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Addition of Kamchatka Leaf Warbler Phylloscopus examinandus and Sakhalin Leaf Warbler P. borealoides to Thailand's Avifauna

By Philip D. Round, Andrew J. Pierce, Takema Saitoh & Yoshimitsu Shigeta
 
Note: This article was originally published in Bulletin of the Japanese Bird Banding Association 28 (2016) and was kindly submitted by Philip D. Round.

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ABSTRACT
The non-breeding season distributions of some Phylloscopus warblers are poorly known due partly to the difficulties of field identification. We document the addition of Kamchatka Leaf Warbler Phylloscopus examinandus and Sakhalin Leaf Warbler P. borealoides to the avifauna of Thailand based on five individuals and 12 individuals respectively. All P. examinandus were caught and banded on spring (northwards) migration, while the P. borealoides sample included both autumn and spring birds. Their identity was established through assay of the cytochrome c oxidase subunit I (COI) gene (c. 700 bp) of the mitochondrial genome.

We present biometric data for these birds together with representative samples from the respective sibling species, Arctic Warbler P. borealis and Pale-legged Leaf Warbler P. tenellipes. We suggest that both P. examinandus and P. borealoides winter mainly or predominantly in the Sunda Subregion.

INTRODUCTION
The application of molecular methods combined with improved and more widespread recording of vocalizations, and increased sampling by ornithologists and bird banders across the breeding and wintering ranges of Asian birds, has greatly increased understanding of the taxonomy and distribution of sibling species among warbler genera. Among these, the large warbler genus Phylloscopus, with c. 60 species, is possibly the most taxing for identification.

We here document the addition of both Kamchatka Leaf Warbler P. examinandus and Sakhalin Leaf Warbler P. borealoides to the Thai avifauna, based on analysis of mitochondrial DNA recovered from tail feathers of birds trapped for banding and subsequently released.

The wide-ranging Arctic Warbler P. borealis was recently recognized as a complex of three species, including Japanese Leaf Warbler P. xanthodryas (breeding in Honshu to Kyushu), and Kamchatka Leaf Warbler P. examinandus which breeds in Kamchatka, Sakhalin and Hokkaido (Dickinson & Christidis 2014). Although P. examinandus was usually treated as an intergrade between P. borealis and P. xanthodryas (e.g., Ticehurst 1938) it has since been shown to lie in a separate clade from the other two species in the complex, differing in morphology and song (Alström et al. 2011, Saitoh et al. 2006, 2008, 2010).

Arctic Warbler (sens. lat.) is a widespread and common passage migrant and winter visitor in Thailand, mainly in the south and southern central part of the country, south of roughly 15 degrees N latitude, occurring in both lowland terrestrial forests and mangroves (Lekagul & Round 1991). Nominate P. b. borealis was the only taxon listed for Thailand by Deignan (1963) while P. xanthodryas is so far known from a single individual caught and banded on northwards migration in SE Thailand (Round et al. 2015). A small number of exceptionally long-winged "Arctic Warblers", including some specimens, from southern Thailand and Malaysia thought likely to be P. xanthodryas, under which name P. examinandus was previously often subsumed (Medway & Wells 1976, Wells 2007) are perhaps also likely to have been examinandus, but we have not examined these and their identification remains tentative.

The Sakhalin Leaf Warbler P. borealoides was described as a distinct species by Portenko (1950) on the basis of its song, which is distinctively different from that of Pale-legged Leaf Warbler P. tenellipes. It breeds in southern Sakhalin, the Kuril Islands and Japan south to Shikoku (Dickinson & Christidis 2014) and shows different habitat preferences from Ussuri and North Chinese-breeding Pale-legged Leaf Warbler (Weprincew et al. 1989). Understanding of its distribution and movements has been confounded, however, owing its great morphological similarity to the latter species. The Pale-legged Leaf Warbler has long been recognized as a widespread and common visitor in forested areas, including mangroves, throughout most of Thailand, though is commonest in the eastern part of the country and scarcer in the peninsula (Deignan 1963, Lekagul & Round 1991).

Arguably, separating these two species pairs, P. examinandus from P. borealis, and P. borealoides from P. tenellipes, on morphology are among the most difficult identification challenges among Asian Phylloscopus species.

METHODS
Biometrics and one or two tail feathers were collected from samples of Arctic Warblers and Pale-legged Leaf Warblers (sens. lat., both) banded mainly on passage at coastal localities in Thailand. Wing measurements were maximum chords and bill-length was measured to skull. All measurements were taken by either PDR or AJP.

DNA was extracted from tail feathers using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). We sequenced a partial region, the cytochrome c oxidase subunit I (COI) gene (c. 700 bp) of the mitochondrial genome, which is used as a standard DNA barcoding region for most animals (Heber t et al. 2003). The barcoding region was amplified using the following primers: L6697Bird (5'-TCAACYAACCACAAAGAYATCGGYAC-3') and H7390Thrush (5'-ACGTGGGARATRATTCCAAATCCTG- 3') following Saitoh et al. (2015). Neighbour-joining (NJ) trees with bootstrap values (1,000 replications) were constructed from the COI data using the Kimura 2-parameter model (K2P) model and Mega 5.2.2 (Tamura et al. 2011). A breeding season/location P. borealis VK57822 (Japan DNA Data Bank Accession no. LC087227) and a breeding season and location P. examinandus (specimen NSMT-A15083; DDBJ Accession no. AB843059) were used to provide sequences for comparison with Thai migrants of these two species, as was a P. borealoides specimen YIO-62722 (DDBJ Accession no.AB843689) and a P. tenellipes specimen 1998-5165 (DDBJ LC087226; Appendix 1 ). A sequence from P. xanthodryas specimen YIO-64546 (DDBJ accession number AB843687) was used as an outgroup for both NJ trees (Figs. 2 and 3 ).

Kamchatka Leaf Warbler P. examinandus
We were able to determine the DNA sequences of 23 "Arctic Warblers" sampled from 659 to 715 bp in the COI region (DDBJ accession Nos. LC087183, LC87186-88, LC87190-93, LC87195-87202, LC87209, LC87213-18; Appendix 1 ). Among these, five belonged in the P. examinandus clade, and 18 individuals belonged in the P. borealis clade (Fig. 2, Appendix 1 ). All but one of the sampled birds of both taxa, P. examinandus and P. borealis, were caught at locations that indicated they were almost certainly passage migrants: on northward (spring) migration 14 Apr. to 19 May, n = 13 both species combined; and southward (autumn) migration, 9 Sept. – 10 Oct., n = 9, P. borealis only). A presumed overwintering individual, another P. borealis, was sampled in lowland evergreen forest at Khao Soi Dao Wildlife Sanctuary, Chanthaburi Province, South-East Thailand, on 28 Dec. (Fig. 1, Appendix 1 ).

All five P. examinandus were caught at Laem Phak Bia, Phetchaburi Province, Central Thailand (Fig. 1 ), on northward migration: on 9 May 2010 (one); 14 May 2011 (one) and 19 May 2012 (three; Appendix 1 ).

The five P. examinandus were scarcely distinguishable in morphology from P. borealis (Table 1, Appendix 1 ), though tended to be slightly larger and longer-winged. One of these birds (band no. 1A04402) was markedly yellower on the supercilium and throat than typical borealis and brighter green above (Figs. 4a, 4b, and 5 ), apparently agreeing with descriptions of P. examinandus in Ticehurst (1938) and Alstrom et al. (2011). However, another individual, 1A04411 with virtually identical colouration to 1A04402, and caught on the same day (Fig. 6 ) proved to be a borealis on DNA (Appendix 1 ), illustrating the extreme difficulty of reliably separating these two species on morphology.


Fig. 1. Map of Thailand to show capture locations mentioned in the text.

Fig. 2. Neighbour-joining trees of partial COI sequences from "Arctic Warblers". The tree was
rooted using Japanese Leaf Warbler P. xanthodryas as outgroup (DNA accession number
AB843687). Numbers at each node indicate bootstrap support (K2Pmodel, 1,000 replicates).
Sakhalin Leaf Warbler P. borealoides
A total of 24 "Pale-legged Leaf Warblers" were sampled of which we were able to determine the DNA sequences of 21 individuals, sampling 602 to 691 bp in the COI region (Japan DNA Data Bank accession Nos. LC087184 – 85; LC087189, LC87194, LC87203-08; LC87210-12; LC87219-25; Appendix 1, Fig. 3 ). Nine belonged in the P. tenellipes clade, and 12 in the P. borealoides clade (Fig. 3 ). No result could be obtained for three other birds for which no usable DNA was extracted.

Fig. 3. Neighbour-joining trees of partial COI sequences from "Pale-legged Leaf Warblers".
The tree was rooted using Japanese Leaf Warbler P. xanthodryas as outgroup (DNA accession
number AB843687). Numbers at each node indicate bootstrap support (K2Pmodel, 1000 replicates).
Table 1. Summary of biometrics (mm) of trapped Phylloscopus sampled for DNA.

Bill width measured proximally (base of the nares) and bill depth distally (distal edge of the nares). P1 (Outermost
primary) was measured as projection beyond the longest primary covert.

Fig. 4. A Kamchatka Leaf Warbler Phylloscopus examinandus (band no. 1A04402) alongside an Arctic Leaf Warbler P.
borealis
(1A04401) showing the brighter green upperparts and more strongly yellow-tinged supercilium and underparts
of the left individual. (a) Dorsal view; (b) lateral view.

Fig. 5. Kamchatka Leaf Warbler Phylloscopus examinandus (band
no. 1A04402) lateral view.
The P. borealoides were caught as presumed passage migrants, all but two during presumed southward passage, 2 Oct. to 2 Nov., from four localities. Three of the localities (Khok Kham, Samut Sakhon Mangrove Research Station and Laem Phak Bia, all on the shores of the Gulf of Thailand), were mangrove-dominated and the fourth, Khao Dinso, Chumphon Province, Peninsular Thailand was scrub and secondary forest on a 300 m elevation mountain, 1.5 km from the coast (Fig. 1 ). One of the two spring birds was caught in mangroves at Laem Phak Bia (on 19 Mar.) and the other on a forested island, Ko Man Nai, on 13 Apr. (Fig. 1, Appendix 1 ).

Fig. 6. Unusually bright Arctic Leaf Warbler P. borealis, band no,
1A04410, apparently identical in colouration to previous (P.
examinandus
) individual.
The nine undoubted P. tenellipes included five birds that were presumed southwards passage migrants caught during 5 – 20 October from two of the same localities reported for P. borealoides: Khao Dinso (four) and from mangroves at Khok Kham (one). Four further P. tenellipes were caught from inland forest sites in eastern Thailand: three midwinter birds from Khao Soi Dao Wildlife Sanctuary and one bird in mid-April at Khao Yai National Park. It was unfor tunate that this latter bird (band no. 1A03209; Appendix 1 ) was the only one of four putative P. tenellipes caught at Khao Yai (where the species has long been considered to be a common winter visitor; Lekagul & Round 1991) to give an adequate DNA signature. Even so, while the result was sufficient to identify it unequivocally as a P. tenellipes, the full sequence was not registered with DDBJ as many unreadable sections were interspersed within.

The two species are very hard to distinguish, with similar plumage, and a large overlap in wing measurements and wing formulae (Table 1 ). While P. borealoides is longer-winged than P. tenellipes, most birds caught lay in an overlap zone which presumably encompasses longer-winged (male) P. tenellipes and shorter-winged (female) borealoides. The longest-winged P. tenellipes caught in the present sample had a wing-length of 65 mm (Table 1, Appendix 1 ).

DISCUSSION
We have provided details for five records of Kamchatka Leaf Warblers P. examinandus and twelve records of Sakhalin Leaf Warblers P. borealoides in Thailand established through assay of the cytochrome c oxidase subunit I (COI) gene (c. 700 bp) of the mitochondrial genome. We present supporting biometric data for our small samples of both the former species compared with Arctic Warblers P. borealis and Pale-legged Leaf Warblers P. tenellipes (Table 1, Appendix 1 ).

Distinguishing morphologically between P. examinandus and P. borealis, and P. borealoides and P. tenellipes are among the most difficult tasks facing the bird bander in the field. Where canonical discriminant analysis has been used successfully, for example to distinguish between borealis and examinandus (Saitoh et al. 2008, Saitoh et al. 2014), it is best applied to sexed specimens. When larger, longer-winged males and smaller, shorter-winged females are combined in an unsexed sample, as with livetrapped birds, this technique may be less reliable in separating between all the members of either species pair. Our results concerning the separation of P. tenellipes from P. borealoides would suggest that any individuals with wing lengths longer than 65 mm might reasonably be identifiable as P. borealoides. However Bozó & Heim (2015) apparently recorded P. tenellipes with a wing length up to 67.5 mm from a sample of 79 individuals trapped and ringed during autumn passage in the Amur region.

Our birds were mostly caught as passage migrants at coastal sites. It is important to resolve the wintering ranges of the taxa through increased mist-netting and sampling during the midwinter period at a wide range of inland forest locations in Thailand and elsewhere in SE Asia. We consider, based on the location and late timing of the spring passage captures of P. examinandus, that its wintering range most likely lies in peninsular Thailand or further south within in the Sunda subregion (perhaps Malaysia or Sumatra). Supporting evidence for a Sundaic wintering area in P. examinandus comes from long winged "Arctic Warblers" (wings > 71– 73 mm), both specimens and banded birds, captured at localities. further south in peninsular Thailand and Malaysia on likely spring (21 Apr. – 30 May) and autumn (24 Sept.) passage dates (Medway & Wells 1976, Wells 2007).

Dates of all P. borealoides so far identified in Thailand are likewise concordant with migration to a wintering area south of the capture localities, in the Sunda subregion. Additionally, an over-wintering P. borealoides was documented in Singapore (Yap et al. 2014). The midwinter capture dates of three P. tenellipes in Eastern Thailand, on the other hand, together with three further relatively short-winged (59– 63 mm) individuals from which DNA could not be obtained (Appendix 1 ), but which were likewise almost certainly P. tenellipes rather than P. borealoides, suggests that the former species may winter predominantly in that region (where borealoides has not yet been recorded). Since P. tenellipes were also captured on migration alongside P. borealoides in peninsular Thailand, this indicates that its full wintering range certainly extends to the peninsula or elsewhere in the Sunda subregion. Similarly, given our limited sampling, the possibility that some P. borealoides may also winter as yet undetected alongside P. tenellipes in eastern or central Thailand cannot yet be ruled out.

Increased sampling of migrants may also resolve the differences in timing of passage between P. examinandus and P. borealis on the one hand, and P. borealoides and P. tenellipes on the other. Larger samples of the latter species-pair, particularly if both wing-formula and vocalizations are recorded, may yield more reliable methods of distinguishing them since the call of P. tenellipes is markedly higher in frequency than that of P. borealoides (Weprincew et al. 1989, Yap et al. 2014). P. examinandus is also distinguishable on call from P. borealis (Alström et al. 2011).

ACKNOWLEDGEMENTS
We are grateful to the Department of National Parks, Wildlife and Plants Conservation (Thailand) for access to bird bands and for permission to band birds in protected areas. We also thank the Laem Phak Bia Environmental Research and Development Project, and the Department of Marine and Coastal Resources (Thailand) for permission to operate at sites they administer. Wichyanan Limparungpatthanakij, Sontaya Manawattana, Jonathan Murray, and Dr. Wangworn Sankamethawee provided assistance in the field. Philip Round is supported by The Wetland Trust.
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Saitoh, T., Sugita, N., Someya, S., Iwami, Y., Kobayashi. S., Kamigaichi, H., Higuchi, A., Asai, S., Yamamoto, Y. & Nishiumi, I. (2015). DNA barcoding reveals 24 distinct lineages as cryptic bird species candidates in and around the Japanese Archipelago. Molecular Ecology Resources 15: 177 – 18.

Svensson, L. (1992). Identification Guide to European Passerines, 4th Edition. Published by the Author, Stockholm.

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Weprincew, B. N., Leonowitsch, W. W. & Netschajew, W. A. (1989). Zur Lebensweise von Phylloscopus borealoides Portenko und Phylloscopus tenellipes Swinhoe. Mitteilungen aus dem Zoologischen Museum in Berlin 65 (Suppl.): 71 – 80.

Yap, F., Yong, D. L., Low, B., Cros, E., Foley, C., Lim, K. K. & Rheindt, F. E. (2014). First wintering record of the Sakhalin Leaf Warbler in South East Asia, with notes on vocalisations. BirdingASIA 21: 76–81.
Kindly submitted by:

Philip D. Round, Department of Biology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Email: philip.rou@mahidol.ac.th

Andrew J. Pierce, Conservation Ecology Program, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkhunthien, Bangkok 10150, Thailand. Email: Andyp67@gmail.com

Takema Saitoh, Division of Natural History, Yamashina Institute for Ornithology, 115 Konoyama, Abiko, Chiba 270-1145, Japan.Email: saitoh@yamashina.or.jp

Yoshimitsu Shigeta, Division of Avian Conservation (Migration Research Center), Yamashina Institute for Ornithology, 115 Konoyama, Abiko, Chiba 270-1145, Japan. Email: shigeta@yamashina.or.jp
   
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