620 ShortCommunications [Auk, Vol. 107

Analysis of Mitochondrial DNA of Pacific Black Brant ( bernicla nigrica•s)

GERALD F. SHIELDS Instituteof ArcticBiology, University of -Fairbanks, Fairbanks,Alaska 99775-0180 USA

Brant (Brantabernicla) have become the object of (Birky et al. 1983), and historical reductions in the increasedconcern to managersof waterfowl popu- sizesof populationsas well asfounder events should lations. The numbers of wintering Brant have de- be revealed more clearly by analysisof mtDNA than creasedmarkedly from a peak of 55,000 wintering by analysisof nuclearDNA, which is diploid (Wilson in the United Statesin 1958 to approximately et al. 1985).Finally, given the assumptionthat mtDNA 5,000in 1972(Management Plan for PacificCoast Brant approximatesa molecularclock marking time via suc- 1981). Correspondingreductions in the numbers of cessivemutations occurring at somewhatregular in- breeding Brantin Alaska have alsooccurred (Lensink tervals, one can usedivergence valuesbetween DNAs 1987). The numbers of wintering birds in traditional to approximateboth the extentand rate of separation Brantareas in BritishColumbia, Washington, Oregon, from a common ancestor. and have declined during periods when Nineteen individuals were available from five sites the numbers of wintering birds in Mexico have in- (Fig. 1). We purified mtDNA from the kidneys and creased.Together, these observationsimply that a spleensof all birds. Tissueswere processedfresh, variety of factorsmay influence Brant numbers in any except for birds from Melville Island, which were localethroughout the year.Reductions in the number banded on Melville as young of the year and then of Brant that traditionally winter in Canadaand the collectedat Padilla Bay,Washington, in winter. Their western United Statesmay simply be associatedwith kidneys were preservedin mannitol/sucrose/EDTA habitat deterioration or disturbance, which forced the buffer on wet ice and sent to Fairbanks where mtDNA birds to winter farther south in Mexico. waspurified upon receipt. Mitochondrial DNA of birds Reductionsin the sizes of breeding populations from the Yukon-Kuskokwim Delta, Anderson River, may be accompaniedby a correspondingreduction and Victoria Island was purified accordingto the in geneticdiversity. Accordingly, we studied the mi- methods of Lansman et al. (1981) and Cann (1982). tochondrial DNA (mtDNA) of individuals from five We purified mtDNAs of samplesfrom the North Slope separatelocations across the breeding range of B. ber- of Alaska and from Melville Island accordingto the nicla.We included four "gray-bellied" birds from an more efficient methodsof Carr and Griffith (1987). We apparent distinct breeding population on Melville used AvaI, EcoRI, HincII, HindIII, HpaI, PvuII, AvaII, Islandof the CanadianArctic. Local populations char- BstUI, HhaI, HinfI, and HpaII to monitor DNA se- acterized by unique mtDNA may suggestan older quence variability in all birds of this study. Mito- radiation followed by reductionsin the numbersof chondrial DNAs of the birds from the Yukon-Kus- individuals. Alternatively, homogeneity among kokwim Delta, Anderson River, and Victoria Island mtDNA may indicate that the Brant have recently were alsodigested with DdeI and RsaI.Mitochondrial expandedtheir range dramaticallyor that the muta- DNAs of birds from the North Slope of Alaska and tion rate in Brant mtDNA is low. Melville Island were alsodigested with BamHI, BanI, Becauseof its simplicity,haploid composition,lack BgIII, ClaI, NarI, NciI, NcoI, SpeI,and StyI. Fragments of recombination,transmission through the maternal of mtDNA were end-labeled with radioactivephos- germ-line, and rapid rate of evolution, mtDNA has phorous,and their sizeswere comparedelectropho- been useful in resolving questionsabout population retically using either agarose(1.0-2.0%) or 5% poly- dynamics and in making predictions about phylog- acrylamideon long vertical gels. We used HindIII to enies (Wilson et al. 1985, Avise 1986). Analysis of digest DNA from phage lambda into fragmentsof mtDNA has been important to biologistswho study known length that were then used as size standards waterfowl (Shields and Wilson 1987b, Van Wagner on each gel. Statisticsof hornologyfrom which the 1987) becauseunlike most other groups of verte- percentagesof nucleotidesequence divergence were brates, female geese and ducks generally establish calculated were from Nei and Li (1979). breedingsites near their natal sitesand return to them On average, 88 fragmentswere analyzed for each year after year to reproduce(Greenwood and Harvey of the 19 individuals.This level of analysisis typical 1982, Rohwer and Anderson 1988). Thus, fidelity to of comparisonsof this type for birds (Ball et al. 1988, natal sites and transmissionof mtDNA through the Shields and Helm-Bychowski 1988). All Brantsstud- germ-linesof thesefemales provides a molecularrec- ied fell into eitherof two typesbased on their mtDNAs. ord of the patternsof their dispersaland phylogenetic Birdsfrom the Yukon-KuskokwimDelta, North Slope relationships.Moreover, effective rates of mitochon- of Alaska, Anderson River of the Yukon Territory, drial gene flow among populationsshould be ap- and Victoria Island had essentiallyidentical mtDNAs. proximately one quarter of those of nuclear genes In birds from the Anderson River, one of the three July1990] ShortCommunications 621

MelvilleIsland•' 5oo •ooo ./• 6557 4361

•akeTes•kpuk AndersOn•vJ • •lslan• 2322 2027 mml --. I • I -- mmmmm m ,..,, I

YukOn-KuskOkw' •

Fig. 1. Collection locations for Brant of this study.

Fig. 2. Autoradiographof BrantDNA digestedwith individualslacked an RsaIfragment of approximately the restriction enzyme BstUI. Numbers on the left 1,250base pairs but possesseda unique fragment of refer to the length in nucleotidesof lambda phage approximately 1,200 base pairs, which all other in- DNA digestedwith the enzyme HindIII and used as dividuals lacked. a size standard. Sample lanes: 1-4, North Slope; 5, The birds from Melville Island were distinct from Anderson River; 6-7 Victoria Island; and 8-11, Mel- all othersof this study.These birds had unique frag- ville Island. ment patterns for 7 (BstUI, AvaII, PvuII, BanI, NcoI, StyI, and NarI) of the 20 restrictionenzymes used to study them. Accordingly, they were 0.74%divergent kwim Delta, North Slope, Anderson River, and Vic- from the birds at all other sites.The restrictionfrag- toria Island. The intermediacyof Melville Brant belly ment patternsfor mtDNAs digestedwith the enzyme coloration and breeding distribution relative to ni- BstUI emphasizethe uniquenessof the Melville Is- gricansand hrotamight imply that Melville birds are land birds (Fig. 2). The four Melville birds (Fig. 2) hybrids.The 0.74%divergence in mtDNA of Melville possessa fragment of approximately1,200 nucleo- birds from nigricansis significant and approximates tides in length (right arrow) which is replacedin all the 0.8% divergence between the RossGoose (Chen other Brant by two fragmentsof approximately750 rossii)and the Snow (C. caerulescens)(Shields and 460 nucleotides (left arrows). Within the Melville and Wilson 1987a). This divergence makes a recent birds, one individual (No. 825) differed from others hybrid origin for Melville birds unlikely. The hybrid in fragment patterns for two enzymes (Table 1). origin hypothesiscan be testedin another way. For- The homogeneity of mtDNAs of Brant from the tunately, data on restrictionfragments of mtDNA of Yukon-KuskokwimDelta, North Slopeof Alaska,An- a single hrotaare available (Van Wagner 1987). If Mel- derson River, and Victoria Island could be caused ville birds are recent hybrids, they should possessa either by a very recent radiation and movement into mtDNA characteristicof the subspeciesof female or previously unoccupiedterritory, or by a decelerated femalesinvolved in the hybridization. Melville birds rate of changein Brant mtDNA relative to other ver- tebrates.Decelerated rates seem unlikely becausethe rateof mtDNA evolutionin geese(Branta, Chen, Anser) B.b. ntaricans (n-15) is ca.2.0% per million years(Shields and Wilson 1987a), similar to most other vertebrates that have been stud- ied in this way (Wilson et al. 1985). -- Melville Brant (n-4) Female Brants exhibit strong philopatry to their B.b. hrota (n-l) natal sites. Such reproductive behavior will eventu- ally promote genetic differentiation among birds t 0.5 oI t•YR which are distributed as widely as Brant. However, 2 t 0 Percent with the exception of the Brant on Melville Island, divergence the mtDNAs of birds of this study were essentially Fig. 3. Phylogenetic tree for Brant constructedby homogeneous.It seemslikely that Branthave recently the midpoint method. We assumea 2.0% per million expanded their range to include the Yukon-Kusko- year rate of evolution for goosemtDNA. 622 ShortCommunications [Auk, Vol. 107

TABLE1. Sizesin basepairs of fragmentsgenerated by digestionof Brant mtDNA with five restriction endonucleases.

Fragmentsize (bp) AvaII Atlantic 7,900 -- -- 2,800 1,800 1,500 900 800 Melville -- -- 6,500 -- 1,800 1,500 900 -- Pacific -- 6,800 -- 2,800 1,800 1,500 -- -- PvuII Atlantic 16,200 .... Melville (A) -- 11,800 -- -- 4,800 Melville (B) -- -- 11,000 5,800 -- Pacific -- 11,800 -- -- 4,800 HhaI Atlantic -- 3,150 1,700 -- 1,500 Melville (A) 3,900 -- 1,700 1,600 1,500 Melville (B) 3,900 -- 1,700 1,600 1,500 Pacific (A) -- 3,150 1,700 -- 1,500 Pacific (B) -- 3,150 1,700 -- 1,500 Atlantic 1,250 1,175 -- 1,050 Melville (A) 1,250 1,175 1,100 -- Melville (B) -- 1,175 1,100 -- Pacific (A) -- 1,175 1,100 1,050 Pacific (B) 1,250 1,175 1,100 -- BstUI Atlantic 4,300 -- -- 2,700 1,700 1,500 Melville -- 3,400 3,300 -- 1,700 1,500 Pacific -- 3,400 3,300 -- 1,700 1,500

Atlantic 1,200 960 870 -- 550 Melville 1,200 960 870 -- 550 Pacific -- 960 870 750 550 460

HindIII Atlantic 4300 4,000 3,900 3,100 2,300 -- -- Melville -- 4,000 3,900 3,100 2,300 1,500 1,000 Pacific -- 4,000 3,900 3,100 2,300 1,500 1,000

shareno patternswith hrotaand only two of the five originatedfrom SouthhamptonIsland and Foxe Basin patternswith nigricans(Table 1). The factthat Melville (T. Barry,K.L. Abraham,A. Reed,unpubl.). Another Branthave unique fragment patterns for mostof these relativelypale-bellied form, breedingchiefly on Mel- enzymesargues against a recenthybridization event. ville Island, was found to winter in Puget Sound, If mtDNA of geeseevolves at a rateof 2.0%per million Washington;and the majorityof dark-belliedwestern years,then Melville birdshave been isolated repro- Brant from northwestern Canada and Alaska win- ductivelyfrom Pacific Black Brant for approximately tered farther southon the Pacificcoast, principally in 400,000years (Fig. 3). Mexico (seeBellrose 1980)." Thus, it appearsthat Mel- My observationson the mtDNA of Brant are com- ville Brantare morphologically distinct and generally plementedby bandingstudies. Boyd et al. (1988:1) separatedfrom other Brant on their wintering terri- statedthat "It usedto be thought that the Brant of toriesin PugetSound (Boyd et al. 1988).Their mtDNA were of just two populationswinter- is unique.A studyof a largersample of Atlantic Brant ing onthe Pacific and Atlantic coasts respectively: the and pale-bellied Brant that breed in the northeastern former were dark-bellied (B.b. nigricans)and the latter CanadianArctic and winter in seemsjustified. werepale-bellied (part of B.b. hrota). During the 1970s RobertBromley, James Hawkings, Austin Reed,and andearly 1980s,ringing and colour-markingof Brant JamesSedinger kindly collectedBrant for this study. in the Queen Elizabeth Islandsand Foxe Basinshowed Dirk Derksenencouraged this studyand offeredad- that thesituation was more complex. Pale-bellied Brant vice. Andrea M. Schmiechenand Judy Matherly iso- breedingin the northeasternCanadian arctic were lated some of the DNA and carried out some of the found to winter in Ireland (Maltby-Prevettet al. 1975), fragmentanalyses. James Sedinger, Dale Guthrie, and whereas those wintering on the US Atlantic coast Edward Murphy have commented on this manu- July1990] ShortCommunicatio• 623

script.Carol Van Wagner and Allan Baker kindly al- es to measure mitochondrial DNA sequencere- lowed us to cite their unpublished data on B. hrota. latednessin natural populations.Ill. Techniques This researchwas funded by an Angus Gavin Migra- and potentialapplications. J. Molec. Evol. 17:214- tory Waterfowl ResearchGrant to Shields through 226. the University of AlaskaFoundation. LF2qSINIC,C.J. 1987. Numbersof BlackBrant nesting on the Yukon-Kuskokwim Delta have declined

LITERATURE CITED by morethan 60%.U.S. Fish and Wildlife Service Research Information Bulletin: 87-126. AvISE,J.C. 1986. Mitochondrial DNA and the evo- NEI, M., & W. H. LI. 1979. Mathematical model for lutionary geneticsof higher .Phil. Trans. studyinggenetic variation in termsof restriction R. Soc. London B 312: 325-342. endonucleases. Proc. Natl. Acad. Sci. USA 76: BALL,R. M., JR., F. C. JAMES,S. FREEMAN,E. BIRMING- 5269-5273. HAM, & J. C. AVISE. 1988. Phylogenetic popu- ROHWER, F. C., & M. G. ANDERSON. 1988. Female- lation structure of Red-winged Blackbirds as- biasedphilopatry, monogamy, and the timing of sessedby mitochondrial DNA. Proc. Natl. Acad. pair formationin migratorywaterfowl. Pp. 187- Sci. USA 85: 1558-1562. 221 in Current ornithology,vol. 5 (R. F. Johnston, BELLROSE,F. C. 1980. Ducks, geeseand swans of Ed.). New York, Plenum Press. North America.Harrisburg, Pennsylvania, Stack- SHIELDS,G. F., & K. M. HELM-BYCHOWSKI. 1988. Mi- pole Books. tochondrialDNA of birds. Pp. 273-295 in Current BIRKY,C. W., JR., T. MARUYAMA, & P. FUERST. 1983. ornithology, vol. 5 (R. F. Johnston,Ed.). New An approachto population theory for genes in York, Plenum Press. mitochondriaand chloroplasts,and someresults. --, & A. C. WILSON. 1987a. Calibration of mi- Genetics 103: 513-527. tochondrialDNA evolutionin geese.J. Mol. Evol. BOYD, H., L. S. MALTBY-PREvETT, & A. REED. 1988. 24: 212-217. Differences in the plumage patterns of Brant --, & 1987b. Subspeciesof the Canada breedingin high Arctic Canada.Progress Notes, Goose(Branta canadensis) have unique mitochon- Can. Wild. Ser. 174: 1-9. drial DNAs. Evolution 41: 662-666. CANN, R. L. 1982. The evolution of human mito- VAN WAGNER,C.E. 1987. Geneticand morphometric chondrial DNA. Ph.D. dissertation, Berkeley, evolution in Canada Geese. Ph.D. dissertation, Univ. California. Toronto, Canada, Univ. of Toronto. CARR,S. M., & O. M. GRIFFITH.1987. Rapid isolation WILSON, A. C., R. L. CANN, S. M. CAP,R, M. GEORGE, of mitochondrial DNA in a small fixed- U. B. GYLLEr•STEN,K. M. HELM-B¾cHOWS•CI,R. G. angle rotor at ultrahigh speed.Biochem. Gen. 25: HIGUCHI, S. R. PALUMIBI,E. M. PRAGER,R. D. SAGE, 385-390. & M. STONEKING. 1985. Mitochondrial DNAand GREENWOOD,P. J., & P. H. HARVEY. 1982. The natal two perspectiveson evolutionarygenetics. Biol. and breedingdispersal of birds.Annu. Rev.Ecol. J. Linn. Soc. 26: 375-400. Syst. 13: 1-21. LANSMAN, R. A., R. O. SHADE, J. F. SHAPIRA,•r J. C. Received5 February1990, accepted 7 March 1990. AvISE. 1981. The use of restriction endonucleas-

Song FeaturesBirds Use to Identify Individuals

D•i• M. W•RY, • K•q J. NORMS,•D J. BRUCEFALLS 2 EdwardGrey Institute of FieldOrnithology, South Parks Road, OxfordOX1 3PS, UnitedKingdom

The ability of birds to discriminatebetween indi- nation. Only two relevant experiments have been viduals on the basisof song has been widely dem- performed(Brooks and Falls 1975,Nelson 1989),both onstrated(Falls 1982).Despite its prevalence,little is of which examinedonly variation within a single known about how the birds perform this discrimi- song type. The ability of birds to discriminate be- tween the songsof neighborsand strangersin play- back experimentsdecreases as the repertoiresize of • Present address: Department of Psychology, the speciesincreases (Falls 1982).This suggeststhat Queen'sUniversity, KingstonK7L 3N6, Canada. a repertoireof song typesis lessrecognizable than a 2 Presentaddress: Department of Zoology,Univer- single song. sity of Toronto, Toronto N5S 1A1, Canada. Male Great Tits (Parusmajor) have an averagerep-