Taxonomy of the Narcissus Flycatcher Ficedula Narcissina Complex: an Integrative Approach Using Morphological, Bioacoustic and Multilocus DNA Data

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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 integrative approach utilizing morphology, songs and mitochondrial and nuclear DNA for all taxa. All taxa were diagnosably different in plumage, and there were also structural differences among them, although the northernmost populations of owstoni (sometimes recognized 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. narcissina 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% (narcissina–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 subspecies 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.

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).

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 & Ericson 2010) and has priority Inc., Ann Arbor, MI, USA), and every point muta- over F. hodgsonii (Zuccon 2011). We used PHASE v. tion was checked for authenticity. The sequences 2.1.1 (Stephens et al. 2001, Stephens & Donnelly were aligned with CLUSTAL W in MEGA v.5.0 (Tam- 2003) implemented in DNASP v. 5 (Librado & Ro- ura et al. 2011) and refined manually. Six Ficedula zas 2009) to infer the haplotypes among heterozy- species (Taiga Flycatcher Ficedula albicilla, Red- gous sites for each nuclear locus and individual. breasted Flycatcher Ficedula parva, Collared The haplotype with the greatest posterior

probability was used for calculation of genetic using Avisoft-SAS Light (Avisoft Bioacoustics, diversity. Models of nucleotide substitution were Glienicke, Germany). Duration of strophe (DS), selected using JMODELTEST v.0.1.1 (Guindon & maximum frequency (Fmax), minimum frequency Gascuel 2003, Posada 2008), based on the Akaike (Fmin) and frequency range (Fr) were measured for information criterion (AIC). The best-fitting sub- each song strophe with Avisoft-SAS Light. Mean stitution model varied among the genetic markers, values of all measured song strophes of an individ- TrN93+ I+Γ for ND2 and COI, TrN93+I for ual were calculated and used in analyses. One-way ND3, TrN93+Γ for ODC, HKY+I for CHDZ15, ANOVA was conducted with multiple comparisons and K80 for MYO. The concatenated sequences on the basis of non-normal distribution in owstoni, partitioned by locus were analysed in MRBAYES and Tamhane’s T2 test was used for pairwise com- v.3.1.2 (Huelsenbeck & Ronquist 2001, Ronquist parisons due to non-homogeneity of variances. & Huelsenbeck 2003). When specific models Acoustic differentiation among the different taxa selected by JMODELTEST were not implemented in was analysed by discriminant function analysis MRBAYES, the closest model in MRBAYES was used. (DFA). All statistical analyses were performed with Two independent simultaneous Markov chain SPSS v. 20.0 (SPSS Inc., New York, NY, USA). Monte Carlo (MCMC) analyses, each with six chains, were run for 5 million generations and Morphology sampled every 1000 steps. Convergence towards the stationary distribution was confirmed in TRACER Plumage characteristics were studied at the Insti- v.1.4.1 (Rambaut & Drummond 2007), with tute of Zoology, Chinese Academy of Sciences, ESS > 200, and by ensuring that the deviation of Beijing (IOZ) (13 adult/first-summer males of nar- split frequencies between the two independent cissina, c. 100 elisae of both sexes); Natural History runs was < 0.01. The first 1250 trees were dis- Museum, Tring, UK (BMNH) (c. 100 narcissina of carded as burn-in. A species tree was reconstructed both sexes, one adult male owstoni and seven using *BEAST (Heled & Drummond 2010). *BEAST adult/first-summer males narcissina/owstoni); and a estimates gene trees and species trees simulta- loan to the BMNH from the American Museum neously, incorporating multi-locus data from mul- of Natural History (AMNH) (three adult males, tiple individuals. Default settings were used and two first-winter males, one first-summer male and different substitution models were assigned to each four females owstoni (‘jakuschima’)). In addition, locus based on the results from ‘jModelTest’.In many individuals of all taxa and in different total, 1 9 108 MCMC generations were run, plumages were observed in the field, mainly in sampled every 1000 generations. Furthermore, the China and Japan. concatenated data were analysed by maximum We measured 158 male specimens at the IOZ: likelihood bootstrapping (1000 replicates) using 66 zanthopygia,13narcissina and 79 elisae.Mea- RAXML-HPC2 v. 7.6.3 (Stamatakis 2006, Stamatakis surements of the following characters were taken et al. 2008) on the Cipres portal (Miller et al. using callipers, to the nearest 0.1 mm: bill-length 2010). The data were partitioned by locus, and (from bill tip to skull), head-length (including bill) GTRCAT was used for the bootstrapping phase; and tarsus-length. Flattened wing-length and tail- GTRGAMMA was used for the final tree inference. length (tail tip to base of under tail-coverts) were To estimate genetic divergences among taxa, measured by ruler with 1-mm precision. In addi- uncorrected p-distances and best-fitting model tion, six F. n. owstoni males were measured from genetic distances were calculated for COI in MEGA the Yamashina Institute for Ornithology and wing- 5.0 following the recommendations of Fregin et al. length was measured for five F. n. owstoni males (2012). (of which three were labelled jakuschima) from the AMNH. The Kolmogorov–Smirnov test was used to assess normality of the morphometric data. Vocalizations One-way ANOVA and discriminant function analyses Recordings of 57 individuals, comprising 851 song (DFAs) were conducted to test for differences in strophes, belonging to all taxa in the complex, the morphometric space among taxa, and the were analysed (Supporting Information Table S3). least-significant difference (LSD) test was used for Most of the recordings were the same as those used pairwise comparison based on homogeneity of var- by Zhang et al. (2006). Sonograms were produced iance for the characters. We combined adults and

first-spring males in the analyses, as a previous genetic diversity in the mitochondrial loci and study found no evidence of age-related differences MYO than the other taxa and similar diversity to for any character (Wei 2010). All statistical analy- zanthopygia in ODC and CHDZ15 (Table 1). The ses were performed with SPSS v. 20.0 for Windows. taxon narcissina had considerably higher nucleotide A P < 0.05 value was considered significant for all diversity than the other taxa across most loci with analyses. the exception of at the COI locus (Table 1). All taxa in the F. narcissina–F. zanthopygia complex were recovered as reciprocally monophyletic RESULTS with high statistical support in analyses where all loci were included (Fig. 2). Ficedula zanthopygia Phylogeny and genetic divergence was found to be a sister to the other taxa with We obtained up to 3543 base pairs (bp) of com- strong support. The tree was further divided into bined DNA sequence data: ND2 (957 bp), ND3 one branch representing elisae and its sister clade and flanking regions (414 bp), COI (576 bp), comprising narcissina and owstoni, with both clades ODC (449 bp), MYO (646 bp) and CHDZ15 strongly supported. The *BEAST phylogeny was (501 bp). Because of the low quality of feather- congruent with the tree derived from concatena- extracted DNA, sample sizes varied among the loci tion, with strong support for nearly all inferred (Table S1). No stop codons were observed in the relationships (Fig. 2). The single-locus analyses mitochondrial genes. The taxon owstoni had lower resulted in well-supported, congruent topologies

Table 1. Genetic diversity within the Ficedula narcissina–Ficedula zanthopygia complex. Sample size for the three nuclear markers (ODC, MYO and CHDZ15) was counted as phased sequences.

Locus zanthopygia narcissina owstoni elisae

ND2 n 15 15 6 15 p (%) 0.189 Æ 0.03 0.424 Æ 0.067 0.056 Æ 0.018 0.147 Æ 0.033 h 7122 7 D 0.895 0.971 0.535 0.781 ND3 n 12 14 6 13 p (%) 0.429 Æ 0.106 0.501 Æ 0.095 0 0.325 Æ 0.075 h 6615 D 0.758 0.78 0 0.769 COI n 7937 p (%) 0.347 Æ 0.101 0.193 Æ 0.077 0.116 Æ 0.055 0.43 Æ 0.073 h 3424 D 0.667 0.583 0.667 0.81 ODC n 24 10 8 18 p (%) 0.725 Æ 0.083 1.831 Æ 0.16 0.851 Æ 0.34 1.319 Æ 0.139 h 14 10 5 14 D 0.957 1 0.786 0.967 MYO n 20 20 6 22 p (%) 0.674 Æ 0.074 0.697 Æ 0.115 0.155 Æ 0.1 0.181 Æ 0.038 h 14 14 2 6 D 0.937 0.916 0.333 0.719 CHDZ15 n 22 12 10 24 p (%) 0.502 Æ 0.093 1.415 Æ 0.118 0.656 Æ 0.137 0.44 Æ 0.053 h 7105 9 D 0.818 0.97 0.822 0.902

n, sample size; p (%), nucleotide diversity; h, number of haplotypes; D, haplotype diversity.

(Supporting Information Fig. S1) with the excep- (owstoni–narcissina) to 8.2% (narcissina–elisae), tion that narcissina was paraphyletic with respect and within the F. narcissinia–F. zanthopygia com- to owstoni for all three nuclear markers (owstoni plex reaching up to 13% (zanthopygia–narcissina) haplotypes monophyletic). (Table 2). In comparison, pairwise divergences Among the outgroup Ficedula species, F. parva– among the outgroup Ficedula sister species ranged F. albicilla, F. albicollis–F. hypoleuca and F. mugi- between 2.0% (hypoleuca–albicollis) and 6.7% maki–F. sordida were inferred to be sisters with (mugimaki–sordida) (Table 2). As expected, the strong support in all analyses (Figs 2 and S1). The uncorrected-p genetic distances were slightly lower ﬁrst two of these pairs formed a well-supported than the corrected distances (Table 2). clade in all analyses, whereas the sister relationship between these and the third pair was poorly sup- Vocalizations ported in all analyses except in the concatenated multilocus analysis. The songs of the taxa in the F. narcissina complex The pairwise COI distances under the are markedly different. The differences are easily TrN93+I+Γ model varied among the three subspe- audible and clearly visible on sonograms. Ficedula cies in the F. narcissina complex: from 2.8% zanthopygia sings short strophes consisting of a

F. n. elisae E1 1.00 E18 F. n.owstoni E11 1.00 E40 1.00 E4 F. n.narcissina E6 E21 F. zanthopygia 1.00/100 E59 el6 1.00 F. sordida el7 el1 F. mugimaki el2 F. n. elisae el3 F. hypoleuca 1.00/100 el4 1.00 el5 F. albicollis o7 1.00 1.00/100 o1 o5 1.00 F. albicilla o2 F. n. owstoni 1.00/100 o4 F. parva o3 na1 Tarsiger cyanurus UW2 1.00/97 S2 Larvivora cyane S4 0.004 na4 na5 N2 na6 1.00/- HN1 S1 S3 F. n. narcissina na2 na3 N1 N3 B1 B2 B3 B4 B7 0.99/- B5 B6 B9 1.00/100 UW4 B11 z1 UW1 F. zanthopygia UW3 z2 z3 1.00/100 F. parva 0.99/85 1.00/100 F. albicilla 1.00/100 F. albicollis F. hypoleuca 1.00/100 0.99/97 F. mugimaki F. sordida Tarsiger cyanurus Larvivora cyane

0.3

Figure 2. Bayesian inference tree for the Ficedula narcissina–Ficedula zanthopygia complex with outgroup species, based on partitioned analysis of concatenated sequences from three mitochondrial genes (COI, ND2 and ND3) and three nuclear loci (CHDZ15, MYO and ODC). Inset: species tree, reconstructed by *BEAST using the same data as in the other analysis. Numbers above branches indicate Bayesian posterior probabilities (ﬁrst) and bootstrap values generated from maximum likelihood (second, on gene tree) (only values > 0.70/70% given). A photo of an adult male of each taxon is shown. Ficedula narcissina elisae © Dongsheng Guo. Ficedula narcissina owstoni © Pete Morris, Ficedula narcissina narcissina, and F. zanthopygia © Nial Moores/Birds of Korea (www.birdskorea.org).

Table 2. Pairwise COI distances in the Ficedula narcissina– and minimum frequency were the most useful Ficedula zanthopygia complex and outgroup Ficedula sister variables for discriminating elisae from the other species. three taxa, and duration and minimum frequency were the most important characters separating nar- Taxa Uncorrected p TN93+I+Γ cissina, zanthopygia and owstoni. The DFA cor- F. n. elisae–F. n. narcissina 7.0 Æ 1.0 8.2 Æ 1.3 rectly classified 100% of the songs (Table 4). The F. n. elisae–F. n. owstoni 6.6 Æ 1.0 7.6 Æ 1.3 variables that contributed most to discriminating – Æ Æ F. n. owstoni F. n. narcissina 2.6 0.7 2.8 0.8 among the groups were frequency range and mini- F. zanthopygia–F. n. elisae 8.9 Æ 1.1 10.7 Æ 1.6 F. zanthopygia–F. n. narcissina 10.4 Æ 1.1 13.0 Æ 1.8 mum frequency (Table S5A). F. zanthopygia–F. n. owstoni 9.4 Æ 1.1 11.3 Æ 1.7 F. albicilla–F. parva 5.9 Æ 0.9 6.6 Æ 1.1 F. albicollis–F. hypoleuca 1.9 Æ 0.6 2.0 Æ 0.6 Morphology – Æ Æ F. mugimaki F. sordida 5.9 0.9 6.7 1.2 All taxa were diagnosably different in adult male and female plumages (Table 5), although the series of low, melodious whistles (Fig. 3, Z1–Z16; plumage of adult male owstoni appeared to vary Table 3). The song of elisae is a soft and clear, clinally; the southernmost birds (from Iriomote) though a slightly harsh warble with more variation were most different from narcissina and the in frequency, consisting of usually long phrases northernmost birds (from Yakushima) approached separated by short pauses (on average slightly narcissina (female owstoni not studied throughout longer than the strophes) (Fig. 3, E1–E5; Table 3). range). Three adult and five first-summer males The song of narcissina is more varied, with fre- collected in southeast China in April and May quent repetitions, more frequent and more irregu- (BMNH; presumably on migration and thus of lar pauses and more dramatic changes in speed unknown provenance) were somewhat intermedi- and pitch than in elisae (Fig. 3, N1–N5; Table 3). ate between narcissina and owstoni: blackish-grey The syllables of owstoni are basically as for narcissi- above with a faint greenish tinge and orange- na, but much simpler and shorter, with less varia- yellow on the throat/upper breast, and some of tion in frequency (Fig. 3, O1–O12; Table 3). them showed grey-fringed lesser coverts. Significant differences were found in most pair- First-summer (second-calendar year) male nar- wise comparisons among the taxa, although cissina and zanthopygia resembled adult males, F. zanthopygia and F. n. owstoni were only signifi- although with contrastingly brown retained juve- cantly different in minimum frequency (Tam- nile outer greater coverts, alula, primary coverts hane’s T2 test, P < 0.001) (Supporting and remiges. Most individuals also showed some Information Table S4A). In particular, the songs of retained olive-brown first-winter feathers above, zanthopygia and owstoni were shorter, with lower particularly on the nape. In contrast, first-summer maximum frequency than the others, and elisae male owstoni were closer to females than to adult had considerably higher maximum frequency and males. However, extensive individual variation was larger frequency range than the others (Table 3). observed in first-summer male owstoni: some indi- The DFA based on the five song characters viduals were female-like, but with some male showed a clear separation among the taxa, with characters (e.g. a trace of a yellow supercilium and the first discriminant function accounting for a little yellow on the throat, breast and rump), 96.5% of the total variation (Wilks’s lambda = whereas others were more evidently males, show- 0.022, P < 0.001, Fig. 4). The frequency range ing extensively yellow throat. The development of

Table 3. Song measurements in the Ficedula narcissina–Ficedula zanthopygia complex (mean Æ 1 SD).

F. zanthopygia F. n. narcissina F. n. owstoni F. n. elisae

DS 0.83 Æ 0.04 3.49 Æ 1.94 0.71 Æ 0.11 2.99 Æ 0.93

Fmax 4.42 Æ 0.13 5.20 Æ 0.45 4.53 Æ 0.43 7.70 Æ 0.60 Fmin 1.84 Æ 0.06 1.70 Æ 0.34 2.29 Æ 0.27 1.45 Æ 0.10 Fr 2.58 Æ 0.13 3.49 Æ 0.38 2.24 Æ 0.31 6.25 Æ 0.63

DS, duration of strophe (s); Fmax, maximum frequency (kHz); Fmin, minimum frequency (kHz); Fr, frequency range (kHz).

Figure 3. Spectrograms of single song strophes in the Ficedula narcissina–Ficedula zanthopygia complex. Z, zanthopygia;E,elisae; N, narcissina;O,owstoni. Strophes separated by dotted lines are from the same individual. All others are from different individuals.

male plumage appeared to be clinal in owstoni, between narcissina and owstoni mentioned above with southern birds being on average less advanced were all basically adult male-like. In contrast to all than northern ones. The first-summer plumage of other taxa, first-summer male F. n. elisae resem- the northernmost populations of owstoni (‘jakuschi- bled females (rarely with a few distinct male char- ma’) was as advanced as in narcissina. The five acters, such as some newly moulted blackish first-summer males with intermediate plumage rectrices, a few yellow rump feathers or a short

Table 4. Classification accuracy (%) of the Ficedula narcissina–Ficedula zanthopygia complex obtained through a discriminant function analysis based on five song characters (before slashes) and five morphometric characters (after slashes). The taxa being com- pared are listed in the rows and columns.

F. zanthopygia F. n. narcissina F. n. owstoni F. n. elisae

F. zanthopygia 100/78.3 0/4.3 0/6.5 0/10.9 F. n. narcissina 0/0 100/100 0/– 0/0 F. n. owstoni 0/16.7 0/0 100/83.3 0/– F. elisae 0/13.7 0/8.2 0/– 100/78.1

yellow supercilium). They showed distinct whitish owstoni (8.2%) was higher than the pairwise com- tips to the retained juvenile greater coverts, in parisons between the other Ficedula sister species common with first-summer females, but unlike in the present analysis: F. albicilla vs. F. parva adult females. This was confirmed in the field, 6.6%, F. albicollis vs. F. hypoleuca 2.0%, F. mugi- with many observations of singing birds in female- maki vs. F. sordida 6.7%. The divergence between type plumage. F. n. narcissina and F. n. owstoni (2.8%) was Significant differences were found in pairwise higher than between F. albicollis and F. hypoleuca. comparisons of wing-length (except owstoni vs. The very shallow divergence between F. zanthopy- zanthopygia) and tail-length (except narcissina vs. gia and F. n. narcissina found by Outlaw and Vo- elisae). Ficedula narcissina had a significantly elker (2006, 2008) was not supported by our data. shorter bill than the other three taxa (Tables 5 They listed two F. narcissina samples (UWBM and S4B). However, in tarsus-length and head- 46988 and 51116), which were said to have iden- length, the significant difference was only observed tical sequences, but the latter of these is in fact between owstoni and other taxa (except zanthopy- F. zanthopygia, as confirmed by Sharon Birks (in gia vs. elisae in head-length; Table S4B). Within litt.) and resequencing of ND2, cyt b and ND3 for owstoni there were indications of decreasing wing- these two samples confirmed that they have differ- length from north to south (Table 5). The DFA of ent sequences, matching narcissina and zantho- morphometrics found significant differences among pygia, respectively. The F. narcissina sequences the taxa, with the three discriminant functions submitted to GenBank in Outlaw and Voelker accounting for 100% of the total variance, mainly (2006) were identical to those we obtained from due to the contributions of wing-length and tail- UWBM 51116 (in fact F. zanthopygia) but not to length (Wilks’s lambda = 0.44, P < 0.001, Sup- UWBM 46988 (F. narcissina). Thus, the shallow porting Information Fig. S2, and Table S5B). In divergence demonstrated between F. narcissina addition, the wing formula differed significantly and F. zanthopygia by Outlaw and Voelker (2006) among all taxa (Table 5). actually represented intraspecific divergence in F. zanthopygia and not interspecific divergence. Our data support the treatment of F. n. elisae DISCUSSION as a distinct species, as proposed by Zhang et al. Analyses of vocalizations, morphology and mito- (2006), Brazil (2009), Zheng (2011), and Gill and chondrial DNA congruently identified four distinct Donsker (2013). It breeds allopatrically with nar- lineages in the F. narcissina–F. zanthopygia com- cissina and is in widespread sympatry with plex, representing zanthopygia, elisae, narcissina F. zanthopygia, without any evidence of inter- and owstoni. The nuclear DNA corroborated these breeding between them. In view of the morpho- results, except that owstoni was recovered in a logical distinctness of elisae, it may seem surprising clade that was nested within narcissina (see that it has been treated as conspecific with F. nar- below). In both the concatenation and the species cissina. However, as remarked by Vaurie (1954, tree analyses, continental F. zanthopygia was inferred 1959), Eck (1998) and Topfer€ (2006), owstoni to be sister to the others, and the continental (including ‘jakuschima’ and ‘shonis’) appears to F. n. elisae wassistertotheinsularF. n. narcissina bridge the morphological gap between elisae and and F. n. owstoni. The corrected COI distance narcissina, as it is somewhat intermediate between between F. n. elisae and F. n. narcissina/F. n. these taxa in plumage, wing- and tail-length and,

Table 5. Morphological characteristics in the Ficedula narcissina–Ficedula zanthopygia complex. Values are in millimetres and repre- sent means Æ SD, with ranges in parentheses and sample size where appropriate.

F. zanthopygia F. n. narcissina F. n. owstoni F. n. elisae

Adult male Colour of White Orange-yellow Yellow Yellow supercilium Length/shape Long, wide Very long, wide Very long, wide Short, narrow of supercilium Crescent Absent Absent Absent Yellow below eye Crown-mantle Black Black Dark green-grey Dull greyish-green Rump patch Large, including Large, including Large, including back Smaller, usually back back not including back Orange tinge Usually faint or Usually strong Faint or lacking Lacking to throat lacking, rarely rather strong Belly Yellow Whitish or pale Whitish or pale yellowish Yellow yellowish Lesser coverts Black Black Grey-fringed Largely grey Remiges Black Black Black, indistinct Dark grey, edged greenish-grey edges pale greenish or brownish Pattern on Long, broad white Unpatterned (little Short white outer edge Unpatterned base of longest outer edge white concealed tertial by greater coverts) Plumage 1st-sum ad male 1st-sum ad male 1st-sum female 1st-sum female maturation (rarely ad male)a in male Adult female Upperparts Greenish grey-brown; Grey-brown; Grey-brown with Greyish-green dark grey uppert-covs; rufous-tinged greenish tinge bright yellow rump uppert-covs Underparts Whitish with pale Whitish, with faint Whitish, with faint Mainly yellow yellow tinge or pale bufﬁsh tinge on bufﬁsh or yellowish yellow throat/breast tinge Wings Broad white tips to Indistinct pale Indistinct pale Indistinct pale inner med + gr covs; tips/edges to tips/edges to tips/edges to distinct white outer med + gr covs + tert med + gr covs + tert med + gr edge to longest tert covs + tert Morphometrics (males only) Wing-length 70.98 Æ 1.62 (67–75; 64) 77.30 Æ 1.49 (75–81; 13) Okinawa: 69.31 Æ 1.44 72.70 Æ 1.89 (66.5–71.5; 7); (68–79; 79) Yakushima: 76.3 Æ 0.29 (76–76.5; 3) Tarsus-length 16.15 Æ 0.41 (15.2–17.0; 65) 16.04 Æ 0.53 (14.9–16.8; 12) 16.91 Æ 0.80 (16.0–18.0; 6) 15.88 Æ 0.46 (13.4–16.8; 77) Tail-length 44.33 Æ 1.68 (42–53; 66) 47.69 Æ 1.31 (45–50; 13) 42.0 Æ 1.34 (40–44; 6) 47.24 Æ 2.07 (40–52; 79) Head-length 31.51 Æ 0.98 (30.1–33.6; 49) 31.38 Æ 0.66 (30.2–32.5; 7) 30.20 Æ 1.11 (28.3–31.4; 6) 31.25 Æ 0.80 (28.8–33.0; 75) Bill-length 10.32 Æ 0.43 (9.2–11.4; 54) 9.79 Æ 0.51 (9.1–10.4; 11) 10.63 Æ 0.60 (9.8–11.2; 6) 10.30 Æ 0.50 (9.2–12.2; 75) Wing formula (both sexes) Wing formula P9 = P5 or = P4/P5 P9 = P5/P6 P9 = P4/P5 P9 = P4/P5 or = P4 Emarginations P6–8P6–8P6–8P5–8 on primaries

1st-sum, ﬁrst-summer plumage (i.e. 2nd calendar-year); ad, adult; P, primary (numbered descendently); med covs, median coverts; gr covs, greater coverts; uppert-covs, uppertail-coverts; tert, tertial. aMost individuals were more female- than male-like, but many showed deﬁnite male characters, particularly in the north of the range (see main text for details).

different from narcissina, although the somewhat F. zanthopygia intermediate northern populations of owstoni (‘ja- F. n. narcissina ’ 4 F. n. owstoni kuschima ) complicate the picture, and we have F. n. elisae only examined a few individuals. The song appears to differ strongly from narcissina (as also pointed out by Brazil 2009), although all of our recordings 2 of owstoni are from Okinawa. Ficedula owstoni and Function 2 F. narcissina are reciprocally monophyletic in the trees based on mitochondrial sequences and all loci combined, although larger sample sizes of owstoni 0 might have revealed a different picture (all our samples of owstoni are from Iriomote Island, Table S1). The divergence between owstoni and narcissina is the shallowest in the F. narcissina–F. zanthopygia –2 complex, as evidenced both in the phylogeny and by the pairwise genetic distances. The paraphyly of narcissina with respect to owstoni in nuclear mark- –6 –3 0 3 6 ers may be due to incomplete lineage sorting, Function 1 which is more likely in nuclear than in mitochondrial markers due to the larger effective population Figure 4. Discriminant function analysis based on five acous- size of the former (e.g. Avise 1994, Zink & tic parameters in the Ficedula narcissina–Ficedula zanthopygia Barrowclough 2008), although hybridization complex. between these two parapatric taxa cannot be rejected. Again, larger sample sizes would be desir- as shown here, also in wing formula and acquisi- able to investigate this further. As stressed by Als- tion of adult male plumage. Moreover, there trom€ et al. (2011), the reciprocal monophyly in the appears to be clinal variation in all these aspects *BEAST species tree is an unfortunate necessary out- within owstoni, with northern birds (‘jakuschima’) come of the method, as *BEAST requires all prede- most approaching narcissina and southern birds, fined taxa to be monophyletic. which are geographically closest to China, most In our opinion, the congruent differences approaching elisae. However, adult male owstoni between owstoni and narcissina in multiple suites actually differ in a larger number of morphological of traits suggest that these taxa have had indepen- characters from adult male elisae than adult male dent histories for a substantial period of time, and narcissina do from adult male F. zanthopygia. support the treatment by Zhang et al. (2006), Bra- We tentatively agree with Vaurie (1954) and zil (2009) and Zheng (2011) of owstoni as a dis- Mayr and Cottrell (1986) in synonymizing jakus- tinct species. We propose the following chima and shonis with owstoni. However, at least arrangement: (1) F. zanthopygia (Hay, 1845), (2) ‘jakuschima’, the northernmost lineage, is perhaps F. elisae (Weigold, 1922), (3) F. narcissina (Tem- better considered an intergrade between owstoni minck, 1836) and (4) F. owstoni (Bangs, 1901). and narcissina. As our sample of ‘jakuschima’ was very small, further research is needed. The differ- Conservation status of F. elisae and ences in length and shape of the wings between F. owstoni northern and southern populations within narcissina–owstoni might reflect differences in migratory Ficedula elisae is subject to habitat loss and frag- behaviour, and conform to general trends in passe- mentation in both its restricted breeding range rines in which long-distance migrants have longer in northeast China and its winter range in the and more pointed wings than short-distance Thai-Malay Peninsula. Its known breeding range migrants (Norberg 1989, Marchetti et al. 1995, is similar to the global range of the Brown Baldwin et al. 2010). Eared-pheasant Crossoptilon mantchuricum and to The taxonomic rank of owstoni is somewhat sub- the breeding range of Grey-sided Thrush Turdus jective as it is parapatric with narcissina. At least feae, which are both globally threatened southern populations are diagnosably morphologically (Vulnerable) because of their small and declining

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Zink, R. & Barrowclough, G. 2008. Mitochondrial DNA under Table S5. Standardized discriminant function coef- siege in avian phylogeography. Mol. Ecol. 17: 2107–2121. ﬁcients of: (A) vocal DFA, DS, duration of strophe Zuccon, D. 2011. Taxonomic notes on some Muscicapidae. (s); F , minimum frequency (kHz); F ,frequency Bull. Brit. Orn. Club 131: 196–199. min r range (kHz), and (B) morphometric DFA. Received 12 February 2014; Audio S1. Ficedula zanthopygia song. Beijing revision accepted 30 December 2014. including Z5–Z7 in Figure 3, China. Associate Editor: Martin Collinson. Audio S2. Ficedula zanthopygia song. Beijing including song Z9–Z13, China. SUPPORTING INFORMATION Audio S3. Ficedula narcissina song. Taiwan including song N1 and N2, China. Additional Supporting Information may be found Audio S4. Ficedula narcissina song. Honshu in the online version of this article: including song N4. Japan. Figure S1. Bayesian haplotype tree of three Audio S5. Ficedula owtoni song. Okinawa mitochondrial genes (A: COI, B: ND2, C: ND3) including song O1–O3. Japan. and individual trees of three nuclear markers (D: Audio S6. Ficedula owtoni song. Okinawa ODC, E: MYO, F: CHDZ15). Posterior probabil- including song O4–O6. Japan. ity >50% is indicated at the nodes. Audio S7. Ficedula owtoni song. Okinawa Figure S2. Scatter plots of discriminant function including song O7–O12. Japan. analysis of morphometric traits in the Ficedula nar- Audio S8. Ficedula elisae song. Beijing including cissina–zanthopygia complex. song E1. China. Table S1. Samples used in the genetic analyses Audio S9. Ficedula elisae song. Beijing including with GenBank accession numbers. song E2. China. Table S2. Primer sequences used in this study. Audio S10. Ficedula elisae song. Beijing includ- Table S3. Songs of the Ficedula narcissina– ing song E3. China. F. zanthopygia species complex used in vocal analyses. Audio S11. Ficedula elisae song. Beijing includ- Table S4. Pairwise comparisons of vocal (A) and ing song E4–E5. China. morphometric characters (B). Appendix S1. Online supplementary references.