Ibis (2015), 157, 312–325 Taxonomy of the Narcissus Flycatcher Ficedula narcissina complex: an integrative approach using morphological, bioacoustic and multilocus DNA data 1 1 € 2,3 1 4 5 LU DONG, MIN WEI, PER ALSTROM, XI HUANG, URBAN OLSSON, YOSHIMITSU SHIGETA, YANYUN ZHANG1* & GUANGMEI ZHENG1 1Ministry of Education Key Laboratory for Biodiversity and Ecological Engineering, College of Life Sciences, Beijing Normal University, Beijing, China 2Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Chaoyang District, Beijing, 100101, China 3Swedish Species Information Centre, Swedish University of Agricultural Sciences, Box 7007, SE-750 07, Uppsala, Sweden 4Section of Systematics and Biodiversity, Biology and Environmental Sciences, University of Gothenburg, Box 463, SE-405 30, Gothenburg, Sweden 5Yamashina Institute for Ornithology, 115 Konoyama, Abiko, Chiba, 270-1145, Japan The taxonomy of the Narcissus Flycatcher Ficedula narcissina–Yellow-rumped Flycatcher Ficedula zanthopygia complex from East Asia has long been debated. Most authors recog- nize two species: F. narcissina, with the subspecies narcissina (most of Japan and Sakha- lin Island), owstoni (south Japanese islands) and elisae (northeast China) and F. zanthopygia (monotypic), although species status has been proposed for elisae and sometimes for owstoni. Here, we revise the taxonomy of this complex based on an inte- grative approach utilizing morphology, songs and mitochondrial and nuclear DNA for all taxa. All taxa were diagnosably different in plumage, and there were also structural dif- ferences among them, although the northernmost populations of owstoni (sometimes rec- ognized as jakuschima and shonis) were somewhat intermediate in plumage, structure and male plumage maturation between southern populations of owstoni and narcissina. All taxa had different songs, and a discriminant function analysis of four song variables correctly classified 100% of all songs. A strongly supported phylogeny was recovered based on three mitochondrial genes and three nuclear introns (total of 3543 bp), reveal- ing a sister relationship between F. zanthopygia and the other taxa, between F. n. narcis- sina and F. n. owstoni, and between F. n. elisae and F. n. narcissina + F. n. owstoni. The corrected COI distances among the three F. narcissina subspecies ranged from 2.8% (nar- cissina–owstoni) to 8.2% (narcissina–elisae). We suggest that the congruent differences in multiple independent traits and the deep genetic divergences among the four taxa in the F. narcissina–F. zanthopygia complex support treatment of all of these taxa as separate species. However, we acknowledge the paucity of data for F. owstoni and recommend further studies of this taxon. We suggest listing both F. elisae and F. owstoni, which have small and fragmented populations, as globally threatened. Keywords: DNA analysis, East Asia, Ficedula, morphology, phylogeny, vocalization. The Narcissus Flycatcher Ficedula narcissina breeds (Russia) to the Ryukyus (Japan) and is disjunctly on East Asian islands extending from Sakhalin distributed in northeast China (Fig. 1). Three sub- species are usually recognized: narcissina, owstoni and elisae (e.g. Vaurie 1959, Watson et al. 1986, *Corresponding author. Email: [email protected] Clements 2000, Dickinson 2003, del Hoyo et al. © 2015 British Ornithologists’ Union Taxonomy of the Narcissus Flycatcher complex 313 2006; Fig. 1). The taxa jakuschima (Tanegashima 2009), but no quantitative analysis has yet been & Yakushima, northern Ryukyu Islands) and shonis undertaken. (Amami & Okinawa, central Ryukyu Islands) are Ficedula narcissina elisae is a rare and poorly usually considered synonymous with owstoni known breeder in northeast China, in Beijing, (Vaurie 1954, Watson et al. 1986, del Hoyo et al. Hebei and Shanxi Provinces (Cai 1987, Cheng 2006, Ornithological Society of Japan 2012). The 1987, Wang et al. 2008), which winters on the Yellow-rumped Flycatcher Ficedula zanthopygia Thai-Malay Peninsula (Robson 2000, Wells 2007). has also been treated as a subspecies of F. narcissi- It has been proposed as a distinct species based on na (e.g. Hartert 1907, Delacour 1947, Flint et al. the analysis of bioacoustic and phenotypic charac- 1984), although it is more commonly treated as teristics (Zhang et al. 2006), although this was specifically distinct (Inskipp et al. 1996, Dickinson questioned by Eck (1998) and Topfer€ (2006) 2003, del Hoyo et al. 2006). Some authors have based on the somewhat intermediate appearance considered F. zanthopygia to form a superspecies (plumage, wing- and tail-length) of owstoni/jakus- with F. narcissina (Watson et al. 1986, del Hoyo chima in relation to elisae and narcissina, and lack et al. 2006), although that was rejected by Eck of analysis of the vocalizations of owstoni. More- (1996) based on the documentation that F. zantho- over, a first-summer (second calendar-year) male pygia and F. narcissina elisae are sympatric. elisae was inadvertently described as a new species, Recently, Outlaw and Voelker (2006, 2008) sug- Ficedula beijingnica (Zheng et al. 2000), as pointed gested that F. zanthopygia and F. narcissina narcis- out by Eck and Topfer€ (2005) and Topfer€ (2006) sina diverged only 200 000 years ago based on based on morphology, and by Zhang et al. (2006) mitochondrial sequence data. based on vocalizations. The breeding and wintering ranges of F. n. nar- We here aim to clarify the taxonomic status cissina cover exclusively East Asian islands, but it and phylogenetic relationships among the taxa in regularly occurs on the East Asian mainland dur- the F. narcissina–F. zanthopygia species complex ing migration. In the Russian far east, it is a com- based on analyses of mitochondrial and nuclear mon breeder on Sakhalin and also nests on the DNA, morphology and vocalizations. We also dis- southern Kuril Islands (Dement’ev & Gladkov cuss the conservation status of these taxa. 1951–1954, Flint et al. 1984); according to Brazil (2009), it also breeds in the Russian Far East METHODS (Ussuriland). In Japan, it breeds on all of the main islands and on Tsushima (Austin & Kuroda 1953, DNA sample collection Brazil 1991, Ornithological Society of Japan 2012). Its main wintering range appears to be in Fifty-one blood, feather or muscle samples of the the Philippines and on Borneo, although Cheng four taxa were obtained: 15 each of elisae, narcissi- (1987) reported that it winters also on Hainan na and zanthopygia and six of owstoni (Supporting and occasionally on Taiwan. On passage, it has Information Table S1). DNA was extracted from been recorded from southern Primorye, Russia blood and feather samples using the TIANamp (Dement’ev & Gladkov 1951–1954), to east and Genomic DNA Kit (Tiangen Biotech, Beijing, south China (Cheng 1987) and Indochina (Robson China) and resuspended in TE buffer. The supe- 2003). The parapatric F. narcissina owstoni rior umbilicus containing a blood clot or the whole (including jakuschima and shonis) breeds in Japan, calamus was used to extract DNA from feathers on Yakushima, Tanegashima and Tokara Islands, (Horvath et al. 2004). and rarely also on Iriomote and the Ryukyu Islands (Brazil 1991, Ornithological Society of Phylogenetic analyses Japan 2012). It is thought to be resident in the Ryukyu Islands (Ornithological Society of Japan Six loci were sequenced: the mitochondrial 2012); there is one record from mainland China NADH dehydrogenase 2 (ND2), NADH dehydro- (Wang & Cui 2007), and it has occurred in Tai- genase 3 (ND3) and cytochrome c oxidase subunit wan as a vagrant (Severinghaus et al. 2012). It has I (COI) genes; the autosomal ornithine decarboxy- been suggested that F. n. owstoni should be treated lase (ODC) introns 6 and 7 and myoglobin as specifically different from F. narcissina based on (MYO) intron 2; and the Z-linked chromo-heli- morphological and vocal traits (Otani 2002, Brazil case-DNA-binding gene intron 15 (CHDZ15). © 2015 British Ornithologists’ Union 314 L. Dong et al. 50°N 30°N 10°N 0 500 1000 km 100°E 120°E 140°E Figure 1. Distribution of the Ficedula narcissina complex. Green: Ficedula narcissina narcissina (dark: breeding, pale: wintering), orange: Ficedula narcissina elisae breeding, blue: Ficedula zanthopygia breeding (north) and non-breeding (Sumatra), pale purple: sympatric wintering range of F. n. elisae and F. zanthopygia, brown: Ficedula narcissina owstoni. Polymerase chain reactions (PCRs) were con- Flycatcher Ficedula albicollis, European Pied Fly- ducted following Friesen et al. (1999) and Dong catcher Ficedula hypoleuca, Mugimaki Flycatcher et al. (2013) with taxon-specific primers (Table Ficedula mugimaki and Slaty-backed Flycatcher S2). PCR products were purified with a WizardTM Ficedula sordida) and two more distantly related PCR Preps DNA purification kit (Promega Inc., Muscicapidae species (Siberian Blue Robin Larvi- Madison, WI, USA) and subsequently sequenced vora cyane and Red-flanked Bluetail Tarsiger cyanu- with each of the primers on an ABI 3730 auto- rus) were used as outgroups based on Sangster mated sequencer using a BigDye kit according to et al. (2010). Ficedula hodgsonii (J. Verreaux, recommended protocols (Applied Biosystems, 1871) was replaced by F. sordida (Godwin-Austen, Foster City, CA, USA). The chromatogram of 1874) because Muscicapella hodgsoni (Moore, each sequence was proofread by eye with the aid 1854) has been shown to be nested within Ficedu- of the program SEQUENCHER v. 4.0 (GeneCodes la (e.g. Zuccon &