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

Phylogenetic Relationships in based on nucleotide sequences of mitochondrial and nuclear marker genes

Michael Wink, Hedi Sauer-Gürth & Marc Fuchs

ABSTRACT Together with morphological and vocal characters, nucleotide sequences help to define phylogenetic relationships in owls. Since owls are nocturnal their morphological traits are often very similar a systematic differentiation or speciation event can easily be overlooked. A recent example is sumbaensis from the island of Sumba. An unknown that had been observed there had been assumed to be a member of Otus. DNA data unequivocally showed (Olsen et al. 2002) that it belongs to the Ninox. It was named Ninox sumbaensis. About 80 of the Strigidae and 16 species of Tytonidae have been studied so far in our laboratory and a phylogeny based on cytochrome b data has been published (Wink & Heidrich 1999). We have enlarged our cytochrome b data base and additionally have sequenced a nuclear marker (LDHb intron DNA). Basically, the ncDNA data support the results obtained from mtDNA.

INTRODUCTION Since owls are nocturnal their morphological traits ^re often very similar (König et al, 1999) a systematic differentiation or speciation event can easily be overlooked. Together with morphological and vocal characters nucleotide sequences of marker genes help to define phylogenetic relationships in owls. Nucleotide sequences of the mitochondrial cytochrome b gene have already been employed to study the systematics and evolution of diurnal raptors and owls (Griffiths 1997; Mindell 1997; Groombridge et al. 2002, Krucken-hauser et al, 2003, Riesing et al, 2003; from our laboratory: Seibold et al, 1993, 1996; Heidrich & Wink 1994, 1998; Heidrich étal., 1995a,b, 1996; Wink 1995, 1998, 2000; Wink & Seibold 1996; Wink & Sauer-Gürth 2000; Wink et al. 1996, 1998; Wink & Heidrich 1999, 2000; Seibold & Helbig 1995, 1996; Olsen et al. 2002). 517 In our studies we regularly amplify and sequence the mitochondrial cytochrome b gene. Since this gene shows a good resolution at the genus level and since we already have quite a large data base for this gene, we use this gene as a platform for our owl project which aims to cover all species of owls. Nuclear genes, such as intron sequences, need to be analysed to corroborate findings of mtDNA, especially relationships between genera. About 80 taxa of the Strigidae and 16 of Tytonidae have been studied so far in our laboratory and a phylogeny based on cytochrome b data has been published (Wink & Heidrich 1999, 2000). We have enlarged our cytochrome b data base and have additionally sequenced a nuclear marker (intron of LDHb-DNA) for all groups that were critical. Basically, the ncDNA data support the results obtained from mtDNA.

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, G. Ehlers, O. Hatzofe, R. Krahe, D. Reynolds, A. Kemp, C. Fentzloff, W. Grummt, C. König, J. Yom-Tov, D. Ristow, H.H. Witt, E. Thaler, B. Etheridge, D. Engelbrecht and U. Schneppat, J. Penhallurick, J. Olsen). The cytochrome b gene was amplified by PCR (primer sequences in Wink & Sauer-Gürth 2000) and sequenced by using AlfExpress (Amersham Pharmacia Biotech) or 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 The original data set (Heidrich & Wink 1998, Wink & Heidrich 1999, 2000) contained cytochrome b sequences from at least two specimens of each species; in several instances 10 and more individuals were sequenced to assess the intraspecific sequence variation. The present analysis to which a large number of new sequences were added, covers approximately 45% of all owl species and approximately 70% of all owl genera. We have performed Maximum Parsimony (MP), Neighbour Joining (NJ) and Maximum Likelihood (ML) analyses. A strict consensus MP cladogram is shown in Figure 1 to illustrate the overall phylogenetic relationships of owls based on nucleotide sequences of the cytochrome b gene. The following clades were recognised unequivocally (bootstrap support usually higher than 90%) by all three methods of tree reconstruction: • monophyletic subfamily Tytonidae • monophyletic subfamily Strigidae • monophyletic genus Tyto • monophyletic genus Strix • monophyletic genus Aegolius

518 • monophyletic genus Athene • monophyletic genus Glaucidium • monophyletic genus Ninox • paraphyletic Bubo complex which comprises the genera Bubo, Scotopelia, Ketupa and Nyctea • polyphyletic Otus complex, in which Old and New World members of the genus Otus form independent monophyletic groups.

Figure 1. Molecular phylogeny of owls inferred from nucleotide sequences of the cytochrome b gene Reconstruction with maximum parsimony; representation as a strict consensus cladogram. A. MP Bootstrap Gallus Struthio. camelus Phodilus. bad/us. 6222 Tyto. alba.deliculata20871 ") 95 Tyto.alba.javanica.9578 Ï Australasia Tyto.alba.sumbaensis.20666 J 94 Tyto. alba.erlangeri. ISR6090 88 100 Tyto.atba.guttata D19729 49 Tyto. alba, affinis. GAMBIa 10409 Europe, Africa Tyto.soumagnei. 363192

83 iod Tyto. alba.hauchekorni Chite8619 Tyto a. pratincola New worid 87 100 Tyto.alba.furcata20859 Tyto. tongimembris. capensis. 21656 95 100 C Tyto.longimembris.chinensis.9579C4 TOO101 j Tyto. castanops. 20995 69 Tyto . novaehollandiae. 20988 >\ustralasia

49 Tyto. tenebricosa. 20993 100 Tyto. multipunctata. 20984 J A egolius.acadicus. 6242 77 Bubo. b. bubo. BNM.C J. 6252 100 Strix. nebulosa.NW261 7

519 B. MP Bootstrap Gallus Struth io.camelus j— Phodilus.badius.6222 1— Tyto.alba.deliculata 20871 } Tytonidae 100— Asio.flammeus.465 94 ri— Asio.capensis.8232 — Asio.o.otus.DE6945 } Asio I Asio.clamator.8231 I— Ptilopsis.granti.6226 "Ir,., 1— Ptilopsis.ieucotis. 10407T rtliopSlS Bubo.b. ascalaphus.9566 Bubo.b.interpositus.lSRAEL.6077 Bubo.b.bubo.BNM.C J.6252 Bubo.bubo.yenisseensis.9581 Bubo.bubo.sibiricus.9580 Bubo.bubo.hispanicus.9120 Bubo. bubo, swinhoei.7.9571 Bubo.turcomanum.6115 Bubo.capensis.mackinderi.9292 Bubo.c.capensis.6116 Bubo.lacteus.Gambia10408 Bubo.bengalensis.Z.A.6118 } Bubo Bubo africanusl Bubo v. Iagophonusl Bubo.virginianus.nacurutu.8229 Bubo magellanicus Bubo (Nyctea) scandiaca4 Bubo.nipalensis.6095 Bubo sumatranus u».— Bubo.vosseleri.8236 55! ^— Bubo (ScotopeIia) peli —[100— Bubo (Ketupa).ketupa.6094 1— Bubo (Ketupaj.zeylonensis.9577 ' Strix.nebulosa.NW2617 -, Strix aluco wilkonskii ISR1 Strix aluco sylvatica ScotH 3 Strix a.aluco DE6 Strix.uralensis.FL6329 Strix butleri2 Strix Strix.w.woodfordii.6112 Strix.woodfordii.nigricantor.8235 Strix.rufipes.6299 Otus.asio.mccallii2848 Otus a. hasbroukiil Otus guatemalae2 Otus atricapillus5 Otus watsonii usta2 Otus roboratus Otus sanctaecatarinae Otus hoyi262 Megascops Otus pefersoni2 0tus.kennicotti.6170 Otus albogularis2 Otus choliba4 Otus flammeolus Pulsatrix p. perspicillata 1 PnIcatriY Pulsatrix.koeniswaldiana.6055 J ulaauiA Otus.brucei.DUBAI.9569 •> Otus.scops.13 77.6251 Otus.sunia.malayamus.9575 Otus Ionglcornis Otus.lettia.erythrocampe.8.9572 Otus.l.lempiji.8863 OtUS Otus lettia.glabripes Otus bakkamoenai I— Otus.megalotis.nigrorum.9559 1 Otus megalotisl Otus.spilocephalus 9574 ÄiuÄsiitenumi} Athene/Glaucidium

520 Struthio.camelus U89170 Micrathene whitneyi Ninox scutulata 10( Ninox sumbaiensis20415 Ninox.novaeseelandiae.5681 Ninox 89 Ninox.connivens.20999 Ninox.rufa.20997 -C Ninox.strinua.21012 Asio.flammeus.465 Asio.capensis.8232 Asio.o.otus.DE6945 Asio Asio.clamator.8231 Aegolius.acadicus.6242 Aegolius Aegolius harisii Aegolius funereus3 Athene.n.indigena GR2877 noctua.Iilith K300lsr Athene.noctua. vida/ii.8228 Athene n.noctua8 DE Athene.brama.6212 Athene Athene cunieularia.hypogaea 8AJA.9027 Athene c.cunicularia NEArgent Athene (Nirtox) .superciliaris.MADAG.10415 Glaucidium brasilianuml1 Glaueidium tucumanuml Glaucidium peruanum2 Glaueidium griseiceps Glaucidium nanuml Glaucidium bolivianum3 ^ Glaucidium Glaucidium j. jardinii4 New World Glaucidium hardyil Glaucidium gnoma Glaucidium californicumlj Glaucidium passerinuml Glaucidium tephronotum Glaucidium Glaucidium.perlatum.TRANSV.6074 Old World Glaucidium.eapense.KENIA.6288 Glaucicium.cuculoides.9576 Surnia.ulula.NW2593 Surnia Phodilus.badius.6222 98 -C Tyto.alba.deliculata20871 } TYTONIDAE

521 Whereas these clades were identified by all reconstructions, differences were found in some instances regarding the phylogenetic relationships between clades, although an Athene, Surnia, Glaucidium cluster, usually including Aegolius or a Strix/Bubo cluster was recovered by all programmes. A few new results have been obtained (as compared to data shown in Wink & Heidrich (1999, 2000): Recently, a new owl was discovered on Sumba island which was assumed to be a member of the genus Otus. DNA analysis revealed unequivocally that it is a member of the genus Ninox. It was described as Ninox sumbaiensis (Olsen et al. 2002). Ninox superciliaris which occurs on Madagascar clusters clearly in the clade of Athene, which would also agree with its plumage characters. Members of the genus Tyto show a high degree of genetical divergence. A number of taxa from the Australasian region have been added, indicating the existence of several Tyto species. Some of them have been attributed species rank already (König et al. 1999); others, such as T.a. deliculata, T.a. sumbaiensis could easily raised to species rank according to the genetic divergences found. A major surprise of the cytochrome b analysis was the finding that the genus Bubo was paraphyletic (it included the genera Ketupa, Nyctea and Scotopelia ) and the polyphyly of Otus. Otus shows three clades, the screech owls of the New World, the Scops owls of the Old World and the white-faced Scops owls of Africa. Critics could argue that cytochrome b represents a maternal lineage only and that hybridisations might have distorted the phylogeny. In order to assess this argument we have amplified and sequenced the nuclear LDHb intron gene (Fig. 2). The results obtained corroborate the cyt b findings, in that Bubo comprises Ketupa and Nyctea ( Scotopelia was not available for the LDH analysis). The three Otus clades are polyphyletic also in the LDH data set (Fig. 2). White faced Scops owls again cluster as a sister group to the genus Asio. Figure 2 gives a LDHb phylogram of the Bubo/Otus/Strix assemblage. In a next step we have combined our cytochrome b and LDHb datasets. A short version of a phylogram is shown in Figure 3. Also this reconstruction confirms previous findings, i.e. the paraphyly of the Bubo group and the polyphyly of the Otus assemblage.

522 Figure 2. Molecular phylogeny of owls inferred from nucleotide sequences (516 base pairs) of the LDHb intron DNA LDH-b Intron NJ • Phodilus badius 6223 _ Bubo africanus 6231 Bubo africanus 6232 " Bubo ascalaphus 6079 Bubo b. swinhoei 9571 Bubo ascalaphus 9565 Bubo capensis 9292 Bubo benealensis 6118 Bubo bubo yeniss 9581 Bubo bengalensis 6219 Bubo interpositus 6077 Bubo bubo 2449 MBub o b sibericus 9583 Bubo bubo 2193 Eagle owls Bubo b. sibiricus 9580 Bubo virginianus 6239 Rnbn virffiniam iQ h3D7 ]Bubo"scandiaca 6097 Bubo scandiaca 6162 Nyctea Bubo scan diaca 6131 —I Bubo lacteus o/zl 'Bubolacteus 6220 J Bubo nipalensis 6095 Bubo sumatranus 6340 M Bubo zeyionensis ôvm RBubo zeyionensis 6341 Ketupa H1Bubo zeyionensis 9577 Bubin opnprisi zeyionensis s 609-———3 j Otus guatemalae 6154 "Otus guatemalae 615 Otus atricapillus 6052 Otus kennicotti 6170 Otus sanctaecatarinae y6129 j- Otus hoyi 6123 1 Otus hoyi 6124 , . , Megascops Otus asio hasbroukn 6-,.::8™6 L Otus asio hasbroukn 1280 LiOtus choliba 6243 Otus choliba 6056 J -r As1Io OtUS 2191 rt Asio otus 6132 U Asio flammeus 6084 A SÌ" nammpiig 6139 r Ptilopsis leucotis 6171 Ptilopsis RTanti 6224 Ptilopsis -Ll StflX Uf iflgftSlg 6329 Strix uralensis 6100 n J'Strix aluco 2457 _ '—r Strix aluco 6235 lStrix aluco 6163 jStrix woodfordii 8235 Strix woodfordii 6112 IStrix rufipes 6299 l_JStrix nebulosus 3639 Strix nebulosus 3637 Ottlc S lémpjji 3643 TI^IUJ icuipiji i/WTi.' -COtue s lempiji 6272 "Otus megalotus 9561 Scops owls -T Otus scops 3552 n—Otus scops 3513 [SuuuSurnia ululululd249a 25902 Surnia ulula 6322 -I Tyto alba 2866 Tyto alba 2873 - 0.005 substitutions/site

523 Figure 3. Molecular phytogeny of owls inferred from a combined data set of nucleotide sequences of cytochrome and LDHb

MP • Gallus - Aegolius acadicus 6242 • Aegolius funereus 6183 Athene noctua 2877 Cyt b+ -Athene brama 6212 r- Glaucidium nanum 6058 Glaucidium tucumanum 6310 Glaucidium brasilianum 6126 JL Glaucidium peruanum 6143 I— Glaueidium griseiceps 6153 p Glaucidium jardinii 6146 J I— Glaueidium bolivianum 6147 '— Glaueidium hardyi 6150 _l— Glaucidium californicum 6169 — Glaueidium gnoma 6149 I Glaueidium passerinum 6167 I Giaucidium passerinum 6180 I- Surnia ulula 2492 L Surnia ulula 2592 Ninox novaeseelandiae 21010 Ninox connivens 21000 Ninox rufa 21189 Ninox strenua 21012 — Ninox scutulata 6271 — Asio otus 2191 Asio flammeus 465 Ptilopsis Ieueotis 6171 Ptilopsis qranti 6224 PtiIopsis Strix uralensis 6329 Strix aluco 2457 Strix rufipes 6299 Strix nebulosus 3637 Strix woodfordii 8235 I Bubo africanus 6232 n Bubo bengalensis 6219 Bubo ascalaphus 6079 T1-Bubo bubo 2193 L- Bubo capensis 9292 Bubo virginianus 630: Bubo virginianu§Ji229 I Bubo scandfëca 6097 Bubo seandiaca 6162 —Bub Bubo nipaiensio Iaeteus 62260905 Bubo sumatranus 6340 Bubo poensis 8236^ ____ I Bubo zeyloner^ts-3935 Bubo zeyionensis 6093 utus guatemalae eit>4 Otus kennicotti 6170 I OtuI s atricapillus 6052 J I— Otus sanctaecatarinae 6129 T— Otus hoyi 6123 Megascops - Otus asio 6286 — Otus choliba 6056 Otus lempiji 6270 Otus megalotus 9561 CltiiQ Otus scops 3552 3 • Phodilus badius 6223 I Tyto alba 2866 I Tyto alba 2873 • 50 changes

524 CONCLUSIONS A few taxonomic conclusions can be drawn (Penhallurick & Wink 2004) which imply to change the following names: New World Otus D Megascops New World Glaucidium 0 Phalaenopsis Otus leucotis • Ptilopsis leiieotis Scotopelia Q Bubo Ketupa 0 Bubo Nyctea D Bubo The results presented here are still preliminary as several species are not yet represented.

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525 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.SOc, 868-882. WINK, M. 1998. Application of DNA-Markers to study the ecology and evolution of raptors. In Holarclic 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 Risk, (R.D. Chancellor & B.-U. Meyburg, Eds.) WWGBP/Hancock House, pp 831-844. WINK, M. & P. HEIDRICH 1999. Molecular evolution and systematics of owls (Strigiformes). In: (C. König, F. Weick & J.H. Becking Eds) Owls of the World. Pica Press: Sussex. WINK, M. & P. HEIDRICH 2000. Molecular systematics of owls (Strigiformes) based on DNA sequences of the mitochondrial cytochrome b gene. In Raptors at Risk, (R.D. Chancellor & B.-U. Meyburg, Eds.) WWGBP/Hancock House, London, pp 819-828. 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/HancockHouse. 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., P. HEIDRICH & C FENTZLOFF 1996. A mtDNA phylogeny of sea eagles (genus Haliaeetus) based on nucleotide sequences of the cytochrome b gene. Biochemical Systematics and 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 Falconiformes). 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, Universität Heidelberg Biotechnologi, Universität Heidelberg INF 364, 69120 Heidelberg, Germany INF 364, 69120 Heidelberg, E-mail: [email protected] Germany

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