THE AUK A QUARTERLY JOURNAL OF ORNITHOLOGY

VOL. 105 APRIL 1988 NO. 2

GENETIC VARIATION IN SYMPATRIC WILLOW FLYCATCHERS ( TRAILLII) AND FLYCATCHERS (E. ALNORUM)

GILLESSEUTIN 1 AND JEAN-PIERRESIMON D•partementde sciencesbiologiques, Universit• de Montreal,90 VincentD'Indy, Outremont,Quebec H2V 2S9, Canada

ASSTRACT.--Weused allozyme electrophoresisto estimatethe level of genetic variability and differentiation of allopatric and sympatricpopulations of the Willow (Empidonaxtraillii) and Alder (E. alnorum)flycatchers from southeasternCanada. We reasonedthat, if hybrid- ization occurs,sympatric populations should be more variable and interspecificallyless dif- ferentiatedthan allopatricpopulations. These predictions were not supportedby our results. Therefore, we concluded that in the surveyed region the two speciesare reproductively isolated.This conclusionis weakenedby the fact that samplesizes were relatively small and that geneticdifferentiation of the two speciesis low. Theseconditions render interbreeding difficult to detect if it occursat a low frequency. Received3 June1987, accepted 16 November 1987.

THE Tyrannidae form the largest and most lation of two taxa, however, cannot be inferred diversefamily of in the New World (Tray- from the mere presenceof differences in their lot and Fitzpatrick 1982). Many genera com- vocalizations. For instance, in Myiarchusswain- prise groups of similar species, a number of soniand in M. tuberculifersome conspecific pop- which are consideredsuperspecies (e.g. Mayr ulations have different vocalizations, and allo- and Short 1970,A.O.U. 1983). Reproductive iso- patric birds do not recognize foreign songs as lation between sympatric members of these being conspecific.Nevertheless, they hybridize speciesgroups is often achieved through hab- freely when sympatric(Lanyon 1978). itat isolation (e.g. Johnson 1963, 1980). If eco- Ornithologists usually have relied on anal- logical preferences of two speciesare similar, ysis of morphometric and plumage characters however, their isolation depends either on the to determine if potentially hybridizing taxa are existenceof a postmating isolating mechanism genetically isolated (e.g. Rising 1983). It is dif- or on the ability of individual birds to discrim- ficult to study hybridization between sibling inate between heterospecificsand conspecifics speciesusing phenotypic characteristics,how- before mating (Mayr 1963). ever. Electrophoretictechniques provide orni- In birds, song differencesare thought to be thologistswith a potentially powerful tool for of prime importanceto ensurereproductive iso- the investigation of such cases(Barrowclough lation in many pairsof sympatricsibling species 1983,Matson 1984).In a few instancesthey were (Becker1982), particularly in the tyrannids (e.g. usedto survey contactzones between taxa that Lanyon 1963, Johnson 1980). Reproductive iso- are known or suspectedto hybridize (Martin and Selander 1975, Corbin et al. 1979, Barrow- clough 1980, Braun 1981, Corbin 1981, Johnson • Presentaddress: Department of Biology, Queen's and Zink 1983, Braun et al. 1984, Braun and University, Kingston, Ontario K7L 3N6, Canada. Robbins 1986, Grudzien and Moore 1986). A1-

235 The Auk 105: 235-243. April 1988 236 SEUTINAND SIMON [Auk,Vol. 105 lozyme traits are of great interest for the anal- Braunand Parker 1985).To maximizethe reliability ysis of hybridization becausethey are discrete of our estimatesof geneticvariability and differen- characters,generally inherited in a simpleMen- tiation, a specific effort was made to detect hidden delian fashion,and are usuallythought to be alleles(Coyne et al. 1979).For mostloci assayed, a set selectivelyneutral (Kimura 1983,Barrowclough of individual samplesof both specieswas run in dif- et al. 1985).Alleles of different frequenciesin ferent gels and buffer systems.The gel-buffer com- bination that provided the clearestbanding pattern allopatricpopulations of two taxamay be good and highestvariability was selectedfor the analysis markersto assessgene flow acrossa contactzone. of the remaining individuals (Table 2). Moreover, simple models make it possibleto Staining techniqueswere similar to thosedescribed predictthe behaviorof somegenetic parameters by Harris and Hopkinson (1976) and Barrowclough through a hybrid zone (Workman 1969,Endler and Corbin (1978).GLUD wasstained by the method 1977,Corbin 1981). These predictionscan then of Brewer (1970). Two esterases(Es-1 and Es-D) were be used to detect hybridization between sym- characterizedusing 4-methylumbelliferylacetate (4- pattic sibling species. Mg) as substrate;Es-3 and Es-4 were stained using We usedthis approachto determine if Willow a-naphthyl acetate. On the basis of heterozygote (EmpidonaxtraiIlii) and Alder (E. aInorum)fly- banding patterns, Es-D and Es-4 were dimeric, and the other two were monomeric.Three globins (Gb) catchershave hybridized in areasof sympatry were stained using the benzidine test (Gordon 1969), in southeasternCanada. These are closely re- and three general proteins (Ptm) were characterized lated sibling species(Stein 1958, 1963;Zink and with CoomassieR-250 (Righetti and Drysdale 1974). Johnson 1984; Shields et al. 1987) that can be Thirty-six presumptivegenetic loci were scored.We separated almost exclusively by advertising alsoattempted to analyze GDH (E.C no. 1.1.1.47),GR songs. Their phenotypic resemblance and the (1.6.4.2),NP (2.4.2.1), HK (2.7.1.1), ACP (3.1.3.2), HEX closesimilarity of their ecologicalpreferences (3.2.1.3.),ALD (4.1.2.13),and FH (4.2.1.2.),but they in southeastern Canada (Barlow and Mc- proved to be unscorable.When more than one locus Gillivray 1983)make them good candidatesfor wasobserved for a particularenzyme, they were num- hybridization. bered sequentially,beginning with the most anodal. Alleles at variableloci were codedby letters,begin- ning with "a" for the most anodal. MATERIALS AND METHODS Presumptiveindividual genotypeswere deduced from banding patterns on stained gels. From these We analyzedthe geneticvariability of 94 specimens we derived a table of allelic frequencies.Observed from 6 Willow or Alder flycatcherpopulations. Birds heterozygosity(Hobs) was determined by directcount were collectedduring the summersof 1985and 1986. for eachspecimen and then averagedfor eachsample Geographiclocation of the populationsamples, their (Corbin 1983).The BIOSYS-1program (Swoffordand status(sympatric or allopatric), and sample sizesare Selander 1981) was used to compute the proportion givenin Table1. Mostspecimens from sympatricpop- of polymorphicloci, the mean numberof allelesper ulationswere collectedat locationswhere syntopy locus,the expectedheterozygosity (Hexp), F-statistics occurred.Specific identity was established by a play- (Wright 1965,1978),and Nei's (1978)and Rogers'(1972) backexperiment before collection (Seutin 1987a).Pro- genetic distances.For each sample genotypic distri- ceduresfor collectionand storageof tissuesamples bution at eachlocus was examined for departurefrom followed those describedby Johnsonet al. (1984). Hardy-Weinberg expectations,using a X2 test (Sokal Sampleswere transportedfrom the field on dry ice and Rohlf 1981). When more than two alleles oc- and stored at -80øC in the laboratory. Extractsof curredat a locus,the actualgenotypes were grouped breastand heart muscleswere preparedseparately; into three classes,according to the procedure pro- liver and kidney sampleswere combinedto produce posedby Swoffordand Selander(1981). This reduced a third extract.Tissue samples were homogenizedwith the number of classeswith low frequencyand less- an equal volume of the buffer solution (Karig and enedthe probabilityof generatingartificially inflated Wilson 1971)and centrifugedat 23,000g for 20 min test values. Among-sampleheterogeneity in allelic at 4øC. frequencydistributions was analyzed using the G test. Electrophoreseswere carried out on horizontal 10% This test was used because it is less sensitive than the starch gels or on vertical polyacrylamideslab gels X2 test to the presence of rare classesin the distri- (Chapel et al. 1974) that varied in acrylamidecon- butions, and it usually better approximatesthe the- centration from 7% to 10% (Seutin 1987b). A discon- oretical X2 distribution (Sokal and Rohlf 1981). The tinuous buffer system(Ornstein 1964) was used for SPSS(Nie et al. 1975) and BMDP (Dixon 1981) com- electrophoresesconducted in acrylamidegels, and a puter packageswere usedto compute theseand other 25-30-mA current was applied for 4-7 h. Four buffer statistics. systemswere usedfor starchgels (Selander et al. 1971, We usednonmetric multidimensional scaling (MDS) April1988] Willowand Alder Flycatcher Genetics 237

T^BLE1. Geneticvariability at 36 loci in 6 flycatcherpopulations. Acronyms in parenthesescorrespond to those used in other tables and in Fig. 1.

% of Mean poly- no. of mor- al- phic leles/ Populationa Status n locib locus Hobs+ SE Hexp+ SEc /•sa Empidonaxalnorum St.-Jean-Vianney(Ea-SJV) Allopatric 19 22.2 1.56 0.082 + 0.009 0.083 + 0.030 -0.025 Mont-Tremblant(Ea-MtT) Allopatric 16 16.7 1.44 0.063 + 0.007 0.073 + 0.030 0.048 Montreal (Ea-Mtl) Sympatric 16 19.4 1.50 0.081 + 0.010 0.084 + 0.030 -0.001 Brighton (Ea-Br) Sympatric 14 19.4 1.44 0.070 + 0.009 0.075 + 0.028 0.021 Empidonaxtraillii Montreal (Et-Mtl) Sympatric 16 27.8 1.44 0.082 + 0.013 0.090 + 0.029 0.006 Brighton(Et-Br) Sympatric 13 22.2 1.42 0.069 + 0.009 0.076 + 0.026 0.063 Coordinatesof locationsare given by Seutin (1987a). Frequencyof mostcommon allele -< 0.95. Unbiased estimate (Nei 1978). Mean Fi•over all polymorphicloci. and correspondenceanalysis (CA) ordination tech- was calculated for each polymorphic locus in niques to summarize genetic relationshipsamong each population. Values ranged from -0.524 to populations. The matrix of Rogers' distance values 1.000. However, Chi-square tests for conform- was subjectedto a principal coordinateanalysis (Le- ance to Hardy-Weinberg equilibrium revealed gendre and Legendre1983) to producean initial con- only 4 cases,from a total of 69, for which ob- figuration of samplesfor MDS. MDS was then per- formedto optimizethe fit of datain the reducedspace served and expecteddistributions of genotypes (Kruskal 1964, Legendre and Legendre 1983), using were significantlydifferent (P < 0.05). For Es-4 the MDSCALEroutine in the NT-SYScomputer pack- in Montreal Alder Flycatchers,Lgg-2 in Bright- age (Rohlf et al. 1982). on Alder Flycatchers,and Lgg-1 in Brighton Correspondenceanalysis (CA) directly producesan Willow Flycatchers,F•, was positive, indicating ordination from a contingencytable. CA preserves, a deficiency of heterozygotes;only for Gb-1 in in the reducedspace, the x2 distancesbetween rows Montreal Alder Flycatcherswas F•,negative. The or columnsof the contingencytable (Legendreand average F•, of the samples,calculated over all Legendre 1983, Greenacre 1984). The ordination was polymorphic loci, ranged from -0.025 to 0.063 producedby the ACOR computerprogram (Cl•roux (Table 1). These values imply that all popula- undated),using the table of actual(not relative) allele frequenciesat polymorphic loci. tions are closeto Hardy-Weinberg equilibrium and, therefore, are panmictic. RESULTS Geneticvariation amongpopulation samples.- The 6 sampleswere similar in allelic frequen- Geneticvariation within population samples.--Of cies at the 36 loci studied. No fixed allelic dif- the 36 loci scored, 16 (44.4%) were variable in ferenceswere found between the two species. at leastone sample(Table 2). The percentageof All allelesthat were limited to one specieswere polymorphicloci, averagenumber of allelesper rare alleles and, owing to small sample sizes, locus,and mean observedheterozygosity of the cannot be considered good species markers population samplesare given in Table 1. Values (Gregorius 1980). Contingency analyses re- resembledthose reported by Zink and Johnson vealedthat populationsdiffered significantly in (1984) for the samespecies. There were no sig- allelic frequenciesat 4 loci: Pgm-2, Es-D, Es-3, nificant differencesamong populations in the and Gb-1 (Table 3). Interspecificdifferences seem percentageof polymorphicloci (G test;Gaaiustea = to be responsible for the results observed at 0.740,P > 0.975), averagenumber of allelesper Pgm-2, Es-D, and Es-3. No significant hetero- locus (Kruskal-Wallis test; H ...... a = 0.684, P = geneities were observed at these loci when 0.984), and mean observed heterozygosity groups of conspecificsamples were analyzed (Kruskal-Wallis test; H ..... •a = 4.450, P = 0.487). separately(Table 3). The inbreeding coefficient,F•, (Wright 1965), F•,values, a measureof geneticdifferentiation 238 SEUTINAND SIMON [Auk,Vol. 105

TABLE2. Allelic frequenciesand electrophoreticconditions for polymorphicprotein loci.a

Locusb Populationsample (E.C. no.) Ea-SJV Ea-MtT Ea-Mtl Ea-Br Et-Mtl Et-Br Tissue, buffer c Adh a 0.026 0.031 L, TC I (1.1.1.1) b 0.974 0.969 1.000 1.000 1.000 1.000 Gpd a 0.789 0.844 0.688 0.750 0.844 0.885 M, TC II (1.1.1.8) b 0.211 0.156 0.312 0.250 0.156 0.115 Icd-1 a 0.139 0.187 0.063 0.031 0.115 L, TC 7.5 (1.1.1.42) b 0.416 0.344 0.375 0.429 0.406 0.423 c 0.056 0.125 0.031 0.107 0.157 0.077 d 0.389 0.344 0.531 0.464 0.406 0.385 Pgd a 0.026 0.031 L, TC I (1.1.1.44) b 0.974 1.000 0.969 1.000 1.000 1.000 Pgm-1 a 1.000 1.000 0.969 0.964 1.000 1.000 L, TC 7.5 (2.7.5.1) b 0.031 0.036 Pgm-2 a 0.031 0.219 0.115 L, TC 7.5 b 0.974 1.000 0.969 1.000 0.781 0.885 c 0.026 Es-D a 0.079 0.031 0.071 0.187 0.231 L, LiOH (3.1.1.X) b 0.921 1.000 0.969 0.929 0.813 0.769 Es-3 a 0.079 0.031 H, TGly b 0.158 0.094 0.250 0.179 0.031 c 0.053 0.125 0.125 0.036 0.063 0.038 d 0.263 0.125 0.125 0.321 0.281 0.308 e 0.447 0.562 0.469 0.393 0.625 0.654 f 0.063 0.031 0.071 Es-4 a 0.028 0.063 0.100 0.038 0.067 H, TGly b 0.972 0.937 0.900 0.962 0.933 1.000 Lgg-1 a 0.026 L, LiOH (3.4.11.X) b 0.053 0.063 0.067 0.071 0.031 0.077 c 0.921 0.937 0.933 0.929 0.969 0.923 Lgg-2 a 0.053 0.031 0.033 0.071 0.038 L, LiOH b 0.921 0.969 0.967 0.893 0.906 0.962 c 0.026 0.036 0.094 La-1 a 0.105 0.063 0.036 L, TC 7.5 (3.4.11.X) b 0.895 1.000 0.937 0.964 1.000 1.000 Ada a 0.031 0.038 M, TC II (3.5.4.4) b 1.000 0.969 1.000 1.000 0.937 0.962 c 0.063 Gpi a 0.036 0.063 0.077 H, TGly (5.3.1.9) b 1.000 0.969 0.969 0.964 0.937 0.923 c 0.031 0.031 Gb-1 a 0.100 0.042 H, TGly b 0.763 0.375 0.656 0.893 0.700 0.875 c 0.026 d 0.211 0.625 0.344 0.107 0.200 0.083 Ptm-3 a 1.000 1.000 1.000 1.000 1.000 0.962 M, TGly b 0.038

• The following loci were monomorphicin all individualsassayed (E.C. no., tissue,and buffer systemin parentheses):Sdh (1.1.1.14,L, TC I), Ldh-1 and -2 (1.1.1.27,L, TGIy), Mdh-1 and -2 (1.1.1.37,L, TGly), Me-1 (1.1.1.40,L, TC I), Me-2 (H, TC I), Icd-2 (L, TC 7.5),Gd (1.1.1.47,L, TGIy), Glud (1.4.1.3,M, TC II), Sod-1and -2 (1.15.1.1,H, TGly), Got (2.6.1.1,H, TGly), Es-1(L, LiOH), La-2 (L, TC 7.5), V1 (3.4.11.X,L, TGIy), Gb-2 and -3 (H, TGIy), Ptm-1 and -2 (M, TGly). bAbbreviations follow Harris and Hopkinson (1976);lowercase letters indicate alleles. cM = breastmuscle, H = heart, L = liver and kidney; TGIy = discontinuoustris-HCl and tris-glycinebuffer, from Ornstein (1964),TCI = tris- citratepH 6.3, from Selanderet al. (1971);TC II = tris-citratepH 8.1, from Selanderet al. (1971);TC 7.5 = tris-citratepH 7.5, from Braunand Parker (1985);LiOH = lithium-hydroxide,from Selanderet al. (1971). April1988] Willowand Alder Flycatcher Genetics 239

TAnrE3. F•t for polymorphic loci (Wright 1978).To- thoseobtained by Zink and Johnson(1984; D N = tal population is formed either by the 6 samplesor 0.009, DR = 0.041). by conspecificsamples. Loci showing significant Becauseall distance values were extremely among-sampleheterogeneities in allelic frequency distribution are indicated by asterisks(G tests).a small, we recomputedgenetic distancesusing only polymorphic loci (Table 4). These values Locus 6 samples E. alnorum E. traillii are overestimated,but they are easier to inter- Adh 0.020 0.015 -- pret and to process.The matricesof D N and DR Gpd 0.027 0.018 0.004 values were highly correlated(r = 0.769), im- Icd-1 0.016 0.020 0.005 plying that bothprovide a similarpicture of the Pgd 0.020 0.015 -- relationshipsamong populations.All pairwise Pgm-1 0.023 0.017 -- Pgm-2 0.106'* 0.018 0.019 comparisonsamong Alder Flycatchersamples Es-D 0.075'* 0.023 0.003 revealed similar levels of genetic differentia- Es-3 0.041 * 0.025 0.002 tion, with no tendency for the sympatric pop- Es-4 0.022 0.014 0.034 ulationsto have divergedsignificantly from al- Lgg-1 0.005 0.002 0.010 Lgg-2 0.021 0.013 0.027 lopatric ones. Similarly, both allopatric and La-1 0.048 0.031 -- sympatricpopulations of the Alder Flycatcher Ada 0.032 0.024 0.015 were equally differentiated from Willow Fly- Gpi 0.024 0.014 0.001 catcherpopulations (/5 R = 0.078vs. 0.080). Gb-1 0.152'** 0.167'** 0.034 Ptm-3 0.032 -- 0.020 Principalcoordinate analysis of the matrix of Mean 0.051'** 0.043 0.011 Rogers' distances explained 78.71% (axis 1: 49.42%, axis 2: 29.29%) of the total variation ' * = P -< 0.05, ** = P < 0.01, *** = P -< 0.001. among samplesin two dimensions.Using the resultingcoordinates as an initial configuration, among populations, were calculated for each nonmetric multidimensional scaling (MDS) polymorphic locus, consideringthe total pop- achieveda final stressof 0.000 (perfectfit) (Fig. ulationto be eitherthe sixsamples or eachgroup 1). Matrix correlationwith the original distance of conspecificsamples (Table 3). All valueswere matrix was 0.9901, suggestinga nearly perfect low, indicatingthat mostof the geneticvariance representationof the original relationshipsin is found within populations.At most loci, and the reducedspace. on average,values were slightly higher when The ordination produced by the correspon- the heterospecificgroup was analyzed, than dence analysis (CA) method was almost iden- when only conspecificpopulations were stud- tical to that generated by MDS. Only the po- ied. A hierarchical analysis with F-statistics sition of the Montreal and Mont-Tremblant (Wright 1978),with localitiesand speciesas the samplesof the Alder Flycatcheralong the first levels, revealed that only 12.9%of the genetic axiswere interchanged(Seutin 1987b). The first variancefound among the six sampleswas dis- two axesof the CA ordination explained70.81% tributed between the species. (axis 1: 46.80% axis 2: 24.01%) of the total vari- Mean values of Nei's (DN) and Rogers' (DR) ance of the table of allelic frequencies. geneticdistances among Alder Flycatchersam- The close resemblance of both ordinations pleswere/5 N = 0.002and/SR = 0.027,and those was verified by the strongcorrelation between betweenWillow Flycatchersamples were DN = the matrices of euclidean distances between 0.000 and DR = 0.021. Mean interspecificdis- samples in each of the reduced spaces(r = tances(/5 N = 0.004,/5R = 0.035)were similarto 0.8466).In both ordinationspopuJations are dis-

TABLE4. Geneticdistances based on 16 polymorphicloci between6 flycatcherpopulations. Above diagonal: Nei's (1978) distances;below diagonal:Rogers' (1972) distances.

Ea-SJV Ea-MtT Ea-Mtl Ea-Br Et-Mtl Et-Br Ea-SJV -- 0.011 0.000 0.000 0.003 0.002 Ea-MtT 0.069 -- 0.006 0.021 0.014 0.023 Ea-Mtl 0.052 0.065 -- 0.001 0.007 0.012 Ea-Br 0.041 0.083 0.054 -- 0.004 0.002 Et-Mtl 0.072 0.083 0.086 0.072 -- 0.000 Et-Br 0.068 0.088 0.093 0.067 0.048 -- 240 SEUrINAND SIMON [Auk, Vol. 105

1,5- leles in the two species.Only for three loci were

o. Ea, MtT there significantdifferences in allelic frequency 1,0- distributions that could be attributed to inter- specificdifferences (Table 3). This number of 0.5- .-.Et. Mtl ..' • :'e: t differentiated loci is probably not sufficientfor ..' Et.B; ." • a significantincrease in p and Hobsto be seen if 0.0- ':.•...... •' '/ ...... g..•i' ' - : Ea.sav •..' hybridization is rare. That we found no signif- icant differences in the level of genetic vari- ability of the populationsdoes not indicate the -1.0 completeabsence of hybridizationbetween the -1.5 -1.0 -0.5 0.0 0.5 1. 1.5 two species,but this suggeststhat interbreeding AXIS 1 does not occur at a high frequency in the sur- Fig. 1. Projectionof 6 flycatcherpopulations along veyed areas. the first two axes of a nonmetric multidimensional In hybrid populations increased heterozy- scaling analysisof the matrix of Rogers' distances. The initial configuration consistedof the population gositywill be particularlynoticeable at loci that coordinatesderived from a principal coordinateanal- show substantialinterspecific differences in al- ysisof the distancematrix. Final stress= 0.000;matrix lelic frequencies.This may result in deviations correlation = 0.9901. of the observedgenotypic distributions at these loci from those expected under Hardy-Wein- berg equilibrium (Workman 1969). In our sym- tributed along the first axis accordingto their pattic samplesFis values for the three loci with taxonomicstatus. The large dispersionof Alder significantinterspecific differences were close Flycatcher samplesalong axis 2 of both ordi- to zero and usually were slightly positive. This nationsseems to be due to the heterogeneityof reflectsa deficiencyin heterozygotes,which is their allelic frequencydistributions at the Gb-1 oppositeto what we expectedto observeif the locus(Seutin unpubl. data). two taxawere hybridizing. Chi-squaretests fur- ther revealed that differences between the ob-

DISCUSSION servedand expectedgenotypic distributions at theseloci were not significant. Geneticvariability of the populationsamples.- Geneticdifferentiation of the populationsam- The amount of genetic variability within sam- ples.--Thecalculated genetic distances between plesof both specieswas within the normal range Willow and Alder flycatchersare among the for avian breeding populations(Barrowclough lowest reported for avian species. The mean 1983, Corbin 1983).All populationssampled ex- Nei's distance between heterospecificsamples hibited nearly the same numbers of polymor- (l•N = 0.004)is an orderof magnitudelower phic loci and levels of heterozygosity.Because than the averagedistance calculated by Barrow- founder eventsusually result in reducedpoly- clough(1980) for congeners([}N = 0.044,based morphism and heterozygosity(Nei et al. 1975, on 71 comparisons).The close genetic resem- Selander 1976), we conclude that this was not blanceof the two speciesis alsoreflected by the a factorin the spreadof the Willow Flycatcher small Fst values found at most loci. The mean to northern latitudes in northeastern America Fstover all polymorphic loci was 0.051 when (Stein 1963, Barlow and McGillivray 1983). the six samples were pooled to form the total Hybrid populations are formed by the ad- population. This is only slightly higher than mixture of differentiatedgene poolsand should valuesfor conspecificpopulations of other pas- show increased genetic variability. The per- setines(Barrowclough 1983), and it impliesthat centageof polymorphicloci (p) will increaseif only 5.1%of the total geneticvariation is found the parental forms are fixed for alternative al- among populations. leles, or if one form is fixed at some loci and Becausecare was taken in choosingelectro- the other is not. As a result of the increasein p phoretic conditions that yielded the highest and in the number of alleles showing inter- protein variation, our estimatesof interspecific mediate frequencies,observed heterozygosity differentiation should be viewed as represen- should also be higher in hybrid populations tative of the actual differentiation of the two than in pure parental stocks(Corbin 1981). We taxaat the protein level. This conclusionis sup- found no locus that was fixed at alternative al- ported indirectly by the fact that genetic dis- April 1988] Willowand Alder Flycatcher Genetics 241 tances calculated for conspecificsamples are havioraland ecologicalstudies showed that they similar to thosereported for populationsof oth- probably are reproductively isolated.Our re- er species(Barrowclough 1980). We did not suitssupport that contention.Our samplesizes include allopatric samplesof the Willow Fly- were relatively small and genetic differentia- catcher in the analyses,however, so our esti- tion between the two speciesis very low, how- matesof the geneticdifferentiation between the ever, making it difficult to detecthybridization two speciesshould be viewed as minimal be- if it occurs at a low frequency. Much larger causeof the possibilitythat the sympatricWil- samplesand, probably,other types of genetic low Flycatchersamples are of partly hybrid or- markers will be needed to assess the extent of igin. gene flow, if any, between the two species. Extremely small interspecific genetic dis- tances (D N < 0.01 or D• < 0.05) seem not to be ACKNOWLEDGMENTS unusual in birds (e.g. Avise and Aquadro 1982). We aregrateful to GenevieveCanzani, Martine Jean, Possiblereasons for this were discussedby Sib- and especiallyColette Trickey, who performedmany ley and Ahlquist (1982) and Avise (1983). The of the electrophoreticanalyses. B. A. Coups,M. Gos- small geneticdifferentiation of Willow and Al- selin, and two anonymousreviewers offered valuable der flycatchersat the protein level should not commentson the manuscript.Financial supportfor be taken as evidence for conspecificity. field and laboratorywork was provided by the A. B. We found no single allele whose presenceor Kelly Memorial Fund of the Province of QuebecSo- frequencydistribution amongsamples could be ciety for the Protectionof Birds, the Universit• de used to detect hybrids in sympatricsamples of Montreal (Fond CAFIR No. 30), the Canadian Wildlife the Alder Flycatchers.Therefore, we usedmul- Service(University Research Support Fund), and the tilocus statistics that summarize differences at Natural Sciencesand Engineering ResearchCouncil of Canada.Logistical support from the National Mu- all loci. The ordination producedby a nonme- seum of Natural Sciences(Ottawa) was greatly ap- tric multidimensional scaling analysis of the preciated. matrix of Rogers'distances (Fig. 1) showed that the two specieswere geneticallydistinct. Of the LITERATURE CITED two sympatricAlder Flycatchersamples, only AMERICAN ORNITHOLOGISTS' UNION. 1983. Check-list the one from Brighton showed signs of inter- of North American birds, 6th ed. Washington, mediacy,and was closerto Willow Flycatchers D.C., Am. Ornithol. Union. than any other Alder Flycatchersample. The AVISE,J.C. 1983. Commentary.Pp. 262-270 in Per- confidenceintervals of our genetic distancees- spectivesin ornithology(A. H. Brushand G. A. timatesare relatively large, however (Nei and Clark Jr., Eds.).Cambridge, England, Cambridge Roychoudhury1974), and, therefore, the posi- Univ. Press. tion of the samplesin the reducedspace may ß, & C. F. AQUADRO.1982. A comparativesum- imperfectly reflect their actual relationships.It mary of geneticdistances in the vertebrates.Evol. would be premature to infer from the only Biol. 15: 151-185. slightly intermediateposition of the Brighton BARLOW,J. C., & W. B. McGILLIVRAY.1983. Foraging and habitat relationshipsof the sibling species Alder Flycatchersample that hybridization oc- Willow Flycatcher(Empidonax traillii) and Alder curs,especially because the other sympatricAl- Flycatcher(E. alnorum)in southernOntario. Can. der Flycatcherpopulation (Montreal) is clearly J. Zool. 61: 1510-1516. not intermediate. A low level of hybridization BARROWCLOUGH,G.F. 1980. Geneticand phenotypic will have very limited influence on geneticdis- differentiation in a wood warbler (genus Den- tances,especially if hybrids exhibit reducedvi- droica)hybrid zone. Auk 97: 655-668. ability of fertility, and our results cannot be 1983. Biochemical studies of microevolu- taken as indicative of the complete absenceof tionary processes.Pp. 223-261 in Perspectivesin interspecificgene flow. Moreover, becausewe ornithology (A. H. Brush and G. A. Clark Jr., included no allopatricWillow Flycatcherpop- Eds.). Cambridge, England, Cambridge Univ. Press. ulations in our analysis,it is possiblethat uni- , •r K. W. CORBIN. 1978. Genetic variation and directional introgression, toward the Willow differentiation in Parulidae. Auk 95: 691-702. Flycatcher, occurs. , N. K. JOHNSON,& R. M. ZINK. 1985. On the Willow and Alder flycatchershave long been nature of genic variation in birds. Pp. 135-154 in treated as subspeciesof the Traill's Flycatcher Current ornithology, vol. 2 (R. F. Johnston,Ed.). (E. traillii), but Stein's (1958, 1963) extensive be- New York, Plenum Publ. Co. 242 SEUTINAND SIMON [Auk,Vol. 105

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