Molecular evidence suggests radical revision of species limits in the great speciator white-eye genus Zosterops

Abstract

White-eyes (Zosterops spp.) are a group of small passerines distributed across the Eastern Hemisphere that have become a textbook example of rapid speciation. However, traditional taxonomy has relied heavily on conservative plumage features to delimit white-eye species boundaries, resulting in several recent demonstrations of misclassification. Resolution of confused taxonomy is important in order to correctly delimit species and identify taxa which may require conservation, particularly in Asia where the songbird trade is decimating wild populations. In this study, we aim to untangle multiple instances of confused taxonomic treatment in three large, widespread Asian wastebasket species complexes of white-eye (Oriental White-eye Zosterops palpebrosus, Japanese White-eye Zosterops japonicus and Mountain White-eye Zosterops montanus) renowned for their conservative morphology. Using mitochondrial DNA from 173 individuals spanning 42 taxa, we uncovered extensive polyphyly in Z. palpebrosus and Z. japonicus and propose some radically revised species limits under which former members of Z. palpebrosus and Z. japonicus would be reassigned into four and two different species, respectively. The revised taxonomy results in a net loss of two previously recognized species and a net gain of two newly recognized species, leading to significant taxonomic change but a lack of additional species-level diversity. One of the newly elevated species, Zosterops melanurus from Java and Bali, is also the world’s most heavily traded songbird and requires urgent conservation attention.

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Vol.:(0123456789)
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Journal of Ornithology
https://doi.org/10.1007/s10336-018-1583-7
ORIGINAL ARTICLE
Molecular evidence suggests radical revision ofspecies limits
inthegreat speciator white‑eye genus Zosterops
BryanT.M.Lim1· KerenR.Sadanandan1· CarolineDingle2· YuYanLeung2· DewiM.Prawiradilaga3·
MohammadIrham3· HidayatAshari3· JessicaG.H.Lee4· FrankE.Rheindt1
Received: 10 November 2017 / Revised: 22 May 2018 / Accepted: 6 July 2018
© Dt. Ornithologen-Gesellschaft e.V. 2018
Abstract
White-eyes (Zosterops spp.) are a group of small passerines distributed across the Eastern Hemisphere that have become a
textbook example of rapid speciation. However, traditional taxonomy has relied heavily on conservative plumage features to
delimit white-eye species boundaries, resulting in several recent demonstrations of misclassification. Resolution of confused
taxonomy is important in order to correctly delimit species and identify taxa which may require conservation, particularly in
Asia where the songbird trade is decimating wild populations. In this study, we aim to untangle multiple instances of con-
fused taxonomic treatment in three large, widespread Asian wastebasket species complexes of white-eye (Oriental White-eye
Zosterops palpebrosus, Japanese White-eye Zosterops japonicus and Mountain White-eye Zosterops montanus) renowned for
their conservative morphology. Using mitochondrial DNA from 173 individuals spanning 42 taxa, we uncovered extensive
polyphyly in Z. palpebrosus and Z. japonicus and propose some radically revised species limits under which former mem-
bers of Z. palpebrosus and Z. japonicus would be reassigned into four and two different species, respectively. The revised
taxonomy results in a net loss of two previously recognized species and a net gain of two newly recognized species, leading
to significant taxonomic change but a lack of additional species-level diversity. One of the newly elevated species, Zosterops
melanurus from Java and Bali, is also the world’s most heavily traded songbird and requires urgent conservation attention.
Keywords Cryptic speciation· Phylogenetics· Wastebasket species· Polyphyly· Taxonomy
Zusammenfassung
Molekulare Belege erfordern eine radikale Revision der Artgrenzen innerhalb der „Superartbildner“-
Brillenvogelgattung Zosterops
Brillenvögel (Zosterops spp.) sind eine Gruppe kleiner Singvögel der östlichen Hemisphäre, die zu einem Paradebeispiel
für schnelle Artbildung geworden sind. Allerdings stützte sich die traditionelle Taxonomie bei der Abgrenzung der
Brillenvogelarten bisher vorwiegend auf konservative Gefiedermerkmale, was zu verschiedenen in neuerer Zeit aufgedeckten
Falschklassifikationen führte. Eine Entwirrung der Taxonomie ist wichtig für eine korrekte Artabgrenzung und die Ermittlung
von Taxa mit besonderem Schutzbedarf, speziell in Asien, wo der Handel mit Singvögeln die Wildpopulationen stark
dezimiert. Ziel dieser Untersuchung war es, verschiedene Fälle verworrener taxonomischer Einordnung bei drei großen,
weitverbreiteten asiatischen “Sammelsurium-Artkomplexen” von Brillenvögeln (Gangesbrillenvogel Z. palpebrosus,
Japanbrillenvogel Z. japonicus und Gebirgsbrillenvogel Z. montanus) aufzulösen, welche für ihre konservative Morphologie
Communicated by M. Wink.
Bryan T. M. Lim and Keren R. Sadanandan contributed equally to
this work.
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s1033 6-018-1583-7) contains
supplementary material, which is available to authorized users.
Extended author information available on the last page of the article

Journal of Ornithology
1 3
bekannt sind. Anhand von mitochondrialer DNA von 173 Individuen aus 42 Taxa entdeckten wir ein beträchtliches Maß an
Polyphylie bei Z. palpebrosus sowie Z. japonicus und empfehlen eine radikale Revision einiger Artgrenzen, durch welche
vormalige Angehörige der Arten Z.palpebrosus und Z. japonicus neu zu jeweils vier beziehungsweise zwei verschiedenen
Arten gerechnet würden. Diese überarbeitete Taxonomie resultiert insgesamt in dem Verlust zweier vormals anerkannter Arten
sowie einem Hinzugewinn von zwei neu etablierten Arten, was zwar zu einer signifikanten taxonomischen Veränderung,
jedoch nicht zu zusätzlicher Diversität auf Artebene führt. Eine der neu anerkannten Arten, Z. melanurus von Java und Bali,
ist zudem der meistgehandelte Singvogel der Welt und bedarf dringender Schutzmaßnahmen.
Introduction
The genus Zosterops, commonly referred to as ‘white-eyes’,
is a group of small passerines comprising approximately
100 species with a distribution spanning the Eastern Hemi-
sphere (Africa to Australasia) (van Balen 2017a). The genus
is famously recognized for having one of the fastest diver-
sification rates amongst birds, and possibly terrestrial ver-
tebrates, and is thus known as a ‘great speciator’ (Diamond
etal. 1976; Moyle etal. 2009; Cornetti etal. 2015). This trait
has made the group a popular model for diversification and
island biogeography studies (Diamond etal. 1976; Warren
etal. 2006; Moyle etal. 2009; Cox etal. 2014; Cornetti etal.
2015; Linck etal. 2016; Wickramasinghe etal. 2017).
Despite their fast genetic diversification rate and a distri-
bution spanning across land masses and islands around the
entire Indian and Western Pacific oceans, the morphology
and vocalisations of many Zosterops taxa have remained
conserved (Eaton etal. 2016). Owing to similarities in
plumage and song, species identification is often challenging
especially with captive birds of unknown provenance, and
different treatises have applied varying species delimitations
(e.g. Eaton etal. 2016; Wells 2017a, b; van Balen 2017a). In
this work, we adopt one of the more conservative Zosterops
treatises (van Balen 2017a) as a baseline taxonomy, with the
exception of the taxon auriventer: the latter taxon is widely
considered a Sundaic subspecies of the Oriental White-eye
Zosterops palpebrosus by van Balen (2017b) and other tra-
ditional inventories, but Wells (2017a, b) recently demon-
strated that auriventer is the proper senior name applying
to Sundaic populations of Everett’s White-eye Zosterops
everetti, as circumscribed by van Balen (2018b), thereby
replacing more junior names such as wetmorei. In return, the
Oriental White-eye subspecies previously named auriventer
assumes its next most senior name erwini (Wells 2017a, b).
Cryptic species-level lineages may lurk in the form of
taxa currently classified as subspecies across multiple large
wastebasket species complexes within Zosterops. Three such
wastebasket species complexes are the Oriental White-eye
Zosterops palpebrosus, the Japanese White-eye Zosterops
japonicus, and the Mountain White-eye Zosterops monta-
nus. As currently circumscribed, the Oriental White-eye is
one of the most widespread species within Zosterops and
is conventionally divided into 11 subspecies ranging from
those of the Indian subcontinent eastwards to Sundaland and
the Lesser Sundas (Fig.1a) (van Balen 2017b). The Japanese
White-eye, ranging from the Japanese Archipelago through
China and northernmost Southeast Asia, is conventionally
divided into eight subspecies (Fig.1a) (van Balen 2017c).
Nine subspecies of the Mountain White-eye are currently
widely accepted, ranging from Sumatra, Java and the Lesser
Sundas northwards to Sulawesi and the Philippine archi-
pelago (Fig.1a) (van Balen 2017d).
These three wide-ranging species complexes have
been identified as requiring more taxonomic attention.
For instance, molecular sampling of two disjunct Oriental
White-eye subspecies has demonstrated that the nominate
form palpebrosus from India and unicus from the Lesser
Sundas are not closely related within the genus (Moyle etal.
2009). These two taxa occupy the extreme ends of the dis-
tribution of the complex, leaving taxonomic arrangements
across the Sundaic region largely unknown and intimating
that the current Oriental White-eye complex constitutes a
non-monophyletic composite. Molecular sampling in the
Japanese White-eye has so far only covered Korea and Japan,
leaving the affinities of populations across the rest of con-
tinental Asia largely unknown (Nishiumi and Kim 2004;
Nagata and Kanetsuki 2006). The evolutionary affinities of
taxa within the widespread Mountain White-eye Z. monta-
nus complex have also received little attention over recent
years except for a study that shed light on the complicated
relationships among populations within the Philippine archi-
pelago (Jones and Kennedy 2008).
With the taxonomy of many Asian white-eyes in disar-
ray, conservation may become a question of urgency in rare
and endangered species-level taxa that may have mistak-
enly languished at the subspecific level so far. The primary
threat to white-eyes is over-harvesting for the songbird trade
coupled with widespread habitat destruction (Eaton etal.
2015). In Indonesia alone, the centuries-old tradition of bird-
keeping has critically endangered many avian species in the
wild (Eaton etal. 2015; Lee etal. 2016), with white-eyes
being among the most popular cage bird species affected.
To satisfy this demand, Oriental White-eyes are commonly

Journal of Ornithology
1 3
poached from their natural habitats across Sundaland, and
massive numbers are sold in wildlife markets across the
region (Chng etal. 2015; Chng and Eaton 2016; Lee etal.
2016; Eaton etal. 2017). Consequently, the Oriental White-
eye and other Zosterops taxa have been identified as being at
high risk of trade-driven local extinction (Lee etal. 2016),
but the taxonomic uncertainty within these species com-
plexes thwarts meaningful conservation attempts.
In the present study, we investigated taxonomic relation-
ships within each of the three Asian white-eye wastebasket
species complexes—Z. palpebrosus, Z. japonicus and Z.
montanus. We used a combination of novel sequence data
and published GenBank material for two mitochondrial
genes in order to test conspecificity of taxa within each of
the three complexes. Our genetic analysis is supplemented
by qualitative information on key morphological characteris-
tics in order to more accurately estimate and define bounda-
ries between and within taxa.
Materials andmethods
DNA sampling
DNA samples were obtained from a mixture of both live
samples and museum loans (TableS1). In total, we obtained
27 samples from live birds, including ten blood samples of
Zosterops japonicus simplex that we caught in Hong Kong
and 17 blood samples of initially unidentified individuals
of Zosterops from Wildlife Reserves Singapore’s captive
collection (TableS1). The latter samples had either been
donated to Wildlife Reserves Singapore by members of the
Singaporean public or had been confiscated.
The museum specimens, in the form of either blood,
feather or breast muscle tissue, were loaned from the Lee
Kong Chian Natural History Museum (Singapore), the
Museum Zoologicum Bogoriense (Cibinong, West Java,
Indonesia), and the Burke Museum of Natural History and
Culture (Seattle, Washington, DC). In total, 57 museum
samples representing ten taxa were obtained for this study
(TableS1). These ten taxa included an optimized selection
of members of the three focal species complexes and sympa-
tric and/or neighbouring congeneric taxa that may be closely
related.
Our final sequence alignments included a total of 140
sequences of nicotinamide adenine dinucleotide (reduced;
NADH) dehydrogenase subunit 2 (ND2) and 103 sequences
of cytochrome b (cytb) across 42 taxa of Zosterops white-
eyes (Tables1, S1 and S2). In total, 54% of ND2 and 73%
of cytb sequences constituted novel sequences generated
for this study (TableS1). The remaining sequences were
sourced from other Zosterops publications (National Center
for Biotechnology Information GenBank) to ensure a more
robust sampling (TableS2). The sequences of the two
loci from the outgroup species, Yuhina brunneiceps, were
retrieved from GenBank as well (TableS2).
Laboratory procedures
DNA extractions were performed with a Qiagen DNeasy
Blood and Tissue Kit following the manufacturer’s protocol
for blood, tissue and feather samples in ethanol. Targeted
Fig. 1 a Range map of Zosterops japonicus, Zosterops palpebro-
sus, Zosterops montanus, Zosterops citrinella, Zosterops salvadorii
and Chlorocharis emiliae based on traditional taxonomic classifica-
tion (following van Balen 2017b, c, d, e, f, 2018a; Wells 2017a, b). b
Range map of revised species distributions based on the results of this
study—Zosterops palpebrosus, Zosterops japonicus, Zosterops mela-
nurus, Zosterops simplex, Zosterops citrinella and Zosterops emiliae

Journal of Ornithology
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Table 1 Summary list of all the taxa and number of sequences generated for this study
Species’ identi-
fying number
Recognised taxa
(van Balen 2017a; Wells 2017a, b)
No. of samples Proposed reclassification from this studya
Nicotinamide
adenine dinucleo-
tide dehydrogenase
subunit 2 (ND2)
Cytochrome b (cytb)
Novel GenBank Novel GenBank
1Zosterops palpebrosus palpebrosus – 2 – 1 Zosterops palpebrosus palpebrosus
2Zosterops palpebrosus nilgiriensis – 9 – 12 Zosterops palpebrosus nilgiriensis
3Zosterops palpebrosus salimalii – – – – Zosterops palpebrosus salimalii
4Zosterops palpebrosus egregius – 7 – – Zosterops palpebrosus egregius
5Zosterops palpebrosus siamensis – 1 – – Zosterops palpebrosus siamensis
6Zosterops palpebrosus nicobaricus – – – – Zosterops palpebrosus nicobaricus
7Zosterops palpebrosus williamsoni – – – – Zosterops simplex williamsonib
8Zosterops palpebrosus erwini 27 – 28 – Zosterops simplex erwinib
9Zosterops palpebrosus buxtoni 5 – 5 – Zosterops melanurus buxtonib
10 Zosterops palpebrosus melanurus 3 – 2 1 Zosterops melanurus melanurusb
11 Zosterops palpebrosus unicus – 2 – – Zosterops citrinella unicusb
12 Zosterops japonicus japonicus 1 – 1 8 Zosterops japonicus japonicus
13 Zosterops japonicus simplex 15 3 16 6 Zosterops simplex simplexb
14 Zosterops japonicus hainanus – – – – Zosterops simplex hainanusb
15 Zosterops japonicus loochooensis – – – – Zosterops japonicus loochooensis
16 Zosterops japonicus daitoensis – – – – Zosterops japonicus daitoensis
17 Zosterops japonicus stejnegeri – – – – Zosterops japonicus stejnegeri
18 Zosterops japonicus alani – – – – Zosterops japonicus alani
19 Zosterops japonicus insularis – – – – Zosterops japonicus insularis
20 Zosterops salvadorii 1 – 1 – Zosterops simplex salvadoriib
21 Zosterops citrinella citrinella 13 1 12 – Zosterops citrinella citrinella
22 Zosterops citrinella harterti 5 – 5 – Zosterops citrinella harterti
23 Zosterops citrinella albiventris – – – – Zosterops citrinella albiventris
24 Zosterops montanus montanus 5 2 5 – Zosterops japonicus montanusb
25 Zosterops montanus whiteheadi – 2 – – Zosterops japonicus whiteheadib
26 Zosterops montanus halconensis – – – – Zosterops japonicus halconensisb
27 Zosterops montanus parkesi – – – – Zosterops japonicus parkesib
28 Zosterops montanus pectoralis – 2 – – Zosterops japonicus pectoralisb
29 Zosterops montanus diuatae – 2 – – Zosterops japonicus diuataeb
30 Zosterops montanus vulcani – – – – Zosterops japonicus vulcanib
31 Zosterops montanus difficilis – – – – Zosterops japonicus difficilisb
32 Zosterops montanus obstinatus – 2 – – Zosterops japonicus obstinatusb
33 Zosterops chloris – 2 – – Zosterops chloris
34 Zosterops atrifrons – 1 – – Zosterops atrifrons
35 Zosterops atricapilla – 2 – – Zosterops atricapilla
36 Zosterops luteus – 1 – – Zosterops luteus
37 Zosterops ceylonensis – 4 – – Zosterops ceylonensis
38 Chlorocharis emiliae – 1 – – Zosterops emiliaeb
39 Zosterops abyssinica – 1 – – Zosterops abyssinica
40 Zosterops erythropleura – 1 – – Zosterops erythropleura
41 Zosterops nigrorum – 1 – – Zosterops nigrorum
42 Zosterops virens – 1 – – Zosterops virens
43 Zosterops maderaspatanus – 1 – – Zosterops maderaspatanus
44 Zosterops senegalensis – 1 – – Zosterops senegalensis
45 Zosterops metcalfii – 1 – – Zosterops metcalfii

Journal of Ornithology
1 3
mitochondrial genes were amplified via polymerase chain
reaction. The ND2 gene was amplified using the primers
L5219Met (5′-CCC ATA CCC CGA AAA TGA TG-3′) and
H6313Trp (5′-CTC TTA TTT AAG GCT TTG AAGGC-3′)
(Sorenson etal. 1999), and the cytb gene was amplified
using L14833 (5′- CAG GCC TAA TAA AAG CCT A-3′) and
H15487 (5′- GAT CCT GTT TCG TGG AGG AAGGT-3′)
(Cibois etal. 1999; Dong etal. 2010). Samples were cycle-
sequenced using the BigDye Terminator version 3.1 Cycle
Sequencing Kit. Sequences were obtained by capillary elec-
trophoresis using an Applied Biosystems 3730 96-capillary
Genetic Analyzer.
Phylogenetic analysis
The forward and reverse sequences of both mitochondrial
genes were assembled using CodonCode Aligner version
7.0 (CodonCode). The datasets were exported to MEGA7
(Kumar etal. 2016) for alignment via ClustalW (Thomp-
son etal. 2002). Three datasets were used for phylogenetic
analysis—the ND2 gene (trimmed to 888 base pairs), the
cytb gene (trimmed to 588 base pairs), and a concatenated
gene sequence with 1476 base pairs.
RAxML (Stamatakis 2014) was employed to build phy-
logenetic trees using maximum likelihood (ML) with a
GTR + Inverse + Gamma model on each of the three data-
sets. Each dataset was run for 1000 rapid bootstrap repli-
cates and ten runs of thorough ML search. For Bayesian
analysis, we employed both PartitionFinder version 1.1.1
(Lanfea etal. 2012) and jModelTest 2.1.10 (Guindon and
Gascuel 2003; Darriba etal. 2012) to determine the best
evolutionary model for each locus and codon position. Par-
titionFinder resulted in the following models: ND2 gene
(codon position 1 and 2—HKY + Inverse + Gamma; codon
position 3—GTR + Gamma); cytb gene (codon position
1—K80 + Inverse; codon position 2—F81 + Inverse; codon
position 3—HKY + Gamma), while jModelTest provided
a TIM2 + Inverse + Gamma model for the ND2 gene and
an HKY + Gamma model for the cytb gene (Darriba etal.
2012; Tamura and Nei 1993; Hasegawa etal. 1985). As
trees resulting from jModelTest and PartitionFinder models
were identical in topology and extremely similar in branch
support, we only report on the results of jModelTest. In
total, 1000,000 generations were run on MrBayes version
3.2 (Ronquist and Huelsenbeck 2003) for each dataset. The
analysis sampled every 1000 steps, with 25% of the samples
discarded as burn-in. The phylogenetic trees generated were
then combined and modified using FigTree version 1.4.3
(Rambaut 2006). Additionally, DnaSP version 5.10.1 was
used to calculate pairwise sequence divergences (Librado
and Rozas 2009).
Dating analysis
We employed BEAUti and BEAST (Drummond etal. 2012)
to estimate the divergence times within our sampled taxa.
We selected the ND2 gene dataset for this dating analysis
as it encompassed the largest sample size with the highest
number of taxa. We assumed a Yule speciation process (Yule
1925; Gernhard 2008) for this model together with a relaxed
a Taxon names under the proposed taxonomic revision following the results of this study
b Taxon reclassifications recommended based on this study (no. of samples in italic)
Table 1 (continued)
Species’ identi-
fying number
Recognised taxa
(van Balen 2017a; Wells 2017a, b)
No. of samples Proposed reclassification from this studya
Nicotinamide
adenine dinucleo-
tide dehydrogenase
subunit 2 (ND2)
Cytochrome b (cytb)
Novel GenBank Novel GenBank
46 Zosterops ugiensis – 1 – – Zosterops ugiensis
47 Zosterops vellalavella – 1 – – Zosterops vellalavella
48 Zosterops fuscicapillus – 1 – – Zosterops fuscicapillus
49 Woodfordia superciliosa – 1 – – Woodfordia superciliosa
50 Zosterops flavifrons – 1 – – Zosterops flavifrons
51 Zosterops luteirostris – 1 – – Zosterops luteirostris
52 Zosterops splendidus – 1 – – Zosterops splendidus
53 Zosterops rendovae – 1 – – Zosterops rendovae
54 Zosterops lateralis – 1 – – Zosterops lateralis
55 Zosterops rennellianus – 1 – – Zosterops rennellianus
56 Zosterops murphyi – 1 – – Zosterops murphyi

Journal of Ornithology
1 3
molecular clock with lognormal distribution and a calibrated
rate of 4.94%/M years obtained from the age of Ranongga
Island for Zosterops splendidus (Moyle etal. 2009). We ran
Markov chain Monte Carlo chains for 10 million generations
and discarded the first 25% as burn-in. Finally we employed
Tracer version 1.6 (Rambaut etal. 2014) to ensure stationar-
ity and examine respective parameters, including the effec-
tive sample size value.
Results
The final datasets included 140 samples for the ND2 align-
ment and 103 samples for the cytb alignment, plus one out-
group sequence for each alignment. All three analyses (ND2,
cytb, and concatenated) resulted in similar tree topologies,
with no conflict among highly supported clades (Fig.2,
S1). Based on consensus trees, both Z. palpebrosus and Z.
japonicus constitute polyphyletic species (Fig.2, S1). Deep
pairwise nucleotide divergences within these traditional spe-
cies complexes yielded additional support for their poly-
phyly (Table2).
In the Zosterops palpebrosus species complex, we dis-
covered deep divergences between the nominate subspecies
group (represented by palpebrosus, egregius, nilgiriensis
and siamensis, ranging from India and Sri Lanka to North
Vietnam) and all other races in Southeast Asia. We found
the Greater Sundaic race erwini [sensu Wells (2017a, b);
formerly auriventer sensu van Balen (2017b)] to be closely
related to Zosterops salvadorii from Enggano Island and
to Zosterops japonicus simplex from mainland China. The
Javan races Zosterops palpebrosus melanurus and Zosterops
Fig. 2 Bayesian tree topology for nicotinamide adenine dinucleotide
(reduced) dehydrogenase subunit 2 (ND2; left) and cytochrome b
(cytb; right). Tip labels for newly sequenced samples (this study) are
denoted by the terminal taxon name and an identifier corresponding
to TableS1. Tip labels for Genbank-derived samples have a numeral
identifier corresponding to TableS2 unless they are unique for their
species. The colour-shaded areas of the figure indicate the newly pro-
posed species delimitation as presented in the central column. Tra-
ditional species delimitation (as in, e.g. van Balen 2017b, c, d, e, f,
2018a, b; Wells 2017a, b) is indicated in the two columns bordering
the central column to the left and to the right, respectively, with stip-
pled lines assisting in delineating some of the species blocks. Branch
support values are indicated by an asterisk at each node if high
(Bayesian posterior probability > 0.95/maximum likelihood > 0.7) but
omitted otherwise. Each terminal taxon name at the tip of the tree is
either the full scientific name or a subspecies epithet (for the key spe-
cies complexes). The initially unidentified Zosterops samples (JBP)
from Wildlife Reserves Singapore were identified to species level in
this tree from the results obtained

Journal of Ornithology
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Table 2 Average pairwise nucleotide divergences between Zosterops taxa in this study (%)
Zos-
terops
palpe-
brosus
palpe-
brosus
Zos-
terops
palpe-
brosus
nilgir-
iensis
Zos-
terops
palpe-
brosus
egre-
gius
Zos-
terops
palpe-
brosus
siamen-
sis
Zos-
terops
simplex
erwini
Zos-
terops
simplex
simplex
Zos-
terops
simplex
salva-
dorii
Zos-
terops
cit-
rinella
cit-
rinella
Zos-
terops
cit-
rinella
har-
terti
Zos-
terops
cit-
rinella
unicus
Zos-
terops
mela-
nurus
mela-
nurus
Zos-
terops
mela-
nurus
buxtoni
Zos-
terops
emiliae
Zos-
terops
japoni-
cus
japoni-
cus
Zos-
terops
japoni-
cus
white-
headi
Zos-
terops
japoni-
cus
diuatae
Zos-
terops
japoni-
cus
pecto-
ralis
Zos-
terops
japoni-
cus
monta-
nus
Zos-
terops
japoni-
cus ssp.
Zosterops
palpe-
brosus
palpe-
brosus
2.21 – – 4.59 4.59 5.27 4.59 4.08 – 4.59 4.93 – 4.25 – – – 4.08 –
Zosterops
palpe-
brosus
nilgir-
iensis
1.80 – – 5.78 5.44 6.12 4.76 4.25 – 4.42 4.42 – 5.10 – – – 5.27 –
Zosterops
palpe-
brosus
egre-
gius
0.90 1.58 – – – – – – – – – – – – – – – –
Zosterops
palpe-
brosus
siamen-
sis
1.13 2.03 0.90 – – – – – – – – – – – – – – –
Zosterops
simplex
erwini
6.99 6.98 6.98 7.43 1.36 1.36 5.10 4.59 – 5.10 5.44 – 4.08 – – – 4.25 –
Zosterops
simplex
simplex
7.21 7.32 7.21 7.66 2.59 2.04 5.44 4.93 – 4.76 5.10 – 4.42 – – – 4.59 –
Zosterops
simplex
salva-
dorii
6.87 6.31 6.87 7.32 1.46 2.59 5.78 5.27 – 5.44 5.78 – 4.42 – – – 4.59 –
Zosterops
cit-
rinella
cit-
rinella
5.63 5.18 5.41 5.86 6.76 7.32 6.64 0.51 – 3.74 4.08 – 4.42 – – – 4.59 –
Zosterops
cit-
rinella
harterti
5.41 4.96 5.18 5.63 6.76 7.32 6.64 1.13 – 3.23 3.57 – 3.91 – – – 4.08 –

Journal of Ornithology
1 3
Table 2 (continued)
Zos-
terops
palpe-
brosus
palpe-
brosus
Zos-
terops
palpe-
brosus
nilgir-
iensis
Zos-
terops
palpe-
brosus
egre-
gius
Zos-
terops
palpe-
brosus
siamen-
sis
Zos-
terops
simplex
erwini
Zos-
terops
simplex
simplex
Zos-
terops
simplex
salva-
dorii
Zos-
terops
cit-
rinella
cit-
rinella
Zos-
terops
cit-
rinella
har-
terti
Zos-
terops
cit-
rinella
unicus
Zos-
terops
mela-
nurus
mela-
nurus
Zos-
terops
mela-
nurus
buxtoni
Zos-
terops
emiliae
Zos-
terops
japoni-
cus
japoni-
cus
Zos-
terops
japoni-
cus
white-
headi
Zos-
terops
japoni-
cus
diuatae
Zos-
terops
japoni-
cus
pecto-
ralis
Zos-
terops
japoni-
cus
monta-
nus
Zos-
terops
japoni-
cus ssp.
Zosterops
cit-
rinella
unicus
5.52 5.07 5.29 5.74 6.31 6.64 6.19 1.46 1.46 – – – – – – – – –
Zosterops
mela-
nurus
mela-
nurus
5.52 5.07 5.29 5.74 6.87 7.88 7.21 4.96 4.73 4.39 1.02 – 4.08 – – – 4.59 –
Zosterops
mela-
nurus
buxtoni
5.18 4.73 4.96 5.41 6.76 7.77 6.87 4.62 4.39 4.05 0.34 – 4.42 – – – 4.93 –
Zosterops
emiliae 6.42 6.19 6.19 6.64 5.29 6.53 5.52 6.19 6.19 5.86 6.42 6.08 – – – – – –
Zosterops
japoni-
cus
japoni-
cus
6.08 5.63 5.86 6.31 4.28 5.74 4.62 5.41 5.18 5.29 5.97 5.63 4.62 – – – 1.53 –
Zosterops
japoni-
cus
white-
headi
5.97 5.52 5.74 6.19 4.73 5.97 4.84 5.52 5.29 5.29 5.86 5.52 4.96 1.69 – – – –
Zosterops
japoni-
cus
diuatae
6.08 5.41 5.63 6.08 5.18 6.19 5.29 5.18 5.18 5.29 5.63 5.29 4.84 1.80 1.91 – – –
Zosterops
japoni-
cus
pecto-
ralis
5.74 5.29 5.52 5.74 4.73 5.74 4.84 5.07 4.84 4.96 5.86 5.52 4.62 1.35 1.69 1.13 – –

Journal of Ornithology
1 3
palpebrosus buxtoni were discovered to constitute a distinct
lineage not closely related to other “Oriental white eye” taxa.
The Lesser Sundaic race Zosterops palpebrosus unicus, on
the other hand, emerged as closely related with and poorly
diverged from members of Zosterops citrinella (Fig.2;
Table2).
In the Z. japonicus species complex, we uncovered
deep divergence between the continental Asian race sim-
plex and the nominate subspecies group from the Japanese
archipelago. The continental Z. japonicus simplex emerged
as closely related to Z. palpebrosus erwini and Z. salva-
dorii, with high support and shallow divergence (Table2;
Fig.2). On the other hand, the Japanese nominate subspecies
emerged as embedded within the Mountain White-eye Z.
montanus with high support (Fig.2).
The results obtained from the BEAST dating analysis
provided approximate divergence times within the species
complexes (Fig.3). Various members of the traditional Z.
palpebrosus species complex emerged as polyphyletic and
were generally ~ 1.2–2 million years removed from one
another. Similarly, the two main taxon groups forming the
traditional Z. japonicus species complex were relatively dis-
tantly related and ~ 1.4 million years apart from each other.
We caution that these estimates can only be interpreted as
rough approximations because they depend on a molecular
clock rate calibrated by using island age estimates for diver-
gences between Solomon island taxa of Zosterops (Moyle
etal. 2009). This particular clock rate (4.94%/million years)
is the only one thus far presented in the literature for Zos-
terops evolution, but is ~ 2.4 times slower than the widely
used passerine mitochondrial clock rate of 2.1%/million
years (e.g. Weir and Schluter 2008; Lovette 2004; Peterson
2006), implying that our actual divergence estimates may be
twice as old as here presented. More importantly, our phylo-
genetic tree’s topology and nodal support demonstrate that
these Zosterops wastebasket species are in fact composed
of artificially merged lineages which are often not closely
related to one another within the genus.
Discussion
Taxonomic revision
The resulting phylogenetic trees provide new insights into
the diversification of white-eyes across Asia, suggesting the
non-monophyly of multiple wide-ranging Zosterops waste-
basket species. Unsurprisingly, low support values were
obtained on some of the basal nodes of the phylogenetic
trees, reflecting the explosive and sudden nature of Zoster-
ops diversification (Moyle etal. 2009). This pattern mir-
rors the results of whole-genome studies of the evolutionary
Table 2 (continued)
Zos-
terops
palpe-
brosus
palpe-
brosus
Zos-
terops
palpe-
brosus
nilgir-
iensis
Zos-
terops
palpe-
brosus
egre-
gius
Zos-
terops
palpe-
brosus
siamen-
sis
Zos-
terops
simplex
erwini
Zos-
terops
simplex
simplex
Zos-
terops
simplex
salva-
dorii
Zos-
terops
cit-
rinella
cit-
rinella
Zos-
terops
cit-
rinella
har-
terti
Zos-
terops
cit-
rinella
unicus
Zos-
terops
mela-
nurus
mela-
nurus
Zos-
terops
mela-
nurus
buxtoni
Zos-
terops
emiliae
Zos-
terops
japoni-
cus
japoni-
cus
Zos-
terops
japoni-
cus
white-
headi
Zos-
terops
japoni-
cus
diuatae
Zos-
terops
japoni-
cus
pecto-
ralis
Zos-
terops
japoni-
cus
monta-
nus
Zos-
terops
japoni-
cus ssp.
Zosterops
japoni-
cus
monta-
nus
5.86 5.63 5.63 5.63 4.73 5.97 5.07 4.96 4.96 5.07 6.19 5.86 4.84 1.35 1.91 1.35 0.90 –
Zosterops
japoni-
cus ssp.
5.97 5.52 5.74 5.97 5.07 6.08 5.07 5.52 5.07 5.41 6.31 5.97 4.73 1.69 2.03 1.46 1.01 1.24
Cells above the diagonal refer to cytb and cells below the diagonal refer to ND2. A dash represents cells in which the taxon was not represented for the gene in question

Journal of Ornithology
1 3
history across avian families, which have similarly found
low support or unresolved branches during periods of rapid
diversification (Jarvis etal. 2014; Prum etal. 2015).
Traditional Zosterops taxonomy has doubtless been based
on an over-reliance on conservative plumage features, with-
out taking vocal or molecular evidence into account. Our
molecular results redefine species limits for five tradition-
ally circumscribed species, which are re-arranged into five
newly circumscribed species: the ‘palpebrosus’, ‘simplex’,
‘melanurus’, ‘citrinella’ and ‘japonicus’ groups, with taxon
ranges based on van Balen (2017b, c, d, e, f) and Wells
(2017a, b) (Fig.1b; Table1).
The ‘palpebrosus’ group
The Oriental White-eye Zosterops palpebrosus was first
described by Temminck in 1824 on the basis of a type speci-
men originating from Bengal, India. A further 11 taxa have
been classified as its subspecies (van Balen 2017b) rang-
ing from South Asia eastwards to Sundaland and the Lesser
Sundas. However, our mitochondrial DNA (mtDNA) analy-
sis revealed deep divergences and non-sister relationships
among South Asian taxa and most races further east (Fig.2;
Table2), corroborating and considerably extending earlier
results (Moyle etal. 2009; Wickramasinghe etal. 2017).
Among the Southeast Asian subspecies we sampled, only
siamensis clustered closely with palpebrosus, whereas all
other Southeast Asian subspecies do not emerge as embed-
ded within or even remotely sister to the palpebrosus clus-
ter, and hence must be separated from it (Fig.2; Fig. S1).
Geographically, the newly circumscribed Z. palpebrosus
ranges from Arabia through India eastwards to North Viet-
nam. A single GenBank sample (AMNH DOT10981) from
North Vietnam that had been lodged as Zosterops japonicus
simplex emerged within the palpebrosus cluster, doubtless
constituting a misidentified individual of Zosterops palpe-
brosus siamensis. The sample did not align with the other
18 genuine simplex samples sequenced in this study and
was collected in late May when wintering simplex should be
absent from Vietnam whilst the breeding siamensis should
be present.
For South Asian taxa, close relationships were confirmed
among Zosterops palpebrosus palpebrosus, Zosterops pal-
pebrosus egregius and Zosterops palpebrosus nilgiriensis
(Fig.2) (Warren etal. 2006; Wickramasinghe etal. 2017),
corroborating their conspecificity. The sole remaining
unsampled taxon Zosterops palpebrosus salimalii, which
is embedded within the range of Zosterops palpebrosus
egregius, is unlikely to be an exception and is provisionally
retained under Z. palpebrosus because of morphological
similarity and geographic proximity.
The taxonomic status of the subspecies Zosterops pal-
pebrosus nicobaricus is more contentious, but we here pro-
visionally retain it under Zosterops palpebrosus given an
Fig. 3 The divergence times of Zosterops taxa with horizontal blue bars indicating 95% highest posterior density intervals. Taxa in bold are the
focal species of this study. Yuhina brunneiceps was the outgroup species for this analysis

Journal of Ornithology
1 3
absence of DNA material and pending its incorporation into
phylogenetic research. This taxon is restricted to the Nico-
bar Islands; nearby populations on the adjacent Andaman
Islands are currently considered undescribed but may belong
with nicobaricus (Rasmussen & Anderton 2012). Bioge-
ographically, the Nicobar and Andaman Islands exhibit
a greater similarity with Southeast Asia than with South
Asia (Woodruff 2010), implying that nicobaricus may share
close affinities with the adjacent taxon erwini from Sumatra
and the Thai-Malay Peninsula rather than with the nomi-
nate India-centred Z. palpebrosus complex as here defined.
Plumage descriptions of this poorly known taxon imply that
nicobaricus should have a less yellow-bright and bronzier
(= more olive) body coloration than Indian taxa (Rasmussen
and Anderton 2012), which would equally suggest a closer
affinity with erwini than with Indian palpebrosus. A merger
of nicobaricus with erwini would have great nomenclatural
consequences as nicobaricus would become the oldest name
(proposed by Blyth in 1845) among all the taxa known to be
closely related to erwini (including erwini itself). Our phy-
logeny demonstrates conclusively that erwini, along with a
number of other East Asian taxa, is not even closely related
to Z. palpebrosus across the genus (see below). Even so,
it seems premature to base the species name for this East
Asian species on a taxon that has not even been included in
the study. Hence, we provisionally retain nicobaricus under
Zosterops palpebrosus but note that it may soon become
necessary to merge it with the East Asian taxa erwini, sim-
plex etc. (see below), rendering the name of that newly con-
stituted species Zosterops nicobaricus.
We provisionally include the following subspecies within
the newly circumscribed Z. palpebrosus:
• Zosterops palpebrosus—Indian White-eye.
• Zosterops palpebrosus palpebrosus Temminck, 1824—
southeast Arabia through northern India east to south-
west Sichuan, Yunnan, and Myanmar.
• Zosterops palpebrosus nilgiriensis Ticehurst, 1927—
southern Western Ghats in southwest India.
• Zosterops palpebrosus salimali Whistler, 1933—south-
ern Eastern Ghats in southeast India.
• Zosterops palpebrosus egregius Madarász, 1911—low-
lands of peninsular India and Sri Lanka.
• Zosterops palpebrosus siamensis Blyth, 1867—southern
Myanmar to northwest Indochina and northern Vietnam.
• Zosterops palpebrosus nicobaricus Blyth, 1845—Anda-
man Islands and Nicobar Islands.
The ‘simplex’ group
The Sundaic taxon erwini [sensu Wells (2017a, b); formerly
named auriventer sensu van Balen (2017b)], previously
subsumed under the Oriental White-eye Z. palpebrosus,
emerged as deeply diverged from the palpebrosus cluster and
in a polyphyletic placement on the tree (Fig.2; Table2). In
fact, Z. palpebrosus as here redefined (see above), emerged
as more closely related to a number of African species with
high support (Figs.2, 3), demonstrating the inappropriate-
ness of retaining erwini as a member of Z. palpebrosus.
Instead, erwini turned out to be closely related to simplex,
a taxon predominantly from China previously classified
erroneously under the Japanese White-eye Z. japonicus.
While a deep division between simplex from the Chinese
mainland and japonicus from the Japanese Archipelago has
previously been proposed on a tentative basis (van Balen
2017c), the association between Chinese simplex and erwini
from Southeast Asia is a novel insight. mtDNA sequence
divergence between erwini and simplex is extremely low
(Table2). Given that they are also united by close pheno-
typic and vocal similarities (personal observations), the most
conservative course of action appears to be a merger of these
two forms into one species, the senior name of which would
be simplex (described by Swinhoe in 1861).
Further to these two forms, we assume that williamsoni
from the Gulf of Thailand also falls within this complex,
based on geographical adjacency to and close morphological
similarity with erwini (Wells 2017a, b). The taxon salva-
dorii from Enggano Island west of Sumatra is also revealed
to have an extremely low mtDNA divergence from erwini
and simplex (Table2) and to be embedded within those two
(Fig.2). It has long been considered a weak monotypic spe-
cies, Enggano White-eye, with a poorly known morpho-
logical distinctness (Eaton etal. 2016). Its unremarkable
differentiation strongly suggests its incorporation with Z.
simplex, shifting the burden of proof to those who would
wish to continue to regard it at the species level.
We have commented above on the contentious status of
nicobaricus, whose DNA has never been included in any
phylogenetic research. To remain conservative, we have
provisionally included this taxon with Z. palpebrosus (see
above), despite contrary circumstantial evidence based on
biogeography and morphology. It may well form a species-
level lineage of its own or necessitate inclusion in the pre-
sent Z. simplex. In the second case, the species name would
need to change to Z. nicobaricus because of the priority of
the latter name.
In summary, we propose the following taxonomic
arrangement for Z. simplex:
• Zosterops simplex—Swinhoe’s White-eye.
• Zosterops simplex simplex Swinhoe, 1861—eastern
China, Taiwan and extreme northeast Vietnam; non-
breeding in Thailand and Indochina.
• Zosterops simplex hainanus E. J. O. Hartert, 1923—
Hainan.

Journal of Ornithology
1 3
• Zosterops simplex erwini Chasen, 1934—Thai-Malay
Peninsula, lowland Sumatra, Riau Island, Bangka,
Natuna Island and lowland Borneo.
• Zosterops simplex williamsoni Robinson & Kloss,
1919—Gulf of Thailand coast.
• Zosterops simplex salvadorii A. B. Meyer & Wigles-
worth, 1894—Enggano Island.
The ‘melanurus’ group
Mitochondrial evidence revealed melanurus and buxtoni,
from eastern and western Java, respectively, to form a tight-
knit group that is deeply diverged from and distantly related
to both Z. palpebrosus and Z. simplex (see above; Figs.2,
3; Table2). Instead, the Javan forms are embedded within a
clade that comprises mostly Malenesian and eastern Indone-
sian species (Fig.3). The vocal impression of Javan popula-
tions in the field is also different from that of adjacent erwini
from Sumatra (personal observation). This Javan subspecies
group displays an atypical range of diversity in phenotype,
with two distinct belly colours—yellow (in melanurus) and
grey (in buxtoni). These two phenotypes have long been
recognised to meet in a hybrid zone in West Java, where
flocks with pure and intermediate phenotypes are wide-
spread around the city of Bogor and adjacent areas. mtDNA
analysis shows extremely limited divergence between the
grey and yellow-bellied forms on Java, suggesting conspeci-
ficity within one polytypic species, Zosterops melanurus.
This newly constituted species, Z. melanurus, may indeed
be endemic to Java and Bali. The West Javan subspecies
buxtoni is reported to extend in range into montane Sumatra
(van Balen 2017b), but photographic evidence of popula-
tions in montane Sumatra strongly suggests the presence
of a population of the montane species Hume’s White-eye,
Zosterops auriventer [sensu Wells (2017a, b); equals Zos-
terops everetti tahanensis sensu van Balen (2018b)], that
has hitherto been misidentified (unpublished data), and we
follow this preliminary evidence here in excluding montane
Sumatran populations from the range of buxtoni. The sepa-
ration of Z. melanurus (including buxtoni) as an independ-
ent species has important conservation implications as this
taxonomic treatment renders it the most heavily trapped bird
species on Earth (Chng etal. 2015; Chng and Eaton 2016;
Lee etal. 2016; Eaton etal. 2017).
In summary, we propose the following taxonomic
arrangement for Zosterops melanurus:
• Zosterops melanurus—Sangkar White-eye.
• Zosterops melanurus melanurus Hartlaub, 1865—eastern
and central Java, Bali.
• Zosterops melanurus buxtoni Nicholson, 1879—West
Java.
The ‘citrinella’ group
The Ashy-bellied White-eye Zosterops citrinella, native to
the eastern Lesser Sunda Islands (East Nusa Tenggara), was
revealed to exhibit an extremely shallow mtDNA divergence
from Zosterops palpebrosus unicus, a former member of the
‘Oriental White-eye’ complex that was found to be unrelated
to Z. palpebrosus already by Moyle etal. (2009) (Table2).
The traditional classification of unicus has clearly been erro-
neous. Based on our mtDNA data, we propose to unite uni-
cus with Z. citrinella, of which it is an allopatric vicariant
in the region. Its yellowish belly colour sets it apart from
other more ashy-bellied members of Z. citrinella. However,
this difference in belly colour amongst members of the same
species is not unusual in Zosterops as seen in the Java/Bali
endemic Z. melanurus (see above) and Indochinese popula-
tions of Z. palpebrosus siamensis (Robson 2005).
In summary, we propose the following taxonomic
arrangement for Zosterops citrinella:
• Zosterops citrinella—Ashy-bellied White-eye.
• Zosterops citrinella citrinella Bonaparte, 1850—Sumba,
Savu, Timor, Semauand Roti.
• Zosterops citrinella harterti Stresemann, 1912—Lem-
bata and Alor.
• Zosterops citrinella albiventris Reichenbach, 1852—
Gunungapi, Wetar, Romang, Damar, Teun, Kisar, Leti,
Moa, Luang, Sermata, Babar, Tanimbar Islands; islands
in Torres Strait and islets off extreme north-eastern Aus-
tralia.
• Zosterops citrinella unicus E. J. O. Hartert, 1897—Sum-
bawa and Flores.
The ‘japonicus’ group
Our analyses corroborate prior preliminary suggestions
(e.g. van Balen 2017c) of a division of the former Japa-
nese White-eye Z. japonicus into an archipelagic group
centred around the Japanese nominate japonicus and a
Chinese mainland group centred around the subspecies
simplex. Extending these preliminary suggestions, we
demonstrated and discussed above that the new Z. sim-
plex additionally includes Southeast Asian taxa that had
erroneously been subsumed under Z. palpebrosus or had
been treated as independent species. However, the status
of the Japanese nominate group is also more complicated
than merely a simple split. Our mtDNA analysis revealed
members of the nominate subspecies group of Z. japoni-
cus from the Japanese archipelago, South Korea and an
introduced population in Hawaii to be embedded with
samples of the Mountain White-eye Z. montanus (on the
basis of ND2) or in a shallow sister relationship with it

Journal of Ornithology
1 3
(based on cytb; Fig.2). Price etal. (2014) reported on a
deep split between Z. japonicus and Z. montanus, but a
search regards their sample’s locality showed it to be from
eastern China, thereby confirming its identity as Z. sim-
plex, leaving the actual relationship between japonicus and
montanus uninvestigated until now. Merging japonicus and
montanus results in a new composite species, Zosterops
japonicus, according to nomenclatural priority. Beyond
the results of our mtDNA analysis, this merger is also sug-
gested by vocal impressions [distinct flight call (personal
observation)] and their shared pale iris, which is unusual
in most other Zosterops species in the region. The Moun-
tain White-eye as traditionally circumscribed (‘Zosterops
montanus’) is known to have great dispersal capabilities,
with a fairly uniform morphology across an insular moun-
tain distribution from Sumatra all the way to the Moluccas
and Philippines. Therefore, an extension of this vast range
northwards to include the Japanese archipelago is unsur-
prising. All the remaining unsampled subspecies refer to
insular forms within the nominate cluster of Z. japonicus
or to Philippine island subspecies of former Z. montanus
which are unlikely to fall outside of their respective main
clade, prompting us to retain them with their traditional
taxonomic alliances.
In summary, we propose the following taxonomic
arrangement for Z. japonicus:
• Zosterops japonicus—Mountain White-eye.
• Zosterops japonicus japonicus Temminck & Schlegel,
1845—Sakhalin, Japan and coastal Korean Peninsula.
• Zosterops japonicus insularis Ogawa, 1905—extreme
northern Ryukyu Islands.
• Zosterops japonicus loochooensis Tristram, 1889—main
Ryukyu Islands.
• Zosterops japonicus daitoensis Nagamichi Kuroda,
1923—Borodino Island.
• Zosterops japonicus stejnegeri Seebohm, 1891—Oshima
(Izu Island) south to Torishima (Nanpo Archipelago).
• Zosterops japonicus alani E. J. O. Hartert, 1905—Iwo
(Volcano) Island.
• Zosterops japonicus montanus Bonaparte, 1850—moun-
tains in Sumatra, Java, Bali, Lesser Sundas, Sulawesi and
southern Moluccas.
• Zosterops japonicus whiteheadi E. J. O. Hartert, 1903—
highlands of Luzon.
• Zosterops japonicus halconensis Mearns, 1907—Mind-
oro.
• Zosterops japonicus parkesi duPont, 1971—mountains
of Palawan.
• Zosterops japonicus pectoralis Mayr, 1945—Negros.
• Zosterops japonicus diuatae Salomonsen, 1953—north-
ern Mindanao.
• Zosterops japonicus volcani E. J. O. Hartert, 1903—cen-
tral Mindanao.
• Zosterops japonicus difficilis Robinson & Kloss, 1918—
Mount Dempo in south Sumatra.
• Zosterops japonicus obstinatus E. J. O. Hartert, 1900—
Ternate, Tidore, Bacan, Seram.
Generic reassignment ofChlorocharis emiliae
The Bornean Mountain Black-eye Zosterops emiliae was
first described in 1888 by Sharpe and was initially given
monotypic genus status under Chlorocharis. Our results
confirmed previous findings (Moyle etal. 2009) showing
this Bornean endemic to be embedded within Zosterops, and
basal to our newly circumscribed Z. japonicus and Z. simplex
species (Fig.2), with which it is allopatric.
Conservation implications
Our taxonomic rearrangements have led to the elevation
of threatened lineages to species level, foremost Zosterops
melanurus. In Java, the white-eye trade has already led to
the endangerment of previously recognised endemic spe-
cies such as the Javan white-eye Zosterops flavus (Eaton
etal. 2015), which is suffering a decreasing population
trend with a current International Union for Conservation
of Nature (IUCN) 2016 status of ‘Vulnerable’ (BirdLife
International 2018). With its elevation to species level, Z.
melanurus urgently requires IUCN classification for conser-
vation priority. Z. melanurus must be the most heavily traded
bird species on Earth with records of 2393 individuals in
single market surveys around West Java (Chng etal. 2015;
Eaton etal. 2015; Chng and Eaton 2016). As the common
white-eye on the island, this species seems to fuel Java’s
deeply entrenched tradition of bird keeping (Eaton etal.
2015). Given the documented exploitation levels (Chng etal.
2015; Chng and Eaton 2016), we believe a ~ 30% reduction
in global population size over the past 10years is a reason-
able—if not conservative—assumption, and hence propose
Vulnerable A2d status under the 2001 IUCN Red List cri-
teria (version 3.1; available at www.iucnr edlis t.org) for Z.
melanurus.
On the small island of Singapore, both the native Z. sim-
plex erwini and escaped individuals of the locally traded Z.
simplex simplex (Jeyarajasingam 2012; Eaton etal. 2017)
are found in the wild, as confirmed by our analyses (Fig.2).
Under previous taxonomic treatments, erwini and simplex
would have been considered members of different species
(Oriental White-eye Z. palpebrosus and Japanese White-eye
Z. japonicus, respectively). However, our genetic data show
that these two taxa are closely related to each other, and the
possibility of secondary gene flow in the wild is very likely
(Fig.2; Table2). Future studies using nuclear DNA sources

Journal of Ornithology
1 3
will be necessary to assess the level of gene flow between
native and introduced white-eyes on Singapore.
Conclusion
Using mitochondrial data, we have uncovered the non-
monophyly of several wide-ranging white-eye species com-
plexes across Australasia—Z. palpebrosus, Z. japonicus and
Z. montanus. Our molecular results redefine species limits
for five traditionally circumscribed species, which were
previously based predominantly on conservative plumage
features. The new taxonomic classification may help direct
conservation attention to relevant groups, particularly Z.
melanurus from Java and Bali, which represent the most
heavily traded bird species on earth and may be at risk of
trade-driven extinction.
Acknowledgements We thank C.Y. Gwee, N.S.R. Ng, E.Y.X. Ng,
Q. Tang, G.W.J. Low, P. Baveja, J.Y. Lim, H.Z. Tan, D.Y.J. Ng,
R.Y.C. Teo, Y.F. Chung, J.E.H. Ang, A.Y.H. Tan and J.S.M.
Soh for field support, lab support and/or valuable feedback on this
manuscript. We acknowledge grants from Wildlife Reserves Singapore
(R-154-000-A05-592) and Wildlife Reserves Singapore Conservation
Fund (R-154-000-A99-592) for funding this project. We are indebted
to Wildlife Reserves Singapore, the Lee Kong Chian Natural History
Museum (Singapore), the Burke Museum of Natural History and Cul-
ture (Seattle, WA), Museum Zoologicum Bogoriense (Cibinong, West
Java, Indonesia) and the Hong Kong Bird Watching Society Ringing
Team for providing samples for this study. The National Parks Board of
Singapore is acknowledged for facilitating fieldwork. The experiments
comply with the laws of the Republic of Singapore and Hong Kong.
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Aliations
BryanT.M.Lim1· KerenR.Sadanandan1· CarolineDingle2· YuYanLeung2· DewiM.Prawiradilaga3·
MohammadIrham3· HidayatAshari3· JessicaG.H.Lee4· FrankE.Rheindt1
* Frank E. Rheindt
dbsrfe@nus.edu.sg
1 Department ofBiological Sciences, National University
ofSingapore, 16 Science Drive 4, Singapore117558,
Singapore

Journal of Ornithology
1 3
2 School ofBiological Sciences, Kadoorie Biological Sciences
Building, University ofHong Kong, Pok Fu Lam Road,
HongKongSAR, China
3 Research Center forBiology-LIPI, Cibinong, WestJava,
Indonesia
4 Wildlife Reserves Singapore, 80 Mandai Lake Road,
Singapore729826, Singapore