Rocamora, G.J. and Richardson, D.S. 2003. Genetic and ...

Ibis (2003), 145 (online), E34–E44Blackwell Science, LtdGenetic and morphological differentiation betweenremnant populations of an endangered species: thecase of the Seychelles White-eyeGÉRARD J. ROCAMORA 1 *† & DAVID S. RICHARDSON 2 †1 Seychelles White-eye Recovery Programme, Ministry of Environment, Seychelles/C.R.B.P.O. Museum Nationald’Histoire Naturelle, 75005 Paris, France2 Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UKThe Seychelles White-eye Zosterops modestus is a critically endangered species that survivesin two remnant populations on the islands of Mahé and Conception. Multilocus minisatelliteDNA fingerprinting and morphometric measurements were used to assess the levels of variationbetween these populations. Mahé White-eyes are on average significantly larger than Conceptionbirds, as are males compared to females. The mean level of bandsharing (c. 60%)indicates low levels of genetic variability within both populations. Bandsharing is significantlylower between populations (32%), suggesting that the two populations are geneticallyisolated from each other, and that one is not a subsample from the other. Bothpopulations should therefore be considered as equally important genetic reservoirs thatdeserve to be safeguarded. Although each population appears locally adapted to its particularisland environment, such limited differentiation is not considered of major taxonomicsignificance. Recombining genetic variation by mixing individuals from both populationsmay be beneficial for the species in view of future island transfers. This illustrates the importanceof investigating differentiation between remnant populations of a threatened speciesto orientate future conservation and management measures.The Seychelles White-eye Zosterops modestus is acritically endangered species endemic to the Seychellesarchipelago (Collar et al. 1994, BirdLifeInternational 2000) where it was, until recently,known only from the main island of Mahé. Duringthe 19th century (Newton 1867) and the early 20thcentury (Nicoll 1906) the White-eye was locallyabundant on Mahé. It then underwent a drasticdecline and was thought to be extinct for severaldecades (Crook 1961), until it was rediscovered(Loustau-Lalanne 1962). In the 1970s White-eyeswere estimated to be fewer than 100 in numberand declining (Watson in Collar & Stuart 1985).Between 1995 and 1997, intensive searches coordinatedby the ministry of Environment andBirdLife International showed that the numberand distribution of White-eyes had declined byapproximately 50% since 1975–77 (Rocamora*Corresponding author.Email: g_rocamora@hotmail.com†Equal co-authors.1997b). The Mahé population was estimated at c. 50individuals.In 1996, following repeated public appealsthrough the media, a population was reported onConception (4°39′S, 55°22′E), a small island (63 ha)1.6 km off Mahé (4°38′S, 55°25′E). The existence ofthis population was confirmed (L. Chong-Seng pers.obs.) and the island was estimated to host 30–50breeding pairs and at least 200 birds (Rocamora1997a, 1997b). This discovery brought renewedhope for the future of this species and a two-yearresearch project was started in 1998 as the first phaseof the Seychelles White-eye Recovery Programme.This study provided basic information on the ecologyand breeding biology of the Conception population(Rocamora & François 2000). Some informationis also available from the small population on Mahé(e.g. Loustau-Lalanne 1962, Feare 1975, Mellanby et al.1996, Rocamora 1997a, 1997b), where the veryhigh rate of nest failure – mainly due to introducedrats and birds – has been identified as the main causeof its decline (Rocamora & François 2000).© 2003 British Ornithologists’ Union

Variation between remnant populations of Zosterops modestusE35It is important to determine whether differencesexist between the Conception and Mahé Whiteeyes,as this will have implications for the futuremanagement and conservation of this species. TheSeychelles White-eyes’ history of isolation, thesevere population bottleneck that occurred duringthe last century and the limited size of both populationssuggest that this species is highly inbred andmay contain limited genetic variation. It is importantto quantify genetic variation as reduced variation canlead to inbreeding depression and reduced populationviability, and is believed to increase the probabilityof extinction in small populations (e.g. vanNoordwijk 1994; Frankham 1998, Saccheri et al.1998). Levels of genetic variation shared by the twopopulations will also help us understand their historyand relationship, i.e. is the Mahé populationa subset of the Conception population maintainedby interisland immigration? Does the Conceptionpopulation originate from a few Mahé birds thatcolonized the island in recent times?In this paper, we investigate morphological differences(including sexual dimorphism) and levels ofgenetic variation within and between the tworemaining populations of Seychelles White-eyes.METHODSBirds were caught in mist-nets placed next tofruiting trees (Conception) or lured into the netsusing a tape playing previously recorded White-eyevocalizations (both islands). Body measurements(wing length, maximum tarsus length (Redfern &Clark 2001), bill, head plus bill, tail and mass) andplumage coloration were recorded. We were unableto sex and age birds in the hand from plumage ormorphometric differences.Birds on Conception (192) were ringed with ParisMuseum rings whereas those on Mahé (35 since1996) were ringed with British Trust for Ornithologyrings. All birds were also colour-ringed. Colour-ringcombinations used on Mahé and Conception weredistinctive so that a bird from Conception recapturedor resighted on Mahé could always be identified easily.Approximately 25 µL of blood was taken fromcaptured birds by piercing the brachial vein witha small hypodermic needle and then drawing offthe blood using a graduated capillary tube. Thissampling technique has been shown to have no illeffects on birds (Stangel 1986). The blood wasimmediately mixed with at least five volumes of100% ethanol.Mean, standard deviation and confidence intervalswere calculated for all measurements. To avoidobserver error, measurements were taken by a singleobserver (G.J.R.). Statistical analyses were doneusing S-plus, Statistica or Excel.Morphometric comparisonTo compare the morphometric measurementsbetween the two islands and between sexes, threedifferent tests and a multivariate analysis were used.The Welch modified two-sample t-test comparesthe means of two normally distributed variableswhen the variances are unequal (Mood et al. 1974,Snedecor & Cochran 1980). Given that we carried outrepeated t-tests for the two groups of birds (one foreach of the seven variables measured), we appliedBonferroni correction (Keppel 1991) to the standardsignificance level to increase confidence in thesignificance of the results. To guard against possibledeviations from normality, a Monte-Carlo test(Efron & Tibshirani 1993, Davison & Hinkley 1997)was applied. A third way of comparing the samplesis to look at the expected difference betweenindividuals chosen at random in each sample. Thisspecifically addresses the probability that a birdfrom Mahé is larger than a bird from Conception.A Monte-Carlo procedure was devised by randomsampling with replacement pairs of birds (one fromeach of the original samples) and taking the differencebetween them. The resulting distribution ofdifferences was used to estimate the average differencebetween individual birds and associated probabilitystatements.Principal Component Analysis (Cooley & Lohnes1971, Venables & Ripley 1994) summarizes the relationshipsbetween the different measurements. Weused the first two components of a PCA to determinethe variables that best explain the observeddifferences between individuals, and to provide agraphical representation of population separation.The extraction of the principal components wasbased on a covariance matrix, without rotation ofthe components.Genetic analysisMultilocus minisatellite DNA fingerprinting (Jeffreyset al. 1985) was used to assess the levels of geneticvariation within and between the populations. Thistechnique was used in preference to microsatellitemarkers as it provides a quicker and more cost© 2003 British Ornithologists’ Union, Ibis, 145 (online), E34–E44

E36G.J. Rocamora & D.S. Richardsonefficient way of investigating levels of genetic variation,especially in species for which microsatelliteshave not already been isolated or characterized. Ithas also been applied successfully to studies of othersmall isolated island populations (Gilbert et al.1990, Degnan 1993, Kappa 1998) with which theSeychelles White-eye can be directly compared.DNA extractions were conducted as follows. Asmall (2 mm × 2 mm) piece of hardened blood wasremoved from the ethanol using a sterilized woodentoothpick, air dried for 10 min and then added toproteinasing solution (1 M TRIS-HCl, pH 8.0; 0.1 MNaCl; 1 mM EDTA) containing 0.5% SDS and 5units of proteinase K (Sigma). The DNA requiredfor fingerprinting was then extracted using standardphenol extraction methods (Sambrook et al. 1989,Bruford et al. 1998). The smaller samples of DNArequired for sexing were extracted using a quickerammonium acetate extraction method as follows.The blood/proteinasing solution was digested (withconstant agitation) at 55 °C for 3 h; 250 µL of 4 Mammonium acetate was added, then the solution wasvortexed and left at room temperature for 15 min.The sample was centrifuged at 14 000 rev/min for10 min and the supernatant decanted into a clean,labelled eppendorf tube. To precipitate the DNAfrom the supernatant, 2 volumes of 100% EtOHwere added and the sample was centrifuged at14 000 rev/min for a further 10 min. The supernatantwas decanted and the pellet was rinsed in 1 mL of70% EtOH and air-dried for 30 min. The pellets(from both extraction methods) were dissolved in300 µL of 1 × T 10E 0.1(10 mM Tris, 0.1 mM EDTA)by leaving them at room temperature overnight.Molecular sexing was undertaken using the PCRmethod (primers P2 and P8) devised by Griffithset al. (1998). PCR products were run on a 3% agarosegel (containing ethidium bromide) for 60 min at100 V and visualized under UV at 254 nm. Femalesare the heterogamatic sex in birds and products fromboth the CHD-w and the CHD N-w genes areamplified in this PCR reaction; consequently, twoproducts of different sizes are visualized on theagarose gel. Males are homogamatic and only producethe band corresponding to the CHD N-w gene.The Seychelles White-eye has a complex cooperativebreeding system (Rocamora & François2001). Therefore, a subset of birds for DNA fingerprintingwas chosen from each island according tostrict criteria (only the main adult male and femalefrom a group) to minimize the chance of directlyrelated birds being used in the analyses. Twenty-twoputatively unrelated birds were sampled on Conceptionand 18 on Mahé. Samples were analysed usingstandard multilocus minisatellite DNA fingerprintingmethods following Bruford et al. (1998). DNAwas digested with HaeIII and probed with radioactivelylabelled 33.15 (Jeffreys et al. 1985).Fingerprints were scored (by D. Richardson)following Bruford et al. (1998). Two gels, each containing20 individuals, were run. Individuals from thesame island were run in adjacent pairs and pairs fromthe two islands were alternated. In this way it waspossible directly to compare the bandsharing of adjacentdyads of birds (i) from within Mahé, (ii) fromwithin Conception and (iii) one individual fromeach of the populations. As each individual was usedonly once in within- and between-population comparisons,all comparisons are independent. In eachcase, calculation of the bandsharing coefficient wasbased on Bruford et al. (1998). The mean withinpopulationbandsharing coefficient (similarity) wascalculated by averaging the bandsharing coefficientsof all pairs of individuals sampled in a population.Similarity of bandsharing between populations,corrected for within-population similarity, was calculatedusing Lynch’s (1991) formula:S ij= 1 + S′ ij− ((S i+ S j)/2)where S iis the mean proportion of bands sharedamong individuals of population i and S′ ijis the meanproportion of bands shared between random pairs ofindividuals across populations i and j.RESULTSMorphometryDifferences between populationsBirds on Mahé have, on average, significantly longerwings, tails and tarsi than birds on Conception(Table 1). For these three measurements, there is ahigh probability of birds from Mahé being largerthan from Conception (varying between 70% and85%). Mass and bill length are also slightly greateron average on Mahé (0.40 g and 0.30 mm, respectively),the differences being statistically significant(P < 0.03) only with the Monte-Carlo test. Only ‘billplus head’ length showed no statistically significantdifference. After applying Bonferroni correction tothe seven t-tests conducted (using P = 0.05/7), differencesin wing length, tail and tarsus measurementswere still statistically significant, while differences in© 2003 British Ornithologists’ Union, Ibis, 145 (online) E34–E44

Variation between remnant populations of Zosterops modestusE37Table 1. Comparison of Seychelles White-eye measurements between Mahé and Conception.Sample statistics aVariable Mahé Conception t-test bDifferencebetweenmeans cDifferencebetweenindividuals dMass 12.41 12.01 t: 1.92 0.39 0.41(g) (1.04) (0.78) df: 49.04 (0.02–0.82) P(M > C): 0.53N = 32 N = 65 P = 0.06 P = 0.018Wing length 61.96 59.84 t: 6.93 2.12 2.08(mm) (1.51) (1.44) df: 64.16 (1.54–2.69) P(M > C): 0.77N = 35 N = 74 P < 0.001 P < 0.001Tail 40.63 39.18 t: 5.35 1.45 1.45(mm) (1.36) (1.04) df : 47.72 (0.93–1.97) P(M > C): 0.70N = 33 N = 75 P < 0.001 P < 0.001Tarsus 1 17.75 16.88 t: 4.90 0.86 0.85(mm) (0.94) (0.57) df: 42.72 (0.54–1.21) P(M > C): 0.78N = 35 N = 34 P < 0.001 P < 0.001Tarsus 2 19.94 18.97 t: 5.24 0.98 0.97(mm) (0.88) (0.66) df: 62.85 (0.63–1.33) P(M > C): 0.80N = 33 N = 75 P < 0.001 P < 0.001Bill 15.69 15.39 t: 1.95 0.29 0.29(mm) (0.77) (0.63) df: 51.98 (0.005–0.57) P(M > C): 0.67N = 34 N = 74 P = 0.057 P = 0.025Bill & head 29.05 28.88 t:1.47 0.17 0.17(mm) (0.61) (0.47) df: 51.87 (−0.06–0.40) P(M > C): 0.56N = 32 N = 72 P = 0.148 P = 0.087a Values in parentheses indicate standard deviation σ. N = number of individuals sampled.b t : value of the t-statistic; df : degrees of freedom; P: level of significance of the test.c Values are the mean of the differences from the bootstrap procedure; values in parentheses represent the lower and upper limits of a95% confidence interval for the difference between means.d Average difference between individuals; P(M > C): Probability that an individual from Mahé is bigger than an individual ofConception island.Table 2. Factor loadings relative to each measurement variable for the three first components of a PCA, showing how each axis isrelated to the morphological traits.Component 1 Component 2 Component 3Wing length 0.812 0.498Tarsus articulation (Tarsus 1) 0.230 0.505 −0.291Tarsus plus tibia (Tarsus 2) 0.257 0.510 −0.567Mass 0.211 −0.178Tail 0.394 −0.647 −0.253Bill 0.144 −0.220 −0.244Bill plus head 0.125 −0.340bill, bill plus head, and mass were not. However,for all the variables compared, there is an overlapbetween individual measurements as shown by thestandard deviations (Table 1). In general the resultsof the three tests are consistent, with the exceptionof some differences in P-values reported by the t-testand the more robust bootstrap procedure for thosevariables that depart most from the normal distribution.Repeatability values could not be calculatedfor the different morphological measurements, asWhite-eyes were only measured once during theircapture, and few individuals were recaptured.The first two components (or axes) of the PrincipalComponent Analysis together explain 73.3% ofthe total variability (59.3% and 14.0%, respectively).Table 2 shows the factor loadings relative to eachvariable, i.e. how each of the first two axes is relatedto the morphological traits. The vectors on Figure 1are the variables plotted according to their relativecontribution on each of the two first components.© 2003 British Ornithologists’ Union, Ibis, 145 (online), E34–E44

E38G.J. Rocamora & D.S. RichardsonTable 3. Comparison between male and female Seychelles White-eye measurements (from both Mahé and Conception islands).Sample statistics aVariable Male Female t-test bDifferencebetweenmeans cDifferencebetweenindividuals dMass (g) 12.12 11.88 t: 1.251 0.25 0.27(0.72) (0.70) df: 39.84 (−0.12–0.62) P(M > F): 0.52(N = 37) (N = 20) P = 0.22 P = 0.10Wing length 60.85 59.82 t: 2.502 1.03 1.13(mm) (1.45) (1.62) df: 39.19 (0.24–1.82) P(M > F): 0.63(N = 41) (N = 22) P = 0.016 P = 0.008Tail (mm) 39.76 39.40 t: 1.121 0.36 0.35(1.55) (0.96) df: 58.38 (−0.27–0.95) P(M > F): 0.45(N = 39) (N = 22) P = 0.267 P = 0.13Tarsus 1 16.96 17.23 t: −1.958 −0.28 −0.30(mm) (0.69) (0.42) df: 60.04 (−0.55–0.01) P(M > F): 0.28(N = 41) (N = 22) P = 0.055 P = 0.024Bill 15.57 15.54 t: 0.136 0.03 −0.005(mm) (0.64) (0.68) df: 40.94 (−0.32–0.37) P(M > F): 0.47(N = 40) (N = 22) P = 0.893 P = 0.436Bill & head 28.92 28.90 t: 0.156 0.02 0.017(mm) (0.45) (0.56) df: 36.06 (−0.24–0.29) P(M > F): 0.51(N = 41) (N = 22) P = 0.877 P = 0.432a Values in parentheses indicate standard deviation σ. N = number of individuals sampled.b t : value of the t-statistic; df: degrees of freedom; P: level of significance of the test.c Values are the mean of the differences from the bootstrap procedure; values in parentheses represent the lower and upper limits ofa 95% confidence interval for the difference between means.d Average difference between individuals; P(M > F): Probability that a male individual is bigger than a female individual.Despite the overlap, individuals from both islandsappear well separated in this lower-dimensionprojection, in particular along the first component,which is most heavily influenced by wing length.This axis shows that wing length, tail length andtarsus length best exemplify the differentiationobserved between individuals from the two islands.The second axis opposes mainly birds (from bothislands) with longer tails to birds with longer tarsibut, with only 14% of the total variance, its importanceis minor. Mahé and Conception populationsappear clearly differentiated with respect to each ofthe two major principal components (t = −9.31,df = 52, P < 0.001 and t = −4.41, df = 46, P < 0.001,respectively; values not departing significantly fromnormality). The third component (11.8% of the totalvariability) and all the others are of less importance(eigenvalues < 1; Kaiser criterion).Sexual dimorphismMale Seychelles White-eyes usually have longerwings than females (63% probability of c. 1.0 mmlonger, P < 0.02 with t-test, P < 0.01 with MonteCarlo test). However, Bonferroni correction for thet-tests suggests that this difference might not bestatistically significant. Tarsus length tends to beslightly longer for males on average (0.27 mm,P < 0.05 with Monte Carlo test). No other morphologicalmeasurements indicated a significant differencebetween sexes (Table 3). This difference wasnot observed on Mahé, although the number offemales captured (seven) was very low.DNA analysisSexingOut of 106 birds captured, 74 (70%) were males and32 (30%) were females. On Mahé, 72% (18/25)were males, while 69% (56/81) were male on Conception;the difference was not statistically significant.Population geneticsThe numbers of ‘scorable’ bands and the bandsharingcoefficients within and between populations aresummarized in Table 4. A mean of 17.9 ± 0.54 sefragments in the size range of 3–20 kb was scored perindividual. Significantly fewer scorable bands werepresent in individuals from the Mahé population© 2003 British Ornithologists’ Union, Ibis, 145 (online) E34–E44

Variation between remnant populations of Zosterops modestusE39Table 4. Average bandsharing coefficients within and between populations of the Seychelles White-eye detected by multilocusminisatellite probe 33.15.Scorable bandsBandsharing coefficientsN Mean se Comparisons Mean seWithin populationsMahé 18 16.45 0.76 9 0.61 0.03Conception 22 19.72 0.51 10 0.59 0.03Combined 40 17.93 0.54 19 0.60 0.02Between populationsMahé × Conception – – – 18 0.32 0.02DISCUSSIONMorphological differencesTARSUS1TARSUS2Figure 1. Factorial plan of a Principal Component Analysisshowing the separation between Mahé () and Conception ()birds from their body measurements. The vectors are thevariables plotted according to their relative contribution on eachof the two components.compared with those from Conception (16.45 ± 0.76se vs. 19.72 ± 0.51 se, t = 3.4, df = 38, P < 0.005). Themean level of bandsharing was high within both Mahé(0.61 ± 0.03 se) and Conception (0.59 ± 0.03 se)indicating that there is low genetic variability withineach population. There was no difference betweenthe populations in the level of within-populationbandsharing (P = 0.61). However, within-populationbandsharing was significantly higher than betweenpopulationbandsharing (see Fig. 2, 0.60 vs. 0.32,respectively, t-test = 3.4, df = 38, P < 0.001). Betweenpopulationbandsharing, after taking into considerationthe within-population bandsharing, was 0.72.Significant morphological differences exist betweenthe two populations of White-eyes. The MahéWhite-eyes have, on average, larger bodies and aboutan 80% probability of having longer wings, tarsus andtail than Conception White-eyes. Although thedifference between the means of these variables isstatistically significant, the actual differences arerelatively small (wing 3.4%, tail 3.6%, tarsus 4.9%).The overlap between the measurements from theislands makes it impossible to identify accurately theorigin of an individual just from its measurements.Differences in size between populations of thesame species across an archipelago are knownfrom other species, and probably occur frequently(e.g. Galapagos finches Geospiza sp. Lack 1968,Grant & Grant 1989; Australian Silvereyes, Zosteropslateralis Degnan 1993). Smaller body-sizes onConception could be due to founder effects.Alternatively, the size differences could indicatelocal adaptation towards the environment onConception or be due to phenotypic plasticity andreflect, for example, differences in food availability.The smaller size of the Conception White-eyes isalso consistent with the general theories of islandbiogeography that predict smaller bodies in islandthan in mainland populations, as this allows higherpopulation sizes in limited island habitats and henceincreased survival (MacArthur & Wilson 1967,Blondel 1986).No significant plumage differences could bedetected between Mahé and Conception Whiteeyes.However, we noted that birds from Conceptiontended to have more uniformly grey upper-partscompared with ones from Mahé, which sometimesshow greenish or reddish tones on their back and© 2003 British Ornithologists’ Union, Ibis, 145 (online), E34–E44

E40G.J. Rocamora & D.S. RichardsonFigure 2. DNA bandsharing coefficients within and between the two populations of Seychelles White-eye (Conception/Mahé).wings (unpubl. data). No sex- or age-related plumagedifferences were detected.There is a significant, but small (1.7%), differencebetween the average wing length of male and femaleSeychelles White-eyes although the degree of overlapmakes it impossible to distinguish the sexes bythis. The sex ratio of the samples from Mahé andConception did not differ significantly from eachother and was not therefore responsible for theaverage differences measured between the two islands.The large skew in sex ratio observed on both Mahéand Conception is probably due to a trapping bias.Almost all birds that were sampled were caught bytape luring, to which males are more likely torespond than females. However, there may also be areal deficit of females, as observed in many criticallyendangered species including Seychelles Magpie-Robin, Copsychus sechellarum (Millett et al. 1999),Mauritius Pigeon Columba mayeri (C. Jones & B.Burn pers. comm.), Echo Parakeet Psittacula equesecho (Diamond 1987), and Black Robin Petroicatraversi (Butler & Merton 1992).Population geneticsAlthough the mean number of scorable bands wassignificantly less on Mahé than on Conception,within-population bandsharing did not differ significantlybetween islands. Both populations had highwithin-population bandsharing coefficients (0.59and 0.61, respectively) compared with levels generallyreported for outbred avian populations (range0.20–0.30, Burke & Bruford 1987, Westneat 1990).These high within-population bandsharing coefficientsare comparable with those found in a numberof other inbred island populations (see Table 5).Table 5. Comparisons of within- and between-population bandsharing coefficients (BSC) for a range of endangered bird species.SpeciesAuthorsWithinpopulationBSCBetweenpopulationBSCSeychelles White-eye Zosterops modestus This study 0.59–0.61 0.72Seychelles Warbler Acrocephalus sechellensis Kappa (1998) 0.46–0.52 0.97Australian Silvereye Zosterops lateralis chlorocephala Degnan (1993) 0.31–0.67 0.99Chlorocephala vs. familiaris race 0.26Brown Skua Catharacta lonnbergi Millar et al. (1994) 0.77–0.76 naBlack Robin Petroica traversi Ardern and Lambert (1997) 0.84 naNew Zealand Bush Robin Petroica australis Ardern and Lambert (1997) 0.45–0.61 naPukeko Porphyrio porphyrio melanotus Lambert et al. (1994) 0.60–0.40 na© 2003 British Ornithologists’ Union, Ibis, 145 (online) E34–E44

Variation between remnant populations of Zosterops modestusE41The results indicate that Mahé and Conceptionboth contain low levels of genetic variability. Thiscould be attributed to one or a combination of thefollowing factors: (1) recent founding, (2) small populationsizes, and (3) geographical isolation and consequentlylow, or no, migration between populations(reviewed in Lande 1999). Indeed, there is goodhistorical evidence that the White-eye underwenta severe genetic bottleneck during the first half of thecentury on Mahé (Crook 1961, Loustau-Lalanne1961, 1962) and both populations remain veryrestricted in size and could be considered still to beundergoing population bottlenecks. The Mahépopulation is estimated at c. 50 individuals whileConception may contain up to 330 individuals.However, the effective sizes of these populations arelikely to be much lower due to the co-operativebreeding system that appears to exist on both islands(Grieg-Smith 1979, Rocamora & François 2000).The effective population size can only be calculatedaccurately if DNA parentage studies are used todetermine the genetic mating system of this species.The Mahé and Conception populations are geneticallydifferentiated, with a significantly lower meanbandsharing between (0.32) than within populations(0.60). This indicates that the populations areisolated from each other, as only one or two migrantsbetween the two populations per generation wouldbe sufficient to prevent this differentiation (Lande1999). The similarity of bandsharing between populationscorrected for within-population similaritywas 0.72, lower than the 0.97 observed betweenthe original Seychelles Warbler population and adaughter population, established on another islandin 1988 (Kappa 1998). In a study of AustralianSilvereyes Zosterops lateralis (Degnan 1993) a similarityof 0.99 was observed between populations withinthe Capricorn Islands (race chlorocephala). But whenthe Capricorn group was compared to the mainlandrace (familiaris) the similarity index was just 0.26(see Table 5). However, caution must be taken wheninterpreting the differences between the presentstudy and that on the Australian Silvereyes as ourstudy comprises only one between-species comparison.Evolutionarily significant units (ESUs) can bedefined as populations that are reproductively isolatedover an evolutionarily significant timescale andare evolving independently of other populations(Moritz 1994). The concept of identifying suchESUs using neutral molecular marker data has beenthe focus of much debate (e.g. Hedrick et al. 2001,reviewed in Crandall et al. 2000, Pearman 2001).The DNA fingerprinting techniques used in the presentstudy mainly reflect recent demography more thanevolutionary differentiation and may therefore beinadequate to separate evolutionary significant units.Our results suggest that, while the SeychellesWhite-eye populations are genetically differentiated,the level of similarity between populations(0.32 when no correction for within-populationsimilarity is applied) is only slightly higher than thatnormally found within outbred avian species (range0.20–0.30, Burke & Bruford 1987, Westneat 1990)and may not be as extreme as that found betweendifferent races of a species. This shallow level ofgenetic differentiation over a short evolutionarytimescale indicates that the two populations couldbe considered as separate management units but notas separate ESUs (Moritz 1994).Higher levels of interpopulation bandsharingwould be expected if one population had recently(since the near extinction) founded the other.Furthermore, one of the populations should havea higher within-population bandsharing than theother as a result of undergoing both the originalbottleneck and a subsequent one as the new populationwas founded. As this is not the case, the twopopulations may have separated before the speciesdeclined and each contains different fragments fromthe genome of the original healthy population.The populations may have separated at any timesince the end of the last glaciation 10 000–12 000 years BP, when sea-level rise divided theislands. However it is likely that, before the arrivalof humans, White-eyes were plentiful and coveredall of Mahé and Conception (and other satelliteislands). White-eyes are strong flyers (G.J. Rocamorapers. obs.) and the short distance between Mahé andConception (1.6 km) would probably not preventmigration and gene exchange between the populations.It appears likely that the White-eye populations havebeen isolated since anthropogenic disturbance reducedthe population on Mahé 100–200 years ago. Thisconcurs with the results of the Silvereye study(Degnan 1993) where two populations, separated bya maximum of 3000–4000 years, showed a muchhigher degree of genetic differentiation than in thisstudy (see above).There are some ecological differences betweenthe two populations of Seychelles White-eyes. OnMahé, White-eyes are flexible in habitat preference.Most remaining territories are in residential areas,clearings or forest edges with small-scale agriculture,but some can also be found in tall forests of© 2003 British Ornithologists’ Union, Ibis, 145 (online), E34–E44

E42G.J. Rocamora & D.S. Richardsonintroduced Sandragon Pterocarpus indicus, orAlbizzia Paraserianthes falcataria (Watson 1984,Rocamora 1997a, 1997b). In all these places, nonnativeplants largely dominate the vegetation,which suggests that the species is very adaptable(Mellanby et al. 1996; Rocamora & François 2000).On Conception, breeding territories occur throughoutthe dense mixed woodland found on the island,dominated by Cinnamon Cinnamomum verum(exotic), Takamaka Calophyllum inophyllum (native)and Cashew Anacardium occidentale (exotic), withabundant indigenous fruiting trees providing berriesand seeds for the White-eyes.Differences have also been found with regardto breeding biology. Birds on Conception are cooperativebreeders with up to nine birds contributingto the same nest, which may contain up to seven eggs(Rocamora & François 2000). The composition ofnesting groups is unstable, as adult birds contributingto a particular nest are often observed on nests fromother neighbouring breeding groups during the sameseason. Such a complex social breeding system hasnever been reported from Mahé (Feare 1975, Grieg-Smith 1979, Rocamora 1997b) where only two eggsper nest and smaller co-operative breeding groups(up to five) have been observed. These differencesmay be due to the extreme scarcity of the species onMahé, but could also be an adaptation to the highdensity and saturated environment faced byWhite-eyes on Conception.In conclusion, the limited morphological andgenetic differentiation found between the twopopulations, combined with our knowledge of thespecies’ former distribution and history, indicate thatthe two populations studied are reproductivelyisolated, probably as a consequence of recent anthropogenicdisturbance, and should not, in our view, beconsidered as two separate races or evolutionarysignificant units (Crandall et al. 2000). However, theobserved differences in morphology, ecology andbreeding behaviour suggest that each may be locallyadapted to its particular environment.Conservation implicationsThe most important conclusion from this study, interms of population management, is that both populationsare important genetic reservoirs, and equallydeserve to be safeguarded. Consequently, the SpeciesAction Plan 2001–06 (Rocamora & Henriette2002), which includes transfers of individuals fromeach population to suitable islands, suggests thateach population should be treated as a separatemanagement unit but not as an evolutionarily significantunit. Birds of Conception and Mahé origin maytherefore be mixed in one of these island transfers.Since the two populations probably retain differentgenetic fragments of the original White-eye population,recombining this genetic variation may bebeneficial for the species in a novel environment.Increasing genetic variation has been shown tohave positive effects on small, isolated, inbredpopulations with low genetic variability, e.g. GreaterPrairie Chickens Tympanuchus cupido pinnatus(Westemeier et al. 1998), the Topminnow Poeciliopsismonacha (reviewed in Vrijenhoek 1994), Viperaberus (Madsen et al. 1999), and the method has beenassessed for use as a conservation measure in theFlorida Panther Felis concolor coryi (Hedrick 1995).Benefits include a better capacity to adapt to newenvironmental constraints (of particular importancefor translocated individuals) and reduced vulnerabilityto extinction due to the negative effects ofinbreeding.If the mixing of the Seychelles White-eye populationson a new island proved successful, with nonegative fitness consequences and with bothpopulations interbreeding and contributing towardsthe genetic variation of the population, then theseWhite-eyes would be used for future transfers tonew islands. However, because gene flow may alsoreduce population fitness within locally welladaptedpopulations (reviewed in Storfer 1999) themixing of the two original populations is not recommendedon either Conception or Mahé.This study illustrates the importance of investigatingdifferentiation between remnant populationsof a threatened species as a source of informationto orientate future conservation and managementmeasures. The Seychelles Fody Foudia sechellarum,another threatened species reduced to just four smallpopulations, warrants similar study.We are grateful to Dr Alejandro Anganuzzi and Prof. TerryBurke for helping with the statistics and early commentson the manuscript, Prof. Denis Couvet, Prof. Chris Feare,Dr Carl Jones, Vikash Tatayah and Adrian Skerrett forproviding references and suggestions, and Andy Krupa forhelp in the lab. Special thanks to all the staff of Conservationsection of the Division of Environment, especiallyJoseph François, Perley Constance, Roland Nolin, JohnNevill and Maurice Loustau-Lalanne, and to two anonymousreviewers who provided very valuable comments.We wish to thank Helicopter Seychelles, Marine ParkAuthority and Thérèse Island, who helped with transportationto Conception, and the Rassool-Savy family forallowing access to their island. This study was funded by© 2003 British Ornithologists’ Union, Ibis, 145 (online) E34–E44

E44G.J. Rocamora & D.S. RichardsonRocamora, G. (ed.) 1997b. Rare and Threatened Species, Sitesand Habitats Monitoring Programme in Seychelles. ProjectG1 EMPS. Final Report 1. Monitoring methodologies & priorityactions. Ministry of Environment/Birdlife International/European Union.Rocamora, G. & François, J. 2001. Seychelles White-eyenews. World Birdwatch 23 (2): 7.Rocamora, G. & François, J. (coordinators). 2000. SeychellesWhite-eye Recovery Programme. Phase 1. Saving the SeychellesGrey White-eye. Final report. Ministry of Environment& Transport/Dutch Trust Fund/IUCN.Rocamora, G. & Henriette, E. 2002. Seychelles White-eyeConservation Assessment and Action Plan. SeychellesWhite-eye Recovery Programme. Phase 2. Ministry ofEnvironment/BirdLife Seychelles. Ministry of Environment/IUCN/Dutch Trust Fund.Saccheri, I., Kuussaari, M., Kankare, M., Vickman, P.,Fortelius, W. & Hanski. I. 1998. Inbreeding and extinction ina butterfly metapopulation. Nature 392: 491–494.Sambrook, J., Fritsch, E.F. & Maniatis, T. 1989. MolecularCloning: a Laboratory Manual. New York: Cold Spring HarbourLaboratory Press.Snedecor, G.W. & Cochran. W.G. 1980. Statistical Methods,7th edn. Ames, Iowa: Iowa State University Press.Stangel, P.W. 1986. Lack of effects from sampling blood fromsmall birds. Condor 88: 244–245.Storfer, A. 1999. Gene flow and endangered species translocations:a topic revisited. Biol. Conservation 87: 173–180.Venables, W.N. & Ripley, B.D. 1994. Modern Applied Statisticswith S-Plus. New York: Spinger-Verlag.Vrijenhoek, R.C. 1994. Genetic diversity and fitness in smallpopulations. In Loeschcke, V., Tomiuk, J. & Jain, S.K. (eds)Conservation Genetics: 65–76. Basel: Birkhäuser-Verlag.Watson, J. 1984. Land birds: endangered species on the graniticSeychelles. In Stoddart, D.R. (ed.) Biogeography and Ecologyof the Seychelles Islands: 513–528. The Hague: Dr W. Junk.Westemeier, R., Brawn, J., Simpson, S.A., Esker, T.L.,Jansen, R.W., Walk, J.W., Kershner, E.L., Bouzat, J.L. &Paige, K.N. 1998. Tracking the long-term decline and recoveryof an isolated population. Science 282: 1695–1698.Westneat, D.F. 1990. Genetic parentage in the Indigo Bunting: astudy using DNA fingerprinting. Behav. Ecol. Sociobiol. 27: 67–76.Received 21 August 2001; revision accepted 4 November 2002© 2003 British Ornithologists’ Union, Ibis, 145 (online) E34–E44