Genetic Evidence for Philopatry in a Colonially Nesting Seabird, the Fairy Prion (Pachyptila Turtur)

GENETIC EVIDENCE FOR PHILOPATRY IN A COLONIALLY NESTING SEABIRD, THE FAIRY PRION (PACHYPTILA TURTUR)

J. R. OVENDEN,• A. WUST-SAUCY?3 R. BYWATER,• N. BROTHERS,2 AND R. W. G. WHITE • •FishResearch Group, Department of Zoology,University of Tasmania,G.P.O. Box252C, Hobart, Tasmania, Australia 7001, and 2TasmanianDepartment of Parks,Wildlife and Heritage, G.P.O. Box 44A, Hobart, Tasmania, Australia 7001

ABsTRaCT.--Philopatryis the selectivereturn of individual birds to breed closeto the site of their own hatching.This phenomenoncan greatly influence the operationof evolutionary forceswithin and amongpopulations of a species.Detection of philopatrynormally involves long-term mark, release,and recapturestudies. As an alternative, we have applied restriction enzyme analysis of mitochondrial DNA to a colonially nesting seabird species,the Fairy Prion (Pachyptilaturtur), as a test of philopatry. As the mitochondrialgenome is strictly maternally inherited, each colony in a philopatric speciesshould have a unique combination of mitochondrialhaplotypes. Twenty-one prions taken from one colonyall had identical mitochondrial genomes,and we argue that juveniles as well as experiencedadults return to the colony to breed. Philopatry within this colony does not explain the lack of mtDNA sequencevariation. The most likely explanation is the recent occurrenceof either a bottleneck or founder event within the colony probably involving a maximum of four femalesand an unspecifiednumber of male prions. Two additional coloniesof prions were not genetically homogeneousand did not have significantly different combinationsof mitochondrial DNA lineages.It was not possibleto confirm the existenceof philopatry in thesecolonies. Received 4 September1990, accepted18 February1991.

AMONGthe myriad of breedingsites potentially genetic tag (Linet al. 1990) recoverablefrom available, the majority of birds choseto breed all individuals. Unlike nuclear DNA, its com- closeto the site of their own hatching (Green- ponents are not subject to recombination be- wood and Harvey 1982). This is true for both tween generationsand its nucleotide sequence migratory and nonmigratory species (Green- is highly polymorphicamong individuals (Avise wood 1987),although it doesnot apply to spe- et al. 1987).Together, these characteristics mean cies occupying unpredictable habitats such as that maternallyrelated groupsof malesand fe- inland Australia.The amount of philopatty dis- malesare highly likely to sharemitochondrial played by a specieswill determine,in part, the genomes with the same nucleotide sequence. degreeof geneticsubdivision between popu- Groupsof animals that are not maternally re- lations that modulatesthe operation of evolu- lated are unlikely to shareidentical mitochon- tionary forcessuch as random mutation, genetic drial genomes.For example, Avise and Nelson drift, and natural selection. (1989) found that geographicallycontinuous Estimatingthe degreeof philopatryexhibited populationsof the SeasideSparrow (Ammodra- by avian speciesin the field has invariably in- mus maritimus)on the Atlantic and Gulf coasts volved long-term mark and recapturestudies of eastern North America possesseddifferent of large numbers of individuals. We applied setsof mitochondriallineages. restriction-site analysis of the mitochondrial The Fairy Prion (Pachyptilaturtur, Procellari- DNA (mtDNA) of breedingadults sampled from idae) is a small, tube-nosed seabird distributed three Australianprion coloniesto testfor philo- around the 40øSconvergence in the oceansof patry. the southern hemisphere.It is widespreadat Mitochondrial DNA is a maternally inherited sea around Australasia, and in the southern At- lantic and Indian oceans.The speciesbreeds in densecolonies from Augustto Februaryon off- 3 Present address:Bassenges 2, CH-1024 Ecublens, shore islands around southern Australia and Switzerland. New Zealand and on the Chatham, Snares, and

688 The Auk 108: 688-694. July 1991 July1991] PhilopatryinPachyptila 689

Antipodes islands (Lindsey 1986). Australian Fairy Prionsuse their coloniesas foraging bases outsidethe breedingseason. Although banding Albatross• • studiesof individuals in New Zealand suggest fidelity to island coloniesand the formation of relatively stable pair bonds (Harper 1976), such studieshave not beenperformed on FairyPrions breeding in Australia. In a speciesthat is not in a state of flux, and which has geographically discrete breeding groupsor colonies,philopatry should leave an unambiguousstamp on the pattern of mtDNA --42os 42os • sequencevariation among and within colonies. A This pattern will have developed over evolutionary time by the independent stochasticcre- ation and extinctionof mtDNA lineagesin each breedinggroup (Avise et al. 1984).Each philopatric colony will have its own combinationof mtDNA lineagesthat is significantlydifferent to that possessedby anotherphilopatric colony...... • - v TasmanIsland

These unique combinationsmay consistof as FlatTop Island / 147øE few as one or asmany as thousandsof lineages, I depending on long-term effectivesize of each Fig. 1. Locationsof Albatross,Flat To•, and Tas- colony. man •s•ands,&ore w•c• •;•ons we;e s•ed •; t•s stud•. MATERIALS AND METHODS

We collectedprions from Flat Top Island (43ø38'S, ter. Lettering began with A and proceededthrough 146ø23'E,n = 19), Tasman Island (43ø14'S, 147ø56'E,n the alphabet.No attempt was made to assignconsec- = 21), and Albatross Island (40ø23'S,144ø39'E, n = 21) utive letters to the most similar restriction patterns. duringthe southernsummer breeding season of 1988- In all cases,the number of fragmentsin the pattern 1989(Fig. 1). Males and femaleswere taken arbitrarily was assumedto be equalto the numberof restriction from accessiblenesting sites on eachisland. One Ant- sites in that genome for that enzyme. The relative arctic Prion (Pachyptiladesolata) was sampled from gain or lossof restrictionsites between sampleswas MacquarieIsland (52øS,158øE). The liver was dissected determined by the additive lossor gain of appropri- from each bird and stored in liquid nitrogen. Mito- ately sized fragments.The haplotype, or mitochon- chondrial DNA was extracted from thawed liver sam- drial genotype,of eachbird was a summaryof letters ples by a modification(Ovenden et al. 1988) of the correspondingto the fragmentpatterns produced from method of Chapman and Powers (1984). that samplefor each of the nine restrictionenzymes. The mtDNA sampleswere digestedwith two 5.33 We estimatedsequence divergence between pairs classrestriction enzymes (Ava I, and BanI) and seven of mitochondrial genomes,with standard errors, by 6.0 classrestriction enzymes (Barn HI, BglI, Cla I, Hin the maximum likelihood method of Nei and Tajima dIII, Nco I, Pst I and Xho I). Each sample was digested (1983). We also estimated the average sequencedi- with up to a tenfold excessof enzyme to ensure com- vergencebetween individual genomesfrom different plete cleavage,but otherwise we followed the sup- coloniesand betweengenomes from the samecolony plier's (New England Biolabs, U.S.) directions. Re- (Nei and Jin 1989). The standard errors of these es- strictionfragments were radiolabelledwith alpha-32P timatesproduced by this method are free of the bias or 35S-deoxycytosinetriphosphate using the exonu- introduced by the nonindependenceof the individ- cleaseand polymeraseactivity of the Klenow frag- ual pairwisedivergence measurements. Student's t-test ment of DNA polymerase I (Ovenden et al. 1989). was used to test the null hypothesisthat the mean Fragments were visualized by autoradiography in sequencedivergence between genomes collected from dried 1.4% agarosegels. Fragment sizes were esti- one colonywas significantlydifferent from that from matedby comparisonwith the mobility of fragments another colony. of known size produced by a Hin dIII digest of bac- We comparedobserved and expectedhaplotype teriophage lambda DNA. frequenciesto detectpossible population subdivision Samplesthat produced unique fragment patterns amongisland colonies, by the Chi-squaretest. A Mon- with a particular enzyme were assigneda unique let- te-Carlo test was used to determine the significance 690 OVENDENET ^L. [Auk, Vol. 108

A•al of the Chi-squarevalue (Roff and Bentzen 1989), as expected classsizes were often <5. As population AI subdivisioncauses localized excesses of homozygotes, a gene diversity analysis on groups of prions was Barn HI done by taking nucleotidesubstitutions (Takahata and 14389 I 45O5 Palumbi 1985) as alternate alleles. The statistic calculated(GsT) can be interpretedas the proportionof 18894 overall geneticvariation that can be attributed to the Ban I presenceof subpopulationsamong which gene flow is limited. The significanceof the GsTvalue was as- A sessedagainst 1,000 pseudo-GsT values obtained from B I randomrearrangements of the raw data (Palumbi and 14200 I I Wilson 1990). If population subdivisionis present, c the true GsTvalue will be greater than 95% of the pseudo-GsTvalues. The cladisticrelationship between D prion haplotypeswas explored using MacClade (ver- B,ql I sion 2.1, Wayne Maddisonand David Maddison,Har- vard University). A 10202 26392499 182411731 B 10202 2639 2499 3672 RESULTS c 10202 2639 4323 1731 We identified 1-5 morphs from 9 restriction D enzymesamong the 62 prion mitochondrialgenomes(Fig. 2). The Antarctic Prion samplewas E 6259I 39432639 4323 1731 not analyzedwith Nco I or Xho I. Itwas assigned Cla I the Fairy Prion commonmorph (A) for each of theseenzymes. The approximatesum of the sizes A 6639 6168 2991 1995 of restrictionfragments from the mtDNA from B 6639 9159 1995 the Fairy Prion was 18,900 nucleotides. c 6639 3269 2899 2991 1995 We scoredeleven haplotypesamong the 62 I I prionsin our surveyof 33 restrictionsites (Table 1). Eachhaplotype was definedby the presence A 12715 of between 26 to 30 of these sites. Twenty-one restrictionsites were presentin all haplotypes, B 12715 nine were presentin one haplotype only, and three sites (Ban I site 1, Bgl I site 1, and Nco I Nco I site 1) were presentin more than one--but not A all--haplotypes. All prions collectedfrom the B 18894 breeding site on Albatross Island possessed 15218148 I haplotype 1. This haplotype was found in 7 Pst I birds from Flat Top Island and 12 from Tasman Island (Table 1). The remaining 12 individuals from Flat Top Island had four different haplo- Xho I types (2, 3, 4, and 5). Four birds from Tasman Island shared two of these haplotypes(2 and A[ 18894 I 5). Haplotypes3 and 4 were found only on Flat Fig. 2. The fragment compositionof the restric- Top Island. The remaining five birds from Tas- tion site morphsidentified amongFairy and Antarctic man Island possessedhaplotypes found only prion mitochondrial genomesby nine restrictionen- within this colony. zymes. Fragment sizeswere scaledso that their sum Fairy Prion haplotypes2 and 4-10 differed is equal to the overall mean genome size of 18,894 by onerestriction-site gain or lossfrom the most nucleotidepairs. Fragments were placedon the linear common haplotype 1 (Fig. 3). The Antarctic mtDNA mapsto show restrictionsite gainsor losses Prion (haplotype 11) differed by three restric- betweenthem. The relative positionof the fragments was not determined absolutely. tion-site gains or lossesfrom Fairy Prion hap- 1otype5. Fairy Prion haplotype 8 was one restriction-sitemutation from either haplotype5 July1991] Philopatryin Pachyptila 691

TABLE1. Numbers of prion haplotypes scoredfrom each of three island colonies.Each haplotype is composedof the morph designationsfor the restrictionenzymes Ava I, BarnHI, BanI, BglI, Cla I, Hin dIII, Nco I, Pst I, and Xho I.

Locality Alba- Flat Tas- tross Top man (n= (n= (n= BglII/• ' site Haplotype 21) 19) 21) Total Bani•.•te 1 Fairy Priori 1 AAAAAAAAA 21 7 12 40 [ • • Hi•d!11 2 AAAAAABAA 0 1 2 3 3 AABBABBAA 0 2 0 2 4 AAAABAAAA 0 2 0 2 5 AAACAAAAA 0 7 2 9 6 AACAAAAAA 0 0 1 1 7 ABAAAAAAA 0 0 1 1 8 AAACAABAA 0 0 1 1 9 AABAABAAA 0 0 1 1 10 AAADAAAAA 0 0 1 1

Antarctic Priori T s"e' site2 site3 11 AADECAAAA 1

(Fig. 3A) or haplotype2 (Fig. 3B). Fairy Prion haplotype3 was three restriction-sitemutations from either haplotype9 (Fig. 3A) or haplotype 2 (Fig. 3B). The mean(_+ SD) sequence divergence among the mitochondrialgenomes from 19 prionsfrom Flat Top Islandwas 0.51 _+0.25%. This wassig- nificantlydifferent from the genomessampled Fig. 3. The two mostparsimonious, unrooted net- from the TasmanIsland colony(0.28 + 0.15%, works (A, B) describingthe relationshipbetween ten t = 3.86, t[P = 0.05, df = 38] = 2.02, two-tailed). Fairy (no. 1-10) and one AntarcticPriori (no. 11) hap- The magnitudeof mtDNA sequencedivergence ]otypes.Labelled barsacross lines joining hap]otypes on Flat Top Island samplediffered from Tasman indicatethe relative gain or lossof restrictionsites. Island because of the exclusive occurrence of Sitesthat were presentor absentin only one haplo- two birds that possessedthe divergent haplotype are representedby an open bar. Sitesthat were presentor absentin more than one, but not all, hap- type 3 within the Flat Top Island sample, lotypesare representedby a shadedbar if they appear The net mtDNA sequencedivergence be- in the network more than once and by a solid bar if tween birds from the Flat Top and Tasman is- they appearonly once.Barn HI site 1 waslost by Barn land colonieswas not significantlydifferent HI morph A. BanI site 1 was gained by BanI morph from zero (0.0087 _+0.0192%). The frequencies D, and BanI sites2 and 3 were lost by morphsC and of haplotypessampled from the two islandswere B, respectively.Bgl I morph B lost BglI site 2, while alsonot significantlydifferent (X 2 = 13.36,P = morph C lost site 1. BglI site 4 was gained by morph 0.05 + 0.0195). Finally, the amount of overall D and site 5 was gained by morph E. Cla I morph B mitochondrialgene diversity that was due to lost Cla I site 1 and morph C gained site 2. Hin dIII geneticsubdivision among island colonies (GsT) site 1 was gainedby morph B, while NcoI site 1 was lost by morph B (Fig. 2). was 0.17. This value doesnot provide evidence for a lack of gene [low betweenisland colonies as it was greater than only 55.4%of 1,000pseu- One Fairy Prion haplotype (no. 3) was as dif- do-GsTvalues. ferent from the remaining Fairy Prion haplo- The mitochondrialgenome of the Antarctic types (range of estimated sequencediversity Prion was similar to those of the Fairy Prion. (+SE), 1.00 + 0.59% to 1.70 _+0.80%) as was the 692 ow•D• ET^L. [Auk, Vol. 108

Antarctic Prion (1.05 + 0.62% to 1.78 + 0.84%). philopatric. Our sampling regime, in terms of The carcassesof the two prions that had hap- either numbers of individuals or numbers of lotype 3 were not morphologically different restrictionsites surveyed per genome,may have from the remaining 17 birds collectedfrom Flat been inadequateto identify significantdiffer- Top Island (Barton 1989). We estimatedthe se- encesin mtDNA profiles,especially if the col- quencedivergence between the Antarctic Prion onies have not experiencedbottlenecks, foun- haplotypeand Fairy Prionshaplotype 3 at 2.54 der events, or the completion of stochastic + 1.03%. lineage sorting. Both colonies are larger than the AlbatrossIsland colony, which would slow DISCUSSION the processof lineage sorting.However, from the length of time eachisland hasbeen isolated All 21 birds from Albatross Island had the from mainland Tasmania,the coloniesmay be samemtDNA haplotype.Although this cannot older than the colonyon AlbatrossIsland. Some be taken to indicate absolutelythat all individ- evidencethat philopatty operateswithin each ualson the islandare homogeneousfor the mi- of the coloniesis given by the significantdif- tochondrial genome sampled, we believe that ferencein the magnitudeof mtDNA sequence this haplotypemust predominatein the colony. divergence within each colony. Different If all birds we examinedwere part of a single amounts of mtDNA sequencevariation in each panmictic population and the haplotypeswere colonywould not accumulateif a large amount present in the entire population in ratios rep- of gene flow occurredbetween them. resentedin the total sampleexamined here, the We suggestthat the observedmitochondrial probability of choosing21 birds all with hap- genome homogeneity within the AlbatrossIs- lotype 1 is only 0.00014. land colony indicates philopatty. Philopatry Thereare approximately 10,000 breeding pairs doesnot, however, necessarilycause homoge- of Fairy Prionson AlbatrossIsland (N. Brothers neity of haplotypes.There are at leastfour pos- unpubl. data). Prions forage over many hun- sible explanationsfor haplotype homogeneity dred square kilometers of the open ocean and within the AlbatrossIsland colony: a founder it is likely that the foragingrange of prionsfrom effect,a bottleneckevent, stochasticsorting of the three coloniessampled in this study would matriarchiallineages, and selection. overlap. Within their foraging range, individ- A founderevent occurs when a new population uals would encounter other breeding colonies is establishedfrom the offspring of a few in- of prions,as well as unusedpotential breeding dividualsin a rangeremote to the original pop- sites. mtDNA is strictly maternally inherited ulation. The Albatross Island colony must be and the best explanation for the current exclu- relatively young and may have experienceda sive occurrenceof haplotype1 amongthe male recent founder event. From 30,000 to at least and female prions sampledfrom the Albatross 8,000years B.P., the sealevel aroundTasmania Island population is strict philopatty of both wasat least100 m lower than it is today.During sexeswith no immigrationfrom geneticallydis- that time Albatross Island would have been an tinct colonies.It is likely that juvenile prions elevated area well inland from the coast and chosemates from other juveniles encountered unsuitable habitat for prions. In contrast, Flat on the island. There is evidence that visual (Ser- Top andTasman islands are surrounded by deep, venty et al. 1989),auditory (V. Bretagnollepers. precipitouswaters, and any land link between comm.),and olfactorycues (Grubb 1974) are used them and the Tasmanian mainland at lower sea by seabirdsto guide their return to specificcol- level would have been of shorter duration and onies. Analysis of the mtDNA of prions from fractured earlier than one between the main- other island colonies in western BassStrait, Black land and Albatross Island. Pyramid Island for example,is necessaryto test If the prion colony on AlbatrossIsland was the hypothesisof island--as opposedto re- establishedrecently, the mtDNA haplotypesof gional--philopatry. the colony would probably not be homoge- The mtDNA profilesprovide no information neous unless the haplotypesof the founding on philopatty of birds from the colonieson Flat femaleswere identical. Assuming that the fre- Top and Tasman islands. It is, however, un- quency of haplotype 1 in the original popula- likely that the birds from thesetwo southerly tion was the sameas it is now, it is likely that islands behave differently from their conspe- the foundinggroup consisted of no more than cificson AlbatrossIsland. They are presumably four females and their mates.With strict philo- July1991] Philopatryin Pachyptila 693 patry and abundantresources, only a few dozen eagesthat have mtDNA lesssusceptible to this generationswould have been required to reach type of decay during the life of a somaticcell the current colony size. may be more long-livedand henceleave more An alternative explanation for the observed offspringthan other lineages.Although there homogeneityof mitochondrialgenomes on Al- are many good reasonswhy selectionmay op- batross Island could be a recent bottleneck event. erateon the mitochondrialgenome, the concept A bottleneckevent is similar to a founder event, hasnot been widely accepted(MacRae and An- exceptthat a new population is producedfrom derson 1988). a few individuals within the range of the orig- The range of intraspecificmtDNA variation inal population, which is assumedto be extinct for FairyPrions is similarto that foundin other now. For all of the prions to have the same vertebrate species.However, interspecific ge- mtDNA, the maximumnumber of genetically netic variation between the Fairy and Antarctic identical female prions that survived the bot- prions is low compared with measurements tleneckto repopulatethe colonymay againhave made between other congenericavian species. been four or less. The range of diversity measurementsbetween The derivation of an entire population from the Antarcticand Fairy prion haplotypes(0.011- a small number of individuals can lead to re- 0.025) is below that for Anas (0.004-0.088), Ay- duced genetic variation, not only in the mito- thya (0.025-0.043),Melospiza (0.026-0.030) and chondrial genomebut also in nuclear genes. Dendroica(0.031-0.055) (Kesslerand Avise 1985). Nei et al. (1975) have shown that this will occur Futureanalyses of mtDNA may explainthis low if the populationdoes not expandrapidly after interspecific genetic divergence. a founder or bottleneck event. Extreme philo- Basedon our finding of mtDNA homogeneity patry can also lead to the loss of genetic vari- in a single colony of Fairy Prions, we suggest ability becauseof random geneticdrift in a fi- that juveniles that were hatched on the island nite population (Greenwood 1987). It may be and experiencedadults that bred at least once possibleto assessthe degree of inbreeding of in the colony return continually to the colony the prion colonyon AlbatrossIsland by a survey to breed. The presenceof philopatry alone can- of heterozygosityat allozyme loci and by mea- not explain the lack of mtDNA sequencevari- surements of fitness that could include fecun- ationin the colony.The mostlikely explanation dity estimates,hatching rates, longevity, and is the recent occurrence of either a bottleneck morphologicalvariation. or founder event within the colony, possibly Theoretically,monophyly or homogeneityof assistedby selection for mitochondrial ge- mtDNA lineagescan occurwithin a population nomes, which increased the fitness of birds in the absence of either founder or bottleneck within certain maternal lineages. The bottle- events. Avise et al. (1984) calculated that the neck or founder event may have involved a processesof stochasticlineage extinction and maximumof only four femalesand an unspeci- creationin a stable-sizedpopulation at carrying fied number of males. The other two colonies capacitycould lead to monophyly in 4n gen- of prions examined appear not to have expe- erations,where n is the female population size. rienced such severe bottleneck or founder The generationtime, or the meanage of a breed- events. Confirmation that philopatry operates ing adult for Fairy Prions is ca. 5 yr. The female within these two colonieswill rely on the de- populationsize for the AlbatrossIsland colony tailed analysisof the mtDNA from large num- is ca. 10,000. Assuming complete philopatry, bersof individuals.Finally, becausethe amount 200,000yr would be neededto homogenizethe of mtDNA sequencedivergence between spe- mtDNA lineages.This argument, therefore, is ciesof prionsis low, the phylogenyof the genus not supportablegiven the short history of the couldbe investigatedby applying cladisticand colony. phenetic analysisto the presenceand absence Natural selectionoperating directly on the of restriction sites in their mtDNA. mtDNA or its gene productsis another way of removing genetic variation in a population. It has been suggestedthat inefficiencyof mito- ACKNOWLEDGMENTS chondrial genome repair and replication in so- We than Jenny Jarrett and Wayne Kelly for assis- matic cells during the lifetime of an individual tancein the laboratory.The NAVAIDS sectionof the is responsiblefor aspectsof aging and death TasmanJanDepartment of Transport and Neil Smith (Linnane et al. 1989). Members of maternal lin- assistedin the collectionof samples. 694 OWNDENE'r AL. [Auk, Vol. 108

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