620 ShortCommunications [Auk, Vol. 107 Analysis of Mitochondrial DNA of Pacific Black Brant ( Branta bernicla nigrica•s) GERALD F. SHIELDS Instituteof ArcticBiology, University of Alaska-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 birds 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 California 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 Goose (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