October 1982] Short Communications 767

EnlargedSex Chromosomesof Woodpeckers(Piciformes)

GE•LD F. SmELDS,GORDON H. J.•a•cc, .•ND ECXZ.•B•TrtR•DRV•'• '• Instituteof ArcticBiology and Divisionof Life Sciences,University of Alaska, Fairbanks, Alaska 99701 USA

The evolutionof sexchromosomes in birds is poorly terize not only woodpeckersbut alsoother piciform understood.In part, this is becausethe W chromo- families. some in most speciesresembles microchromosomes, Collectionof specimensfor this study has been which are lessthan 2/x in length and thereforedif- piecemeal in that, for the most part, we have ana- ficultto characterize.Autoradiographic studies of the lyzed the karotypesof variouswoodpeckers as the chickenby Schmid(1962) indicated that the W chro- birds becameavailable. The Toronto specimenswere mosomeis the last to incorporatetritiated thymidine taken as adultsor eggs,and at leastthree malesand during the synthesisphase of the cell cycle. Thus, three femalesof each specieswere used. All karyo- late replication of the DNA of the W chromosome types of thesebirds were preparedfrom acetoorcein- characterizesthis elementin the karyotype.More re- stainedslides of culturedkidney cellsgrown in CMRL cently, the staining of large amountsof constitutive 1415+15% fetal calf serum.We verified the presence (C-) heterochromatinon W chromosomesof birds by of enlargedZ chromosomesin AlaskanHairy Wood- C-banding establishedanother procedurefor iden- peckersand flickers.These specimens (3 femaleand tification of the W chromosome(Stefos and Arrighi 2 male Hairy Woodpeckersand 3 femaleand 3 male 1971).Neither of thesetechniques has been used ex- flickers)were taken as embryosand culturedin es- tensively, however. The Z chromosomeof birds ap- sentiallythe sameway as the Torontobirds. pearsto have been conservedin evolutionto the ex- Adult lesser Spotted Woodpeckers (3 females, 1 tent that it consistently comprises 8-9% of the male), 2 female Greater Spotted Woodpeckers(D. chromatinof the entire genome, that it is typically major), and a male Black Woodpecker(Dryocopus the fourth or fifth largest chromosomeof the com- martius)were shot near As, Norwayand processed plement, and that the centromereis consistentlyme- as above. diocentric (Ohno et al. 1964, Ohno 1967). Ohno has The tentativeidentification by the Torontogroup referred to this, and a similar chromosome in ad- of an enlargedW chromosomein femaleflickers from vanced snakes, as the "original Z" to emphasize its the Torontoarea made this speciesthe logicalchoice conservative evolution. for autoradiographicidentification of the W chro- We describea deviationfrom Ohno's originalZ in mosome.Nestlings of both sexeswere used. Kidney somewoodpeckers (Piciformes: Picidae) and believe cultureswere labelledwith tritiatedthymidine (2 that this phenomenon is widespread within the ml, 2,233/xC//xM), and treatment was continuous to group. Our interestin woodpeckersex chromosomes fixationat 4, 43/4,and 51/2h. The cellswere pretreated was stimulated by the unpublished observationsof with colchicine(1 /xg/ml) for 2 h. Metaphase cells Drs. E. Sexsmith, E. Redrupp, and B. Thorneycroft were photographedand then autoradiographedwith in the laboratoryof ProfessorK. H. Rothfelsof the Kodak AR-10 strippingfilm. After 26 h exposure,the Department of Botany, University of Toronto from labelledmetaphases showed 8-400 grains,depend- 1965-1967. These researchers studied chromosomes ing on the time spentsynthesizing DNA in the pres- of the CommonFlicker (Colaptesauratus), the Yel- enceof the isotope.All metaphasecells previously low-belliedSapsucker (Sphyrapicus varius), the Hairy photographedwere scored for totalgrains, grains over Woodpecker (Picoidesvillosus), and the Downy the sexchromosomes, and the two pairs of metacen- Woodpecker(P. pubescens).They observedthat the tric autosomes.These chromosomeswere easily Z chromosomesof thesespecies are extremelylarge, identified and were divided into three or four seg- nearly twice the size of Z chromosomesof the ma- ments for counting. C-banding of Lesser Spotted jority of other birds. Additionally, they demonstrat- Woodpeckercells was done by the methodof Sum- ed that the W chromosome of the flicker could be ner (1972). identifiedby its late replicationpattern. Partial karyotypes of the four North American We have now verified the presenceof largeZ chro- woodpeckersstudied are shownin Fig. 1. Chromo- mosomesin Alaskan flickers and Hairy Woodpeck- somesof femalesare arrangedin descendingorder ers. Additionally, we have observedlarge Z chro- of size, and Z chromosomesof malesof eachspecies mosomesin three speciesof Europeanwoodpeckers are inset. and have identified the W chromosome of the Lesser (a) CommonFlicker (Colaptes auratus): The Z chro- Spotted Woodpecker (Dendrocoposminor) by mosomes have an arm ratio of 1:3.5. The second and C-banding. Enlarged Z chromosomesmay charac- fourth pairs of chromosomesare metacentric. The probablediploid numberfor this speciesis 90, with • Presentaddress: Department of BiologicalSciences, University of the fundamentalnumber (total number of arms)being Western Ontario, London, Ontario, Canada. 96. Late labellingof centromereswas not observed 768 ShortCommunications [Auk,Vol. 99

i 2 3 4 5 6 7 ß 9 10....4 5.46

Cß111ptesIlllrlltlls 21l:9ß,f.L:96

! w Picoidesvillßsus 211:92,[11.:96

iP.pubescertst II2U II:92, f.U. II= 96 tl ,, ,.., ,, ,, ......

lW Sphyrapicusvllrius 211--92,f.11.-- lO6

Fig. 1. Partialkaryotpyes of femalewoodpeckers showing enlarged Z chromosomes.Paired Z chromo- somesof males of each speciesare inset. in the flicker. The W chromosome was late to label (d) Yellow-bellied Sapsucker(Sphyrapicus varius): in both the shortand long arm, but this effectwas The Z chromosomes have an extreme arm ratio of 1: not as pronouncedas in mammaliancells (e.g. late 5, and the secondpair of chromosomesis almost ex- X in Chinese hamster). DNA replication of the Z actly metacentric.This i• iollowed by five sets of chromosomesin both sexeswas synchronouswith chromosomeshaving various centromerelocations. the autosomes. There was no difference between la- The diploadnumber appearsto be 92, with the fun- belling patternsof the two Z chromosomesin males. damental number of 106. (b) Hairy Woodpecker (Picoidesvillosus): the Z Partial karyotypesof the three European wood- chromosomeof this specieshas a median centromere peckersare shown in Fig. 2. with an arm ratio of 1:1.5. The karyotypeis alsodis- (e) Lesser Spotted Woodpecker (Dendrocopusmi- tinguishedby the presenceof a secondlarge chro- nor): the Z chromosomeof this speciesis again the mosome pair (number 2) with a sub-median centro- largestof the complement,its arm ratio being 1:1.4. mere (arm ratio 1:2.5). The remainder of the The W chromosomeis conspicuouslylarge and has chromosomesare acrocentric. The dipload number an arm ratio of 1:1.8. The secondpair of chromo- appears to be 92, with the fundamental number of somesis sub-metacentric,while the remainingpairs 96. appear acrocentric.The entire chromosomecomple- (c) Downy Woodpecker(P. pubescens):the Z chro- ment of this speciesis shown in Fig. 3a. The dipload mosomeof this speciesis the largestof the comple- number appears to be 108 while the fundamental ment, being almosttwice the sizeof the next largest number is 112.Figure 3b showsa C-banded cell from pair. The Z has an arm ratio of 1:2. Exceptfor the the same bird. The W chromosome is heterochro- metacentricfourth pair, all other chromosomesare matic, while the negativelystaining Z chromosome acrocentric.The diploadnumber appearsto be 92 and is unpaired in the complement. the fundamental number 96. (f) GreaterSpotted Woodpecker: (D. major):the Z October1982] ShortCommunications 769

I 2 3 4 5 6 7 8 9 10

Z W Dendrocopos minor

Z W D. major

Z Z D__ryocopos martius

Fig.2. Partialkaryotypes of threespecies of woodpeckers.Enlarged Z chromosomesarethe largest of the complements. chromosomeis large but with a differentarm ratio embryowhose sex was eitherdifficult or impossible (1:2.5)than the Z of the LesserSpotted Woodpecker. to determine. If the individual were a male, chro- The remainderof the karyotypeis similarto that of mosomesidentified by Hammar as Z and W might the LesserSpotted Woodpecker. be paired with a number of autosomeswhose struc- (g) BlackWoodpecker (Dryocopus martius): a single ture is similar. As Hammar's descriptiondid not in- male of this specieswas analyzed, so we cannot un- clude autoradiographyor C-banding and as, accord- equivocallyidentify the Z chromosomes.Because the ing to our scheme,the largestchromosomes of his Z chromosomesare the largestchromosomes in the karyotypeare similarto the Z chromosomesof other otherspecies, however, it is likelythat the largest chro- woodpeckers,we proposethis alternativeinterpre- mosomesin the BlackWoodpecker are also the Z tation. chromosomes; their arm ratio is 1:4.0. The second The absence of C-bands in the Z chromosome of pair in thisspecies is nearlymetacentric (1:1.1). This the LesserSpotted Woodpecker indicates that en- pair is followedby anothernearly metacentric (1:1.2) largementof this chromosomehas not been accom- but smallerpair and by three other pairs that are plished by the addition of C-heterochromatin.We submetacentric.The remainingchromosomes are ac- intend to determine whether or not G- (Giesma) rocentric.The diploid numberappears to be 88, with banding sequencesin original Z chromosometypes the fundamental number being 98. also occurin enlargedZ chromosomesof Alaskan Table 1 summarizesthe available karyotypic data woodpeckers. for piciformbirds. All speciesof Picidae,except the While the size of Z chromosomesin woodpeckers Green Woodpecker(Picus viridis) discussedbelow, is generallyuniform (Table 1), the positionof the possessZ chromosomesthat are the largestof the centromereis not. Even speciesof the same genus complements.Barbets (Capitonidae) also possess large (P. villosusand P. pubescensas well asD. minorand Z chromosomes.Other piciform families have trot D. major)have Z chromosomesthat differ in position been studied. of the centremere.This variability is probably the That large Z chromosomeswere describedinde- result of intrachromosomalrearrangement (e.g. peri- pendentlyby severalresearchers studying diverse taxa centric inversions or centric shifts). supportsour contentionthat large Z chromosomes The enlargedZ chromosomeis not unique to Pi- characterizeall woodpeckers.The karyotypeof the ciformes.The karyotypesof 20 speciesof the family GreenWoodpecker is a possibleexception. Hammar Accipitridae (Falconiformes)have been described (1970:44) describesthe Z and W chromosomesfrom (Takagi and Sasaki1974, de Boer 1976, Misra and this speciesas acrocentricand as being the fourth Srivastava 1976, Williams and Benirschke 1976), and and tenthlargest in size, respectively.The possibil- these speciespossess enlarged Z and W chromo- ity existsthat the karyotypewas preparedfrom an somes. The Z chromosomes resemble those of the 770 ShortCommunications [Auk, Vol. 99

Fig. 3(a). Entirekaryotype of a femalelesser spotted woodpecker. Z and W sexchromosomes are indicated by arrows.(b) C-banded(constitutive heterochromatin) karyotype of a femaleLesser Spotted Woodpecker. The heavilystained W chromosomeis obviousas is the unpairedZ chromosome. woodpeckersin that they, too, are the largestof the of dimorphic sex chromosomesin ratites (Takagi et complement.Moreover, Bulatova(1973) reported en- al. 1972) and in all but the advancedophidian rep- largedZ chromosomesin three speciesof lark (Alau- tiles complicatesOhno's interpretation of a homol- didae) near Novosibirsk, USSR. We know of no case ogousZ chromosomein these separateclasses. in birdsin which the Z chromosomeis enlargedwhile We thank Prof. Klaus H. Rothfelsof the Depart- the W is not. This suggeststhat a mechanismfor ment of Botany,University of Torontoand his stu- enlargement operateson both sex chromosomesin dents, BruceThorneycroft and ElizabethSexsmith, birds. While Ohno's original Z hypothesischarac- for bringing their observationson enlargedsex chro- terizes perhaps the majority of avian lineages, de- mosomesin woodpeckersto our attention. Also, we rived conditions,such as describedhere, may be thank all of theseresearchers for allowing us to cite common.Stock and Mengden (1975)did not describe and describetheir unpublisheddata. Leonard Pey- homologybetween avian Z chromosomesand chro- ton locatedand helped collecteggs and young of mosomesof bold snakes,a primitive family without Alaskanwoodpeckers. Hans Staalandand Knut Rudd dimorphic sex chromosomes.The apparentabsence helpedcollect the Norwegianwoodpeckers. Finally,

TABLE1. A stunmaryof sex-chromosomemorphology and chromosomenumbers in Piciformes.

Species Z • Z b W c 2n f.n. Reference

Picidae Picoidespubescens 23.3 1:2.0 10.8 92 96 This paper P. villosus 15.6 1:1.5 10.0 92 96 This paper P. mahrattensis 16.0 1:3.1 5.1 84 98 Kaul and Ansari (1978) Dendrocoposminor 19.1 1:1.4 12.0 108 112 This paper D. major 18.0 1:2.5 9.6 108 112 This paper Picusviridis 9.2? n.r. a 6.3? 94 102 Hammar (1970) Dryocopusmartius 19.7 1:4.0 ? 88 98 This paper Dinopiumbenghalense 14.8 1:3.0 ? 92 94 Kaul and Ansari (1978) Colaptesauratus 17.1 1:3.5 11.7 90 96 This paper Sphyrapicusvarius 21.7 1:5.0 6.4 92 106 This paper Capitonidae Megalaimazeylanica 15.6 1:2.6 10.7 96 126 Kaul and Ansari (1979) M. haemacephala 17.3 1:2.5 2.9 90 106 Kaul and Ansari (1978) Relative length of Z chromosomein relation to total macrochromosomelength. Ratioof the lengthof the longarm of the chromosometo the lengthof the shortarm of the chromosome. Relativelength of W chromosomein relationto total macrochromosomelength. n.r.--not reported. October 1982] Short Communications 771 we thank the Government of Norway for financial --, C. StENIUS, L. C. CI4RIStI.•N, W. BEc.•c, & support,tissue culture materials, and laboratoryspace M. L. BECAme.1964. Chromosomaluniformity during the seniorauthor's stay there. in the avian subclass Carinatae. Chromosoma 15: 280-288. Scream, W. 1962. DNA replication patterns of the L•TERATURE CITED heterochromosomesin Gallusdomesticus. Cyto- genetics 1: 344-352. BOER, L. E.M. 1976. The somatic chromosome S•EFOS, K., & F. ARR•½I4I. 1971. Heterochromatic complementsof sixteenspecies of Falconiforrnes nature of the W chromosomeof birds. Exp. Cell. (Aves) and the karyologicalrelationships of the Res. 68: 228-231. order. Genetica 46: 77-113. STock, A.D., & G. ME•½VE•. 1975. Chromosome BULATOVA,N. S. 1973. Unusually large sex chro- banding pattern conservatismin birds and non- mosomes in some larks (Aves: Alaudidae). Mammal. Chromos. Newsl. 14: 150. homologyof chromosomebanding patternsbe- tween birds, turtles, snakes and amphibians. H•a•aAR, B. 1970. The karyotypes of thirty-one Chromosoma 50: 69-77. birds. Hereditas 65: 29-58. K•uL, D., & H. A. A•SARi. 1978. Chromosome SU•a•ER,A. T. 1972. A simple techniquefor dem- onstrating centromeric heterochromatin. Exp. studies in three speciesof Piciforrnes(Aves). Cell. Res. 75: 304-306. Genetica 48: 193-196. TAr,•½•, N., & M. SASA•CI.1974. A phylogenetic ß & -- 1979. Chromosomalpolymor- study of bird karyotypes. Chromosoma(Berlin) phism in a natural population of Northern Green 46: 91-120. Barbet, Megalaimazeylanica caniceps (Franklin) --, M. Itoi4, & M. SASA•C•. 1972. Chromosome (Piciforrnes:Aves). Genetica 49: 1-5. studiesin four speciesof Ratitae (Aves). Chro- MISRA, M., & M.D. L. SRIVAStAVA. 1976. The mosoma 36: 281-291. karyotypesof two speciesof Falconiforrnes.Cy~ W•LLi•s, R. M., & R. J. BE•mscmc•. 1976. The tologia41: 313-317. OI4•O, S. 1967. Sex chromosomes and sex-linked chromosomesof four speciesof Falconiformes. Experientia 32: 310-311. genes.New York, Heidelberg, Berlin, Springer- Verlag. Received22 October1981, accepted 18 February1982.

Individuality of Vocalizationsin Dunlin: a PossibleAcoustic Basis for Recognitionof Parentby Offspring

MYRON CI4ARLES BA•ER Departmentof Zoologyand Entomology, Colorado State University, Fort Collins, Colorado 80523 USA

Calls of parentscan be recognizedby the young "purring" vocalization.The purr vocalizationis also in severalspecies of the avian order Charadriiformes given by a parent when gatheringup its young for (Beer 1970a,b; Evans 1970a,b). It can be predicted brooding.As the chickswander over the prime feed- that parent-offspringvocal recognitionmay evolve ing areas,two or more broodsmay mingle and sep- in precocial specieswith the potential of young of arate. different parentagebecoming mixed during foraging Observationsat this time indicatethat adultsgive activities. purr callsalmost constantlyand the young move to- In the eastern Canadian Arctic, I made tape re- ward an adult that is producing these calls. These cordings of breeding Dunlin (Charadriiformes, observations raise questions about the possible Scolopacidae, Calidris alpina). From these I at- acousticbasis for recognition of the correct parent. tempted to establish the acousticproperties of a It is possibleto discountthe importanceof visual certain vocal signal that may allow young chicks recognition,because the uneventerrain usually pre- to recognizetheir parents. vents the tiny chicksfrom seeingthe parent after a The precocialyoung Dunlin leave the nest cup few metersof separation.I did not verify the occur- within a few hoursafter hatching and beginpecking at small adult Diptera resting on vegetation.In the renceof vocalrecognition with individuallymarked first two or three days, a parent broodsits offspring, birds, but I assumedthat the young re-assortwith particularly in cold or rainy times. One or both of the correctparent. This assumptionis parsimonious the parentsaccompanies the young, often "leading" and logical from an evolutionary point of view. them by walking aheadand emiting a low-intensity, Broodingspace under an adult is limited, and the