Genetic Differentiation and Hybridization Between Greater and Lesser Spotted Eagles (Accipitriformes:Aquila Clanga, A

J Ornithol (2005) 146: 226–234 DOI 10.1007/s10336-005-0083-8 ORIGINAL ARTICLE Andreas J. Helbig Æ Ingrid Seibold Æ Annett Kocum Dorit Liebers Æ Jessica Irwin Æ Ugis Bergmanis Bernd U. Meyburg Æ Wolfgang Scheller Michael Stubbe Æ Staffan Bensch Genetic differentiation and hybridization between greater and lesser spotted eagles (Accipitriformes:Aquila clanga, A. pomarina) Received: 16 December 2004 / Revised: 14 February 2005 / Accepted: 15 February 2005 / Published online: 28 April 2005 Ó Dt. Ornithologen-Gesellschaft e.V. 2005 Abstract Greater and lesser spotted eagles (Aquila samples of individuals in which mtDNA haplotype and clanga, A. pomarina) are two closely related forest eagles phenotype agreed. This indicates that mismatched birds overlapping in breeding range in east-central Europe. In were either F1 or recent back-cross hybrids. Mitochon- recent years a number of mixed pairs have been ob- drial introgression was asymmetrical (no pomarina served, some of which fledged hybrid young. Here we haplotype found in clanga so far), which may be due to use mitochondrial (control region) DNA sequences and assortative mating by size. Gene flow of nuclear markers AFLP markers to estimate genetic differentiation and was estimated to be about ten times stronger than for possible gene flow between these species. In a sample of mtDNA, indicating a sex-bias in hybrid fertility in 83 individuals (61 pomarina,20clanga, 2 F1-hybrids) we accordance with Haldane’s rule. Hybridization between found 30 mitochondrial haplotypes which, in a phylo- the two species may be more frequent and may occur genetic network, formed two distinct clusters differing much further west than hitherto assumed. This is sup- on average by 3.0% sequence divergence. The two spe- ported by the recent discovery of a mixed pair producing cies were significantly differentiated both at the mito- at least one fledgling in NE Germany. chondrial and nuclear (AFLP) genetic level. However, five individuals with pomarina phenotype possessed Keywords Amplified fragment length clanga-type mtDNA, suggesting occasional gene flow. polymorphism Æ Haldane’s rule Æ Hybridization Æ Surprisingly, AFLP markers indicated that these ‘‘mis- Hypervariable control region 1 Æ Mitochondrial matched’’ birds (originating from Germany, E Poland introgression and Latvia) were genetically intermediate between the Communicated by F. Bairlein Introduction A. J. Helbig (&) Æ I. Seibold Æ A. Kocum Æ D. Liebers Institute of Zoology, Greifswald University, Among the hawk-like birds of prey (Accipitriformes) Vogelwarte Hiddensee, Zum Hochland 17, only a few cases of species forming a ‘‘zone of overlap 18565 Kloster, Germany and hybridization’’ (sensu Short 1969) are known, and E-mail: [email protected] even fewer of them have been well documented. One J. Irwin Æ S. Bensch example is the contact zone of western and eastern Molecular Population Biology Lab, marsh harrier (Circus aeruginosus, C. spilonotus)in Department of Animal Ecology, Lund University, Siberia (Fefelov 2001). In Europe, a possible case where 22362 Lund, Sweden the frequency of hybridization may have been underes- U. Bergmanis timated concerns the greater spotted eagle (A. clanga) Toces-3, 4862 Laudona, Madonas raj, Latvia and the lesser spotted eagle (A. pomarina), two similar B. U. Meyburg species of Palearctic forest eagles. A. pomarina is Wangenheimstr 32, 14193, Berlin restricted as a breeding bird to temperate parts of Cen- W. Scheller tral and eastern Europe and winters in Africa. A. clanga Danschowstr 16, 17155 Teterow, Germany breeds from eastern Europe to eastern Asia and winters M. Stubbe mainly on the Indian subcontinent and in SE Asia, but Institut fu¨ r Zoologie, Universita¨ t Halle-Wittenberg, also in the Middle East and NE Africa (Cramp et al. Domplatz 4, 06108 Halle, Germany 1980). Breeding ranges of the two species overlap in an 227 area several hundred kilometres in width between the species and to identify hybrids (Bensch et al. 2002a, Baltic republics, eastern Poland, Belarus, and Ukraine 2002b; Wang et al. 2003). If ancestral polymorphism was (Hagemeijer and Blair 1997). In the entire area of responsible for the lack of complete segregation between sympatry, A. clanga is much rarer than A. pomarina mtDNA and phenotype, we would expect ‘‘mismatch’’ (Meyburg et al. 2001). However, both an assessment of birds (individuals with phenotype of one, but mt hap- the distribution and relative abundance of the two spe- lotype of the other taxon) to be clearly assignable, cies as well as documentation of possible hybridization genetically, to one or the other taxon. Alternatively, if have been hampered by difficulties of field identification. such mismatches were due to recent hybridization, Reliable characters for distinguishing the species in the AFLP markers should indicate a genetically intermedi- field have only become known since about the mid-1970s ate position of mismatched individuals. (Porter et al. 1974; Svensson 1975; for an up-to-date summary, see Forsman 1999). Studies of museum specimens have indicated that, in Methods the zone of sympatry between the two species, a con- siderable proportion of individuals may show interme- Sampling diate phenotypic characters (Zhezherin 1969). Whether this is due to hybridization and whether such hybrid- Blood samples were taken of 61 A. pomarina individ- ization leads to gene flow (back-crossing), remained uals (adults and nestlings) in Germany (13 in Meckl- unknown. Only in recent years have a few cases of mixed enburg-Vorpommern, 11 from Hakel forest, Sachsen- pairs and successful hybridization been described Anhalt), eastern Poland (3), Belarus (4) and Latvia (Bergmanis et al. 2001,Lo˜ hmus and Va¨ li 2001; Domb- (30). Aquila clanga was sampled on the breeding rovski 2002;Va¨ li and Lo˜ hmus 2004). These were dis- grounds in Poland (9) and Belarus (4) as well as on covered mainly during the course of ringing work at the migration or wintering grounds in Turkey (2), Saudi nests, an activity that started in Latvia in 1985 (nes- Arabia (2) and Israel (3). Only individuals not directly tlings) and 1994 (adults) and was intensified in Germany related through the maternal line were included in this from 1990 onwards (nestlings and adults), partly in the analysis (i.e. no known mother-offspring pairs or sib- course of satellite telemetry studies (Meyburg et al. lings). In addition, two putative F1 hybrids were in- 1995). In eastern Poland, mixed pairs were first discov- cluded, i.e. offspring of mixed pairs whose mothers ered when attempts were made to capture adults for were of the clanga phenotype: one from near Greifs- satellite telemetry (from 1992 onwards). Recent surveys wald, Mecklenburg-Vorpommern, NE Germany (year in Estonia showed that hybridization probably occurs 2003; this individual was not scored for AFLP) and regularly, but the extent of possible back-crossing is still one from NE Poland (Biebrza, year 2001). Since eagles unknown (Va¨ li and Lo˜ hmus 2004). are known to be faithful to their nest-sites over many Here, we investigate the genetic differentiation and years (Meyburg et al. 2004), we sampled each territory potential gene flow between the two eagle species using only once in order to avoid the inclusion of siblings or sequences of the mitochondrial control region and nu- half-siblings. clear amplified fragment polymorphism (AFLP) mark- Nestlings were sampled shortly before fledging and ers. In particular, we were interested to see if unisexual were identified based on plumage features (size of pale (female) hybrid sterility might restrict mitochondrial spots on median wing-coverts, width of barring on gene flow in the contact area. If interspecific hybrids remiges) and structural measurements (bill height, length show reduced viability or fertility compared to the of middle toe, emargination on seventh primary) using parental species, the heterogametic sex (female in birds) criteria outlined by Bergmanis (1996) and Forsman usually suffers more than the homogametic sex, a pat- (1999). Care was also taken to safely identify both adults tern known as ‘‘Haldane’s rule’’ (Haldane 1922). In attending a nest. birds, several cases conforming to this pattern have been documented (Tegelstro¨ m and Gelter 1990; Wu and Davis 1993; Helbig et al. 2001). mtDNA If unisexual hybrid sterility was operating among spotted eagles, we would expect mitochondrial gene flow Total DNA was isolated from the blood samples using to be restricted or completely interrupted between the a standard salting-out procedure (Miller et al. 1988). A taxa, because sterility should preferentially occur in fe- large fragment of mitochondrial DNA including the males, i.e. the sex transmitting the mitochondrial gen- entire control region (cr), ND6 gene, and pseudo- ome. If, alternatively, segregation of mitochondrial control region was amplified on a Perkin Elmer ther- haplotypes between the two taxa is found to be incom- mocycler (Geneamp 4200) with primers UUL (Liebers plete, this could either be due to ancestral polymorphism et al. 2001) and DDL (Helbig and Seibold 1999) using or ongoing gene flow. To decide between these alterna- the Expand TM Long Template PCR System (Boeh- tives, we investigated anonymous nuclear genomic ringer Mannheim) as described in Liebers et al. (2001). markers by AFLP (Vos et al. 1995), a method which has Primer UUL binds to the tRNA-Thr gene immediately previously been used to distinguish populations within a upstream of the cr, DDL binds to the 12sRNA gene 228 downstream of the pseudo-control region. Note that Analysis Aquila eagles possess mitochondrial gene order C as described by Mindell et al. (1998). The PCR products From the 30 HVR-1 haplotypes a median-joining net- were of variable lengths due to variable numbers of work (Bandelt et al. 1999) was constructed using the tandem repeats in the pseudo-control region (cf. Va¨ li program NETWORK 4.1 (Ro¨ hl 2004). Genetic differ- 2002). The hypervariable part of the control region entiation between species (F ST) was calculated using (HVR-1), in most birds consisting of approximately ARLEQUIN v.

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