Ibis (2016), 159, 204–216 Phylogenetic relationships, song and distribution of the endangered Rufous-headed Robin Larvivora ruficeps 1,2 € 1,3,4 5,6 7 1,2 1 1 MIN ZHAO, PER ALSTROM, RUOCHENG HU, CHAO ZHAO, YAN HAO, FUMIN LEI & YANHUA QU * 1Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 2University of Chinese Academy of Sciences, Beijing 100049, China 3Department of Animal Ecology, Evolutionary Biology Centre, Uppsala University, Norbyvagen€ 18D, 752 36 Uppsala, Sweden 4Swedish Species Information Centre, Swedish University of Agricultural Sciences, Box 7007, Uppsala SE-750 07, Sweden 5Research Centre for Nature and Society, School of Life Sciences, Peking University, Beijing 100871, China 6Shanshui Conservation Centre, Beijing 100871, China 7Cloud Mountain Conservation, Dali, Yunnan 671003, China The Rufous-headed Robin Larvivora ruficeps is one of the world’s rarest and least known birds. We summarize the known records since it was first described in 1905 from Shaanxi Province, central China. All subsequent Chinese records are from seven adjacent localities in nearby Sichuan Province. We studied its phylogenetic position for the first time using mitochondrial and nuclear markers for all species of Larvivora and a broad selection of other species in the family Muscicapidae. Our results confirmed that L. rufi- ceps is appropriately placed in the genus Larvivora, and suggested that it is sister to the Rufous-tailed Robin Larvivora sibilans, with these two forming a sister clade to a clade comprising both the Japanese Robin Larvivora akahige and Ryukyu Robin Larvivora komadori. Siberian Blue Robin Larvivora cyane and Indian Blue Robin Larvivora brunnea form the sister clade to the other Larvivora species. In contrast, song analyses indicated that the song of L. ruficeps is most similar to that of L. komadori, whereas the song of L. sibilans is relatively more similar to that of L. akahige, and songs of L. cyane and L. brunnea closely resemble each other. We used ecological niche modelling to estimate the suitable habitats of L. ruficeps based on the records from breeding grounds, suggest- ing that north and central Sichuan, south Gansu, south Shaanxi and south-east Tibet are likely to contain the most suitable habitats for this species. Keywords: Luscinia ruficeps,MAXENT, phylogeny, principal component analysis, taxonomy, vocalizations. described from there in 1905 by Hartert (Hartert INTRODUCTION 1907a,b, Li & Zhang 1986, Collar et al. 2001, The Rufous-headed Robin Larvivora ruficeps is one Clement & Rose 2015, Collar 2016; Fig. 1). There of the world’s least known birds. It is an endemic are only three records outside China: an adult male breeder in China, where it is thought to breed in was caught and ringed on 15 March 1963 in Mount temperate mixed coniferous and deciduous forest at Batu Brinchang, Cameron Highlands, Malaysia 2400–3000 m asl in southern Shaanxi and northern (McClure 1963); a first-winter female was observed Sichuan Provinces, although it has not been in Phnom Penh, Cambodia, on 16–20 November observed in the former province since it was first 2012 (Mahood et al. 2013, Clement & Rose 2015); and an adult female was photographed at Genting *Corresponding author. Highlands, Malaysia, on 20 April 2014 (www.sites. Email: [email protected] google.com/site/mnsbccrc/Home/record-status-1/2014) © 2016 British Ornithologists’ Union Rufous-headed Robin 205 Figure 1. Records of the Rufous-headed Robin Larvivora ruficeps. ▲ Breeding grounds: (1) Taibai Mountain, Qinling Mountains, Shaanxi Province, China; (2–5) Nuorilang Waterfall, Pearl Shoal Waterfall, Keze Valley and Primeval Forest of Jiuzhaigou Nature Reserve, Sichuan Province, China; (6) Baihe, Sichuan Province, China; (7) Huanglongsi Nature Reserve, Sichuan Province, China; (8) Wanglang Nature Reserve, Sichuan Province, China. ■ Possible migratory stopover: (9) Phnom Penh, Cambodia. ● Possible wintering grounds: (10) Mount Batu Brinchang, Cameron Highlands, Malaysia; (11) Genting Highlands, Selangor, Malaysia. Map was obtained from the National Geomatics Center of China. Inset: Adult male, Jiuzhaigou, Sichuan Province, 17 May 2013. Photo: Pete Morris. [Colour figure can be viewed at wileyonlinelibrary.com] (Fig. 1, Table S1). All three are thought to represent has only recently been described properly (Mahood birds in migration. Larvivora ruficeps is categorized as et al. 2013, Clement & Rose 2015), and the females Endangered (EN) by IUCN due to its small popula- are more cryptic. The rich and beautiful song has tion, which is assumed to be declining as a result of been described briefly in Collar (2016) and more habitat loss and degradation (BirdLife International fully in Clement and Rose (2015), and sound 2013). There is also evidence of males being illegally recordings are available on Xeno-canto (www.xeno- captured and sold in the cagebird market (e.g. an canto.org; restricted access) and AVoCet (www. informal Chinese website for the cagebird keepers: avocet.zoology.msu.edu). www.bbs.18888.com). The genus Larvivora comprises six species: The adult male L. ruficeps has strikingly coloured Larvivora ruficeps, Rufous-tailed Robin Larvivora plumage (Fig. 1, Figure S1). The female plumage sibilans, Indian Blue Robin Larvivora brunnea, © 2016 British Ornithologists’ Union 206 M. Zhao et al. Siberian Blue Robin Larvivora cyane, Ryukyu at 2630 m asl on 25 June 2015, and a blood sam- Robin Larvivora komadori and Japanese Robin ple was obtained before the bird was released Larvivora akahige (Sangster et al. 2010, Dickin- (sample ID: IOZ20891). son & Christidis 2014, Gill & Donsker 2015). The inclusion of L. ruficeps in this genus was Sequencing and phylogenetic analyses tentative, as it was only based on overall per- ceived ‘similarity of its structure, song and beha- DNA was extracted using TransGen 2*EasyTaq viour to those of L. brunnea and L. cyane’ PCR SuperMix Kit following the manufacturer’s (Sangster et al. 2010). protocol. We amplified and sequenced the mito- Larvivora is included in the family Muscicapi- chondrial cytochrome b (Cytb, 1077 bp) and the dae, along with various groups of chats, flycatchers nuclear lactate dehydrogenase intron 3 (LDH, and other small passerines (Sangster et al. 2010, 535 bp), ornithine decarboxylase introns 6–7 Zuccon & Ericson 2010, Clement & Rose 2015, (ODC, 725 bp), and Myoglobin intron 2 (Myo, Collar 2016). Sangster et al. (2010) and Zuccon 675 bp), c. 3 kb in total. Primer pair H16065 and and Ericson (2010) analysed phylogenetic relation- L14990 (Desjardins & Morais 1990) was used for ships of this group using multiple molecular mark- Cytb, OD6 and OD8R (Allen et al. 2003) for ers, and identified several clades. They showed, for ODC, and Myo3F and Myo2 (Kimball et al. 2009) example, that the genus Luscinia (sensu Dickinson for Myo. Primer pair for LDH (LDHP5: 2003) was non-monophyletic, and Sangster et al. GCTTGCTCTGGTTGAYGTTATGG, LDHP6: (2010) proposed a revised classification by dividing CACATTCCTCTGCACYAGGTTGAG) was Luscinia into Calliope, Larvivora, Tarsiger and Lus- designed by Urban Olsson, with annealing temper- cinia sensu stricto. This taxonomic revision has sub- ature of 58 °C. Sequences were later assembled in sequently been accepted by Dickinson and DNAStar LASERGENE 7.1 SEQMAN (Burland 2000), Christidis (2014), Gill and Donsker (2015) and and checked for homologues in NCBI Nucleotide Clement and Rose (2015). BLAST (www.blast.ncbi.nlm.nih.gov/Blast.cgi). Using multilocus data, the present study aims We obtained sequences from 35 additional spe- to test the hypothesis of Sangster et al. (2010) that cies of Muscicapidae from GenBank, representing L. ruficeps belongs in the genus Larvivora. We also primary clades within the family (based on Sangster describe its song and compare it with its close rela- et al. 2010), with Turdus merula and Myadestes uni- tives, use ecological niche modelling to estimate color from the sister family Turdidae as outgroups. the suitable habitats and summarize the known ODC sequences from Monticola cinclorhynchus and records of this poorly known species. Monticola rufiventris were unavailable. See Table S2 for GenBank accession numbers. Sequences were aligned using MEGA 5 (Tamura METHODS et al. 2011), and necessary manual adjustment was Information about records of L. ruficeps (including carried out to the alignment. Substitution models field reports, photographs, sound-recordings and were determined by the Bayesian information cri- specimens) was obtained from GBIF (www.gbi- terion both in PARTITIONFINDER (Lanfear et al. f.org), eBird (www.ebird.org), BirdLife 2012): HKY + I + G for Cytb, separating (www.birdlife.org/datazone/species), IUCN 1st + 2nd codon positions from 3rd codon posi- (www.iucnredlist.org), the Internet Bird Collection tion, and in JMODELTEST (Guindon & Gascuel (www.ibc.lynxeds.com), Oriental Bird Club 2003, Darriba et al. 2012) on each locus: GTR + I Images (www.orientalbirdimages.org), Xeno-canto + G for Cytb, LDH and Myo, and GTR + G for (www.xeno-canto.org), AVoCet (www.avocet.zo- ODC. ology.msu.edu), the literature, and personal obser- Trees were reconstructed in MRBAYES 3.2 vations and contacts. China Bird Report (Ronquist & Huelsenbeck 2003), both single locus (www.birdreport.cn) and BirdTalker Records Cen- analyses and all loci concatenated. The concate- ter (www.birdtalker.net) were also checked, but nated sequences were partitioned by locus, and for neither of them included any records of L. rufi- Cytb in two codon partitions (1st + 2nd codon ceps. positions and 3rd codon position). Markov chains A single adult male L. ruficeps was captured in with an incremental heating temperature of 0.2 Baihe, Sichuan Province, China (33°20N, 104°60E), were run for 10 million generations and sampled © 2016 British Ornithologists’ Union DEL in R unique strophes fromtinuously every singing song malesspecies were were (Tables generated 1 used. andwere Sonograms S3). chosen Only of to recordings represent of con- the author P.A. or downloaded from Xeno-canto. and songs of Province (see Table S3).six We from compared Jiuzhaigou these and with one from Baihe, Sichuan M every 1000th generation.