Conservation Genetics (2006) 7:105115 DOI 10.

1007/s10592-005-8704-6

Springer 2006

Population genetic structure and conservation of the Galapagos petrel (Pterodroma phaeopygia)
Vicki L. Friesen1,*, Jose A. Gonzalez2 & Francisco Cruz-Delgado3
1

Department of Biology, Queens University, Kingston, Ontario, K7L 3N6, Canada; 2Agencia Espanola de Cooperacion Internacional Programa Araucaria, Francisco Salazar E12-73 y Toledo, Quito, Ecuador; 3 Parque Nacional Galapagos, Ocina Tecnica San Cristobal, Galapagos, Ecuador (*Corresponding Author: Phone: +1-613-533-6156; Fax: +1-613-533-6617; E-mail: friesenv@biology. queensu.ca)
Received 24 March 2005; accepted 14 June 2005

Key words: conservation, Galapagos Islands, Galapagos petrel, management unit, population genetic structure, Pterodroma phaeopygia

Abstract The Galapagos petrel (Pterodroma phaeopygia) is endemic to the Galapagos archipelago, where it is known to breed only on ve islands. The species has been listed as critically endangered due to habitat deterioration and predation by introduced mammals. Signicant morphological and behavioural dierences among petrels nesting on dierent islands suggest that island populations may dier genetically. Furthermore, nesting phenology suggests that genetically dierentiated seasonal populations may exist within at least one island. We analysed variation in six microsatellite loci and part of the mitochondrial ATPase 6/8 gene in 206 Galapagos petrels sampled from all ve islands. No evidence of genetic structuring within islands was found, although statistical power was low. In contrast, signicant dierences occurred among island populations. For the microsatellite loci, private alleles occurred at all islands, sometimes at high frequency; global and pairwise estimates of genetic dierentiation were all statistically signicant; Bayesian analysis of genotypes frequencies provided strong support for three genetic populations; and most estimates of migration between populations did not dier signicantly from zero. Only two ATPase haplotypes were found, but the geographic distribution of haplotypes indicated signicant dierentiation among popula tions. For conservation purposes, populations from Floreana, Santa Cruz, San Cristobal and Santiago should be regarded as separate genetic management units. Birds from Isabela appear to be derived recently from the Santiago population, and the population on San Cristobal appears to be a mixture of birds from other populations. However, considering ecological and behavioural dierences among birds from dierent islands, we recommend that all ve populations be protected.

Introduction Evolutionary processes leading to high endemism are characteristic of oceanic archipelagos. The Galapagos Islands are world renowned as a storehouse of unique terrestrial and marine biological diversity and as a natural laboratory for evolution and speciation. At a global scale, the archipelago supports a low diversity of ora and fauna, but the

percentage of species that are endemic is exceptionally high, reaching 32% of vascular plants and 59% of land vertebrates (Tye et al. 2002). Interisland variation is also very high, with various islands harbouring genetically distinct populations, races and subspecies, reecting dierent stages of genetic radiation (CDF-WWF 2002; e.g. Petren et al. 1999; Freeland and Boag 1999; Cio et al. 2002; Beheregaray et al. 2004).

106 The Galapagos petrel (Pterodroma phaeopygia) breeds only in the Galapagos archipelago, and only on ve islands: Floreana, Isabela, San Cristobal, Santa Cruz and Santiago (Figure 1). Nests are located in highland wet zones where vegetation is dense, 300700 m a.s.l., mostly in burrows in the soft soil of water gullies or in deep holes in the slopes of craters (Harris 1970; Coulter 1984). All breeding populations have been seriously reduced through predation by introduced mammals and destruction of nesting habitat (Coulter et al. 1985; Tomkins 1985; Cruz and Cruz 1987, 1990). Given these conservation concerns, the restricted distribution, and small population sizes, the Galapagos petrel was added to the IUCN Red Data Book as a Critically Endangered species (Hilton-Taylor 2000). Determining the genetic relationships among breeding populations of the Galapagos petrel is key to developing an eective conservation strategy for this species. Until recently, Galapagos and Hawaiian (P. sandwichensis) petrels were considered subspecies of the dark-rumped petrel (Pterodroma phaeopygia phaeopygia and P. p. sandwichensis, respectively) (Warham 1990). Sibley and Monroe (1993) used geographical separation and morphological and behavioural dierences to elevate these taxa to species. This separation was supported by subsequent comparisons of allozyme variation (Browne et al. 1997). However, inter-island dierences in timing of breeding, morphology, egg size, and vocalizations, and a lack of documented dispersal among colonies (Cruz and Cruz 1990; Tomkins and Milne 1991), raise the possibility that island populations of Galapagos petrels also are genetically distinct. Dierences in breeding chronology have been associated with reproductive isolation, morphological and/or genetic divergence, and/or cryptic species in band-rumped storm-petrels (Oceanodroma castro) in the Galapagos and Atlantic islands (Monteiro and Furness 1998; A. Smith et al. unpubl.), and in Leachs storm-petrels (O. leucorhoa) in Baja California (Ainley 1980; M. Atkey et al. unpubl.). Furthermore, Tomkins and Milne (1991) suggested that seasonally segregated breeding populations may exist on San Cristobal, raising the possibility that two genetically dierentiated populations exist within this island. Browne et al. (1997) and Nunn and Anderson (1999) compared allozymes and sequences of the mitochondrial cytochrome b gene among Galapagos petrels from four islands. However, variability was low, and little or no population genetic structure was found. We predicted that, if behavioural and morphological dierences are a result of recent isolation of colonies, population genetic structure would be evident in more variable markers such as microsatellites (Browne et al. 1997). We therefore compared variation in six microsatellite loci and the mitochondrial ATPase gene among Galapagos petrels from all ve islands to test the hypotheses that island and seasonal populations dier genetically.

Figure 1. Map of the Galapagos islands, with locations of sampling sites for Galapagos petrels.

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Table 1. Locations, numbers and dates of sample collections from Galapagos petrels Islands Number of birds sampled Adults Floreana Isabela San Cristobal Santa Cruz Santiago 51 10 35 53 27 Nestlings* 0 1 13 0 16 Total 51 11 48 53 43 Number of colonies surveyed 1 4 8 8 4 Collection dates

MayJun. 2003 Jun.Aug. 2003 Oct. 2002Feb. 2003 June 2003 MayAug. 2003

*Nestlings were only sampled when no adult bird was found at the nest-burrow.

Methods Collection of blood samples Blood samples were collected from 206 Galapagos petrels from all ve breeding islands (Table 1). Nest-burrows were located during monitoring activities conducted by the Galapagos National Park Service. Other likely breeding sites also were surveyed from October 2002 to August 2003 for additional petrel nests. Once a nest was located, it was checked with a torch and mirror. Birds were captured either by hand, or using a device developed by Cruz-Delgado (unpubl.) if the nest chamber was hard to reach by hand. After capture, 1 cc blood sample was drawn with a hypodermic syringe from the brachial vein, and the petrel was banded with an A4 numbered aluminium band. A hood was used to minimize bird stress. Blood samples were placed into separate tubes with Queens lysis buer (Seutin et al. 1991; for San Cristobal) or ethanol (for the other four islands), and refriger ated. Samples from San Cristobal included six individuals from the warm season population (breeding from November to March), 37 from the cool season population (breeding from April to June), and ve of uncertain seasonal origin. Laboratory analyses Microsatellite primers specic for Pterodroma petrels are not presently available. Therefore, a subset of four individuals from each island was screened for amplication and variation using PCR primers developed for 76 microsatellite loci for other species of birds, primarily procellariiforms. Amplications were conducted in 10 ll volumes containing 1 QIAgen (Valencia, California) PCR buer with 2.5 lM MgCl2, 0.6 lM dNTPs, 0.3 lM

each primer (tailed with the M13 forward primer), and 0.25U QIAgen Taq DNA polymerase. Amplications involved an initial cycle of 94 C for 2 min, 48 C for 1 min and 72 C for 2 min, ve cycles of 94 C for 1 min, 48 for 45 s. and 72 C for 1 min, and 30 cycles of 94 C for 30 s, 50 C for 30 s. and 72 C for 45 s, followed by a 10 min extension at 72 C. Thirty-seven loci yielded visible bands on agarose gels. For these loci, uorescently labelled M13 forward primer was included in a second set of PCRs, and the 20 test samples were screened for length variation using a Licor 4200 automated DNA sequencer (Lincoln, Nebraska). Six loci were suciently variable for large-scale analyses: Dc16 (Burg 1999), Dpm-01 (Dawson et al. 1997), Paequ10 (Techow and ORyan 2004), RBG18, RBG29 (Given et al. 2002) and Cco5-21 (Friesen unpubl.). A 650 bp fragment of subunits 6 and 8 of the mitochondrial ATPase gene was amplied using primers A8PWL and CO3HMH (G. Seutin and E. Bermingham unpubl.). Amplications were conducted in 20 ll volumes containing 1 QIAgen PCR buer (with 1.5 mM MgCl2; QIAgen), 0.6 lM dNTPs, 0.15 lM each primer, and 0.25U QIAgen Taq DNA polymerase. PCRs involved one cycle of 94 C for 2 min, 50 C for 1 min and 72 C for 1.5 min, ve cycles of 94 C for 1 min, 50 C for 1 min and 72 C for 1 min, and 30 cycles of 94 C for 30 s, 52 C for 30 s and 72 C for 45 s, and a nal extension of 72 C for 10 min. Samples were screened for sequence variation using single stranded conformational polymorphisms (SSCPs; Hayashi 1991) following standard protocols (Friesen et al. 1997). At least four representatives of each haplotype from each island, as well as individuals with unusual SSCP proles and all individuals from Isabela, were sequenced directly: PCR products were harvested from 1%

108 agarose gels, bands were puried using QIAgen Gel Extraction kits, and DNA was sequenced using Thermosequenase cycle sequencing kits (USB Corp., Cleveland, Ohio) according to the manufacturers suggested protocol. Statistical analyses ARLEQUIN (version 2.0, Schneider et al. 2000) was used to calculate expected heterozygosities (HE) for microsatellite loci, and nucleotide diversities (p, Nei 1987) for ATPase sequences. Microsatellite variation was tested for deviations from Hardy Weinberg equilibrium using exact tests based on contingency tables (Guo and Thomson 1992), and for deviations from linkage equilibrium with likelihood ratio tests (Slatkin and Excoer 1996) using ARLEQUIN. Variation in ATPase was too low to test for deviations from neutrality (see Results). On four islands, birds were collected from several colonies separated by distances up to 6.7 km. To conrm whether samples from dierent colonies within the same island could be combined, we tested for microgeographic structure within San Cristobal, Santa Cruz and Santiago (sample sizes were too small for tests within Isabela). Specically, estimates of FST from microsatellites were tested for signicance, and the number of genetic populations was estimated using STRUCTURE (see below for methodological details). Although statistical power for these tests was low (sample sizes ranged from 5 to 32 birds, and only six loci were screened), none of the estimates of either global or pairwise FST were statistically signicant (all P > 0.10), and results from STRUCTURE strongly rejected more than one genetic population on any island (all P < 0.001). Samples from dierent colonies within each island therefore were pooled for further analyses. The extent of dierentiation in microsatellites among island or seasonal (San Cristobal) populations was indexed using Wrights (1931) FST, which assumes the innite alleles model of mutation. Although an analog of FST that incorporates stepwise mutation has been developed for microsatellites (Slatkin 1995), simulations indicate that FST is a better index when the number of loci is less than $20 (Gaggiotti et al. 1999). For mitochondrial sequences, population genetic structure was indexed using FST (Excoer et al. 1992) using Kimuras 2-parameter mutation model, which accommodates unequal base representation and a biased transition:transversion ratio (Kimura 1980). Statistical signicance of FST and FST was tested by randomization using 10,000 permutations of the data in ARLEQUIN. Sequential Bonferroni corrections were applied to pairwise comparisons of populations (Rice 1989). Given the low variability of the ATPase genes, no further analyses were conducted on mitochondrial data. For the microsatellite data, populations were grouped in dierent ways in hierarchical analyses of molecular variance (AMOVAs; Excoer et al. 1992) to nd the grouping that maximized the proportion of variation due to dierences among groups (FCT) and minimized the proportion of variation due to dierences among populations within groups (FSC; Stanley et al. 1992). To test for isolation by distance, the shortest geographic distance between populations was calculated using EARTH (Byers 1999; Table 2). Linearized estimates of FST between population pairs were then tested

Table 2. Pairwise estimates of shortest geographic distance (km, lower number below diagonal), FST (for microsatellite loci, upper number below diagonal) and FST (for ATPase sequences, above diagonal) for ve populations of Galapagos Petrels Isabela Isabela Floreana Santa Cruz Santiago San Cristobal 0.20** 86 0.23** 83 0.07** 74 0.14** 176 Floreana 0.35 Santa Cruz )0.06 0.18* 0.18** 72 0.17** 123 0.09** 118 Santiago 0.16 0.00 0.12 0.26** 67 0.14** 98 San Cristobal 0.08 0.03 0.08 )0.02 0.12** 155

*P < 0.05; **P < 0.01.

109 for correlation with the natural log of geographic distance using Mantels tests (Smouse et al. 1986) in ARLEQUIN; signicance was tested by randomization with 10,000 permutations of the data. A population tree was generated by neighbour-joining (Saitou and Nei 1987) on Cavalli-Sforza and Edwards (1967) chord distance (DC) using POPULATIONS (version 1.2.28; Langella 2002). Support for nodes was derived by bootstrapping across individuals with 100 pseudoreplications. Microsatellite variation also was analyzed using STRUCTURE, which uses a Bayesian maximum likelihood analysis to delineate genetic populations, independent of sampling site, on the basis of deviations from HardyWeinberg and linkage equilibrium (Pritchard et al. 2000; Falush et al. 2003). The program was run under the no admixture model with correlated allele frequencies, a burn-in of 10,000 iterations and 100,000 replications after the burn-in; sampling location was not used as a priori information, k was set at 1 (the default value), and a was allowed to vary (Pritchard et al. 2000, pers. comm.). The program was run ve times (to verify the consistency of results) for each value of k (number of populations) between 1 and 5, and signicance was calculated from the posterior probabilities as described by Pritchard and Wen (2003). Traditional methods of estimating migration using molecular markers make several assumptions, such as migration/drift equilibrium, negligible mutation rate, and/or an n-island model of dispersal (e.g. Beerli 1999; Hedrick 1999), some of which may not hold for petrels (e.g. Congdon et al. 2000). In contrast, molecular assignments using programs such as BAYESASS make few assumptions (reviewed in Manel et al. 2005). Migration rates therefore were estimated using BAYESASS 1.1, which applies a Bayesian approach to multilocus genotypes (Wilson and Rannala 2003). 3,000,000 MCMC iterations were run, with a burn-in of 1,000,000 iterations and a sampling frequency of 2000. Delta was set to 0.15 (the default value).
Table 3. Frequencies of ATPase haplotypes and microsatellite alleles, sample sizes (N) and estimates of nucleotide diversity (p in %, for ATPase) and heterozygosity (HE, for microsatellite loci) for Galapagos Petrels from ve islands Allele Isabela Floreana Santa Santiago San Cruz Cristobal

Results Microsatellites Between two and 14 alleles were found for each of the six microsatellite loci (Table 3). Three

ATPase A B N p Dc16 127 129 131 133 135 139 N HE Dp 01 145 147 N HE Paequ10 152 154 N HE RBG29 145 147 149 151 153 155 157 159 N HE RBG18 183 185 187 191 193 195 197 199 201 209 211 213

0.82 0.18 11 0.05 0.05 0.86 0.05 0.00 0.00 0.05 11 0.34 0.27 0.73 11 0.5 0.68 0.32 11 0.45 0.00 0.50 0.00 0.50 0.00 0.00 0.00 0.00 11 0.52 0.00 0.00 0.00 0.05 0.00 0.09 0.77 0.00 0.00 0.05 0.00 0.05

1.00 0.00 50 0 0.00 0.02 0.46 0.49 0.03 0.00 45 0.57 0.25 0.75 51 0.37* 0.37 0.63 46 0.47 0.16 0.59 0.00 0.18 0.08 0.00 0.00 0.00 51 0.60 0.00 0.00 0.00 0.04 0.00 0.01 0.82 0.04 0.01 0.04 0.04 0.00

0.80 0.20 51 0.05 0.00 0.39 0.61 0.00 0.00 0.00 53 0.49 0.89 0.11 53 0.20 0.41 0.59 53 0.50 0.02 0.56 0.05 0.23 0.12 0.00 0.02 0.01 53 0.63 0.02 0.01 0.00 0.00 0.00 0.00 0.85 0.05 0.00 0.02 0.06 0.00

0.98 0.02 43 0.01 0.00 0.86 0.14 0.00 0.00 0.00 44 0.24 0.11 0.89 44 0.20 0.51 0.49 42 0.53 0.01 0.64 0.08 0.13 0.02 0.11 0.00 0.00 42 0.57 0.00 0.00 0.00 0.01 0.00 0.04 0.81 0.13 0.00 0.01 0.00 0.00

0.96 0.04 46 0.01 0.01 0.34 0.65 0.00 0.00 0.00 48 0.27* 0.33 0.67 46 0.43 0.66 0.34 37 0.41 0.03 0.67 0.04 0.17 0.07 0.00 0.02 0.00 48 0.58 0.00 0.04 0.01 0.00 0.01 0.03 0.82 0.02 0.00 0.00 0.02 0.02

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Table 3. Continued Allele Isabela Floreana Santa Cruz 0.00 0.00 53 0.30 0.02 0.00 0.00 0.00 0.00 0.00 0.46 0.00 0.50 0.00 0.01 0.00 0.00 0.01 52 0.55 Santiago San Cristobal 0.01 0.01 46 0.35 0.00 0.02 0.01 0.01 0.02 0.04 0.12 0.17 0.43 0.11 0.06 0.01 0.00 0.00 42 0.78*

219 221 N HE Cco521 129 135 137 139 141 143 145 147 149 151 153 155 161 165 N HE

0.00 0.00 11 0.41 0.00 0.00 0.00 0.00 0.00 0.00 0.23 0.00 0.77 0.00 0.00 0.00 0.00 0.00 11 0.44

0.00 0.00 51 0.32 0.00 0.00 0.00 0.01 0.01 0.13 0.37 0.06 0.35 0.03 0.03 0.00 0.01 0.00 52 0.72

0.00 0.00 42 0.35 0.00 0.00 0.00 0.00 0.00 0.09 0.33 0.06 0.44 0.04 0.03 0.03 0.00 0.00 40 0.70

*Signicant homozygote excess after Bonferroni correction.

deviations from HardyWeinberg expectations were found; all involved homozygote excesses, but no consistent patterns occurred either across loci or across populations (Table 3). Thus, there was no evidence for null alleles or Wahlund eects. No signicant deviations from linkage equilibrium were found (all P > 0.10). No evidence of genetic dierentiation was found between seasonal populations on San Cristobal: FST was low (0.00) and not signicantly dierent from zero (P = 0.47); results from STRUCTURE provided strong support for a single genetic population (P > 0.9999), and BAYESASS indiciated signicant migration from the cool- to the warm-season population (mean = 0.29, sd = 0.04, P < 0.01). Data from seasonal populations were therefore pooled for inter-island comparisons. Note however that the number of birds sampled from the warm season population was low and only six loci were assayed. Private alleles were found for all islands. Most private alleles occurred at low frequency, but allele 155 for RBG29 occurred in 29% of birds from Santiago, and allele 133 for Dc16 occurred in 49% of birds from Floreana (Table 3). The global

estimate of FST was high (0.16), and signicantly greater than zero (P < 0.0001). All estimates of FST for pairwise comparisons of populations were signicantly greater than zero, ranging from 0.07 (between Isabela and Santiago) to 0.26 (between Santa Cruz and Santiago; Table 2). In hierarchical AMOVAs, FCT never attained statistical signicance and FSC always retained a signicant component of variation, indicating that variation is best explained when populations are not grouped. Neighbour-joining on chord distances placed petrels from Isabela with those from Santiago with 92% condence; the relationships among this pair and the other three populations were unclear (Figure 2). Mantels test did not detect signicant isolation by distance (P > 0.50). Results from STRUCTURE provided strong support for three genetic populations (P > 0.9999999) and strongly rejected a single genetic population (P < 0.0000001). Most petrels from Isabela and Santiago (64% and 77%, respectively) had high probabilities (>90%) of belonging to Genetic Population 2, whereas most of those from Floreana (75%) had high probabilities of belonging to Genetic Population 1, and most from Santa Cruz (72%) had high probabilities of belonging to Genetic Population 3 (Figure 3). Most birds from San Cristobal (69%) could not be assigned to any one population with high certainty, but eight (17%) appeared to be from Genetic Population 1 and six (13%) appeared to be from Genetic Population 2. Results from BAYESASS indicated that migration among these ve islands is very low: most estimates were not signicantly dierent from zero (Table 4). The only exception was between Santiago and Isabela.

Floreana 54 San Cristobal 92 Santiago Isabela Santa Cruz

DC = 0.01

Figure 2. Neighbour-joining tree for island populations of Galapagos Petrels based on Cavalli-Sforza and Edwards (1967) chord distance (DC) for microsatellite variation. Numbers are bootstrap support indices.

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Figure 3. Proportion of times in 100,000 replications that petrels from dierent islands were assigned to dierent genetic populations by STRUCTURE. Red = Genetic Population 1; green = Genetic Population 2; blue = Genetic Population 3.

ATPase Only two ATPase haplotypes, diering by a single base pair, were found. Nucleotide diversity was very low, ranging from 0.00% at Floreana to 0.05% at Isabela (Table 3). Haplotype frequencies diered among the ve islands (Table 3), with haplotype A occurring in 20% of petrels from Isabela and Santa Cruz but only 24% of birds from San Cristobal and Santiago and none of 50 birds from Floreana. AMOVA indicated signicant population genetic structure (FST = 0.096, P < 0.001). Estimates of FST for pairwise comparisons of populations ranged from )0.06 to 0.35; only the estimate for Floreana versus Santa Cruz was signicantly greater than zero after Bonferroni corrections, but statistical power was low (Table 2).

Discussion Although statistical power was low, we found no evidence that seasonal populations of Galapagos petrels on San Cristobal dier genetically. This result contrasts with ndings of signicant mor-

phological and/or genetic dierentiation among seasonal populations of Leachs and band-rumped storm-petrels on various islands (Harris 1970; Ainley 1980; Monteiro and Furness 1998; M. Atkey et al. unpubl.; A. Smith et al. unpubl.). However, it agrees with recent surveys of Galapagos petrels, which failed to nd clear seasonal separation in breeding phenology at this island (Cruz-Delgado unpubl.). In contrast, strong population genetic structure exists among island populations: private microsatellite alleles occurred at all islands, sometimes at high frequency (Table 3); indices of population genetic structure both for microsatellites and for ATPase were high and signicantly greater than zero; pairwise estimates of FST were all statistically signicant, with a range up to 0.26 (Table 2); results from STRUCTURE provided strong support for three genetic populations, and strongly rejected a single population; and most estimates of migration derived by BAYESASS did not dier signicantly from zero (Table 4). Population structure in mi crosatellites in Galapagos petrels is higher than in any other species of seabird that has been studied to date (Table 5), and is especially high given the species restricted breeding range. Furthermore,

Table 4. Estimates of migration rates (proportion of individuals) among island populations of Galapagos petrels, derived by BAYESASS Migration From Isabela Migration Into Isabela Floreana Santa Cruz Santiago San Cristobal Floreana 0.016 0.003 0.003 0.003 0.004 0.004 0.009 0.008 Santa Cruz 0.032 0.005 0.01 0.032 Santiago 0.24** 0.005 0.003 0.037 San Cristobal 0.026 0.007 0.004 0.013

**Signicantly greater than zero at a = 0.01.

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Table 5. Estimates of population genetic structure based on microsatellite variation in various species of seabirds Common Name Adelie penguin wandering albatross Grey-headed albatross Black-browed albatross Shy albatross White-capped albatross Great cormorant Audouins gull Yellow-legged gull Common murre Pigeon guillemot Scientic Name Pygoscelis adeliae Diomedea exulans Thalassarche chrysostoma T. melanophris T. cauta T. steadi Phalacrocorax carbo Larus audouinii L. michahellis Uria aalge Cepphus columba Distribution Circum-Antarctica Circum-Antarctica Circum-Antarctica Circum-Antarctica Tasmania Tasmania/New Zealand Europe Mediterranean Europe/N. Africa North Atlantic Northeastern Pacic FST or RST1 0.0007 0.02 [0.020] [0.028*] 0.09* 0.012 [0.069*] )0.011 [0.032*] 0.005* 0.09* Reference Roeder et al. 2001 Burg and Croxall 2004 Burg and Croxall 2001 Burg and Croxall 2001 Abbott and Double 2003 Abbott and Double 2003 Goostrey et al. 1998 Genovart et al. 2003 Pons et al. 2004 Riault et al. 2005 Friesen, unpubl.

*P < 0.05. 1Square brackets indicate mean across pairwise population comparisons.

the global estimate of FST is at least as high as estimates for sister species of albatrosses (mean = 0.14 among black-browed albatrosses Thalassarche spp., Burg and Croxall 2001; mean = 0.09 among wandering albatrosses Diomedea spp., Burg and Croxall 2004; 0.054 between shy T. cauta and white-capped T. steadi albatrosses, Abbott and Double 2003) and gulls (0.069 among white-headed gulls Larus spp., Crochet et al. 2003). Molecular results for Galapagos petrels are generally consistent with dierences in morphology and behaviour among island populations. Tomkins and Milne (1991) found that a combination of laying dates and seven morphological measurements separated Galapagos petrels from four islands into three groups: (1) birds from Santa Cruz plus birds that lay eggs in AprilJune on San Cristobal, (2) birds from Santiago plus birds that lay eggs in NovemberMarch on San Cristobal, and (3) birds from Floreana. (This particular grouping was not strongly supported by the present data, however.) Signicant inter-island dierences in calls have been found, reecting that dialects probably exist within the archipelago. And banding data from 1500 adults and juveniles indicated that little or no migration occurred among islands; even movement between colonies on the same island was rare, with adult petrels returning repeatedly to the same island-specic colonies to breed (Cruz and Cruz 1990). Although all island populations of petrels were found to dier from each other, petrels from Isabela and Santiago were the most similar: these islands grouped together in the population trees

(Figure 2); they had the lowest pairwise FST estimate (Table 2); individuals from both were placed primarily in Genetic Population 2 by STRUCTURE; and BAYESASS detected signicant migration from Santiago to Isabela (Table 4). The rst active nests on Isabela were only found in 1996 (Jimenez and Wiedenfeld 2002), and the number of nests located to date is very small. Petrels from Isabela and Santiago appear to share a common foraging area (F. Cruz, com. pers.), which also could explain the apparent existence of relatively high migration rates between these islands (Table 4). These results together suggest that Isabela may have been colonized recently by birds from Santiago. There are no obvious physical barriers to dis persal among islands for Galapagos petrels. More likely, the genetic dierences result from high mate- and breeding site delity, and rigid annual breeding (Cruz and Cruz 1990). Many species in the Procellariidae are highly faithful to their natal island (Warham 1990; Tomkins and Milne 1991), and this trait would be an eective isolating mechanism. Once isolation exists, genetic drift, and natural and sexual selection can produce population dierences (Lack 1966). Results of the present study dier from those for Galapagos marine iguanas (Amblyrhynchus cristatus), which show marked dierences among islands in cytochrome b sequences (global FST = 0.68) but little population genetic structure in micro- or minisatellites (global FST = 0.10; Rassmann et al. 1997). Rassmann et al. argued that the existence of signicant population genetic structure in mitochondrial but not nuclear DNA in marine iguanas results from

113 male-biased dispersal among islands. Results for Galapagos petrels are more similar to terrestrial vertebrate species: for example, Galapagos giant tortoises (Geochelone nigra) and Darwins nches (Geospizinae) show signicant genetic dierences among conspecic populations from dierent islands (Freeland and Boag 1999; Petren et al. 1999; Cio et al. 2002; Beheregaray et al. 2004). Several studies reported that population dierentiation increased with the age of the island groups, being lowest for the western islands (including Isabela [$0.7 My old], and Santiago [$ 1.2 My]) and highest for the central and eastern islands (including Santa Cruz [$2.2 My] and San Cristobal [$3.2 My]; Rassmann et al. 1997; Cio et al. 2002; Beheregaray et al. 2004). Interestingly, the lowest divergence in microsat ellites in Galapagos petrels was between populations on the two youngest islands: Isabela and Santiago. Populations of giant tortoises on these islands also were genetically most similar (Cio et al. 2002). Otherwise, an eect of island age was not obvious with the petrels. According to our results, populations on Floreana, Santa Cruz and Santiago should be regarded as separate genetic management units (sensu Moritz 1994). If resources are limited, these populations should be given the highest priority for conservation to preserve the maximum amount of genetic variability. The San Cristobal population appears to represent, from a strictly-genetic point of view, a mixture of birds from other islands. This nding is supported by morphological data. However, based on substantial ecological and behavioural dierences (Tomkins and Milne 1991), we recommend that this population also be managed as a separate conservation unit (sensu Crandall et al. 2000), as there is strong evidence for adaptive distinctiveness. The birds from Isabela are genetically similar to the Santiago population, but we also recommend to manage them as a separate conservation unit at present, given the very dierent ecological characteristics of the island, the critically small number of nests found to date, and the absence of behavioural and morphological information. As a general rule, a species composed of several populations should be less susceptible to global extinction than a species consisting of only one population. However, in the case of the Galapagos petrel, the loss of any island population will result in a loss of genetic variability and, eventually, the interruption of evolutionary processes that might be occurring (e.g. Templeton 2001; Reed and Frankham 2003). Managers must incorporate these considerations into their conservation plans, making eorts to preserve viable breeding populations on the ve islands where the species occurs so that natural patterns of adaptive diversity are not lost and long-term evolutionary processes are not aected. Acknowledgments We are grateful to the Galapagos National Park Service sta, and especially to Washington Tapia, who facilitated the research permits, and the park rangers Rommel Aguas, Nelson Garc a, Paul Rodr guez (San Cristobal Island), Klever Aguilar, Wilman Valle, Patricio Cadena (Santa Cruz and Santiago Islands), Anibal San Miguel, Milton Chugcho (Floreana Island), and Maximo Mendoza, Daniel and Lenin Tenelema and Javier Jaramillo (Isabela Island), who conducted eld surveys to locate nests and helped with the collection of blood samples. We also thank Virna Cedeno and Leandro Patino (University of Guayaquil), who performed the DNA extractions, and Candace Scott, Tim Birt and Theresa Burg for help with laboratory analyses. David Wiedenfeld, Felipe Cruz and James Austin provided valuable comments on earlier drafts of this paper. Malcolm Coulter helped with initial coordination of the study, and provided insightful comments on the manuscript. Finally, we are grateful for the resources and support oered from the Charles Darwin Research Station during the start of re search in San Cristobal. Financial support was provided by Instituto Galego de Cooperacion Iberoamericana (IGACI) and Xunta de Galicia, the Araucaria Programme of the Spanish Agency for International Cooperation (AECI), and NSERC Canada (Discovery Grant held by V.L.F).

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