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A Molecular Phylogeny of the Australian Skink Genera Eulamprus, Gnypetoscincus and Nangura

A Molecular Phylogeny of the Australian Skink Genera Eulamprus, Gnypetoscincus and Nangura

CSIRO PUBLISHING www.publish.csiro.au/journals/ajz Australian Journal of Zoology, 2003, 51, 317–330

A molecular phylogeny of the Australian genera , Gnypetoscincus and

D. O’ConnorA,B,D and C. MoritzA,C

ACooperative Research Centre for Tropical Ecology and Management, Department of Zoology and Entomology, University of Queensland, Qld 4067, . BPresent address: School of Biological Sciences, University of Sydney, NSW 2006, Australia. CPresent address: Museum of Vertebrate Zoology, University of California, Berkeley, CA 94720-3160, USA. DTo whom correspondence should be addressed. Email: [email protected]

Abstract from the genera Eulamprus, Gnypetoscincus and Nangura are a prominent component of the fauna of the mesic forests of the east coast of Australia and have been the subject of numerous ecological studies. Highly conserved morphology and the retention of ancestral traits have limited our understanding of the relationships within and among these genera beyond an initial identification of groups within Eulamprus. To address this deficit and to explore the relationships between Eulamprus and the monotypic genera Nangura and Gnypetoscincus, sections of two mitochondrial genes (ND4 and 16S rRNA) were sequenced and subjected to Bayesian phylogenetic analysis. This phylogenetic analysis supports recognition of the three species groups proposed for Eulamprus (murrayi, quoyii and tenuis) and indicates that this is paraphyletic, with Gnypetoscincus and Nangura being proximal to basal lineages of the tenuis group. To resolve these and broader problems of paraphyly, we suggest that each of the species groups from ‘Eulamprus’ should be recognised as a distinct genus. The phylogenetically and ecologically distinct water skinks of the quoyii group would be retained within Eulamprus and the diverse species of the tenuis group allocated to . We suggest placing the monophyletic murrayi group, endemic to the of central eastern Australia, in a new genus (yet to be formally described). The sequencing data also revealed the existence of a genetically divergent but morphologically cryptic lineage within E. murrayi and substantial diversity within E. quoyii. There is evidence for two major shifts from rainforest towards drier , one leading to the quoyii group and the second defining a of three species within the tenuis complex. These ecological transitions may represent adaptations to general drying across eastern Australia during the late Miocene–Pliocene. Each of the major areas of east coast tropical or subtropical rainforest contains multiple phylogenetically diverse endemic species, reflecting the long-term persistence and high conservation value of wet forest habitats in each area.

ZO02050 PhyD. logO’Coenetnnorics andand C. evol Mutorionitz of Eulamprus

Introduction The Australian genus Eulamprus is part of the lineage of lygosomine skinks and its members are characterised by relatively large body size, fully developed limbs and viviparous reproduction (Greer 1989, 1992). Members of the genus occur in mesic habitats along the east coast of Australia and several species, especially those in rainforests, have very restricted distributions (Fig. 1, Table 1). Greer (1989) split the genus into three species groupings based on morphological characters, the tenuis, quoyii and murrayi groups, although the first of these, like the genus as a whole, was not defined by any derived trait and was not proposed to be monophyletic. With subsequent taxonomic revisions (Greer 1992; Hutchinson and Rawlinson 1995; Sadlier 1998) seven species are recognised within the tenuis complex, three within the murrayi complex and five within the quoyii group (Table 1). Each group has a broad geographic range across eastern Australia, yet each also includes one or more species with very narrow geographic ranges (Fig. 1).

© CSIRO 2003 10.1071/ZO02050 0004-959X/03/040317 318 Aust. J. Zoology D. O’Connor and C. Moritz a E. sokosoma E. brachysom E. murrayi E. tryoni E. sp. E. luteilateralis Eulamprus murrayi species group E. tenuis E. martini E. amplus E. frerei E. tigrinus Eulamprus tenuis species group Nangura Gnypetoscincus Gnypetoscincus and Nangura E. heatwolei E. leuraensis species groups (from Cogger 2000). Eulamprus South east Australia Mid-east Queensland E. heatwolei E. quoyii E. kosciuskoi E. tympanum Wet Tropics Tropical and subtropical rainforests and eachthe of Cape York Nangura , species group Map of mainland eastern Australia showing distributions of tropical and subtropical rainforest (adapted from Adam 1992), Adam from (adapted rainforest subtropical and tropical of distributions showing Australia eastern mainland of Map Gnypetoscincus Fig. 1. Fig. and evolution of Eulamprus Aust. J. Zoology 319

Table 1. Species groupings proposed by Greer (1989) and habitat characteristics of these species as well as Nangura spinosa and Gnypetoscincus queenslandiae (as per Cogger 2000)

Species Habitat E. tenuis group E. amplus Among rocks or at the base of trees beside steams in rainforest E. brachysoma Rocky areas in rainforest, sclerophyll forests and sub-tropical woodlands E. frerei Granite tors and boulders in high montane rainforest E. martini Clearings in rainforests and vine thickets, moister sclerophyll forests and woodlands E. sokosoma Dry rainforest, vine thickets, moist sclerophyll forests and sub-tropical woodlands E. tenuis Rock slopes primarily in rainforest and wet and dry sclerophyll forests E. tigrinus Rainforest E. murrayi group E. luteilateralis Litter and logs in montane rainforest E. murrayi Rainforest and wet sclerophyll forests E. tryoni Rainforest and wet sclerophyll forests E. sp. Rainforest and wet sclerophyll forests (pers. obs.) E. quoyii group E. heatwolei Open upland forests and grasslands. Swamps, lagoons and creeks E. kosciuskoi Beside creeks in montane forests and woodlands and in alpine bogs and marshes E. leuraensis Riparian and swampy areas in montane forests and heaths E. quoyii Most wet habitats in a wide variety of vegetation types E. tympanum Rocks and logs along small creeks N. spinosa Creek banks in seasonally dry rainforest G. queenslandiae Logs in rainforest

Due to the paucity of informative morphological variation there has not been any further refinement of phylogenetic relationships among these species. Not only are the relationships within the genus currently unresolved, but there also remains uncertainty about the relationships of the monotypic genera Nangura and Gnypetoscincus to each other and to Eulamprus. Gnypetoscincus queenslandiae may be closely related to Eulamprus on the basis of its well developed limbs, viviparity, east coast distribution and chin scalation (Greer 1989). G. queenslandiae also has inguinal fat bodies, a character which, within the broader Sphenomorphus lineage of skinks, is known only in the tenuis group and three species of Glaphyromorphus. From morphological analysis, Nangura spinosa appears to be a primitive member of the Sphenomorphus group but differs significantly in two characters (Covacevich et al. 1993). Its karyotype is 2n = 28 whilst all genera tested within the present Sphenomorphus grouping have 2n = 30. The parietal scales of N. spinosa are totally separated by the interparietal, a character state otherwise found only in Eulamprus quoyii. However, N. spinosa lacks a postorbital bone, a character state present in Eulamprus. The species’ keeled scales are unusual and resemble those of G. queenslandiae. Whilst the current evidence supports N. spinosa being placed in the Sphenomorphus lineage, it is still uncertain what its relationships are with other members of the group and, given several unique traits, it is possible that the genus represents a relatively ancient divergence. In order to gain a better understanding of relationships within Eulamprus and to explore the phylogenetic affinities of Nangura and Gnypetoscincus, we have generated mtDNA 320 Aust. J. Zoology D. O’Connor and C. Moritz

sequence data and from these estimated a phylogeny. This is intended to generate a broad framework for interpreting historical biogeography and speciation within the genus and the habitats they occupy (e.g. Moritz et al. 2000; Stuart-Fox et al. 2001), as well as a template for interpreting differences in ecology and life history (e.g. Daniels et al. 1987; Daniels and Heatwole 1990; Schwarzkopf and Shine 1991; Schwarzkopf 1993, 1996, 1998; Shine and Harlow 1993; Doughty and Shine 1997, 1998; Sumner et al. 1999, 2001).

Materials and Methods Where available, two samples of each of the 12 currently recognised Eulamprus species were sequenced, covering as broad a geographic range as possible (see Appendix for details). In addition, Nangura spinosa, Gnypetoscincus queenslandiae and two outgroup species, Egernia frerei and rawlinsoni, were sequenced. E. frerei represented the phylogenetically distinct Egernia group and C. rawlinsoni a divergent lineage within the Sphenomorphus group (Greer 1989). DNA was extracted following a standard chelex/proteinaseK extraction, amplified following standard PCR protocols and sequenced using ABI protocols. A 540-bp region of the 16S rRNA mitochondrial gene was amplified using the primers 16SaR (5′-CGCCTGTTTATCAAAAACAT-3′) and 16SbR (5′-TGCACTAGACTCAAGTCTGGCC-3′) (Palumbi 1996). Due to amplification difficulties, the Eulamprus amplus 16S rRNA sequence was supplied by T. Reeder. A 954-bp region of the mitochondrial ND4 gene was amplified using the primers ND4 (5′-TGACTACCAAAGCTCATGTAGAAGC-3′; Bovine 11173–11196, modified from Arevalo et al. 1994) and tLEU2 (5′-TTTTACTTGGATTTGCACCA-3′; Bovine 12086–12105, modified from the Arevalo et al. 1994 LEU primer). Of this region, the first 400 bp from the ND4 end was sequenced in all taxa and used to estimate the phylogeny. Sequences were aligned in ClustalX (Higgins et al. 1992). Alignments were manually checked, with reference to reading frames for ND4. The final ND4 alignment was translated to check for stop codons and frameshift mutations. The aligned sequences were analysed phylogenetically using a combination of methods (likelihood, parsimony) with emphasis on the MCMC-Bayesian approach (Huelsenbeck et al. 2001; reviewed in Holder and Lewis 2003) implemented in the program Mr Bayes (Huelsenbeck and Ronquist 2001). For this analysis, the model of sequence evolution (GTR + Γ) was selected using Mr ModelTEST Ver. 3.06 (Posada and Crandall 1998). We conducted two independent analyses to check for consistency of results, each with multiple (n = 4) chains, a random starting tree and 2000000 generations, with trees sampled every 100 generations to estimate likelihood and sequence evolution parameters. Inspection of the output files indicated that likelihood and other parameters reached asymptotes well before 100000 generations (1000 trees); accordingly, we base our inference on a consensus of the 19000 trees sampled after this burn-in period. As a complement to the Bayesian analysis, we also conducted parsimony searches with weighting of transversions over transitions (10:1) or exclusion of 3rd codon positions within the ND4 sequence, all with 1000 bootstrap pseudoreplicates. Phylogenetic hypotheses were compared using likelihood ratio tests, specifically the Shimodaira and Hasagawa (SH) test which is appropriate for testing the maximum- likelihood tree against alternatives (Goldman et al. 2000). These analyses were performed using PAUP 4.0b8 (Swofford 2001). All sequences have been deposited in GenBank (accession numbers AF530191–AF530264).

Results Sequence evolution and variation Of the 400 ND4 sites aligned, 213 were variable and 176 were phylogenetically informative. No stop codons were observed when the sequence was protein translated. For 16S rRNA, 499 sites were aligned with 149 variable sites, of which 106 were parsimony informative. Comparing the base compositions of the taxa revealed them as being adenosine rich with adenosine constituting 33% of the nucleotides. Percentages of cytosine (26%) and thymine (24%) were approximately equal, with guanine (17%) being the least represented. Rates of substitution estimated from the Bayesian analysis indicate an 8–10-fold excess of transitions over transversions. These parameters are typical for mtDNA (Moritz et al. Phylogenetics and evolution of Eulamprus Aust. J. Zoology 321

1987). Most nucleotide changes occur at the third codon position (15%, 6.5% and 31.5% of sites variable for 1st, 2nd and 3rd positions respectively) and the Bayesian estimate of the overall shape parameter for the gamma distribution was α = 0.23. Levels of DNA sequence divergence (model: GTR + Γ; α = 0.23) between species ranged from 3.3% (ND4 7.1%; 16S 1.0%; between E. sokosoma and E. brachysoma) to >20% in most pairwise comparisons (Table 2). Between the quoyii and tenuis species groupings, as defined by Greer (1989) (see Table 1), the average level of divergence was 26.6% (ND4 55.4%; 16S 17.5%). This compares with 13.6% (ND4 40.0%; 16S 6.2%) within the tenuis group, 12.2% (ND4 44.9%; 16S 5.8%) within the murrayi group and 13.0% (ND4 28.4%; 16S 9.2%) within the quoyii group. G. queenslandiae and N. spinosa differed from Eulamprus species by a minimum of 13.3% (ND4 36.8%; 16S 6.7%; E. murrayi) and 13.0% (ND4 40.3%; 16S 5.6%; E. tenuis), respectively. Two species were found to include individuals with highly divergent mtDNAs. Samples from Cambridge Plateau in north-eastern New South Wales identified as E. murrayi in the field were 10.5% (ND4 23.0%; 16S 4.3%) divergent from E. murrayi and 15% (ND4 55.5%; 16S 4.2%) from E. tryoni. This contrasts markedly with otherwise low sequence divergence across the range of E. murrayi (average of 1.3% for ND4 and cytochrome b combined, unpublished data). Here we refer to this population as Eulamprus spp. pending formal description. The second example concerns the northern-most sample of E. quoyii that was >20% divergent from other samples of this species, again suggesting the possibility of cryptic lineages within this widespread taxon.

Phylogenetic analyses The majority-rule consensus across the 19000 trees sampled from the Bayesian analysis (subsequent to the burn-in period) revealed several strongly supported (Fig. 2). Two major species groupings suggested by Greer (1989), the quoyii and murrayi clades, are clearly defined in the present molecular analysis (100% and 99%, respectively). The quoyii clade forms a sister group to the remaining species in the analysis, the closer relationship between the murrayi and tenuis groups being firmly inferred from the data (97%). Within the quoyii clade the analysis indicates close affinity between E. kosciuskoi and E. leuraensis, and of E. quoyii to these; otherwise, the relationships among species within this group remain obscure. Within the murrayi clade, the new taxon from Richmond Range (Eulamprus sp.) and E. murrayi are identified as sister taxa and there is some indication (83%) that E. tryoni, the third species endemic to this region, is more closely related to these than is E. luteilateralis. Each of the relationships described above, except for (quoyii (kosciuskoi, leuraensis)), were also present in the parsimony analyses, though often with lower support levels, as indicated by bootstrapping. The remaining species – all members of Greer’s tenuis group along with the monotypic genera Gnypetoscincus and Nangura – form a single clade, though with slightly less (91%) support. Within this third clade, there is strong support (98%) for grouping of tigrinus, brachysoma, sokosoma and martini, and 100% support for a subclade of the last three species. There is reasonable confidence (91–92%) for inclusion of two other members of the tenuis complex (E. tenuis, E. amplus) in this lineage, though because of uncertainty about the position of E. frerei, it appears paraphyletic with Nangura and possibly Gnypetoscincus as well. Overall, there is a tendency for relationships among the more basal elements of this clade (Gnypetoscincus, Nangura, E. frerei, and E. amplus) to be less clearly resolved. The same trends are evident in parsimony analyses – the majority-rule consensus supports the topology (tenuis (tigrinus (martini (sokosoma, brachysoma)))), but 322 Aust. J. Zoology D. O’Connor and C. Moritz n4 Etym2 Equo1 Equo2 Equo3 Ehea1 Ehea2 Ekos3 Eleu2 Eleu3 Craw 29 0.247 0.226 0.284 0.265 0.262 0.238 0.238 0.224 0.269 0.265 022 206 0.193 304 0.301211 0.148 0.210230 0.124 0.225233 0.179 0.137 0.235238 0.180 0.086 0.240 0.052 264 0.159 0.090 0.255 0.143244 0.170 0.144 0.113 0.247 0.150244 0.154 0.151 0.122 0.246 0.141 0.011 0.167 0.128 0.121 0.162302 0.131 0.167 0.175 0.314 0.136 0.162 0.123 0.174 0.299 0.136 0.123 0.312 0.090 0.134 0.351 0.329 0.091 0.301 0.000 0.325 0.313 0.342 0.344 0.282 = 0.23)across 899 bp of ND4andgenes 16SrRNA Γ Table 2.Table Estimatessequence of divergence (GTR + Emar1 Emar3 Etig1 Etig2 Esok1 Esok2 Ebra2 Ebra3 Ebra4 Eamp Nspi1 Nspi2 Efre1 Efre2 Etry1 Etry2 Espp1 Espp2 Emur1 Emur2 Gqld2 Gqld3 Elut1 Elut2 Eten3 Ete Emar1 Emar3Etig1 0.015 Etig2 0.125Esok1 0.116 0.125Esok2 0.111 0.098Ebra2 0.005 0.086 0.089 0.147Ebra3 0.076 0.142 0.104 0.136Ebra4 0.096 0.132 0.122Eamp 0.156 0.002 0.109 0.151 0.111Nspi1 0.147 0.033 0.099 0.139 0.179Nspi2 0.028 0.132 0.035 0.177 0.124 0.167Efre1 0.033 0.189 0.029 0.172 0.146 0.046 0.185Efre2 0.180 0.025 0.143 0.164 0.176 0.036 0.198Etry1 0.149 0.154 0.189 0.006 0.190 0.150 0.198 0.196 0.173Etry2 0.216 0.173 0.202 0.184 0.199 0.153 0.217 0.168Espp1 0.183 0.227 0.191 0.145 0.167 0.177 0.163 0.218Espp2 0.165 0.168 0.163 0.158 0.156 0.173 0.189 0.163 0.171 0.189Emur1 0.145 0.188 0.169 0.211 0.187 0.168 0.192 0.169 0.191 0.195 0.196Emur2 0.182 0.160 0.213 0.030 0.194 0.207 0.172 0.204 0.182 0.178 0.199Gqld2 0.183 0.200 0.163 0.237 0.216 0.196 0.158 0.207 0.168 0.205 0.183 0.230Gqld3 0.203 0.176 0.139 0.239 0.172 0.213 0.177 0.172 0.253 0.185 0.135 0.232Elut1 0.200 0.197 0.163 0.164 0.198 0.188 0.152 0.160 0.256 0.198 0.152 0.017 0.149 0.186Elut2 0.211 0.168 0.154 0.200 0.200 0.168 0.147 0.191 0.208 0.169 0.212 0.174 0.189Eten3 0.187 0.166 0.180 0.208 0.206 0.173 0.210 0.166 0.193 0.173 0.178 0.191 0.177 0.186Eten4 0.197 0.214 0.175 0.211 0.173 0.182 0.188 0.179 0.188 0.146 0.181 0.215 0.168 0.189Etym2 0.180 0.005 0.188 0.157 0.161 0.143 0.165 0.207 0.182 0.183 0.130 0.165 0.188 0.149 0.136Equo1 0.159 0.191 0.198 0.170 0.164 0.128 0.156 0.229 0.194 0.160 0.142 0.153 0.172 0.225 0.158 0.138Equo2 0.156 0.147 0.234 0.193 0.162 0.138 0.172 0.282 0.213 0.174 0.144 0.150 0.101 0.271 0.214 0.158Equo3 0.129 0.162 0.285 0.194 0.167 0.001 0.145 0.101 0.274 0.266 0.185 0.142 0.158 0.105 0.312 0.189 0.155Ehea1 0.136 0.101 0.235 0.272 0.175 0.143 0.152 0.104 0.319 0.269 0.175 0.172 0.159 0.103 0.228 0.273 0.160 0.148Ehea2 0.133 0.105 0.308 0.270 0.185 0.179 0.156 0.233 0.271 0.172 0.214 0.138 0.193 0.106 0.307 0.290 0.233 0.147Ekos3 0.137 0.248 0.292 0.187 0.216 0.151 0.150 0.009 0.291 0.325 0.226 0.236 0.146 0.189 0.234 0.278 0.211 0.268 0.153Eleu2 0.129 0.262 0.317 0.155 0.234 0.156 0.160 0.216 0.318 0.206 0.262 0.275 0.115 0.195 0.245 0.306 0.169 0.273 0.158Eleu3 0.132 0.219 0.283 0.150 0.270 0.264 0.120 0.213 0.359 0.271 0.197 0.268 0.140 0.260 0.182 0.177 0.261 0.147 0.262 0.256Craw 0.193 0.257 0.275 0.199 0.284 0.157 0.281 0.197 0.235 0.313 0.181 0.255 0.252 0.260 0.206 0.215 0.257 0.158 0.283 0.060 Erfre 0.246 0.204 0.237 0.252 0.183 0.284 0.263 0.282 0.243 0.310 0.284 0.170 0.272 0.247 0.202 0.218 0.206 0.215 0.144 0.276 0.243 0.249 0.246 0.239 0.324 0.150 0.273 0.253 0.239 0.220 0.280 0.224 0.155 0.271 0.281 0.247 0.194 0.196 0.330 0.139 0.253 0.249 0.239 0.241 0.231 0.281 0.240 0.146 0.220 0.248 0.247 0.191 0.267 0.359 0.292 0.268 0.284 0.191 0.234 0.290 0.243 0.131 0.200 0.230 0.239 0.189 0.354 0.281 0.229 0.263 0.242 0.198 0.214 0.306 0.253 0.003 0.237 0.269 0.285 0.188 0.357 0.269 0.210 0.269 0.228 0.168 0.213 0.319 0.253 0.234 0.235 0.239 0.183 0.323 0.294 0.251 0.295 0.242 0.180 0.199 0.323 0.274 0.201 0.209 0.228 0.188 0.310 0.342 0.247 0.286 0.261 0.168 0.191 0.246 0.235 0.245 0.221 0.242 0.314 0.276 0.224 0.298 0.232 0.169 0.227 0.265 0.225 0.258 0.242 0.261 0.268 0.247 0.228 0.281 0.285 0. 0.224 0.251 0.265 0.261 0.274 0.233 0.313 0.266 0.265 0.261 0.288 0. 0.272 0.209 0.206 0.268 0.285 0.289 0.246 0.268 0.270 0.262 0.297 0.220 0.234 0.208 0.289 0. 0.258 0.214 0.258 0.284 0.264 0.245 0.237 0.246 0.262 0.286 0.213 0.220 0.250 0. 0.245 0.212 0.242 0.284 0.253 0.248 0.283 0.253 0.188 0.269 0.227 0.246 0. 0.261 0.289 0.213 0.180 0.273 0.256 0.249 0.290 0.281 0.217 0.235 0. 0.241 0.213 0.287 0.291 0.250 0.238 0.261 0.217 0. 0.276 0.235 0.239 0.264 0.249 0.279 0.249 0.234 0. 0.236 0.326 0.249 0.223 0.253 0.321 0. 0.214 0.249 0.319 0.236 0. 0.249 0.243 0.288 0.2 0.288 0. Phylogenetics and evolution of Eulamprus Aust. J. Zoology 323

100 Emar1 Emar3 100 97 Esok1 100 Esok2 Ebra2 98 66 100 Ebra3 Ebra4 92 100 Etig1 Etig2 Eten3 91 100 Eten4 tenuis Eamp 62 100 Nspi1 Nspi2 91 100 Efre1 Efre2 100 Gqld2 Gqld3 97 100 Etry1 Etry2 83 100 Espp1 100 Espp2 murrayi 99 100 Emur1 Emur2 67 100 Elut1 Elut2 Etym2 Equo1 100 Equo2 99 Equo3 100 Ekos3 quoyii 100 100 Eleu2 Eleu3 100 Ehea1 Ehea2 Craw Erfre Fig. 2. Phylogenetic hypothesis obtained from the consensus of 19000 trees sampled from a Bayesian search of tree space. The three major species groups suggested by Greer (1989) are indicated on the right. Numbers above the branches indicate the proportion of trees in which the clade to the right was present and can be interpreted as the level of statistical confidence. relationships of the other two tenuis group species (E. amplus and E. frerei), G. queenslandiae and N. spinosa are unresolved. To investigate further the ambiguity regarding the placement of G. queenslandiae and N. spinosa, in particular, we tested alternative hypotheses using likelihood. The consensus from the Bayesian analysis (Fig. 2) has a likelihood (using the same values for sequence evolution parameters) of ln L = –6710.7. Alternative topologies in which G. queenslandiae and N. spinosa form a polytomy that is either (i) immediately basal to the tenuis group, (ii) basal to both the tenuis and murrayi groups, or (iii) outside of all Eulamprus, have likelihoods of lnL = –6716.4, –6721.8 and –6731.8, respectively. The Shimodaira and 324 Aust. J. Zoology D. O’Connor and C. Moritz

Hasagawa test (using 1000 bootstraps with full-optimisation option) indicated that the first alternative is not significantly worse than the best tree (one-tailed SH tests, P = 0.36). However alternative (ii) is marginally rejected (P = 0.075) and alternative (iii) more clearly so (P = 0.02). Thus, from the molecular data alone, we can be confident that Eulamprus is paraphyletic with respect to the monotypic genera Gnypetoscincus and Nangura and we suggest, but cannot demonstrate conclusively, that they are most closely related to, and perhaps within, the tenuis species group. In addition, we also tested the inter-group relationships proposed by Greer (1989) in which the tenuis group represents a basal grade and the quoyii and murrayi groups are sisters – this had a substantially and significantly lower likelihood (lnL = –7596.85, SH test, P < 0.001) and can be confidently rejected in favour of the topology with the quoyii group basal and murrayi and tenuis groups as sister clades.

Discussion Phylogeny and implications for The mitochondrial sequence data support and refine the broad species groups within Eulamprus as hypothesised by Greer (1989), but also suggest a different interpretation of relationships among these groups and paraphyly of the genus in relation to Gnypetoscincus and Nangura. Before considering the biogeographic and evolutionary implications of the new hypothesis of relationships, we first explore consistency of key morphological changes considered by Greer with the new phylogenetic hypothesis (Fig. 3) and indicate changes to taxonomy that could follow. As before, the monophyly of the quoyii group is supported by two derived character states – grooved subdigital lamellae and the distal dorsal scales being in a single row. Likewise, the species within the murrayi group are united by having the postmental in contact with one infralabial. More at issue is interpretation of two other characters previously used as evidence for monophyly of the quoyii + murrayi species groups. According to Greer (1989), members of the tenuis group together with Gnypetoscincus (and three species of Glaphyromophus) are unusual among Sphenomorphus group of lygosomine skinks in having inguinal fat bodies, a trait also widespread within the Eugongylus group. These fat bodies are also present within Nangura (P. Couper, personal communication) and we suggest that this represents a synapomorphy for the clade (tenuis group + Gnypetoscincus + Nangura) rather than the ancestral condition, as proposed by Greer (1989). The second proposed synapomorphy for the clade of (quoyii + murrayi groups) was the presence of 5 scales separating the 3rd pair of enlarged chin scales, rather than the 3 scales seen in all other Australian representatives of the Sphenomorphus group skinks. Again, the new phylogenetic hypothesis reverses the polarity, suggesting that the presence of 5 scales as a synapomorphy for the genus (and Gnypetoscincus and Nangura) with a reversal to 3 scales in the tenuis group. A corollary of this interpretation is that Gnypetoscincus and Nangura are supported as a sister taxa to the species of the tenuis group. This hypothesis (Fig. 3) differs from the molecular tree in the placement of E. frerei relative to Nangura, but is not rejected relative to the Bayesian tree (Fig. 2) in a likelihood test (lnL = –6715, SH RELL bootstrap test P = 0.20). We take this reconciliation of morphological and molecular traits as our working hypothesis of relationships for the group. The strong support from the molecular analysis (and some morphological traits) for paraphyly of Eulamprus relative to Gnypetoscincus and Nangura raises the question of whether the former genus should be split or whether Gnypetoscincus and Nangura should Phylogenetics and evolution of Eulamprus Aust. J. Zoology 325

Drier woodland habitat

tenuis

3 scales separate 3rd chin scale

Fat bodies present

Rainforest

Postmental contacts one infralabial.

5 scales separate murrayi 3rd chin scale

Single dorsal scales distal on digits Subdivided lamellae grooved Wet microhabitat in sclerophyll forests quoyii

Fig. 3. Mapping of key morphological traits and habitat shifts on the phylogeny modified from Fig. 2 (see text). be subsumed within Eulamprus. Greer (1989) notes that the only trait uniting Eulamprus is ovoviviparity, a condition also shared with Gnypetoscincus and a taxonomically broader mtDNA phylogenetic analysis, including one species from each of the three Eulamprus species groups, shows the genus to be polyphyletic relative to other genera of Australian skinks of the Sphenomorphus group (Reeder 2003). The quoyii group is shown here to be phylogenetically cohesive and highly divergent from the remaining species. In contrast to the other clades, this group occurs primarily in south-eastern Australia and also is somewhat ecologically distinct, being more strongly heliothermic and occurring in association with water in drier forests – hence the colloquial name ‘water skinks’ (Greer 1989). Considering the concordance across the phylogenetic, ecological and biogeographic evidence, we suggest that the quoyii group be considered as a separate genus from the 326 Aust. J. Zoology D. O’Connor and C. Moritz

remainder. As the type species for the genus is E. quoyii (Fitzinger 1843), the quoyii group must remain within Eulamprus. The phylogenetic hypothesis (Fig. 3) indicates that the remaining species of Eulamprus are paraphyletic with respect to Gnypetoscincus and Nangura, a result supported and extended in the broader analysis of Sphenomorphus group relationships (Reeder 2003). Accordingly, we suggest that the tenuis and murrayi groups, each of which appears to be monophyletic, should be placed into separate genera. The species of the former could be allocated to Concinnia, a genus proposed by Wells and Wellington (1983), E. tenuis being the type species. In addition to tenuis group species, Concinnia, as proposed by Wells and Wellington also contains species from Glaphyromorphus (fuscicaudis and mjobergi), for which there is no evidence of monophyly with the tenuis group, as well as members of the murrayi group. Further research is therefore necessary to determine the appropriate limits for Concinnia and which, if any, species from genera other than Eulamprus should be included (see also Reeder 2003). Until this is completed we propose that only species from the tenuis group be assigned to Concinnia. There is no existing generic name applicable to the species of the murrayi group and a formal description of this genus is underway. We expect that this proposed arrangement will be stable, but clearly there is a need for further phylogenetic analysis of relationships among species of the Sphenomorphus group (Greer 1989; Hutchinson 1993; Reeder 2003). The current molecular analysis has also revealed substantial diversity and possible cryptic species within some currently recognised species. Intraspecific phylogeography has previously been studied intensively within three of the species examined here: G. queenslandiae (Moritz et al. 1993; Schneider et al. 1998), ‘E.’ amplus (Stuart-Fox et al. 2001) and ‘E.’ murrayi (O’Connor, Mousalli and Moritz, unpublished). The highest level of divergence observed was between two parapatric lineages within the Wet Tropics endemic, G. queenslandiae, corresponding to 6% divergence across the gene segments examined here. This is substantial, but is still much less than the >10% divergence observed between the individuals of ‘E.’ murrayi from Cambridge Plateau and surrounding populations. The aberrant population, here referred to as ‘E.’ sp. probably represents a narrowly endemic cryptic species and is being investigated further. Even more dramatic is the divergence of >20% between the Wet Tropics sample of E. quoyii and others (Border Ranges, south-east Queensland and Sydney, New South Wales) from this widely distributed species. An ongoing analysis of phylogeography within E. quoyii, as currently recognised, has confirmed the presence of multiple distinct lineages and some cryptic species (I. Scott, personal communication).

Implications for historical biogeography and ecology The mesic forest environments occupied by most of these species have been present on the east coast, with varying patchiness and extent, for millions of years and probably covered much of the continent prior to the general drying from the mid–late Miocene to the present (Adam 1992; Hope 1994; Kershaw et al. 1994). The deep molecular divergence within and between major clades of Eulamprus (sensu lato) is consistent with diversification of these skinks within these habitats over this long history, with the major lineages probably separating well back into the Miocene. Further support for the long history of Eulamprus comes from the recent finding (Mackness and Hutchinson 2000) of an early Pliocene fossil attributable to the quoyii group from northern Queensland. The current phylogeny suggests two major shifts in habitat among the lineages studied here. The more ancient divergence was that between the moist forest species and the mainly Phylogenetics and evolution of Eulamprus Aust. J. Zoology 327

saxicoline and creek-dwelling water skinks. Within the clade consisting of the tenuis and murrayi groups, plus Gnypetoscincus and Nangura, the basal lineages are rainforest specialists (though Nangura is found in drier vine thickets) with the more recently derived clade in the tenuis group consisting of ‘E.’ martini, ‘E.’ brachysoma and ‘E.’ sokosoma representing a shift back towards rocky or creekline habitats within drier and more open woodlands. Australia has an extremely rich reptile fauna with a large number of habitat generalists occurring in rainforests, but relatively few rainforest endemics (reviewed in Williams et al. 1996). For both flora and fauna, rainforest contractions since the mid-Miocene may have caused the extinction of rainforest specialists (Heatwole and Taylor 1987; Busby and Brown 1991; Hope 1994; Archer et al. 1994) or, for species persisting in now drier forests, promoted ecological shifts towards mesic microhabitats. The shifts towards such habitat preferences in both the quoyii and martini/sokosoma/brachysoma lineages (Fig. 3) may reflect this trend. In broad terms, the coastal wet forests occupied by most of these species can be split into four biogeographic regions (from north to south): the Wet Tropics, mid-east Queensland, a region incorporating south-east Queensland and north-east New South Wales, and south of the Hunter Valley (e.g. Keast 1981; Adam 1992). Each of the three major clades revealed here spans multiple regions but has its diversity focused in one or two (Fig. 1). The quoyii clade is primarily a southern radiation, although E. quoyii itself (as currently recognised) is widely distributed. Three of the four species in the murrayi clade are from the rainforests of south-east Queensland and north-east NSW, the fourth being endemic to the mid-east Queensland rainforest. Finally, the clade consisting of the tenuis group plus Gnypetoscincus and Nangura includes three endemic species from the Wet Tropics rainforests, one from mid-east Queensland rainforests and one from south-east Queensland as well as the three drier forest taxa distributed from mid-east to south-eastern Queensland. Collectively, these relationships indicate deep historical connections between the now isolated rainforests of mid-east Queensland and the Wet Tropics, on one hand, and south-eastern Queensland, on the other. Whether the phylogenetic affinities of the mid-east Queensland rainforests are primarily to the north or south requires phylogenetic analysis of additional groups with multiple, narrowly endemic species (e.g. snails: Hugall et al. in press; plants: Crisp et al. 2001). Whatever the general pattern of biogeography, the considerable divergence (>15%) between each Eulamprus endemic to mid-east Queensland and their nearest relatives, together with recent evidence for substantial local endemicity and genetic diversity within this region (e.g. Couper et al. 2000; Stuart-Fox et al. 2001) emphasises the antiquity of this fauna and high conservation value of their rainforest habitats, as well as those of the better known Wet Tropics and the border region of Queensland and New South Wales.

Acknowledgments We thank Michael Cunningham and Chris Schneider for assistance with sample collection and advice on the molecular sequencing component; Tod Reeder for supplying the Eulamprus amplus 16S sequence; Andrew Hugall and Shawn Kuchta for help with data analysis; and Teena Browning, Patrick Couper, Allen Greer, Michael Kearney, Rick Shine and two anonymous referees for comments on drafts of the manuscript. References Adam, P. (1992). ‘Australian Rainforests.’ (Clarendon Press: Oxford.) Archer, M., Hand, S., and Godthelp, H. (1994). Patterns in the history of Australia's mammals and inferences about palaeohabitats. In ‘History of the Australian Vegetation: Cretaceous to Recent’. (Ed. R. Hill.) pp. 80–103. (Cambridge University Press: Cambridge.) 328 Aust. J. Zoology D. O’Connor and C. Moritz

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Manuscript received 15 August 2002; accepted 2 September 2003 330 Aust. J. Zoology D. O’Connor and C. Moritz

Appendix. Details of the sequenced specimens of Eulamprus species

Species Genetic code Sample code Voucher specimen Location

E. amplus E. amp1 CJS 673 Mt Blackwood, Qld E. amplus E. amp2 CJS 740 Finch Hatton Gorge, Qld E. brachysoma E. bra2 CJS 645 Boulder Ck, Eungella, Qld E. brachysoma E. bra3 DOC 61 Chillagoe Caves, Qld E. brachysoma E. bra4 DOC 10 Chillagoe Caves, Qld E. frerei E. fre1 Q1037 Mt Bartle Frere, Qld E. frerei E. fre2 CON Mt Bartle Frere, Qld E. heatwolei E. hea1 NR88L AMS R133188 15 km N of Kanangra Walls, NSW E. heatwolei E. hea2 NR92 AMS R133191 15 km N of Kanangra Walls, NSW E. kosciuskoi E. kos3 94 Cathederal Rock, New England NP, NSW E. leuraensis E. leu2 NR 3875 Blue Mtns, NSW E. leuraensis E. leu3 NR 3876 Blue Mtns, NSW E. luteilateralis E. lut1 CJS 754 Dalrymple Rd, Eungella, Qld E. luteilateralis E. lut2 A35 Eungella, Qld E. martini E. mar1 D5 Gambubal SF, Qld E. martini E. mar3 DOC 39 Lamington NP, Qld E. murrayi E. mur1 DOC 718 Nightcap NP, NSW E. murrayi E. mur2 DOC 717 Pt Lookout, NSW E. quoyii E. quo1 T81 QM J47602 Charmillon Ck, Qld E. quoyii E. quo2 Q910 Lamington NP, Qld E. quoyii E. quo3 1142 Oxford Falls, Sydney, NSW E. sokosoma E. sok1 DHR1 Hervey Range, Townsville, Qld E. sokosoma E. sok2 DHR4 Hervey Range, Townsville, Qld E. tenuis E. ten3 DOC 106 Bulburin SF, Qld E. tenuis E. ten4 CHDD 230 Mt Nebo, D’Aguilar Range, Qld E. tigrinus E. tig1 CJS 869 Lake Eacham, Qld E. tigrinus E. tig2 CJS 799 Massey Ck, Atherton Tbld, Qld E. tryoni E. try1 DOC 760 Lamington NP, Qld E. tryoni E. try2 DOC 751 Lamington NP, Qld E. tympanum E. tym2 NR 3950 AMS R148525 Kosciusko NP, NSW E. sp. E. spp1 BP 121 1.5 km N of Cambridge Plateau, Richmond Range, NSW E. sp. E. spp2 BP 131 1.5 km N of Cambridge Plateau, Richmond Range, NSW G. queenslandiae G. qld 2 JJ 136 Massey Ck, Atherton Tbld, Qld G. queenslandiae G. qld 3 Q 193 Mt Lewis, Qld N. spinosa N. spi 1 N. spi Oakview SF, Qld N. spinosa N. spi 2 Q925 QM 7247 Nangur SF, Qld Egernia frerei Er. fre CJS 676 Mt Blackwood Ctenotus rawlinsoni C. raw N29869 Cape Flattery

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