Phasianidae, Aves) Using Multilocus Sequence Data

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Zoological Journal of the Linnean Society, 2015, 175, 429–438. With 3 ﬁgures

Revival of the genus Tropicoperdix Blyth 1859 (Phasianidae, Aves) using multilocus sequence data

DE CHEN1, YANG LIU2, GEOFFREY W. H. DAVISON3, LU DONG1, JIANG CHANG4, SHENGHAN GAO5, SHOU-HSIEN LI6 and ZHENGWANG ZHANG1*

1Key Laboratory for Biodiversity Science and Ecological Engineering, College of Life Sciences, Beijing Normal University, Beijing, 100875, China 2State Key Laboratory of Biocontrol and College of Ecology and Evolution, Sun Yat-sen University, Guangzhou, 510275, China 3National Biodiversity Centre, National Parks Board, 1 Cluny Road, 259569, Singapore 4State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China 5Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China 6Department of Life Science, National Taiwan Normal University, Taipei, 10610, Taiwan

Received 14 July 2014; revised 13 March 2015; accepted for publication 19 March 2015

Although Tropicoperdix has been considered to be either a full genus or a species complex within the Phasianid genus Arborophila (hill partridges), there is long-standing uncertainty regarding the degree of difference that war- rants generic separation, including reported anatomical cranial differences. In addition, the intra-generic taxonomy remains under dispute. Most studies hypothesize that Tropicoperdix comprises three species, while others postulate from one to four species. However, no molecular study has been performed to clarify the systematic and taxonomic uncertainties surrounding Tropicoperdix. In the present study, we performed a series of molecular phylogenetic analyses of Tropicoperdix and Arborophila taxa based on two mitochondrial genes and ﬁve nuclear introns. All the results are consistent with the ﬁnding that Tropicoperdix and Arborophila are phylogenetically distinct and distant genera, although the precise phylogenetic position of Tropicoperdix remains undetermined. Retrospective examination of external characteristics also supports the generic separation, as well as providing evidence of re- markable multiple character convergence. We propose that Tropicoperdix comprises at least two full species based on mitochondrial data obtained from museum specimens by using a next-generation sequencing method.

ADDITIONAL KEYWORDS: Arborophila – generic separation – historical DNA – intra-generic taxonomy – molecular systematics – multilocus – next-generation sequencing.

INTRODUCTION of taxonomic revisions caused by upgrades of subspecies to full species, changes at higher taxonomic levels Recent studies in avian molecular genetics have re- have also been reported (Fjeldså, 2013). For example, vealed a number of disputes about traditional classi- Elachura formosa, previously considered a babbler in ﬁcation and phylogenies at various taxonomic levels the family Timaliidae, is in fact the sole member of a (Hillis, 1987; Hackett et al., 2008). Apart from the ﬂux separate evolutionary lineage and therefore it has been proposed as being its own family, Elachuridae (Alström et al., 2014). The genus Ptilopachus was misplaced *Corresponding author. E-mail: [email protected] in Phasianidae for more than 150 years until a

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 175, 429–438 429 430 D. CHEN ET AL. molecular study showed that it should be placed in the species, namely A. charltonii, A. chloropus and A. merlini family Odontophoridae (Cohen et al., 2012). These (Peters, 1934; del Hoyo, 1994; Madge & McGowan, 2002; notable examples highlight the complexity of the avian Zheng et al., 2002; Clements et al., 2014) (see Fig. 1 tree of life and suggest similar unexpected relation- for their distributions) from museum specimens by using ships to be discovered in birds. next-generation sequencing (NGS). The hill partridges (Arborophila Hodgson 1837), found in Asia from the Himalayas eastwards to Taiwan and south to Java, constitute the second largest genus of MATERIALS AND METHODS Phasianidae (Johnsgard, 1988; Madge & McGowan, 2002). However, their precise systematics is some- TAXON SAMPLING, DNA EXTRACTION what confusing and controversial (del Hoyo, 1994; Madge AND SEQUENCING & McGowan, 2002). Particular confusion surrounds the Our taxon sampling was based on the available A. chloropus complex. The intra-generic taxonomy of multilocus dataset generated by Wang et al. (2013). To this species complex is controversial, with one to four ensure that no relevant taxa were overlooked, we ob- species having been proposed (Peters, 1934; Davison, tained the sequence data of most Phasianid genera, 1982; Johnsgard, 1988; Madge & McGowan, 2002; except for grouse, from GenBank (supplementary Table Dickinson & Remsen, 2013; del Hoyo & Collar, 2014). S1 lists the accession numbers of all samples ana- The affinity of this species complex to Arborophila is lysed). The blood sample of a single A. chloropus was also sometimes called into question. obtained from Mengla County in Yunnan Province, When Blyth (1859) established the name Tropicoperdix south-west China. Toe pads of A. charltonii and for the newly discovered taxon T. chloropus A. merlini were excised from two specimens archived (= A. chloropus), he simultaneously transferred Perdix in the Zoological Reference Collection (ZRC) of the Na- charltonii (= A. charltonii) to this new genus. tional University of Singapore (catalogue numbers ZRC Ogilvie-Grant (1895) accepted the validity of 3.1513 for A. charltonii and ZRC 3.1478 for A. merlini) Tropicoperdix because both T. chloropus and T. charltonii because fresh tissues are difficult to obtain from field have a single supraorbital bone, rather than a chain research. of four supraorbital bones found in Arborophila species. Blood sample DNA was extracted using the TIANamp Besides, members of Tropicoperdix have yellowish legs blood genomic DNA extraction kit (Tiangen). We am- and white axillary feathers, whereas members of plified two mt genes, cytochrome b (CYTB) and NADH Arborophila usually have red legs and their axillary dehydrogenase subunit 2 (ND2), as well as five nuclear feathers are grey (Ogilvie-Grant, 1895; Morony, Bock introns, i.e. clathrin heavy polypeptide intron 7 (CLTC), & Farrand, 1975; Cheng, 1978). However, Davison (1982) clathrin heavy chain-like 1 intron 7 (CLTCL1), included Tropicoperdix in Arborophila because of the eukaryotic translation elongation factor 2 introns 5 and high degree of morphological and vocal similarities 6 (EEF2), ovomucoid intron G (OVOG) and ovalbumin between the two genera (for details see Davison, 1982), intron C (SERPINB14), of A. chloropus using the primers a position then widely accepted in various world bird listed in Table S2. A single touchdown PCR protocol checklists (Johnsgard, 1988; Madge & McGowan, 2002; was used for all loci (for details see Wang et al., 2013). Zheng et al., 2002; Dickinson & Remsen, 2013; Clements Each round of PCR also included one negative control et al., 2014; del Hoyo & Collar, 2014; Gill & Donsker, to check for contamination. Both strands of each PCR 2014). Nevertheless, the difference in the number of product were sequenced on a 3730 DNA sequencer supraorbital bones appears to be a non-negligible trait (Applied Biosystems). The sequences were visually com- because skull morphology is an important and clas- pared with the original chromatograms to avoid reading sical characteristic in avian taxonomy and systemat- errors and were also checked against published DNA ics (Huxley, 1807; Simonetta, 1960; Zusi, 1993) and has sequences. The sequences were then assembled using been highlighted in several recent studies (James et al., MEGA version 5 (Tamura et al., 2011). 2003; Chiappe & Bertelli, 2006). The toe pads were cut into small pieces and pre- Therefore, the objectives of this study were to de- treated as described by Hung et al. (2013). The Qiagen termine the validity of Tropicoperdix and to investi- DNeasy Tissue Kit was used to extract historical DNA gate its controversial intra-generic taxonomy as a (hDNA). Approximately 200 ng hDNA was used for preliminary to further investigations. Phylogenetic re- single-end short-read sequencing (Nextera DNA Sample lationships between A. chloropus and several other Preparation Kits, Illumina) according to the instruc- Arborophila species, as well as additional representa- tions of the manufacturer. Single-end 90-bp sequence tive Phasianid genera, were reconstructed based on two reads were obtained from a separate lane of a HiSeq mitochondrial (mt) genes and five nuclear introns. We 2000 Genome Analyzer (Illumina) for each sample. Berry also compared genetic distances based on mt genes Genomics performed the single-end library prepara- between three conventionally considered Tropicoperdix tion and sequencing. A total of 72 360 071 (A. charltonii)

Figure 1. Approximate geographical distributions of Tropicoperdix species, according to del Hoyo & Collar (2014). Here, we followed the conventional taxonomy to treat three species; by contrast, del Hoyo & Collar split off the forms tonkinensis (from T. chloropus) and graydoni (from T. charltonii) as species, and treated merlini as a subspecies of T. chloropus. and 85 299 440 (A. merlini) NGS reads were generat- quality-controlled (Q ≥ 30) with FastQC v0.11.2 ed. The duplicated reads were 15 767 966 (21.79%) and (Andrews, 2010) for NGS assembly. SOAPdenovo2 r240 7 244 030 (8.49%), separated and then removed from (Luo et al., 2012) was then used to carry out the as- the sequencing data before mapping to the reference. semblies in continuous K-mer sizes from 17 to 63. All The PCR-based mt genes of A. chloropus were provid- the contigs were combined and further assembled with ed as references to extract the homologous mt genes CAP3 version 10/15/07 (Huang & Madan, 1999) for full- from A. charltonii and A. merlini. We used Bowtie2 length sequences. Finally, the mt genes were re- v2.2.4 (Langmead & Salzberg, 2012) for mapping NGS identiﬁed using BLAST from the assembled contigs. reads to the references, with the ‘local’ and ‘very- Furthermore, to enhance the taxonomy between these sensitive’ search strategy for better tolerance of genetic three taxa, we also ampliﬁed the cytochrome oxidase variation. Next, the mapped reads were extracted and I (COI) barcode (Hebert, Cywinska & Ball, 2003; Hebert

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 175, 429–438 432 D. CHEN ET AL. et al., 2004) of A. chloropus and extracted the COI bar- tances among the Arborophila species. The pairwise codes from A. charltonii and A. merlini reads. Se- distances and standard errors (SE) were calculated using quences collected in this study have all been deposited a bootstrap method with 1000 replicates under the in GenBank (KP968265-KP968278, Table S1). Tamura–Nei model using the program MEGA version 5 (Tamura et al., 2011). We also compared the COI divergences (in %) between each pair of the three SEQUENCE ALIGNMENT Tropicoperdix taxa. We combined the sequence alignments generated by Muscle (Edgar, 2004) included in MEGA version 5 (Tamura et al., 2011) into a complete concatenated data EXTERNAL CHARACTERISTICS matrix to clarify the systematic relationship between Qualitative comparisons were made based on exter- Tropicoperdix and Arborophila, and incorporated CYTB nal morphologies and plumage characteristics ob- and ND2 sequences extracted from the two museum tained from colour photographs of live birds representing specimens into a mt-only data matrix to determine the the majority of Arborophila and Tropicoperdix taxa col- phylogenetic relationships within Tropicoperdix. lected on the website http://www.orientalbirdimages.org, including multiple images of the adults of most taxa and the juveniles from a few taxa. Additional images CONCATENATED DATA ANALYSES of A. orientalis were obtained from http://www Phylogenetic analyses were performed using Bayes- .ibc.lynxeds.com and A. campbelli images were sup- ian inference (BI) and maximum-likelihood (ML) analy- plied by Con Foley (pers. comm.). ses. BI was performed in BEAST v1.8.0 (Drummond Quantitative comparisons were made based on the & Rambaut, 2007) with a random starting tree and ratio of the length between tarsus, toe and claw of the a Yule tree prior. The best-fitting nucleotide substitu- various taxa of Arborophila and Tropicoperdix from tion model for each of the five nuclear introns was se- specimens in the Natural History Museum (NHM) at lected using the Akaike information criterion (AIC) by Tring, UK, and (for A. diversa) data in Riley (1930). jmodeltest v2.1.4 (Darriba et al., 2012) and mt genes Measurements were taken with sliding vernier calli- were partitioned by codon position (Yang, 1996). The pers, to the nearest 1 mm for tarsus and toes and the log-likelihood values and effective sample size were nearest 0.5 mm for hind toe and claws. Toe lengths checked using Tracer v1.5 (Rambaut & Drummond, consisted of the sum from measurements of each 2009) to confirm the stationary of the chains. The final phalanx in their respective digits, beginning from the analysis was run for 500 million generations with trees base of the lowest tarsal scute to the base of the claw. sampled every 1000 generations. TreeAnnotator v1.8.0 Claw measurements were taken as the straight-line was then used to discard the first 20% of trees and chord from the tip to the base where it joins the last to generate a consensus tree with Bayesian posterior dorsal scute of the toe. No test for repeatability of meas- probability (PP). ML analysis was performed in RaxML urement was carried out. v8.0.26 (Stamatakis, 2014) using the same strategy as BI. Bootstrap values (BS) were calculated using 1000 replicates. RESULTS ANALYSES FROM MOLECULAR DATA SPECIES TREE ANALYSIS Average sequence coverage was 437 (COI), 491 (CYTB) We constructed a coalescent-based species tree using and 481 (ND2) for A. charltonii and 76 (COI), 82 (CYTB) *BEAST (Heled & Drummond, 2010) within BEAST and 59 (ND2) for A. merlini, suggesting these se- v1.8.0 (Drummond & Rambaut, 2007). The species tree quences were correct. The complete concatenated data analysis was carried out using all six partitions (five matrix aligned by Muscle was 5234 bp in length, in- nuclear introns and one linked mt partition) and all cluding 2135 bp of the mt genes and 3099 bp from the species (without A. charltonii and A. merlini) with a nuclear introns. The concatenated BI and ML analy- UPGMA starting tree and a Yule tree prior. The final ses from all data matrices indicate that Tropicoperdix analysis was run for 500 million generations with trees forms a separate clade away from Arborophila (Figs 2, sampled every 1000 generations, of which the first 20% 3). This is further supported by our species tree analy- was discarded as burn-in. sis (Fig. S1). We located Tropicoperdix within the ‘Chick- ens and allies’ clade, whereas other Arborophila were monophyletic and placed in the ‘Arborophilinae’ clade GENETIC DISTANCES WITHIN TROPICOPERDIX (Fig. 2). However, the Chickens and allies clade was We calculated pairwise distances based on the mt genes only well supported in the complete concatenated BI CYTB and ND2 between each pair of the three tree (PP = 0.99, Fig. 2); other analyses showed Tropicoperdix taxa, and compared these with the dis- extremely low support or even failed to reveal this clade

Figure 2. Consensus tree with support from the complete concatenated data matrix. The taxa representing the clade of interest are highlighted in different colours. Numbers above nodes are Bayesian posterior probability (PP) represented by percentage and ML bootstrap support (BS). *100% support.

(Figs 2, 3, Fig. S1). Furthermore, our results indicat- the ML analyses (Fig. 3). In addition, the pairwise dis- ed a Tropicoperdix cluster formed as a separate clade tances of the concatenated mt genes CYTB and ND2 within the Chickens and allies clade (Fig. 3), but the between T. charltonii and T. chloropus was 0.061 (± 0.005 relationships between the Tropicoperdix clade and four SE), which was double that between A. ardens and other well-supported clades remain controversial. Al- A. crudigularis at 0.031 (± 0.004 SE). However, the though the Tropicoperdix clade is shown to be a sister pairwise genetic distance between T. chloropus and group to the peafowl clade (Pavo, Afropavo and T. merlini was only 0.014 (± 0.003 SE). The COI di- Argusianus) in the complete concatenated BI tree vergence between T. charltonii and T. chloropus is 7.70%, (PP = 0.96, Fig. 2), other analyses showed low support but only 1.33% between T. chloropus and T. merlini. or even failed to uncover this relationship (Figs 2, 3, Fig. S1). Within Tropicoperdix, the mt trees showed that DATA ANALYSIS ON EXTERNAL CHARACTERISTICS T. chloropus and T. merlini group together and are sister In addition to previously published distinctions for to T. charltonii with full support from both the BI and the colours of axillary feathers and legs, additional

Figure 3. Consensus tree with support from mt genes CYTB and ND2. The taxa representing the clade of interest are highlighted in different colours. Numbers above nodes are Bayesian posterior probability (PP) represented by percentage and ML bootstrap support (BS). ‘–’ indicates no support for that clade, ‘*’ indicates 100% support for that clade. characteristics such as the plumage patterns of greater in 13 Arborophila species, the ratio of hind claw to hind secondary coverts (Fig. S2) and the colours of other bare toe (digit I) length is 0.39–0.75, whereas in Tropicoperdix parts, including the bill, claw and orbital ring, show this is a little longer, from 0.72 to 0.83 (Table 1). recognizable differences between Arborophila and Tropicoperdix (overall, bill: black versus yellow, claw: red versus yellow, orbital ring: red versus grey, see Table DISCUSSION S3 for details). In 13 Arborophila species, the ratio of the length of SYSTEMATIC STATUS OF THE TROPICOPERDIX GROUP inner toe (digit II) to tarsus ranges from 0.47 to 0.62, In the present study, we performed a series of mo- whereas in Tropicoperdix this is slightly shorter, i.e. lecular phylogenetic analyses to determine the sys- from 0.40 to 0.44 (Table 1). The ratio of the length of tematic position of Tropicoperdix. Together with the middle toe (digit III) to tarsus is 0.63–0.70 in qualitative and quantitative morphological compari- Tropicoperdix, also slightly shorter than that in sons between Tropicoperdix and other Arborophila Arborophila, from 0.71 to 0.90 (Table 1). In addition, species, our results contradict the prevailing view that

Table 1. Ratios derived from comparative measurements of Arborophila and Tropicoperdix taxa based on specimens in NHM Tring and (for A. diversa) data in Riley (1930) (see Table S4 for details)

Inner Mid Hind Mid Hind Taxon (and sample size) toe/tarsus toe/tarsus toe/tarsus claw/mid toe claw/hind toe

A. atrogularis (1) 0.500 0.761 0.190 0.406 0.437 A. brunneopectus (9) 0.534 0.837 0.209 0.361 0.555 A. cambodiana (1) 0.547 0.785 0.226 0.424 0.631 A. crudigularis (2) 0.609 0.878 0.219 0.416 0.611 A. diversa (1) – 0.845 – – – A. gingica (5) 0.534 0.767 0.197 0.424 0.588 A. hyperythra (6) 0.511 0.777 0.177 0.342 0.625 A. javanica (5) 0.466 0.711 0.177 0.453 0.625 A. mandelli (3) 0.619 0.904 0.214 0.342 0.555 A. orientalis (5) 0.478 0.739 0.195 0.411 0.722 A. rubrirostris (2) 0.477 0.772 0.181 0.382 0.750 A. rufipectus (2) 0.533 0.755 0.200 0.411 0.388 A. rufogularis (7) 0.575 0.825 0.200 0.393 0.687 A. torqueola (12) 0.511 0.800 0.200 0.361 0.555 T. charltonii (6) 0.395 0.627 0.209 0.425 0.833 T. chloropus (4) 0.441 0.697 0.209 0.400 0.722 places Tropicoperdix within Arborophila (Davison, 1982; Clues to the distinctiveness of Tropicoperdix can be Johnsgard, 1988; Madge & McGowan, 2002; Zheng et al., found in the morphological comparisons with 2002; Dickinson & Remsen, 2013; Clements et al., 2014; Arborophila, although these differences are subtle. The del Hoyo & Collar, 2014; Gill & Donsker, 2014), but colours of the bare parts (bill, orbital ring, legs and support the revival of Tropicoperdix as a separate genus claws) (Table S3) show recognizable differences between (Blyth, 1859; Ogilvie-Grant, 1895; Morony et al., 1975). Tropicoperdix and Arborophila. These bare-part colour More interestingly, our analyses indicate that differences are shown to be associated with interspecific Tropicoperdix and Arborophila are distinct and dis- differences in their life histories and habitat use (Olson tantly related taxa, residing in two different major & Owens, 2005). Tarsus, toe and claw measurements Phasianid clades, the Chickens and allies clade and also indicate subtle differences, with proportionately the Arborophilinae clade (Fig. 2). longer inner and mid toes in Arborophila than in While the Chickens and allies clade, to which Tropicoperdix, and a proportionately shorter claw on Tropicoperdix belongs, received mixed support in our the hallux of Arborophila than of Tropicoperdix (Table 1). analyses as well as in numerous earlier studies (re- Such differences between the hind limb structures may viewed by Wang et al., 2013; Kimball & Braun, 2014), also indicate different foraging techniques and habitat there is strong support for Tropicoperdix being within use (Keast & Saunders, 1991; Carrascal, Moreno & the clade that includes all Phasianidae minus Valido, 1994; Einoder, Richardson & Briskie, 2007). Arborophila (Figs 2, 3, Fig. S1). The uncertainty about However, Tropicoperdix and Arborophila are sympatric the Chickens and allies clade was eventually support- and both inhabit evergreen forests in tropical south- ed by the use of large amounts of ultraconserved east Asia (Davison, 1982), masking their possible habitat element (UCE) data (Sun et al., 2014). Furthermore, difference. Furthermore, Tropicoperdix and Arborophila four monophyletic clades have been identified within are all small, round-bodied partridges with dark brown the Chickens and allies clade (Wang et al., 2013; Kimball cryptic plumage, skulking behaviour, with long, sharp & Braun, 2014; Sun et al., 2014). Our results suggest and nearly straight claws, performing ventriloqual and strongly that Tropicoperdix should not be included in antiphonal duets, and building domed nests of dead any of these four clades but forms another separate leaves on the forest floor (Davison, 1982; Johnsgard, monophyletic clade (Fig. 3). Similar to the uncertain- 1988). Individually each of these features is exhibit- ty surrounding the Chickens and allies clade, our ed by at least one other genus, for example domed nests smaller dataset failed to resolve the precise phylogenetic in Rollulus and Caloperdix, antiphonal duets in position of the Tropicoperdix clade. Additional nuclear Rhizothera and Odontophorus, and long sharp claws loci, such as UCEs, are likely to resolve the exact po- in Odontophorus and Dactylortyx. These seem conver- sition of Tropicoperdix in the Phasianid phylogeny gent in tropical forests but not all are shared by (Kimball & Braun, 2014; Sun et al., 2014). all such genera, and the examples cut across

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 175, 429–438 436 D. CHEN ET AL. subfamilies. All these features come together in Dr Limin Feng for their help in sample collection, Mr Tropicoperdix and Arborophila, and although not unique Kevin Lim’s permission to collect tissues from the Lee to that pair, would encourage the traditional view that Kong Chian Natural History Museum at the Nation- they are congeneric. Overall, our molecular phylogenetic al University of Singapore, and the Natural History results indicate that Tropicoperdix is genetically dif- Museum at Tring (UK) for access to specimens for meas- ferent from Arborophila, although their appearance, urement. We also thank the editors and anonymous vocalization and habitat selection are so similar that reviewers for their comments on the manuscript. this similarity has misled many ornithologists into think- ing that they are closely related. 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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. Species tree analysis from the complete concatenated data matrix. The taxa representing the clade of interest are highlighted in different colours. Numbers above nodes represent Bayesian posterior probability (PP). Figure S2. Sketches of the exposed tips of the greater secondary coverts (left wing) of Arborophila and Tropicoperdix, from photographs. Upper row from left to right: A. brunneopectus, A. rufogularis, A. crudigularis and A. torqueola (reversed from right wing). Lower row from left to right: T. charltonii, T. chloropus and T. merlini. Table S1. GenBank accession number for each sequence used in this study. Table S2. Names, location and primer sequences of the eight regions. Table S3. Colours of bare parts between the Arborophila and Tropicoperdix taxa. Table S4. Measurements (mm) of various taxa of Arborophila and Tropicoperdix based on specimens in NHM Tring and (for A. diversa) data in Riley (1930).