Chancellor, R. D. & B.-U. Meyburg eds. 2004 Raptors Worldwide WWGBP/MME

Phylogenetic Relationships in the Hierofalco Complex (Saker-, Gyr-, Lanner-, Laggar )

Michael Wink, Hedi Sauer-Giirth, David Ellis & Robert Kenward

ABSTRACT Saker, Gyr, Lanner and Laggar represent large that belong to the Hierofalco complex. We have amplified and sequenced the mitochondrial cytochrome b gene of all representatives of this clade, including several hundreds of Sakers from Kazakhstan and Mongolia. DNA data show that these taxa are very closely related. Within the Saker falcons 3 genetic lineages can be detected. No evidence could be obtained that the Altai falcon or the other Saker can be recognised at the DNA level. Within the two lineages are evident that correspond to the subspecies F. b. biarmicus or F. b. feldeggii. The Australian F. subniger appears to be associated with the Hierofalco complex.

INTRODUCTION Although Gyr, Lanner and Saker falcons are apparently closely related, they show a high degree of plumage polymorphism and have puzzled taxonomists for some time. 0. Kleinschmidt (1901, 1958) and Meinertzhagen (1954) had placed them in a single , Falco hierofalco, and distinguished F. h. islandus Brünn, F. h. norvegicus Schleg., F. h. cherrug Gray and F. h. feldeggi Schleg. F. hierofalco has been split since and today some authorities include F. rusticolus, F. cherrug, F. biarmicus, F. jugger and F. mexicanus in the subgenus Hierofalco and consider them as a superspecies (Amadon & Bull 1988; del Hoyo et al. 1994; Sibley & Monroe 1990; Cramp 1980). Within the species some subdivision, mostly concerning geographical forms or even morphs, has been considered (Niethammer 1938; Dementiev 1960; Weick 1980; Amadon & Bull 1988; del Hoyo et al. 1994; Sibley & Monroe 1990):

499 • F. rusticolus L.: Several subspecies; F. r. rusticolus (Scandinavia and northern Russia); F. r. uralensis Menzbier (Russia east of Petschora); F. r. islandus Brünnich (on Iceland); F. r. candicans Gmelin (on Greenland) • F. cherrug: Several subspecies; F. c. cherrug Gray (E Europe, Russia to Altai mountains and Yenisey) and F. c. milvipes Jerdon (Siberia, Mongolia, China) (del Hoyo et al., 1994). Weick (1980) distinguishes: F. c. cherrug, F. c. cyanopus Thiemann (Bohemia to Wolga River), F. c, saceroides (Bianchy) (Kirghiz steppe Saja, Altai, Tarbatagai, NW Mongolia), F. c. coatsi Dementiev (Tian Shan west to NE ), F. c. hendersoni Hume (Pamir, Himalayas to Kansu), F. c. milvipes Jerdon (Mongolia, Manchuria). Weick includes F. c. altaicus in F. c. milvipes and also concludes that F. c. saceroides, F. c. coatsi and F. c. hendersoni could be colour morphs and would then be treated as F. c. milvipes. • F. altaicus Menzbier: a dark coloured falcon of central Asia (Altai region, NW Mongolia, W China); treated also as a morph, either closer to F. rusticolus or F. cherrug. Ellis (1995) provided arguments that F. altaicus is a good species. • F. biarmicus: 5 geographically defined subspecies; F. b. biarmicus Temminck (southern ), F .b. abyssinicus Neumann (western, central and eastern Africa), F. b. tanypterus (northern Africa, Israel, Iraq), F. b. erlangen Kleinschmidt (NW Africa), F. b. feldeggii Schleg. (southern Italy, E to Armenia and Azerbaijan) • F. jugger : monotypic (Indian subcontinent) • F. mexicanus: (North America) We have started to use molecular genetic techniques to elucidate the complex relationships in the Hierofalco complex. We mainly rely on nucleotide sequences of the mitochondrial cytochrome b gene that shows a good resolution on genus and even species level. Nucleotide sequences of the mitochondrial cytochrome b gene have already been employed to study the systematics and evolution of diurnal raptors (Griffiths 1997; Mindell 1997; from our laboratory: Groombridge et al. 2002, Kruckenhauser et al. 2003; Riesing et al. 2003; Seibold et al. 1993, 1996; Seibold & Helbig 1995, 1996; Helbig et al. 1994; Wink 1995, 1998, 2000; Wink & Seibold 1996; Wink & Sauer-Gürth 2000; Wink et al. 1996, 1998a,b;). First and preliminary results have been published already on the phylogenetic relationships of Hierofalcons. The first publications based on the PhD thesis of I. Seibold (Seiboldl994) (Seibold et al. 1993; Wink 1995b, 1998; Wink & Seiboldl996; Wink et al. 1998) presented a mistaken picture, since nuclear copies of the cytochrome b genes had been sequenced by chance (in Hierfalcons but not for the other falcons), which placed the Hierofalcons at the base of the falcon tree. The mistake was identified when new PCR primers and new sequencing techniques were employed. Later phylogenies unequivocally showed that F. rusticolus, F. biarmicus, F. cherrug and F. jugger form a closely related complex that clusters as a sister group to the peregrines (Wink 2000; Wink & Sauer-Gürth 2000). F. mexicanus is not a member of this clade but shares ancestry with both peregrines and hierofalcons. A closer relationship with peregrines had been postulated by Schmutz & Oliphant (1987).

500 We have amplified and sequenced the mitochondrial cytochrome b gene of all representatives of this clade, including several hundreds of Sakers from Kazakhstan and Mongolia. Samples are included that were identified by scientists and falconers as "Altai falcons". Although the present study is still incomplete, since important samples from several populations/subspecies are still missing, a few conclusions can be drawn and a new hypothesis put forward on the phylogeny and of this falcon complex.

MATERIAL AND METHODS We have isolated total DNA from feather, blood or tissue samples (see Wink 2000) which had been kindly supplied by several colleagues (W. Bednarek, J. Bragin, F. Nittinger, A. Gamauf, R. Pfeffer, D. Ristow, J. Penhallurick, M. Heidenreich). The cytochrome b gene was amplified by PCR (primer sequences in Wink & Sauer-Gürth 2000) and sequenced by using AlfExpress (Amersham Pharmacia Biotech) or a capillary sequencer ABI 3100 (Applied Biosystems) instruments. Sequences of 1000 and more base pairs were aligned manually and analysed with the software packages PAUP* (Swofford, 2002) and MEGA2 (Kumar et al. 2001) (see Wink 2000; Wink & Sauer-Gürth 2000; Wink et al. 2002; Broders et al. 2003 for further details).

RESULTS AND DISCUSSION Figure 1 shows a molecular phylogeny of the Hierofalco complex based on a reconstruction with Maximum Parsimony (NJ and ML lead to similar results; trees are not shown because of limited space). The overall topology shows that: • The peregrines (F. peregrinus, F. pelegrinoides) form a sister group to the Hierofalcons that include F. rusticolus, F. cherrug, F. biarmicus, F. jugger and apparently F. subniger. • F. mexicanus is not a member of the Hierofalco group sensu stricto; thus Schmutz & Oliphant (1987) were right in their conclusions that F. mexicanus is closer to F. peregrinus than to F. rusticolus. • F. subniger from Australia clusters at the base of the Hierofalco clade; sequences of more are needed to corroborate this surprising finding. Since members of the Hierofalco complex were found on all continents except Australia, this finding would make sense. • Members of the Hierofalco clade are closely related; the interspecific genetic distances between Gyr, Saker and Lanner falcons are in the range of 0.4 to 2.0%, indicating that they have evolved during the last 200,000 to 1 million years (assuming a 2% divergence = 1 million years; Wilson et al. 1987; Tarr & Fleischer 1993). This is the divergence range of young species; in some families taxa with such small genetic distances would be considered as subspecies. Thus the concept of O. Kleinschmidt (1901) or Meinertzhagen (1954) to lump these taxa into a single species Falco hierofalco had some merits and justification. Our data allow some comments on the intraspecific genetic variation of F. rusticolus, F. cherrug and F. biarmicus: • The Gyr falcons appear as a monophyletic group, often clustering at the base of the Gyr/Saker/Lanner assemblage but no birds from Siberia or North America have been studied so far. • Lanner falcons are represented in our study by two subspecies: F. b. 501 biarmicus from southern Africa and F. b. feldeggii from southern Italy. Both groups are genetically divergent and differ by 1.0 to 1.4% (p-distance) indicating that they are genetically separated for almost 500,000 years. Figure 1. Molecular phylogeny of hierofalcons inferred from nucleotide sequences of the cytochrome b gene Reconstruction with maximum parsimony; representation as a phylogram (left) and 95% major consensus cladogram.

Gallus J F.cherrug ER A 7 —H3C Gallus 'F ch« rug .EM . 2033 — F cherrug.ER A7 -v. IF cherrug ER C5 — F cherrug EM.2033 ^ rr F di er rug ER CS ~F cherrug.ER C5 I'- F. cherrug. ER.£3 —F.cherrug ER C8 F cherrug.Mtaj 433Fche —F cherrug.ER .E3 jF.chernjg.Altai.2019 —F. cherrug. ER 64 F.cherrug. EM.2001 — F.cherrug.Efl D5 F cherrug. KK. N6.95 — Fcherrug.EM 2012 - F cherrug.KK N15 95 — F Chwrug EM.2014 F.cherrug.KK.N1295 —F. cherrug. Altai 2019 F.cherrug.KK. N13 95 — F.cherrug EM 2001 F.cherrug. ER. 64 —F.cherrug EM 2006 • - F. cherrug.EM .2012 —F.cherrug EM 2837 Saker III F.cherrug.EM.2006 — F eher rug. EM.2008 - F. cherrug. EM.2007 —F cherrug.KK.N6 95 Fcc2114857 —F.cherrug KK.N16 95> r Fcc4 L14857 — F.cherrug KK.M595 Fcc5 L14857 — F.cherrug KK 04 - F.cherrug.Tu t19Fche — F.ch«rrug.KK.Nl295 IF cherrug ER D5 F cherrug KK.N1395 IrF cherrug EM.2008 — F chwrug Mat 433Fche Fcherrug.KK N16.95 — Fcc2L14857 F.cherrug EM.2014 — Fcc4 L14857 - F cherwg KK D4 u — Fcc5 114857 r F cherrug.ER B6 TO F chetrug.TU ÎF&ç/119F L F.cherrug.ER 3285 C7 —F cherrug ER F cherrug.ER.06 —F cherrug.ER 3285 C7 Fcftefru9.ER.3285.D9 — F.cherrug.ER.06 F.cherrug KK.T4.95 -Fchectvig ER 3286 09 J- F cherrug.KK.T9.95 — Fcherrug.KK.T4 95 ^ F. cherrug.KK. N2.95 — Fcheriug.KK T5 95 F.eher tug.KK.T10.95 —F.cherrug KK T9.95 Fcherrug.KK.N1.95 —F.cherrug.KK. N2 96 F.cherrug. KK. 512.94 — F.cherrug.KK T1096 F.cherrug KK. 06.94 — Fcherrug KKT1595 F.eherrug.KK.D8 94 — Fcherrug.KK.T16.95 Saker II F.cherrug KK Tl.94 —F chwtug.KK.Nl 95 > F.cherrug Alta 3608 3 — F.cherrug KK N7 95 F cherrug KK.T5.95 — Fcherrug.KKN8.95 F.cherrug.KK T15.95 —Fcherrug Altai.AI.94 F.cherrug KKT16 95 — F cherrug.KK.B 12.94 •F.cherrug KK.N7.95 —F cherrug.KK.D6.94 F.cherrug KK.NS.95 — F.cherrug.KK 08 94 F.cherrug.Altai.AI 94 — F.cherrug KK T1 94 J r F.biarmcus.feldeggi Sl 1 Pbi — F cfiwaig.fctta 3e08-r F.biarmicus.2Fbi —F bi armi eus iddeggi -F.biarmicu6.2Fbi • F.biarmicus P0.3859Fb feldeggii _r F.biafmicus. 16495 —F.biarmicus.PO.385! P F. biarmicus. 16496 —F.biarmicus.16496 LTJ T. '— Fb .biarmicußS AF.241 Fbi. biarmicu! -F.cherrug.ER.C10 -F.cherrug.ER.C1—F.b.biarmicus.SAFeus.SA0F. P' P • F.ûh9rrug.ER.Q3 —F.cherrug.ER D3 • F cherrug.ER.E6 — F.cherrug.ER 69 F cherrug. ER. E1 -F.cherrug EM.2002 I—F. cherrug.ER.69 -F.cherrug.ER E6 ^ F.cherrug. EM ,2002 -F.cherrug.EFl.Cl • F cherrug.ER.C1 -Fcherrug ER C4 •F. cherrug. Altai.2005 -Fcherrug ER E1 F.cherrug. EM.2010 -F. cherrug. Altai.2005 F.ch»wg.KK.T\.95 -F.cherrug EM 2010 F.cherrug. KK.T2.95 —F.cfterrug.KK.T1.95 Saker 1 -F.cherrug ER C4 -Fcherrug KK T2.95 > F.ch«fug.KK.TÖ.95 -F.cherrug.KK T8 95 F.chertyg.Wta.fcS 94 -F cherrug.Altai.A5 94 F cherrug. Altai. A8.94 -Fcherrug Altai.AS 94 F. cherrug. KK 03.94 —Fchera19.KK.D394 F.cherrug.KU 6 AK -Fcherrug KU 6.AK F.eheriug KU 26 AK -Fcherrug KU 26 AK - F eher rug. Altai. 3608.2 F.cherrug Tij 117Fche F cherrug Altai.3603 2 J F. rusticolus 17074 •F.ru6tioolus 17074 J-— F.rustocdus. 5705 F. rusticolus. 5705 -J1 Frusticolus.TM2605 F rusticolus TM2605 F.rusticdus 9692 F. rusticolus 9692 I F rusticolus TM.2473 F rusticdu6.TM.2473 —j F.rusticolus TM 2476 -F rusticdus.TM.2476 > Gvr ] F.rusticdus OS. t -6 F.rusöcdusOS. 1 Frusticdus TM 2472 F rusticdus.TM.2472 — F rusticdu9691 F. rusticolus.9691 _jF rusticolus.23707 _J F rustiedus. 23707 T rustiedus 23708 ' F. rustiedus 23708 • F.jugger. 28 e jugger z: Falco, subniger 070108 -Falco subniger.070108 F peregnnus.brookei 2tâ2- — F peregrinus broc*ei 2192 r F perFaicoperognnu« s 2126) -FaIcoperegnnus 21268 Falcopelegnroides — Falco pelegrmoides 945 -Lr-F. - F mexicanus. NA. IFmex — F mexicanus 5 changes

502 The Sakers are most diverse. At least three well-supported clades (termed as Saker I, II & III) are evident, which do not show a good geographical correlation. The Saker III clade, for example, comprises birds from Hungary (i.e. F. c. cherrug) and also birds from Kazakhstan and Mongolia (i. e. F. c. milvipes). Saker I and Saker II clades also contain birds that would be attributed to either F. c. cherrug or F. c. milvipes. Thus a clear geographical correlation, as seen in the Lanner, is not evident in the . Sakers, that were classified as "Altai falcons" because of their dark plumage, are found in all three Saker clades, indicating that they are morphs and not valid subspecies or even species (F. altaicus). A similar conclusion can be found already in Sibley & Monroe (1990) or del Hoyo et al., (1994) (but see Ellis 1995). In most reconstructions, the Saker does not cluster as a monophylum, but rather as a paraphyletic group. As can be seen in Fig. 1, the Lanners cluster as a sister group to Saker II and III. How to explain the genetic and morphological diversity in Sakers? Since the haplotypes do not correlate with populations we speculate that the clades are the result of old hybridisations (not recent; according to the genetic distances found we have to think in terms of 100,000 to 200,000 years), of an ancient Saker with Gyr and Lanner falcons. The plumage polymorphism seen in Sakers would also be a plausible consequence of such early hybridisations. Other genetic markers, such as microsatellite analyses, are necessary to elucidate and corroborate this hypothesis. Gyr and Saker falcons can be easily hybridised in captivity; since several of these hybrids have already escaped to the wild, the future situation might become even more complicated if these birds start to interbreed with wild birds. In addition, genomic fingerprinting with ISSR-PCR (results not shown; for the method see Wink et al. 2000; Wink 2000), which usually shows significant differences between species, exhibits only few differences between the Saker and Gyr falcons, thus confirming their close relatedness.

REFERENCES AMADON, D., & J. BULL 1988. Hawks and owls of the world. Proc. W. Found. Vertebr. Zool. 3, 297-357 BRODERS, O., T. OSBORNE & M. WINK 2003. A mtDNA phylogeny of bustards (family Otitidae) based on nucleotide sequences of the cytochrome b gene. J. Ornithol. 144, 176-185. CRAMP, S. 1980. The Birds of the Western Palearctic. Vol. II. Oxford University Press. DEL HOYO, J., A. ELLIOTT & J. SARGATAL 1994. Handbook of the Birds of the World. Vol. 2, Lynx edition. Barcelona DEMENTIEV, G.P. 1960. Der Gerfalke. Neue Brehm Bucherei. A. Ziemsen Verlag. Wittenberg, Germany. ELLIS, D. H. 1995. What is Falco altaicus Menzbier? J. Raptor Res. 29:15-25. GRIFFITHS, C S. 1997. Correlation of functional domains and rates of nucleotide substitution in cytochrome b. Moi Phylog. Evol. 7,352-365. GROOMBRIDGE, J.J., C.G. JONES, M.K. BAYES, A.J. VAN ZYL, J. CARRILLO, R.A. NICHOLS &M.W. BRUFORD 2002. A molecular phylogeny of African kestrels with reference to divergence across the Indian Ocean. Mot. Phyiogenet. Evol. 25, 267-277 KLEINSCHMIDT, O. 1901. Der Formenkreis Falco Hierofalco und die Stellung des ungarischen Würgfalken in denselben. Aquila 8: 1-49. KLEINSCHMIDT, O. 1958. Raubvögel und Eulen der Heimat. A. Ziemsen Verlag, Wittenberg- Lutherstadt, Germany. KRUCKENHAUSER, L., E. HARING, W. PINSKER, M.J. RIESING, H. WINKLER, M. WINK & A. GAMAUF 2003 Genetic versus morphological differentiation of old world buzzards (genus Buteo; Accipitridac). Zoologica Scripta (in press). KUMAR, S., K. TAMURA, I. B. JAKOBSEN & M. NEI 2001. MEGA2: Molecular Evolutionary Genetics Analysis software , Arizona State University, Tempe, Arizona, USA. MEINERTZHAGEN, R. 1954. Birds of Arabia. Oliver and Boyd, Edinburgh and London. MINDELL, D.P. 1997. Avian molecular evolution and systematics. Academic Press San Diego. NIETHAMMER, G. 1938. Handbuch der deutschen Vogelkunde. Vol. 2., Akad. Verlg. Ges., Leipzig 503 RIESING,M.J.; L. KRUCKENHAUSER, A. GAMAUF & E. HARING 2003. Molecular phylogeny of the genus Buteo (Aves: Accipitridae) based on mitochondrial marker sequences. MoL Phyiogenet. Evol. 27 (2), 328-342 (2003) SCHMUTZ S.M. & W. OLIPHANT 1987. Chromosome study of peregrine, prairie, and with implications for hybrids. J. Heredity 78, 388-390. SEIBOLD, I. 1994. Untersuchungen zur molekularen Phylogenie der Greifvögel anhand von DNA- Sequenzen des mitochondriellen Cytochrom b Gens. PhD Dissertation, Heidelberg University. SEIBOLD, I. & A. HELBIG 1995. Evolutionary history of New and Old World vultures inferred from nucleotide sequences of the mitochondrial cytochrome b gene. Phil. Transact. Roy. Soc. London Series B, 350, 163-178. SEIBOLD, I. & A. HELBIG 19%. Phylogenetic relationships of the sea eagles (genus Haliaeetus)-. Reconstructions based on morphology, allozymes and mitochondrial DNA sequences. J. Zool. Syst. Evol. Res. 34, 103-112. SEIBOLD, I., A. HELBIG, B.-U. MEYBURG, J. NEGRO & M. WINK 1996 Genetic differentiation and molecular phylogeny of European Aquila eagles according to cytochrome b nucleotide sequences, pp. 1-15, •In : B.-U. Meyburg & R. Chancellor Eagle Studies. WWGBP, Berlin, London & Paris. SEIBOLD, I., A. HELBIG & M. WINK 1993. Molecular systematica of falcons (family ). Naturwissenschaften 80, 87-90. SWOFFORD, D. L. 2002. PAUP-Phylogenetic analysis using parsimony. Version PAUP*4.0bl(). TARR, C.L. & R.C. FLEISCHER 1993. Mitochondrial DNA variation and evolutionary relationships in the amakihi complex. Auk 110, 825-831 WEICK, F. 1980. Die Greifvögel der Welt , P. Paray, Hamburg. WILSON, A.C., H. OCHMAN & E.M. PRAGER 1987. Molecular time scale for evolution. Trends Genetics 3, 241-247 WINK, M. 1995. Phylogeny of Old and New World vultures (Aves: Accipitridae and Cathartidae) inferred from nucleotide sequences of the mitochondrial cytochrome b gene. Z. Naturforsch.5()c, 868-882. WINK, M. 1996 Molecular phylogeny of the Saker Falcon (Falco cherrug). Proc. Middle East Falcon Workshop, Abu Dhabi 1995, 178-202. WINK, M. 1998. Application of DNA-Markers to Study the Ecology and Evolution of Raptors. In Holarctic Birds of Prey. (R.D. Chancellor, B.-U.Meyburg, J.J. Ferrerò, Eds), Adenex & WWGBP, Berlin, pp 49-71. WINK, M. 2000. Advances in DNA studies of diurnal and nocturnal raptors. In Raptors at RiskX R.D. Chancellor B.-U. Meyburg, Eds.) WWGBP/Hancock House, pp 831-844. WINK, M. & H. SAUER-GÜRTH 2000. Advances in the molecular systematics of African raptors In Raptors at Risk,( R.D. Chancellor B.-U. Meyburg, Eds.) WWGBP/Hancock House, pp 135-147 WINK, M. & I. SEIBOLD 1996. Molecular phylogeny of Mediterranean raptors (Families Accipitridae and Falconidae). In: Biology and Conservation of Mediterranean raptors, 1994. (Muntaner, J. & J. Mayol, Eds), SEO/BirdLife, Madrid Monografia 4: 335-344. WINK, M, D. GUICKING & U. FRITZ 2000. Molecular evidence for origin of Mauremys iversoni Pritchard et McCord, 1991 and Mauremys pritchardi McCord, 1997 (Reptilia: Testudines:Bataguridae). Zoologische Abhandlungen, Staatl. Mus. Tierkunde Dresden 51, 41-49. WINK, M., P. HEIDRICH & C FENTZLOFF 1996. A mtDNA phylogeny of sea eagles (genus Haliaeetus) based on nucleotide sequences of the cytochrome b gene. Biochemical Systenuitics ami Ecology 24,783-791. WINK, M., H. SAUER-GÜRTH & E. GWINNER 2000. A molecular phylogeny of stonechats and related turdids inferred from mitochondrial DNA sequences and genomic fingerprinting by ISSR-PCR. British Birds 95, 349-355 WINK, M., I. SEIBOLD, F. LOTFIKHAH & W. BEDNAREK 1998. Molecular systematics of holarctic raptors (Order ). In Holarctic Birds of Prey. (R.D. Chancellor, B.-U.Meyburg, J.J. Ferrerò, Eds), Adenex & WWGBP; pp. 29-48.

Prof. Dr. M. Wink Hedi Sauer-Gürth Institut für Pharmazie und Molekulare Institut für Pharmazie und Molekulare Biotechnologie Biotechnologie Universität Heidelberg Universität Heidelberg INF 364, 69120 Heidelberg, Germany INF 364, 69120 Heidelberg, E-mail: [email protected] Germany

Dr David Ellis Dr Robert Kenward USGS Sonoran Desert Research Station, CEH & IUCN-European Sustainable Use School of Renewable Natural Resources Specialist Group; The University of Arizona, 125 Biological Centre for Ecology & Hydrology, Sciences East1Tucson, Arizona 85721; Winfrith, Dorchester DT2 8ZD, UK Email: [email protected] Email: [email protected] 504