Ibis (2016), doi: 10.1111/ibi.12377 The phylogenetic position of the world’s smallest passerine, the Pygmy Bushtit Psaltria exilis ULF S. JOHANSSON,1* PER G. P. ERICSON,1 JON FJELDSA2 & MARTIN IRESTEDT3 1Department of Zoology, Swedish Museum of Natural History, Box 50007, Stockholm 10405, Sweden 2Center for Macroecology, Evolution and Climate, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark 3Department of Bioinformatics and Genetics, Swedish Museum of Natural History, Box 50007, Stockholm 10405, Sweden The Pygmy Bushtit is confined to the montane forests of Java. It is the world’s smallest passerine and morphologically resembles a small, drab long-tailed tit or bushtit (Aegithali- dae). In its behaviour the Pygmy Bushtit show similarities with the members of the Aegit- halidae, but owing to its small size and isolated geographical distribution relative to the other members of the Aegithalidae, it has always been placed in a monotypic genus within the family. The affinities of the Pygmy Bushtit have never been tested in a phylogenetic context and the species has to date not been included in any molecular studies. In this study we use sequence data from four different genetic markers to place it in the passerine phylogenetic tree. Our results confirm the inclusion of the Pygmy Bushtit in the Aegithali- dae, but rather than being an isolated lineage, our results strongly suggest that it is nested in the Aegithalos clade, and most closely related to the Black-throated Bushtit Aegithalos concinnus. The range of the Black-throated Bushtit extends south into subtropical Indo- china, with an isolated subspecies occurring in southern Vietnam. The Black-throated Bushtit contains several morphologically and genetically distinct lineages, which could rep- resent distinct species, but the phylogenetic relationships within this complex are poorly resolved and partly in conflict with current taxonomic treatment based on morphology. Keywords: Aegithalidae, Aves, biogeography, Passeriformes, phylogeny. The Pygmy Bushtit Psaltria exilis is a Javan ende- lack of a territorial song (Harrap & Quinn 1996, mic confined to montane forests in the highlands Harrap 2008). Based on this resemblance it has of western and central Java. It is a nondescript long been placed in the Aegithalidae (e.g. Snow drab grey bird with whitish underparts, whitish iri- 1967, Sibley & Monroe 1990, Dickinson 2003, des and yellow legs and feet (Harrap & Quinn Dickinson & Christidis 2014). As it is smaller and 1996, Harrap 2008). In spite of its long tail, it is duller than other aegithalids and is the only tropi- only 8.5 cm in length, which makes the Pygmy cal member of the clade, the Pygmy Bushtit has Bushtit the smallest passerine species (Harrap & been placed in the monotypic genus Psaltria. Quinn 1996). Morphologically it resembles a The genera Aegithalos, Psaltriparus and Psaltria small, dull bushtit and although very little is have been grouped together in most classifications known about its biology, it also appears to behave since the mid-1800s. Until recently this group was very much as other members of the Aegithalidae. in turn often associated with the tits and chick- For instance, it lives in small family groups that adees (Paridae). Once designed a separate family, move actively through the vegetation, builds a Aegithalidae (Vaurie 1957), the bushtits neverthe- ‘pouch-like’ nest and resembles other long-tailed less remained linked with the parids due to the tits and bushtits in its contact calls and apparent lack of evidence suggesting an alternative place- ment. It was not until molecular data were avail- fi – *Corresponding author. able, rst DNA DNA hybridization (Sibley & Email: [email protected] Ahlquist 1990) and later DNA sequencing © 2016 British Ornithologists’ Union 2 U. S. Johansson et al. (Alstrom€ et al. 2006, Johansson et al. 2008, Fregin western Java on 12 April 1920 (NRM 571647). et al. 2012), that the relationship with parids was We also extracted DNA from additional specimens rejected and it was shown that Aegithalidae are of the Black-throated Bushtit complex including instead most closely related to leaf warblers (Phyl- two specimens of the subspecies A. concinnus loscopidae) and bush warblers (Cettiidae) in the annamensis and representatives of the subspecies Sylvoidea. These latter studies also revealed that A. c. talifuensis and A. c. concinnus (Table 1). the more warbler-like Leptopoecile species are part As the DNA obtained from old museum study of the Aegithalidae and sister taxa to Aegithalos skins is generally heavily degraded, the laboratory and Psaltriparus. However, the systematic position work was conducted under strict conditions to of the Pygmy Bushtit has been addressed in few avoid contamination, following the procedures systematic studies and to date it has not been described in Irestedt et al. (2006, in press). We included in any molecular phylogeny. specifically designed primers that PCR-amplified Aegithalos, with between six and nine species, the individual loci in short c. 200-bp fragments has the largest distribution of the four genera in the (Supporting Information Table S1). We sequenced family. Members of the genus extend from the Ibe- four loci, the mitochondrial cytochrome-b gene, rian Peninsula and the British Isles across the and three nuclear intron regions: ornithine decar- Palaearctic to Kamchatka and Japan, and south to boxylase (ODC) introns 6 and 7, myoglobin intron the Himalayas and Indochina. This large distribu- 2, and transforming growth factor beta 2 (TGFB2) tional range, however, comprises primarily a single intron 5. species, the Long-tailed Tit Aegithalos caudatus. Another fairly widespread species is the Black- Phylogenetic analyses throated Bushtit Aegithalos concinnus, which is dis- tributed in the Himalayan foothills and over much We first aimed to evaluate the phylogenetic position of eastern China and along mountain ridges in tropi- of the Pygmy Bushtit relative to other passerine cal Indochina. The remaining species in the clade all birds to ascertain its relationships with the Aegithal- have rather restricted distributions confined to small idae, as suggested by behavioural and morphological regions in the Himalayas or in the mountains of data. To cover as broad a range of potential closely southwestern China. The two Leptopoecile species related groups as possible, we made use of a previ- inhabit the mountains north and east of the Tibetan ously published dataset of passerine birds (Johans- Plateau, primarily above 3000 m, whereas the sole son et al. 2008; Supporting Information Table S2). member of the genus Psaltriparus, the American This dataset consists of myoglobin and ODC Bushtit Psaltriparus minimus, inhabits western sequences from a broad representation (90 taxa) of North America, extending from southwestern primarily Passerida lineages, with the densest sam- Canada to southern Mexico and Guatemala. pling within the Sylvioidea, including all Aegithali- Here we address the systematic position of the dae genera (Aegithalos, Psaltriparus and Pygmy Bushtit of Java by including it in a molecu- Leptopoecile). We also included additional Aegithalos lar phylogenetic study for the first time. We first sequences from GenBank: White-throated Bushtit verify its position among the bushtits by including Aegithalos niveogularis (KJ454741, GU434033), it in a large phylogeny of passerine bird species, Black-browed Bushtit Aegithalos bonvaloti and then specifically address its relationships to (KJ790385, GU434032) and Aegithalos concinnus the other species in the Aegithalidae in an analysis iredalei (DQ008570, GU434027). with a comprehensive coverage of the taxa cur- While this first analysis confirmed the place- rently recognized within this family. ment of the Pygmy Bushtit in the Aegithalidae, it also suggested a previously unrecognized affinity for this species within the genus Aegithalos near METHODS A. concinnus. We therefore performed an extended analysis of the relationships within this family DNA extraction, PCR-amplification and based on three markers (ODC, TGFB2 and cyto- sequencing chrome-b) and additional sequences primarily DNA for this study was obtained from toe-pads of obtained from P€ackert et al. (2010) (Table 1). museum study skins. The DNA from P. exilis was However, as recent studies of the A. concinnus obtained from a specimen that was collected in complex (P€ackert et al. 2010, Dai et al. 2011) © 2016 British Ornithologists’ Union Table 1. GenBank accession numbers for the samples used in the three gene analysis of this study. GenBank accession no. Species Subspecies Locality Specimen no. Tgfb2 Ref. cytb Ref. ODC Ref. Psaltria exilis Indonesia, western NRM 571647 KX014777 1 KX014769 1 KX014783 1 Java Aegithalos concinnus concinnus Taiwan NRM 571736 KX014774 1 KX014766 1 Failed Aegithalos concinnus concinnus China, Anhui NRM 571732 KX014773 1 KX014765 1 KX014780 1 province Aegithalos concinnus talifuensis Vietnam, Lao Cai NRM 571740 KX014775 1 KX014767 1 KX014781 1 province, Sapa Aegithalos concinnus talifuensis Vietnam, Lao Cai NRM 571743 KX014776 1 KX014768 1 KX014782 1 province, Sapa Aegithalos concinnus annamensis Vietnam, Lam Dong NRM 571725 KX014771 1 KX014763 1 KX014778 1 province, Da Lat Aegithalos concinnus annamensis Vietnam, Dac Lac NRM 571729 KX014772 1 KX014764 1 KX014779 1 province, B’sre Aegithalos fuliginosus China, Shaanxi, Taibai MAR809 GU434014 2 GU244444 2 GU434031 2 Shan, Houzhenzi Aegithalos bonvaloti China, Yunnan, Jizu MAR3232