Current Status of the Northern Goshawk Accipiter Gentilis in Japan Based on Mitochondrial DNA

Ornithol Sci 7: 143–156 (2008)

ORIGINAL ARTICLE Current status of the Northern Goshawk Accipiter gentilis in Japan based on mitochondrial DNA

Shigeki ASAI1,#, Daisuke AKOSHIMA2, Yoshihiro YAMAMOTO3, Yoshimitsu SHIGETA1, Masahiko MATSUE4 and Hiroshi MOMOSE4,*

1 Yamashina Institute for Ornithology, 115 Konoyama, Abiko, Chiba 270–1145, Japan 2 Laboratory of Wild Animals, Department of Applied Biophilia, Faculty of Agriculture, Tokyo University of Agriculture, 1737 Funako, Atsugi, Kanagawa 243–0034, Japan 3 Department of Genetics, Hyogo College of Medicine, 1–1 Mukogawa, Nishinomiya, Hyogo 663–8501, Japan 4 Landscape and Ecology Division, Environment Department, National Institute for Land and Infrastructure Management, 1 Asahi, Tsukuba, Ibaraki 305–0804, Japan

Abstract Although the Northern Goshawk Accipiter gentilis is not designated a ORNITHOLOGICAL threatened species in Japan, it is thought that its population once experienced a de- SCIENCE crease. To evaluate the current status of the goshawk, we sequenced the mitochondrial © The Ornithological Society DNA control region and determined its variation among individuals. Considering that of Japan 2008 part of the Japanese population migrates or moves seasonally, we divided the samples into two categories (breeding and non-breeding season, based on sampling dates) and then calculated indices of genetic diversity and statistics for each category. Among 145 samples, we found ten haplotypes, of which two were dominant in both frequency and range. Haplotype diversity and nucleotide diversity were 0.630.04SD and 0.00180.0014SD, respectively. Comparing this diversity with those of other species, we concluded that the status of the Northern Goshawk in Japan is neither urgent nor secure. Signiﬁcant genetic distance was not detected between the breeding and non-breeding groups, thus we could ﬁnd no evidence of seasonal movement. The long-term effective female population size was estimated at 3,000 to 30,000 individuals. A recent population decline was not detected from the mismatch distribution. Therefore, the past population decline of the goshawk may not have been very serious. Future studies should consider the genetic structure of goshawks that inhabit other areas near Japan.

Key words Accipiter gentilis, Control region, Genetic diversity, Goshawk, Mito- chondrial DNA

The Northern Goshawk Accipiter gentilis was des- result of urbanization of this species, or true growth ignated a threatened species in the Red Data Book of (e.g. Research Division, Wild Bird Society of Japan Japan (Ministry of the Environment 2002). However, 1984; Ohba 1988; Endo 1989; Koita et al. 1997; the government of Japan eliminated this species from Kawakami & Higuchi 2003). It is unknown to what the threatened species list in 2006, because the popu- extent the goshawk declined in the past, due to a lack lation size was estimated to be sufficiently larger than of comparable data on population sizes. Nevertheless, it was two or three decades ago. The growth in the many researchers believe that this species was rare in estimated population size could result from previous the past (Ohba 1988; Endo 1989; Koita et al. 1997). underestimations due to insufficient questionnaire In general, it is assumed that genetic diversity in a surveys, recent high incidences of observation as a diminished population of a threatened species is low due to genetic drift. A decline in genetic diversity (Received 18 June 2008; Accepted 29 October 2008) would reduce the adaptability of a population to envi- # Corresponding author, E-mail: [email protected] ronmental fluctuation (Primack & Kobori 1997; * Present Address: Eastern Region Research Subteam for Wildlife Management, National Agricultural Research Center, 3–1–1 Frankham et al. 2002). If the Goshawk had under- Kannondai, Tsukuba, Ibaraki 305–8666, Japan gone a population decline with reduced genetic diver-

143 S. ASAI et al. sity, it could still be considered threatened. Therefore, variation (Baker & Marshall 1997). A primer set to it is necessary to determine genetic diversity to assess amplify the CR is a prerequisite for genetic studies, the true current status of this species. Our aim is to and thus we ﬁrst had to sequence a region of mtDNA document the genetic structure of the goshawk in containing the CR in a preliminary experiment to de- Japan, based on variation among mitochondrial DNA sign the primers (unlike the cytochrome b region, for sequences. which universal primers can be used). We determined In Japan, the goshawk is a resident of Hokkaido, the complete DNA sequence of the mitochondrial Honshu, Shikoku, and Kyushu (Committee for genome. Check-list of Japanese Birds 2000). However, in the Total cellular DNA was extracted from a tissue southwestern part of the range, breeders are uncom- sample of a goshawk preserved at the Yamashina In- mon while wintering birds are common (Morioka et stitute for Ornithology (Accession ID: 1999-0198). al. 1998; Committee for Check-list of Japanese Birds The complete 18,266-bp nucleotide sequence (DDBJ 2000). In addition, this species is observed annually Accession No. AP010797) was determined using de- at Cape Irago, a famous stopover point over which scribed procedures (Yamamoto et al. 2000). The PCR raptors migrate (e.g. Tsuji 1988; Kawakami & primers AG50 (5-GGC GGT TGC TAT GAG GGT Higuchi 2003). Kudo (2008) reported that two satel- TAG AAG GAG AAT GAT GC-3) and AG30 (5- lite-tracked individuals migrated from Hokkaido to CAG AAT GAT ATT TCC TAT TCG CAT ACG Honshu. In Europe and North America, Northern CTA TCC TAC-3) were made from the cytochrome Goshawks move southward in winter, even if they are b sequences of a goshawk previously registered in the not always migratory (Cramp & Simmons 1979; DDBJ (Accession No. X86738), in order to amplify Kenward 2006). Some goshawks that breed in Utah the rest of the mitochondrial genome. The total se- migrate there from 100–613 km away (Sonsthagen et quence was then determined using the M13 shotgun al. 2006), whereas other birds are considered resi- method. dents (Underwood et al. 2006). From the data avail- In contrast to the gene order of Gallus gallus (Oki- able it seems that at least part of the goshawk popula- moto et al. unpublished), the CR of the goshawk is tion in Japan moves seasonally. located between tRNA-thr and tRNA-pro. At the po- Genetic structure may reveal seasonal movements. sition where the CR is found in many other birds, the For example, if haplotypes found in the breeding sea- goshawk has a non-coding region (pseudo-CR) be- son are replaced by others in the non-breeding sea- tween tRNA-glu and tRNA-phe, as in other raptors son, it is plausible that breeders emigrate and individ- including Common Buzzard Buteo buteo (Haring et uals breeding elsewhere immigrate. Regarding ge- al. 1999), Peregrine Falcon Falco peregrinus (Min- netic diversity, we must also discriminate wintering dell et al. 1998), and Mountain Hawk Eagle Spizaetus birds; not doing so could lead to overestimates of ge- nipalensis (Asai et al. 2006). netic diversity of the original Japanese population. We discuss the seasonal movement of the goshawk 2) DNA samples and determination of haplo- based on sampling dates and the phylogeny of each types haplotype. Samples were mainly collected from four sources: If the Japanese goshawk experienced a relatively 29 blood samples collected by the National Institute recent bottleneck, this should be evident in its genetic for Land and Infrastructure Management from structure. We also discuss a recent demographic event Ichikai-machi, Tochigi Prefecture, between 1999 and in the Japanese population, by calculating the effec- 2001 (two of them were considered as one sample be- tive population size and depicting the frequency of cause the two individuals were deemed to be broth- pairwise sequence differences. ers); 37 feathers collected by the River Bureau, the Ministry of Land, Infrastructure, Transport, and MATERIALS AND METHODS Tourism, and the Incorporated Administrative Agency, Japan Water Agency, between 2000 and 1) Determination of the complete mtDNA se- 2006; tissues from carcasses deposited at the Ya- quence mashina Institute for Ornithology; and blood samples Due to its high variability, the control region (CR) from injured birds at conservation centers. Because of mitochondrial DNA (mtDNA) is thought to be some feathers collected at the same time resulted in most appropriate for detecting intraspeciﬁc genetic the same haplotype, we considered them to be from

144 Status of Goshawk based on mtDNA the same individual, and thus dealt with them as one wood et al. 2006). Therefore, it is reasonable to as- sample. Excluding samples whose collecting sites sume that samples collected from March to July rep- and dates were unknown, we analyzed a total of 145 resent the breeding distribution in Honshu. We then samples. calculated indices of genetic diversity and statistics We extracted DNA from the samples using a for each category. We considered samples from Sep- DNeasy Tissue Kit (QIAGEN) according to the man- tember in Hokkaio to represent breeding distribution ufacturer’s guidelines. We designed a primer set (Ag- (no samples were collected during either March or ContRegFa 5- TCT TCC CAC TAA CCG GAG April in Hokkaido). We considered three categories CCC -3 and AgContRegRa 5- CCT GAA GCT of breeding season: long (February to July), standard GGG AAC GTA GGG -3) to amplify a 625-bp frag- (March to July), and short (April to July). ment containing a highly variable part (domain I; de- fined as a 448-bp region from the 5-end of the CR to 4) Haplotype analysis the 5-adjacent site of the F box, a conserved se- To construct phylogenetic trees, appropriate substi- quence block in the CR; see Baker & Marshall 1997), tution models for the datasets were determined based based on the complete mtDNA sequence. PCR was on the Akaike Information Criterion (AIC; Akaike conducted following standard procedures using Ex 1974) calculated using MrModeltest2 (Nylander Taq (TaKaRa) at an annealing temperature of 62°C. 2004) in conjunction with PAUP* (Swofford 2003). PCR products were separated on 1% agarose gels The selected model for CR was the HKY model (SeaKem LE agarose by BMA) and then purified by (Hasegawa et al. 1985) with an estimated proportion the phenol-chloroform procedure or using a minicol- of invariant sites (I; Gu et al. 1995) (HKYI). Then, umn kit (Wizard® SV Gel and PCR Clean-up System, we constructed phylogenetic trees using the neighbor- Promega). Purified PCR products were sequenced joining (NJ) method, maximum parsimony (MP), and using a BigDye Terminator (Applied Biosystems) maximum likelihood (ML) using PAUP*. The phylo- with either the AgContRegFa or AgContRegRa genetic trees were rooted with eight CR haplotypes of primer following the manufacturer’s recommenda- goshawks captured in Utah, United States (Sonstha- tions. Sequence data were read with a 3100 Genetic gen et al. 2004). Branch supports were assessed by Analyzer (Applied Biosystems). bootstrapping with 1,000 replicates. The domain I region was trimmed from the se- Gene diversity (h; Nei 1973) and nucleotide diver- quence data. Multiple alignments of CR domain I se- sity (p; Nei & Tajima 1981) were calculated using quence were performed using GENETYX to deter- Arlequin 2.001 (Schneider et al. 2000), to examine mine the haplotype of each sample. the genetic diversity of the current population. To examine the difference in genetic structure be- 3) Breeding season and annual movement tween breeding and non-breeding seasons, we calcu-

In Tochgi Prefecture, central Japan, goshawks lated the pairwise FST (Wright 1969) using the fol- begin building nests in early February at the earliest lowing formula (Nei 1987): (Endo et al. 1987), and in Kyoto Prefecture, western HS Japan, one pair began nesting in early March (the FST 1, same pair had been observed in the territory in Febru- HT ary; Ezaki et al. 2000). The chicks ﬂedged during late where HT is the heterozygosity over all seasons, and June to early July in Tochigi (Endo et al. 1987), and HS is the average heterozygosity of each season. The in late July in Kyoto (Ezaki et al. 2000). A radio- signiﬁcance of FST was examined by randomization tracking study revealed that two adults and three ju- tests that generated 1,000 randomly re-ordered data veniles left the breeding site around one month after sets, holding sample sizes constant at the observed

fledging, i.e., in early to mid-August (Ueta et al. values. For the same purpose, we also calculated F ST 2006). In Hokkaido, two satellite-tracked individuals (Excoffier et al. 1992) including mutation steps be- left on 30 September and 20 October and then re- tween haplotypes, and tested significance with the turned to their former nesting sites on 26 March and randomization tests using Arlequin. 9 April (Kudo 2008). In Utah, United States, at al- To determine whether the population experienced a most the same latitude as Japan, migratory individu- bottleneck in the recent past, we calculated the effec- als leave their breeding sites from August to Decem- tive population size. Wilson et al. (1985) suggested ber, and then return in February and March (Under- that the number of generations that have passed since

145 S. ASAI et al. two randomly selected genes had a common ancestor sudden expansion model was tested using Arlequin is expected to approximate the long-term effective (Schneider & Excoffier 1999). A distribution that population size. We estimated the long-term effective does not fit the model would suggest that some his- female population size, Nf , from the following equa- torical events may have interfered with simple popu- tion (Wilson et al. 1985): lation expansion. 6 Nf 10 p/sg , RESULTS AND DISCUSSION where p is the mean pairwise difference (%), equal to the percent of nucleotide diversity, p; s is the diver- 1) Overview of haplotype frequency and phylo- gence rate (%/genome/Myr); and g is the generation genetic analyses time (years). We adopted 20.8% as the divergence We found ten haplotypes with nine polymorphic rate (s) of the CR domain I (Quinn 1992). For the sites from a total of 145 samples (Table 1). The maxi- same purpose, however, Moum and Árnason (2001) mum of pairwise differences among haplotypes was adopted 3.3%/Myr, based on the ratio of substitution five substitutions (Table 1). Over all samples, h and p rates between the CR domain I and cytochrome b were 0.590.02SD and 0.00170.0014SD, respec- among Razorbill Alca torda and Common Guillemot tively (Table 2). Uria aalge, assuming a divergence rate of cy- Three phylogenetic trees using NJ, MP and ML tochrome b of 2.0%/Myr. The divergence rate may be different between avian taxa (Crochet & Desmarais Table 1. Sequence differences and frequencies of ten haplo- 2000). In the same manner as Moum and Árnason types in the mtDNA control region of the Northern Goshawk. (2001), we then adopted 3.1%/Myr as the divergence rate (s) of the CR domain I of the Accipitridae, based Site position on a comparison between Spizaetus nipalensis and S. Haplotype N alboniger (Yamamoto & Asai unpublished data). 88394182 232 253 290 291 309 Other studies have shown that juveniles do not breed, I—CCTCTCGC62 or only do so occasionally (Kenward 2006). There- II ·····C···69 fore, we adopted g2. III G · · · ·C···2 To examine demographical changes in the IV G ········2 goshawk, we calculated the frequency of pairwise V·T···C···2 sequence differences (mismatch distribution). If the VI ····TC··T4 population had consistently expanded since arriving VII ···C·C·A·1 VIII ······T··1 on the Japan mainland, the mismatch distribution IX G ·····T··1 would be expected to fit the sudden expansion model X··T··C···1 (Rogers & Harpending 1992). Goodness of fit for the

Table 2. Statistics calculated from samples categorized according to sampling month (see text for deﬁnitions). One sample with haplotype VII was excluded from the calculations except for the category of ‘all seasons,’ because it was clear that the sample was derived from an accidental visitor (see text for details). FST and F ST were calculated for three pairs: Feb–Jul vs. Aug–Jan, Mar–Jul vs. Aug–Feb, and Apr–Jul vs. Aug–Mar. No match-ups were signiﬁcantly different. Nf s were calculated with two divergence rates (s) (see text for details).

N No. of f Category N h p F F haplotypes ST ST s0.208 s0.031

All seasons 145 10 0.59330.0222SD 0.0017200.001402SD 4,135 27,742 Feb–Jul 89 9 0.62820.0311SD 0.0018990.001505SD 0.01198 0.00766 4,565 30,629 Aug–Jan 55 3 0.52660.0218SD 0.0012990.001180SD 3,123 20,952 Mar–Jul 66 8 0.63260.0355SD 0.0017560.001434SD 0.00115 -0.00723 4,221 28,323 Aug–Feb 78 6 0.55110.0273SD 0.0014050.001234SD 3,377 22,661 Apr–Jul 51 6 0.61730.0399SD 0.0018210.001475SD 0.00701 0.00753 4,377 29,371 Aug–Mar 93 7 0.57150.0278SD 0.0015870.001334SD 3,815 25,597

146 Status of Goshawk based on mtDNA

Fig. 1. Phylogenetic tree using the maximum likelihood (ML) method with the HKYI model. Bootstrap values (50) of NJ (left), maximum parsimony (MP; middle), and maximum likelihood (ML; right) analyses are indicated. The outgroup was derived from data of Northern Goshawks in Utah (Sonsthagen et al. 2004). were identical (Fig. 1). One lineage of haplotype VII was discriminated from the others, with a bootstrap consensus of more than 50%. In addition, a cluster consisting of haplotypes I, IV, VIII, and IX was found (Fig. 1). Haplotype I (43%) and II (48%) were dominant in frequency and range (Figs. 2, 3). These two haplotypes were found almost evenly throughout the seasons, whereas the other haplotypes were found in only one to four samples (Fig. 2). Fig. 2. Frequency of each haplotype according to month. 2) Minor haplotypes and sampling dates Haplotype numbers are listed in the legend on the right of the Only one individual with haplotype VII was pres- graph, whereas the numbers of haplotypes found in a single ent on Haha-jima, Ogasawara Islands, on 13 March sample are indicated to the right of each bar in the graph. 1998, and died later (Accession ID: 1998-0042). In the Ogasawara Islands, the goshawk has been should be considered an accidental visitor. In addi- recorded as a passing migrant or an irregular visitor, tion, phylogenetic analysis suggested that this indi- never as a breeder (Momiyama 1930; Hasuo 1969; vidual with haplotype VII had either immigrated Takano et al. 1970; Bureau of Environmental Pollu- from outside Japan, or been imported to Japan and tion, Tokyo Metropolitan Government 1975; then released. Hasegawa 1977; Senba 1977; Nakane et al. 1980; Samples from a radio-tracking study in Tochigi Chiba & Funazu 1991). Therefore, this individual Prefecture (Ueta et al. 2006) found haplotypes II and

147 S. ASAI et al.

III. Haplotypes IX and X were found only in March compared genetic structure between the breeding and and July, respectively. Haplotypes IV, V, and VIII, non-breeding seasons, both FST and F ST did not differ sampled in February, may have been migrants which signiﬁcantly (Table 2). Although these data seem to had bred further north (Fig. 2). However, haplotypes suggest that the goshawk is not migratory in Japan, IV and V were also sampled from March to July (Fig. we cannot reject the existence of migratory birds, be- 2), and haplotypes IV and VIII were clustered in the cause we could not reject the possibility that a north- phylogenetic analysis (Fig. 1). These data suggest ern population outside Japan with the same haplo- that the Japanese population retained haplotypes I, II, types as those that we found, moves southward to III, IV, V, VIII, IX, and X in the frequencies shown in winter in Japan. In addition, no genetic variation be- Figure 2. tween northern and southern groups in Japan would One individual with haplotype VI was sampled in lead to the same consequence for small-scale migra- Chiba, central Japan, in January, i.e., during the win- tion within Japan. Takaki et al. (2008) indicated that tering season, and two were sampled in Hokkaido in genetic variation was very small between Hokkaido September when migratory birds are thought to move (northern group) and Kanto (south group), using mi- southward. These results imply that the northern pop- crosatellite DNA polymorphism. If haplotype VI had ulation winters in central Japan, although this haplo- been derived from wintering birds, it would have type was also sampled from Chiba in March. Accord- been detected in the northern population. Further ing to Kudo (2008), two individuals, which bred in studies are necessary to reveal the genetic structures Hokkaido and stayed there until September, migrated of neighboring populations, especially in China and to winter in Honshu. We were unable to ﬁnd evidence Russia. of a population breeding outside Japan and wintering in Japan. The distribution of haplotype VI was not 4) Genetic diversity substantially different from those of haplotypes I and In samples collected from March to July, h and p II. were 0.630.04SD and 0.00180.0014SD, respectively (Table 2). The indices of genetic diversity did 3) Seasonal movement not substantially differ among the three categories of Haplotypes I and II were sampled from a large area breeding season (i.e., long, standard, or short; see during all seasons, with no seasonal bias (Figs. 2, 3). Table 2). Comparing the h value to those of large en- The numbers of other haplotypes were too small to dangered birds (Bonelli’s Eagle Hieraaetus fasciatus, detect any seasonal patterns. In addition, although we Crested Ibis Nipponia nippon, and Spanish Imperial

Fig. 3. Sampling sites of haplotype I (left), haplotype II (middle), and the other haplotypes (right). In the left and middle ﬁgures, open triangles and open circles indicate the sampling sites from March to July (breeding season) and from August to February (non-breeding season), respectively. In the right ﬁgure, an open triangle, two closed triangles, a closed square, four open circles, and an open square indicate the sampling sites of haplotypes III, IV, V, VI, and X, respectively. Haplotypes III, V, VIII, and IX were found at the sampling site indicated by a star. Haplotype VII was found in the far south in the Ogasawara (Bonin) Islands (not shown).

148 Status of Goshawk based on mtDNA Source n, although the region did not al- N lues using the information in cited paper. 0.0017SD 72 Cadahía et al. (2007) 0.0007SD0.00013SD0.019284SD0.00068SD 57 360.00055SD 250.001823SD 34 Moum & Árnason (2001) Zhang et al. (2004) 33 et al. (1994) Wenink 49 Martínez-Cruz et al. (2004) Jiang et al. (2007) Sonsthagen et al. (2004) 0.0015SD 22 Moum & Árnason (2001) 0.00413SD 68 Asai et al. (2006) 0.003021SD0.0017SD0.0012SD 20 42 81 Murata et al. (2004) Moum & Árnason (2001) Moum & Árnason (2001) p 0.005061SD0.0015SD0.00024SD 290.006210SD 25 60 Hasegawa et al. (1999) 25 Solórzano et al. (2004) Martínez-Cruz et al. (2004) et al. (1994) Wenink 0.11SD 0.0054 0.046SD 0.0024 0.07SD0.074SD0.0362SD0.0420SD0.010SD 0.0048 0.00069 0.036779 0.0561SD 0.00548 0.00616 0.002465 0.013SD 0.00741 0.1076SD0.02SD0.03SD 0.005489 0.0173 0.0097 0.0704SD0.0730SD 0.008724 0.0231SD 0.00098 0.010388 h 0.00470SD 0.0171 0.87 0.0026 10 Marshall & Baker (1997) 0.89 0.129 42 Marshall & Baker (1997) 0.612 0.134 190 Merilä et al. (1997) 0.69 0.0039 25 Baker et al. (1994) 0.61 0.0032 105 Roques & Negro (2005) 0.71 0.72 0.95 0.88 0.542 0.386 0.992 0.935 0.9333 0.7790 0.7185 0.6947 0.7808 0.3215 0.9567 0.8180 Fringilla montifringilla Hieraaetus fasciatus Nippoina nippon Calidris alpina Aquila heliaca Syrmaticus ellioti Accipiter gentilis chloris Carduelis Uria aalge Fringilla coelebs Spizaetus nipalensis Calidris canutus Uria aalge Ciconia boycinia Alca torda Alca torda Milvus milvus Grus japonensis mocinno Pharomachrus Aquila adalberti interpres Arenaria Genetic diversity of the mtDNA control region other species. These values were based on a part or whole regio Species Scientific name Brambling Bonelli’s Eagle Bonelli’s Crested Ibis Dunlin* Eastern Imperial Eagle Pheasant Elliot’s Northern Goshawk* European Greenfinch Common Guillemot Common Chaffinch Hodgson’s Hawk-eagle Hodgson’s Red Knot Common Guillemot Oriental Stork* Razorbill Razorbill Red Kite Red-crowned Crane* Resplendent Quetzal Spanish Imperial Eagle Ruddy Turnstone able 3. T ways correspond to the domain I region, which we referred to in this paper. In species with an asterisk, we recalculated the va ways correspond to the domain I region, which we referred in this paper.

149 S. ASAI et al.

Eagle Aquila adalberti; Table 3), the goshawk should not be considered imminently endangered. When we recalculated samples from Utah (Son- sthagen et al. 2004), h and p were 0.720.06SD and 0.00250.0018SD (N49), respectively. The haplotype diversity (h) of the Japanese population was slightly lower than that of the population in Utah, where the goshawk is not considered threatened (Boyce et al. 2006). It is remarkable that the h and p values of the last Japanese population of Oriental Stork (Murata et al. 2004), which is now extinct, are no lower than those of the current goshawk population, even though it is unclear whether the decline of genetic diversity affected the extinction. Also, the h values of the non-endangered species European Greenfinch Carduelis chloris (Merilä et al. 1997), Fig. 4. Mismatch distribution depicted by samples collected Red Knot Calidris canutus (Baker et al. 1994), and from March to July. The observed frequency was not signifi- Red Kite Milvus milvus (whose population has de- cantly different in the sudden expansion model. clined because of human persecution; Roques & Negro 2005) are as low as that of the Japanese bution of the minor haplotypes, the majority of goshawk population (Table 3). Thus, from the per- goshawks might have survived in central Japan spective of genetic diversity, the status of the (Kanto District). If the recent recovery of goshawks goshawk in Japan is neither urgent nor secure. is derived from dispersal from the Kanto District, however, this would not explain the selective expan- 5) Did the Northern Goshawk in Japan undergo sion of haplotypes I and II. Alternatively, a popula- a population decline? tion outside Japan might have included haplotypes I The long-term effective female population size and II, and individuals from that population might ranged from 3,000 to 30,000 (Table 2). In many have recently immigrated to Japan. This remains cases, the real number of individuals is approxi- speculative until the genetic structures of surrounding mately ten times larger than the estimated effective populations can be studied. However, the possibility population size (Frankham et al. 2002). Therefore, is supported by a preliminary experiment with one the results of a previous questionnaire survey of some sample collected from Mongolia, which was found to hundreds to thousands of individuals (Research Divi- have haplotype II (Asai et al. unpublished data). sion, Wild Bird Society of Japan 1984; Koita et al. Takaki et al. (2008) indicated that gene flow occurred 1997) evidently underestimated the population size. between Japan and Central Asia, using variation of The null hypothesis that the mismatch distribution microsatellites. fits the sudden expansion model was not rejected (Sum of squared devistion 0.00929, P 0.11; Fig. ACKNOWLEDGMENTS 4). Therefore, since a few founders reached Japan, the goshawk may have expanded consistently. Be- This study would have been impossible without so many cause parameter t on the adopted model was 0.943, samples to analyze. Therefore, we would like to express our the time of the expansion was calculated to be 10,120 gratitude to the River Bureau, the Ministry of Land, Infrastruc- ture, and Transport, and the Incorporated Administrative years for a divergence rate of 0.208, or 67,900 years Agency Japan Water Agency, for the samples they provided. for a divergence rate of 0.031 (see Rogers & Harp- We also greatly appreciate the help of H. Asuka, T. Ichikita, F. ending 1992). Although these values may indicate the IIzuka, H. Inada, Y. Kashiwagi, T. Kobayashi, S. Kuroda, T. period when the goshawk arrived in to Japan, no re- Mano, Y. Matsumaru, H. Murata, N. Nishi, N. Onozawa, H. cent population decline was detected from the mis- Sakai, Y. Shimazaki, H. Suzuki, K. Takenami, M. Tanaka, T. match distribution. Even if the population decline did Tomioka, T. Umeyama, and T. Yasuda and Dr. Gombobaatar in fact occur, however, it could not have been very Sundev; and the following organizations and offices: Ecosys- tem Conservation Society Saitama, Environmental Protection serious, because the effective female population size Division, Shizuoka Prefecture, Forest Conservation Division, was rather large. Considering the geographical distri- Kyoto Prefecture, Forestry Division, Nishi-Tama Economic

150 Status of Goshawk based on mtDNA

Office, Tokyo Metropolitan, Gyotoku Bird Watching Cottage, Tokyo Metropolitan University (In Japanese). Green and Nature Division, Saitama Prefecture, Ikeya Animal Committee for Check-list of Japanese Birds (2000) Hospital, Kanagawa Prefectural Natural Environment Conser- Check-list of Japanese birds, sixth revised edition. vation Center, Kumamoto Prefectural Animal Protection Cen- Ornithological Society of Japan, Obihiro. ter, Kushiro Zoo, Niigata Prefectural Bird Protection Center, Cramp S & Simmons KEL (eds.) (1979) The birds of Nogeyama Zoo, Raptor Conservation Center, and Ueno Zoo. the western palearcitc, Vol. II. Oxford University We analyzed some data from studies by the National Institute for Land and Infrastructure Management (NILIM), and by the Press, Oxford. Ministry of the Environment. I. Nishiumi and T. Saitoh helped Crochet P-A & Desmarais E (2000) Slow rate of evolu- in preparing the NILIM DNA samples. We thank N. Fukai, T. tion in the mitochondrial control region of gulls. Mol Hirayama, Y. Obata, M. Ueta, H. Uchida, and Y. Yamada for Biol Evol 17: 1797–1806. their help in capturing goshawks. We appreciate the helpful Endo K (1989) Status and protection of goshawks in comments of an anonymous referee. We also thank Mr. T. Japan. Strix 8: 233–247 (In Japanese). Saitoh who kindly supported us to complete the manuscript. Endo K, Wakasugi S, Takamatsu T & Nakayama M This study was supported in part by a Grant-in-Aid for Scien- (1987) Nasunogahara ni okeru otaka no hansyoku tific Research by the Ministry of Education, Culture, Sports, seitai (Breeding ecology of goshawks in Nasunoga- Science, and Technology. hara). Jpn J Ornithol 36: 111 (In Japanese). Excoffier L, Smouse PE & Quattro JM (1992) Analysis REFERENCES of molecular variance inferred from metric distances among DNA haplotypes: application to human mito- Akaike H (1974) A new look at the statistical model chondrial DNA restriction data. Genetics 131: 479– identification. IEEE Trans Automat Contr 19: 491. 716–723. Ezaki Y, Hashiguchi D, Kanazawa M, Imahori R & Asai S, Yamamoto Y & Yamagishi S (2006) Genetic di- Ikeda Y (2000) Breeding and wintering of goshawks versity and extent of gene flow in the endangered in an isolated forest of southern Kyoto. Jpn J Ornithol Japanese population of Hodgson’s hawk-eagle, Spiza- 48: 267–279 (In Japanese with English abstract). etus nipalensis. Bird Conserv Intl 16: 113–129. Frankham R, Ballou JD & Briscoe DA (2002) Introduc- Baker AJ & Marshall HD (1997) Mitochondrial control tion to conservation genetics. Cambridge University region sequences as tools for understanding evolu- Press, Cambridge (Japanese version by M Nishida, tion. In: Mindell DP (ed.) Avian molecular evolution Bun-ichi Sogo Shuppan, Tokyo). and systematics. pp 51–82. Academic Press, San Gu X, Fu YX & Li WH (1995) Maximum likelihood es- Diego and London. timation of the heterogeneity of substitution rate Baker AJ, Piersma T & Rosenmeier L (1994) Unravel- among nucleotide sites. Mol Biol Evol 12: 546–557. ing the intraspecific phylogeography of knots Calidris Haring E, Riesing MJ, Pinsker W & Gamauf A (1999) canutus: a progress report on the search for genetic Evolution of a pseudo-control region in the mitochon- markers. J Ornithol 135: 599–608. drial genome of Palearctic buzzards (genus Buteo). J Boyce Jr. DA, Reynolds RT & Graham RT (2006) Zool Syst Evol Res 37: 185–194. Goshawk status and management: what do we know, Hasegawa H (1977) A note on land-birds observed in what have we done, where are we going? Stud Avian the early winter in Chichijima and Hahajima, the Biol 31: 312–325. Ogasawara Islands. Misc Rep Yamashina Inst Or- Bureau of Environmental Pollution, Tokyo Metropolitan nithol 9: 280–283. Government (ed.) (1975) Tokyo no tori—Tokyoto-san Hasegawa M, Kishino H & Yano T (1985) Dating of the chorui mokuroku (Birds of Tokyo—Checklist of birds human-ape splitting by a molecular clock of mito- inhabiting Tokyo). Wild Bird Society of Japan, Tokyo chondrial DNA. J Mol Evol 22: 160–174. (In Japanese). Hasegawa O, Takada S, Yoshida MC & Abe S (1999) Cadahía L, Negro JJ & Urios V (2007) Low mitochon- Variation of mitochondrial control region sequences drial DNA diversity in the endangered Bonelli’s eagle in three crane species, the red-crowned crane Grus (Hieraaetus fasciatus) from SW Europe (Iberia). J japonensis, the common crane G. grus and the Ornithol 148: 99–104. hooded crane G. monacha. Zoo Sci 16: 685–692. Chiba H & Funazu T (1991) Chichijima Hahajima no Hasuo Y (1969) Ogasawara shoto no dobutsu—chorui chorui (Birds of Chichijima and Hahajima). In: Ono honyurui o tyushin to shite— (Fauna of the Bonin Is- K, Kimura M, Miyashita K & Nogami M (eds.) Re- lands—birds and mammals—). In: Ogasawara shoto port of the second general survey on natural environ- shizen keikan chosa hokokusyo (Reports of natural ment of the Ogasawara (Bonin) Islands. pp 135–147. landscape research in Ogasawara islands). pp

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APPENDIX 1. Sample list.

ID Tissue Date Latitude Longitude District Haplotype

014 muscle 1996.9.6 35°01 135°46 Kyoto 1 017 muscle 1995.6.9 36°12 139°17 Saitama 4 018 muscle 1997.3.26 35°44 139°18 Tokyo 5 022 muscle 1996.8.18 34°44 135°49 Kyoto 2 026 muscle 1999.5.20 37°55 139°13 Niigata 2 027 muscle 1995.5.30 35°42 139°45 Tokyo 1 028 muscle 2000.2.21 35°42 139°45 Tokyo 4 029 muscle 2000.2.7 36°33 140°06 Tochigi 5 060 feather 2000.5.23 34°40 136°11 Mie 1 061 feather 2002.6.4 36°31 139°40 Tochigi 3 062 feather 2002.5.31 36°31 139°40 Tochigi 2 063 blood 2000.2.4 36°33 140°06 Tochigi 2 064 blood 2000.2.5 36°33 140°06 Tochigi 1 065 blood 2000.2.5 36°33 140°06 Tochigi 2 066 blood 2000.2.5 36°33 140°06 Tochigi 2 067 blood 2000.2.5 36°33 140°06 Tochigi 8 068 blood 2000.2.5 36°33 140°06 Tochigi 2 069 blood 2000.2.6 36°33 140°06 Tochigi 1 070 blood 2000.2.6 36°33 140°06 Tochigi 2 072 blood 2000.2.8 36°33 140°06 Tochigi 2 073 blood 2000.3.11 36°33 140°06 Tochigi 1 074 blood 2000.3.11 36°33 140°06 Tochigi 2 075 blood 2000.3.12 36°33 140°06 Tochigi 1 076 blood 2000.3.12 36°33 140°06 Tochigi 2 077 blood 2000.3.13 36°33 140°06 Tochigi 2 078 blood 2000.3.13 36°33 140°06 Tochigi 9 079 blood 2000.3.14 36°33 140°06 Tochigi 2 080 blood 2000.3.15 36°33 140°06 Tochigi 2 081 blood 1999.1.25 36°33 140°06 Tochigi 1 082 blood 1999.1.25 36°33 140°06 Tochigi 2 083 blood 1999.1.25 36°33 140°06 Tochigi 1 084 blood 1999.1.25 36°33 140°06 Tochigi 2 085 blood 1999.1.26 36°33 140°06 Tochigi 2 086 blood 1999.2.8 36°33 140°06 Tochigi 2 087 blood 1999.2.9 36°33 140°06 Tochigi 2 088 blood 2001.7.11 36°33 140°06 Tochigi 2 089 blood 2001.7.13 36°33 140°06 Tochigi 3 090 blood 2001.7.17 36°33 140°06 Tochigi 2 092 blood 2001.7.18 36°33 140°06 Tochigi 2 093 muscle 1998.3.13 27°04 142°13 Tokyo 7 094 blood 1999.7.4 35°27 139°38 Kanagawa 1 097 muscle 1996.6.23 34°52 137°04 Aichi 1 098 muscle 2001.1.22 35°42 139°45 Tokyo 1 099 muscle 2002.1.27 35°39 139°54 Chiba 1 100 muscle 2002.1.6 35°30 140°07 Chiba 6 101 muscle 2003.2.3 35°50 140°09 Chiba 2 102 feather 2002.7.5 34°09 133°42 Kagawa 2 104 muscle 2003.2.19 34°50 137°40 Shizuoka 2 105 feather 2003.2.12 43°30 142°30 Hokkaido 2 106 blood 2002.11.3 35°27 139°38 Kanagawa 2 107 blood 1998.8.11 34°44 135°49 Kyoto 2

154 Status of Goshawk based on mtDNA

Appendix 1. (Continued)

ID Tissue Date Latitude Longitude District Haplotype

108 blood 2000.3.8 35°01 135°46 Kyoto 2 109 blood 2000.7.25 35°01 135°46 Kyoto 1 110 muscle 1999.2.6 35°42 139°45 Tokyo 1 111 unknown 2003.4.11 35°49 139°43 Saitama 1 113 muscle 2003.7.21 35°47 139°16 Tokyo 2 114 blood 2003.9.16 35°37 139°44 Tokyo 2 115 muscle 1997.11.10 42°02 140°49 Hokkaido 1 118 blood 2003.10.11 35°27 139°38 Kanagawa 1 120 unknown 2004.4.9 36°15 139°11 Saitama 1 126 blood 2004.10.2 37°55 139°02 Niigata 2 127 unknown 2005.1.21 34°45 139°21 Tokyo 2 130 feather 2004.1.29 42°02 140°49 Hokkaido 2 131 blood 2004.9.12 43°33 144°58 Hokkaido 6 132 blood 2004.9.2 44°01 143°46 Hokkaido 6 133 feather 2003.7.2 36°31 139°40 Tochigi 1 134 feather 2003.7.3 36°31 139°40 Tochigi 1 135 feather 2003.10.1 35°59 139°05 Saitama 2 137 feather 2003.9.4 35°29 136°34 Gifu 1 138 feather 2003.9.3 42°35 142°08 Hokkaido 2 141 feather 2004.7.8 35°59 139°05 Saitama 2 149 feather 2004.7.15 35°59 139°05 Saitama 10 172 feather 2004.7.29 34°46 137°23 Aichi 2 173 feather 2004.7.28 34°46 137°23 Aichi 1 175 feather 2004.7.29 34°51 137°25 Aichi 1 179 feather 2004.7.8 34°09 133°42 Kagawa 2 182 feather 2004.7.22 42°35 142°08 Hokkaido 2 190 blood 2005.5.21 34°58 135°25 Osaka 1 191 blood 2004.12.13 36°19 139°00 Gunma 1 192 blood 2004.9.27 36°19 139°00 Gunma 1 194 blood 2004.11.7 37°57 139°20 Niigata 1 195 blood 2005.3.16 35°43 140°06 Chiba 2 196 blood 2004.9.6 36°23 139°04 Gunma 1 197 blood 2004.12.3 38°16 139°32 Niigata 2 199 blood 2004.7.13 34°35 133°03 Hiroshima 1 200 blood 2004.8.26 36°16 139°04 Gunma 1 201 blood 2003.9.28 35°27 139°38 Kanagawa 1 202 blood 2004.2.12 34°24 134°50 Hyogo 1 204 blood 2005.1.19 34°49 134°41 Hyogo 1 205 blood 1995.8.29 36°34 136°39 Ishikawa 2 206 blood 2005.2.4 33°58 130°56 Yamaguchi 2 207 blood 2004.1.29 42°02 140°49 Hokkaido 2 208 blood 2004.12.27 36°19 139°00 Gunma 1 209 blood 2003.3.10 36°34 136°39 Ishikawa 1 210 blood 2005.3.7 35°34 139°22 Kanagawa 1 211 blood 2005.2.16 35°43 139°47 Tokyo 1 212 blood 2004.11.5 35°14 139°06 Kanagawa 2 213 blood 1996.4.8 34°43 136°30 Mie 2 214 blood 1996.4.12 34°29 136°33 Mie 1 216 feather 2005.7.22 34°09 133°42 Kagawa 2 218 feather 2005.7.17 34°54 137°30 Aichi 1 220 feather 2005.6.22 34°46 137°23 Aichi 2 241 feather 2005.5.11 36°46 139°42 Tochigi 1 244 muscle 2005.6.28 36°39 139°03 Gunma 1 245 muscle 2005.7.28 36°58 140°03 Tochigi 2

155 S. ASAI et al.

Appendix 1. (Continued)

ID Tissue Date Latitude Longitude District Haplotype

247 muscle 2001.6.6 35°33 138°54 Yamanashi 1 248 muscle 2005.12.28 35°53 140°15 Ibaraki 1 249 muscle 2005.5.29 42°35 141°56 Hokkaido 2 250 muscle 2005.9.1 44°01 144°16 Hokkaido 1 251 muscle 2006.3.1 35°52 139°59 Chiba 6 254 blood 2005.8.31 33°57 131°15 Yamaguchi 2 255 blood 2005.9.6 33°57 131°15 Yamaguchi 1 256 blood 2005.9.18 33°57 131°15 Yamaguchi 2 257 blood 2005.9.27 33°57 131°15 Yamaguchi 2 258 blood 2005.12.3 33°57 131°15 Yamaguchi 1 259 blood 2005.12.27 33°57 131°15 Yamaguchi 2 262 blood 2006.9.24 35°25 140°21 Chiba 2 263 blood 2005.11.17 35°34 139°22 Kanagawa 2 264 blood 2005.7.15 35°29 139°24 Kanagawa 1 265 blood 2005.12.13 35°23 139°13 Kanagawa 2 266 blood 2006.7.27 35°24 139°19 Kanagawa 1 267 blood 2006.8.11 35°20 139°21 Kanagawa 2 271 feather 2006.7.12 34°46 136°08 Mie 2 274 feather 2006.7.22 36°31 139°40 Tochigi 2 275 blood 2006.7.9 35°40 140°04 Chiba 2 276 blood 2006.6.29 36°20 138°53 Gunma 2 277 blood 2005.9.11 35°59 140°09 Ibaraki 2 278 blood 2003.8.30 35°59 140°29 Ibaraki 1 279 blood 2007.2.10 35°17 140°15 Chiba 1 280 blood 1997.5.19 37°39 140°57 Fukushima 1 281 blood 1998.1.24 37°22 140°12 Fukushima 2 282 blood 2006.2.9 37°03 140°53 Fukushima 1 283 blood 2007.3.23 35°09 139°07 Kanagawa 1 284 blood 2004.12.27 36°19 139°00 Gunma 2 285 blood 2004.12.13 36°19 139°00 Gunma 1 286 blood 2005.8.17 36°16 138°53 Gunma 1 287 blood 2006.2.23 34°24 135°20 Osaka 2 289 blood 2004.5.10 33°53 130°53 Fukuoka 1 291 muscle 2007.10.27 36°19 139°00 Gunma 1 294 unknown 2002.8.20 35°26 133°20 Tottori 1 295 unknown 2003.7.2 39°23 140°03 Akita 1 296 unknown 2005.1.14 39°38 141°57 Iwate 2 297 unknown 2005.8.31 38°55 139°50 Yamagata 1 298 unknown 2006.8.24 38°55 139°50 Yamagata 2 301 unknown 2003.9.21 38°55 139°50 Yamagata 1

156