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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Molecular Phylogenetics and Evolution 52 (2009) 530–537 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Short Communication Multigene phylogeny of Malagasy day geckos of the genus Phelsuma S. Rocha a,b,c,*, M. Vences d, F. Glaw e, D. Posada c, D.J. Harris a,b a CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Instituto de Ciências Agrárias de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal b Departamento de Zoologia e Antropologia, Faculdade de Ciências, Universidade do Porto, Praça Gomes Teixeira 4099-002, Portugal c Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo, Vigo 36310, Spain d Technical University of Braunschweig, Zoological Institute, Spielmannstr. 8, 38106 Braunschweig, Germany e Zoologische Staatssammlung München, Münchhausenstr. 21, 81247 München, Germany article info Article history: Received 19 November 2008 Ó 2009 Elsevier Inc. All rights reserved. Revised 18 March 2009 Accepted 29 March 2009 Available online 9 April 2009 1. Introduction chromatic characters and not well defined. In several cases color transitions/polymorphisms can be observed and thus some species Geckos of the genus Phelsuma are amongst the most conspicu- may represent artificial taxa based on local color morphs. Previous ous lizards of the Malagasy region. The genus contains around 43 studies, based on phenetic characters (Loveridge, 1942; Mertens, extant species, of which ca. 29 occur in Madagascar, and 25 of 1962; Glaw et al., 1999; Van Heygen, 2004), have led to recognition these are endemic to this island. Rhoptropella ocellata, endemic to of eight species groups of Malagasy species. Close relationships be- a small region of Namibia and South Africa is, together with Lygo- tween some of the groups were postulated (e.g. P. guttata- and P. dactylus sp., the closest relative of Phelsuma, which is considered a madagascariensis-group and the Mascarene Islands species with monophyletic genus (Austin et al., 2004). With Madagascar as their the P. modesta-group) but some species were not assigned to any main centre of diversity, Phelsuma are also present on almost all particular phenetic group (e.g. P. vanheygeni). neighbouring islands and locally at the East African coast (Fig. 1). The main potential difficulty with Phelsuma taxonomy is thus The Comoros harbour seven species (five of them endemic), the its reliance on highly variable coloration characters, which has Mascarenes have seven endemic extant species (two extinct spe- led to the description of a large number of species and subspecies. cies are known: P. gigas and P. edwardnewtoni), with one subspe- Taxon sampling has always been incomplete in previous molecular cies endemic to Agalega island (1100 km north of Mauritius), one studies of Phelsuma (Radtkey, 1996; Austin et al., 2004; Sound (non-endemic) species in two islands of the Aldabra archipelago, et al., 2006; Rocha et al., 2007; Raxworthy et al., 2007; Harmon two endemic species in the granitic islands of the Seychelles, one et al., 2008), making it difficult to evaluate intrageneric relation- endemic species in the island of Pemba off the East African coast ships. Additionally, the validity of many taxa is yet to be assessed of Tanzania, and one species in the Andaman islands, off Myanmar, using molecular markers. in the Bay of Bengal. Within Madagascar, Phelsuma species are Here, we present the most comprehensive molecular phyloge- found in a variety of habitats spanning the island, mainly in pri- netic analysis of Phelsuma to date, based on a near-complete taxon mary forest regions, either dry southern seasonal forests (e.g. P. sampling of all but two species, as well as most subspecies, and on breviceps), scrublands (e.g. P. mutabilis, P. modesta, P. hielscheri), a multilocus dataset including both fast-evolving mitochondrial western coastal forests (e.g. P. abbotti), low and mid-altitude hu- genes and more moderate-to-slow-evolving nuclear DNA markers. mid forests (P. madagascariensis, P. guttata, P. lineata ssp., P. quad- riocellata ssp.) and even in some high-altitude regions (P. 2. Materials and methods barbouri, P. malamakibo). Despite extensive work on Phelsuma taxonomy, ecology, bioge- 2.1. Biological material, DNA isolation and sequencing ography and ethology, even its alpha-taxonomy is not fully under- stood. Many taxa, especially subspecies, are based only on Tissue samples (tail tips) of 104 specimens of Phelsuma and five additional taxa (outgroup) were obtained during fieldwork in Madagascar, Seychelles, the Comoros and Tanzania in the * Corresponding author. Address: CIBIO, Centro de Investigação em Biodiversid- period 2000–2006 and preserved in 70–100% ethanol. Additional ade e Recursos Genéticos, Instituto de Ciências Agrárias de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal. Fax: +351 252 661 780. samples were obtained from breeders. All recognized Phelsuma E-mail address: [email protected] (S. Rocha). species, with the exception of P. masohoala and P. guimbeaui, 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.03.032 Author's personal copy S. Rocha et al. / Molecular Phylogenetics and Evolution 52 (2009) 530–537 531 Fig. 1. Distribution of Phelsuma in the Indian Ocean Region and number of species per island/archipelago. The number of endemic species is given in parenthesis, when different from the total. Lighter (non-delimitated) areas correspond to areas of lower sea-level. and most recognized subspecies were represented (Table 1). We 2.2. Sequence alignment and phylogenetic analyses refrained from including partial sequences of P. guimbeaui avail- able from Genbank (cytochrome b, 16S rRNA and C-mos)to Cytochrome b, Rag-1 and Rag-2 gene sequences were aligned avoid large amounts of missing data in our data set, and be- manually using BioEdit (Hall, 1999). Within C-mos, indels were cause this species clearly belongs into the well-studied Masca- present and thus nucleotide sequences were translated to amino rene clade (Austin et al., 2004). For species with ample acid sequences to aid the alignment. The 16srRNA nucleotide data- distributions, an effort was made to include samples from wide- set contained highly variable regions and many indels, and there- spread locations to obtain preliminary information on intraspe- fore ProAlign version 0.5a3 (Löytynoja and Milinkovitch, 2003) cific variation. DNA extraction followed standard salt or phenol– was used. Hidden Markov Model parameters delta and epsilon were chloroform protocols (Kocher et al., 1989). Fragments of two estimated from the data, while remaining parameters were set to mitochondrial (16s rRNA and cytochrome b) and three nuclear their default values. All the columns with posterior probabilities (C-mos, Rag-1 and Rag-2) genes were amplified via PCR. Primers lower than 90 were removed, for a final alignment of 384 bp. All used were 16sA-L and 16sB-H for 16srRNA (Palumbi et al., protein-coding genes were translated to amino acids to check for 1991); forward CBL14753 (Austin et al., 2004) and CBL14841 stop codons that could indicate the presence of pseudogenes. (Austin et al., 2004, modified from Kocher et al., 1989) and re- The concatenated data were filtered to remove redundant se- verse CB3H (Palumbi et al., 1991) and CBH15579 (Rocha et al., quences, resulting in a data set with 84 sequences. The appropriate 2007) for cytochrome b; G73 and G74 (Saint et al., 1998) for C- model of nucleotide substitution for each gene was determined mos; L2408 and H2920 for Rag-1 (Vidal and Hedges, 2004) and using PAUP* 4.0b10 (Swofford, 2002) and Modeltest 3.06 (Posada Lung35F, Lung320R, Lung460R and 31FNVenk (Hoegg et al., and Crandall, 1998) under the AIC criterion (Akaike, 1974). Models 2004) for Rag-2. Amplified fragments were of 545, 714, 344, were determined also for each codon position of the protein coding 473 and 796 bp, respectively, for 16srRNA, Cyt-b,C-mos, Rag-1 genes. v2 tests of sequence composition and likelihood mapping and Rag-2 fragments. All PCR amplifications were conducted analysis (Strimmer and von Haesler, 1997), which allow an a priori in 25 ll reactions using standard conditions. Annealing temper- examination of conflict versus support signal in molecular se- atures varied between 50 °C and 52 °C for 16srRNA, 42 and quence data, were performed using Tree-Puzzle 5.0 (Schmidt 53 °C for Cyt-b, 46 and 56 °C for C-mos, 52 and 57 °C for Rag- et al., 2002). 1 and 53 and 57 °C for Rag-2 and magnesium concentration Bayesian phylogenetic analyses were conducted using MrBayes varied between 1 and 4 mM. Amplification products were di- 3.1.2 (Huelsenbeck and Ronquist, 2001). These began with a ran- rectly purified using a standard enzyme procedure or were dom starting tree and were run for 11,000,000 generations (20 mil- sometimes cut from the gel and then purified using a gel band lion in the case of Cyt-b), and sampled every 1000 generations. purification kit (Amersham Biosciences). Amplified fragments Multiple runs were always performed. Convergence was checked were sequenced on an ABI 3730xl automated capillary DNA se- using AWTY (Wilgenbusch et al., 2004), which provides plots of quencer.