAGGGAGTCCTATCGACCACGAG[CTCGCTTGC] GTCAAAATAGACCATTTCGCT

15160 15180 16100 Mus TACTAATCTCCAGAACCCCGGCTTTGCTAGAGTTGGATAAGAGTTTGAAATTTACCCATTCTTCGGGCCGAG Xen TACTAATCTCCAGAACCCCGGCTTTGCTAGAGTTGGATAAGAGTTTGAAATTTACCCATTCTTCGGGCCGAG Spe TACTAATCTCCAGAACCCCGGCTTTGCTAGAGTTGGATAAGAGTTTGAAATTTACCCATTCTTCGGGCCGAG Ran TACTAATCTCCAGAACCCCGGCTTTGCTAGAGTTGGATAAGAGTTTGAAATTTACCCATTCTTCGGGCCGAG Gas TACTAATCTCCAGAACCCCGGCTTTGCTAGAGTTGGATAAGAGTTTGAAATTTACCCATTCTTCGG-CCGAG Lei TACTAATCTCCAGAACCCCGGCTTTGCTAGAGTTGGATAAGAGTTTGAAATTTACCCATTCTTGCGGCCGAG Nes TACTAATCTCNAGAACCCCGGCTTTGCTAGAGTTGGATAAGAGTTTGAAATTTACCCATTCTTC-GGCCGAG Den TACTAATCTCCAGAACCCCGGCTTTGCTAGAGTTGGATAAGAGTTTGAAATTTACCCATTCTTCGGGCCGAG Cer TACTAATCTCCAGAACCCCGGCTTTGCTAGAGTTGGATAAGAGTTTGAAATTTACCCATTCTTCGGGCCGAG Lat TACTGATCTCCAGAACCCCGGCTTTGCTAGAGTTGGATAAGAGTTTGAAATTTACCCATTCTTCGGGCCGAG

16120 16140 16160 16180 Mus CGACCGCACCTCGGCCC-GCACCTTACGCTCA--CGGATCACCCGGTGAAAACCATTCGTCTTGACCGCGAC Xen CGACCGAACCTCGGCCC-GCACCTTACGCTCGTGCGGATCACCCGGTGAAAACCATTCGTCTTGACCGCGAC Spe CGACCGGACCTCGGCCCCGTACCTTACGCTC--GCGGATCACCCGGTGAAAACCATTCGTCTTGACCGCGAC Ran CGACCGGACCTCGGCCC-GCACCTTACGCTCNNNNNNNTCACCCGGTGAAAACCATTCGTCTTGACCGCGAC Gas CGACCGGACCTCGGCCCCGCACCTTACGCTC--GCGG-TCACCCGGTGAAAACCATTCGTCTTGACCGCGAC Lei CGACCGGACCTCGGCCC-GCACCTTACGCTCNNNNNGATCACCCGGTGNNNNNNNNNNNNNNNNNNNN Nes CGACCGGACCTCGGCCC-GCACCTTACGCTC--GCG--TCACCCGGTGAAAACCATTCGTCTTGACCGCGAC Den CGACCGCACCTCGGCCC-GCACCTTACGCTCT-GCGGATCACCCGGCGAAAACCATTCGTCTTGACCGCGAC Cer CGACCGGACCTCGGCCC-GCACCTTACGCTCG-GCG--TCACCCGGTGAAAACCATTCGTCTTGACCGCGAC Lat CGACCGAACCTCGGCCC-GCACCTTACGCTCA--CGGATCACCCGGTGAAAACCATTCGTCTTGACCGCGAC

17100 17120 17140 Mus GCCCTACTTGGCTTGCGGCCCAATTCCGCGGGCTACGGCTGCGAGTAGTCTGGGGTCTTTTCCACAACCAAC Xen GCCCTACTTGGCTTGCGGCCCAATTCCGCGGGCTACGGCTGCGAGTAGTCTGGGGTCTTTTCCACAACCAAC Spe GCCCTACTTGGCTTGCGGCCCAATTCCGCGGGCTACGGCTGCGAGTAGTCTGGGGTCTTi'CCACAACCAAC Ran GCCCTACTTGGCTTGCGGCCCAATTCCGCGGGCTACGGCTGCGAGTAGTCTGGGGTCTTTTCCACAACCAC Gas GCCCTACTTGGCTTGCGGCCCAATTCCGCGGGCTACGGCTGCGAGTAGTCTGGGGTCTTTTCCACAACCAAC Lei NNNN MN NNNNNNNNNNNNNNTACGGCTGCGAGTAGTCTGGGGTCTTTTCCACAACCAAC Nes GCCCTACTTGGCTTGCGGCCCAATTCCGCGGGCTACGGCTGCGAGTAGTCTGGGGTCTTTTCCACAACCAAC Den GCCCTACTTGGCTTGCGGCCCAATTCCGCGGGCTACGGCTGCGAGTAGTCTGGGGTCTTTTCCACAACCAAC Cer GCCCTACTTGGCTTGCGGCCCAATTCCGCGGGCTACGGCTGCGAGTAGTCTGGGGTCTTTTCCACAACCAAC Lat GCCCTACTTGGCTTGCGGCCCAACACCGCGGGCTACGGCTGCGAGTAGTCTGGGGTCTTTTCCACAACCAAC

17160 17180 33160 Mus TATATCTGTCGTCCTGCCACCGGTACCTTCAGCTACACTAAAGACGGGTCACGAGACTTACAGTTTCACT Xen TATATCTGTCGTCCTGCCACCGGTACCTTCAGCTACACTAAAGACGGGTCACGAGACTTACAGTTTCACT Spe TATATCTGTCGTCCTGCCACCGGTACCTTCAGCTACACTAAAGACGGGTCACGAGACTTACAGTTTCACT Ran TATATCTGTCGTCCTGCCACCGGTACCTTCAGCTACACTAAAGACGGGTCACGAGACTTACAGTTTCACT Gas TATATCTGTCGTCCTGCCACCGGTACCTTCAGCNNNNNTGNNNNNNNNNNMNNMAGNNNNNNNNNNNNNN Lei TATATCTGTCGTCCTGCCACCGGTACCTTCAGCTACACTAAAGACGGGTCACGAGACTTACAGTTTCACT Nes TATATCTGTCGTCCTGACACCGGTGCCTTCAGCTACACTAAAGACGGGTCACGAGACTTACAGTTTCACT Den TATATCTGTCGTCCNGCCACCGGTACCTTCAACTACACTAAAGACGGGTCACGAGACTTACAGTTTCACT Cer TATATCTGTCGTCCTGCCACCGGTACCTTCAGCTACACTAAAGACGGGTCACGAGACTTACAGTTTCACT Lat TATATCTGTCGTCCTGCCACCGGTACCTTCAGCTACAC TAAAGAC GG GTCAC GAGAC TTACAGTT TCACT

FIG. 1.-Continued. 1993]19931 HERPETOLOGICAL MONOGRAPHS 125

33180 34100 34120 34140 Mus TCTTTAAGTTACTTCGCGCCCATTTGCCGCCCTCATTGATACTGAGAGAATTCCATCGGTTTACGGAGCAGT Xen TCTTTAAGTTACTTCGCGCCCATTTGCCGCCCTCATTGATACTGAGAGAATTCCATCGGTTTACGGAGCAGT Spe TCTTTAAGTTACTTCGCGCCCATTTGCCGCCCTCATTGATACTGAGAGAATTCCATCGGTTTACGGAGCAGT Ran TCTTTAAGTTACTTCGCGCCCATTTGCCGCCCTCATTGATACTGAGAGAATTCCATCGGTTTACGGAGCAGT Gas NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN Lei TCTTTAAGTTACTTCGCGCCCATTTGCCGCCCTCATTGATACTGAGAGAATTCCATCGGTTTNNNNNNNNNN Nes TCTTTAAGTTACTTCGCGCCCATTTGCCGCCCTCATTGATACTGAGAGAATTCCATCGGTTTACGGAGCAGT Den TCTTTAAGTTACTTCGCGCCCATTTGCCGCCCTCATTGATACTGAGAGAATTCCATCGGTTTACGGAGCAGT Cer TCTTTAAGTTACTTCGCGCCCATTTGCCGCCCTCATTGATACTGAGAGAATTCCATCGGTTTACGGAGCAGT Lat TCTTTAAGTTACTTCGCGCCCATTTGCCGCCCTCATTGATACTGAGAGAATTCCATCGGTTTACGGAGCAGT

34160 34180 35100 35120 Mus AGATTAATCACTGCGCGTACTTACCTACTTGCTCTAAGGGTGACAGGGATGGATGATAGGTCGCTTTGGTGT Xen AGATTAATCACTGCGCGTACTTACCTACTTGCTCTAAGGGTGACAGGGATGGATGATAGATCGCTTTGGTGT Spe AGATTAATCACTGCGCGTACTTACCTACTTGCTCTAAGGGTGACAGGGATGGATGATAGATCGCTTTGGTGT Ran AGATTAATCACTGCGCGTACTTACCTACTTGCTCTAAGGGTGACAGGGATGGATGATAGATCGCTTTGGTGT Gas NNNNNNNNNNNNNNNNNNACTTACCTACTTGCTCTAAGGGTGACAGGGATGGATGATAGATCGCTTTGGTGT Lei NNNNNNNNCACTGCGCGTACTTACCTACTTGCTCTAAGGGTGACAGGGATGGATGATAGATCGCTTTGGTGT Nes AGATTAATCACTNNNCGTACTTACCTACTTGCTCTAAGGGTGACAGGGATGGATGATAGATCGCTTTGGTGT Den AGATTAATCACTGNNNNTACTTACCTACTTGCTCTAAGGGTGACAGGGATGGATGATAGATCGCTTTGGTGT Cer AGATTAATCACTGCGCGTACTTACCTACTTGCTCTAAGGGTGACAGGGATGGATGATAGATCGCTTTGGTGT Lat AGATTAATCACTGCGCGTACTTACCTACTTGCTCTAAGGGTGACAGGGATGGATGATAGATCGCTTTGGTGT

35140 35160 35180 Mus CGGTTCCCTTGCCCGAACCGCCTTAGTCGCCCCTTTC--TTCTGGGACAACTCGAACTGAGATCAGACCGTG Xen CGGTTCCCTTGCCCGAACCGCCTTAGTCGCCCCTTTC--TTCTGGGACAACTCGAACTGAGATCAGACGTTG Spe CGGTTCCCTTGCCCGAACCGCCTTAGTCGCCCCTTTC--TTCTGGGACAACTCGAACTGAGATCAGACGTTG Ran CGGTTCCCTTGCCCGAACCGCCTTAGTCGCCGCAGTCGCCTTTGGGACAACTCGAACTGAGATCAGNNNNNN Gas CGGTTCCCTTGCCCGAACCGCCTTAGTCGCCCCTTTC--TTCTGGGACAACTCGAACTGAGATCAGACGTTG Lei CGGTTCCCTTGCCCGAACCGCCTTAGTAGCCCCTTTC--TTCTGGGACAACTCGAACTGAGATCAGACGTTG Nes CGGTTCCCTTGCCCGAACCGCCTTAGTCGCCCCTTTC--TTCTGGGACAACTCGAACTGAGATCAGACGTTG Den CGGTTCCCTTGCCCGAACCGCCTTAGTCGCCCCTTTC--TTCTGGGACAACTCGAACTGAGATCAGACNNNN Cer CGGTTCCCTTGCCCGAACCGCCTTAGTCGCCCCTTTC--TTCTGGGACAACTCGAACTGAGATCAGACGTTG Lat CGGTTCCCTTGCCCGAACCGTCTTAGTCGCCCCTTTC--TTCTGGGACAACTCGAACTGAGATCAGACCGTG

36100 36120 36140 36160 Mus CCACTTCTCTGTACTCTCCACATCTTATTCACCCTCCGGG[GGCCGCGGGCCGGGGCAGGAGCGCAGCCC] Xen ACACTTCTCTGTACTCTCCACATCCTATTCACCCTCCGGG[GGCGCGAGCAGCGTTTCCCCG] Spe ACACTTCTCTGTACTCTCCACATCCTATTCACCCTCCGGG[GGNAGCGAGCACGCAC] Ran NNNNNNNNNNNNNNNNNNNNNNNNCTATTCACC-TC-GGG [CCCTCCGGGTGCGGGAGCGCCACCCCG] Gas ACACTTCTCTGTACTCTCCACATCCTATTCACCNNNNNNNI I Lei ACACTTCTCTGTACTCTCCACATCCTATTCACCCTCCGGG[GGCAGGCGCCCT] Nes ACACTTCTCTGTACTCTCCACATCCTATTCACCCTCCGGG[GGCGGGCGCAGGGGCAGTGGGCCCGGGCC] Den NNNNNNNNNNNNACTCTCCACATCTTATTCACCCTCCGGG[CGGG] Cer ACACTTCTCTGTACTCTCCACATCCTATTCACCCTCCGGA[GGCGAGTCAGGGGGAGCGCCCAGCCGCTT] Lat ACACTTCTCTGTACTCTCCACATCCTATTCACCCTCCGGA[GGCAGCNAGGGACCGATCCGACT]

36180 37100 37120 Mus [CAGCCCCGTGCGGCCGGAGCGCC]CGGCGGCCACTTTATGGTGATGAGAGTAGCAAAAAAGTGACTGGGC Xen CGGCGGCCACTTTATGGTGATGAGAATAGCAAAAAAGTGAATGGGC Spe GGACGGCCACTTTATGGTGATGAGAATAGCAAAAAAGTGAATGGGC Ran CGGCGGCCACTTTATGGTGATGAGAATAGCAAAAAAGTGAATGGGC Gas NNNNNNCCACTTTATGGTGATGAGAATAGCAAAAAAGTGAATGGGC Lei CGGCGGCCACTTTATGGTGATGAGAATAGCAAAAAAGTGAATGGGC Nes [GTGGCCT] CGGCGGCCACTTTATGGTGATGAGAATAGCAAAAAAGTGAATGGGC Den CNNNNGCCACTTTATGGTGATGAGAATAGCAAAAAAGTGAATGGGC Cer [TCCCCG] CGGCGGCCACTTTATGGTGATGAGAATAGCAAAAAAGTGAATGGGC Lat CGGCGGCCACTTTATGGTGATGAGAATAGCAAAAAAGTGAATGGGC

FIG. 1. -Continued. 126 HERPETOLOGICALHERPETOLOGICAl MONOGRAPHS [No. 7

37140 37160 37180 Mus CACTCCGCCCCCCCGCTCGGGGCTCCCCGAGAGCGAAGACCGCGGTTCGCAG[GCAGGGCGCGCACGC] Xen CACTCCGCCCCCCCGCTCGGGGCTCCCCGAGAGCGAAGACCTGGGTTCGC-G[GGCCGGGGGCG] Spe CACTCCGCCCCCCCGCTCGGGGCTCCCCGAGAGCGAAGACCGAGGTTCGC-G[GGCCGGCANNGT] Ran CACTCCGCCCCCCCGCTCGGGGCTCCCCGAGAGCGAAGACCGAGGTTCGG-G[GCCCCGGGG] Gas CACTCCGNNNNN NNNN [ ] Lei CACTCCGCCCCCCCGCTCGGCG-TCCCCGAGAGCGAAGACCGAGGTTCGCAG[GCCGCG] Nes CACTCCGCCCCCCCGCTCGG--CTNNNNGAGAGCGAAGACCGGCGTTCGNNN[ ] Den CACTCCGCCCCCCCGCTCGG--CTCCCCGAGAGCGAAGACCGAGGTTCGCGG[ Cer CACTCCGCCCCTCCGCTCGGGGCTCCCCGAGAGCGAAGACCAGGGTTCGNGG[ ] Lat CACTCCGCCCCCCCGCTCAGGGCTCCCCGAGAGCTAAGACCACTGTTCGC-G[GCCGAGC]

38100 38120 38140 38160 Mus CCGCCCGCGCTGGGCGAGGCCCCTGTCACGGTCCACCCCTCAAACTGACCCCGCCATGTGGACAGTTTGCCA Xen CGGCCCGCGCTGGGCGAGGCTCCTGTCACCGTCCACCCCTCAAACTGACCCCGCCATGTGGACAGTTTGGCA Spe NNNCGCTCNNNGGGCGAGGCTCCTGTCACCGTCCACCCCTCAAACTGACCCCGCCATGTGGACAGTTTGGCA Ran CAGCCCGCGCTGGGCGAGGCTCCTGTCACCGTCCACCCCTCAAACTGACCCCGCCATGTGGACAGTTTGCCA Gas TTTGGCA Lei CACGGCCGGCTGGGCGAGGCTCCTGTCACCGTCCACCCCTCAAACTGACCCCGCCATGTGGACAGTTTGCCA Nes NNNNNNNNNNNNGGCGAGGCTCCTGTCACCGTCCACCCCTCAAACTGACCCCGCCATGTGGACAGTTTGGCA Den NNNNNNNNNNNNGGCGAGGC-CCTGTCACAGTCCACCCATCAAACTGACCCCGCCATGTGGACAGTTTGGCA Cer NNNNNNNNNNTGGGCGAGGCTCCTGTCACCGTCCACCCCTCAAACTGACCCCGCCATGTGGACAGTTTGGCA Lat CGGCCCACGCTGGGCGAGGCCCCTGTCACCGTCCACCCCTCAAACTGACCCCGCCATGTGGACAGTTTGGCA

38180 39100 39120 39140 Mus TTGCGTCCACAGGATTCCGCTCGAGTCCCTCCTGTCTTTGGAGGGCACCTCGTCTTCCCGTTTTCGAGCGAA Xen TTGCGTCCACAGGATTCCGCTCGAGTCCCTCCTGTCTTTGGAGGGCACCTCGTCTTCCCGTTTTCGAGCGAA Spe TTGCGTCCACAG-ATTCCGCTCGAGTCCCTCCTGTCTTTGGAGGGCACCTCGTCTTCCCGTTTTCGAGCGAA Ran TTGCGTCCACAGGATTCCGCTCGAGTCCCTCCTGTCTTTGGAGGGCACCTCGTCTTCCCGTTTTCGAGCGAA Gas TTGCGTCCACAGGATTCCGCTCGAGTCNCTCCTGTCTTTGGAGGGCACCTCGTCTTCCCGTTTTCGAGCGAA Lei TTGCGTCCACAGGATTCCGCTCGAGTCCCTCCTGTCTTTGGAGGGCACCTCGTCTTCCCGTTTTCGANCGNA Nes TTGCGTCCACAGGATTCCGCTCGAGTCCCTCCTGTCTTTGGAGGGCACCTCGTCTTCCCGTTTTCGAGCGAA Den TTGCGTCCACAGGATTCCGCTCGAGTCCCTCCTGTCTTTGGAGGNCACCTCNTCTTCCCGTTTTCGAGCGAA Cer TTGCGTCCACAGGATTCCGCTCGAGTCCCTCCTGTCTTTGGAGNNCATCTCGTCTTCCCGTTTTCGAGCGAA Lat TTGCGTCCACAGGATTCCGCTCGAGTCCCTCCTGTCTTTGGAGGGCACCTCGTCTTCCCGTTTTCGAGCGAA

39160 39180 40100 Mus CTAGAACTAAAAGTCATGCTTATGTCTGGCACTTTCG--CCCCGGAGTGCTAGGAAGACTGGAAAACCCAAA Xen CTAGAACTAAAAGTCATACTTATGTCTGGCACTTTCG--CCCCGGAGTGCTAGGAAGACTGAAAAACCCAAA Spe CTAGAACTAAAAGTCATACTTATGTTTGGCACTTTTG--GCCCGGAGTGGTAGGAAGAATGCAAAACCCAAA Ran CTAGAACTAAAAGTCATACTTATGTCTGGCACTTT-GCGCCCCGGAGTGCTAGGAAGACTGAAAAACCCAAA Gas CTAGAACTAAAAGTCATACTTATGTCTGGCACTTTCGCCCCNNGGAGTGCTAGGAAGACTGAAAAACCCAAA Lei CTAGAACTAAAAGTCATACTTATGTCTGGCACTTTCG--CCCCGGAGTGCTAGGAAGACTGAAAAACCCAAA Nes CTAGAACTAAAAGTCATACTTATGTCTGGCACTTTCG--CCCCGGAGTGCTAGGAAGACTGAAAAACCCAAA Den CTAGAACTAAAAGTCATACTTATGTCTGGCACTTTCG--CCCCGGANTGCTAGGAAGACTGNAAAACCCAAA Cer CTAGAACTAAAAGTCATACTTATGTCTGGCACTTTCG--CCCCGGAGTGCTAGGAAGACTGGAAAACCCAAA Lat CTAGAACTAAAAGTCATACTCATGTCTGGCACTTTCGCGCCCCGGAGTGCTAGGAAGACTGAAAAACCCAAA

40120 40140 40160 40180 Mus ATTCGTCCTCCACAGTCTTTTCAATGGTGTCCCTATTGACCGAACACCGCCGGTTCGCAAGTATCGCTGCAG Xen ATTCGTCCTCCACAGTCTTTTCAATGGTGTCCCTATTGACCGAACACCGCCGGTTCGCAAGTATCGCTGCAG Spe ATTTGTCCTTCACAGTCTTTTCAATGGTGTCCCTATTGACCGAACACCGCCGGTTCGCAAGTATCGNNNNNN Ran ATTCGTCCTCCACAGTCTTTTCAATGGTGTCCCTATTGACCGAACACCGCCGGTTCGCAAGTATCGCTGCAG Gas ATTCGTCCTCCACAGTCNNNNNNNNNNNNCCGAACACCGCCGGTTCGCAAGTATCGCTGCAG Lei ATTCGTCCTCCACAGTGTTTTCAATGGTGTCCCTATTGACCGAACACCGCCGGTTCGCAAGTATCGCTGCAG Nes ATTCGTCCTCCACAGTCTTTTCAATGGTGTCCCTATTGACCGAACACCGNNNNNNNGCAAGTATNNNNNNNN Den ATTCGTCCTCCACAGTCTTTTCAATGGTGTCCCTATTGACCGAACACCGCCGGTTCGCAAGTATCGCTGCAG Cer ATTCGTCCTCCACAGTCTTTTCAATGGTGTCCCTATTGACCGAACACCGCCGGTTCGCAAGTATCGCTGCAG Lat ATTCGTCCTCCACAGTCTTTTCAATGGTGTCCCTATTGACCGAACACCGCCGGTTCGCAAGTATCGCTGCAG

FIG. 1.-Continued. 1993] HERPETOLOGICAL MONOGRAPHS 127

41100 41120 Mus CGAAAAACTAGGAAGCTACAGCCGAGAAGGATAGTAACACTTCGTCTTAAG Xen CGAAAAACTAGGAAGCTACAGCCGAGAAGGATAGTAACACTTCGTCTTAAG Spe NNN NNN NNN Ran CGAAAAACTAGGAAGCTACAGCCGAGAAGGATAGTAACACTTCNTCTTAAG Gas CGAAAAACTAGGAAGCTACAGCCGAGAAGGATAGTAACACTTCGTCTTAAG Lei CGAAAAACTAGGAAGCTACAGCCGAGAAGGATAGTAACACTTCGTCTTAAG Nes NNAAAAACTAGGAAGCTACAGNNNAGAAGGNTAGTAACACTTCGTCTTAAG Den CGAAAAACTAGGAAGCTACAGCCGAGAAGGATAGTAACACTTCGTCTTAAG Cer CGAAAAACTAGGAAGCTACAGCCGAGAAGGATAGTAACACTTCGTCTTAAG Lat CGAAAAACTAGGAAGCTACAGCCGAGAAGGATAGTAACACTTCGTCTTAAG

FIG. 1.-Continued. the additional presence of phylogenetic DISCUSSION signal among the frogs. The data matrix The clade of that is continued to show only higher frogs significant structure as by both the morpho- the next four brancheswere strongly supported resolved:these and molecular data sets is Pipanura. branches for the logical provided support mono- The morphologicaland our 28S rDNA data phyly of the pipanurans, the two meso- therefore that Archaeobatrachia the ranid agree batrachians, plus the two me- (sensu Duellman, 1975) is not monophy- and the sobatrachians, hylid plus sooglossid. letic, in contrast to the analysis of Of the six Hedges commonly recognized higher and Maxson (1993). There is also support groups of frogs described in the introduc- of the Mesobatrachia from both the 28S our of 28S rDNA tion, analysis provided data and some morphologicalstudies (e.g., independent support for three taxa: Me- Cannatella, 1985; Ford and Cannatella, sobatrachia, Pipanura, and Hyloidea. In 1993), other with recent although morphological (e.g., agreement morphological the data of Duellman and Trueb, 1986) analyses (see Ford and Cannatella, 1993), and molecular and Maxson, our data that Archaeobatrachia (e.g., Hedges suggest 1993) studies do not support this group. is not (sensu Duellman, 1975) monophy- Other comparisons between morpho- letic. our data also do not However, sup- logical and molecular studies show little some of the that are port groups supported in the relationships among Neo- agreement by morphological analyses, namely families. The Hyloidea has no known sup- batrachiaand Ranoidea Dendrobatidae (if port from morphology, and yet appears to is included in the latter group). The morphological data of Duellman 41 and Trueb (1986) conflict with our results MUs by supporting Neobatrachia and Ranoi- 1 3-16 Xenopus dea, but not Mesobatrachia or Hyloidea 7-14 53-60 The tree is 12 Spea (Fig. 3). morphological steps 23-35 Rana long and has a consistency index of 0.917. 1 Gastrotheca The two data sets agree that Pipanura is 5-219 318326 6 Nesomantis monophyletic and that Archaeobatrachia 14-28 J 13-19 Ceratophrys is not. A combined analysis of our molec- 28-31 ular data and the 0141 Allobates corresponding morpho- 1626 logical data from Duellman and Trueb Leiopelma (1986) produces a single most parsimoni- 39-53 Latimeria ous tree that supports the monophyly of FIG. 2.-One of three most parsimonious trees for Pipanura, Mesobatrachia, Neobatrachia, the 28S rDNA data. The two arrows indicate the and but not Archaeobatrachia alternative placement of Allobates in the other two Hyloidea, trees. The or Ranoidea 3). This tree is 395 numbers along the branches show the min- (Fig. steps imum and maximum number of changes that can long and has a consistency index (exclud- occur across all most parsimonious character recon- ing uninformative characters) of 0.797. structions. 128 HERPETOLOGICAL MONOGRAPHS [No. 7

Pipanura receive some support from the ribosomal "Archaeobatrachia" Neobatrachia genes. Hedges and Maxson (1993) also found some for a modified "Mesobatrachia" "Hyloidea" Ranoidea support Hy- I , . I loidea (their Bufonoidea), but only if Rha- cophoridae (which has all the morphological synapomorphies of Ranoidea) was included in the group. Neither the 12S nor the 28S rDNA data support the inclusion of Dendrobatidae in the Ranoidea. If dendrobatids are considered ranoids, then ranoids are either paraphyletic or polyphy- letic in our analysis, and in the tree of Hedges and Maxson (1993) Dendrobatidae is embedded within the hyloids. If we limit our analysis to the frog taxa, the shortest Pipanura tree that would place ranids and dendro- "Archaeobatrachia" "Neobatrachia" I I batids together would require six additional steps (although there are fewer steps between the alternatives if various com- binations of outgroup taxa are added). Morphologists have long disagreed about the relationships of this family, and have been divided about whether or not Den- drobatidae belonged with ranoids (e.g., Duellman and Trueb, 1986; Ford, 1989; Ford and Cannatella, 1993; Griffiths, 1963) or hyloids (Laurent, 1979, 1986; Lynch, 1971, 1973; Noble, 1922, 1931). We see and 4-17 the molecular data (i.e., this paper Pipanura Hedges and Maxson, 1993) as too weak to "Archaeobatrachia" Neobatrachia resolve this controversy satisfactorily, al- do some for Mesobatrachia "Ranoidea" Hyloidea though they provide support f I a relationship of dendrobatids. 0 I . . m hyloid a 'O Perhaps the most surprising relationship IE m E suggested by the 28S rDNA data is the 0o 2 -o a 70 ._ M 0 -0 0E connection between the ranid and the me- 0 I0 0 a. >. 0 -J d: sobatrachians, which suggests that Neo- batrachia (as usually recognized) is not monophyletic. For the 28S rDNA data, the shortest ingroup tree that contains a monophyletic group of taxa that are currently considered to be neobatrachians is 6 steps longer than the most parsimonious tree. (c) v Of if the tree shown in 2 is 6-25 course, Fig. correct and the phylogenetic definitions of FIG. 3.-Comparison and combination of molec- and Ranoidea ular and Neobatrachia, Pipanura, (28S rDNA) morphological (from Duellman used Ford and Cannatella are and Trueb, 1986) data sets. The two outgroup taxa by (1993) in the molecular study were used to root the trees followed, then all three of these names but are not shown here for the sake of simplicity. For would be synonyms. Figure 2 also suggests the morphological data, the outgroups were scored that the possibility of firmisterny as the as having all ancestral states. The content of the six ancestral condition of the major groups of frogs follows common usage, and pipanuran pec- non-monophyletic groups on each tree are enclosed toral girdle should be given consideration. in quotation marks. However, the monophyly of Neobatrachia 1993] HERPETOLOGICAL MONOGRAPHS 129 appears to be well supported by morpho- are encouraged by the level of indepen- logical synapomorphies (Ford and Can- dent support by the 28S rDNA data for natella, 1993), and also is the group most some groups that were suggested originally strongly supported by 18S rDNA sequenc- by morphological studies, and we expect es (Hedges and Maxson, 1993). that a continued parallel development of As can be seen in Fig. 3, our tree is morphological and molecular studies considerably different from the tree based eventually will result in a well supported on earlier morphological data. For the in- phylogenetic hypothesis for frogs. group taxa, the morphological tree would require 19 additional steps to explain the Acknowledgments. -We thank David Cannatella 28S rDNA data to the most and John Wiens for commenting on the manuscript. compared par- Ronald Nussbaum and David Green provided some simonioustree. In cases of conflict between of the tissues for this study. This work was supported multiple data sets, one option is to combine by NSF grant BSR 8796293. This paper was derived the data in a joint analysis (Hillis, 1987; from our presentation in the symposium "Amphibian 1989; 1985). relationships: Phylogenetic analysis of morphology Kluge, Miyamoto, Minimally, and molecules" at the 1990 meeting of the American this permits discovery of which data set Society of Zoologists in San Antonio, Texas. shows the strongest support for its respec- tive conclusions.It is also possiblethat weak LITERATURE CITED but compatible signal in the two data sets historical AJUH, P. M., P. A. HEENEY, B. E. MADEN, AND H. will reveal underlying patterns EDWARD. 1991. Xenopus borealis and Xenopus where none was visible in the separate laevis 28S ribosomal DNA and its complete 40S analyses (Barrett et al., 1991). However, ribosomal precursor RNA coding units of both spe- there is also the possibility that a noisy, cies. Proc. Roy. Soc. London, B, Biol. Sci. 245:65- data set will overwhelm the 71. misleading BARRETT, M., M. J. DONOGHUE, AND E. SOBER. 1991. phylogenetic signal in an otherwise infor- Against consensus. Syst. Zool. 40:486-493. mative data set. Despite these limitations, CANNATELLA, D. C. 1985. A Phylogeny of Primi- we believe the results from the combina- tive Frogs (Archaeobatrachians). Ph.D. Disserta- tion of the and 28S rDNA tion, The University of Kansas, Lawrence, Kansas. morphological DIXON, M. T., AND D. M. HILLIS. 1993. Ribosomal data sets are revealing (Fig. 3). The com- RNA secondary structure: Compensatory muta- bined analysis shows elements of both the tions and implications for phylogenetic analysis. molecular and morphological trees, and is Mol. Biol. Evol. 10:256-267. nearly consistent with the classification DUBOIS,A. 1984. La nomenclature supragenerique Ford and Cannatella des amphibiens anoures. Mem. Museum Nat. d'His- proposed by (1993). toire Naturelle, Ser. A 131:1-64. The only deviations are that this tree pro- 1986. Miscellanea taxinomica [sic] batra- vides support for the monophyly of the chologica (I). Alytes 5:7-95. taxa (which Ford and Cannatella DUELLMAN, W. E. 1975. On the classificationof hyloid Mus. 42: consideredto be paraphyletic),and the two frogs. Occ. Pap. Nat. Hist. Univ. Kansas to be 1-15. included "ranoids"appear paraphy- DUELLMAN,W. E., AND L. TRUEB. 1986. Biology letic. If this tree accurately reflects the of Amphibians. McGraw-Hill Book Co., New York. phylogenetic history of frogs, then it sug- FELSENSTEIN, J. 1985. Confidence limits on phy- gests that firmisterny could be ancestral in logenies: An approach using the bootstrap. Evolu- Neobatrachia. a tree that unites tion 39:783-791. However, FORD, L. S. 1989. The Phylogenetic Position of Rana and Allobates is only three steps away Poison-dart Frogs (Dendrobatidae): Reassessment from the shortest tree in this analysis, so of the Neobatrachian Phylogeny with Commen- the additional synapomorphies of Ra- tary on Complex Character Systems. Ph.D. Disser- noidea discussed Ford and Cannatella tation, The University of Kansas, Lawrence, Kan- by sas. (1993) are probably sufficient to support In press. The phylogenetic position of the the monophyly of this group. dart-poison frogs (Dendrobatidae) among anurans: Obviously, an expansion of the 28S An examination of the competing hypotheses and rDNA data set to include additional taxa their characters. Ecol. 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HerpetologicalMonographs, 7, 1993, 131-159 ? 1993 by The Herpetologists'League, Inc.

SYSTEMATIC STUDIES OF THE COSTA RICAN MOSS SALAMANDERS, GENUS NOTOTRITON, WITH DESCRIPTIONS OF THREE NEW SPECIES

DAVID A. GOOD1 AND DAVID B. WAKE Museum of Vertebrate Zoology and Department of Integrative Biology, University of California, Berkeley, CA 94720, USA

ABSTRACT: Study of allozyme variation, external morphology, and osteology reveals that there are more species of moss salamanders (genus Nototriton) in Costa Rica than the two currently recognized. The three species for which names are available are valid, and new diagnoses are presented for them; three additional species are described. The phylogenetic relationships and biogeography of the six species are investigated. The radiation of Nototriton in present-day Costa Rica has involved miniaturization accompanied by both morphological and ecological specialization. Costa Rican species inhabit moss-mats and leaf-litter; most of the remaining species in the genus are bromeliad-dwellers. The revised genus Nototriton includes two Mexican, one Guatemalan (another, detected in the present study, remains undescribed), two Honduran, and six Costa Rican species. The six Costa Rican species appear to form a monophyletic group, but the phylogenetic relationships of the two northern species groups to each other and to the southern group remain uncertain.

Key words: Salamanders; Plethodontidae; Nototriton; Costa Rica; Allozymes; Morphometrics; Systematics; New species

SALAMANDERSof the genus Nototriton Rica), they can be found easily, but char- (commonly known as moss salamanders) acteristically they are uncommon. Even are inconspicuous components of cloud species that have been known taxonomi- forest faunas from Oaxaca, Mexico, to cen- cally for more than 40 years (e.g., N. ri- tral Costa Rica. Most of the species occur chardi) are represented by fewer than 25 in moss mats hanging in trees or bushes, specimens in the museums of the world. or in moss covering dirt banks, large boul- Typically, species of Nototriton are small; ders, or stumps. Others inhabit bromeliads. none exceeds 40 mm in snout-vent length In a few places (such as on the northeastern and several species are not known to ex- slopes of the Cordillera Central in Costa ceed 30 mm. These salamanders have slen- der bodies, narrow heads, and long, ta- tails that exceed their snout-vent PRESENT ADDRESS:Museum of Natural Science, pering 119 Foster Hall, Louisiana State University, Baton length. Their eyes are small and oriented Rouge, LA 70803, USA. anteriorly, and several of the species have