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
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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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.
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
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 &
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.
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
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,
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
Neighbour-joining trees of partial COI sequences from "Pale-legged
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).
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
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
no. 1A04402) lateral view.
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.
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
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
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,
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
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).
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
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Philip D. Round, Department of Biology, Faculty of Science, Mahidol
University, Rama 6 Road, Bangkok 10400, Thailand. Email: email@example.com
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: firstname.lastname@example.org
Yoshimitsu Shigeta, Division of Avian Conservation (Migration Research
Center), Yamashina Institute for Ornithology, 115 Konoyama, Abiko,
Chiba 270-1145, Japan. Email: email@example.com