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Material and methods
Specimens were collected during fieldtrips across the Indian Ocean and Pacific Islands and additional samples obtained from museums (see Fig. 1 and Appendix 1 for locality details).
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Fig. 1. Sampling localities: Seychelles (1); Aldabra (2); Madagascar (3); Mascarenes (4); Myanmar (5-7); Malaysia (8); Luzon, Philippines (9); Palau (10); Mariana (11); Caroline (12, 13); Papua New Guinea (14); Vanuatu (15); Hawaii (16); French Polynesia (17) and Clipperton Atoll (18) (see Appendix 1 for exact location and details on origin of the samples). Localities harbouring different mtDNA lineages (see results) are colour coded (black versus white dots). |
Genomic DNA was extracted following a standard high-salt protocol (Sambrook et al., 1989) and part of the 16s rRNA gene (517 bp) was amplified and sequenced using primers 16sA-L and 16sB-H (Palumbi et al., 1991). Amplification conditions are described in Harris et al. (1998). Amplified fragments were directly purified with ExoSap-IT (Amersham-Pharmacia Biotech) and sequenced on an ABI 3730xl automated capillary DNA sequencer and aligned with available Gehyra sequences from GenBank. Generated sequences are deposited in GenBank under accession numbers FJ613425-FJ613473 and the full alignment is available on the TreeBASE website (www.treebase.org/treebase; study accession number = S2324). As the pattern of genetic variation was clear, evolutionary history was inferred using the Minimum Evolution method (Rzhetsky and Nei, 1992), with the evolutionary distances computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and presented in units of number of base substitutions per site. One thousand bootstrap replicates (Felsenstein, 1985) were performed to estimate nodal support. The ME tree was searched for using the Close Neighbor Interchange (CNI) algorithm (Nei and Kumar, 2000) at a search level of 2. The Neighbor Joining algorithm (Saitou and Nei, 1987) was used to generate the initial tree. Due to the shorter size of some of the sequences (Genbank sequences DQ857338/Madagascar; DQ270549 and AY517559/Mauritius), positions containing gaps and missing data were eliminated from the dataset (‘complete gap deletion’ option), leaving a total of 318 positions in the final dataset, from which the ME tree topology was inferred. Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007). Additionally, a haplotype median-joining network (Bandelt et al., 1999) was obtained using NETWORK4.5 (Fluxus Engineering, Suffolk, UK), in which the shorter sequences were excluded, indels of more than 1bp were coded as single events, and the full fragment length (517 bp) was considered.