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African Journal of Herpetology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ther20 Phylogeny and phylogeography of the Malagasy -tailed in the ebenaui group Fanomezana Mihaja Ratsoavina a b c , a & Edward E. Louis Jr. c d a Zoological Institute, Technical University of Braunschweig, Braunschweig, Germany b Département de Biologie Animale, Université d'Antananarivo, Madagascar c Center for Conservation and Research, Omaha's Henry Doorly Zoo and Aquarium, Omaha, NE, USA d Madagascar Biodiversity Partnership, Antananarivo, Madagascar Version of record first published: 26 Oct 2012.

To cite this article: Fanomezana Mihaja Ratsoavina, Miguel Vences & Edward E. Louis Jr. (2012): Phylogeny and phylogeography of the Malagasy leaf-tailed geckos in the group, African Journal of Herpetology, 61:2, 143-158 To link to this article: http://dx.doi.org/10.1080/21564574.2012.729761

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Phylogeny and phylogeography of the Malagasy leaf-tailed geckos in the Uroplatus ebenaui group

1,2,3 1 FANOMEZANA MIHAJA RATSOAVINA *, MIGUEL VENCES 3,4 &EDWARD E. LOUIS,JR.

1Zoological Institute, Technical University of Braunschweig, Braunschweig, Germany; 2De´partement de Biologie Animale, Universite´ d’Antananarivo, Madagascar; 3Center for Conservation and Research, Omaha’s Henry Doorly Zoo and Aquarium, Omaha, NE, USA; 4Madagascar Biodiversity Partnership, Antananarivo, Madagascar

Abstract.*Leaf-tailed geckos, Uroplatus, are one of the most prominent endemic groups from Madagascar, but the diversity and diversification of this taxonomic group are not completely understood. Here, we present a phylogenetic reconstruction of the small-sized Uroplatus which are included in the Uroplatus ebenaui group, focusing on the most widespread species of these geckos, , which occupies a large mid-altitude distribution range in the eastern rainforests of Madagascar. Our phylogeny is based on DNA sequences of four mitochondrial genes (12S rRNA, 16S rRNA, COI and ND4), with a total of 2 312 base pairs. Partitioned Bayesian analysis confirmed U. malama from south-eastern Madagascar as the most basal representa- tive of the group, followed by U. ebenaui from northern lowlands, and clades containing various undescribed candidate species as well as U. finiavana from Montagne d’Ambre in the north. Within our main targeted species U. phantasticus, the northernmost population from Zahamena (here considered as unconfirmed candidate species) forms the most basal lineage, while the southernmost populations studied from Marolambo, Ranomafana and Kianjavato form a nested clade, suggesting the species has expanded its range southwards undergoing repeated events of isolation and differentiation of lineages. Within the Ranomafana Kianjavato area, we identified three different lineages, of which the two from Ranomafana are differentiated by 28 mutational steps in a fragment of the ND4 gene and occur on opposing banks of the Namorona River. This result suggests that this relatively small river currently limits dispersal and gene flow in these geckos. However, further downstream the Kianjavato population is more closely related to the population on the opposite side of the Namorona in Ranomafana, indicating that this genetic isolation is not absolute, and the river probably represents only a secondary barrier to gene flow in U. phantasticus.

Key words.*Uroplatus ebenaui group, U. phantasticus, phylogeny, phylogeography, Ranomafana, Kianjavato, river barrier, Madagascar Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012 INTRODUCTION

Madagascar is well known for its high organismal richness and endemism which has resulted in part from the long isolation of this island from continental land masses (Samonds et al. 2012; Crottini et al. 2012). This also applies to amphibians and , with the island considered to be one of the global herpetofaunal diversity

*Corresponding author. Email: [email protected] Online Supplementary Material is available for this article which can be accessed via the online version of this journal available at www.tandf.co.uk/journals/THER.

ISSN 2156-4574 print/ISSN 2153-3660 online # 2012 Herpetological Association of Africa http://dx.doi.org/10.1080/21564574.2012.729761 http://www.tandfonline.com 144 RATSOAVINA ET AL.*Phylogeny and phylogeography of the Uroplatus ebenaui group

hotspots (Raxworthy 2003). Although Madagascar only hosts two endemic reptile families (Opluridae and Xenotyphlopidae), many genera and more than 92% of the over 363 reptile species are found nowhere else on earth (Glaw & Vences 2007). Discovery of new reptile species is ongoing (Nagy et al. 2012) although its rate does not equal that of amphibians (Ko¨hler et al. 2008; Vieites et al. 2009). One of the fascinating groups of Malagasy reptiles is formed by the nocturnal and arboreal of the genus Uroplatus. Since the description of the first species U. fimbriatus by Schneider (1792), these geckos have thrilled researchers and naturalists due to their often unique appearance. The largest species in the genus are especially distinctive, characterised by flat tails, broad heads and lateral skin flaps, thereby becoming almost indistinguishable from the bark of trees as they rest in an adpressed posture during the day. After the last comprehensive taxonomic revision of the genus by Bauer and Russell (1989), new species have been added by Bo¨hme and Ibisch (1990), Nussbaum and Raxworthy (1994, 1995), Bo¨hle and Scho¨necker (2003), Glaw et al. (2006) and Ratsoavina et al. (2011). Furthermore, other authors have published information about the biology, ecology and husbandry of leaf-tailed geckos (e.g., Svatek & Van Duin 2002). Information on the phylogenetic relationships among species of Uroplatus has only recently started to emerge, largely by analyses of DNA sequences (Greenbaum et al. 2007; Raxworthy et al. 2008). These studies agreed in defining the U. ebenaui group of small-sized leaf-tailed geckos characterised by an absence of skin flaps, a triangular head and dermal spines and flaps on the head and eyelid as monophyletic but containing numerous taxonomically undescribed lineages. Ratsoavina et al. (2011) confirmed that this group contains numerous new candidate species in northern Madagascar along with describing a new species in the group. Thus, the U. ebenaui group is currently composed of four nominal species of which two (U. ebenaui and U. finiavana) are restricted to rather small ranges in the north, one (U. malama) to the south-east, and one (U. phantasticus) is widespread in the east. Although Raxworthy et al. (2008) studied the biogeographical patterns of Uroplatus, the diversification mechanisms that generated the current species richness of the genus are still insufficiently known. Various diversification mechanisms have been proposed for Madagascar, and many of these focused on the influence of rivers (summary in Vences et al. 2009). The importance of large rivers as barriers on species distribution in Madagascar was first hypothesised by Martin (1972) and subse- quently analysed in more detail by Pastorini et al. (2003) and Goodman and Ganzhorn (2004) based on distribution patterns of lemurs, and by Knopp et al. (2011) based on the distribution of dung beetles. Wilme´ et al. (2006) instead Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012 proposed a role for watersheds with headwaters in relatively low elevations as refuges for humid-adapted vegetation during past, more arid climate conditions; organisms thus remained isolated and diverged in these watersheds which today mark centres of endemism. Pearson and Raxworthy (2009) found some support for this hypothesis from the analysis of distribution patterns in Malagasy reptiles. A further, complementary pattern in various reptile species and in mouse lemurs has been observed by Yoder and Heckman (2006), Boumans et al. (2007) and Ratsoavina et al. (2011). Within several rainforest species or species complexes, the most basal lineages are concordantly found in northern Madagascar, with an apparent though not unequivocal signal of southwards expansion and differentiation. AFRICAN JOURNAL OF HERPETOLOGY 61(2) 2012 145

In this study, we focus on the species of the Uroplatus ebenaui group, and more specifically on the widespread U. phantasticus, to contribute to a better under- standing of their phylogeny and phylogeography and ultimately of the diversification mechanisms acting on these geckos. We reconstruct a phylogeny based on multiple mitochondrial genes to understand the relationships of U. phantasticus to other species, and among its various intraspecific lineages. Secondly, we use sequences of one mitochondrial gene for a large set of specimens from the Ranomafana region to better understand the small-scale spatial distribution of these lineages and the role of riverine barriers to gene flow in these geckos.

MATERIALS AND METHODS Fieldwork and sampling were searched for at night during opportunistic surveys along forest paths, using flashlights or headlamps. Representative specimens were euthanised by injection of chlorobutanol solution, fixed in 90% and preserved in 70% ethanol. Specimens were deposited at the collections of the De´partement de Biologie Animale of the Universite´ d’Antananarivo (UADBA) in Madagascar and the Zoologische Staatssammlung Mu¨nchen (ZSM) in Germany. Locality data were recorded with Global Positioning System (GPS) devices. Tissue samples and specimens used in this study originate from several field surveys undertaken by the authors in different regions of Madagascar. The following acronyms are from collaborators or localities and are utilised to identify samples and specimens: KAF/BET/ZAH for Kianjavato/ Betampona/Zahamena localities, MPFC for Maciej Pabijan, ACZC for Angelica Crottini, PSG for Sebastian Gehring and DRV for David Vieites. FGZC, ZCMV and FGMV correspond to Frank Glaw and Miguel Vences specimens. Naming of candidate species follows Padial et al. (2010), with the name of the most similar nominal species followed in square brackets by Ca for candidate species and a consecutive number. Naming of biogeographical regions in Madagascar follows Boumans et al. (2007) and Glaw and Vences (2007).

DNA extraction, amplification and sequencing Tissue samples used for molecular analysis were tail tips clipped from subsequently released specimens, or femur muscle for preserved specimens. All tissue samples were preserved in 9599% ethanol. Total genomic DNA was extracted using proteinase-K Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012 digestion followed by a standard salt extraction (Bruford et al. 1992). Standard PCR protocols were followed to amplify four genes of the mitochondrial genome: NADH dehydrogenase subunit 4 plus a short stretch of the adjacent tRNAHis gene (ND4) using primers ND4 5’-CACCTATGACTACCAAAAGCTCATGTAGAAGC-3’ and LeutR- NA 5’-CATTACTTTTACTTGGATTTGCACC-3’ (Are´valo et al. 1994); Cytochrome oxidase I (COI) using primers CO1vertF2 5‘-TCAACCAACCACAAAGA- CATTGGCAC-3’ and CO1vertR1 5’-TAGACTTCTGGGTGGCCAAAGAAT- CA-3’; part of the 16S ribosomal gene (16S rRNA) using 16SAL 5’-CGCCTGTTTATCAAAAACAT-3’ and 16SBH 5’-CCGGTCTGAACTCA- GATCACGT-3’ (Palumbi et al. 1991) and part of the 12S rRNA gene using primers 146 RATSOAVINA ET AL.*Phylogeny and phylogeography of the Uroplatus ebenaui group

12SAL 5’-AAACTGGGATTAGATACCCCACTAT-3’ and 16SHBnew 5’-CCT- GGATTACTCCGGTCTGA-3’. Sequences were resolved on an automated DNA sequencer (ABI 3130 XL, Applied Biosystems), quality checked, and corrected manually if needed, using the program CodonCode Aligner (CodonCode Corpora- tion). Multiple DNA alignments were performed using the Muscle algorithm in MEGA 5 (Tamura et al. 2011) and refined manually. Newly determined sequences from thiswork were submitted to GenBank (accession numbers JX205218JX205296 for 12S, JX205297JX205364 for ND4, JX205365JX205433 for COI and JX205434JX205494 for 16S; see Online Supplementary Material).

Molecular data analysis The concatenated DNA sequences were submitted to Bayesian analyses using MrBayes v3.1.1 (Ronquist & Huelsenbeck 2003). Default priors and analysis parameters were used, except when implementing substitution models previously determined using MrModeltest v2.2 (Nylander 2004). The most appropriate model for each gene and gene partition were chosen under the Akaike Information Criterion. We have designed several partition schemes of the data set run for a preliminary 10 million generations. After each run, harmonic means were calculated using the sump command in MrBayes. According to BF values comparison (Brandley et al. 2005), the partitioning strategy that explained the data set best was the one subdividing our data into four character sets as follows: the two non- coding genes (16S and 12S) were combined and treated as one set; the first and the second codon of all coding genes (ND4 and CO1) were defined as one character set; as well as the third codon as another set, and a short segment of tRNAHis (included in the ND4 DNA fragment) as one set. This best partition of our data set was used in partitioned Bayesian analysis with two runs of 25 million generations (started on random trees) and four incrementally heated Markov chains (using default heating values) each, sampling the Markov chains at intervals of 2 000 generations. Trees sampled, character setting parameters, effective sample size and run convergence were checked using Tracer 3.1 (Rambaut & Drummond 2007). Stabilisation and convergence of likelihood values occurred after three million generations. The first five million generations were conservatively discarded and 10 001 trees were retained for post burn-in and summed to generate a 50% majority rule consensus tree. Uncorrected pairwise genetic distance caclulated from a fragment of ND4 gene between U. phantasticus populations is given in Table 1. A haplotype network of ND4 sequences of U. phantasticus from the Ranomafana region was reconstructed using Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012 the software Network (version 4.6.0.0; Bandelt et al. 1999) after inferring haplotypes (list shown in Table 2) using DnaSP (Version 5.10.3; Librado & Rozas 2009).

RESULTS Phylogeny of the Uroplatus ebenaui group Forty samples from all described species and various undescribed candidate species of the U. ebenaui group were collected along their distribution area and were subject to the following study. The phylogenetic analysis was performed on a concatenated Table 1. Uncorrected pairwise distance calculated from ND4 mtDNA fragment of U. phantasticus populations here represented by one sample from HERPETOLOGY OF JOURNAL AFRICAN each locality and other described species of the U. ebenaui group.

12345678910111213 Anjozorobe FGZC 4384 (U. phantasticus)1 Anosibe an’Ala FGZC 4505 (U. phantasticus) 2 0.076 Marolambo RATF 29 (U. phantasticus) 3 0.075 0.048 Fierenana RAFT 38 (U. phantasticus) 4 0.022 0.079 0.072 Betampona BET 5.15 (U. phantasticus) 5 0.050 0.085 0.067 0.026 Kianjavato KAF 183 (U. phantasticus) 6 0.063 0.051 0.060 0.069 0.077 Ranomafana MPFC 501 (U. phantasticus) 7 0.085 0.060 0.060 0.095 0.082 0.032 Ranomafana URANO 4.28 (U. phantasticus) 8 0.085 0.060 0.060 0.095 0.082 0.032 0.000 Zahamena ZAH 222 (U. phantasticus [Ca10]) 9 0.232 0.227 0.217 0.227 0.257 0.232 0.227 0.227 12 02147 2012 61(2) Zahamena ZAH 257 (U. phantasticus [Ca10]) 10 0.230 0.224 0.215 0.223 0.252 0.228 0.227 0.227 0.010 Nosy Be ACZC 1199 (U. ebenaui) 11 0.305 0.307 0.307 0.314 0.325 0.295 0.312 0.312 0.343 0.343 Beampingaratra MPFC 411 (U.malama) 12 0.267 0.283 0.305 0.278 0.270 0.279 0.293 0.293 0.325 0.320 0.318 Montagne d’Ambre UAMB 5.36 (U. finiavana) 13 0.274 0.299 0.308 0.285 0.294 0.284 0.292 0.292 0.296 0.301 0.352 0.347 Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012 27 October at 06:00 Ratsoavina] Mihaja Fanomezana by [Miss Downloaded 148 RATSOAVINA ET AL.*Phylogeny and phylogeography of the Uroplatus ebenaui group

DNA sequence alignment including a total of 2 312 base pairs of the mitochondrial DNA (mtDNA) genes 12S rRNA, 16S rRNA, ND4 and COI (Fig. 1). The resulting tree revealed a largely resolved phylogenetic hypothesis. High support values (posterior probabilities of 0.951.0) were found for most relationships among species. Our phylogenetic tree shows six major clades with U. malama in a basal position, followed by U. ebenaui. All remaining species and candidate species are a monophyletic group, with a posterior probability (PP) of 0.97. The clade next splitting off is represented by a specimen from Fierenana, but its position is supported by only PP0.9. Based on another, as yet unpublished taxonomic survey of the genus, we list this species here as a confirmed candidate species (CCS) and designate it as U. ebenaui [Ca7]. Subsequently, a clade of specimens from Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012

Figure 1. Bayesian analysis based on the best partition scheme for the concatenated alignment of DNA sequences of four mtDNA fragments (COI, 12SrRNA, 16SrRNA and ND4) from species of the U. ebenaui group. The concatenated sequence alignment consists of 2 312 base pairs and was analysed in MrBayes for 25 million generations. Numbers are posterior probabilities higher than 0.9. The inset map shows the sampled localities of the species U. phantasticus, the blue line representing the Mangoro River. This figure is included in colour in the online version of this article. AFRICAN JOURNAL OF HERPETOLOGY 61(2) 2012 149

mid-elevations around the Tsaratanana Massif in northern Madagascar splits off, representing a CCS here named U. ebenaui [Ca1]. A PP0.95 supports this position and defines a clade of all remaining samples, which is split in two subclades. The first of these contains with maximum support (PP1.0) U. finiavana, plus three candidate species from mainly northern Madagascar: U. ebenaui [Ca2], U. ebenaui [Ca3] and U. ebenaui [Ca4]. The second subclade contains all lineages here assigned to U. phantasticus, plus one sample from Zahamena considered as unconfirmed candidate species (UCS), equally with maximum support.

Phylogeography of Uroplatus phantasticus Within the U. phantasticus subclade, several deep mitochondrial lineages are apparent, and most nodes depicting their relationships have received high support of PP0.971.0. The most basal position corresponds to the UCS from the northernmost population from Zahamena, U. phantasticus [Ca10]. Most basal within U. phantasticus sensu stricto is a sample from a northern central eastern locality (Anosibe an’ala). The remaining samples are divided in two subclades as follows: one from populations in the northern central east, north of the Mangoro River (Betampona, Fierenana and Anjozorobe) and one with populations from the southern central east, south of the Mangoro River (Marolambo, Ranomafana and Kianjavato).

Differentiation of U. phantasticus populations in Ranomafana and Kianjavato ND4 sequences were available for a total of 46 U. phantasticus specimens from the RanomafanaKianjavato area. Among these samples, 16 haplotypes could be distinguished, belonging to three main lineages (Fig. 2; Table 2). Of these three lineages, only two are represented in the multi-gene tree (Fig. 1), but a phylogenetic analysis of ND4 sequences only (not shown) indicated that all three lineages together form a clade. The three lineages occurred in strict allopatry despite a close spatial proximity of their localities. All specimens from Kianjavato (light blue in Fig. 2) were collected in three different forest fragments, falling into two haplotypes (C1 and C2), and make up a first main haplotype lineage. Specimens from Ranomafana Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012 National Park collected south of the Namorona River, with several specimens only a few hundred metres from the river bank, collected mostly during 2004, fall into 13 haplotypes from the second haplotype lineage (dark blue in Fig. 2; not included in Fig. 1). Specimens from sites within and nearby Ranomafana National Park, but on the northern side of the Namorona River, mostly collected in 2010 but including three specimens collected in 2004 (URANO4.19, URANO4.28 and URANO4.29) all had the same, rather strongly differentiated haplotype. The lineages of Ranomafana-south and Kianjavato were separated by a minimum of 10 mutational steps, whereas the two Ranomafana populations were separated by 28 mutational steps. 150 RATSOAVINA ET AL.*Phylogeny and phylogeography of the Uroplatus ebenaui group Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012

Figure 2. (A) Haplotype network of 46 individuals from U. phantasticus populations collected in Ranomafana and Kianjavato, based on 709 base pairs of the ND4 mtDNA fragment. Circle size is proportional to number of individuals belonging to the haplotype. Blue circles labeled A1A13 or A are haplotypes from populations south of the Namorona River while red circles labeled B are haplotypes from populations north of the river. Light blue circles labeled C1C2 or C represents populations from Kianjavato. Grey circles are the median, and transversal lines are mutational steps between haplotypes. In (B) a simplified map of the occurrence of the same 46 individuals from Ranomafana and Kianjavato is shown. Circle size is equivalent to sample size. Black squares indicate major villages. The black line is the national road. This figure is included in colour in the online version of this article. AFRICAN JOURNAL OF HERPETOLOGY 61(2) 2012 151

Table 2. Haplotype list for 46 individuals belonging to U. phantasticus from Ranomafana and Kianjavato populations. See Fig 2 for the corresponding haplotype network and geographic occurrence of samples.

Haplotype Number of Individuals Number per Haplotype Individual Identification A1 10 URANO4.1, URANO4.12, URANO4.14, URANO4.22, URANO4.21, URANO4.24, URANO4.25, URANO4.35, URANO4.39, URANO4.42 A2 3 URANO4.11, URANO4.15, URANO4.37 A3 4 URANO4.16, URANO4.4, URANO4.6, URANO4.8 A4 1 URANO4.17 A5 1 URANO4.18 A6 3 URANO4.20, URANO4.40, URANO4.41 A7 1 URANO4.23 A8 1 URANO4.27 A9 1 URANO4.30 A10 1 URANO4.32 A11 1 URANO4.34 A12 1 URANO4.38 A13 1 URANO4.5 B 9 URANO4.19, MPFC511, MPFC510, MPFC509, MPFC506, MPFC503, MPFC501, URANO4.28, URANO4.29 C1 4 KAF237, KAF209, KAF194, KAF181 C2 4 KAF191, KAF190, KAF184, KAF179

DISCUSSION Phylogeny and biogeography of the U. ebenaui group The diversification of vertebrate clades in Madagascar has resulted in a high proportion of species that are microendemic, i.e. restricted to small ranges, and are consequently of conservation concern (Kremen et al. 2008; Vences et al. 2009). Many such microendemic species are found in northern Madagascar, which for some groups of reptiles and amphibians is an obvious centre of species richness and endemism (Raxworthy & Nussbaum 1995; Wollenberg et al. 2008; Townsend et al. 2009; Ratsoavina et al. 2010; Kaffenberger et al. 2012). The origin of the U. ebenaui Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012 group is equivocal given that the initial split in our tree separates a southern species (U. malama) and a predominantly northern clade (Fig. 1). It needs to be mentioned, however, that U. malama has not been included in previous multi-gene assessments of Uroplatus phylogeny (Greenbaum et al. 2007; Raxworthy et al. 2008), and its very strong differentiation encountered here and in Ratsoavina et al. (2011) suggests that its attribution to the U. ebenaui group requires confirmation from additional studies that include more species of Uroplatus and additional DNA sequences, preferably of nuclear genes. After the split of U. malama, the next species and candidate species splitting off the U. ebenaui group (Fig. 1) all occur in the northern half of Madagascar, and most 152 RATSOAVINA ET AL.*Phylogeny and phylogeography of the Uroplatus ebenaui group

in an area located roughly north of 158 S. This relationship little doubt that the diversification of this clade of geckos has mainly occurred in the environmentally heterogeneous areas of northern Madagascar. That U. phantasticus is phylogeneti- cally nested among exclusively northern lineages suggests that its ancestral population at some point expanded its range southwards, thereby colonising the vast mid-altitude areas of rainforest in the northern and southern regions of central eastern Madagascar (morphological variation shown in Fig. 3).

Phylogeographic pattern of U. phantasticus Our phylogeny suggests a population of small-sized Uroplatus from Zahamena being the sister group of a clade comprising all populations of U. phantasticus. Unfortunately, no morphological data are available for this Zahamena population, but its differentiation is confirmed by a large number of ND4 sequences from additional specimens from this locality (F. Ratsoavina, unpublished data). Taxono- mically, the status of this population is thus difficult to assess based on its high genetic divergence to other U. phantasticus populations (p-distance for ND4: 23.2% to the Anjozorobe and Kianjavato populations and 21.5% to the Marolambo population). We have here designated it as an unconfirmed candidate species. Despite this taxonomic uncertainty, it is relevant from a biogeographic perspective in that it is located north of all other populations attributed to the U. phantasticus clade, and thus provides further evidence for a northern origin and southwards expansion of this species. Within U. phantasticus, phylogenetic arrangement of the main Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012

Figure 3. Morphological chromatic variation of Uroplatus phantasticus females found in Ranomafana (A), in Kianjavato (B) and in Betampona (C). This figure is included in colour in the online version of this article. AFRICAN JOURNAL OF HERPETOLOGY 61(2) 2012 153

mitochondrial lineages also tentatively supports a northsouth expansion, although the signal is mixed. The first lineage splitting off (Anosibe an’ala) is from a region north of the Mangoro River belonging to the northern half of the species’ range, but the population does not represent the northernmost locality. The next node ends with two clades geographically separated by the Mangoro River as northern and southern clades. In this last group, the northernmost population (Marolambo) is again sister to the lineages occurring at the southern limit of the range (Ranomafana and Kianjavato). The existence of deeply differentiated and geographically restricted mitochon- drial lineages in U. phantasticus, together with a weak but regular signal of southern lineages nested among more northern ones, leads us to hypothesise that this species originated in northern Madagascar, and colonised its current distribution area through intermittent episodes of southwards range expansions and genetic isolation. The high level of differentiation among lineages and phylogeographic structure in these geckos indicates that these processes occurred over a considerable amount of time, similar to the pattern found in other geckos, e.g. in the Neotropics (Pellegrino et al. 2005). This pattern is very different from that found in the frogs Mantella baroni and M. nigricans which are equally distributed in mid-altitudes from south-eastern to northern Madagascar, and where a single cytochrome b haplotype is prevalent in all populations, suggesting a very recent expansion history and/or maintenance of gene flow (Rabemananjara et al. 2007). The fact that U. phantasticus populations north and south of the Mangoro River form monophyletic groups suggests that this large riverine barrier might have played a role in causing or maintaining this lack of gene flow among subpopulations (see also Knopp et al. 2011).

Riverine barrier influences on U. phantasticus in Ranomafana and Kianjavato Our intensive sampling in the southern half of central eastern Madagascar around Ranomafana and Kianjavato at localities along the Namorona River provides additional evidence of riverine barriers influencing biogeographic patterns in this species. The Namorona River is a relatively small river and at Ranomafana is only about 20 m wide, with submerged rocks among rapids theoretically making a passage possible by crossing only a few metres of water. Additionally, the narrowness of this river potentially allows for tree branches or large fallen trees to span the river in places along its course. Already this relatively modest barrier apparently impacts Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012 gene flow among populations of U. phantasticus in Ranomafana based on the specimens sampled and sequenced by us, since the two distinct haplotype lineages were exclusively found on either side of the river (Fig. 2). Because U. phantasticus depends on intact rainforest and is difficult to find in many forests, including the northern parcels of Ranomafana National Park, our sampling is limited (9 specimens north and 26 specimens south of the Namorona River). Despite this restriction, our data suggest a barrier function of this river, and future research analysing the differentiation of a variety of amphibian and reptile species occurring in forest parcels on opposite sides of the river may yield important information on their ability to cross such barriers. 154 RATSOAVINA ET AL.*Phylogeny and phylogeography of the Uroplatus ebenaui group

By including the additional mitochondrial lineage from Kianjavato, a locality at considerably lower elevation (ca. 600 m vs. ca. 1 000 m) in the analysis, we could determine that riverine barriers, at least small ones such as the Namorona River, are not insurmountable for leaf-tail geckos. Although Kianjavato is located north of the river, it is inhabited by a mitochondrial lineage that clearly is more closely related to that south of the river in Ranomafana. A more fine-scale population sampling would be needed to compare phylogeographic patterns with those expected in different scenarios of river barrier diversification (Patton et al. 2000; Vences et al. 2009). Given the difficulties of finding leaf-tail geckos, especially in disturbed habitat and with the fast pace of habitat destruction in Madagascar, this task is challenging and will require future intensive fieldwork. The possible specialisation of mtDNA lineages to particular elevations also requires further attention, given that adaptive diversifica- tion thus far has rarely been considered a possible driver of species diversity in Madagascar.

ACKNOWLEDGEMENTS

We are indebted to Shannon Engberg, Gaby Keunecke, Meike Kondermann, Runhua Lei and Eva Saxinger for their help in the laboratory, and to numerous friends and colleagues who have assisted during fieldwork and data analysis, in particular to Angelica Crottini, Marcelo Gehara, Sebastian Gehring, Frank Glaw, Susanne Hauswaldt, Andrew Koraleski, Aure´lien Miralles, Maciej Pabijan, Emile Rajeriarison, Theo Rajoafiarison, Ando Rakotoarison, Franc¸ois Randrianasolo, Roger Randrianiaina, Solohery Rasamison and David Vieites. We are grateful to the Malagasy authorities for issuing research and export permits. We are grateful to anonymous reviewers for comments on an early version of the manuscript. Financial support was provided by the Volkswagen Foundation to FMR and MV and by Omaha’s Henry Doorly Zoo and Aquarium to EL.

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Received: 18 July 2012; Final acceptance: 11 September 2012 AFRICAN JOURNAL OF HERPETOLOGY 61(2) 2012 157

ONLINE SUPPLEMENTARY MATERIAL

List of voucher specimens (marked with *) and tissue samples used as representative sequences of each lineage cited in the study. Accession numbers are from COI sequences submitted to Genbank.

Species/Locality Field number Accession number

U. phantasticus Anjozorobe FGZC 4341* JX205365 Anjozorobe FGZC 4342* JX205366 Anjozorobe FGZC 4384* JX205367 Anjozorobe FGZC 4385* JX205368 Anosibe an’Ala FGZC 4505* JX205369 Marolambo PSG 1348 JX205370 Marolambo PSG 1346 JX205371 Fierenana 2002A30 JX205372 Vohidrazana 2002-986 JX205373 Kianjavato KAF 179 JX205374 Kianjavato KAF 183 JX205375 Kianjavato KAF 184 JX205376 Kianjavato KAF 190 JX205377 Kianjavato KAF 191 JX205378 Kianjavato KAF 194 JX205379 Kianjavato KAF 209 JX205380 Kianjavato KAF 237 JX205381 Ranomafana MPFC 501 JX205382 Ranomafana MPFC 503 JX205383 Ranomafana MPFC 509 JX205384 Ranomafana MPFC 510 JX205385 Ranomafana MPFC 511 JX205386 Ranomafana FGMV 2002.639* JX205387 U. phantasticus [Ca10] Zahamena ZAH 87 JX205388 Zahamena ZAH 88 JX205389 Zahamena ZAH 91 JX205390 Zahamena ZAH 96 JX205391 Zahamena ZAH 118 JX205392 Zahamena ZAH 257 JX205393 U. ebenaui [Ca1] Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012 Analabe ZCMV 12275* JX205394 Analabe ZCMV 12276* JX205395 Analabe ZCMV 12277* JX205396 Analabe DRV 6409* JX205397 Analabe DRV 6418* JX205398 Ambodikakazo ZCMV 12503* JX205399 Ambodikakazo MPFC 562 JX205400 Ambodikakazo MPFC 563 Ambodikakazo MPFC 564 JX205401 Ambodikakazo DRV 6325* JX205402 158 RATSOAVINA ET AL.*Phylogeny and phylogeography of the Uroplatus ebenaui group

(Continued )

Species/Locality Field number Accession number Ambodikakazo DRV 6326* JX205403 Ambinanitelo DRV 6263* JX205404 U. ebenaui [Ca7] Fierenana 2002A31 JX205405 U. ebenaui [Ca2] Tsaratanana ZCMV 12388* JX205406 Tsaratanana ZCMV 12389* JX205407 Tsaratanana DRV 6192* JX205408 Tsaratanana DRV 6248* JX205409 Tsaratanana DRV 6249* JX205410 U. ebenaui [Ca3] Andrevorevo DRV 6280* JX205411 Andrevorevo DRV 6281* JX205412 Marojejy ZCMV 2030* JX205413 U. ebenaui [Ca4] Makira ZCMV 11309* JX205414 U. ebenaui Manongarivo FGMV 2002.826* JX205415 Manongarivo FGMV 2002.2205* JX205416 Manongarivo FGMV 2002.2403* JX205417 Nosy Be ACZC 1199* JX205418 Foreˆtd’Ambre FGZC 3153* JX205419 U. ebenaui [Ca1] Manarikoba 2001F10 JX205420 U. ebenaui [Ca5] Ankarana FGZC 552* JX205421 U. finiavana Joffreville ACZC 1420 JX205422 Joffreville ACZC 1426 JX205423 Montagne d’Ambre FGZC 619* JX205424 Montagne d’Ambre FGZC 621* JX205425 Montagne d’Ambre FGZC 622* JX205426 Montagne d’Ambre FGZC 624* JX205427 Montagne d’Ambre FGZC 625* JX205428 Montagne d’Ambre FGZC 626* JX205429

Downloaded by [Miss Fanomezana Mihaja Ratsoavina] at 06:00 27 October 2012 Montagne d’Ambre FGZC 1096* JX205430 U. malama Beampingaratra MPFC 407 JX205431 Beampingaratra MPFC 411 JX205432 Beampingaratra MPFC 416 JX205433