Methods
Phylogeny estimation. Species were collected from 25 sites that represent the major volcanoes on each of the five main Hawaiian Islands. The analysis included a total of 209 individuals representing 26 species. Each species is represented by a single population with the exception of Laupala paranigra, for which two populations were sampled. All individuals were collected from the wild and transported to the mainland alive. The wings, right hind leg, and genitalia of each individual were retained as voucher specimens. Nearly all individuals in the analysis were male and were therefore identified to species according to Otte1.
For the AFLP assay, genomic DNA was extracted using standard phenol/chloroform extraction procedures. Procedures generally followed that of Vos et al.2. Briefly, restriction enzymes EcoRI and PstI were used to digest genomic DNA, and ligation of EcoRI and PstI adapters took place concurrently with digestion. Two increasingly selective PCR amplifications of the ligated DNA were conducted. For the first amplification, 2 ul of a 1:10 dilution of restriction-ligation product was used as template. Primers for this preamplification were EcoRI and PstI primers with one additional selective nucleotide (EcoRI: 5'-GACTGCGTACCAATTC+A; PstI: 5'-GACTGCGTACATGCAG+A). The second selective amplification was conducted using 2 ul of a 1:400 dilution of preamplification product as template. Primers were the same as in preamplification, but with two additional selective nucleotides; for the selective amplification the EcoRI primer was fluorescently labelled (Qiagen, Inc) for automated fragment analysis. In total, 18 unique combinations of primer pairs were used: EcoRI +AAC was paired with PstI +AAA/ +AAC/ +AAG/ +ACG/ +ACT/ +AGA/ +AGG/ +ATA, and EcoRI +AGC was paired with PstI +AAA/ +AAC/ +AAG/ +ACC/ +ACG/ +ACT/ +AGA/ +AGG/ +AGT/ +ATA.
For automated fragment analysis, selective PCR products were injected with a size standard (GS-500 ROX, Applied Biosystems, Inc.) into an ABIPrism 3100 DNA Analyzer (Applied Biosystems, Inc.). Fragments were sized in GeneScan Analysis v3.7. AFLP data were scored in Genotyper® 2.5 (Perkin-Elmer Corp.) by noting the presence or absence of same-sized fragments, or bands. Bands used in scoring ranged in size from 75 bp to 490 bp. Bands were deemed homologous if the difference in size was less than 1 bp. The assumption that co-migrating (same-sized) AFLP bands represent homologous genetic regions in Laupala has been supported by direct sequencing of bands in previous studies3,4.
A presence/absence matrix ("1" for present, "0" for absent) was generated for all scorable bands for all individuals. This matrix was translated into a distance matrix using the Nei-Li equation for genetic distance. This matrix was used to estimate a neighbour-joining tree in PAUP*(5). A heuristic search using the weighted least squares criterion yielded the same topology. Bootstrap support for each node was estimated based on 1,000 replicates using the NJ algorithm. The tree was rooted with Prolaupala kukui, a member of the putative sister genus to Laupala1. Whereas previous studies of mitochondrial and nuclear DNA sequence analyses were unable to generate a resolution of species relationships sufficient to estimate rates of speciation in this genus6,7, AFLP data yielded a highly resolved phylogenetic tree which is largely consistent with the phylogeny derived from nuclear sequence data7.
Speciation rates. In each of the two main lineages, species endemic to Hawai’i Island form a monophyletic clade; thus, the most recent common ancestor (MRCA) of each of these clades is estimated to be no older than the age of Hawai’i, or 0.43 Myr8. Likewise, the MRCA of the clades that include the Maui-Nui and Hawai’i Island species is no older than the age of the Maui-Nui complex (1.9 Myr)8, and the MRCA of the two main lineages that comprise the genus is no older than the age of O’ahu (3.7 my)8. These dates were used to estimate rates of diversification (Table S1) using the simple estimator:
(1),
where N is the number of extant species, N0 is the number of founding lineages, t is divergence time, and is the rate of diversification. For very recently diverged lineages, a pure birth model with no extinction is assumed9, so is also the rate of speciation. For monophyletic lineages with a single common ancestor, N0 = 1; thus for young, monophyletic clades, the speciation rate is estimated as = SRln = ([lnN]/t)10,11. Units of this estimator are species per million years. Due to a lack of resolution, the species L. hapapa, L. oahuensis, and L. pacifica were considered a single species for purposes of rate estimation. Due to rate heterogeneity among lineages, alternative methods for estimating speciation rates that incorporate the sum of calibrated branchlengths12 were not used. The precision with which the islands can be dated allows a maximum, and therefore conservative, age of each clade to be reliably inferred in the absence of branchlength estimation.
1. Otte, D. The Crickets of Hawaii: Origin, Systematics, and Evolution (Orthoptera Society/Academy of Natural Sciences of Philadelphia, 1994).
2. Vos, P. et al. Nucleic Acids Res. 23, 4407-4414 (1995).
3. Parsons, Y. M. & Shaw, K. L. Mol. Ecol. 10,1765-1772 (2001).
4. Mendelson, T. C., Siegel, A. M. & Shaw, K. L. Mol. Ecol. 13, 3787-3796 (2004).
5. Swofford, D. L. PAUP*, Phylogenetic Analysis Using Parsimony (*and Other Methods) (Sinauer Associates, Sunderland, Massachussetts, 2000).
6. Shaw, K.L. Evolution 50, 256-266 (1996).
7. Shaw, K. L. Proc. Natl. Acad. Sci. USA 99,16122-16127 (2002).
8. Clague, D. A. & Dalrymple, G. B. in Volcanism in Hawaii (eds. Decker, R. W., Wright, T. L., & Stauffer, P. H.) 1-54 (U.S. Geological Survey Professional Paper 1350, U.S. Government Printing Office, Washington DC, 1987).
9. Yule, G. Phil. Trans. R. Soc. Lond. B 213, 21-87 (1925).
10. Coyne, J. A. & Orr, H. A. Speciation (Sinauer Associates, Sunderland, Massachusetts, 2004).
11. McCune, A. R. in Molecular Evolution and Adaptive Radiation (eds Givnish, T. J. & Sytsma, K. J.) 585-610 (Cambridge Univ. Press, New York, New York 1997).
12. Baldwin, B. & Sanderson, M. J. Proc. Natl. Acad. Sci USA 95, 9402-9406 (1998).
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