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Home , Eastern meadowlark, Icterid, Leistes

KARYOTYPES OF SIX OF NORTH AMERICAN BLACKBIRDS (ICTERIDAE: PASSERIFORMES)

HOLLYH. HOBART,1 SCOTTJ. Gu•q•q,2 A•qVJoH•q W. BICKI4AM Departmentof Wildlife and FisheriesSciences, Texas A&M University, CollegeStation, Texas77843 USA

ABSTRACT.--Karyotypesof six speciesof blackbirdswere prepared using in vivo tech- niques.Five speciesare karyotypicallysimilar and may be more closelyrelated than tradi- tional morphologicalcharacters suggest. The EasternMeadowlark ( magna), how- ever, is very differentin karyotypeand may be more distantlyrelated to the other species than has been previouslythought. Karyotypicevidence is consistentwith Beecher'shy- pothesisof a closerelationship between emberizine finches and blackbirds.Received 25 June 1981, accepted18 December1981.

THE family Icteridae (Passeriformes)is a di- mosomalvariation that can revealevolutionary verse group of predominantly tropical New and phylogeneticrelationships. Among the World songbirdsarranged by various investi- karyotypicallybest known vertebrategroups gators into 89-101 species in 25-36 genera are the mammalian orders Chiroptera, Pri- (Storer 1960, 1971; Blake 1968; Short 1968, 1969; mates, and Rodentia and the reptilian order Clements 1978). Beecher (1951) believed that Testudines.These groups show diverse pat- the radiation of the icteridswas an "explosive" terns of variation in the chromosomecomple- event, the family quickly filling all available ment (Capanna and Civitelli 1970, Gorman niches. Later, Beecher (1953) described Icteri- 1973,Matthey 1973,Wilson et al. 1975,Bush et dae as a terminal taxon, anatomicallyand be- al. 1977, Bickham and Baker 1979, Bickham haviorally more complexthan the group from 1981). The mammalianorders exhibit highly which it arose. variable karyotypes, while lower vertebrate Early phylogeneticstudies of the taxa usually are karyotypicallyconservative. were confinedto plumagecoloration and gross Comparedto the karyologyof mammalsand morphological characters of the bill, skull, reptiles, avian karyology is relatively unex- wing, and foot. In 1951, Beecherestablished plored,with lessthan 3% of all specieskaryo- the mostrecent phylogeny of the group, based typed (Takagi and Sasaki 1974, Shields in on jaw and skull musculatureand bill shape. press).Many avian taxa show little or no vari- He hypothesizedthat the icteridsevolved from ation in standardkaryotypes, yet someavian the emberizinefinches, through the primitive genera (e.g. lunco) are composedof species Molothrus,in three phyletic lines: the that share identical chromosomepolymor- Agelaiine, Quiscaline, and Cassicine.Based on phisms(Shields 1973, 1976). Shields (in press) myologicaland foragingstudies, Raikow (1978) has reviewed the amount of variation in chro- believed that Emberizinaeand Icteridae may mosomesof , and studiescomparing the be sistergroups. karyotypesof closelyrelated speciesare Recently, comparative karyological studies rare. The availability of six speciesof icterids have been used to discern patterns of chro- in our area provided an opportunity for com- parative studies. There are two basic strategiesfollowed in karyotypicinvestigations. The firstis to survey • Presentaddress: Department of Ecologyand Evo- relatedforms throughout a taxonin an attempt lutionary Biology,The University of Arizona, Tuc- to determine patternsof karyotype variation son, Arizona 85721 USA. amongsubgroups. The secondis to surveya 2 Presentaddress: Department of Biology,Texas large number of individualsfrom the rangeof A&M University, CollegeStation, Texas77843 USA. a single speciesor speciesgroup to discern

514 The Auk 99: 514-518.July 1982 JULY1982] BlackbirdKaryotypes 515

Fig.1. Brown-headedCowbirdkaryotype. Acomplete chromosome setfrom a male; inset are the sex chromosomesfrom a female.

croscope.Chromosomes werearranged according to whatpatterns of intraspecificvariation and size;terminology for centromereposition follows polymorphismarepresent in naturalpopula- Patton (1967). tions. At least10 metaphasocells of eachspecies except Thepurpose of thispaper is to presentthe I. graduacaudawereexamined todetermine thedip- resultsof our karyotypicstudy of these'six 1oidnumber. Both sexes were examined for each speciesrepresenting fivegenera of Icteridae: the speciesexcept I. graduacauda(male only) and Q. Brown-headedCowbird (Molothrusater), the quiscula(female only). Sturnella magna (Brazos Coun- Great-tailedGrackle (Quigcalus mexicanus), the ty,Texas) and I. graduacauda(Webb County, Texas) CommonGrackle (Q. quiscula),the Red- werecollected during the breeding season; all other individualswere trappedfrom wintering popula- wingedBlackbird ( phoeniceus), the tionsin BrazosCounty. Fifteen individuals of Black-headedOriole (Icterus graduacauda), and aterwere examined, as were at leasttwo each of the the EasternMeadowlark (Sturnella magna). otherspecies except I. graduacauda. Thisrepresents the only cytogenetic informa- Voucherspecimens deposited in theTexas Coop- tionreported for mostof thesespecies, al- erativeWildlife Collection(TCWC) at TexasA&M thoughDNA amounts were reported for Age- Universityare: NI. aterTCWC 11029, 11030, 11031, laiusphoeniceus and Sturnellamagna by 11032,10926; A. phoeniceusTCWC 11033, 11034; Q. Bachmannet al. (1972),and the karyotypeof mexicanusTCWC 11035,11036; Q. quisculaTCWC the formerspecies was reported by Makino 11028;and I. graduacaudaTCWC 10910; 10 specimens and Baldwin (1954). of M. ater,2 specimensof S. magna,1 of Q. mexi- canus,2 of Q. quiscula,and 1 of A. phoeniceusare METHODS uncatalogued.

Somaticmetaphase chromosomes were prepared RESULTS from 27 icteridsusing in vivo bone marrow proce- dures.Individuals were injectedintraperitoneally MOLOTnRUS AT•R (2n = 78--80:Fig. 1), Qms- with a 0.05% solutionof colchicineat 0.3-0.4 ml per CALUSt•EXlCAt•US (2n = 76-78: Fig. 2A), Q. 100g body weight and were sacrificed after a period QCnSCULA(2n = 76: Fig. 2B),AGELAIUS PnO•NI- of 20-30min. The femurswere removed and flushed C•US(2n = 80: Fig. 2C), ICTERUSGRADIL4CAUDA witha hypotonicsolution of 0.075M KCIto obtain (2n = 76-78:Fig. 2D) a suspensionof bone marrow cells, which was in- cubatedin thehypotonic solution at 37øCfor 30 min. Thechromosomal complements of these five The cellswere then fixed (3 partsmethanol to 1 part speciesare similar. The six largest autosomal aceticacid) for 10 min and resuspended two or three pairs(macrochromosomes) aredistinguishable timesin freshfixative to ensurefixation. Slides were bysize from the remaining 31-33 pairs (micro- preparedbydropping three or four drops of cell sus- chromosomes).The largest pair of macrochro- pensionon cleanedglass slides and igniting the mosomesis submetacentric,the nextfour pairs preparation.The slides were then stained for 5 min in a warm 10% Giemsasolution. Preparations were aresubtelocentric, and the sixthlargest pair is observedand photographed on a LeitzDialux mi- acrocentric.Microchromosomes appear to be 516 HOBART,GUNN, AND BICKHAM [Auk,Vol. 09

zw

Fig.2. Partialkaryotypes offive icterids. Only the 10 largest autosomes andsex chromosomes areshown forA. Great-tailed Grackle, female; B., female; C.Red-winged Blackbird, female; D.Black- headedOriole, male; and E. EasternMeadowlark, female.

acrocentricexcept for two pairs of biarmed centric,and the fifth autosomeis acrocentric; chromosomes.Microchromosomes arefigured thus,there are fourpairs of autosomalbiarmed onlyfor M. ater(Fig. 1). Thelack of resolution macrochromosomesas compared to five in the due to smallsize and apparentsimilarity other species.The Z chromosomeis a medium- among species limits the usefulnessof micro- sizedmetacentric, and the W is probablyac- chromosomesfor comparativestudies. We rocentric.There are 32 pairs of microchromo- haveattempted only to enumeratethem. The somes. Z chromosome,which is about the fourth larg- est chromosomein size, is subtelocentricfor DISCUSSION all of these species.The W chromosomeis probably an acrocentricmicrochromosome in Beecher(1951) hypothesized that the icterids all exceptM. ater,in whichit probablyis a evolved from the emberizine finches via the small metacentric chromosome. Makino and genusMolothrus. He describedthree phyletic Baldwin(1954) reported karyotypes for A. linesof Icteridaedescending from Molothrus: phoeniceusand Xanthocephalus xanthocephalus the Agelaiine,containing Agelaius, Sturnella, (theYellow-headed Blackbird) with macrochro- Icterus,and severalgenera not reportedhere; mosomal complementsidentical to those re- the Quiscaline,containing the subgenera portedin this study. (Q. quiscula)and Cassidix(Q. mexi- canus)of thegenus Quiscalus and several gen- STURNELLAMAGNA (2n = 78:Fig. 2E) era not reportedhere; and the Cassicine,none of which is reportedhere. Therefore, Molothrus Thekaryotype of S. magnadiffers from those mightbe expectedto possessa primitive,or describedfor theabove five species in several ancestral, karyotype. respects.The largestautosomal element is sub- In general,the six species show little karyo- telocentric, the fourth autosome is subrecta- typicvariation. The diploidnumbers range JvL¾1982] BlackbirdKaryotypes 517 from 76 to 80 due to interspecificdifferences in for a primitive biarmed W chromosome(the the number of microchromosomes.Five species only character in which M. ater differs from appear to have identical macrochromosomal most other icterids). Certain biochemical stud- complements (Figs. 1 and 2A-D). Sturnella ies, however, indicate that Beecher was correct magnadiffers from these by centromereposi- on this point also. tion in pairs 1, 4, 5, and the Z chromosome. Smith and Zimmerman (1976) investigated Molothrusater differsfrom all speciesstudied the biochemical geneticsof seven speciesof by centromereposition of the W chromosome North American blackbirds representing the (one of the larger microchromosomes).This Agelaiine and Quiscalinegroups. Of the Quis- differencein W chromosomemorphology may caline species, M. ater was the most similar to be significant.The karyotypesof Emberizacit- the Agelaiine species,especially to Agelaius. rinella (Hammar and Herlin 1975),E. fiaviven- This correlates well with Beecher's (1951) tris (Hirschi et al. 1972),and E. bruniceps(Misra groupings, appearing to support the theory and Srivastava 1978) are similar to the M. ater that Molothrusrepresents the primitive group karyotype, differing in centromereposition in that could have given rise to both the Quis- three autosomal pairs. Emberiza citrinella caline and Agelaiine radiations. Another bio- shares the metacentric condition of the W chro- chemical study (Prager et al. 1974) showed a mosome with M. ater. Other emberizine closerelationship between Molothrus and Age- species,however, are known to possessacro- laius, which could alsobe interpretedto sup- centric W chromosomes,such as Junco(Shields port Beecher's(1951) hypothesis. 1973)and Zonotrichia(Thorneycroft 1976). It seemsthat morphologic,karyotypic, and We found no intraspecificpolymorphism in biochemical data are consistent with the the karyotypesof these species,although the groupings of Beecher,with some reservation limited sample sizes leave room for doubt. We concerningS. magnabased on the karyotypic have studied 15 specimensof M. ater from a data. Overall, the chromosomal variation noted Texas wintering population that has been could be attributed to pericentric inversions, shown to be made up of birds that breed as have been observed in the genus Junco throughoutthe MississippiRiver drainageand (Shields 1973) and Zonotrichia albicollis (Thor- beyond(Coon and Arnold 1977),so we may be neycroft 1976), or to additions or deletions of justifiedin believingthat karyotypicpolymor- heterochromatin. The demonstration of ho- phisms do not exist in this species. mologies and the rearrangementsleading to The interspecificdifferences in microchro- variation is not possible without studying mosomenumbers are difficult to interpret, par- differentiallystained chromosomes. These data ticularly in view of the difficulties of optical seem to support Shields' (in press) contention resolution of these very small chromosomes. that chromosomalchanges do not play a major Only the very best of karyotypepreparations role in the speciationprocess of birds. Again, permit the microchromosomesto be seen well this is stated with reservation due to the dif- enough to be accuratelycounted. Use of mi- ferencesnoted for S. magna. crochromosomenumbers and morphologiesin ACKNOWLEDGMENTS cytosystematicstudies awaits further improve- ment of resolutionby advancesin cytogenetic This study was supportedby NSF grantsDEB- technique. 7713467 and DEB-7921519 to J. W. Bickham. We wish The close resemblance of emberizine and ic~ to thank K. Arnold, A. Bivings,J. Bostick,R. Dowler, terid karyotypesis indicative of a relatively M. Engstrom, N. Garza, J. Sites, O. Ward, G. closephylogenetic relationship. Likewise, the Shields, and M. Witmet for providing assistance identical macrochromosomal sets of the icter- during the study and/orreviewing earlier drafts of the manuscript. ids reported, exceptS. magna,indicate a close phylogeneticrelationship. It appearsthat the chromosomal data are consistent with Bee- BACHMANN,K., B. A. H.•WRXNGTON,& J.P. CR_•XG. cher'shypothesis that icteridsevolved from the 1972. Genome size in birds. Chromosoma (Ber- emberizinefinches. The primitive natureof M. lin) 37: 405-416. ater is not well establishedon karyological B•ECH•R,W. J. 1951. Adaptationsfor food-getting grounds, becauseno strongcase can be made in the American blackbirds. Auk 68: 411-440. 518 HOBART,GUNN, AND BICKHAM [Auk, Vol. 99

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