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Zoological Journal of the Linnean Society, 2019, XX, 1–24. With 9 figures. Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlz022/5477306 by University College Dublin, [email protected] on 24 April 2019

A sympatric pair of undescribed white-eye species (Aves: Zosteropidae: Zosterops) with different origins

DARREN P. O’CONNELL1,2*, , DAVID J. KELLY1, NAOMI LAWLESS1, KATIE O’BRIEN1, FIONN Ó MARCAIGH1, ADI KARYA3, KANGKUSO ANALUDDIN3 and NICOLA M. MARPLES1

1 Department of Zoology, School of Natural Sciences, Trinity College Dublin, Dublin DO2 CX56, Ireland 2 School of Biology & Environment Science, University College Dublin, Dublin DO4 N2E5, Ireland 3 Department of Biology and Biotechnology, Universitas Halu Oleo, Kendari, South-east Sulawesi, Indonesia

Received 13 September 2018; revised 15 February 2019; accepted for publication 17 February 2019

Research in the Indo-Pacific region has contributed massively to the understanding of speciation. White-eyes (Aves: Zosteropidae: Zosterops), a lineage containing both widespread ‘supertramp’ species and a high proportion of island endemics, have provided invaluable models. Molecular tools have increased speciation research, but delimiting species remains problematic. We investigated the evolutionary history of Zosterops species in south-east Sulawesi using mitochondrial DNA, morphometric, song and plumage analyses, to draw species limits and assess which techniques offer best resolution. Our investigation revealed a novel Zosterops species, >3000 km from its closest relative. Additionally, we demonstrated unanticipated diversity in the alleged ‘supertramp’ Zosterops chloris and propose the Wakatobi Islands subspecies (Z. c. flavissimus) to be given full species status. Furthermore, we provide the first molecular and phenotypic assessment of the Sulawesi endemic Zosterops consobrinorum. While local populations of this species vary in either genetics or morphometrics, none show consistency across measures. Therefore, we propose no change to Zosterops consobrinorum taxonomy. This study gives insight into one of the great Indo-Pacific radiations and demonstrates the value of using multiple lines of evidence for taxonomic review.

KEYWORDS: birds – evolution – Indonesia – islands – new species – Wallacea.

INTRODUCTION species (Lohman et al., 2010; Irestedt et al., 2013) and elucidating the evolutionary history of island Islands have long been key to our understanding of colonizations (Cibois et al., 2011, 2014, Andersen et al., evolution, providing discrete geographical units to 2013, 2014). However, questions still remain on how study patterns of speciation and the processes that best to delimit species in widespread radiations (Tobias underlie these patterns (Darwin, 1859; Wallace, 1869). et al., 2010; Andersen et al., 2014), and which processes The islands of the Indo-Pacific have been particularly allow some populations to maintain connectivity over important in the last half-century for laying down large distances, while others become isolated endemic many of the fundamental principles underpinning our taxa (Andersen et al., 2015; Pedersen et al., 2018). understanding of island biogeography and speciation One of the lineages of major importance to the study of (MacArthur & Wilson, 1967; MacArthur et al., 1972; avian speciation is the Zosterops white-eyes. Zosterops Diamond, 1974, 1998; Diamond et al., 1976). This have a wide distribution across the Indo-Pacific, South region is home to thousands of islands and several Asia and Africa (Van Balen, 2008). They are supreme widespread species radiations, perfect for studying island colonizers, which are found throughout the evolution in multiple, closely related populations Indo-Pacific, with 73 of the 96 currently recognized (Mayr & Diamond, 2001). Modern molecular tools have bolstered this work, uncovering cryptic Zosterops species being found on islands in this region (Mees, 1961, 1969; Mayr & Diamond, 2001; Warren et al., 2006; Van Balen, 2018a). Zosterops show one of *Corresponding author. E-mail: [email protected] the fastest speciation rates of any vertebrate (Moyle

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, XX, 1–24 1 2 D. P. O’CONNELL ET AL. Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlz022/5477306 by University College Dublin, [email protected] on 24 April 2019 et al., 2009), rivalled only by cichlid fish (Meyer, 1993; the molecular markers, in isolated populations, would Genner et al., 2007; Elmer et al., 2010). This rapid rate give greater insight into this rapidly evolving lineage of diversification has earned them the label as one of (Jønsson et al., 2014) and provide more effective the ‘great speciators’ of the Indo-Pacific, species groups species delimitation (Dong et al., 2015; Liu et al., 2016; marked out by their remarkable speciation rates Wood et al., 2016). (Mayr & Diamond, 2001; Moyle et al., 2009; Cornetti In the heart of the Wallacea region, the south- et al., 2015; Lim et al., 2018). eastern peninsula of Sulawesi provides an excellent Zosterops species embody the paradox of ‘great study system to test the effect of isolation on Zosterops speciators’ (Diamond et al., 1976): how do taxa that species (Fig. 1). There are continental islands (Buton, are sufficiently vagile to be such successful island Muna, Kabaena and Wawonii), which were connected colonizers become isolated and diverge into endemic to Sulawesi at the time of the last glacial maximum, species? Diamond et al. (1976) proposed that this around 20 000 years ago (Voris, 2000; Yokoyama pattern may arise from rapid shifts in dispersal et al., 2000; Clark et al., 2009), and oceanic islands ability in populations. The phylogeographic pattern (the Wakatobi Islands and Runduma Island), which of Zosterops species in Moyle et al. (2009) appeared have never been connected to the Sulawesi mainland consistent with this thesis. Dispersal ability can (Milsom & Ali, 1999; Nugraha & Hall, 2018). The be reduced in island taxa when adaptation to local region has been fruitful for recent speciation research. conditions favours traits other than dispersal ability While the Wakatobi Islands are only separated from (Mayr & Diamond, 2001; Gillespie et al., 2012). Buton by 27 km, they are home to six endemic bird Wright et al. (2016) assessed this phenomenon across subspecies (Kelly & Marples, 2010; Collar & Marsden, nine avian families (including the Zosteropidae) and 2014), a proposed new species of flowerpecker (Aves: found that reduced dispersal ability was associated Dicaeidae; Kelly et al., 2014) potential new subspecies with the lower species richness of predators on small of kingfisher (Aves: Alcedinidae; O’Connell et al., 2019). islands. Additionally, the phenomenon of ‘behavioural Kabaena Island, only 16 km from the mainland, is also flightlessness’, where isolated populations show home to an endemic subspecies of red-backed thrush, reduced propensity to fly across water barriers despite Geokichla erythronota subsp. kabaena (Robinson-Dean being physically capable of doing so, has been observed et al., 2002). to have evolved in multiple island bird and butterfly Current taxonomy identifies two Zosterops species, populations (Diamond, 1972, 1981; Holloway, 1977; the lemon-bellied white-eye, Zosterops chloris Diamond & Gilpin, 1983). Adaptations in island (Bonaparte, 1850), and the pale-bellied white-eye, populations that reduce the tendency to make long Zosterops consobrinorum (Meyer, 1904), in south-east flights may conserve energy in resource-constrained Sulawesi (Van Balen, 2018a). The natural history of islands (Diamond, 1981, 1984). these species is still being studied. Zosterops chloris The Zosterops radiation includes extremely is thought to be a typical ‘supertramp’ species (Mayr widespread species, such as the Japanese white-eye, & Diamond, 2001; Eaton et al., 2016). The designation Zosterops japonicus (Temminck & Schlegel, 1845), ‘supertramp’ was developed by Diamond (1974, 1975) and the Oriental white-eye, Zosterops palpebrosus to describe the island-colonizing behaviour of birds; (Temminck, 1824), containing multiple well-defined it includes the categories: (1) ‘sedentary’ – species races (Lim et al., 2018), ‘supertramp’ edge species such confined to the larger islands, (2) ‘tramps’ – present as the Louisiade white-eye Zosterops griseotinctus on larger islands but also many smaller and more (Gray, 1858), which are found on many small islands, remote islands and (3) ‘supertramps’ species that varying little throughout their range (Mayr & are confined as residents mainly to small islands Diamond, 2001), and a large number of single island and virtually absent from larger islands, apart from endemics (Van Balen, 2008). Recent molecular work edge habitats, such as mangroves, where they avoid has begun to re-draw the taxonomy and evolutionary stronger competitors. Zosterops chloris is found on relationships of widespread Zosterops species (Habel small islands from the east coast of Sumatra to the et al., 2013, 2015a; Cox et al., 2014; Husemann et al., west coast of Papua, and in coastal areas and edge 2015; Round et al., 2017; Wells, 2017; Lim et al., 2018) habitats on larger islands in the Lesser Sundas and even show unexpected divergence in supposedly and on Sulawesi, showing the habitat associations ‘supertramp’ lineages like Z. griseotinctus (Linck et al., of a ‘supertramp’ species (Van Balen, 2018b). The 2016). However, there are still few studies addressing different races of Z. chloris are not thought to be very phenotypic and song evolution, processes key to species distinct, with significant overlap in phenotypic traits isolation (Uy et al., 2009), although see Phillimore et al. (Eaton et al., 2016). The subspecies Z. c. flavissimus (2008), Baker (2012), Potvin (2013), Husemann et al. is found on the Wakatobi Islands and the subspecies (2014) and Habel et al. (2015b). An understanding Z. c. intermedius is found on Buton, Muna and Kabaena of how phenotype and song diverge in comparison to (Van Balen, 2018b). The newly discovered Z. chloris

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, XX, 1–24 SYMPATRIC UNDESCRIBED ZOSTEROPS SPECIES 3 Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlz022/5477306 by University College Dublin, [email protected] on 24 April 2019 population on the mainland of south-east Sulawesi methods, with a particular focus on the undescribed has been proposed to be either Z. c. intermedius ‘Wangi-wangi white-eye’, which has been provisionally (Kelly et al., 2010) or Z. c. mentoris, which is found in assigned to this species, and (3) to estimate divergence northern and central Sulawesi (Trochet et al., 2014). times to gain insight into the evolutionary relationships Zosterops consobrinorum is restricted to the south- of the Zosterops taxa in the region. eastern peninsula of Sulawesi, Buton and Kabaena (Wardill, 2003; Van Balen, 2008; O’Connell et al., 2017). The Buton Island population has been suggested as MATERIAL AND METHODS a potentially separate subspecies (Wardill, 2003). A potentially novel Zosterops species is present on only Study site and sampling the northernmost Wakatobi Island, Wangi-wangi (Fig. Sampling was carried out throughout south-east 1). It has been provisionally assigned as a population Sulawesi (Fig. 1) on research expeditions undertaken of Z. consobrinorum (Van Balen, 2018c) and was between 1999 and 2017 in the months of June–September proposed as a novel species by Eaton et al. (2016), the by NNM, DJK, AK, KA and DOC. Zosterops species ‘Wangi-wangi white-eye’. This Zosterops population were sampled on 12 islands throughout the region was first identified by DJK, NMM and Martin Meads (Fig. 1). For additional details on sampling locations, see in 2003 (Kelly & Marples, 2010) and has been awaiting Supporting Information (Table S1). Mist-nets were used molecular work to confirm its status. The south-east to trap birds for sampling. Birds trapped were colour- Sulawesi study system provides the opportunity, ringed for easy identification if trapped again. Coates first, to clarify the understudied taxonomy of these & Bishop (1997) and Eaton et al. (2016) were used for populations, and, second, to investigate the impact of species identification and ageing of birds trapped. The isolation on a widespread ‘supertramp’ and regional morphometric measurements were taken: wing length endemic Zosterops species. (maximum chord), tarsus length (minimum), bill length To achieve the aims of this study our research (tip of bill to the base of the skull), skull length (base goals were: (1) to assess the ‘supertramp’ Z. chloris by of the bill to the notch at the back of the head), bill comparing populations for divergence in mitochondrial depth (measured at the nares), tail length (longest tail DNA, morphometrics or song, (2) to assess populations of feather from base to tip) and mass (grams) (Svensson, the regional endemic Z. consobrinorum using the same 1992; Redfern & Clark, 2001). All measurements

Sulawesi New Guinea

Borneo

Java Lombok

Menui

Mainland Wawonii South-east Sulawesi

Muna Wakatnds

Wangi- wangi Kabaena Buton O Kaledupa Tomia Lintea Binongk 050 100 150 200 250 km Selatan

Figure 1. Map showing the study region of south-east Sulawesi (main panel) and the Sulawesi region of Indonesia (top right panel). Sites where Zosterops were sampled are indicated by coloured pins: red indicates Z. consobrinorum, blue indicates the ‘Wangi-wangi white-eye’ and green indicates Z. chloris, with the Z. c. flavissimus subspecies indicated by lime green.

© 2019 The Linnean Society of London, Zoological Journal of the Linnean Society, 2019, XX, 1–24 4 D. P. O’CONNELL ET AL. Downloaded from https://academic.oup.com/zoolinnean/advance-article-abstract/doi/10.1093/zoolinnean/zlz022/5477306 by University College Dublin, [email protected] on 24 April 2019 were taken by a single recorder (NMM). Only adult Zosterops species, which would be suitable for binding by birds were included in morphometric analyses. All primers that conformed to the usual principles of primer morphometric data used in these analyses are available design (Lustbader, 2015). The established primer L10755 at https://figshare.com/articles/SE_Sulawesi_Zosterops_ was also used for ND3 (Chesser, 1999). The polymerase morphology/7998299/1. The Zosterops species of south- chain reaction (PCR) procedure was adapted from Kelly east Sulawesi are sexually monomorphic (Van Balen, et al. (2014). All PCR amplifications were performed in

2018b, c), so sexes were not separated for morphometric 20 µL reactions, consisting of 8.1 µL double-distilled H20, analyses. Approximately 5–10 contour feathers were 0.4 µL 10 mM deoxynucleoside triphosphates (dNTPs), plucked from the flank of each bird and stored in sealed 2 µL 10× PCR reaction buffer, 2.4 µL 25 mM MgCl2, 1 µL paper envelopes. Contour feathers were sampled to 10 µM forward primer, 1 µL 10 µM reverse primer, 0.1 µL minimize the risk of injury to the birds and to avoid Taq polymerase (New England Biolabs) and 5 µL template disruption to flight ability and plumage-based visual DNA. All reactions were amplified under the following signals (McDonald & Griffith, 2011). Mist-netting was thermal cycler conditions: 4 min at 94 °C followed by carried out in a variety of habitats used by Zosterops 45 cycles of 1 min at 94 °C, 1.5 min at the gene-specific species, including plantation, forest edge, farmland and annealing temperature (53 °C for ND2, 50 °C for ND3 and mangroves. 55 °C for COI) and 1.5 min at 72 °C, finishing with 5 min Zosterops songs were recorded using a Zoom H2 at 72 °C. Amplified PCR products were screened on 2% Handy Recorder with a Sennheiser Me62 Omni- agarose gels stained with Gel Red. Sanger sequencing was Directional Condenser Microphone Capsule with a carried out in both directions by GATC Biotech (Cologne, K6 power supply. The microphone was mounted on a Germany) using an ABI 3730xl DNA analyser system. Telinga V2 Foldable Parabolic Reflector to minimize All sequences were submitted to GenBank (Benson background noise. Songs were saved in a Waveform et al., 2013). The accession numbers of all sequences are Audio File format for maximum song quality. As the provided in Supporting Information, Table S1. song of the different focal Zosterops species is similar, the microphone operator was accompanied by another team member with binoculars to identify the species of Taxon sampling each individual recorded. Recording was mainly carried In addition to our focal study populations in south-east out just after dawn and just before dusk, at the peaks Sulawesi, sequence information for Zosterops species and of singing activity. To ensure each recording was of a other comparison groups were sourced from GenBank separate individual, the song recording team walked a (Benson et al., 2013) (accession numbers provided in new 1-km transect route during each recording session Supporting Information, Table S1). ND2, ND3 and COI and observed Zosterops flocks to ensure that the same are widely used genes, allowing for comparisons with individuals were not being recorded multiple times. In a large amount of published material to elucidate the addition to these recordings, the xeno-canto bird sound evolutionary history of our target species. ND2 and ND3 collection (http:www.xeno-canto.org) was searched to sequences were concatenated and analysed separately source further recordings of our study species. to COI sequences, due to a much wider sample of Zosterops ND2 and ND3 genes being available on GenBank (Moyle et al., 2009; Wickramasinghe et al., DNA sequencing 2017). Moyle et al. (2009) provided the only published DNA was extracted from feathers using a Qiagen DNeasy sequences for an individual of our focal Zosterops Blood and Tissue Kit (Qiagen, California, USA), following species, a Z. chloris sampled in south Sulawesi. The the protocol of Kelly et al. (2014). We sequenced three ND2/ND3 analyses included 137 samples (56 produced mitochondrial genes: the entire second (1041 bp) and by this study, 81 sourced from GenBank) representing third (351 bp) subunits of mitochondrial nicotinamide 62 species; 51 Zosteropidae along with three Timaliidae, adenine dinucleotide dehydrogenase (ND2 and ND3, four Pellorneidae, one Passeridae, two Leiotrichidae and respectively) and a 615-bp region of the cytochrome c one Muscicapidae to serve as outgroup taxa (Supporting oxidase subunit I (COI) gene. Several novel primers were Information, Tables S1, S3). COI analyses included 108 developed for use in this study to amplify the selected samples (30 produced by this study, 78 sourced from regions (Supporting Information, Table S2). For primer GenBank) representing 22 species; 16 Zosteropidae development, suitable binding sites on either side of the along with four Timaliidae, one Vireonidae and one regions we sought to amplify were identified by inspecting Muscicapidae to serve as outgroup taxa (Supporting published Zosterops whole mitochondrial genomes Information, Tables S1, S4). Genetic sample sizes for (GenBank accession numbers: KX181885, NC_032058, each species are available in Supporting Information, KT194322, NC_027942, KC545407, NC_029146, Tables S3 & S4. All taxonomy was based on current KX181886, NC_032059 and KX181887). This allowed us Handbook of the birds of the world alive designations to identify sites that were conserved between multiple (del Hoya et al., 2018a).

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Phylogenetic and genetic analyses v.1.4.3 (Rambaut, 2016). Annotations were added in Sequences were aligned using ClustalW multiple INKSCAPE v.0.48.5 (Team Inkscape, 2018). alignment in BioEdit v.7.2.5 (Hall, 1999) and the ND2 TCS haplotype networks of the sampled Sulawesi and ND3 genes were concatenated using MESQUITE Zosterops species were constructed with concatenated v.3.40 (Maddison & Maddison, 2018). Only one ND2/ND3 and with COI sequences using POPART representative of each haplotype for ND2/ND3 and COI (Leigh & Bryant, 2015). This allowed the connections was included in the construction of the phylogenetic between populations and haplotype sample sizes to trees for the sampled species; a full list of the be visualized. samples and their haplotypes is provided (Supporting Information, Table S1). The aligned ND2/ND3 samples were partitioned by gene and both concatenated Molecular dating ND2/ND3 and COI were partitioned by codon position We estimated divergence times in BEAST v.2.4.8 for model selection (Angelis et al., 2018). Samples were (Drummond et al., 2002; Bouckaert et al., 2014). partitioned by codon position, to allow for different Concatenated ND2/ND3 sequences were used for substitution rates between positions (Shapiro et al., divergence dating due to the wide array of comparison 2006). Modeltest was performed with MEGA X (Kumar taxa (Moyle et al., 2009). The same partitioning scheme et al., 2018). Using Bayesian Information Criterion and model set was used as in the phylogenetic analysis (BIC) (Jhwueng et al., 2014) implemented in the ‘Find (Supporting Information, Table S5). To allow different best DNA model’ tool (Kumar et al., 2018), the optimal substitution models to be implemented for each nucleotide substitution model for each partition of the partition, nucleotide substitution models were unlinked. concatenated ND2/ND3 and COI data was selected Model calibration was provided by published geological and sequence summary information was produced information (Moyle et al., 2009) and substitution rates (Supporting Information, Table S5). (Lerner et al., 2011). The estimated date of the divergence Using the partitioned model scheme selected of Zosteropidae and Zosterornis (formerly Stachyris) (Supporting Information, Table S5), we carried from related taxa, given as 5.01 Myr (4.46–5.57 Myr) by out Maximum Likelihood analysis and Bayesian Moyle et al. (2009), was used as a point calibration. This Phylogenetic Inference on our concatenated ND2/ND3 calibration was set as a normal distribution with mean and COI data separately. Maximum Likelihood 5.01 and sigma 0.555 (Wickramasinghe et al., 2017). (ML) heuristic tree searches were performed using Rates of evolution were set at 0.029 (lower bound: 0.024, GARLI v.2.01 (Zwickl, 2006). To avoid local optima, upper bound: 0.033) and 0.024 (lower bound: 0.019, 250 independent searches were performed, each upper bound: 0.029) for ND2 and ND3, respectively, starting from a random tree following Andersen et al. representing the number of substitutions per site per (2014). Searches were terminated when no topological million years, derived from estimates produced by improvements were found after 100 000 generations. All Lerner et al. (2011) for honeycreepers, following Linck other parameters were left at default settings. Statistical et al. (2016). As no Zosterops fossil data are available support for the ML topology was assessed with 1000 for calibration, and the available rate calibrations nonparametric bootstrap replicates (Felsenstein, 1985) for the target genes are not from close relatives of and a 50% majority-rule tree was generated in PAUP* Zosterops, strict interpretation of divergence dating 4.0b10 (Swofford, 2002). Bayesian Phylogenetic Inference estimates presented here is not advised. Clock and tree (BI) was carried out using MrBayes v.3.2.6 (Ronquist models were linked between partitions (Drummond & & Huelsenbeck, 2003). We used two independent Bouckaert, 2015). Following Baele et al. (2012), path Markov chain Monte Carlo (MCMC) runs, with four sampling and stepping-stone sampling were carried chains per run, sampling every 1000 generations. out in BEAST to test for clock-like rates, by computing Burnin and convergence were assessed using TRACER the marginal likelihood for each clock model (Lartillot v.1.7.1 (Rambaut et al., 2018), burnin was set at 25% & Philippe, 2006; Xie et al., 2011). A Relaxed Clock with convergence in runs accepted when the average Log Normal clock model was found to have the highest standard deviation in split frequencies (ASDSF) reached marginal likelihood and was selected for use (Baele et al., 0.01 (Ronquist et al., 2012) and the effective sample size 2012, 2013). A Yule speciation process was assumed for (ESS) of model parameters exceeded 200 (Drummond the tree model, following Wickramasinghe et al. (2017). et al., 2006). The model of concatenated ND2/ND3 We ran 10 independent MCMC chains for 100 million reached ASDSF 0.01 and an ESS of >200 for all model generations, sampling every 20 000 generations. We parameters after four million generations. The model assessed burnin and convergence using TRACER v.1.7.1 for COI reached ASDSF 0.01 and an ESS of >200 for (Rambaut et al., 2018) to confirm acceptable mixing, all model parameters after seven million generations. likelihood stationarity and ESS > 200 for all estimated Phylogenetic tree topology was taken from the BI, parameters. Burnin was set at 25% for all runs. We with a 50% majority rule tree produced in FigTree used TreeAnnotator v.2.4.8 (Bouckaert et al., 2014)

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Pairwise distance and molecular species delimitation Morphometric and song analyses Pairwise comparisons were carried out in MEGA X All morphometric and song statistical analyses were (Kumar et al., 2018) to calculate maximum, minimum carried out in R Software v.3.4.2 (R Development and mean uncorrected proportional genetic distances Core Team, 2017). Histograms of each trait were first (p-distances) within and between sampled Zosterops plotted to ensure normal distributions. Two types of populations, for both longer mitochondrial genes: ND2 analyses were carried out for both the morphometric and COI. This outlined the level of within and between and song data (separately), Principal Component species genetic distance to be expected for Zosterops. Analysis (PCA) and Discriminant Function Analysis Molecular species delimitation was carried out (DFA). PCA was carried out to capture the variance for sampled Zosterops populations to highlight in the morphometric and song traits in a smaller populations for further assessment (see section Tobias number of principal components. A PCA was carried scoring of species status). This was done for both ND2 out for each of the analysis groups: (1) Z. chloris and COI using Automatic Barcode Gap Discovery morphometrics, (2) Z. chloris song, (3) Z. consobrinorum (ABGD). ABGD is a distance-based method developed and the ‘Wangi-wangi white-eye’ morphometrics and (4) by Puillandre et al. (2012) utilizing pairwise genetic Z. consobrinorum song. As the traits in each PCA were distance calculations. This method groups individuals on different scales, all were re-scaled for inclusion in so that the distance between the sequences of two the PCA using the scale function in R, such that their groups is always larger than a certain genetic distance means were = 0 and their variances were = 1 (Thomas threshold value termed the barcode gap. The barcode et al., 2017). To test whether the different populations gap is a taxonomic group and gene-specific as it is of our focal Zosterops species differed from each dependent on the level of intraspecific and interspecific other in morphometrics or song, Analysis of Variance variation in the sampled group (Prévot et al., 2013). (ANOVA) was carried out on principal components ABGD was run on the web-server http://wwwabi.snv. with eigenvalues > 1. To ensure that the assumption of jussieu.fr/public/abgd/abgdweb.html using default normality was not violated, Q–Q plots of the residuals settings (Pmin = 0.001, Pmax = 0.1, Steps = 10, of each ANOVA test were inspected. Tukey’s Honest X(relative gap width) = 1.5, Nb bins = 20) and a Significant Difference (HSD) tests were used as post-hoc Kimura-2-Parameter (K2P) model (Kimura, 1980). tests for ANOVAs, which returned significant results. DFA, conducted with package ‘MASS’ (Ripley et al., 2018), was used to identify axes that provided the Song data extraction most effective separation between pre-defined groups. Zosterops’ recordings were separated into calls and For the DFA analyses, the groupings were taken from songs. Songs were selected to be analysed, as they our molecular phylogenies and our analyses assessed are of principal importance in mediating species how well (% grouping accuracy) the morphometric and recognition (Uy et al., 2009). Sonograms were prepared song data for our study populations supported the and analysed using RAVEN PRO v.1.5 (Bioacoustics phylogenetic groupings. DFA was carried out using the Research Program, 2018). Contrast and brightness same groupings as in the PCA. were set to an equal level and the sharpness was set at 2000, all other settings were left at default (Ng et al., 2016). Recordings with clear sonograms, containing Tobias scoring of species status at least two discrete bursts of song, were chosen for To assess the species status of potentially novel analysis. Standard song traits were measured from Zosterops species addressed in this study, a Tobias the sonograms following Tobias et al. (2010): (1) scoring was carried out for any populations showing total number of notes, (2) duration of song, (3) pace potential species-level genetic separation. The Tobias (number of notes divided by duration), (4) maximum scoring system is used by the Handbook of the birds of frequency, (5) minimum frequency, (6) bandwidth the world and Birdlife International for their taxonomic (maximum minus minimum frequency) and (7) peak assessments (del Hoyo et al., 2018a), based on the criteria frequency (the frequency with the greatest amplitude) outlined by Tobias et al. (2010). This system assesses (Fig. 2). To account for intra-individual variation, phenotypic characteristics only (morphology, song and

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Maximum frequency

A single note Bandwidth

Minimum frequency Duraon

Figure 2. Typical Zosterops song burst as viewed in RAVEN PRO 1.5, illustrating some of the traits measured in this study. The individual shown is a Zosterops consobrinorum from Kabaena Island. plumage) and does not take genetic results into account. Sequence production A population must reach a Tobias score of seven to be Sequencing of our focal Zosterops species focuses on considered a separate species. A detailed description of the ND2 and ND3 genes, as they allow for comparison the criteria is supplied in the Supporting Information, with the largest array of published Zosterops sequences Tobias Scoring for potentially novel Zosterops species. (Moyle et al., 2009; Wickramasinghe et al., 2017). All individuals sequenced for ND2 were also sequenced for ND3, with a smaller sample of individuals sequenced Ethics statement for COI (Table 1; Supporting Information, Table S1). The necessary permits and approvals for this study were obtained from Kementerian Riset Teknologi Dan Pendidikan Tinggi (RISTEKDIKTI). Permit numbers: Phylogenetic analyses 0143/SlP/FRP/SM/Vll/2010, 278/SlP/FRP/SM/Vll/2012, 279/SIP/FRP/SM/VIII/2012, 174/SIP/FRP/E5/Dit. Results from our ML and Bayesian analyses produced KI/V/2016, 159/SIP/FRP/E5/Fit.KIVII/2017 and 160/ highly concordant topologies for well-supported nodes SIP/FRP/E5/Fit.KIVII/2017. We obtained prior for both concatenated ND2/ND3 and COI haplotypes. permission from all landowners and no protected species The concatenated ND2/ND3 tree is most informative, were sampled. We are committed to reproducibility because more comparative material is available from and aliquots of the extracted DNA for all sampled GenBank. Zosterops chloris and Z. consobrinorum individuals are available upon request (subject to the are close relatives, sharing a node with the black- Material Transfer policies of Trinity College Dublin, crowned white-eye, Zosterops atrifrons (Vigors & Halu Oleo University and RISTEKDIKTI). Horsfield, 1827) (node support: BI–0.91, ML–84) (Fig. 3; Supporting Information, Fig. S1). For Z. chloris, there is a clear split between the Z. c. flavissimus RESULTS population on the Wakatobi Islands and all other Z. chloris populations in ND2/ND3 (node support: Range extensions BI–1.0, ML–100) (Fig. 3; Supporting Information, Fig. This study provides the first records of Z. chloris on S3). All individuals from mainland south-east Sulawesi Wawonii and Runduma Islands and of Z. consobrinorum and the adjacent continental islands (Buton, Muna, on Muna Island (Van Balen, 2018b, c) (Fig. 1). Kabaena and Wawonii) group closely together. However,

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Table 1. Number of sequences produced for each of our focal species, for ND2/ND3 and for COI. The Sulawesi mainland and its continental islands are highlighted in bold, Wakatobi Islands are highlighted in italics and Runduma is treated as a separate oceanic island. Location for each individual sampled and GenBank accession numbers are provided in Supporting Information, Table S1

Island Zosterops Zosterops Zos. sp. nov. ‘Wangi- chloris consobrinorum wangi white-eye’

ND2/ND3 COI ND2/ND3 COI ND2/ND3 COI

Mainland Sulawesi 12 4 6 2 - - Buton 5 2 5 3 - - Muna 4 - 1 - - - Kabaena 4 2 4 2 - - Wawonii 1 - - - - - Runduma 2 2 - - - - Wangi-wangi 2 2 - - 4 4 Kaledupa 2 4 - - - - Tomia 2 - - - - - Binongko 2 3 - - - - Total 36 19 16 7 4 4 the mainland south-east Sulawesi population shows (node support: BI–1.0, ML–73) and show only minor some divergence from the Z. c. intermedius population divergence from the most isolated Z. consobrinorum in south Sulawesi (ND2: 1.22%, see section Genetic population on Kabaena Island in ND2/ND3. However, distance) (node support: BI–1.0, ML–99). Therefore, the Buton and Muna populations are suggested to the mainland south-east Sulawesi population is not be distinct (node support: BI–1.0, ML–88). In this assigned to this subspecies. The Runduma population population there is a further deeper split between is also distinct from the mainland south-east Sulawesi individuals with the haplotypes hapCO11-12 (N = 2 population (node support: BI–0.91, ML–99), although from Buton) that are more closely related to the the split is shallower than that between the mainland Kabaena and Sulawesi populations, and individuals south-east Sulawesi and south Sulawesi populations. with haplotypes hapCO10 and hapCO13 (N = 3 The four groupings of Z. chloris: (1) Wakatobi from Buton and N = 1 from Muna) that are much Z. c. flavissimus, (2) south Sulawesi Z. c. intermedius, more distinct (node support: BI–1.0, ML–100) (3) south-east Sulawesi mainland and continental (Supporting Information, Fig. S3). Individuals from islands Z. chloris and (4) Runduma Island Z. chloris, both of these divergent Buton/Muna populations are show little within-group variability, but distinct splits found at the same site on Buton (Kusambi, 5.153 S between populations (Fig. 3; Supporting Information, 122.895 E) (Supporting Information, Table S1). The Fig. S3). The majority of the ‘mainland south-east mainland Sulawesi and Kabaena populations show Sulawesi’ individuals share the same haplotype no difference in COI, sharing the same haplotypes (ND2/ND3: hapCH02) (Supporting Information, (Fig. 4; Supporting Information, Fig. S4, Table S1). Fig. S3, Table S1). The COI tree provides additional The COI phylogeny also separates the Buton birds support for the taxonomic pattern seen in Z. chloris from those on mainland Sulawesi and Kabaena (node (Fig. 4; Supporting Information, Fig. S2), with a strong support: BI–1.0, ML–100), but with a shallower split split between the Z. c. flavissimus population on the then for ND2/ND3. Wakatobi Islands and mainland south-east Sulawesi The ‘Wangi-wangi white-eye’ is not closely related populations (node support: BI–1.0, ML–100) and a to Z. consobrinorum, as had been provisionally further shallower split between the Runduma Island suggested (Van Balen, 2018c) (Fig. 3). It is a population and the individuals sampled on mainland highly distinct taxon, most closely related to the Sulawesi and its continental islands (node support: Kolombangara white-eye, Zosterops murphyi BI–1.0, ML–93). There is little within-group variability (Hartert, 1929), the Rennell white-eye, Zosterops between haplotypes and distinct splits between these rennellianus (Murphy, 1929) and Z. griseotinctus; groups (Supporting Information, Fig. S4, Table S1). taxa found in the Solomon Islands (>3000 km distant) Zosterops consobrinorum displays an unusual (node support: BI–0.75, ML–14). The COI tree lacks pattern (Fig. 3; Supporting Information, Fig. S3). All the depth of sampling in the genus Zosterops to mainland Sulawesi individuals group closely together provide any further insight into the evolutionary

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Zosterops fuscicapilla 0.56 Zosterops rendovae 1.0 Zosterops stresemanni 37 99 Zosterops metcalfii Zosterops splendidus 1.0 1.0 Zosterops kulambangrae hapKU02 [Rendovae] 1.0 96 100 Zosterops kulambangrae hapKU03[Tetepare] 0.97 98 Zosterops kulambangrae hapKU01[Kohingo] Zosterops luteirostris 42 Zosterops vellalavella 1.0 Zosterops murphyi 0.99 0.75 100 Zosterops griseotinctus 100 14 Zosterops sp. nov. 'Wangi-wangi hapCX01-03 White-eye' 0.86 Zosterops rennellianus 14 1.0 Zosterops lateralis hapLA01 [Australia] 1.0 100 Zosterops lateralis hapLA03 [New Zealand] 100 Zosterops lateralis hapLA02 [Vanuata] 1.0 Zosterops citrinella 0.80 100 Zosterops palpebrosus hapPA01 [Indonesia] 42 Zosterops luteus 1.0 Zosterops flavifrons 80 Zosterops superciliosus Zosterops erythropleurus hapER01 1.0 Zosterops erythropleurus hapER02 0.87 94 Zosterops atricapilla 0.7 42 Zosterops japonicus hapJA01-02 [Thailand] 0.99 37 Zosterops montanus 64 Chlorocharis emiliae

Zosterops palpebrosus hapPA03-06 [India] 0.84 84 1.0 Zosterops palpebrosus 100 hapPA07-09 [Sri Lanka] 1.0 1.0 Zosterops japonicus hapJA03 [Vietnam] 100 Zosterops palpebrosus hapPA02[Nepal] 74 1.0 Zosterops maderaspatanus Zosterops virens 1.0 77 1.0 Zosterops poliogastrus 67 1.0 100 1.0 Zosterops flavilateralis Zosterops senegalensis hapSE01 [Kenya] 100 100 Zosterops senegalensis hapSE02 [Ghana] Zosterops atrifrons

hapCO01-06 [mainland Sulawesi] 1.0 1.0 73 0.88 70 Zosterops 1.0 hapCO07-08 [Kabaena] consobrinorum 15 1.0 88 hapCO09 [Kabaena] 97 1.0 hapCO11-12 [Buton] 100 0.91 hapCO10 [Buton] 84 hapCO13 [Muna] hapCH01 [South Sulawesi] 1.0 1.0 hapCH02-07 [mainland Zosterops 99 100 South-east Sulawesi and 0.91 chloris 1.0 continental islands] 99 100 hapCH08 [Runduma] hapCH09-12 Zosterops [Wakatobi Islands] chloris flavissimus Zosterops nigrorum Zosterops ceylonensis hapCE01-02

Outgroup

0.02

Figure 3. Bayesian consensus tree for concatenated ND2/ND3 haplotypes, showing Bayesian posterior probabilities (above) and bootstrap values from our Maximum Likelihood analysis (below) for each node. Haplotype number was given when there was more than one representative of a single taxon, with geographic information added with square brackets (single node) or curly brackets (multiple nodes) when that was informative to the pattern seen. Focal species highlighted in colour. Full tree with outgroups shown available in Supporting Information, Fig. S1.

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Zosterops japonicus hapJA01 [China] 0.54 Zosterops erythropleurus hapER01 35 1.0 99 0.9 Zosterops erythropleurus hapER02 56 Zosterops erythropleurus hapER03

1.0 Zosterops japonicus hapJA02-hapJA20 [Japan and Hawaii] 99 Zosterops meyeni 1.0 97 0.52 Zosterops nigrorum hapNI01 0.99 55 Zosterops nigrorum hapNI02 73 Zosterops lateralis 0.55 21 Zosterops pallidus Zosterops palpebrosus 0.56 58 Zosterops atrifrons hapCH01-02 1.0 [Sulawesi mainland + Zosterops 1.0 100 connental islands] chloris 93 hapCH03 [Runduma Island] 0.93 69 Zosterops 1.0 hapCH04-06 chloris 0.94 100 [Wakatobi Islands] flavissimus 18 0.95 hapCO01-02 [Sulawesi 69 and Kabaena] 1.0 Zosterops 100 consobrinorum 0.98 hapCO03-04 [Buton] 88

1.0 Zosterops sp. nov. 'Wangi-wangi 100 hapX01-02 White-eye'

Zosterops poliogastrus 1.0 99 0.95 Zosterops flavilateralis 99 Zosterops senegalensis Outgroup

000.011

Figure 4. Bayesian consensus tree for COI haplotypes, showing Bayesian posterior probabilities (above) and bootstrap values from our Maximum Likelihood analysis (below) for each node. Haplotype number was given when there was more than one representative of a single taxon, with geographic information added with square brackets (single node) or curly brackets (multiple nodes) when that was informative to the pattern seen. Focal species highlighted in colour. Core Z. japonicus lineage collapsed as it was monophyletic. Full tree with outgroups shown available in Supporting Information, Fig. S2. history of the ‘Wangi-wangi white-eye’, but confirms Divergence dating its difference from sequenced taxa (Fig. 4). Our molecular clock shows that the Zosterops radiation In addition to our focal species, the phylogenetic began ~1.8 Mya (Fig. 5) as demonstrated in Moyle et al. analyses illustrated deep separations in the (2009) and Wickramasinghe et al. (2017). Among our widespread species Z. palpebrosus, Z. japonicus and focal species, the ‘Wangi-wangi white-eye’ is estimated the African yellow white-eye, Zosterops senegalensis to have diverged 0.7–1.23 Mya. Precise dating for this (Bonaparte, 1850) (Figs 3, 4). taxon is difficult, as its closest relatives are Solomon

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Islands endemics, separated by a large geographic Sulawesi and Kabaena populations differ little (ND2: distance. Zosterops chloris diverged from Z. atrifrons 0.31%, COI: 0%). The Buton/Muna population shows and Z. consobrinorum 0.77–1.36 Mya. Zosterops high within-group variability for ND2 (ND2: 1.09%) c. flavissimus on the Wakatobi Islands diverged from in comparison to Sulawesi (ND2: 0.12%) and Kabaena Z. chloris mainland Sulawesi populations 0.38–0.8 (ND2: 0.29%) populations. COI is much less variable, Mya. This may mark the colonization of the Wakatobi with Buton populations showing only 0.11% within- Islands by Z. chloris. The south Sulawesi and south- group variation and the undifferentiated Sulawesi and east Sulawesi populations of Z. chloris then diverged Kabaena populations showing 0.08%. 0.17–0.38 Mya, with a later divergence of the Runduma Island population from south-east Sulawesi mainland populations 0.08–0.22 Mya. Zosterops sp. nov. Zosterops consobrinorum diverged from Z. atrifrons The ‘Wangi-wangi white-eye’ is strongly distinct from 0.57–1.21 Mya (Fig. 5). The unusual population all Z. consobrinorum populations (ND2: 6.23% and COI: structure in Z. consobrinorum from Buton and Muna 8.35% at a minimum) and all Z. chloris populations makes estimating divergence dates challenging. (ND2: 5.24% and COI: 7.17% at a minimum). The most Individuals with the ND2/ND3 haplotypes hapCO10 closely related populations are Z. griseotinctus (ND2: and hapCO13 (Buton and Muna) diverged from other 5.08%) and the lowland white-eye, Zosterops meyeni populations 0.22–0.51 Mya. The remaining Buton (Bonaparte, 1850) (COI: 6.78%). The ‘Wangi-wangi individuals diverged 0.08–0.22 Mya. Divergence white-eye’ shows minor within-group variability (ND2: dating of the Kabaena population is also unclear and 0.29%, COI: 0.16%). too shallow to offer sensible estimates.

Molecular species delimitation Genetic distance Automatic Barcode Gap Discovery (ABGD) analysis Calculations of pairwise genetic distance provides finds the barcoding gap between Zosterops species to an indication of the level of divergence between the be 3.5% (COI) and 1.3% (ND2) K2P genetic distance. populations described in our phylogenetic trees. COI For both genes, ABGD groups individuals from our samples were not available for all populations, but focal Zosterops populations in Sulawesi into four COI distances are given where available. Pairwise putative species; distances between all Zosterops species sampled are available in the supplementary material (Supporting 1. Zosterops chloris from mainland south and south- Information, Tables S6, S7). east Sulawesi, the continental islands of Buton, Muna, Kabaena and Wawonii and Runduma Island (ND2 hapCH01-08, COI hapCH01-03; Supporting Zosterops chloris Information, Table S1). Mainland south-east Sulawesi (including continental 2. Zosterops c. flavissimus from the Wakatobi Islands islands) and south Sulawesi populations are divergent (ND2 hapCH09-12, COI hapCH04-06). (ND2: 1.22%) as shown by our phylogenetic work. In 3. Zosterops consobrinorum – all sampled individuals the focal region, mainland south-east Sulawesi and (ND2 CO01-13, COI CO01-04) the Wakatobi population are strongly divergent (ND2: 4. Zosterops sp. nov. ‘Wangi-wangi white-eye’ – all 2.5%, COI: 4.9%). The Wakatobi population also differs sampled individuals (ND2 hapCX01-03, COI from south Sulawesi (ND2: 2.05%) and Runduma hapCX01-02). (ND2: 2.35%, COI: 4.66%). The most closely related population to Runduma is that on mainland south-east Sulawesi (ND2: 0.73%, COI: 2.22%). Each population Morphometric analyses shows low within-group variability; mainland south- A total of 752 Zosterops individuals from 11 islands east Sulawesi (ND2: 0.09%, COI: 0.04%), Runduma were measured for these analyses; 575 Z. chloris, (ND2: 0%, COI: 0%) and Wakatobi Islands (ND2: 139 Z. consobrinorum and 38 ‘Wangi-wangi white- 0.04%, COI: 0.14%). eyes’ (Supporting Information, morphometric trait summaries, Tables S8–S11). The full morphometric database is available at https://figshare.com/articles/ Zosterops consobrinorum SE_Sulawesi_Zosterops_morphology/7998299/1. For The Buton/Muna population differs in ND2 from the analysis, the sampled individuals were grouped along the Sulawesi population (2.1%) and Kabaena population splits provided by the molecular phylogenies. Zosterops (1.9%), although the Buton population shows less chloris individuals were classified into the groupings: difference in COI to Sulawesi/Kabaena (0.59%). mainland (Sulawesi mainland and the continental

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Zosterops flavilateralis Zosterops senegalensis hapSE01 [Kenya] Zosterops poliliogastrus Zosterops senegalensisl hapSE02 [Ghana] Zosterops maderaspad tanus Zosterops viri ens Zosterops japonicus hapJA03 [Vietnam] Zosterops palpebrosus hapPA07-09 [Sri Lanka] Zosterops palpebrosus hapPA02[Nepal] Zosterops palpebrosus hapPA03-06 [India] Zosterops erythropleurus hapER01-02 Zosterops atricapilla Zosterops japonicus hapJA01-02 [Thailand] Zosterops montanus Chlorocharish emiliae Zosterops atrifrons hapCO01-06 [mainland Sulawesi] Zosterops hapCO07-09 consobrinorum [Kabaena] hapCO11-12[Buton] hapCO10 + 13 [Buton + Muna] hapCH01 [South Sulawesi] hapCH02-07 [mainland Zosterops south-east Sulawesi and chloris connental islands] hapCH08 [Runduma] Zosterops hapCH09-12 [Wakatobi Islands] chloris Zosterops citrinella flavissimus Zosterops palpebrosus hapPA01 [Indonesia] Zosterops luteus Zosterops fuscicapilla Zosterops metcallfii Zosterops stresemanni Zosterops rendovae Zosterops sp. nov. 'Wangi-wangi hapCX01-03 White-eye' Zosterops griseonctus Zosterops murphyi Zosterops rennellianuslil Zosterops lateralis hapLA01 [Australia] [New Zealand Zosterops lateralis hapLA02-03 and Vanuata] Zosterops kulambangrae hapKU01 Zosterops kulambangrae hapKU02-03 Zosterops splendidus Zosterops lluteiirostris Zosterops vellalavella Zosterops flavifrons Zosterops superciiliosusi Zosterops nigrorum Zosterops ceylonensis hapCE01-02 Outgroup

2.5 2.0 1.5 1.0 0.5 0 Ma

Figure 5. Divergence dating of Zosterops species based on BEAST analysis on concatenated ND2/ND3 genes. The blue bars indicate 95% Highest Posterior Density (HPD) intervals. islands N = 168), Wakatobi (Z. c. flavissimus from the > 1 and were carried forward for analyses. PC1 was six Wakatobi Islands, N = 362) and Runduma (N = 45). loaded equally between the seven morphometric traits, Zosterops consobrinorum individuals were split into giving a general indicator of body size (Supporting mainland Sulawesi (N = 48), Buton and Muna (N = 68) Information, Table S12). PC2 was largely loaded by and Kabaena (N = 23) groups. ‘Wangi-wangi white- bill length and skull length, giving a general indicator eyes’ (N = 38) were analysed with Z. consobrinorum to of bill to skull ratio. The Z. chloris populations are establish the level of separation between them. significantly different from each other in body size (PC1,

For Z. chloris morphometrics, PC1 (78% of the ANOVA: F2, 572 = 554.5, P < 0.001), with the mainland, variance) and PC2 (8.3% of the variance) had eigenvalues Wakatobi and Runduma populations all significantly

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● Z. chloris Runduma Z. chloris Sulawesi 4 Z. chloris Wakatobi tio)

ra 2

● ● ● ● ● ● ● ● ● 0 ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● −2 ● ● ● ● ● ● PC2 (bill to skull −4

−6 −4 −2 024 PC1 (body size)

Figure 6. Scatterplot of Zosterops chloris morphometric PCA. Black triangles represent individuals from mainland south-east Sulawesi and its continental islands, grey circles represent individuals from Runduma Island, green diamonds represent individuals from the Wakatobi Islands. Variance explained: PC1–78.3%, PC2–8.3%.

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3 Z. consobrinorum Buton/Muna ● Z. consobrinorum Kabaena Z. consobrinorum Sulawesi 2 Zosteropssp. nov. ● ● 1 ●● ●

PC 2 ● ● ● ● 0 ● ● ● ● ● ● ● ● ● ● ● −1 ●

−2 −6 −4 −2 024 PC1 (body size)

Figure 7. Scatterplot of Zosterops consobrinorum and Zosterops sp. nov. (‘Wangi-wangi white-eye’) morphometric PCA. Black triangles represent Z. consobrinorum individuals from Buton and Muna Islands, red squares represent Z. consobrinorum individuals from mainland Sulawesi, green circles represent Z. consobrinorum individuals from Kabaena Island, blue diamonds represent the ‘Wangi-wangi white-eye’. Variance explained: PC1–88.7%, PC2–3.2%.

Z. chloris mainland population differs significantly distinction between Z. consobrinorum populations. from the Z. c. flavissimus Wakatobi population in PC1 The Kabaena Z. consobrinorum is the most accurately (Tukey HSD, P adj. < 0.001) and from the Lombok classified in morphometrics and the Buton/Muna population in PC1 and PC2 (Tukey HSD, P adj. < 0.001 population shows the greatest classification accuracy in and P adj. < 0.05, respectively). The Wakatobi and song, but both show a large degree of overlap with other Lombok populations differ significantly in PC2 and Z. consobrinorum populations. The mainland Sulawesi PC3 (Tukey HSD, P adj. < 0.01 and < 0.05, respectively). population cannot be accurately classified, particularly For Z. consobrinorum song, PC1 (41.7% of the with song traits. More Sulawesi individuals are variance), PC2 (21.8%) and PC3 (16.2%) had eigenvalues classified as belonging to other islands than to Sulawesi. > 1 and were carried forward for analyses (Supporting Information, Table S17). PC1 is most heavily loaded by duration, maximum frequency and bandwidth. PC2 is Tobias scoring most heavily loaded by pace and peak frequency. PC3 is For the Tobias scoring of phenotypic traits, the most heavily loaded by the number of notes, maximum Wakatobi Z. c. flavissimus population is compared frequency and bandwidth. The Z. consobrinorum to the Z. chloris population from mainland south- Kabaena and Buton/Muna populations differ east Sulawesi and its continental islands and significantly in song PC1 (PC1, ANOVA: F2, 66 = 4.133, the ‘Wangi-wangi white-eye’ was compared to P < 0.05; Tukey HSD, P adj. < 0.05) (Fig. 9). There are no Z. consobrinorum. Both Z. c. flavissimus (Tobias other significant differences in Z. consobrinorum song. score: nine) and the ‘Wangi-wangi white-eye’ (Tobias score: seven) are identified as distinct species. Detailed scoring is provided in the Supplementary Classification based on morphometric and song Information (Tables S18, S19). traits Discriminant Function Analysis (DFA) classification of Z. chloris individuals suggests a close match of DISCUSSION morphometric and song traits for the taxonomic groupings identified in our molecular phylogeny (Table Our results present evidence for a new species of 2; Figs 3, 4). The sampling location of the majority of Zosterops and that a subspecies of another Zosterops individuals can be accurately predicted from these traits. should be recognized as a full species, both from the The ‘Wangi-wangi white-eye is 100% distinguishable in same island archipelago in Sulawesi. The ‘Wangi- morphometrics from all Z. consobrinorum populations wangi white-eye’ is a genetically and phenotypically in the DFA analysis (Table 3). There is only a weak distinct species in need of recognition and formal

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4 ● Z. chloris Lombok ● Z. chloris Sulawesi Z. chloris Wakatobi 2 ● )

0 PC2 (song

−2

−4 −2 024 PC1 (song)

Figure 8. Scatterplot of Zosterops chloris song PCA. Black triangles represent individuals from mainland south-east Sulawesi and its continental islands, green diamonds represent individuals from the Wakatobi Islands, blue circles represent individuals from Lombok. Variance explained: PC1–39.8%, PC2–24.0%.

Z. consobrinorum Buton/Muna 4 ● Z. consobrinorum Kabaena Z. consobrinorum Sulawesi

2

) ● ● ● ● ● ● ● ● ● ● ● 0 ●● ● ● ●● ● ● ●

PC2 (song ● ● ● ● ● −2

● −4 −2 024 PC1 (song)

Figure 9. Scatterplot of Zosterops consobrinorum song PCA. Black triangles represent Z. consobrinorum individuals from Buton and Muna Islands, red squares represent Z. consobrinorum individuals from mainland Sulawesi, green circles represent Z. consobrinorum individuals from Kabaena Island. Variance explained: PC1–41.7%, PC2–21.8%. description. It is reciprocally monophyletic from as a full species, Z. flavissimus the ‘Wakatobi white- all other sampled Zosterops species. Zosterops eye’. The proposal to recognize these two new species c. flavissimus proves distinct in genetic, morphometric is also supported by molecular species delimitation and song analyses. The first mention of the Wakatobi (ABGD) and the Tobias taxonomic scoring criteria Z. c. flavissimus population by Hartert (1903) referred (Supporting Information, Tobias scoring, Tables S18, to it as a separate species, Zosterops flavissimus, S19; Tobias et al., 2010; Puillandre et al., 2012). Our although subsequent classifications of the avifauna in results suggest that Sulawesi Z. chloris subspecies are the region subsumed this population into Z. chloris. We in need of further revision. We do not recommend any propose that Z. c. flavissimus be once again recognized change to the taxonomy of Z. consobrinorum, because

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Table 2. Percentage classification accuracy of the DFA for morphometrics and song of Zosterops chloris. Sample sizes given are: N = morphometric sample size / song samples size. A dash indicates no sample available for that population. Results given indicate the % of individuals classified in that category, with morphometric results before the slash (/) and song results after. Shaded grey squares are the expected result, with the percentage in the shaded squares indicating what percentage of individuals from that population were correctly classified in the population from which they were sampled. All seven morphometric traits; wing, tail, tarsus, skull and bill length, bill depth and weight, were used. All seven song traits; number of notes, duration, pace, maximum, minimum and peak frequency and bandwidth, are also used

Z. chloris Z. chloris Z. chloris Z. chloris ‘mainland’ flavissimus ‘Runduma’ maxi ‘Wakatobi’ ‘Lombok’

Z. chloris ‘mainland SE Sulawesi + continental 89.29 / 95.83 8.92 / 4.17 1.79 / - - / 0 islands’ (N = 168 / 24) Z. chloris flavissimus ‘Wakatobi Islands’ (N = 362 / 28) 3.6 / 0 96.4 / 100 0 / - - / 0 Z. chloris ‘Runduma’ 2.22 / - 2.22 / - 95.56 / - - / - (N = 45 / -) Z. chloris maxi ‘Lombok’ - / 0 - / 0 - / - - / 100 (N = - / 2)

Table 3. Percentage classification accuracy of the DFA for morphometrics and song of Zosterops consobrinorum and the ‘Wangi-wangi white-eye’. Sample sizes given are: N = morphometric sample size / song samples size. A hyphen (-) indicates no sample available for that population. Results provided are: % of individuals classified in that category, with morphometric results before the slash (/) and song results after. Shaded grey squares are the predicted result, i.e. the population from which the individual was sampled. Morphometric traits wing, tail, tarsus, skull and bill length, bill depth and weight used. All seven song traits; number of notes, duration, pace, maximum, minimum and peak frequency and bandwidth, are also used

Z. consobrinorum Z. consobrinorum Z. consobrinorum ‘Wangi-wangi ‘mainland’ ‘Buton/Muna’ ‘Kabaena’ White-eye’

Z. consobrinorum ‘mainland 58.33 / 18.18 41.67 / 45.45 0 / 36.36 0 / - Sulawesi’ (N = 48 / 11) Z. consobrinorum ‘Buton/Muna’ 20.6 / 0 77.94 / 74.19 1.47 / 25.81 0 / - (N = 68 / 31) Z. consobrinorum ‘Kabaena’ 4.35 / 0 13.0 / 33.33 82.61 / 66.66 0 / - (N = 23 / 27) ‘Wangi-wangi White-eye’ (N = 38 / -) 0 / - 0 / - 0 / - 100 / - populations of this species do not show consistent descriptions date from the expedition of Heinrich variation between genetic and phenotypic measures. Kühn (1901–02; Hartert, 1903). This single island endemic must have been overlooked. The ‘Wangi-wangi white-eye’ occurs in mixed-species Zosterops sp. nov. – the ‘wangi-wangi flocks with Z. c. flavissimus on Wangi-wangi Island and white-eye’ exhibits the same generalist foraging habits common Due to its unique biogeographic position (Esselstyn to Zosterops (Van Balen, 2008; Kelly, 2014). The et al., 2010), Sulawesi has particularly high endemism ‘Wangi-wangi white-eye’ is a much larger bird than (Michaux, 2010). It also remains relatively poorly Z. chloris (Supporting Information, Tables S9, S11), studied (Cannon et al., 2007) and novel taxa have likely facilitating niche partitioning between these been found on Sulawesi in recent years (Indrawan & congeneric species. It is relatively common on Wangi- Rasmussen, 2008; Esselstyn et al., 2012; Harris et al., wangi: in the 18 mist-netting sessions conducted on 2014). However, these taxa were found in remote that island, 20% of birds caught were ‘Wangi-wangi forested areas or on more isolated islands. The fact white-eyes’ and 39% were Z. chloris. All netting was that the ‘Wangi-wangi white-eye’ occurs on a densely carried out in the scrub and forest edge habitats, populated, environmentally degraded island is which are the most common ecosystems on the island. particularly remarkable. Most Wakatobi bird species ‘Wangi-wangi white-eyes’ shows tolerance of disturbed

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Zosterops ‘Wangi-wangi white-eye’ is amplified by the small size c. intermedius as currently defined includes populations of Wangi-wangi Island (155 km2) and that extensive from south Sulawesi, the continental islands of south- surveys in south-east Sulawesi have shown it to be east Sulawesi and much of the Lesser Sunda Islands the only home of the ‘Wangi-wangi white-eye’ (it is (Van Balen, 2018a). An assessment of the different absent from Oroho and Kapota, the satellite islands of populations currently assigned to Z. c. intermedius Wangi-wangi). The authors recommend the collection and Z. c. mentoris (isolated populations in central and of type specimens so that this species can be officially northern Sulawesi) is needed to clarify the taxonomy named and recognized, coupled with detailed surveys of Z. chloris on mainland Sulawesi. of Wangi-wangi Island to assess its distribution and Within south-east Sulawesi, the Runduma density, and any conservation action required. A series population of Z. chloris (first noted by this study) of photos of this species are supplied in the Supporting represents a recent independent colonization from Information to aid future field identification of this a mainland south-east Sulawesi source population new taxon (Supplementary information, Tobias (Fig. 5), not from the Wakatobi Islands. This was an scoring, Table S19). unexpected discovery, because the shortest distance The provisional classification of the ‘Wangi-wangi between Runduma and the closest mainland population white-eye’ as a population of Z. consobrinorum (Van (Buton) is 123 km (Fig. 1). The distance between Balen, 2018c) is understandable. Both are pale-chested Runduma and its nearest Wakatobi Island neighbour Zosterops separated by a short geographical distance is only 61 km. The Runduma population of another (27 km between Buton and Wangi-wangi). Our work small passerine, the olive-backed sunbird Cinnyris shows the closest relatives of the ‘Wangi-wangi white- jugularis (Linnaeus, 1766), appears to have colonized eye’ are found in the Solomon Islands: Z. murphyi Runduma via the shorter distance from the Wakatobi and Z. rennellianus (single island endemics) and Islands (Kelly, 2014). Given the isolation of Runduma Z. griseotinctus, a ‘supertramp’ species restricted to a and its tiny size (c. 5.5 km2), it was unsurprising that series of small islands (Van Balen, 2018a). These taxa it was colonized much later than the Wakatobi Islands are all >3000 km distant from Wangi-wangi and are (Fig. 5). Runduma Z. chloris are morphologically phenotypically distinct, all having yellow/green chests. distinct from other Z. chloris populations, showing The nodes placing the ‘Wangi-wangi white-eye’ in this the largest body size and longest bill length (Fig. 6; clade have low support (Fig. 3), so its evolutionary Supporting Information, Table S8). Larger bill and origins remain uncertain. Sequencing of other Indo- body size has been repeatedly observed to evolve in Pacific Zosterops species that have not yet had their bird populations as an adaptation to a more generalist genetic data assessed, such as the black-ringed white- niche on small islands (Grant, 1965; Clegg & Owens, eye Zosterops anomalus (Meyer & Wiglesworth, 2002; Clegg et al., 2002; Scott et al., 2003). Runduma 1896) from south Sulawesi, may shed light on this Island is almost entirely covered in coconut plantations situation. The ‘Wangi-wangi white-eye’ may be a and the Z. chloris population has been observed to remnant of an older Zosterops radiation and represent feed on coconut nectar more regularly on Runduma the remaining relict taxon. than elsewhere (DJK, pers. obs.). Thus, the longer bill may be an adaptation allowing the population to take advantage of an abundant resource in an ecologically Zosterops chloris – independent colonizations constrained habitat. Such changes can be rapid and and the ‘Wakatobi white-eye’ quickly come to fixation in a population (Bosse et al., This study clarifies a number of features about 2017). This morphometric difference, coupled with Sulawesi Z. chloris populations, while raising further the pairwise genetic distance (ND2: 0.73%, COI: questions. It appears from our data that white- 2.22%), between mainland Sulawesi and Runduma eyes from the south-east Sulawesi mainland and its populations indicates there may be a subspecies level continental islands form a continuous population, difference between them (Hebert et al., 2004). Future rather than Z. c. intermedius being present on the collection of song recordings and type specimens for continental islands and Z. c. mentoris on the mainland, assessment of more subtle plumage differences might as was suggested by Trochet et al. (2014). The prove useful in determining the taxonomic status of mainland south-east Sulawesi population of Z. chloris this population. is closely related to the south Sulawesi population Zosterops c. flavissimus (Wakatobi Islands) proved (Z. c. intermedius), but shows sufficient divergence the most distinct of the Z. chloris populations sampled. (ND2: 1.22%) that further investigation is required to It appears to have diverged much earlier (0.38–0.8

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Mya) than any of the other Sulawesi populations of relative of Z. consobrinorum among the species sampled Z. chloris (Fig. 5). This was an older date of divergence is not surprising. Zosterops atrifrons is a pale-chested, than that of several recognized Zosterops species (Fig. white-eye endemic to central and northern Sulawesi, 5). Zosterops c. flavissimus is morphometrically distinct showing geographic and phenotypic similarity (Van from other Z. chloris populations (Fig. 6; Supporting Balen, 2008). The unusual patterns of divergence Information, Tables S8, S9), being significantly smaller. between Z. consobrinorum populations emphasize Its song is highly distinct from mainland south-east how incorrect inferences can easily be drawn in Sulawesi Z. chloris (Fig. 8), with a generally higher phylogenetic studies, particularly when using a small maximum frequency and number of notes (Supporting number of mitochondrial genes. Due to unavoidable Information, Tables S13, S14), which would be expected logistical constraints, many phylogeographic studies for a population with a smaller body size (Potvin, have relied on a small number of museum specimens 2013). Zosterops c. flavissimus is also distinct from from each individual population, or a single line of mainland south-east Sulawesi Z. chloris in plumage, evidence, for assessing populations (genetic, phenotypic with a more vibrant yellow head and paler bill or acoustic). While phenotypic and genetic measures (Supporting Information, Tobias scoring, Table S18). often provide the same answer (García et al., 2016), The pairwise difference between Z. c. flavissimus and there are cases where they have been shown to differ mainland south-east Sulawesi Z. chloris (ND2: 2.5%, (Phillimore et al., 2008; Potvin et al., 2013). While the COI: 4.9%) is much larger than the average species Kabaena population of Z. consobrinorum is distinct difference (COI: 2.7%) that Hebert et al. (2004) found in morphometrics and song (Figs 7, 9), it is almost between North American birds, and is much more inseparable from the mainland Sulawesi population than 10 times the intra-group variation. In addition, in mitochondrial DNA (Figs 3, 4). This population has our molecular species delimitation analyses (ABGD) only been separated from mainland Sulawesi since highlights Z. c. flavissimus as a separate species. All of the last glacial maximum (Voris, 2000). Kabaena is this evidence makes a strong case for the recognition the smallest island (873 km2) that Z. consobrinorum of Z. c. flavissimus as a full species. While the gap was found on. This may have presented a more between the Wakatobi Islands and Buton is small (27 ecologically constrained environment for the Kabaena km), differentiation over small, open-water gaps has Z. consobrinorum population in comparison to the been noted many times in the genus Zosterops (Mayr, mainland (Lomolino & Weiser, 2001). The larger body 1942; Diamond, 1998; Mayr & Diamond, 2001). The size of the Kabaena Z. consobrinorum population isolation of the Wakatobi population may have been (Fig. 7) may have been an adaptation to life on a helped by a loss of dispersal ability during adaptation smaller island (Clegg & Owens, 2002). Morphological to the Wakatobi Islands (Supporting Information, adaptation to new environmental conditions can occur assessment of dispersal ability, Fig. S5). Several rapidly in birds and may not be related to change in type specimens of the Wakatobi Zosterops currently neutral genetic markers like mitochondrial DNA, designated as Z. c. flavissimus are in the American particularly over the short time -span Kabaena has Museum of Natural History’s collection (Supporting been isolated (Nussey et al., 2005; Charmantier et al., Information, Table S20) from the expedition of Heinrich 2008; Lande, 2009). As well as adaptation to local Kühn (1901–02; Hartert, 1903), which should facilitate conditions, genetic drift can play a role in phenotypic the promotion of this population to a full species as change in island populations and may lead to rapid Z. flavissimus. change in small populations on islands (Clegg et al., Zosterops c. maxi from Lombok is also significantly 2002; Runemark et al., 2010). different in song from other Z. chloris populations, The unusual population structure of the Buton/ although with a tiny sample size (N = 2). A much Muna Z. consobrinorum is more difficult to explain. larger sample size and investigation of further traits Initial observations of the song and phenotype of the would be needed to form a greater understanding Buton population prompted suggestions it could be an of the relationship of Z. c. maxi to other Z. chloris independent subspecies (Wardill, 2003). This would populations. be unexpected for an island only 6 km from Sulawesi, but not unprecedented (Mayr, 1942; Mayr & Diamond, 2001). This study finds no such differences, but there Zosterops consobrinorum – inconsistent is strong genetic divergence in ND2/ND3 in half of variation between measures the Buton birds and the single Muna bird sampled. By providing the first detailed assessment of That such genetically divergent individuals could be Z. consobrinorum, this study gives a first insight into found at the same site on Buton (Kusambi, 5.153 °S, its evolutionary history and emphasizes the need to 122.895 °E) seems strange. The regular trading of use a combined approach when studying systematics Zosterops species as pets in Indonesia (Harris et al., and evolution. The fact that Z. atrifrons is the closest 2017) may also have confused the pattern. Zosterops

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CONCLUSIONS Studying ‘great speciator’ lineages of the Indo-Pacific, AUTHOR CONTRIBUTIONS like Zosterops white-eyes, provides an excellent DJK, NMM, KA, DOC and AK conceived this study opportunity for both taxonomic revision and the and carried out fieldwork. DOC, NL and DJK carried examination of evolutionary processes. This study out the lab work. DOC and DJK conducted genetic documents unrecorded endemism and different analyses. DOC extracted the song data and led the evolutionary histories in multiple Zosterops taxa writing. DOC and KOB conducted the song and and highlights the utility of using multiple measures morphological analyses. DOC and DJK conducted of divergence to understand speciation. Our study plumage comparisons. FOM screened batches of species included an apparent ‘supertramp’ (Z. chloris), recordings for clear Zosterops songs. All authors a regional endemic (Z. consobrinorum) and a single contributed to revising and improving the manuscript island endemic (‘Wangi-wangi white-eye’) (Eaton et al., 2016). This study found significant geographic structure to Z. chloris populations. However, the panmixia that would be expected of a true ‘supertramp’ REFERENCES was not apparent in Z. chloris, with the Wakatobi Andersen MJ, Oliveros CH, Filardi CE, Moyle RG. 2013. Z. c. flavissimus population deserving recognition as a Phylogeography of the variable dwarf-kingfisher Ceyx separate species. This tallies with the recent assessment lepidus (Aves: Alcedinidae) inferred from mitochondrial and of the classic ‘supertramp’, Z. griseotinctus, by Linck nuclear DNA sequences. The Auk 130: 118–131. et al. (2016), who found significant geographic and Andersen MJ, Nyári ÁS, Mason I, Joseph L, population structure. This may be an illustration of the Dumbacher JP, Filardi CE, Moyle RG. 2014. Molecular rapid shifts in dispersal ability inferred to explain the systematics of the world’s most polytypic bird: the paradox of ‘great speciator’ lineages (Diamond et al., Pachycephala pectoralis/melanura (Aves: Pachycephalidae) 1976; Moyle et al., 2009). Our taxonomic considerations species complex. Zoological Journal of the Linnean Society are given impetus by the discovery of a novel species 170: 566–588. on a small ecologically-degraded island. The ‘Wangi- Andersen MJ, Shult HT, Cibois A, Thibault JC, Filardi CE, wangi white-eye’ (first noted in 2003) still awaits Moyle RG. 2015. Rapid diversification and secondary formal description. This illustrates the administrative sympatry in Australo-Pacific kingfishers (Aves: Alcedinidae: delays that can occur in conservation biology. We hope Todiramphus). Royal Society Open Science 2: 140375.

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher's web-site. Figure S1.Full Bayesian consensus tree for concatenated ND2/ND3 haplotypes with outgroups shown, Bayesian posterior probabilities (above) and bootstrap values from our Maximum Likelihood analysis (below) are provided for each node. Solid grey lines used to indicate posterior probabilities and bootstrap values where there was not room by a node to note this. Haplotype number was given when there was more than one representative of a single taxon, with geographic information added with square brackets (single node) or curly brackets (multiple nodes) when that was informative to the pattern seen. Focal species highlighted in colour. Grey dashed lines used to space out species names. Branch lengths for outgroup root taxa reduced to save space. Figure S2.Bayesian consensus tree for COI haplotypes, showing Bayesian posterior probabilities (above) and bootstrap values from our Maximum Likelihood analysis (below) for each node. Haplotype number was given when there was more than one representative of a single taxon, with geographic information added with square brackets (single node) or curly brackets (multiple nodes) when that was informative to the pattern seen. Focal species highlighted in colour. Core Z. japonicus lineage collapsed as it was monophyletic. Branch lengths for outgroup root taxa reduced to save space. Figure S3. Haplotype network of sampled Sulawesi Zosterops populations samples, based on concatenated ND2/3 sequences. One bar indicates one mutation, black nodes are hypothetical ancestral states and the size of the circles corresponds to the number of sampled individuals sharing that haplotype. Figure S4. Haplotype network of sampled Sulawesi Zosterops populations samples, based on COI sequences. One bar indicates one mutation, black nodes are hypothetical ancestral states and the size of the circles corresponds to the number of sampled individuals sharing that haplotype. Assessment of dispersal ability: an assessment of the dispersal ability of the focal Zosterops populations using wingspan (S) to weight (m) ratio (S3/m) as a proxy of dispersal ability. Includes Figure S5: The wingspan/weight ratio (S3/m) of each Zosterops population identified in this study, providing an indication of their dispersal ability. Table S1. Full list of samples utilized in the phylogenetic analyses, detailing the species, sampling location and museum ID. Haplotypes identified for concatenated ND2 and ND3 sequences and for COI sequences in this study are listed. GenBank accession numbers are provided. Individuals are ordered by ND2/ND3 haplotype. Sulawesi samples collected under licence from Kementerian Negara Riset dan Teknologi (RISTEKDIKTI). Table S2. Novel primers developed for this study. Tables S3 & S4. Summary tables of taxa used in this study. Table S5. Phylogenetic models used for each partition and summary information about the sequence data used. Table S6. Pairwise distances between sampled Zosterops for ND2. Table S7. Pairwise distances between sampled Zosterops for COI. Tables S8–S11. Summary tables for the morphometric data used in this study, providing mean ± standard error and sample size from each island sampled. Tables S13–S16. Summary tables for the song data used in this study, providing mean ± standard error and sample size from each island sampled. Tables S12 & S17. PC trait loadings for PCAs on morphometric and song data. Tobias Scoring for potentially novel Zosterops species: an assessment of the taxonomic status of the potentially novel Zosterops species identified in this study using the Tobias Scoring criteria implemented by the Handbook of the Birds of the World. Includes Tables S18 & S19 featuring photographic comparisons of the potentially novel taxa. Table S20. Existing vouchered specimens of Zosterops chloris from south-east Sulawesi.

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