Molecular Phylogenetics and Evolution 39 (2006) 899–905 www.elsevier.com/locate/ympev Short communication The origin of the East Asian Erithacus robin, Erithacus komadori, inferred from cytochrome b sequence data Shin-Ichi Seki ¤ Kyushu Research Center, Forestry and Forest Products Research Institute, 4-11-16 Kurokami, Kumamoto 860-0862, Japan Received 28 September 2005; revised 27 January 2006; accepted 27 January 2006 Available online 10 March 2006 1. Introduction two subspecies are, hence, also listed as vulnerable species in the Japanese Red List (Ministry of the Environment of The avian genus Erithacus is a member of the chat tribe, Japan, 2002). To make an eVective conservational decision Saxicolini, which is grouped in the Old World Xycatcher about E. komadori, there must be an understanding of its family, Muscicapidae (Sibley and Ahlquist, 1990; Voelker present distribution, in relation to its origin and diversiWca- and Spellman, 2004). The genus presently comprises three tion history of subspecies. species according to Sibley and Monroe (1990): E. rubecula, The phylogenetic relationship among the Erithacus rob- E. akahige, and E. komadori. Erithacus rubecula occurs in ins, however, is still a question under debate (Kajita, 1999; the western palearctic, from humid lowlands, wooded Ornithological Society of Japan, 2000). Some morphological mountains to treeline, and breeds in various types of for- similarity between E. rubecula and E. akahige, particularly ests, parks and gardens with trees, and shrubs (Cramp, the close similarity of feather coloration (Meinertzhagen, 1988). Contrary to this, the other two species are endemic 1951), appears to be the main reason why the East Asian to East Asia (Fig. 1). Erithacus akahige breeds in Sakhalin, Erithacus robins and E. rubecula have been classiWed into the four main Japanese islands, and some other adjacent the same genus by most authors (e.g., Cramp, 1988; Orni- islands. Its preferred breeding habitat is coniferous and thological Society of Japan, 2000; Sibley and Monroe, mixed forest in boreal montane and cool-temperate zones, 1990). On the other hand, some other authors classiWed the except for some small islands in the southern part of Japan, East Asian Erithacus robins into the genus Luscinia, leaving where it breeds in evergreen broad-leaved forests (Kiyosu, E. rubecula as a single species for the genus Erithacus, 1965). Erithacus komadori breeds in lowland evergreen because their feather texture and the form of their tails are broad-leaved forests on small to middle-sized islands in the diVerent from those of E. rubecula, and more closely resem- Ryukyu and Danjo Islands, located in the southwestern ble those of a certain group of Luscinia in East Asia, such as part of Japan (Kawaji and Higuchi, 1989). L. cyane and L. sibilans (Kiyosu, 1965; Ornithological Soci- The distribution of E. komadori is especially restricted ety of Japan, 1975; Yamashina, 1941). The genus Erithacus among these three species (Kiyosu, 1965; Ornithological itself sometimes merged with Luscinia and Tarsiger in the Society of Japan, 2000), and this species has been desig- past (reviewed in Cramp, 1988), but recent molecular stud- nated as a “national natural treasure” and “national ies revealed that western palearctic Luscinia forms a geneti- endangered species of wild fauna and Xora” of Japan. Two cally distinguishable clade from Erithacus (Sibley and subspecies are described in its narrow distribution, E. k. Ahlquist, 1990; Wink et al., 2002). To which group do the komadori in the northern Ryukyu Islands and E. k. namiyei East Asian Erithacus robins belong? Is the discontinuous in the central Ryukyu Islands (Kawaji and Higuchi, 1989). distribution of these similar looking robins a result of the The populations of both subspecies are recently declining in wide distribution of a common ancestor, or just the conver- their major habitats, mainly because of the introduced pre- gence of feather coloration? dators (Kinjou et al., 2003; Sugimura et al., 2003), and the This paper is the Wrst in a series of studies on the phylo- geography of the E. komadori. The aims of this study were: * Fax: +81 96 344 5054. (1) to reveal the molecular phylogenetic relationships E-mail address: [email protected]. between the three species of Erithacus robins and some 1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2006.01.028 900 S.-I. Seki / Molecular Phylogenetics and Evolution 39 (2006) 899–905 Erithacus rubecula Erithacus akahige Hokkaido Honshu Yaku shima Erithacus komadori Nakano-shima Oh-shima&Tokuno-shima Japanese Okinawa-jima Is. Ryukyu Is. Fig. 1. Distribution of the species within the genus Erithacus, showing their approximate range (Cramp, 1988; Nakamura and Nakamura, 1995; Ornitho- logical Society of Japan, 2000) and sampling locations. related Luscinia species, and (2) to examine the origin and Total DNA was extracted from muscle tissue of frozen subspecies diversiWcation history of E. komadori with such specimens using QIAamp Tissue Kits (Qiagen), and also a restricted distribution, based on the mitochondrial cyto- from plucked feathers of live individuals following the pro- chrome b (cyt b) sequence data. tocol described by Baba et al. (2001) using the extraction kit IsoQuick (ORCA). These samples show relatively high 2. Materials and methods concentrations of mitochondrial DNA compared with those from blood, which reduce the possibility of isolating The sampling scheme was designed to identify the ori- nuclear pseudogenes (Lee et al., 1997). SpeciWc fragments gin of E. komadori, and I used 18 samples from eight taxa were ampliWed using the polymerase chain reaction (PCR) in Saxicolini, collected from adult birds mostly during the and one set of primers: AXL149 (5Ј-ACT CCA TCA AAC breeding season. The sampled taxa with sampling location ATC TCA GCT TGA TGA A-3Ј) which was modiWed and sample size are as follows (see also Fig. 1 for sampling from L14841 (Kocher et al., 1989) and AXH160 (5Ј-AGT locations): E. k. komadori (Nakano-shima:1, Oh-shima:1, CTT CAA TCT TTG GCT TAC AAG ACC-3Ј) which was Tokuno-shima:1), E. k. namiyei (Okinawa-jima:3), E. a. modiWed from H15915 (Irwin et al., 1991). All PCRs were akahige (Hokkaido:2, Honshu:1), E. a. tanensis (Yaku- performed in 25 l volumes on a Perkin Elmer 9600 Ther- shima:1), L. calliope (Hokkaido:2), L. cyane (Hokka- mal Cycler, using Taq DNA polymerase (TaKaRa Ex Taq, ido:2), Tarsiger cyanurus. (Honshu:2, wintering individu- Takara). Each reaction mixture included 1 l of puriWed als), and Phoenicurus auroreus (Honshu:2, wintering total DNA template, 2 l of dNTP mixture (2.5 mM each), individuals). Although Kuroda (1923) described the third 2.5 l of 10£ Ex Taq BuVer (Takara), 0.25 l of bovine subspecies for E. komadori, E. k. subrufus, in the southern serum albumin (20 mg/ml), 1.25 l of each primer (2 pmol/ part of the Ryukyu Islands, I did not include samples of l), and 0.5 l of Taq (1.25 U/l). The thermal proWle com- this doubtful subspecies in the present analysis; it was prised an initial denaturing step of 94 °C for 2 min, followed described based on the type captured in autumn, some by a “touch-down” scheme where the annealing tempera- individuals from the northern population of E. k. koma- ture was lowered by 1 °C per cycle, starting from 65 °C until dori is known to overwinter in the Southern Ryukyus, all a temperature of 55 °C was reached. Then 25 additional morphological characters of that individual are well cycles were run at a constant annealing temperature of within the range of E. k. komadori (Kawaji and Higuchi, 55 °C. Denaturation was done at 94 °C for 15 s, annealing 1989), and there are no breeding records of this species in for 30 s, and extension at 72 °C for 90 s, and a Wnal exten- the southern Ryukyus. For the above samples, a large sion step of 5 min was added after the last cycle. The result- portion of the mitochondrial cyt b gene, 1007 base pairs ing products were sequenced using Big Dye Terminator (bp), was sequenced and analyzed. I also included pub- ver3.1 Cycle Sequencing Kit (Applied Biosystems) and lished nucleotide sequences of E. rubecula (GenBank visualized on an ABI PRISM 310 Automated sequencer. Direct Submission: Accession Nos. Y08058 and L78807) All sequences are available from DDBJ nucleotide Data- and L. svecica (Questiau et al., 1998: Accession Nos. bank with Accession Nos. AB236370–AB236376, and Y10061 and Y08045) as ingroups, and Muscicapa infus- AB244005–AB244015. cata (GenBank Direct Submission: Accession No. Sequences were aligned by eye, because no gaps occurred MST299690) and M. striata (Sætre et al., 2001: Accession in the target region. All phylogenetic analyses were per- No. AY206912) as outgroup taxa; Muscicapini which formed using the computer program PAUP* 4.0b10 (Swo- includes Muscicapa is known to be a sister taxa for the Vord, 2003). Maximum-parsimony (MP) analysis in the tribe Saxcolini (Voelker and Spellman, 2004). Of these, the form of heuristic search was performed with 1000 random 887–1007 bp segments of the cyt b gene were aligned to stepwise-addition replicates. The robustness of the phylog- the present sequences. eny was tested by bootstrapping with 1000 replicates with S.-I. Seki / Molecular Phylogenetics and Evolution 39 (2006) 899–905 901 100 random stepwise-addition replicates. In maximum-like- 0.5 mybp (Warren et al., 2003), and divergence rate curve lihood (ML) analysis, I used Modeltest 3.06 Posada for mitochondrial coding region estimated by Ho et al. and Crandall, 1998) with PAUP* to determine the model of (2005) using data from variety of avian lineage and also age evolution and parameter estimates by Akaike Information Cri- of calibration point (d D 1/ (¡ exp(¡ td)+ktd + ), where terion (AIC) value.