Mitochondrial-Dna and Nuclear-Gene Differentiation in North American Prairie Grouse (Genus Tympanuchus)

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The Auk 111(3):661-671, 1994

MITOCHONDRIAL-DNA AND NUCLEAR-GENE DIFFERENTIATION IN NORTH AMERICAN PRAIRIE GROUSE (GENUS TYMPANUCHUS)

DARRELL L. ELLSWORTH,L3 RODNEY L. HONEYCUTT,i NOVA J. SILVY,• KEVIN D. RITTENHOUSE,x AND MICHAEL H. SMITH 2 •WildlifeGenetics Laboratory, Department of Wildlifeand Fisheries Sciences, TexasA&M University,College Station, Texas 77843, USA; and 2SavannahRiver Ecology Laboratory, P.O. DrawerE, Aiken,South Carolina 29801, USA

ABSTP,•CT.--Thefragmentary effects of Pleistoceneglacial activity have been implicated in speciation among avifauna endemic to the Central Plains of North America. The prairie- grousecomplex (genus Tympanuchus),distributed throughout the central United Statesand Canada,contains three sistertaxa believed to have originated from the expansionof late Pleistocenerefugial populations.We assayedmitochondrial-DNA (mtDNA) restriction-site and allozyme variation in prairie grouseobtained from localitiesthroughout their current range in North Americato examinethe nature and timing of eventspromoting differentiation in Tympanuchus.The geneticdata were not consistentwith Pleistoceneisolation of sufficient duration to allow a taxonomicallyor geographicallystructured pattern of genetic variation to emerge. No clear genetic differencesamong specieswere observed.Allozymes could not distinguishpopulations belonging to different speciesand frequencieswere generally similar acrosstaxa. The mtDNA differentiationwas characterizedby a predominanthaplotype shared by all taxa;the remainder(15) were generallyinfrequent and closelyrelated to the prevalent (and presumablyancestral) haplotype. The presenceof unique mtDNA haplotypeswithin speciesand absenceof certain allozyme alleles from particular taxa implied a degree of isolation and restrictionsto gene flow. However, the mtDNA haplotypesdid not sort phy- logenetically,which suggestsrecent commonancestry of the lineagesand may explain the lack of congruencebetween genetic variation and speciesdesignations. Despite the absence of quantitativegenetic differentiation, considerable morphological and behavioraldifferences are apparent among the putative species.Adult male plumage, vocalization structures,and courtshipbehaviors form the basisfor taxonomicdivisions among prairie grouse,but, considering their closeassociation with reproduction,such charactersmay be subjectto sexual selectionand may evolve rapidly relative to mtDNA or allozymes. Received27 January1993, accepted19 August1993.

RANGE FRAGMENTATION and isolation associ- Many subspeciesand speciesin the Great Plains ated with Pleistocene glaciation have been are assumedto have arisen through geographic widely implicated in the differentiation of var- isolationin refugia during Pleistoceneglacial iousclosely related, but largely allopatric,avian maxima (Selander 1965, Hubbard 1973). Con- species(Rand 1948, Mengel 1964, 1970, Selan- versely,the low speciesdiversity of grassland der 1965). The Central Plains of North America, birds and paucity of closely related congeners in particular,have been viewed as an isolating led Mengel (1970) to hypothesizethat there has agent promoting speciation among avian taxa been little pastfractionation of the Central Plains that now occupyeastern deciduous and western into isolated environments. Therefore, it is un- montane forest (Mengel 1964, 1970, Berming- clear as to whether differentiation and specia- ham et al. 1992). However, the role of glacial tion amongbirds of the Central Plains may be activity in the speciationof avifauna endemic attributedto allopatric(due to isolation)or sym- to the Central Plains is not well understood. patric (without geographicsubdivision) mech- anisms. 3 Present address: Center for Demographic and The prairie grouse(genus Tympanuchus) com- Population Genetics,University of TexasHealth Sci- prise an interesting "speciescomplex" for ex- ence Center at Houston, P.O. Box 20334, Houston, amining the competing hypothesesregarding Texas 77225, USA. the fate of the Central Plains during the Pieis- 661 662 ELLSWORTHETAL. [Auk,Vol. 111 tocene and possible modes of differentiation/ gent avian sister taxa believed to have origi- speciationamong birds of the North American nated from the expansion of late Pleistocene grasslands.The prairie-grouse complex con- refugial populations.In this study,we assessed tains three primarily grasslandadapted sister mtDNA andallozyme variation in prairiegrouse taxa distributedthroughout the central United and testedprevious hypotheses regarding dif- Statesand Canada.Two extant formsof T. cupido ferentiation and speciation among the taxa. (Greater Prairie-Chicken) occur primarily in Specifically,we addressedthe following ques- remnant tall-grassprairie in Kansas,Nebraska, tions:What is the nature of genetic differenti- Oklahoma,and SouthDakota (T. c.pinnatus) and ation in the prairie-grouse complex?Are the in smallendangered populations along the Tex- patternsof geneticvariation and estimatedtimes as Gulf Coast (T. c. attwateri; Westemeier 1980). sincedivergence among mtDNA lineagescon- Tympanuchuspallidicinctus (Lesser Prairie-Chick- sistent with late Pleistocene (recent) vicarlance? en) occupies the shrub grasslandsof south- Has sporadic interspecific hybridization in- western Kansassouthward through Oklahoma ducedby human activity influencedthe pattern to eastern New Mexico (Waddell and Hanzlick of differentiationamong taxa?How do levels 1978, Cannon and Knopf 1980, Taylor and of genetic differentiation comparewith mor- Guthery 1980), and T. phasianellus(Sharp-tailed phologicaland behavioraldivergence among Grouse) is resident to the north-central United the putative species? States and central Canada to Alaska (Miller and Graul 1980). METHODS Tympanuchuscupido and T. pallidicinctusare recognized as distinct species(AOU 1983) due Specirnens.--Tissues(brain, heart, liver) were ob- to differences in behavior, habitat affiliation, tained from 86 prairie grouseand frozen on dry ice. and socialaggregation (Grange 1940, Jones 1964, Most birds were harvestedby hunters, but samples Sharpe 1968). However, the differencesare not from the endangered T. c. attwateriwere taken from asgreat asthose seen among other well-defined incidental mortalities at the Attwater Prairie-Chicken grousespecies, causing some researchers (Short National Wildlife Refuge. Collection sites, listed be- 1967, Johnsgard1983) to regard these taxa as low by state and county (USA) or province and mu- nicipality (Canada),were locatedthroughout the cur- allopatricsubspecies. Conversely, T. phasianellus rent range of each taxon: T. c. pinnatus,ILLINOIS, is morphologicallydistinct from the "prairie Jasper(2), Marion (2), KANSAS, Butler (3), Chase(5), chickens." Elongated central and reduced pe- Lyon (2), Shawnee (4), NEBRASKA, Rock (3), Thomas ripheral rectricesas well asrudimentary pinnae (3), OKLAHOMA, Osage(3), SOUTH DAKOTA, Ly- and cervicalapteria distinguish phasianellus from man (6); T. c. attwateri,TEXAS, Colorado(1), Refugio cupidoand pallidicinctus(Ridgway and Fried- (1); T. pallidicinctus,KANSAS, Clark (9), Morton (7); mann 1946). In fact, phasianellus(formerly Pe- T. phasianellus,COLORADO, Routt (2), MANITOBA, dioecetesphasianellus) was once placed in a dis- CANADA, Coldwell (4), Chetfield (2), MINNESOTA, tinct monotypicgenus, but Pedioeceteshas been Aitkin (4), Lake of the Woods (3), NEBRASKA, Tho- synonymized with Tympanuchusto reflect phys- mas(5), Rock (1), NORTH DAKOTA, Billings (1), Bur- leigh (1), Grant (2), McKenzie (1), Morton (1), SOUTH iological (Hudson et al. 1959, 1966) and de- DAKOTA, Campbell (1), Dewey (2), Lyman (2), QUE- mographicsimilarities. BEC,CANADA, Ungava Comt• (3). Wisconsinglaciation has been cited as the Mitochondrial-DNA analysis.--In the laboratory, primary factor promoting subdivisionand di- mtDNA was isolated from frozen tissueand purified vergence among prairie-grouse populations. on cesiumchloride density gradients(Cart and Grif- Hubbard(1973) hypothesized that prairiegrouse fith 1987).Mitochondrial DNA from 51 prairie grouse were continuouslydistributed throughout much (T. c. pinnatus,21; T. c. attwateri,2; T. pallidicinctus,6; of the Great Plains during the Sangamon in- T. phasianellus,22) representativeof localitiesfrom the terglacial,but that considerablerange fragmen- Texas Gulf Coast to Hudson Bay was digested with tation and isolation of populations occurred 12 six-base-recognizing(Barnil I, Cla I, Dra I, EcoRI, EcoRV, Hind III, Nde I, Pst I, SspI, SstI, Stu I, Xba I) during the Wisconsinglacial period. Differen- and 4 four-base-recognizing(Hha I, Msp I, RsaI, Taq tiation within isolated refugia and postglacial I) restriction enzymes. Fragmentswere end-labeled reinvasion of the plains may thus have pro- with 32P-deoxynucleotides,separated by molecular duced the distribution of extant taxa. Therefore, weight on 1.2% agaroseor 4% acrylamide gels, and prairie grouseare useful for characterizingge- visualizedby autoradiography.Fragment sizes were netic variation among morphologicallydiver- estimatedfrom comigrating molecular size standards July1994] DifferentiationinPrairie Grouse 663

composedof lambda DNA and PM2 DNA digested phate dehydrogenase(L) (GAPD; 1.2.1.12);aglycerol- with Hind III. 3-phosphate dehydrogenase (L) (GPD1; 1.1.1.8); Unique fragment patterns produced by each re- sorbitoldehydrogenase (L) (SORD;1.1.1.14); and xan- striction enzyme were designatedalphabetically in thine dehydrogenase(L) (XDH; 1.1.1.204).(2) Con- chronologicalorder of discovery.Restriction sites were tinuous tris citrate II (Selander et al. 1971): aconitase easily inferred from the fragment data and used to 1, soluble, aconitase 2, mitochondrial (L) (ACO1, assigneach individual a compositehaplotype that ACO2;4.2.1.3); glutamate dehydrogenase (L) (GLUD; reflecteda unique pattern of site variation acrossall 1.4.1.2);isocitrate dehydrogenase 1, soluble(L) (IDH1; restrictionenzymes. The compositehaplotypes were 1.1.1.42);lactate dehydrogenase A (L) (LDHA; 1.1.1.27); arbitrarily assignednumerical designations.To de- malate dehydrogenase,NAD (soluble), malate de- termine the most-parsimoniousrelationships among hydrogenase,NAD (mitochondrial) (H) (MDH1, the compositehaplotypes, a presence/absencematrix MDH2; 1.1.1.37);phosphogluconate dehydrogenase of the inferred restrictionsites was analyzedusing (L) (PGD; 1.1.1.44);and phosphoglucomutase1,2 (L) the HEURISTIC-SEARCHoption in PAUP 3.0n(Swof- (PGM1, PGM2; 5.4.2.2). (3) Lithium hydroxide (Se- ford 1990). A strict-consensustree was constructed lander et al. 1971):esterase (L) (ES;3.1.1.1). (4) Poulik containing information commonto all equally-par- (Poulik 1957):albumin (L) (ALB);mannose phosphate simonioussolutions. Haplotypes unique to a single isomerase(L) (MPI; 5.3.1.8);nucleoside phosphory- specieswere projectedonto a map of North America lase(L) (NP; 2.4.2.1);dipeptidase A, C (L) (PEPA,PEPC; and a minimum-mutationnetwork summarizingthe 3.4.13.11);peptidase E (L) (PEPE; 3.4.11.1); and su- consensustree was constructedby linking the hap- peroxide dismutase1, soluble (L) (SOD1; 1.15.1.1).(5) lotypes in an unrooted phylogenetic network so as Tris-maleate (Manlove et al. 1975): adenosine deam- to minimize the number of mutational steps. inase (L) (ADA; 3.5.4.4); creatine kinase, brain form, The proportion of shared restriction siteswas used creatine kinase, muscle form (L) (CKBB, CKMM; to estimatethe extent of nucleotidesequence diver- 2.7.3.2);glutamic-oxaloacetic transaminase 1, soluble, genceamong the haplotypes(Upholt 1977). Within and glutamic-oxaloacetictransaminase 2, mitochon- each species,two indices of mtDNA variation were drial (L) (GOT1, GOT2; 2.6.1.1). calculated:(1) nucleotidediversity (•r; Nei and Tajima The commonallele at each locuswas designated 1981),which is a measureof mtDNApolymorphism• 100 or -100 to denote respectiveanodal or cathodal acrossall individuals; and (2) nucleon diversity (h; migration. Other alleleswere labeled numerically by Nei and Tajima 1981) reflecting the diversity of expressingthe electrophoreticmobility of their pro- haplotypes within each taxon. Mitochondrial-DNA tein products relative to the common allele. Indices differentiationin the prairie-grousecomplex was es- of diversityand polymorphismincluding mean het- timated by determining the proportion of mtDNA erozygosityover all loci(/•) andthe percentageof variation attributable to differencesamong (versus polymorphic loci (P) were calculated from the allo- within) populations(GAT; Takahata and Palumbi 1985). zyme data.Genetic distances among populations and Due to small sample sizes, we combined some geo- among specieswere calculatedaccording to Rogers graphicallyproximate populations in the Gsranalysis. (1972),and patterns of similaritybased on the genetic The GaTstatistic was interpreted by a comparisonwith distanceswere delineatedby UPGMA clusteranalysis values derived from 100 permutation testsin which (Sheath and Sokal 1973). individualswere drawn randomly from the entire data set (from any species)and assignedto hypo- thetical populations with the samedimensions as the field-sampledpopulations. RESULTS Allozymeanalysis.--Horizontal starch-gel electro- phoresis(Selander et al. 1971,Harris and Hopkinson Mitochondrial-DNA differentiation.--From 1976)conducted at the SavannahRiver EcologyLab- fragmentprofiles with comigratingmolecular oratory was used to resolve30 presumptivegenetic size standards,we estimatedthe length of the loci in 60 prairie grousefrom localitiesthroughout mtDNA moleculein prairie grouseto be 16,600 the United Statesand Canada.Appropriate tissues basepairs (bp), which is typicalfor avianspecies from the endangeredT. c. attwateriwere unavailable for the allozymeanalysis. The five buffersystems and (Shieldsand Helm-Bychowski1988). All indi- tissuetypes (H = heart, L = liver) employedto vi- viduals possesseda single mtDNA haplotype sualize specific proteins (designatedaccording to and no length variation was observed.Sixteen McAlpine et al. [1989]and followed by International compositehaplotypes (Table 1) eachconsisting Union of Biochemistry[1984] EC numbers) are indi- minimally of 122 inferred siteswere identified. cated. (1) Amine-citrate (Clayton and Tretiak 1972): Of the 12 six-base-recognizingendonucleases, fumarate hydratase (L) (FH; 4.2.1.2); glucose dehy- 7 produced only one fragment profile in all drogenase(L) (GDH; 1.1.1.47); glucose phosphate individuals. The remaining 5 six-base-cutting isomerase(L) (GPI; 5.3.1.9);glyceraldehyde-3-phos- enzymes,as well as all four-base-recognizing 664 ELLSWORTHET ^L. [Auk, Vol. 111

TABLE1. Mitochondrial DNA haplotypes in prairie grousegenerated by 12 six-base-recognizingand 4 four-base-recognizingrestriction enzymes. Frag- ment patterns for each restriction endonuclease designatedalphabetically and combined as com- posite haplotypes acrossall restriction enzymes. Consecutivealphabetical designationsdo not imply genetic relatedness.

n Compositehaplotype a T. cupido 8 1 AAAAAAAAAAAA AAAA 6 2 ...... B 1 5 ...... B ...... B 1 4 ...... B.. 1 5 ...... B... 2 6 ...... C ...... B 1 ? ...... B ...... I 8 ...... B ...... CB I 9 .B ...... D. I tO .B ...... T. pallidicinctus

3 10 .B ...... 1 11 .B ...... B. T. phasianeHus 17 i ...... I 12 ...... B ...... 1 13 . .B ...... 1 14 ...... C ...... Fig. 1. Parsimonynetwork interrelating all mtDNA I 15 ...... C... haplotypes observed in prairie grouse. Haplotypes 1 16 .... B ...... identified by number asin Table 1. Haplotypes shared amongtaxa (1 and 10)indicated by circles;those unique "Compositehaplotypes numbered consecutively. Letters within hap- 1otypesfrom left to right representfragment profiles for following to a given taxon are depicted symbolically:oval, T. restrictionendonucleases: (six-base-recognizing enzymes) BamH I, Cla cupido;diamond, T. pallidicinctus;square, T. phasianel- I, Dra I, Eco RI, EcoRV, Hind III, Nde I, Pst I, SspI, Sst I, Stu I, Xba I; lus. Cross bars on lines interconnecting haplotypes (four-base-recognizingenzymes) Hha I, Msp I, RsaI, TaqI. show inferred number of restriction-site differences (gains or losses)between them. endonucleases,generated more than one pattern. Prairie-grousemtDNA haplotypes were not The HEURISTIC-SEARCH option in PAUP partitionedalong species boundaries. Estimates for the most-parsimonious relationship(s) of nucleotide-sequencedivergence within spe- amongthe haplotypesdetermined six equally cies exceededinterspecific values in many in- parsimonioustrees of 16 steps(consistency in- stances. The percent sequence divergence dex = 0.938). In the minimum-mutation net- amongall haplotypesaveraged 0.22 and ranged work (Fig. 1), the majority of haplotypes, from 0.07 to 0.54. The minimum sequencedi- whether speciesspecific or sharedamong taxa, vergencebetween two haplotypesunique to dif- differedfrom a prevalenthaplotype by a single ferent taxa (0.14%)was very low even by avian mutationalevent (a restriction-sitegain or loss). standards. The extent of differentiation was The centralhaplotype in Figure I (number 1) greater among haplotypesunique to T. cupido predominatedin abundanceand geographic (P averaged0.25%) than among haplotypesre- distribution. This particular haplotype was stricted to T. phasianellus(P = 0.15%). Tympa- common to all three prairie-grouse species, nuchuscupido also possessed greater nucleon di- identified at mostlocalities, and presentin 53% versity and nucleotidediversity than T. phasi- of the 51 individuals examined. A secondhap- anellus (Table 2). lotype (number 10) was sharedby T. cupidoand The level of mtDNA differentiation in prairie T. pallidicincfusin Kansas,but the otherswere grouseindicated a high degreeof similarity be- unique to a given taxon (Fig. 2). tween geographically-distantconspecific pop- July1994] DifferentiatwninPrairie Grouse 665

CurrentGeographic Species-specific Distribution mtDNA Haplotype [] T. cupido C) [] T.pallidicinctus T. phasianellus [] Fig.2. Geographicorientation of species-specificmtDNA haplotypes in prairiegrouse. Haplotypes iden- tifiedby numberas in Table1. Haplotypes2 and 6 (notshown) were observed in morethan one T. cupido population.Original range of extinctHeath Hen (T. c. cupido) indicated along northeastern Atlantic coast (H). ulations, as well as between groups of popu- heterozygosityand polymorphismwithin spe- lationsbelonging to different species.The Gsr cies (Table 2) were within the range of values statistic(0.282) representingthe proportion of reportedfor othergalliform birds (Corbin 1987, mtDNA variationattributable to geneticdiffer- Gutierrez et al. 1983, Zink et al. 1987, Ellsworth encesamong localitiesdid not exceed95% of et al. 1989). No fixed allelic differences among the Gsr values derived from the permutation the taxa were observed. Overall, the distribu- tests.Therefore, we did not observegeographic tion and frequencyof alleles acrossall prairie subdivision for mtDNA among prairie-grouse grousewere similar (Table 3, Fig. 3). However, populationsand detectedno quantitativedif- severallow-frequency alleles at specificloci were ferentiation among the putative species. absentfrom particularspecies. Although T. cu- Allozymevariation.--Six of the 30 allozymeloci pidoand T. pallidicinctuswere more similarto exhibitedvariation in prairie grouse.Levels of eachother (D = 0.011)than either wasto T. phasi-

T^BLœ2. Indices of mtDNA and nuclear-genediversity in prairie grouse.

mtDNA Allozymes Taxon /• + SE •r + SEa /• + SE P T. cupido 0.826ñ 0.059 0.0013+ 0.0005 0.039+ 0.017 20.0 T. pallidicinctus 0.733+ 0.155 0.0007+ 0.0006 0.054+ 0.026 20.0 T. phasianellus 0.411+ 0.131 0.0004+ 0.0003 0.046+ 0.020 20.0 ß•r values(and SEs)calculated using the SEND programwritten by L. Jin (Nei and Jin 1989). 666 ELLSWORTHETAL. [Auk,Vol. II1

TAnrE 3. Allele frequenciesat polymorphic allo- quiscula;Zink et al. 1991). The limited degree zyme loci in prairie grouse. of mtDNA differentiation in these specieswas attributed to recent and extensiverange expan- Taxon (n) sion, life historiesconducive to dispersal,and T. pallidi- T. phasi- the absenceof long-term zoogeographicbarri- T. cupido cinctus anellus ers to movement that maskthe relationshipbe- Allele (25) (13) (22) tween phylogeny and geography(category IV; A½01 Avise et al. 1987, Avise 1989). However, Red- 121 0.040 0.038 0.227 winged Blackbirdsand Common Grackles ex- 100 0.960 0.962 0.773 hibit greater dispersaltendencies than prairie ADA grouse.Banding-recovery data indicate that 85- 125 -- -- 0.045 100 0.920 0.923 0.955 90%of young Red-wingedBlackbirds and Com- 74 0.080 0.077 -- mon Gracklesmay disperseas much as 60 km, and the remaindermay potentially move much IDttl farther (over 700 km; Moore and Dolbeer 1989). 185 0.318 0.308 0.150 I00 0.682 0.692 0.850 In contrast,dispersal distances in prairie grouse are typically less than 7 km (Hamerstrom and MPI Hamerstrom 1951, Copelin 1963, Robel et al. 106 0.040 -- 0.068 I00 0.940 0.731 0.864 1970, 1972). Additionally, the range of prairie 93 0.020 0.269 0.068 grousehas diminished significantly in recent PEPC timesbecause of human activity(Johnsgard and 104 0.040 0.077 0.091 Wood 1968). I00 0.800 0.846 0.841 For prairie grouse,there are two primary hy- 98 0.040 -- 0.045 pothesesthat may accountfor the high degree 93 0.120 0.077 0.023 of geneticsimilarity amongtaxa despite marked PGD morphologicaland behavioraldifferences. Hy- 117 0.020 0.038 -- pothesisone posits that differentiation among 100 0.940 0.962 0.977 prairie grouseoccurred in geographicisolation 80 0.040 -- 0.023 during Pleistoceneglacial advances, but genetic evidence of such an event has been lost upon secondarycontact due to hybridization (pastor anellus(D = 0.020 and 0.023,respectively), con- on-going)that hasfacilitated gene flow and in- specificpopulations did not clustertogether in trogressionamong taxa. The secondhypothesis UPGMA analysis(Fig. 4). predictsthat subdivisionamong prairie grouse occurredduring the Wisconsin(Hubbard 1973), DISCUSSION which was sufficientlyrecent such that all populations retain (share) ancestral genetic poly- The most striking aspectsof genetic differ- morphismsthat aroseprior to divergence.Mor- entiation in the prairie-grouse complex were phologicaland behavioraldifferentiation among the absenceof phylogenetic resolution among species,therefore, may have proceededrapidly the mtDNA haplotypes,and lack of association relative to the rate of genetic change. between allozyme genotype and the putative We acknowledgethat sporadichybridization species.We observedno clear distinctionsbe- amongprairie grousedoes occur, but we do not tween taxa for either mtDNA or allozymes.For believe it is a significantfactor in determining example, some mtDNA haplotypes observed the high degree of genetic similarity among only in T. cupidowere more closelyrelated to taxa or their phylogeneticstatus. Tympanuchus lineagesunique to T. phasianellusthan theywere cupidoand T. phasianelluswere largely allopatric to other T. cupidohaplotypes. Additionally, al- throughout most of their original (pre-settle- lozymescould not distinguishpopulations be- ment) ranges (Coues 1874) with only narrow longing to different species.The pattern of zones of sympatry (Leopold 1931). The minor mtDNA differentiation in prairie grouse was geographic overlap coupled with ecological similar to that observed in other birds such as separation in areas of sympatry suggestvery the Red-wingedBlackbird (Agelaius phoeniceus; limited pre-settlement hybridization between Ball et al. 1988) and Common Grackle (Quiscalus cupidoand phasianellus.With the settlement of July1994] Differentiationin Prairie Grouse 667

PeptidaseC [] T. cupido 93 104 T.pallidicinctus T. phasianellus 98100 Fig.3. Geographicdistribution of allelefrequencies at peptidasec locusin prairiegrouse. Allele frequencies expressedas proportionalarea of circleoccupied. Species-specific populations denoted by letterswithin each pie diagram:(c) T. cupido;(1) T. pallidicinctus;(p) T. phasianellus. the prairie, agricultural practices and devel- dictsthat haplotypediversity within a histori- opment have increased the incidence of inter- cally isolatedassemblage will be drasticallyre- specifichybridization (Bent 1932,Evans 1966, duced (Ferris et al. 1983) due to substantial Johnsgardand Wood 1968), which may have fluctuationsin populationsize and the signif- facilitatedrecent gene flow and introgression icanceof effectivepopulation sizesin models amongtaxa. However, we sampledlocalities far of stochasticmtDNA lineagesurvivorship (Av- from potential zonesof hybridizationsuch as Colorado and Quebec, Canada for T. phasianel- Rogers'(1972) GeneticDistance lus, and the Attwater Prairie-Chickensubspe- ciesof T. cupidoin Texas.Due to our findings 7' cupido(Illinois) that(1) a mtDNA haplotypecommon to all three T cupido(Kansas) speciesoccurred at most localities (including • T. pallidi• incms(Kansas 1) geographicallydistant sites),(2) many unique T. phasianellus(Mtmlesota) haplotypeswere presentwithin species,and (3) • T. phasianellus(Nebraska) certain low-frequencyalleles were absentfrom T. phasianellus(South Dakota) particulartaxa, we believe hybridization and T. cupido(Nebraska) current gene flow have had little effect on the T. phasiantllus(Mmfitoba) overall pattern of genetic differentiation in T. cupido(South Dakota) prairie grouse. T. pallidicinctus(Kansas 2) The genetic data do not provide evidence of Fig. 4. UPGMA dendrogramdepicting allozyme an early Pleistoceneseparation of sufficientdu- similarityamong populations of prairie grouse.Tym- ration to allow a taxonomicallyor geographi- panuchuspallidicinctus collected from: (1) Clark Co., cally ordered pattern of genetic variation to Kansas;and (2) Morton Co., Kansas.Cophenetic cor- emerge.The conceptof historicalrefugia pre- relation was 0.856. 668 ELLSWORTHETAL. [Auk, Vol. 111 iseet al. 1984).If prairie grousewere subdivided of divergencetimes among lineages.The dif- during the early Pleistocene,we may expect ferencesamong mtDNA haplotypesin prairie stochasticlineage loss to have occurredwithin grousemay reflectevolutionary change occur- isolatedrefugia and an accumulationof genetic ring both prior and subsequentto "speciation," differences between separated populations making it difficult to estimate accuratelythe (taxa). However, mtDNA variation in prairie time sincedivergence among Tympanuchusspe- grouse(Fig. 1) was characterizedby a predom- cies. Nevertheless,the greatestnucleotide-se- inant haplotypeshared by all taxaand a number quencedivergence values suggest that the max- of others that differed from the common hap- imum time elapsed since divergence among lotype by singlemutational events. The pattern lineageshas been approximately270,000 years. of differentiationsuggests that the less-frequent More recent "speciation"is highly likely be- lineageswere derived from the prevalent and causethe greatestsequence divergence values presumablyancestral lineage. The putative an- reflect intraspecific differences. Thus, the cestralhaplotype was likely present in prairie mtDNA data are roughly consistentwith a late grouseprior to subdivisionbecause it is broadly Pleistoceneseparation among the taxa. distributedgeographically and is sharedby all Although prairie grouseare geneticallysim- taxa. Most of the infrequent haplotypes were ilar and appear to have been subdivided re- species-specific,indicating that they may have cently, considerablemorphological and behav- arisen after "speciation." ioral differentiation is apparent among the The presenceof species-specificmtDNA hap- species,particularly between the "prairie chick- lotypesand absenceof severalallozyme alleles ens" and T. phasianellus.Prairie grouseare sex- from certain taxa imply a degree of isolation ually dimorphic and malesexhibit complexste- and restrictionto gene flow amongthe species. reotypedbehaviors associated with reproduction However, we detected no clear phylogenetic (Johnsgard1983). Adult male plumage,vocal- resolutionamong the mtDNA haplotypesand ization structures, and courtship behaviors no congruencebetween allozyme variation and comprise the primary differencesamong the speciesdesignations. These observationssug- taxa.Thus, several explanations may account for gestsubdivision among taxamay have occurred the morphologicaland behavioraldivergence relatively recently. Recentlyspeciated taxa may among prairie grouse despite the absenceof exhibit a pattern of mtDNA differentiation diagnosticgenetic differences.First, morpho- where sequencedivergence within speciesex- logical and behavioral differentiation may be ceeds interspecificvalues becausemtDNA re- attributable to sexual selection. Male prairie flectschange that has accumulatedsince the grouseaggregate and aggressivelydefend small time of last common female ancestorirrespec- display territories(leks; Hamerstrom and Ham- tive of the time since speciation (Avise et al. erstrom 1973, Ballard and Robel 1974) and 1983).It is highly probablethat speciesdefined overtly competewith one anotherfor matesby by morphological characteristicswill appear ritualized display behaviors that include tail polyphyletic in terms of mtDNA lineages for fanning, erection of specialized neck feathers many generationsfollowing a speciationevent (pinnae), and vocalizationsproduced by inflat- (Avise et al. 1984, Neigel and Avise 1986). able esophagealair sacs(Sharpe 1968, Hjorth Therefore, prairie-grousemtDNA lineagesmay 1970). Dominant males occupythe central ter- not have had sufficient time to achieve a con- ritorieson the "booming/dancinggrounds" and dition of reciprocalmonophyly (i.e. to sort phy- participatein the greatmajority of matings(Bal- logenetically)such that each taxon constitutes lard and Robel 1974).The displaystructures of a distinct lineage.' males form the basis for taxonomic distinctions Correspondencebetween estimated times of among prairie grouse, but considering their divergenceamong the prairie-grousemtDNA prominent role in reproduction, they may be lineagesand the timing of Wisconsinglaciation subjectto sexualselection and thus may evolve would be consistent with Hubbard's (1973) vi- rapidly. Second,the morphologicalattributes cariance-speciationhypothesis. However, pre- that differ among taxa may, to an extent, be dicted datesof geologicalevents in earth'shis- ecophenotypic.James (1983) has demonstrated tory are merely approximations,and calibrations a nongenetic component to geographic char- of a molecular clock for mtDNA in birds (Shields acter variation in Red-winged Blackbirds.As and Wilson 1987) permit only crude estimates prairie-grouse taxa have distinctly different July1994] DifferentiationinPrairie Grouse 669 habitat requirements (Johnsgard 1983), mor- The manuscript was improved by helpful com- phologicaldifferentiation may, in part, reflect ments from and insightful discussionswith Robert adaptation to regional environmental condi- Zink, JosephNeigel, Thomas Dowling, Scott Davis, tions.A third possibilityis that genesrespon- Steven Palumbi, Ronald Westemeier, and Fred Zwick- siblefor morphologyand behaviorevolve much el. Paul Johnsof the SavannahRiver EcologyLabo- ratory graciouslyhelped conductthe allozymic por- more rapidly than mtDNA (or allozymes).Ra- tion of the study.Laboratory assistance was provided cial differentiation in color and size of House by Caren Eckrich. This researchwas supportedby Sparrow(Passer domesticus) populations has been grants from the National ScienceFoundation (BSR- shownto occurextremely rapidly (Johnstonand 9016327, Grant for Improving Doctoral Dissertation Selander 1964). Similarly, geographic differ- Research, to D. L. Ellsworth and BSR-8918445 to R. L. entiation in prairie grouse may have occurred Honeycutt),the Welder Wildlife Foundation(contri- over a time span that was too short for mtDNA bution #425), the SavannahRiver EcologyLaboratory restrictionsite or isozymedifferences to accrue. (Departmentof Energycontract DEAC09-76SR00-819), In summary,the genetic data were not con- and the AmericanOrnithologists' Union (Alexander sistent with a Pleistocene vicariance of suffi- WetmoreFund). The seniorauthor was partially sup- ported by a GraduateDeans' Merit Fellowshipand a cient durationto allow substantialgenetic dif- Thomas Baker Slick Senior Graduate Research Fel- ferentiation between isolated populations lowship from TexasA&M University. (species)to accumulate.However, the presence of unique mtDNA haplotypeswithin species and absenceof specificalleles from particular LITERATURE CITED taxa imply some degree of separation.Mito- AMERICAN ORNITHOLOGISTS' UNION. 1983. Check-list chondrial-DNA and allozymevariation in prai- of North American birds, 6th ed. Am. Ornithol. rie grousesuggest that gene flow is not respon- Union, Washington, D.C. sible for the high degreeof similarity among AVISE,J. C. 1989. Gene trees and organismalhisto- taxa or the absenceof geographicallyand tax- ries: A phylogeneticapproach to populationbi- onomically structuredpatterns of genetic dif- ology. Evolution43:1192-1208. ferentiation. Rather, recent population frag- AVISE,J. C., J. ARNOLD,R. g. BALL,JR., E. BERMINGHAM, mentation and isolation may account for the T. LAME, J. E. NEICEL, C. A. REEE,AND N. C. lackof cleargenetic distinctions among species. SAUNDERS.1987. Intraspecificphylogeography: The mitochondrialDNA bridge between popu- Morphological and behavioral differentiation lation geneticsand systematics.Annu. Rev. Ecol. amongprairie grouse has probably been driven Syst.18:489-522. by sexual selectionand appearsto have pro- AVtSE,J. C., J. E. NEIGEL,AND J. ARNOLD. 1984. De- gressedrapidly relativeto either mtDNA or al- mographic influences on mitochondrial DNA lozymes. lineage survivorship in animal populations. J. Mol. Evol. 20:99-105. AVISE,J. C., J. F. SHAPIRA,S. W. DANIEL,C. F. AQUADRO, AND R. 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