Genes Genet. Syst. (2010) 85, p. 55–63 Genetic diversity and phylogeny of the endangered Okinawa , okinawae

Kiyoaki Ozaki,1* Yoshihiro Yamamoto2 and Satoshi Yamagishi1 1Yamashina Institute for Ornithology, 115 Konoyama, Abiko, Chiba 270-1145, 2Department of Genetics, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan

(Received 6 November 2009, accepted 28 January 2010)

Genetic diversity of the wild population of the endangered Okinawa Rail, Gallirallus okinawae, was revealed by analyzing haplotypes in the mitochondrial control region for 177 individuals. We found 6 haplotypes with nucleotide differ- ences at 6 sites. The four major haplotypes, Type 1 to Type 4, were present in 121 (68.4%), 21 (11.9%), 8 (4.5%) and 25 individuals (14.1%), respectively. Type 5 and Type 6 were each found in one individual. The gene diversity (h) and nucle- otide diversity (π) of Okinawa Rail were calculated to be 0.499 ± 0.040 and 0.00146 ± 0.00098, respectively. Gene diversity in Okinawa Rail is higher than that found in other endangered avian species, but the relative nucleotide diversity is lower due to few nucleotide differences among the haplotypes. Our sample of 177 indi- viduals represents 20–25% of the total population, and thus allows a rigorous esti- mate of the population structure of Okinawa Rail, and makes it unlikely that more haplotypes would be found with additional sampling. The low nucleotide diver- sity in the control region may indicate that Okinawa Rail has gone through a recent bottleneck. The minimal span network of haplotypes, and the distribution pattern of sampled individuals, indicate that the number of with rare haplo- types, Type 5 and 6, decreased during the recent population decline caused by loss and introduced predators. Our results are relevant to the current conservation program for the endangered Okinawa Rail, and perhaps for other species of flightless rails.

Key words: genetic diversity, Gallirallus okinawae, mitochondrial DNA, Okinawa Rail, phylogenetic analysis

other pests (Kishida, 1931). In addition, habitat frag- INTRODUCTION mentation due to anthropogenic causes also contributed In 1981, a new avian species was discovered in the to the population decline. Similarly, other species of northern part of , Japan (Yamashina and island-dwelling flightless rails have declined as a result of Mano, 1981). This endemic species was named the and (Taylor, 1998). Okinawa Rail, Gallirallus okinawae, and is nearly flight- Recently, the Yamashina Institute for Ornithology ini- less and nests on the ground (Harato and Ozaki, 1993). tiated the Mitochondrial Genome Project with the goal of The population size of Okinawa Rail was estimated to be sequencing the entire mitochondrial genome of endan- 1,800 individuals in 1986 (Hanawa and Morishita, 1986), gered birds of Japan (Yamamoto et al., 2005). With this however, surveys in 2004 and 2005 revealed that the pop- information, it is possible to identify nucleotide variations ulation had dropped to 810 and 720 individuals, respec- in mitochondria and haplotypes, and elucidate current tively (Ozaki et al., 2006). This decline was primarily population structure. For the Okinawa Rail, we also due to predation by the Asian mongoose, Herpestes used the sequence of the mitochondrial genome to mea- javanicus (Ozaki et al., 2002), introduced in 1910 to con- sure nucleotide diversity and conduct a phylogenetic trol the venomous snake, Protobothrops flavoviridis, and analysis. Phylogenetic analyses, based on mitochondrial sequence data, have been conducted for some rail species Edited by Hidenori Tachida in order to better understand their evolution of flightless- * Corresponding author. E-mail: [email protected] ness (Trewick, 1997; Slikas et al., 2002). These authors Note: Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession num- concluded that the evolution of flightlessness in rails was bers AP010821, AP010822 and AP010823. rapid and independent. Houde et al. (1997) analyzed 56 K. OZAKI et al. phylogenetic relationships between birds, ples were preserved in 100% ethanol. After washing including rails, using DNA sequences of mitochondrial samples twice with DNA extraction buffer containing 50 12S-rRNA genes. Also, a phylogeny of five core families mM Tris-HCl (pH 8.0), 10 mM EDTA and 100 mM NaCl, in Gruiformes was conducted using sequence data of four DNA was extracted using the SDS-protease-phenol mitochondrial and three nuclear genes (Fain et al., 2007). method (Sambrook et al., 1989). A phylogenetic analysis of Okinawa Rail was made to clarify its relationship to other rails using the correspond- Determination of the total mitochondrial genome ing sequences from the published data above. of the Okinawa Rail Some related DNA sequences of Analyses of genetic diversity of wild populations of mitochondrial 12S-rRNA and cytochrome-B (cytB) genes island-dwelling flightless rails have not been previously have been registered in the DNA data bank. ThreeS- 12 conducted, though genetic diversity of captive Guam Rails rRNA genes from Clapper Rail ( longirostris, Acces- (Gallirallus owstoni) has been examined using allozymes sion number: DQ485825), Water Rail (Rallus aquaticus: and minisatellite DNA profiles (Haig and Ballou, 1995). U77149) and Buff-banded Rail (Rallus philippensis In 2004, planning was initiated to develop a captive dieffenbachia: U88026) were aligned by clustalW, and breeding and re-introduction program for the endangered PCR primer ROMT02 was made in a preserved region of Okinawa Rail (Ozaki, 2008). Required baseline data for 12S-rRNA gene. ROMT01 was also designed in the cytB this plan included an estimate of the rail population size gene by comparison with four cytB genes of Clapper Rail and its genetic diversity. To evaluate genetic diversity, (Rallus longirostris: DQ485908), Water Rail (Rallus we determined the nucleotide sequences of the mitochon- aquaticus: U77172), Takahe (Porphyrio mantelli drial control region in 177 wild individuals. Here, we hochstetteri: U77167) and Sooty Crake (Porzana tabuensis: describe our haplotype analysis of the mitochondrial con- U77170). ROMT01 and ROMT02 primers were expected trol region, and describe the population structure of the to amplify the coding region between cytB and 12S-rRNA endangered Okinawa Rail. These results should also be genes of the Okinawa Rail, and their sequences are shown useful in conserving other flightless rails. in Table 1. Long PCR was carried out with ROMT01 and ROMT02 primers using total cellular DNA of Okinawa Rail as a MATERIALS AND METHODS template and the TaKaRa LA-PCR kit (Takara Bio. Co., Samples and DNA extraction DNA was extracted Shiga, Japan). The components of the procedure were: from 177 samples: 61 blood samples were obtained from denaturing the DNA at 96°C for 3 min, followed by 30 wild Okinawa Rails either captured for banding, or unin- cycles of denaturing at 98°C for 10 sec, primer annealing tentionally caught in mongoose traps; 20 tissue samples and elongating at 68°C for 15 min, and an additional elon- were collected from un-hatched dead embryos; and 96 gation at 72°C for 10 min. A DNA fragment, approxi- muscle samples were taken from dead birds, of which 67 mately 13 kb in size, was amplified and purified by had been killed by cars and 18 by predators. Samples, agarose gel electrophoresis. The DNA sequence of the and thus haplotypes, were collected from the northeast- fragment was determined using the M13 shotgun method ern portion of Okinawa Island (Fig. 4). All tissue sam- as described by Yamamoto et al. (2000). Next, ROMT03

Table 1. PCR primers used in the genetic analysis of Okinawa Rail

Primer Sequence (5’-3’) Position2 ROMT011 GCCTGAAATAGCCTCTAGAAGGAGGATTTAGCAG 5617–5650 ROMT021 GTGTCTGCGGTATAGTGTATGGCTAGTAGTAGGC 179 –146 ROMT03 CCTAGAATCATTCGCCATCTCAGCCCTCACTATC 18340 –18373 ROMT04 CTACGTCTTGGTGCTAAGTGCACCTTCCGGTAC 5826 –5794 ROD01 CACCGCGGCATGTAATCATGTAC 2609 –2631 ROD02 GGACGAAGTCCATTGATGCTCAC 3274 –3252 ROD03 GGATCACCGACAGATCCTCGCTC 3156 –3178 ROD04 GTCTTTCGAACATTAACTAACATG 3664 –3641 1 The underscores in ROMT01 and ROMT02 primers show mismatched nucleotides compared with the mitochondrial genome of Okinawa Rail. Two and three mismatches were found because the primers were made from cytB and 12S- rRNA genes of related species. 2 Positions indicate corresponding sequence positions of the mitochondrial genome of Okinawa Rail (Accession No. AP010821). Genetic diversity and phylogeny of Okinawa Rail 57 and ROMT04 primers (Table 1) were made at both ends of the 13 kb DNA to amplify the rest of the mitochondrial genome. PCR conditions were: denaturation at 96°C for 3 min, 30 cycles of denaturing at 98°C for 10 sec, primer annealing and elongating at 68°C for 6 min, and an addi- tional elongation at 72°C for 10 min. The long-PCR product was about 5 kb in length, and its DNA sequence was determined using the procedures described above. Total DNA sequence was obtained by combining both sequences, and was found to be 18,404 bp. The total mitochondrial genome sequence of Okinawa Rail was reg- istered with the DNA data bank under accession number Fig. 1. Organization of mitochondrial control region of Okinawa AP010821. After determination of the entire mitochon- Rail. Repeat A, 649 bp, contains 4.36 repeats of a unique 149 drial genome, the first long PCR primers, ROMT01 and bp sequence. Repeat B, 841 bp, contains 10 repeats of 84 bp. ROMT02, were found to have 2 and 3 mismatches, respec- Repeat C, 222 bp, contains 4.4 repeats of 50 bp. PCR1 is ampli- tively (see Table 1). However, those mismatches were fied with ROD01 and ROD02 primers, while PCR2 with ROD03 and ROD04. Haplotypes were compared in 1,009 bp region. considered to have no effect on amplification of the 13 kb product under the PCR conditions outlined above. Two rRNA, 13 protein-coding genes, and 22 tRNA genes of Okinawa Rail were identified by similarity comparisons with other avian mitochondrial genes. We also determined the whole nucleotide sequences for the mitochondrial genomes of Banded Crake (Rallina eurizonoides sepiaria: AP010822) and Swinhoe’s Rail (Coturinicops exquisitus: AP010823) using the same procedures. These results were produced over the past several years by the Mitochondrial Genome Projects of Endangered Birds in Japan, based at the Yamashina Institute for Ornithology. The mitochondrial genome of Banded crake is 16,942 bp, while that of Swinhoe’s Rail 17,136 bp.

Haplotype analysis in the control region The con- trol region (CR) of Okinawa Rail has 3 repeat regions: one Fig. 2. Relationships of Okinawa Rail and related species. in front of CR domain I, and two others downstream of CR Based on mitochondrial DNA sequences of cytochrome-b (1,143 domain III (Fig. 1). Four PCR primers, ROD01, ROD02, bp), 12S-rRNA (980 bp) and 16S-rRNA (1,590 bp), analyzed by ≥ ROD03 and ROD04, were made between domains I and maximum likelihood. Numbers indicate bootstrap values ( 50%). III (Table 1). The 666 bp DNA fragment was amplified by PCR with ROD01 and ROD02, while the 509 bp frag- ment with ROD03 and ROD04. The 2 PCR fragments Table 2. Haplotypes and nucleotide differences of Okinawa had a 119 bp overlap, therefore, the amplified region is Rail1 1,056 bp in total length. Each PCR fragment was Haplotype 174 193 248 282 658 940 Sample number treated with a pre-sequencing kit (Takara Bio. Co., Shiga, Japan) to remove the remaining PCR primers, and then 1 T T T C T G 121 directly sequenced in both directions using PCR primers 2 C – – T – – 21 and the BigDye Terminator cycle-sequencing kit. DNA 3 – – C T – – 8 sequences were determined using an Applied Biosystems 4 – C – T C A 25 3130 Genetic Analyzer (Applied Biosystems Japan, 5 – – – T – – 1 Tokyo, Japan). After cutting the primer sequence, DNA 6 – C – T – – 1 sequences were combined in each sample of Okinawa Rail 1 to produce a haplotype. The sequenced region was 1,009 Numbers of nucleotide differences between haplotypes are bp in each sample and they were compared using GENE- positioned the top A of sequencing region 1,009 bp as one after removal of PCR primer sequences, which corresponds TYX software (GENETYX Co., Tokyo, Japan). to 2,632 of the mitochondrial genome of Okinawa Rail (Accession No. AP010821). A total of 177 samples were Phylogenetic analysis We constructed phylogenetic analyzed. 58 K. OZAKI et al. trees using the maximum likelihood (ML) method imple- den Markov model. mented in PHYLIP ver. 3.6 (Felsenstein, 2004). We com- bined DNA sequences of cytochrome-b, 12S-rRNA, and 16S- Mismatch distribution and haplotype network con- rRNA genes of 7 Rallidae from the DNA data bank: Purple struction Gene diversity (h; Nei, 1973) and nucleotide Swamphen (Porphyrio porphyrio: DQ485905, DQ485822, DQ485858), Rufous-sided Crake (Laterallus melanophaius: DQ485906, DQ485823, DQ485859), Buff-banded Rail (Gallirallus philippensis: DQ485907, DQ485824, DQ485860), Clapper Rail (Rallus longirostris: DQ485908, DQ485825, DQ485861), Sora Rail (Porzana Carolina: DQ485909, DQ485826, DQ485862), American Coot (Fulica Americana: DQ485910, DQ485827, DQ485863) and Moorhen (Gallinula chloropus: DQ485911, DQ485828, DQ485864), to analyze the phylogenetic relationship by ML method, using American finfoot (Heliornis fulica: DQ485902, DQ485819, DQ485857) and Red-crowned Crane (Grus japonensis: U27550, DQ485807, DQ485845) as outgroups. We also determined the whole nucleotide sequ- ences of the mitochondrial genome in Banded Crake (Rallina eurizonoides sepiaria: AP010822) and Swinhoe’s Rail (Coturnicops exquisitus: AP010823), and the same 3 Fig. 3. Minimum spanning network of 6 haplotypes of Okinawa gene sequences as above were analyzed together. All Rail. The relationship between 6 haplotypes of the mitochon- sequences were aligned by Clustal W program (Thompson drial control region was analyzed by TCS 1.21(Clement et al. et al., 1994), and then analyzed with ‘dnaml’ program in 2000). Numbers next to circles represent individuals belonging PHYLIP ver. 3.6 (Felsenstein, 2004) package using a Hid- to each haplotype.

Table 3. Genetic diversity of the mitochondrial control region in avian species. In some cases, genetic diversity was calculated from only a portion of the control region

Species Scientific name Source h π N note Sage Grouse Centrocercus urophasianus Kahn et al. 1999 0.06 0.0011 31 Small-bodied population Red-crowned Crane Grus japonensis Hasegawa et al. 1999 0.25 0.0047 15 Japanese population Golden Eagle Aquila chrysaetos japonica Masuda et al, 1998 0.26 0.0017 14 Spanish Imperial Eagle Aquila adalberti Martínez-Cruz et al. 2004 0.3215 0.00098 60 Crested Ibis Nipponia nippon Zhang et al. 2004 0.386 0.00069 36 Okinawa Rail Gallirallus okinawae This paper 0.499 0.00146 177 Bonelli’s Eagle Hieraaetus fasciatus Cadahia et al. 2007 0.542 0.0024 72 Red Kite Milvus milvus Roques & Negro 2005 0.61 0.0032 105 Greenfinch Carduelis chloris Merilä et al. 1997 0.612 0.134 190 Knot Calidris canutus Baker et al. 1994 0.69 0.0039 25 Common Guillemot Uria aalge Moum & Árnason 2001 0.71 0.0054 22 West Atlantic population Common Guillemot Uria aalge Moum & Árnason 2001 0.72 0.0048 57 East Atlantic population Eastern Imperial Eagle Aquila heliaca Martínez-Cruz et al. 2004 0.7790 0.00548 34 Red-crowned Crane Grus japonensis Hasegawa et al. 1999 0.78 0.0087 29 East Asia population Resplendent Quetzal Pharomachus mocinno Solorzano et al. 2004 0.8180 0.0171 25 Brambling Fringilla montifringilla Marshall & Baker 1997 0.87 0.0026 10 Razorbill Alca torda Moum & Árnason 2001 0.88 0.0097 81 East Atlantic population Common Chaffinch Fringilla coelebs Moum & Árnason 2001 0.89 0.129 42 Hodgson’s Hawk-Eagle Spizaetus nipalensis Asai et al. 2006 0.935 0.00741 68 Razorbill Alca torda Moum & Árnason 2001 0.95 0.0173 42 West Atlantic population Turnstone Arenaria interpres Wenink et al 1994 0.9567 0.010388 25 Elliot’s Pheasant Syrmaticus ellioti Jiang et al. 2007 0.992 0.00616 33 Genetic diversity and phylogeny of Okinawa Rail 59 diversity (π; Nei and Tajima, 1981) were calculated using The CR (D-loop) of the Okinawa Rail is found between the Arlequin 2.000 (Schneider et al., 2000). Mismatch distri- tRNA-Glu and tRNA-Phe genes, a common location in bution values between 177 rails were also calculated using avian mitochondrial genomes, however, it also displays Arlequin 2.000 (Schneider et al., 2000) to compare with some unique characteristics (Fig. 1). the sudden expansion model (Rogers and Harpending, The CR (D-loop) of the Okinawa Rail is 2,874 bp long 1992). In addition, Tajima’s D-test (Tajima, 1989) was and displays an interesting organization (Fig. 1). In the performed using Arlequin 2.000 (Schneider et al., 2000), CR of Okinawa Rail, the first repeat region, named and a cladogram of haplotype sequences was estimated Repeat A, is 649 bp long, lies in front of domain I, and con- using TCS 1.21 free software (Clement et al., 2000). sists of the 149 bp unique sequence repeated 4.4 times. In the fifth repeat of the 149 bp sequence contained in Repeat A, only the first 53 bp are repeated. The second RESULTS repeat region, Repeat B, is 841 bp long and contains 10 The novel structure of the CR of Okinawa Rail repeats of the unique 84 bp unit downstream of domain The position and direction of 2 rRNA genes, 13 protein- III. The third repeat region, Repeat C, is 220 bp long, coding genes and 22 tRNA genes in the mitochondrial and has 4.4 repeats of 50 bp in front of the tRNA gene for genome of Okinawa rail are similar to those of many phenylalanine. The fifth repeat, within Repeat C, con- avian species, such as the chicken (Gallus gallus var. tains only the first 20 bp of the 50 bp unit. As those domesticus) (Desjardins and Morais, 1990) and Oriental repeats were heteroplasmic, with a variable number of White Stork (Ciconia boyciana) (Yamamoto et al., 2000). repeat units within an individual, we selected the central

Fig. 4. Geographic distribution of haplotypes of 177 Okinawa Rails. Symbols indicate each haplotype of Okinawa Rail. Symbols that originally hid others, were moved slightly to eliminate overlap. 60 K. OZAKI et al. non-repetitive region, 1,009 bp, for PCR amplification and lower than simulated values. Tajima’s D value (Tajima, sequencing to determine haplotypes of Okinawa Rails. 1989) was estimated to be 0.848 (p = 0.824) using Arle- quin 2.000 (Schneider et al., 2000). Phylogenetic analysis of the mitochondrial genome of Okinawa Rail We determined the entire nucleotide DISCUSSION sequence of the mitochondrial genome of Okinawa Rail (18,404 bp), Swinhoe’s Rail (17,136 bp), and Banded Long CR with long repeat regions The CR (D-loop) Crake (16,942 bp). We chose the longer sequences of of the Okinawa Rail is 2,874 bp long, about 3-times the cytochrome-b (1,143 bp), 12S-rRNA (980 bp), and 16S- length found in most . Some avian mitochon- rRNA (1,590 bp) genes of 7 Rallidae species in DNA data drial CR have the repeat sequence region(s) downstream bank for ML analysis, using American Finfoot and Red- from domain III and hence have a longer CR as seen in crowned Crane as outgroups. Accordingly, DNA sequ- the Oriental Stork (Ciconia boyciana: AB026193), Turkey ences of 3,266 bp in total were analyzed. Using the ML Vulture (Cathartes aura: AY463690), Black-browed Alba- method, we were then able to describe the phylogenetic tross (Diomedea melanophrys: AY362763) and Magpie relationship of 10 Rallidae species (Fig. 2). Goose (Anseranas semipalmata: AY309455). The CR of The grouping of Okinawa Rail, Banded Rail, and Little Penguin (Eudyptula minor: AF362763) also has a Clapper Rail into one clade was supported by bootstrap long repeat region, including 9.4 repeats of 25 bp, and analysis (1,000 replicates). Okinawa Rail was found to be 20.9 repeats of 7 bp, however, they are downstream of more closely related to Buff-banded Rail than to Clapper domain III, as in other avian CR. In contrast, the CR of Rail, and supports the current placement of Okinawa Rail Okinawa Rail has its first repeat of 649 bp, Repeat A, in and Buff-banded Rail in the Gallirallus, and the front of domain I, and the length of the repeat unit of Clapper Rail in the genus Rallus. Ten Rallidae species unique sequence is 149 bp. The other repeat regions, formed a large group distinguished from the outgroup spe- Repeat B and C, also have long repeat units of 84 bp and cies, American Finfoot and Red-crowned Crane (Trewick, 50 bp, respectively. The unusual position of Repeat A, 1997; Fain et al., 2007). long unique repeat units, and the longest CR, make the mitochondria of Okinawa Rail unique among avian mito- Haplotype analysis and genetic diversity of Okinawa chondrial genomes studied thus far. The nucleotide Rail We examined 177 individual rails and identified 6 sequences of the three repeat units have no similarity haplotypes having nucleotide differences at 6 sites (Table with each other, and no homologous sequence was found 2). Type 1 was the major haplotype, containing 121 indi- in the DNA data bank. In CR of Swinhoe’s Rail, we iden- viduals (68.4%), while Types 2, 3, and 4 contained tified a small repeat region of 179 bp downstream from 21(11.9%), 8(4.5%) and 25(14.1%), respectively. Two domain III, which is composed of 5.4 repeats of a 33 bp other haplotypes were found in one individual each. For unit. However, no repeat sequence was found in the CR haplotype analysis, we used the nucleotide sequence of of Banded Crake. 1,009 bp between domain I and III, which contains most Genetic divergence of Clapper Rail and King Rail has of CR without repetitive sequences. However, only 6 been described previously using restriction site variation variable sites in the sequenced region were found in 177 in mitochondrial DNA (Avise and Zink, 1988). The rails, indicating low nucleotide diversity in the Okinawa authors estimated the size of the mitochondrial DNA from Rail population. There appeared to be no sampling bias Clapper and King Rails to be about 17.9 kb. They also due to tissue type because all major haplotypes were found mitochondrial DNA size differences in 7 Clapper found in all tissue types at similar rates. The minimum Rails and 10 King Rails, and concluded the species were span network of 6 haplotypes is shown in Fig. 3. Gene heteroplasmic. The observation of large mitochondria diversity (h) and nucleotide diversity (π) was 0.499 ± size and heteroplasmy in rails may be a function of the 0.040 and 0.00146 ± 0.00098 (Table 3), respectively. CR containing long repeat regions with variable repeat The geographic distribution of rail haplotypes shows number. As shown in mitochondrial CR organization of that Types 1 and 3 are widely distributed across the Okinawa Rail, many species in the genus Gallirallus may range of the Okinawa Rail, while Type 2 is found in the have large repeat regions in the CR. However, there is northern part of the range, and Type 4 in the central part currently no information on the nucleotide sequence of (Fig. 4). mitochondrial CR, and its structure, for other rails. The mismatch distribution of haplotype sequences of Okinawa rails was analyzed using Arlequin 2.000 Phylogenetic analysis of Swinhoe’s Rail Three mito- (Schneider et al., 2000) to compare with the sudden chondrial gene sequences, cytB, 12S-rRNA, and 16S- expansion model (Rogers and Harpending, 1992). The rRNA, in 10 Rallidae species were analyzed by ML mismatch distribution pattern is shifted to the left and method (Fig. 2). Swinhoe’s Rail appears to be more mismatch values with one and three differences are much closely related to Rufous-sided Crake than to Banded Genetic diversity and phylogeny of Okinawa Rail 61

Crake, and was once thought to be a subspecies of Yellow Rail (Coturnicops noveboracensis) (Swinhoe, 1873). A partial DNA sequence, 633 bp, of mitochondrial CO1 gene of Yellow Rail (DQ433553) was compared to the equiva- lent sequence of mitochondrial genome of Swinhoe’s Rail. Nucleotide differences were found at 46 sites (7.3%), indicating that Swinhoe’s Rail is probably best viewed as an independent species.

Genetic diversity and haplotypes Gene diversity (h) and nucleotide diversity (π) of mitochondrial CR in birds is shown in Table 3. The gene diversity (h) of Okinawa Rail is higher than that of Sage Grouse (Centrocercus urophasianus: small-bodied population), Red-crowned Crane (Grus japonensis: Japanese population), Golden Eagle (Aquila chrysaetos japonica), Spanish Imperial Fig. 5. Mismatch distributions histogram of CR (D-loop) nucle- Eagle (Aquila adalberti) and Crested Ibis (Nipponia otide sequences of Okinawa Rails. Black bars represent nippon), which are listed in the IUCN Red List of Threat- observed values of pairwise mismatches between 177 individu- ened Species in categories ranging from Least Concern to als, while open circles show simulated values estimated using the sudden population expansion model (Rogers and Harpending Endangered, mainly due to their small population sizes 1992). (IUCN, 2009). Okinawa Rail is listed as endangered because the population size has declined rapidly in recent years. Although the gene diversity of Okinawa Rail is model, and suggests that a population bottleneck may high relative to other , this does not have occurred (Fauvelot et al., 2003). The mismatch dis- mean that Okinawa Rail population is more diverse than tribution pattern of Okinawa Rail in Fig. 5 resembles that other endangered avian species. The gene diversity of damselfishes, and suggests that Okinawa Rails may value depends on the length of the DNA sequence ana- have passed through a recent population bottleneck. In lyzed, the number of samples, along with other factors. addition, mismatch values with one and three differences The sample of 177 Okinawa Rails is large relative to that are much lower than simulated values in Okinawa Rails, of other endangered species (Table 3). Sequences equiv- indicating that some haplotypes may have been lost alent to the CR region without repeated sequences were recently due to habitat fragmentation. Tajima’s D value used for analysis in Okinawa Rail, but only shorter for haplotypes of Okinawa Rail was 0.848 using Arlequin domain I sequences were analyzed in other endangered 2.000 (Schneider et al., 2000). Negative values of D are species. On the contrary, nucleotide diversity (π) of indicative of a recent population bottleneck (Tajima, Okinawa Rail is low compared to the endangered species 1989). A discrepancy between the mismatch distribution listed above, and other avian species in general (Table and D-value was also observed in whales (Patenaude et 3). We found only 6 haplotypes having nucleotide differ- al., 2007). ences at 6 sites (Table 2) in 177 individual rails. The Based on the minimum span network of 6 haplotypes recent population decline may also have affected nucle- (Fig. 3), it appears that all other haplotypes are derived otide diversity through the loss of other haplotypes, from Type 5, even though Type 1 has the highest fre- including rare ones. We suggest that the low nucleotide quency in the population. The other three major haplo- differences in the control region may be the result of a types, Types 2, 3, and 4, appear to have formed from Type recent population bottleneck. As seen in Fig. 5, the mis- 5. This atypical network may be the result of habitat match distribution pattern closely fits the simulated one fragmentation and recent loss of Type 5, Type 6, and from a sudden expansion model (Rogers and Harpending, other haplotypes in the southern portion of the rail’s 1992). The same left-shifted pattern of mismatch distri- range, where mongooses and feral cats have invaded. bution of mitochondrial D-loop sequences has been The distribution of sampled individuals (Fig. 4) indi- described in damselfishes (Fauvelot et al., 2003) and cates that most samples were from the eastern and cen- whales of the Indo-Pacific ocean (Patenaude et al., 2007). tral part of the rail’s range. All 4 major haplotypes were However, mismatch distribution of whales in the Indo- found in both of these areas while Types 5 and 6, were Pacific ocean has secondary peaks at other sites, and only found in the eastern seaside area. The abundance Patenaude et al. (2007) concluded that the mismatch dis- of Types 5 and 6 individuals may have decreased rapidly tribution does not fit the sudden expansion model. In as the population declined. Because we sampled 20–25% damselfishes, Chrysiptera glause or Pomacentrus pavo, of the population, it seems unlikely that new haplotypes the mismatch distribution fits well to a sudden expansion would be found with increased sampling. Here, we 62 K. OZAKI et al. reported our findings for wild rails, but we have con- sea-level change. Evolution 57, 1571–1583. ducted additional studies on captive rails and have not Felsenstein, J. (2004) PHYLIP (Phylogeny Inference Package) identified other haplotypes. version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle. The recovery program for the Okinawa Rail, initiated Haig, S. M., and Ballou, J. D. (1995) Genetic diversity in two by the Japan Ministry of Environment and Okinawa Pre- avian species formerly endemic to Guam. Auk 112, 445– fecture, may have begun in time to protect the genetic 455. diversity of the rail population, as indicated by minor Hanawa, S., and Morishita, E. (1986) The Okinawa Rail’s distri- haplotypes still surviving in the population. The recov- bution and estimated number of individuals. In: Survey on Special Birds for Protection. Environmental Agency of ery plan recommends creation of a captive breeding pro- Japan. pp. 43–61 [in Japanese]. gram, and future re-introduction of these birds into the Harato, T., and Ozaki, K. (1993) Roosting behavior of the wild. In addition, it calls for removal of predators of Okinawa Rail. J. Yamashina Inst. Ornithol. 25, 40–53. rails, such as mongooses and feral cats, along with the Hasegawa, O., Takada, S., Yoshida, M. C., and Abe, S. (1999) protection of the rail’s habitat. Stabilization of the Variation of mitochondrial control region sequences in three crane species, the red-crowned crane Grus japonensis, the Okinawa Rail population, and retention of its genetic common crane G. grus and the hooded crane G. monacha. diversity, is needed to prevent the extinction of this island Zoo Sci. 16, 685–692. endemic. Houde, P., Cooper, A., Leslie, E., Strand, A. E., and Montãno, G. A. (1997) Phylogeny and evolution of 12S rDNA in Grui- This study was supported by the Grant-in-Aid for Scientific formes (Aves). In: Avian Molecular Evolution and Syste- Research by the Ministry of Education, Culture, Sports, Science matics (ed.: D.P. Mindel), pp. 121–158. Academic Press, San Diego, CA. and Technology, and also by the Environmental Technology IUCN (2009) IUCN Red List of Threatened Species. Version Development Fund of the Ministry of the Environment, 2009.1. [www.iucnredlist.org] Downloaded on 10 July 2009. Japan. Yambaru Wildlife Center provided samples from dead Jiang, P. P., Ge, Y. F., Lang, Q. L., and Ding, P. (2007) Genetic Okinawa Rails. N. Kotaka and H. Shichiri of the Center, and ucture among wild populations of Elliot’s Pheasant T. Nagamine and Y. Nakaya of the Conservation & Wel- str Syrmaticus ellioti in China from mitochondrial DNA analy- fare Trust, helped with sampling. S. Komeda and T. Baba of the ses. Conserv. Internatn. 17, 177–185. Yamashina Institute for Ornithology, and Y. Toguchi, T. Harato, Kahn, N. W., Braun, C. E., Young, J. R., Wood, S., Mata, D. R., and M. Kinjyo assisted with field work. We thank P. Sievert for and Quinn, T. W. (1999) Molecular analysis of genetic vari- his review of the manuscript. ation among large- and small-bodied sage grouse using mitochondrial control-region sequences. Auk 116, 819– REFERENCES 824. Kishida, K. (1931) Professor Watase and the import of Asai, S., Yamamoto, Y., and Yamagishi, S. (2006) Genetic diver- mongoose. Zoological Science 44, 70–78 [in Japanese]. sity and extent of gene flow in the endangered Japanese Marshall, H. D., and Baker, A. J. (1997) Structural conservation population of Hodgson’s hawk-eagle, Spizaetus nipalensis. and variation in the mitochondrial control region of fring- Bird Conserv. Internatn. 16, 113–129. illine finches (Fringilla spp.) and the Greenfinch (Carduelis Avise, J. C., and Zink, R. M. (1988) Molecular genetic divergence chloris). Mol. Biol. Evol. 14, 173–184. between avian sibling species: King and Clapper rails, Long- Martínez-Cruz, B., Godoy, J. A., and Negro, J. J. (2004) Popula- billed and Short-billed dowitchers, Boat-tailed and Great- tion genetics after fragmentation: the case of the endan- tailed glackles, and Tufted and Black-crested titmice. Auk gered Spanish imperial eagle (Aquila adalberti). Mol. Ecol. 105, 516–528. 13, 2243–2255. Baker, A. J., Piersma, T., and Rosenmeier, L. (1994) Unraveling Masuda, R., Noro, M., Kurose, N., Nishida-Umehara, C., the intraspecific phylogeography of knots Calidris canutus: Takechi, H., Yamazaki, T., Kosuge, M., and Yoshida, M.C. a progress report on the search for genetic markers. J. (1998) Genetic characteristics of endangered Japanese Ornthol. 135, 599–608. golden eagles (Aquila chrysaetos japonica) based on mito- Cadahía. L., Negro, J. J., and Urios, V. (2007) Low mitochon- chondrial DNA D-loop sequences and karyotypes. Zoo Biol. drial DNA diversity in the endangered Bonelli’s eagle 17, 111–121. (Hieraaetus fasciatus) from SW Europe (Iberia). J. Orni- Merilä, J., Björklund, M., and Baker, A. (1997) Historical 8thol. 14 , 99–104. demography and present day population structure of the Clement, M., Posada, D., and Crandall, K. A. (2000) TCS: a com- greenfinch, Carduelis chloris –An analysis of mtDNA con- puter program to estimate gene genealogies. Molecular trol-region sequences. Evolution 51, 946–956. Ecology 9, 1657–1660. Moum, T., and Árnason, E. (2001) Genetic diversity and popula- Desjardins, P., and Morais, R. (1990) Sequence and gene organi- tion history of two related seabird species based on mito- zation of the chicken mitochondrial genome. J. Mol. Biol. chondrial DNA control region sequences. Mol. Ecol. 10, 212, 599–634. 2463–2478. Fain, M.G., Krajewski, C., and Houde, P. (2007) Phylogeny of Nei, M. (1973) Analysis of gene diversity in subdivided popula- “core Gruiformes” (Aves: Grues) and resolution of the tions. Proc. Natl. Acad. Sci. USA 70, 3321–3323. Limpkin-Sungrebe problem. Mol. Phylogen. Evol. 43, 515– Nei, M., and Tajima, F. (1981) DNA polymorphism detectable by 529. restriction endonucleases. Genetics 97, 145–163. Fauvelot, C., Bernardi, G., and Planes, S. (2003) Reductions in Ozaki, K. (2008) Conservation effort, Okinawa Rail. In: Summa the mitochondrial DNA diversity of coral reef fish provide Ornithologica (eds. F. Akishinonomiya and Y. Nishino), pp. evidence of population bottlenecks resulting from Holocene 495–508. The University Museum. The University of Tokyo Genetic diversity and phylogeny of Okinawa Rail 63

[in Japanese]. 456. Ozaki, K., Baba, T., Komeda, S., Kinjyo, M., Toguchi, Y., and Swinhoe, R. (1873) On three new species of birds from Chefoo Harato, T. (2002) The declining distribution of the Okinawa (North China). Ann. Mag. Nat. Hist. 40, 373–377. Rail Gallirallus okinawae. J. Yamashina Inst. Ornithol. Taylor, B. (1998) Rails: A guide to the rails, crakes, gallinules, 34, 136–144 [in Japanese with English summary]. and coots of the world. Yale Univ. Press, New Haven, CT. Ozaki, K., Baba, T., Komeda, S., Hiroi, T., Harato, T., Toguchi, Thompson, J. D., Higgins, D. G., and Gilson, T. J. (1994) Y., and Kinjyo, M. (2006) The declining distribution and CLUSTAL W: improving the sensitivity of progressive population of the Okinawa Rail. The summaries of the pre- multiple sequence alignment through sequence weighting, sentations for 2006 annual meeting of the Ornithological positions-specific gap penalties and weight matrix. Nucleic Society of Japan. p.71 [in Japanese]. Acids Res. 22, 4673– 4680. Patenaude, N. J., Portway, V. A., Schaeff, C. M., Bannister, J. L., Tajima, F. (1989) Statistical method for testing the neutral Best, P. B., Payne, R. S., Rowntree, V. J., Rivarola, M., and mutation hypothesis by DNA polymorphism. Genetics 123, Baker, C. S. (2007) Mitochondrial DNA diversity and popu- 585–595. lation structure among Southern Right Whales (Eubalaena Trewick, S. A. (1997) Flightlessness and phylogeny amongst australis). J. Heredity 98, 147–157. endemic rails (Aves: Rallidae) of the New Zealand region. Rogers, A. R., and Harpending, H. (1992) Population growth Philos. Trans. R. Soc. Lond. B. Biol. Sci. 352, 429– 446. makes waves in the distribution of pairwise genetic differ- Yamamoto, Y., Murata, K., Matsuda, H., Hosoda, T., Tamura, ences. Mol. Biol. Evol. 9, 552–569. K., and Furuyama, J. (2000) Determination of the complete Roques, S., and Negro, J. J. (2005) MtDNA genetic diversity and nucleotide sequence and haplotypes in the D-loop region of population history of a dwindling raptorial bird, the red kite the mitochondrial genome in the oriental white stork, (Milvus milvus). Biol. Consv. 126, 41–50. Ciconia boyciana. Genes Genet. Syst. 75, 25–32. Sambrook, J., Frisch, E. F., and Maniatis, T. (1989) Molecular Yamamoto, Y., Kakizawa, R., and Yamagishi, S. (2005) Mitochon- Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor drial genome project on endangered birds in Japan: 1. Ancient Laboratory Press, New York. Murrelet, Synthliboramphus antiquus. J. Yamashina Inst. Schneider, S., Roessli, D., and Excoffier, L. (2000) Arlequin: A Ornithol. 37, 20–29. software for population genetics data analysis. Ver 2.000. Yamashina, Y., and Mano, T. (1981) A new species of rail from Genetics and Biometry Lab, Dept. of Anthropology, Univer- Okinawa island. J. Yamashina Inst. Ornithol. 13, 1–6. sity of Geneva. Wenink, P. W., Baker, A. J., and Tilanus, M. G. (1994) Mitochon- Slikas, B., Olson, S. L., and Freischer, R. C. (2002) Rapid, inde- drial control-region sequences in two shorebird species, the pendent evolution of flightlessness in four species of Pacific turnstone and the dunlin, and their utility in population islands rails (Rallidae): an analysis based on mitochondrial genetic studies. Mol. Biol. Evol. 11, 22–31. sequence data. J. Avian Biol. 33, 5–14. Zhang, B., Fang, S.-G., and Xi, Y.-M. (2004) Low genetic diver- Solórzano, S., Baker, A. J., and Oyama, K. (2004) Conservation sity in the endangered crested ibis Nipponia nippon and priorities for resplendent quetzals based on analysis of mito- implications for conservation. Bird Conserv. Internatn.14, chondrial DNA control region sequences. Condor 106, 449– 183–190.