Transpacific species are particularly prominent among shore fishes (species with relatively sedentary adult phases that live along the shoreline), in which 95 species (roughly 18% of all tropical EP reef fishes) are thought to have resident, locally breeding, populations on both sides of the EPB (Robertson et al. 2004
). We studied 20 such tropical species (). We collected 5–25 individuals (mean sample size 15.65) per species in the EP and 6–25 (mean sample size 16.15) in the CP. All but three species were collected from more than one locality in each region (see figure 1 in electronic supplementary material). Genomic DNA was extracted from each individual, and an 842
bp fragment of the ATPase8 and ATPase6 gene regions of mitochondrial DNA was amplified and sequenced. Details of the methods are provided in the electronic supplementary material.
Table 1 Analysis of molecular variance (AMOVA; Excoffier et al. 1992), comparing variation within and between the eastern (EP) and the central Pacific (CP) and nucleotide diversity within each region. (Values in parentheses in Acanthurus triostegus are from calculations (more ...)
Program TCS v. 1.18 (Clement et al. 2000
) was used for the construction of statistical parsimony (Templeton et al. 1992
) networks with the confidence of connection limits set at 95%. Program Arlequin
v. 2.0 (Schneider et al. 2000
) was used for calculations of analysis of molecular variance (Excoffier et al. 1992
) and FST
statistics. To generate the null distributions for assessing the significance of the FST
000 permutations of haplotypes between populations were used.
To distinguish recent population splitting from recurrent gene flow and to determine direction of gene flow, we employed Bayesian estimation, based on coalescence, according to the procedure developed by Nielsen & Wakeley (2001)
and by Hay & Nielsen (2004)
. The method uses gene genealogies to estimate effective population size of ancestral and daughter populations, the time since their initial separation (i.e. the time since vicariance or the last massive invasion) and the migration rate in each direction. We used Program IM (Hay & Nielsen 2004
) to estimate the times of separation t
(number of generations, scaled by mutation rate, μ
) between EP and CP populations, θ
is the effective population size of the ancestral and the two daughter populations, each estimated separately) and the scaled migration rate m
is the proportion of migrants arriving into a population per generation) in each direction. As coalescence estimations assume that each population is effectively panmictic, we pooled samples of the same species from different localities within a region only if FST
statistics (table 1 in electronic supplementary material) under the island model indicated that they exchanged more than one female per generation (if FST
values were less than 0.33 or if they were not significant). Populations with more restricted intra-regional gene flow were compared separately to those on the other side of the barrier. Analyses were implemented assuming that base substitution followed the Hasegawa et al. (1985)
model. Details of the IM runs and of the methods used to estimate whether differences between parameter estimates were statistically significant are presented in the electronic supplementary material. As IM makes a number of simplifying assumptions regarding population history and as our data consist of a single locus, we regard the results of this procedure as hypotheses to be further tested with additional data.
From the results of IM, twice the number of females moving through the barrier per generation (M
) was calculated as M
/2 (where mf
is the female migration rate). The time since separation was converted from number of generations scaled by mutation rate to number of years using a mutation rate estimate of the sequenced fragment from six other fish genera, with species-pairs, the members of which were likely to have been separated by the closure of the Isthmus of Panama (see table 2 in electronic supplementary material), 3.1
Myr ago (Coates & Obando 1996
). These transisthmian genera were selected on the basis of their similar amounts of divergence in cytochrome oxidase I, as determined by Bermingham et al. (1997)
. ATPase8 and -6 were amplified and sequenced from a minimum of two individuals per species on each side of the isthmus, with the methods described above, then the divergence in six species pairs was averaged to obtain a rate of 1.3×10−8
substitutions per site per year or a substitution rate per branch for the entire fragment of 5.49×10−6
substitutions per year (see electronic supplementary material).