Headed Robin Larvivora Ruficeps

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. ruficeps 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)

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,

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 perceived ‘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

every 1000th generation. Default priors in 19.84 11.31 11.97 28.90 7.48 MRBAYES were used. We also used the coalescent- 8.97 – – – – – – based species tree method *BEAST, which simulta- Speed neously co-estimates gene trees and the species (notes/s) tree within which they are embedded (Heled & Drummond 2010) to estimate the phylogeny, with 25 4.78 13 3.13 20 1.83 68 9.37 23 2.56 9 2.41 – – – – – 50 million generations sampled every 1000th gen- – Number eration. In all Bayesian analyses, convergence to of notes the stationary distribution of the single chain was inspected in TRACER 1.6.0 (Rambaut et al. 2014) 5.81 5 2.07 4 4.31 3 2.58 19 4.82 5 2.76 2 – – – – – –

using a minimum threshold for the effective sam- (kHz) ple size (ESS). The joint likelihood and other Bandwidth parameter values indicated large ESSs (> 300, generally > 1000). Good mixing of the MCMC and 1.52 0.75 2.07 0.69 5.22 0.86 3.38 0.69 4.08 1.55 2.04 0.86 – – – – – search reproducibility were established by multiple – Delta runs from independent starting points. Topological time (s) convergence was examined by eye and by the average standard deviation of split frequencies (< 0.01). The ﬁrst 25% of the generations were discarded and the posterior probability was esti- 3.79 0.88 7.06 0.76 4.50 0.99 8.10 1.50 4.13 0.73 7.24 0.64 (kHz) – – – – – mated for the remaining generations. –

Single-locus and concatenated trees were also Peak frequency estimated in RAXMLGUI 1.5 (Silvestro & Michalak 2012), a Graphical User Interphase of RAXML 8 0.76 3.79 1.09 1.89 13 10.88 0.18 2.93 1.46 1.13 7 5.05 0.86 5.06 2.43 2.41 9 3.64 0.27 3.79 2.25 1.55 37 16.84 0.47 4.85 2.68 3.27 11 3.97 ﬁ 0.36 3.27 1.20 1.72 5 3.85 3.28 2.07 6.94 2.81 4.37 2.58 7.15 3.27 4.20 2.24 6.84 2.76 (Stamatakis 2014), using the models de ned by JMO- – – – – – – (kHz) DELTEST.ML+ Rapid bootstrap was used, and 1000 Mean frequency replicates were run for each analysis. GTRGAMMAI was used for Cytb, LDH, Myo and concatenation analyses, and GTRGAMMA for ODC. We also used the gene-tree-based coalescent 4.55 2.20 9.73 3.11 6.31 2.81 9.46 4.60 6.53 2.38 9.60 2.81 (kHz) – – – – – method MP-EST (Liu et al. 2010) to estimate a –

phylogeny with 1000 bootstrap calculation, on the Top frequency Species TRee Analysis Web server (Shaw et al. 2013), based on the rooted ML gene trees gener- 2.02 3.28 5.29 4.55 2.62 3.73 4.87 7.00 2.20 3.30 4.08 4.02 – – – – – ated by RAXML. Trees were viewed in FIGTREE v. – (kHz) 1.3.1 (Drummond 2012). Bottom frequency 2.35 5.56 4.18 1.35 3.65 2.50 2.54 6.71 4.90 1.92 4.80 3.37 2.63 8.61 5.66 et al. 1.62 4.66 3.16

Song analysis types We analysed songs of seven individuals of L. ruﬁceps, Strophe six from Jiuzhaigou and one from Baihe, Sichuan Province (see Table S3). We compared these with songs of L. sibilans, L. cyane, L. brunnea, L. akahige and L. komadori, which were either recorded by Total no. of strophes author P.A. or downloaded from Xeno-canto. Songs of at least eight individuals per species 7 98 53 0.94 were chosen to represent the ﬁve other Larvivora 8 67 43 1.05 10 197 66 1.57 10 93 3 1.25 11 108 67 1.62 14 101 9 1.13 species (Tables 1 and S3). Only recordings of con-

tinuously singing males were used. Sonograms of Characteristics of songs. Minimum, maximum and median values are given. See Figure S4 for detailed information. unique strophes from every song were generated ceps in RAVENPRO v. 1.5 (www.birds.cornell.edu/raven) ﬁ L. ru L. cyane L. sibilans L. brunnea L. komadori ﬁ L. akahige Table 1. with a xed spectrogram window size and Species Songs

brightness and contrast adjusted by the same We also calculated two indices, Blomberg’s observer (M.Z.). RAVENPRO settings were: window Kappa (Blomberg et al. 2003) and Pagel’s Lambda size = 256 samples; window type = Hann; 3 dB (Pagel 1999), in R studio (Munkem€ uller€ et al. filter bandwidth = 270 Hz; overlap = 50%; hop 2012) to measure and test phylogenetic signal to size = 2.67 ms; DFT size = 256 samples; grid check whether the PC values and transformed spacing = 188 Hz. Top frequency, bottom fre- acoustic measurements exhibited significant phylo- quency, peak frequency (the frequency at which genetic signal. PC values and transformed acoustic the strongest power occurs), bandwidth 90% (the measurements were averaged over all the individu- difference between the 5% and 95% frequencies, als from the same species for this analysis. hereinafter referred to as bandwidth), note number (the number of notes in one strophe) and delta Ecological niche modelling time (the difference between the Begin Time and End Time) were measured for each strophe in MAXENT v. 3.3.3k (Phillips et al. 2006) was used RAVENPRO according to the RavenPro User’s Man- to perform the ecological niche modelling (ENM) ual (Charif et al. 2010). These acoustic measure- of L. ruficeps, based on the eight breeding localities ments used in our study are shown in Figure 2. All in Sichuan and Shaanxi, China (Fig. 1). In this statistical analyses, e.g. normality tests of values case, MAXENT was chosen as it has been shown to and calculations of standard deviations, were con- work better than other approaches with limited ducted in PAST (Hammer et al. 2001). sample sizes (Phillips & Dudık 2008, Wisz et al. Principal component analysis (PCA) was per- 2008). formed by PAST, based on the correlation matrix of We obtained 27 environmental and bioclimatic the measurements of six characters: top frequency, variables for projection (Table S4): 19 bioclimatic bottom frequency, peak frequency, delta time, variables were downloaded from the WorldClim note number and average speed (note number/ 1.4 database (Hijmans et al. 2005), elevation, delta time). Measurements used in PCA were slope and aspect from the Shuttle Radar Topogra- averaged over all the strophes from the same indi- phy Mission (SRTM) image (Hof et al. 2012), dis- vidual. Further data transformation included loga- tance to water, soil type, distance to road and rithm transformation and mean subtraction. distance to railway from the Institute of Geogra- All the sound measurements and analyses were phy and Resource Research, Chinese Academy of carried out by the same person (M.Z.). Sciences, and land use type from the National

6 Single-element Note Introductory Multi-element Note 4 Element

Silent Pause Trill Delta Time kHz S 0.4 0.8 1.2 1.6

10 Top Frequency 95% Introductory Elements 8 Bandwidth 90%

6 5% Bottom Frequency 4 Trill 2 Delta Time

kHz S 0.4 0.8 1.2 1.6 2 2.4 2.8 3.2

Figure 2. Sound terminology, using one strophe from Larvivora ruﬁceps (top) and L. cyane (bottom), respectively, as examples. Consecutive strophes are separated by silent pauses.

Geomatics Centre of China (National Geomatics S4). This clade was sister to the L. akahige and Center of China 2013). L. komadori clade, and the sister relationship Ten bootstrap replicates were run in MAXENT, between these two clades was strongly supported. with random assignment of occurrences as training The Larvivora clade was sister to the Brachypteryx and testing data. The average of these 10 repli- clade, with high support. Single-locus analyses cated analyses was computed as output. Default were generally less well resolved and supported, settings and logistic output format (ranging from 0 although without strongly supported conflicts with to 1) were used in MAXENT. respect to Larvivora (data not shown). A distribution map was generated in ARCGIS 10.2.1. Elevation (2400–3000 m asl) and land use Songs type (woodland) were preset as filters. Habitats with predicted suitability lower than 0.5 were The song of L. ruficeps (Fig. 4) was built up of omitted from the map. short (0.88–2.07 s; median 1.46 s; n = 98) strophes (Table 1), each consisting of a single- or multi-element trill, which was usually immediately RESULTS preceded by one or more different-type notes and occasionally also succeeded by a single different- Phylogeny type note or a second type of trill. A single, All of the analyses inferred L. ruficeps to be sister drawn-out introductory note was frequently given, to L. sibilans, with good support (Fig. 3, Figs S2– clearly separated from the rest of the strophe. The

*/*/*/92 Myrmecocichla aethiops */86/0.96/66 Oenanthe leucopyga */*/*/95 Saxicola caprata 0.95/76/0.61/57 Saxicola torquata */*/*/99 Phoenicurus auroreus */97/0.95/68 Phoenicurus phoenicurus // */98/*/95 Monticola cinclorhynchus 0.99/58/- Monticola rufiventris */*/*86 Ficedula strophiata 0.9/-/-/- Ficedula superciliaris */*/*/99 Calliope calliope 0.89/-/-/- Calliope pectoralis */61/0.95/- Enicurus ruficapillus 0.84/-/-/- Myophonus caeruleus */97/*/50 Heinrichia calligyna 0.8/-/-/- Rhinomyias insignis */77/*/- Luscinia megarhynchos Luscinia svecica 0.57/-/-/- 0.95/88/0.91/77 Larvivora akahige Larvivora komadori */*/*/99 Larvivora ruficeps */99/0.98/68 0.98/59/0.94/85 Larvivora sibilans 0.86/-/-/- */*/*/* Larvivora brunnea */98/0.99/82 Larvivora cyane */*/*/95 Brachypteryx leucophrys 0.83/55/0.97/- Brachypteryx montana */*/*/85 Tarsiger chrysaeus Tarsiger cyanurus */88/-/50 */98/0.99/55 Cossypha cyanocampter Erithacus rubecula */*/*/* */96/*/- Cyornis rubeculoides Niltava davidi */91/*/75 Cercotrichas leucophrys Muscicapa striata */*/*/* Myadestes unicolor Turdus merula

0.04

Figure 3. Phylogeny of representatives for the main clades in the family Muscicapidae according to Sangster et al. (2010), based on MRBAYES analysis of mitochondrial cytochrome b, and nuclear LDH, myoglobin and ODC sequences (c. 3 kb in total). Larvivora ruﬁ- ceps and its congeners are highlighted in grey. Values at nodes represent Bayesian posterior probabilities and maximum-likelihood bootstrap values in the order MRBAYES/RAXML/*BEAST/MP-EST. *, posterior probability 1.00 or 100% bootstrap support. –, support below 0.5/50%.

6 … … L. akahige 4 2

KHZ S 2 4 6 8 10 12

6 4 …… … … 2

KHZ S 2 4 6 8 10 12 14 L. komadori 6 4 …… … …… 2

KHZ S 2 4 6 8 10 12 14

4 …… … … ……… L. ruficeps 2 KHZ S 2 4 6 8 10 12 14

4 … … … … … 2

KHZ S 24 6 810 12 14

6 L. sibilans 4 … 2

KHZ S 24 6 810

6 … … L. brunnea 4 2

KHZ S 2 4 6 8 10 12 14 16

6 … … …

4 L. cyane 2 KHZ S 2 4 6 8 10 12 14

4 … …

KHZ S 2 4 6 8 10 12 14

Figure 4. Sonograms of Larvivora ruficeps and other Larvivora species. The phylogenetic tree is based on the topology in Figure 3. Representative strophes were chosen for each species. Pauses between strophes of the same individual have been shortened. Stro- phes from different individuals are separated by vertical bars: L. akahige (XC155761 (L. a. tanensis), XC194981 (L. a. akahige), XC285835 (L. a. tanensis)), L. komadori (XC286176, XC286177, XC286191, XC156015), L. ruficeps (XC312266, XC312118, XC312264), L. sibilans (XC312115, XC149249, XC267834), L. brunnea (XC312269, XC312270, XC312278, XC312134), L. cyane (XC312348, XC312111, XC285651). See Table S2 for further information (locality, date, recordist, etc.) of each song, and Figure S5 for more sonograms of each species. song was rather low- and evenly pitched (top fre- n = 98), with a rather narrow bandwidth (0.69– quency 3.28–4.55 kHz; bottom frequency 0.94– 2.07 kHz; median 1.13 kHz; n = 98) and a strong, 2.02 kHz; mean frequency 2.50 kHz; sd = 0.18; ‘pan flute-like’ quality. Each male had a large

repertoire of different strophe types (10–24; mean (17.5%) was mainly correlated to delta time and 16, sd = 7, n = 3 (only long recordings used)). number of notes. The PCA plot (Fig. 5) largely Different strophe types were not created by chang- confirmed the similarities and differences among ing the syntax (i.e. by using the same notes but the species perceived audibly and in sonograms changing their sequence), although some notes at (Fig. 4). It also suggested that the song of the end of a strophe were sometimes dropped L. akahige of the northern, nominate, subspecies and/or added. The strophe types were given in a was more similar to the song of L. sibilans than to seemingly random succession rather than in a fixed that of the subspecies L. akahige tanensis of the order. The same strophe type was only exception- Izu Islands on PC1 and PC2, but not on PC3. ally given twice in succession. A significant phylogenetic signal was only found The song of L. ruficeps resembled that of sibi- in transformed delta time (Table S5), and the lans (Fig. 4), although the latter’s song was on other categories did not exhibit a significant signal average higher-pitched (top frequency 3.30– or any phylogenetic signal. 6.53 kHz; bottom frequency 1.05–2.20 kHz; mean frequency 3.16 kHz; sd = 0.36; n = 67), with Distribution and records slightly broader frequency band (0.86–2.76 kHz; median 1.72 kHz; n = 67), and the strophes There have been only a handful of records of L. rufi- lacked a time-separated introductory element. ceps since it was first described in 1905 (Table S1). The songs of L. akahige and L. sibilans (Fig. 4) All subsequent Chinese records are from seven adja- were simpler, consisting of longer trills (especially cent localities in the nearby Sichuan Province in the latter), with many more, much thinner ele- (Fig. 1), and in the last 25 years it has only been ments, which made the trills sound like dry rattles. observed at two sites in Sichuan (Jiuzhaigou and The strophes usually began with one or two more Baihe). There have been no records from the type drawn-out, fuller elements in the former species, locality, Taibai Mountain, Shaanxi Province, since and a short series of slightly more drawn-out, the original record, and only three records outside thicker elements in the latter species (‘introductory the breeding grounds, as described above. No obser- trill’). L. sibilans often sounded as if it ‘shifted vations are known from China in 2016. gear’ when it changed from the introductory trill In the ENM, the average value of the areas to the main trill. Songs of L. akahige akahige from under the curve (AUC) computed from the 10 Russia and the main Japanese islands bore more independent ENM replicates was 0.981 (Fig. S6). resemblance to those of L. sibilans, whereas indi- The distribution map based on the ENM analysis viduals from Miyake Island, Izu Islands, Japan, rec- (Fig. 6) suggested that the most suitable habitats ognized as L. a. tanensis, featured overall higher for L. ruficeps are likely to be in the Minshan pitch, broader bandwidth and shorter strophes Mountains (southern Gansu and northern (Fig. 4). Sichuan), Qionglai Mountains (central Sichuan), The songs of L. brunnea and L. cyane (Fig. 4) Daliang Mountains (southern Sichuan), Daba were basically similar to each other. They began Mountains (southern Shaanxi), Qinling Mountains with a slow series of short elements (thinner in the (southwestern Shaanxi) and eastern Himalayas. latter), which progressively increased in volume, and then switched over to a single- or multi-ele- DISCUSSION ment trill (on average less complex in the latter), which was frequently introduced by one (in the Our phylogenetic analyses confirm that L. ruficeps latter species) or one or two (in the former spe- belongs in the Larvivora clade. It should therefore cies) more drawn-out elements. be placed in this genus, as hypothesized by Sang- PCA indicated that most of the variance ster et al. (2010). There is strong support for a sis- (96.4%) was concentrated in the first three princi- ter relationship between L. ruficeps and L. sibilans. pal components (Fig. 5): principal component 1 Within Larvivora, there is further strong support (PC1) explained 46.9% of the total variance, inter- for sister relationships between L. komadori and preted mainly as the bottom frequency, top fre- L. akahige and between L. brunnea and L. cyane, quency and peak frequency; principal component as previously shown by Sangster et al. (2010). 2 (PC2) explained 31.9%, interpreted mainly as Provided that the inferred phylogeny is true, the note number and average speed; and PC3 the similarities in songs between L. ruficeps and

(a) 1.6 1.6 Larvivora ruficeps 1.2 1.2 Larvivora sibilans 0.8 0.8

0.4 0.4 Larvivora akahige akahige 0.0 0.0 Larvivora akahige tanensis PC2 –0.4 PC3 –0.4 Larvivora brunnea –0.8 –0.8

–1.2 –1.2 Larvivora cyane

–1.6 –1.6 Larvivora komadori –2.0 –2.0 –3.0 –2.5 –2.0 –1.5 –1.0 –0.5 0.0 0.5 1.0 1.5 –3.0 –2.5 –2.0 –1.5 –1.0 –0.5 0.0 0.5 1.0 1.5 PC1 PC1

(b) PC1 PC2 PC3 1.0 0.96 0.99 0.94 0.84 0.86 0.9 0.92 0.94 0.80 0.84 0.72 0.8 0.64 0.60 0.7 0.48 0.48 0.6 0.47 0.36 0.5 0.32 0.24 0.4 0.16 0.18 0.12 correlation correlation correlation 0.35 0.3 0.00 0.27 -0.08 0.00 –0.04 0.2 –0.16 –0.12 -0.22 –0.12 0.1 –0.19 –0.32 –0.23 0.02 -0.38 –0.24 0.0 Top Top Top Note Note Note Peak Peak Time Peak Time Time Speed Speed Speed Bottom Bottom Bottom

Figure 5. (a) PCA plot, based on mean values for six song parameters for the six Larvivora species, Rufous-headed Robin L. ruficeps, Rufous-tailed Robin L. sibilans, Japanese Robin L. akahige, Ryukyu Robin L. komadori, Indian Blue Robin L. brunnea and Siberian Blue Robin L. cyane. The two Japanese Robin subspecies (Larvivora akahige akahige and L. a. tanensis) are indicated with different symbols. Each symbol represents one individual. (b) PCA loadings for the six parameters of the first three principal components. [Colour figure can be viewed at wileyonlinelibrary.com] komadori and between L. akahige and L. sibilans L. komadori and their increased complexity com- cannot be explained by shared history, unlike the pared with those of their congeners would most similarity between the songs of L. brunnea and likely be due to parallel evolution. L. cyane. This can be seen in the sonograms Alternatively, two cases of parallel evolution (Fig. 4) and in the overall low level of phyloge- could be invoked to explain the similarities netic signal in the acoustic data (Table S5). There between the songs of L. ruficeps and L. komadori are at least two plausible explanations for the and between L. akahige and L. sibilans. The longer observed pattern. It seems most parsimonious that and less complex strophes in the most northerly the most recent common ancestor (MRCA) of the distributed species, L. sibilans, L. cyane and extant Larvivora species had a fairly simple song L. akahige (especially the northern, nominate, sub- with one or more introductory elements followed species), compared with the more southerly ones, by a trill, as that is the case in L. akahige, L. sibi- agree with the general pattern found in the family lans, L. brunnea and L. cyane. The complexity of Phylloscopidae (leaf warblers) (Tietze et al. 2015), the individual strophes might have been further and this might support the double parallel evolu- reduced in the MRCA of the clade containing tion scenario. L. ruficeps, L. komadori, L. akahige and L. sibilans, Although beyond the scope of this paper, our so that the song of the MRCA of these four spe- analysis of the songs of the two subspecies of cies resembled those of the two latter. In that case, L. akahige suggests pronounced differences the similarity of the songs of L. akahige and sibi- between these. Seki et al. (2012) found coexis- lans would then be plesiomorphic, whereas the tence of markedly different mitochondrial haplo- resemblance of the songs of L. ruficeps and types (inferred to have diverged 1.1 million years

Figure 6. Potential distribution of Rufous-headed Robin Larvivora ruficeps based on the ecological niche modelling in MAXENT. Habitat suitability ranges from 0.5 to 1. [Colour figure can be viewed at wileyonlinelibrary.com] ago) within the range of L. a. tanensis in the Izu unlikely to be found when not singing. Our ENMs Islands, and suggested that this was the result of suggest that suitable breeding habitat for L. rufi- ancient introgression of L. a. akahige haplotypes ceps exists over a considerably larger area than into the Izu Islands population. These observations where it is presently known. Extensive surveys of suggest that more research is warranted on the suitable habitats in Sichuan, adjacent southern geographical variation of L. akahige. Gansu (especially) and Shaanxi and southeastern Although all breeding period records from the Tibet during May and June would probably reveal 20th and 21st centuries are from Jiuzhaigou, the presence of L. ruficeps in at least a few more Sichuan, except for two mid-1980 records and places. Another rare robin, the Blackthroat Cal- two recent records from the neighbouring Baihe, it liope obscura, had only been seen a handful of seems highly likely that L. ruficeps occurs else- times since it was first described in the 1890s, where in Sichuan. It is also probable that it still until a breeding population was discovered in the breeds in the Qinling Mountains in Shaanxi, Qinling mountains in 2011 (Song et al. 2014) and although there have been no records from there another recently discovered potential breeding area for more than 100 years. To our knowledge, no in southern Gansu province (Alstrom€ et al. 2016). dedicated surveys have been carried out there. Our study used eight confirmed localities of Most birdwatchers go to places where they can be L. ruficeps to estimate its breeding range, but such sure to find their target species, and exploration of a small sample size can reduce the accuracy of this a ‘new site’ is not so common. Moreover, L. rufi- prediction (Stockwell & Peterson 2002), especially ceps is an extremely secretive bird and is highly the reliability of predicting occurrences in

southeastern Tibet, where there is at present no BirdLife International. 2013. Luscinia ruficeps. The IUCN known occurrence. However, MAXENT has been Red List of Threatened Species 2013: e.T22709711A49017546. http://dx.doi.org/10.2305/ shown to work better than other approaches with – ı IUCN.UK.2013-2.RLT2400 3000S.T22709711A49017546.en limited sample sizes (Phillips & Dud k 2008, Wisz (accessed 9 December 2015). et al. 2008) (AUC = 0.981 in our case), including Blomberg, S.P., Garland, T. & Ives, A.R. 2003. Testing for sample sizes as small as five (Hernandez et al. phylogenetic signal in comparative data: behavioral traits are 2006). more labile. Evolution 57: 717–745. ’ Based on two records of L. ruficeps from Malay- Burland, T.G. 2000. DNASTAR s Lasergene sequence analysis software. Methods Mol. Biol. 132:71–91. sia, it seems plausible that it winters in the mon- Charif, R.A., Waack, A.M. & Strickman, L.M. 2010. Raven tane zone of that area. The record from Pro 1.4 User’s Manual. 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Figure S5. Sonograms of all the representative Table S3. Sound-recordings used in our study. strophe types, generated by RavenPro, based on all ‘No. inds’ is the abbreviation for the number of the Larvivora songs we have analysed. individuals, and ‘No. strs’ for the number of stro- Figure S6. The average value of the areas under phes we extracted in each song. Two or three the curve (AUC) computed from 10 independent sound recordings from the same individual were ENM replicates. considered as one song. Table S1. Records of Rufous-headed Robin Table S4. Environmental and bioclimatic vari- Larvivora ruﬁceps. ables used in ecological niche modelling. Table S2. GenBank accession numbers of the Table S5. Blomberg’s Kappa and Pagel’s sequences analysed. ODC sequences from Monti- Lambda based on PC values and transformed cola cinclorhynchus and Monticola ruﬁventris were acoustic measurements. unavailable.