A Reappraisal of the Systematic Affinities of Socotran, Arabian And

Ibis (2013), doi: 10.1111/ibi.12041

A reappraisal of the systematic afﬁnities of Socotran, Arabian and East African scops owls (Otus, Strigidae) using a combination of molecular, biometric and acoustic data ^ JEAN-MARC PONS,1,2* GUY M. KIRWAN,3 RICHARD F. PORTER4 &JER OME FUCHS5 1UMR7205 ‘Origine, Structure et Evolution de la Biodiversite’,Departement Systematique et Evolution, Museum National d’Histoire, Naturelle, 55 Rue Buffon, Paris, 75005, France 2Service de Systematique Moleculaire, UMS 2700, Museum National d’Histoire Naturelle, 43 rue Cuvier, Paris, 75005, France 3Field Museum of Natural History, 1400 South Lakeshore Drive, Chicago, IL 60605, USA 4BirdLife International, Wellbrook Court, Girton Road, Cambridge CB3 0NA, UK 5Department of Ornithology and Mammalogy, California Academy of Sciences, 55 Music Concourse Drive, San Francisco, CA 94118, USA

We investigated phylogenetic relationships among Otus scops owls from Socotra Island, the Arabian Peninsula and East Africa using molecular, vocalization and biometric data. The Socotra Scops Owl Otus senegalensis socotranus, currently treated as a subspecies of the African Scops Owl Otus senegalensis, is more closely related to the Oriental Scops Owl Otus sunia and to the endemic Seychelles Scops Owl Otus insularis. Considerable mitochondrial genetic distance and signiﬁcant morphological differentiation from its two closest relatives, as well as its distinctive vocalizations compared with O. insularis, strongly support recognition of Socotra Scops Owl as a full species. Unexpectedly, two taxa from the Arabian Peninsula, Pallid Scops Owl Otus brucei and African Scops Owl Otus senegalensis pamelae, represent very distinct lineages; O. brucei is basal to a clade that includes taxa found in the Indo-Malayan region and on Indian Ocean islands. In contrast, O. s. pamelae occupies a well-supported basal position within a clade of continental Afro-Palaearctic taxa. The uncorrected-p genetic distance between O. s. pamelae and its closest relatives (other populations of senegalensis from mainland Africa) is c. 4%. As O. s. pamelae is also well differentiated phylogenetically, morphologically and vocally from O. s. senegalensis, we recommend its elevation to species status, as Otus pamelae. Among mainland African O. senegalensis subspecies, Ethiopian populations appear to represent the most divergent lineage, whereas other lineages from Somalia, Kenya and South Africa are poorly differentiated. The large genetic distance between the Ethiopian haplotype and other African haplotypes (3.2%) suggests that the Ethiopian Otus may represent a cryptic taxon, and we recommend that more individuals be sampled to assess the taxonomic status of this population. Keywords: biogeography, Indian Ocean Islands, Indo-Malaya, phylogeny, Socotra Island, taxonomy.

Scops owls (Otus, Strigidae) represent a genus of on small and remote islands. Despite high species small to medium-sized nocturnal birds with strong richness, the morphological diversity encountered colonizing capability, suggested by their presence within the genus is limited, with the occurrence of only two primary plumage colour types: rufous or *Corresponding author. grey. This lack of major plumage differences is Email: [email protected] common among members of the Strigidae and is

usually explained by the need to remain inconspic- insularis formed a well-supported clade with uous during the day as a defence against predators Oriental Scops Owl Otus sunia (Fuchs et al. or from being mobbed by other birds (Marks et al. 2008). 1999). As a consequence of their highly conserved Samples of Socotra Scops Owl, currently con- morphology, it is unsurprising that the systematics sidered to be a subspecies of O. senegalensis, were of the genus Otus, as traditionally defined, have unavailable to Fuchs et al. (2008), and the same been challenged by molecular data (Fuchs et al. was true of several taxa found on the Arabian Pen- 2008, Wink et al. 2009, Miranda et al. 2011). insula (Pallid Scops Owl Otus brucei, and Otus sen- These studies suggested not only that the genus egalensis pamelae) and East Africa (northern Otus is polyphyletic, because the White-faced Somalia, Ethiopia, Kenya), where several subspe- Scops Owl Otus leucotis is sister to the genus Asio, cies of O. senegalensis have been described on the whereas the New World Screech Owls have affini- basis of differences in voice, plumage coloration ties with the genera Bubo, Strix and the Asio–Otus and size (Chapin 1930, Meinertzhagen 1948, leucotis clade (Fuchs et al. 2008), but also that Keith & Twomey 1968) but are not generally rec- species limits in some groups must be reconsidered ognized in the modern literature (Kemp 1988, due to either divergent mtDNA sequences Marks et al. 1999, Dickinson 2003, Konig€ et al. (Miranda et al. 2011) or paraphyly of traditionally 2008). The affinities of these taxa are still poorly recognized species (e.g. Collared Scops Owl O. let- known and this uncertainty is reflected by frequent tia; Fuchs et al. 2008). changes in their taxonomic treatment (Marks et al. As currently understood, the genus Otus has its 1999, Konig€ et al. 2008). Since Sclater (1924), the centre of diversity in the Palaearctic and Oriental Socotran population has usually been treated as a (Indo-Malayan) regions (26 species). Secondary subspecies of O. senegalensis (Otus senegalensis soco- radiations have occurred on the Indian Ocean tranus), but it is sometimes placed together with islands, which are occupied by six species, and in Pallid Scops Owl O. brucei (e.g. Kemp 1988, Africa with four mainland species (Eurasian Scops Konig€ et al. 1999), whose range otherwise encom- Owl Otus scops, African Scops-Owl Otus senegal- passes the eastern part of the Arabian Peninsula ensis, Sokoke Scops Owl Otus ireneae, Sandy extending east to western Pakistan and north to Scops Owl Otus icterorhynchus) and two insular southeastern Turkey and Central Asia (Marks et al. species (S~ao Tome Scops Owl Otus hartlaubi, 1999). This taxon’s treatment as a subspecies of Pemba Scops Owl Otus pembaensis) (Marks et al. O. senegalensis appears to have been influenced by 1999). the report ‘that the song on Socotra is identical to Fuchs et al. (2008) suggested that the rapid that in Kenya’ (A. Forbes-Watson in Marshall divergence following the initial colonization of the 1978). However, recently Konig€ et al. (2008) and Indian Ocean islands (Madagascar and the Como- Porter and Aspinall (2010) considered socotranus ros archipelago) led to distinctive genetic lineages, to represent a separate species. and this result has favoured the recognition of Similarly, scops owls from southern Arabia, cur- each insular Otus taxon as a distinct species rently recognized as Otus senegalensis pamelae, (Fig. 1). Intriguingly, molecular DNA sequence have also been suggested to be closely related to data suggest that several species have strikingly O. brucei (Marks et al. 1999), although Marshall different affinities than recognized under tradi- (1978) already noted that this taxon’s vocalizations tional hypotheses, suggesting that morphology and are ‘intermediate between the stutter of Otus sen- vocalizations are weak characters with which to egalensis senegalensis and the purr of O. sunia di- define phylogenetic relationships in scops owls. stans of Thailand.’ Furthermore, the systematic For example, O. pembaensis proved not to be con- affinities of O. brucei remain unclear. It has been specific with Madagascar Scops Owl Otus rutilus, treated either as a subspecies of Otus scops (Mein- contrary to previous hypotheses (Marks et al. ertzhagen 1948) or Otus sunia prior to vocal stud- 1999), but instead is closely related to mainland ies that supported its specific status (Marshall O. senegalensis (Fuchs et al. 2008). Likewise, the 1978, Roberts & King 1986). S~ao Tome endemic Otus hartlaubi was found to We here examine further the patterns of pheno- have strong affinities with O. senegalensis. Even typic differentiation among scops owl taxa, more surprisingly, the Seychelles Scops Owl Otus and discuss these with respect to phylogenetic

Afro-Palaearctic clade Indo-Malayan/Indian Ocean clade

Figure 1. Map depicting the geographical range of Otus taxa in the region of interest. Afro-Palaearctic clade: A1 = Otus senegalensis pamelae;A2= Otus scops;A3= Otus pembaensis;A4= Otus hartlaubi;A5= Otus senegalensis. Indo-Malayan/Indian Ocean clade: i1 = Otus brucei;i2= Otus moheliensis;i3= Otus pauliani;i4= Otus capnodes;i5= Otus mayottensis;i6= Otus rutilus; i7 = Otus madagascariensis;i8= Otus senegalensis socotranus;i9= Otus insularis; i10 = Otus sunia. relationship highlighted by our analyses of mito- Two mitochondrial markers, nicotinamide chondrial and nuclear sequence data. dehydrogenase subunits 2 and 3 (ND2, ND3), and two nuclear introns (myoglobin intron-2 (MB) and TGFb2 intron-5 (TGFb2)) were METHODS amplified via the polymerase chain reaction We collected blood samples for the Socotra Scops (PCR) and sequenced. ND2 and ND3 were Owl and used toe-pads from museum specimens PCR-amplified and sequenced using primer pairs held at the Natural History Museum, Tring, UK, L5219/H6313 (Sorenson et al. 1999) and for O. brucei, O. s. pamelae and O. s. senegalensis L10755/H11151 (Chesser 1999), respectively. collected in Ethiopia, Somalia and Kenya. In addi- The MB nuclear intron was PCR-amplified and tion, our taxonomic sampling was completed with sequenced using MYO2/MYO3F (Slade et al. several species included in Fuchs et al. (2008; 1993, Heslewood et al. 1998). TGFb2 was PCR- Table 1). amplified and sequenced using primer pairs TG5/ TG6 (Bures et al. 2002). The following internal primers were designed to amplify and sequence Laboratory procedures DNA obtained from the toe-pads: ND2intL1 5′- Total genomic DNA was extracted from blood pre- CATYAARTRYTTCCTAGTACAAGC-3′; ND2int served in EDTA using a CTAB-based protocol L2 5′-CAGCYATTGCAATAAAACTAGGAC-3′; (Winnepenninckx et al. 1993). DNA was extracted ND2intR1 5′-GGACAATGRGACATCACACAA from toe-pads using a DNeasy Tissue Kit (Qiagen -3′; ND2intR2 5′-CCTAAAYCAAACACAAATC Inc., Valencia, CA, USA) adding 20 lL of dith- CGA-3′; ND3intL 5′-CTVCCATTCTCARTMC- iothreitol (DTT; 0.1 M). Genomic extractions of GATTCTTC-3′; ND3intR 5′-CCTATTCCTYCTR toe-pads were performed separately from the DNA TTY GAYCTRG-3′;MYO2intL5′-TTATTTAC extractions of fresh tissues of Socotra Scops Owl. TGWTGGCTAGTTGG-3′;MYO2intR5′-GGTA Prior to each extraction, the workstation and all TGTGAATATCCAAGTTTAGA-3′. We obtained equipment were cleaned using a bleach solution and DNA sequences of 500 and 320 bp from toe-pad UV light was used to help prevent contamination. samples for ND2 and MB, respectively. To enable

Table 1. List of samples used and GenBank accession numbers for the four loci analysed. Taxonomy follows Marks et al. (1999). New sequences: (T) individuals for which Pons J.-M. the source of DNA was a toe-pad from a historical museum specimen; (B) individuals for which the source of DNA was blood.

Species Voucher/Tissue number Geographical origin Myoglobin TGFB2 ND2 ND3 tal. et Bubo bubo MNHN 24-55 France EU601069 EU600949 EU601029 EU600992 Otus capnodes MNHN 30-10J Anjouan EU601078 EU600957 EU601038 EU601000 Otus hartlaubi MNHH 32-04G Sao~ Tome EU601072 EU600952 EU601032 EU600995 ’ Union Otus insularis D. Currie 5H21863 Mahe, Seychelles EU601059 EU600940 EU601022 EU600983 Otus insularis D. Currie 5H21866 Mahe, Seychelles EU601060 EU600941 EU601023 EU600984 Otus ireneae MNHN 32-06J (M. Virani C30577) Kenya EU601077 EU600956 EU601037 EU600999 Otus lettia lettia MNHN JF142 Laos EU601073 EU600953 EU601033 EU600996 Otus lettia ussuriensis UWBM 75379 Russia EU601075 EU600955 EU601035 EU600998 Otus longicornis FMNH 433020 Luzon, Philippines EU601084 EU600952 EU601043 EU601005 Otus longicornis ZMUC 114206 Isabela, Philippines EU601063 No sequence EU6010526 EU600987 Otus mayottensis MNHN R22 Mayotte EU601087 EU600965 EU601046 EU601008 Otus megalotis FMNH 433019 Luzon, Philippines EU601083 EU600961 EU601041 EU601004 Otus megalotis ZMUC 114208 Isabela, Philippines EU601064 EU600944 EU601027 EU600988 Otus mirus FMNH 357429 Mindanao, Philippines EU601099 EU600978 EU601057 EU601020 Otus moheliensis MNHN E-135 Moheli EU601086 EU600964 EU601045 EU601007 Otus pauliani MNHN R24 Grande Comore EU601100 EU600979 EU601058 EU601021 Otus pembaensis MNHN uncatalogued Pemba Island EU601090 EU600967 EU601048 EU601010 Otus pembaensis MNHN uncatalogued Pemba Island EU601091 EU600968 EU601049 EU601011 Otus madagascariensis FMNH 393149 Madagascar EU601082 EU600960 EF198290 EU601003 Otus rutilus FMNH 431150 Madagascar EU601068 EU600948 EF198307 EU600991 Otus scops MNHN 23-5F France EU601079 EU600958 EU601039 EU601001 Otus senegalensis MVZ uncatalogued South Africa EU601098 EU600976, EU600977 EU601053 EU601019 Otus spilocephalus MNHN 15-58 China EU601080 EU600980 EU601040 No sequence Otus sunia MNHN 6-98 Thailand EU601081 EU600959 EU601041 EU601002 Strix aluco MNHN 24-27 France EU601070 EU600950 EU601030 EU600993 New sequences Otus brucei BMNH, 1979.2.22 (T) Oman KC138811 No sequence KC138817 KC138825 Otus brucei BMNH, 1979.11.06 (T) Oman KC138811 No sequence KC138818 KC138826 Otus s. pamelae BMNH, 1977.21.11 (T) Saudi Arabia KC138812 No sequence KC138819 KC138827 Otus s. pamelae BMNH, 1937.4.17 (T) Saudi Arabia KC138812 No sequence KC138820 KC138828 Otus s. senegalensis BMNH, 1912.12.23.44 (T) Orr Valley, Kenya KC138813 No sequence KC138821 KC138829 Otus s. senegalensis BMNH, 1965.M.5013 (T) North Somalia KC138814 No sequence KC138822 KC138830 Otus s. senegalensis BMNH, 1923.8.7.7553 (T) North Somalia KC138815 No sequence KC138822 KC138830 Otus s. senegalensis BMNH, 1887.11.11.44 (T) Abyssinia, Ethiopia No sequence No sequence KC138823 KC138831 Otus s. socotranus MNHN Uncatalogued (B) Mer Koh, Socotra Island KC138816 KC138810 KC138824 KC138832 Otus s. socotranus MNHN Uncatalogued (B) Mer Koh, Socotra Island KC138816 KC138810 KC138824 KC138832 Otus s. socotranus MNHN Uncatalogued (B) Mer Koh, Socotra Island KC138816 KC138810 KC138824 KC138832

BMNH, Natural History Museum, Tring; FMNH, Field Museum of Natural History, Chicago; MNHN, Museum National d’Histoire Naturelle, Paris; MVZ, Museum of Vertebrate Zoology, Berkeley; UWBM, University of Washington, Burke Museum, Seattle; ZMUC, Zoological Museum, University of Copenhagen. Systematics of Otus scops owls 5

detection of possible contamination, negative con- prior for estimation of the branch-lengths. In all trols were included during PCR-amplification of MRBAYES analyses, four Metropolis-coupled Markov DNA extracts, which utilized the Illustra Hot Start chain Monte Carlo runs, one cold and three Mix RTG (Buckinghamshire, UK). heated, were conducted for 20 million iterations each, with trees sampled every 1000 iterations. Two independent Bayesian runs initiated from ran- Determining the phase of alleles dom starting trees were performed for each dataset, We used PHASE V2.1.1 (Stephens et al. 2001, and the log-likelihood values and posterior proba- Stephens & Donnelly 2003) to infer the associa- bilities were checked to ascertain that the chains tion among heterozygous sites for each nuclear had reached stationarity. We ensured that the locus and individual. Three runs using different potential scale reduction factor (PSRF) approached seed values were performed and results were 1.0 for all parameters and that the average standard compared across runs. We used the recombina- deviation of split frequencies converged towards tion model and ran the iterations of the final zero. For the concatenated data, we use the con- run 10 times longer than for the other two runs. sensus sequences from the two alleles for the two The best haplotype estimate was used for the nuclear loci, using the IUPAC code for the hetero- phylogenetic analyses. zygous sites. The two independent Bayesian runs were combined to estimate the consensus topology and posterior probabilities of clades. Phylogenetic analyses We used TRACER v1.5 (Rambaut & Drummond Phylogenetic analyses including estimation of indi- 2007) to determine that our sampling of the pos- vidual gene trees and a concatenated approach terior distribution had reached a sufficient effective were conducted using maximum likelihood (ML) sample size (ESS > 200) for meaningful parameter and Bayesian inference (BI), as implemented in estimation. RAXML V7.0.4 (Stamatakis 2006, Stamatakis et al. 2008) and MRBAYES 3.1.2 (Ronquist & Huelsen- Genetic distance beck 2003), respectively. The most appropriate models of nucleotide substitution were determined Uncorrected-p genetic distances among taxa were using TOPALI v2.5 (Milne et al. 2009) and the calculated using the concatenated mitochondrial Bayesian information criterion (BIC). Maximum sequences (ND2 and ND3). According to evolu- likelihood and Bayesian analyses under the concate- tionary rates estimated from complete mitochon- nated approach were performed, allowing substitu- drial genomes for Hawaiian honeycreepers (Lerner tion model parameters to differ among loci et al. 2011), the ‘standard’ cytochrome-b gene (Nylander et al. 2004). For the gene tree analyses, evolves at a slower rate (0.014 substitutions per we ran several preliminary analyses by changing site per million years) than ND2 (0.029 s/s/my) the branch-length prior, from unconstrained:exp and ND3 (0.024 s/s/my) used in the present study (10) to unconstrained:exp(200), and the heating to calculate genetic distances among taxa. temperature from 0.2 to 0.1. Better mixing was always achieved using a temperature of 0.1 instead Morphological data of the 0.2 default value. The best-fit brlens prior was selected using the Bayes factor (BF; Nylander For museum specimens of O. senegalensis, O. s. et al. 2004). A value > 4.6 for lnBF was considered pamelae, O. s. socotranus and O. sunia (Supporting to be strong evidence against the simpler model Information Table S1), we obtained the following (Jeffreys 1961). For MRBAYES 3.1.2, we used biometric measurements: wing length (flattened) default priors for the base frequency and substitu- and tail length, using a standard metal wing-rule tions models. For the gene tree analyses, we with a perpendicular stop at zero (measured to observed better mixing of the chains and higher 0.5 mm), and culmen length (to skull), culmen likelihood using the unconstrained:exp(10) branch- width (at the feathers) and tarsus length (mea- length prior for the mtDNA data and uncon- sured from the tarsal joint to the last complete strained:exp(150) or unconstrained:exp(200) for scute before the toes diverge), using digital calli- nuclear data. Analyses of the concatenated data pers (to a precision of 0.01 mm). All measure- were performed using an unconstrained:exp(100) ments were taken by G.M.K.

All measurements were log-transformed to Total evidence normalize the distribution and to allow for The analyses of the concatenated dataset resulted allometry. However, morphological variables in a 50% majority consensus rule tree that was very were not size-standardized because size is an similar to the mtDNA topology. However, most important taxonomic criterion (Helbig 2000, nodes received better support values (Fig. 2). The Kaboli et al. 2007) and must be considered with African species Otus ireneae, endemic to the coastal shape in studies of morphological characters forests of Kenya and Tanzania, was found to be the (Mosimann & James 1979). Sexual dimorphism sister-taxon to all of the remaining Otus scops owls was tested using analysis of variance (ANOVA). (Fig. 2). The phylogenetic tree was then divided Principal component analyses (PCA) using corre- into two clades. One Indo-Malayan clade included lation matrices were performed with five biomet- Mountain Scops Owl Otus spilocephalus, Otus lettia ric variables to visualize patterns of covariation and Philippine Scops Owl Otus megalotis, whereas in a multivariate space. In addition, discriminant the other clade consisted of all the western Indian function analyses (DFA) were performed using a Ocean taxa as well as O. scops and all African priori defined groups. Groups included in each mainland and insular taxa. This clade can be DFA were chosen on the basis of our phyloge- divided further into two highly supported sub- netic results: O. s. socotranus was compared with clades, which comprised Indo-Malayan/Indian its closest relatives (O. sunia, O. insularis) and Ocean taxa and Afro-Palaearctic taxa (Fig. 2). O. s. pamelae was compared with five O. sen- The two Arabian taxa belonged to different egalensis populations from eastern Africa. All sta- clades: O. brucei was recovered with moderate tistical analyses were conducted using STATISTICA support to be the first offshoot in the Indo-Mala- version 6 (StatSoft Inc, Tulsa, OK, USA). yan/Indian Ocean (ML = 80, BI = 0.74), whereas O. s. pamelae grouped with the Afro-Palaearctic taxa in the second clade. Socotra Scops Owl Vocalization O. s. socotranus grouped with Oriental Scops We qualitatively compared sound recordings Owl and Seychelles Scops Owl (ML = 100, (n = 38) of O. senegalensis, O. s. pamelae and O. BI = 1.0), but the relationships among these insularis. Localities and sample sizes are pre- three species were not resolved. The latter clade sented in Supporting Information Table S2. was closely related to the other Indian Ocean Recordings were obtained from two online data- taxa (ML = 88, BI = 0.96). Two species endemic bases, Xeno-canto (XC; www.xeno-canto.org) to the Philippines (Luzon Scops Owl Otus longi- and the Cornell Lab of Ornithology Macaulay cornis and Mindanao Scops Owl Otus mirus) Library (ML; http://macaulaylibrary.com/), com- were the sister-group to this mostly Indo-Mala- mercially available recordings (Chappuis 2000), yan clade (ML = 100, BI = 1.0). The African from colleagues or from personal recordings. Scops Owl was not monophyletic because the Sonograms were created using WAVESURFER Arabian taxon O. s. pamelae was sister to the 1.8.8p4 (http://sourceforge.net/projects/wavesur- clade formed by O. scops, O. s. senegalensis, O. fer/) under the following parameters: FFT win- hartlaubi and O. pembaensis (ML = 99, BI = 1.0). dow length: 2048 points; window type: There was substantial mitochondrial genetic Hamming; analysis bandwidth: 57 Hz; and win- divergence among samples of O. s. senegalensis; dow length: 768 points. Vocalizations are com- the individual collected in Ethiopia differed by mented upon in the Discussion with respect to up to 3.2% from other O. s. senegalensis individ- phylogenetic relationships. uals included in our study.

mtDNA RESULTS The ﬁnal mtDNA data retained for the analyses included 30 individuals and 1392 bp (ND2: Phylogeny 1041 bp, ND3: 351 bp). Four individuals were The length of the alignment, number of individu- not included in the analyses because they had the als sequenced, DNA substitution models, likeli- same haplotype as another conspeciﬁc individual hood scores and clock model selected for each (the two Seychelles Scops Owls, the three Socotra individual locus are indicated in Table 2. Scops Owls and the two Pemba Scops Owls). The

Table 2. Length of the alignment and DNA substitution models selected for each individual locus.

Locus ND2 ND2 ND3 mtDNA MB TGFb2 Concatenated

Length (bp) 1041 500 351 1392 723 593 2708 No. of individuals (haplotypes) 34 (29) 34 (29) 34 (29) 34 (30) 33 (26) 27 (33) 32 Substitution model TrN+Γ HKY +Γ K81uf +Γ +I partitioned TrnEf K81 + Γ partitioned ML score À6208.53 À2958.01 À2066.58 À8276.17 À1459.86 À1474.96 À10943.43 BI harmonic mean À6256.55 À3019.23 À2138.1 À8351.17 À1514.25 À1565.65 À11098.72

Figure 2. Bayesian phylogenetic tree obtained with the concatenated data set (ND2, ND3, MB, TGFb2). Values close to nodes represent maximum likelihood bootstrap percentages (ML) and Bayesian posterior probabilities (PP). As outgroups, we used Eurasian Eagle Owl Bubo bubo and Tawny Owl Strix aluco (not shown on the ﬁgure). tree resulting from the concatenated mtDNA data Nuclear data had uneven support, with most of the relation- The two nuclear loci were phased prior to the ships among species of recent origin having low phylogenetic analyses and only unique alleles were support (BI = 0.50) whereas the backbone rela- retained for tree reconstruction. Sharing of allele tionships of the Otus clade often received strong sequences was widespread among closely related support (ML = 100, BI = 1.0) (Supporting Infor- species. Three alleles were shared among species mation Fig. S1). for MB, the ﬁrst being found among several

members of the Indo-Malayan/Indian Ocean clade Vocalization (O. insularis, O. mirus, O. rutilus, O. longicornis, Karthala Scops Owl O. pauliani and O. s. socotr- Otus s. socotranus anus). The second and third alleles were shared Although our sample size was of only three differ- among species of the Afro-Palaearctic clade ent recordings, G.M.K. and R.F.P. have considerable (O. pembaensis, O. s. pamelae and O. s. senegalen- field experience with O. s. socotranus and its vocal- sis; and O. scops and O. s. senegalensis, respec- izations (G.M.K. during 1 week of intensive general tively). For TGFb2, alleles were only shared avifaunal fieldwork in spring 1993 and R.F.P. during between O. rutilus and Mayotte Scops Owl Otus 11 visits to the island between 1993 and 2011, for a mayottensis. Overall, there were a greater number total of 6 months). R.F.P. has heard a total of 68 dif- of alleles for TGFb2 than for MB (33 vs. 26). ferent socotranus individuals singing as part of The tree resulting from the BI of the MB alleles focused fieldwork on the taxon designed to eluci- recovered the same five primary lineages high- date its general biology, natural history and popula- lighted by the mtDNA (O. irenenae, O. brucei, tion size. The song is a repeated, low-pitched woop Indo-Malayan/Indian Ocean clade, Afro-Palaearctic woop da-pwoorp…woop woop da-pwoorp…woop woop clade and Indo-Malayan clade) but relationships da-pwoorp, each phrase lasting c. 2 s with similar- among these five lineages were not supported. length intervals between them (Supporting Infor- Relationships within these clades formed a polyto- mation Audio File S1). The two woop notes are my (Supporting Information Fig. S2). much less audible, except at close range. This is very The TGFb2 tree exhibited greater structure. similar to at least some songs of O. sunia, but less so Otus ireneae diverged at the base of the Otus to O. senegalensis over most of mainland Africa. We clade (ML = 80, BI = 1.0) and members of the found no evidence of close vocal similarities ‘Indo-Malayan clade’ were paraphyletic with between O. s. socotranus and other populations of respect to the ‘Indo-Malayan/Indian Ocean’ and O. senegalensis, including those in Arabia. Across ‘Afro-Palaearctic’ clades. Members of the two lat- much of eastern and southern mainland Africa, sen- ter clades were recovered in a general polytomy egalensis gives a soft, purring trill, kjurrrr, lasting up (Supporting Information Fig. S3). Yet, as for to 1 s (but often just 0.5 s). Inter-phrase intervals are other loci, O. s. socotranus clustered with O. su- typically 4–8 s. Sonograms of typical songs are pre- nia and O. insularis (ML = 43, BI = 0.93) sented in Figure 3. whereas O. s. senegalensis clustered with O. pembaensis (ML = 75, BI = 0.96). Sequences from Otus pamelae O. s. pamelae and O. brucei could not be Southern Arabian O. s. pamelae differs from other obtained for this locus. populations of senegalensis. The sound is obviously The concatenation of the nuclear data using higher pitched than that of eastern and southern unphased sequences yielded a 50% majority rule African populations, each phrase lasts c. 1 s, and consensus tree that was very similar to the MB the notes are harsher sounding, with a more and TGFb2 gene trees. A major discrepancy noticeable tremolo. By contrast, inter-phrase inter- involved the relationships of Pallid Scops Owl, vals are similar to those in eastern and southern which clustered with Sokoke Scops Owl at the African populations. The sole recording available base of the Otus clade (ML = 65, PP = 0.89), a to us from the Republic of Somaliland appears to result that was not recovered in any of the sin- present a similar vocal type to those in southern gle gene tree analyses. We attribute this result to Arabia and is the focus of ongoing research (G. the joint effect of MB having limited variation to M. Kirwan & R. F. Porter unpubl. data). We do resolve its relationships and the fact that we not agree with Marshall (1978) that pamelae is could not sequence TGFb2 for O. brucei.The ‘intermediate’ between senegalensis and sunia in its three primary clades within Otus (Indo-Malayan, song; it is clearly similar to the former in most Afro-Palaearctic, Indian Ocean/Indo-Malayan) respects. were all recovered as monophyletic, although support was variable (ML = 58, BI = 0.93; Morphology ML = 62, BI = 0.94; and ML = 90, BI = 1.0, respectively), but no resolution was achieved We did not observe significant sexual dimorphism within each of the clades. in morphometric traits (ANOVA, all comparisons

Figure 3. Examples of sonograms from typical songs of Otus taxa: Otus senegalensis socotranus, Socotra Island, Yemen, March 1993 (recorder: Peter Davidson/Ornithological Society of the Middle East; Supporting Information Audio File S1); Otus insularis, near La Mission, Mahe, Seychelles, December 2008 (recorder: Daniel F. Lane) (XC26204); Otus sunia, Orullathany, near Thattekkad, Kerala, India, December 2009 (recorder: Yong Ding Li) (XC42319); Otus senegalensis pamelae, Ain Hamran, Oman, January 2010 (recorder: Herman van Oosten) (XC44491); Otus senegalensis, Awash National Park, Ethiopia, February 2011 (recorder: David Mar- ques) (XC82660); Otus senegalensis, South Luangwa National Park, Zambia, October 2007 (recorder: Derek Solomon) (XC43146); Otus brucei, Khatmat Milalah, Oman, January 2010 (recorder: Herman van Oosten) (XC44545); Otus scops,Hameenlinna,€ Mustila, Finland, June 2011 (recorder: Juha Honkala) (XC82047).

P > 0.05, results not shown) except for the bill factor loadings for each biometric variable and length of socotranus (F1,17 = 13.92, P = 0.0001). each PCA analysis. The ﬁrst two components All individuals were thus pooled to perform the (PC1, PC2) together accounted for a large amount multivariate analyses. Descriptive statistics for each of the variance (> 70%). taxon are shown in Table 3. Morphological divergence of O. s. socotranus. PC1 PCA explained 54.5% of the variation and described size Table 4 summarizes the eigenvalues, the propor- differences among taxa, each standardized biomet- tion of variance captured by the ﬁrst two PCs and ric variable, except bill width, being roughly equally

Table 4. Eigenvalues, percentage variance explained and factor loadings of morphometric traits for two principal components (PC1, PC2): (A) Otus senegalensis socotranus, Otus

Otus sunia, Otus insularis. (B) Otus senegalensis pamelae, Otus insularis senegalensis senegalensis from eastern Africa.

A – PC1 A – PC2 B – PC1 B – PC2

Eigenvalue 2.73 1.58 2.47 1.39 Variance explained 54.54% 31.54% 49.41% 27.94% Factor loadings Wing 0.88 À0.37 0.90 À0.13 Tail 0.82 À0.46 0.86 À0.34 Tarsus 0.84 0.20 0.79 À0.14 Bill length 0.74 0.59 0.51 0.68 Bill width 0.12 0.91 0.20 0.87

socotranus Otus sunia and positively correlated with PC1. PC2 captured a

Otus senegalensis comparatively minor amount of variation (31.5%) and described differences in shape among taxa, bill width having the highest factor loading. There was clear separation of PC1 and PC2 scores among socotranus, insularis and sunia individuals (ANOVA PC1, F2,22 = 101.46, P < 0.0001; ANOVA PC2, F2,22 = 31.28, P < 0.0001). Socotra Scops Owls had shorter wings and a shorter tail than O. insularis. In addition, PC2 clearly separated O. sunia from socotranus, which possessed a relatively larger and wider bill. PC3 (9% of the total variation, F2,22 = 0.17, P = 0.84) and PC4 (4% of the total variation, F2,22 = 0.29, P = 0.75) did not provide any clear separation among these three taxa. Morphological divergence of O. s. pamelae. As for the previous PCA, each standardized morphometric variable was positively correlated with PC1 and

Otus senegalensis senegalensis contributed roughly equally to PC1, which explained most of the variation (49.4%). PC2 cap- Afro-Palaearctic clade Indo-Malayan/Indian Ocean clade tured 27.94% of the variance and was positively correlated with bill length and bill width. Otus s. pamelae was partially separated from its African counterparts along PC1 (ANOVA PC1, F5,27 = 6.74, P = 0.0003). Most O. s. pamelae individuals tended to be larger in wing and tail length than their Afri- can counterparts. There was no clear separation of PC2 scores among scops owls whatever their geographical origin. PC3 and PC4, which explained 12 and 8% of the total variation, respectively, did not pamelae 7, 30.3 (1.9) 4, 22.7 (1.5) 3, 24.3 (1) 7, 24.5 (1.48) 6, 25 (2.1) 6, 25 (1.7) 13, 24.4 (2.3) 9, 25 (2) 3, 32.1 (0.7)

7, 137.6 (5.6) 5, 121.4 (2.9) 3, 130.3 (3.5) 7, 132.1 (6.8) 7,134.1 (4.8) 6, 131.7 (6.1) 21, 129.3 (3.8) 17,permit 145.7 (3.8) 3, 164.7 (5.9) further separation of O. s. pamelae from Arabian Peninsula Somalia Ethiopia Kenya Sudan Uganda Socotra India Seychelles Otus senegalensis African taxa (PC3, F5,27 = 1.66, P = 0.18; PC4, F5,27 = 1.38, P = 0.36). Descriptive statistics of morphological variables included in the multivariate analyses. Sample size, mean (sd). DFA Morphological divergence of O. s. socotranus (Fig. 4). length length Tail lengthTarsus 7, 60.2 (3.4) 5, 51.8 (0.4) 3, 57 (3) 7, 57.2 (4.1) 7, 60.1 (3.4) 6, 60.2 (4) 20, 54.1 (4.3) 17, 64.9 (3.3) 3, 77 (4.3) Wing Bill lengthBill width 7, 18.4 (1.4) 7, 7.6 (0.5) 5, 16.9 (0.6) 3, 17.6 5, (0.6) 7.5 (0.7) 7, 18.3 (0.8) 3, 7.4 (0.6) 7, 17.4 (1.2) 7, 8.3 (0.4) 6, 16.7 (1.3) 7, 7.8 (0.4) 21, 19.4 (0.9) 6, 7 (0.5) 16, 18.5 (1.1) 3, 13, 23.5 9.1 (1.3) (0.8) 16, 6.7 (0.7) 3, 9.9 (1.3) Table 3. A multivariate ANOVA was strongly signiﬁcant

(Wilks’ lambda = 0.011, F10,36 = 30.1, P < 0.0001) length than its African counterparts. The classified and fully separated the three taxa. The variables matrix derived from the DFA correctly classified that contributed most to the discrimination 100% of O. s. pamelae with high posterior proba- between groups are wing length (root 1: standard- bilities (PP > 0.99) for five individuals and low ized coefficient = 1.06) and bill length (root 2: stan- posterior probabilities for only two individuals dardized coefficient = À0.71). Socotran birds were (PP < 0.50). shorter in wing length than sunia and insularis, and had a wider and larger bill than sunia. The classified matrix derived from the DFA correctly classified DISCUSSION 100% of socotranus (n = 7) with high posterior probabilities (PP = 1.0). Species limits Morphological divergence of O. s. pamelae (Fig. 5). We recovered the same general topology as that of The first two discriminant functions explained Fuchs et al. (2008), which was expected given that 87% of the variability in the dataset. DFA signifi- the taxonomic and gene sampling was very similar, cantly differentiated biometric characteristics with only a few nodes showing less support. Otus among groups (MANOVA, Wilks’ lambda = 0.080, brucei was recovered with moderate support to F25,86 = 3.50, P < 0.0001). However, there was no occupy a basal position within the Indo-Malayan clear pattern of geographical structure among sub-clade, whereas all senegalensis taxa belonged to mainland African Scops Owls. In contrast, O. s. the Afro-Palaearctic sub-clade. pamelae was clearly separated from the other The Pallid Scops Owl is considered by some groups: O. s. pamelae is larger in wing and tarsus authorities to be a subspecies of either O. scops or

Figure 4. Discriminant function analysis performed on five biometric variables of 25 scops owls from Socotra (Otus senegalensis socotranus), the Seychelles (Otus insularis) and India (Otus sunia). The variables that most contributed to the discrimination between groups are wing length and bill width (standardized coefficients, respectively, 1.06 and À0.40, root 1) and bill length and bill width (standardized coefficients, respectively, À0.71 and À0.65, root 2).

Figure 5. Discriminant function analysis performed on five biometric variables of 33 scops owls from the Arabian Peninsula (Otus senegalensis pamelae) and eastern Africa (Otus senegalensis senegalensis). The variables that most contributed to the discrimination between groups are tarsus length and wing length (standardized coefficients, respectively, 1.02, and 0.89, root 1) and tail length and bill width (standardized coefficients, respectively, À0.77 and 0.61, root 2).

O. sunia, or regarded as distinct, forming a super- insularis and O. sunia, the Socotra Scops Owl forms species with O. senegalensis, O. scops and O. sunia a well-supported monophyletic group nested within (Marks et al. 1999). Our molecular results do not the Indo-Malayan/Indian Ocean clade. Socotra support such systematic arrangements among these Scops Owl vocalizations are reminiscent of O. su- four species. Indeed, the Pallid Scops Owl is not nia, in accordance with the close relationship to the closely related to either O. scops or O. senegalensis, latter taxon highlighted in our phylogenetic tree. but appears to be the most ancient lineage that Mitochondrial uncorrected-p distances from its diverged from the Indo-Malayan and Comorian sister species O. sunia and O. insularis are 4.7 and sub-clade (Fig. 1). Otus brucei is in widespread 3.5%, respectively, indicating that the two taxa are sympatry with O. scops without evidence of inter- clearly genetically distinct. These genetic distances breeding, and also exhibits unique vocal (for sono- based on ND2 and ND3 genes are of the same order grams see Rasmussen & Anderton 2005: 238) and of magnitude as genetic distances usually found in morphological characters (Shirihai 1993) that fully the cytochrome-b gene between sister-species of support specific taxonomic treatment according to raptors (Wink & Sauer-Gurth€ 2000). Socotra Scops the guidelines proposed by Helbig et al. (2002). Owl is clearly distinguishable morphologically from The Socotra Scops Owl is currently treated as a its closest relatives; it is significantly smaller than O. subspecies of O. senegalensis in most of the litera- sunia and O. insularis in wing and tail length. The ture (e.g. Marks et al. 1999, Dickinson 2003, Socotra Scops Owl is further differentiated from O. Clements 2007). This taxonomic treatment is not sunia with respect to its larger bill length and bill supported by our molecular results and our qualita- width. Otus senegalensis socotranus is definitively tive analyses of vocalization data. Together with O. monomorphic (occurring solely in a grey-brown

morph; G.M. Kirwan & R.F. Porter pers. obs.), rected-p mitochondrial distance = 3.2%) from unlike O. sunia, which possess two colour morphs other lineages found in eastern (northern Somalia (rufous and grey-brown) in most or all populations, and Kenya) Africa and in South Africa. We even for most or all insular subspecies, e.g. Otus included only three Ethiopian scops owls in our sunia japonicus (Japan) and Otus sunia leggei (Sri biometric analyses and these were not differenti- Lanka), as well as mainland taxa, e.g. O. s. sunia ated from their African counterparts. The level of (northern Indian subcontinent) and Otus s. stictono- phenotypic distinctness of the Ethiopian popula- tus (Far East Asia) (G.M. Kirwan pers. obs.; tion remains to be established with a larger sample Rasmussen & Anderton 2005, Konig€ et al. 2008). size. Furthermore, as our phylogenetic analyses Overall, our results suggest that this insular taxon only included one individual, collected in the late represents an independent evolutionary lineage. 19th century without precise data concerning its Socotra Scops Owl possesses several independent geographical provenance, we advocate additional diagnostic characters (sensu Helbig et al. 2002) that genetic studies prior to any taxonomic change. strongly favour its status as a species Otus socotr- The two Somaliland scops owls sampled were anus, supporting the treatment adopted by Konig€ genetically poorly differentiated from their Kenyan et al. (2008), Jennings (2010) and Porter and and South African counterparts. However, popula- Aspinall (2010). tions from the highlands of Somaliland are vocally Another taxon that deserves further attention is distinct from other eastern African populations O s. pamelae and its position within the Afro-Pal- and possess a similar song to the phylogenetically aearctic clade (ML = 99, BI = 1.0). This southern distantly related Arabian O. pamelae. In accor- Arabian taxon is highly divergent from African sen- dance with Fuchs et al. (2008) concerning the egalensis (uncorrected-p mitochondrial genetic dis- affinities of the Indian Ocean scops owls, the pres- tance = 4%), making O. senegalensis, as currently ent study affirmed that vocal differences among defined, polyphyletic. The song of pamelae is very Otus taxa do not always reflect evolutionary affini- different from that of O. scops and O. brucei but ties among lineages. more similar to that of O. senegalensis. It neverthe- less differs from the latter’s song in being higher Biogeographical aspects pitched, sounding ‘scratchier’ and having more prolonged notes; the song sounds two-parted, due That Otus socotranus is most closely related to to the much quieter first note (G.M. Kirwan & R. Indo-Malayan (O. sunia) and Seychelles (O. insu- F. Porter pers. obs., Konig€ et al. 2008). In terms of laris) Scops Owls might appear surprising given its biometrics, our results clearly suggest that pamelae geographical location in the Gulf of Aden, closest is longer winged and longer legged than mainland to the African mainland (Fig. 1). Although Socotra African populations of senegalensis. In comparison is very distant from other Indian Ocean islands, with populations of O. senegalensis in continental Socotra Scops Owls are more closely related to Africa, Arabian pamelae is distinguished in being other insular Otus taxa than to their Arabian or paler overall, with less distinct streaking over the African counterparts. Surprisingly, of all western underparts and a less obvious whitish line on the Indian Ocean Islands only Pemba Island, located scapulars (Konig€ et al. 2008). Such plumage char- 50 km off mainland Tanzania, was colonized by acteristics might result from adaptation to arid scops owls from Africa (Fuchs et al. 2008, this habitats, as has been demonstrated for other des- study). Having been above sea level consistently ert-dwelling species (Guillaumet et al. 2008). Our since 31 mya (Braithwaite 1987), Socotra has most study reveals that Arabian Scops Owls possess sev- probably served as a ‘stepping stone’ between eral diagnostic genetic and phenotypic characters. Africa and Arabia for several unrelated organisms We therefore consider the most appropriate taxo- (Wranik 2003). However, in the case of Otus, the nomic treatment is to recognize Arabian Scops Socotra archipelago represents the westernmost Owl as a species, Otus pamelae, and not as a sub- point reached by this Indo-Malayan clade, as it species of O. senegalensis as it was originally seemingly has never successfully colonized eastern described based solely on morphological data Africa. Furthermore, although usually considered (Bates 1937). part of the Afrotropical zoogeographical realm, Among O. s. senegalensis (sensu stricto), the Socotra lies close to this realm’s junction with the Ethiopian lineage is genetically distinct (uncor- Palaearctic and Oriental realms, making it unsur-

prising that flora and fauna representative of both work has also revealed the role played by the Red these regions occur on the archipelago (Cheung & Sea as a more or less effective biogeographical bar- DeVantier 2006, Porter & Kirwan 2010). Socotra’s rier. Our results clearly eliminate a possible African modern diversity is the result of complex origin of Arabian pamelae and underline the impor- biogeographical patterns in which vicariant as well tant biogeographical role of the Red Sea in prevent- as long-distance transoceanic dispersal events have ing any gene flow between African senegalensis and played a major role (Gomez-D ıaz et al. 2012). their Arabian counterparts. Given its long isolation from the Arabian Peninsula Our limited genetic data on the Ethiopian sen- and its ecological characteristics, the archipelago egalensis lineage imply a long period of genetic iso- supports high levels of endemism among several lation from other eastern and southern African zoological and botanical groups, leading to the Scops Owl populations. Additional studies are islands being often referred to as the ‘Galapagos of clearly needed to investigate relationships among the Indian Ocean’. For example, among reptiles, Otus scops owls from the Horn of Africa. How- 90% of species are endemic (Gomez-D ıaz et al. ever, our preliminary results fully agree with previ- 2012). ous observations that the Rift Valley strongly The finding that O. socotranus represents a impacted the phylogeographical structure of sev- distinctive species brings to 10 the number of eral species distributed in this region (e.g. Galerida avian taxa now recognized at the species level as larks, Guillaumet et al. 2008, Ostrich Struthio being endemic to the Socotran archipelago, camelus, Freitag & Robinson 1993, Lanius shrikes, thereby significantly enhancing its importance as Fuchs et al. 2011). The Horn of Africa is an an Endemic Bird Area (Stattersfield et al. 1998). important hotspot of avian endemism in Africa, However, further molecular work on endemic with several Endemic Bird Areas having been iden- Socotran birds is required to elucidate their phylo- tified (Stattersfield et al. 1998). Thirty bird species geography. Although most of these taxa appear to endemic to Ethiopia and Eritrea alone are known be Afrotropical in origin, the limited available data to date (Ash & Atkins 2009, Redman et al. 2009) are as yet largely inconclusive in determining this. and additional cryptic species probably remain to For example, Socotra Sunbird Nectarinia balfouri be identified in this part of Africa. lies outside both clades of Indian Ocean Nectari- niidae and taxon sampling was insufficiently We thank Mark Adams at the Natural History Museum, detailed to resolve its position and closest relatives Tring, for expediting the toe-pad samples of various scops owl populations. For access to relevant specimen (Warren et al. 2003). Furthermore, although the material at the same institution, G.M.K. is indebted to two endemic sparrows, Socotra Sparrow Passer insu- Robert Prys-Jones^ and Mark Adams; at the Smithsonian laris and Abd Al Kuri Sparrow Passer hemileucus Institution, National Museum of Natural History, in have been sampled (Ryan et al. 2010), most of Washington, DC, James P. Dean and Christina A. Geb- their presumed closest relatives in the Passer motit- hard afforded similar assistance, and at the Field ensis complex (Kirwan 2008) have not. On the Museum of Natural History, Chicago, G.M.K. was other hand, the endemic Socotra Buzzard Buteo so- assisted by John Bates, Mary Hennen and David Willard. Fieldwork by G.M.K. in Yemen and Socotra was under- cotranus does indeed appear to be most closely taken within the framework of the 1993 Ornithological related to Afrotropical Buteo (Riesing et al. 2003, Society of the Middle East (OSME) South Yemen expe- Porter & Kirwan 2010). dition, while his museum studies in North America were The two Arabian taxa, O. brucei and O. pamelae, partly supported by a grant from OSME and further were the first taxa to diverge within their respective enabled by a scholarship award from the Field Museum Indo-Malayan/Indian Ocean and Afro-Palaearctic of Natural History (Chicago). Additional vocal material clades. This unexpected finding advocates the need was kindly supplied by Nik Borrow, Peter Davidson, Jon Hornbuckle, Michael Mills and Dave Showler. Willem- for fresh studies to understand the role played by Pier Vellinga (of Xeno-canto) very kindly assisted in pre- the Arabian Peninsula in the evolution of Otus lin- paring the sonograms for this paper. We are also grateful eages, and birds in general. to Celine Bonillo and to the technical staff of the ‘Ser- Many studies of mammalian and reptilian taxa vice de Systematique Moleculaire’ for helping with labo- with disjunct distributions in the Horn of Africa and ratory work. We thank Daniel Pons who helped to draw southwest Arabia have demonstrated that coloniza- Figures 1 and 3 and two anonymous referees, Gary tion most frequently occurred from Africa into the Voelker and Rauri Bowie for helpful comments on an earlier version of the manuscript. This project was Arabian Peninsula (Portik & Papenfuss 2012). Such

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