Molecular Phylogenetics and Evolution 39 (2006) 503–511 www.elsevier.com/locate/ympev Using ancient and recent DNA to explore relationships of extinct and endangered Leiolopisma skinks (Reptilia: Scincidae) in the Mascarene islands J.J. Austin 1, E.N. Arnold ¤ Department of Zoology, Natural History Museum, Cromwell Rd., London SW7 5BD, UK Received 29 August 2005; revised 15 December 2005; accepted 15 December 2005 Available online 10 February 2006 Abstract Phylogenetic analysis, using 1455 bp of recent mtDNA (cytochrome b 714 bp, 12S rRNA 376 bp) and nuclear (c-mos 365 bp) sequence from 42 species and 33 genera of Scincidae, conWrms Leiolopisma telfairii, now conWned to Round island oV Mauritius, is a member of the mainly Australasian Eugongylus group of the Lygosominae. Ancient mtDNA (cytochrome b 307 bp, 12S rRNA 376 bp) was also extracted from subfossils of two other Mascarene taxa that are now extinct: the giant L. mauritiana from Mauritius and Lei- olopisma sp., known only from fragmentary remains from Réunion. Sequence divergences of 4.2–5.7% show that all three forms were distinct and form a clade. There is restricted evidence that L. mauritiana and L. sp. from Réunion were sister species. Monophyly and relationships suggest Leiolopisma arose from a single transmarine invasion of the oceanic Mascarene islands from Australasia, 5600– 7000 km away. This origin is similar to that of Cryptoblepharus skinks and Nactus geckos in the archipelago but contrasts with Phel- suma day geckos, which appear to have arrived from Madagascar where Mascarene Cylindraspis tortoises may also have originated. DiversiWcation of the known species of Leiolopisma occurred from about 2.3–3.4 Mya, probably beginning on Mauritius with later invasion of Réunion. The initial coloniser may have had a relatively large body-size, but L. mauritiana is likely to have become gigan- tic within the Mascarenes. Other relationships supported by this investigation include the following. Scincines: Pamelaescincus + Janetaescincus, and Androngo (Amphiglossus, Paracontias). Lygosomines: Sphenomorphus group—(Sphenomorphus, Lipinia (Ctenotus, Anomalopus (Eulamprus and Gnypetoscincus))): Egernia group—Egernia (Cyclodomorphus, Tiliqua); Eugongylus group—(Oligosoma, Bassiana. (Lampropholis (Niveoscincus, Carlia))). © 2006 Elsevier Inc. All rights reserved. Keywords: Leiolopisma skinks; Island colonisation; Island gigantism; Mascarene islands; Ancient DNA; Mitochondrial DNA; c-mos 1. Introduction 7 (23%) that retain substantial ranges (Arnold, 2000). Among the forms aVected were three large-bodied skinks The Mascarene islands in the Southwest Indian Ocean that are referred to Leiolopisma Duméril and Bibron, (Fig. 1) had the richest oceanic island reptile fauna in the 1839; a genus in which many Australasian species were World, but this was devastated after the arrival of people once included, but which is now restricted to the Masca- and the animals they introduced 500 years ago. Of the 33 rene taxa (Hutchinson et al., 1990). Unlike the Austral- reptile species known to have been present, 15 (46%) are asian species, the Mascarene forms frequently have extinct and 11 (33%) reduced to small relicts, leaving only pterygoid teeth (Arnold, 1980; Hutchinson et al., 1990) and in L. telfairii, the one species in which they can be * checked, there are distinctive features of head scalation Corresponding author. Fax: +44 0 20 7 942 5054. (Hutchinson et al., 1990). Only one of the species, L. telf- E-mail address: [email protected] (E.N. Arnold). W 1 Present address: School of Earth and Environmental Sciences, Univer- airii Desjardins, 1831; survives and is now con ned to the sity of Adelaide, North Terrace, Adelaide, SA 5005, Australia. 150 ha oVshore Round Island, although it was once also 1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2005.12.011 504 J.J. Austin, E.N. Arnold / Molecular Phylogenetics and Evolution 39 (2006) 503–511 Fig. 1. (A) Map of the Indian Ocean and surrounding landmasses showing the location of the Mascarane islands. (B) Mascarane islands showing collec- tion localities for Leiolopisma skinks. present on neighbouring Flat island and subfossil mate- elevated number of premaxillary teeth (11 instead of the 9 rial shows that it used to occur widely on the main island usually present in other lygosomines), lack of a separate of Mauritius as well (Arnold, 1980; Fig. 1). A second spe- postorbital bone and a covered Meckel’s canal in the den- cies found on Mauritius, L. mauritiana (Günther, 1877) is tary bone suggests they are members of the mainly Aus- extinct and was one of the largest skinks known. It tralasian Eugongylus group (Greer, 1979). Other typical reached an estimated snout-vent length of around features of this assemblage not checkable in L. mauritiana 340 mm, compared with 170 mm for the largest Round can be discerned in L. telfairii, including parietal scales in Island L. telfairii and an estimated 200 mm for Mauritian contact behind the interparietal, 28 presacral vertebrae sub-fossils of this species (Arnold, 1980). L. mauritiana and a diploid chromosome number of 30 (Hardy, 1979; was originally assigned its own genus, Didosaurus Gün- Donnellan, 1985). In contrast, the fragmentary nature of ther, 1877, its apparent aYnity to L. telfairii being noted available material of the Réunion skink makes it impossi- later (Arnold, 1980). A third, as yet unnamed, taxon ble to be sure of its aYnities, and at the same time it is not occurred on Réunion island 145 km southwest of Mauri- clear how distinct this taxon is from L. telfairii. To test tius and is also extinct, being known only from fragments hypotheses about the relationships of these Mascarene (isolated post-cranial and skull bones, including dentar- skinks and their relative status, they are investigated here, ies). This form was similar to L. telfairii but was more using recent mitochondrial (cytochrome b and 12S robust and had coarser dentition (Arnold, 1980; Arnold rRNA) and nuclear (c-mos) DNA from L. telfairii, and and Bour, Submitted for publication). ancient mtDNA (cytochrome b and 12S rRNA) from the Leiolopisma telfairii and L. mauritiana are clearly two extinct forms. The results are then used to consider members of the Lygosominae, having the characteristic the history and biogeography of Leiolopisma. The investi- features of this group (fused frontal bones, and a second- gation also incidentally tests other hypothesised relation- ary palate—Greer, 1970). Within this assemblage, their ships within the Lygosominae. J.J. Austin, E.N. Arnold / Molecular Phylogenetics and Evolution 39 (2006) 503–511 505 2. Materials and methods Microcon-30 centrifugal Wlter units (Millipore). DNA extraction attempts from alcohol-preserved museum tissues 2.1. Material involved air drying small, 2–3 mm3, pieces of muscle and washing once in 1 ml of 10 mM Tris–HCl (pH 8.0), followed Tissue samples from members of the following assem- by digestion and extraction using the DNeasy tissue kit blages are included (numbers are given in brackets). Lygos- (Qiagen). ominae—Eugongylus group (13), Egernia group (3), For the ancient DNA, short (103–178 bp) overlapping Sphenomorphus group (6) and Mabuya (2); Scincinae (14); segments of the 12S rRNA (»400 bp total) and cytochrome Feylininae (1) and Acontinae (1). Three taxa, Zonosaurus b (307 bp total) genes were ampliWed via two rounds of sp. (Gerrhosauridae), Pseudocordylus capensis and Cordylus PCR as described by Austin et al. (2002, 2004) using combi- cordylus (Cordylidae) were included as outgroups. Samples nations of the following primers: 12S rRNA, 12Sa (Kocher of Mascarene Leiolopisma comprise four individuals from et al., 1989), 12SH1269 (5Ј-TTTCTTTCATAAGGTAGG the extant population of L. telfairii on Round Island, one CTGAC-3Ј), 12SL1266L (5Ј-GAAACTCAGCCTATATA old alcohol-preserved L. telfairii from the extinct popula- CCGCCG-3Ј), 12SH1382L (5Ј-GTTTCATTGTGCTGTT tion on Flat Island, two subfossil bones of the extinct L. CGTGTTC-3Ј), 12SL1357L (5Ј-GTGTAGCAYATAAA mauritiana collected from diVerent localities on Mauritius, GCGGAAGAG-3Ј), 12Sb (Kocher et al., 1989), cyto- and two subfossil bones of the extinct L. sp. population chrome b, L14841 (Kocher et al., 1989), CBH14957 (5Ј-AA from Réunion (Fig. 1). All species and samples included in GTCATCCGTATTGTACGTCTCG-3Ј), CBH14953L (5Ј-G the study are listed in Table 1. CCGTATTGGACATCCCGGGT-3Ј), CBL14936L (5Ј-C AGCAGACATTTCATCCGCATTCA-3Ј), CBH15039L 2.2. DNA extraction, PCR ampliWcation and sequencing (5Ј-GCCGTAATAAAGGCCCCGACCA-3Ј), CBL15030L (5Ј-GCCTCAATATTCTTYATCTGCMTCTA-3Ј), H15149 DNA sources included ethanol-preserved tissue (tail tips (Kocher et al., 1989). Negative extraction and PCR controls and liver), genomic DNA extracts and subfossil bones (no tissue and no DNA extract, respectively) were included (extinct L. mauritiana from Mauritius and L. sp. from alongside all extract and PCR ampliWcation attempts. PCR Réunion). For extant taxa, DNA was extracted from 2 to products were puriWed and sequenced directly as described 3mm3 of preserved tissue samples using standard Protein- by Austin et al. (2002, 2004). All sequences have been ase K digestion and phenol:chloroform protocols (Car- deposited in GenBank (Accession Nos.: AF280114, ranza et al., 1999). Fragments of two mitochondrial genes, AF280115, AF280117–AF280120, AF280122–AF280124, 12S rRNA (»400 bp) and cytochrome b (714 bp), and one AF280129, AF280130, AF280133–AF280135, AY818735– fragment of the nuclear gene,