Sequences of the CO1 mitochondrial gene fragment in five
individuals across islands showed almost no variation, with two
individuals (one Santa Cruz highland and one Floreana highland) having
an identical single nucleotide substitution (T-G). This supports the
existence of one sampled species across the three islands.
Genetic diversity and differentiation
Probability of identity (PI) analyses showed that the microsatellite
loci had sufficient power and resolution for the analyses. The unbiased
PI value was 1.333-06, and the PI for sibs was 2.610-3.
This equates to one individual in approximately 751 880 having a
non-unique genotype where individuals are unrelated (unbiased), and one
individual in approximately 383 individuals having a non-unique
genotype if all individuals are siblings.
The total number of alleles observed at each locus was as follows;
Pd1 = 4; Pd2 = 3; Pd4 = 4; Pd6 = 5; Pd7 = 3; Pd8 = 4; Pd9 = 3; Pd10 = 3
(Table 2). There was significant genotypic differentiation across the three islands (Fisher's Exact method: X2 =
72.75; df = 16; P < 0.001). Mean allelic richness across loci was
almost identical on each island (Santa Cruz: 3.50; Floreana 3.63;
Isabela: 3.5) and the range of observed heterozygosity across loci was
also similar (Santa Cruz: 0.45–0.70; Floreana: 0.45–0.73; Isabela:
0.44–0.89). The number and size of alleles from each island population
were the same at each locus with two exceptions: there was a unique
allele at locus Pd6 on Isabela (allele frequency = 0.055), and at locus
Pd7 on Floreana (allele frequency = 0.012), which were each detected
only in a single individual. Pairwise Fst analysis showed low, but significant levels of genetic differentiation between Santa Cruz and Floreana (Fst = 0.02, P < 0.02) Isabela and Floreana (Fst = 0.04, P < 0.02), but not between Santa Cruz and Isabela (Fst
= 0.01, P > 0.1). The low genetic differentiation found between
islands was reflected in an AMOVA, which showed that just 2% of the
molecular variance was attributable to variation among islands (sum of
squares (SS) = 14.23; df = 2; variance components (V) = 0.052), 4%
among individuals (SS = 413.06; df = 155; V = 0.133), and 94% within
individuals (SS = 385.5; df = 158; V = 2.44).
Table 2. Allele frequencies for eight microsatellite loci in P. downsi within two genetic clusters.
Combining individual from all islands (n = 158), a clear excess of
heterozygosity (He) relative to the equilibrium heterozygosity (Heq)
was observed, indicative of a population bottleneck under the TPM model
(Wilcoxon sign-rank test; P < 0.01) and under the SMM model (P <
0.01). A mode-shift distortion in the distribution of allele
frequencies was evident (Figure 2).
Figure 2. Distribution of allele frequencies indicating a mode-shift.
Bars represent the proportion of alleles found in each allele frequency
class. Deviation from an L-shaped distribution is indicative of a
mode-shift in allele frequency due to a recent genetic bottleneck.
Bayesian clustering analysis
Individual-based cluster analysis using STRUCTURE did not detect any genetic structuring in P. downsi collected across the three islands (Figure 3a), with individual assignment being evenly proportioned across variable numbers of k.
This implies high levels of inter-island ancestry brought about by
frequent dispersal and subsequent gene flow across the three islands
sampled. However, when incorporating geographic coordinates of sampling
locations into Bayesian analyses using GENELAND, two distinct genetic clusters were consistently found across runs (Figures 3b and 4).
The first cluster includes all individuals sampled from Santa Cruz and
Isabela Islands (n = 62), while the second cluster includes all
individuals sampled from Floreana Island (n = 76). Assignment
probabilities were between 0.98 and 1.0 across all individuals.
Figure 3. Estimated number of populations from STRUCTURE (a) and GENELAND (b) analyses. (a) Mean ( ± SD) probabilities of the data (LnPr [X|k]) over three replicate STRUCTURE runs plotted as a function of the putative number of clusters (k); (b) Posterior density distribution of the number of clusters estimated from GENELAND analysis in three replicates.
Figure 4. Genetic assignment of P. downsi individuals across three islands using Bayesian clustering analysis.
Two genetic clusters are identified: (a) including all individuals from
Santa Cruz (n = 62) (bottom left) and Isabela (n = 9) (centre top), and
(b) all individuals from Floreana Island (n = 76) (bottom right). Black
dots represent independent geographic sampling points (i.e. location of
bird nests). Note that two geographic sampling points on Isabela Island
were within 5 m of each other and are not distinguishable.
Genetic diversity and differentiation among inferred clusters
The two clusters identified by GENELAND displayed
comparable genetic diversity with regard to allelic richness and
differed slightly in heterozygosity across loci (Tables 2 and 3). Although two clusters were detected, measures of genetic differentiation (Fst) between them demonstrated the low divergence between individuals on Floreana Island and those on Santa Cruz and Isabela (Fst
= 0.024; 95% Confidence Interval (CI) = 0.014 – 0.034; P < 0.05).
Tests of departure from HW equilibrium showed no significant deviation
in either of the two clusters across all loci.
Table 3. Genetic variation at the eight microsatellite loci for the two P. downsi populations inferred from cluster analysis in GENELAND.