Molecular Phylogenetics and Evolution xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev A comprehensive molecular phylogeny for the hornbills (Aves: Bucerotidae) ⇑ Juan-Carlos T. Gonzalez a,b, Ben C. Sheldon a, Nigel J. Collar c,d, Joseph A. Tobias a, a Department of Zoology, Edward Grey Institute for Field Ornithology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK b Institute of Biological Sciences, University of the Philippines Los Baños, College, Laguna 4031, Philippines c BirdLife International, Wellbrook Court, Girton Road, Cambridge CB3 0NA, UK d Department of Zoology, Natural History Museum, Tring, Herts HP23 6AP, UK article info abstract Article history: The hornbills comprise a group of morphologically and behaviorally distinct Palaeotropical bird species Received 25 August 2012 that feature prominently in studies of ecology and conservation biology. Although the monophyly of Revised 11 February 2013 hornbills is well established, previous phylogenetic hypotheses were based solely on mtDNA and limited Accepted 13 February 2013 sampling of species diversity. We used parsimony, maximum likelihood and Bayesian methods to recon- Available online xxxx struct relationships among all 61 extant hornbill species, based on nuclear and mtDNA gene sequences extracted largely from historical samples. The resulting phylogenetic trees closely match vocal variation Keywords: across the family but conflict with current taxonomic treatments. In particular, they highlight a new Biogeography arrangement for the six major clades of hornbills and reveal that three groups traditionally treated as Bucerotidae Classification genera (Tockus, Aceros, Penelopides) are non-monophyletic. In addition, two other genera (Anthracoceros, Phylogeny Ocyceros) were non-monophyletic in the mtDNA gene tree. Our findings resolve some longstanding prob- Systematics lems in hornbill systematics, including the placement of ‘Penelopides exharatus’ (embedded in Aceros) and ‘Tockus hartlaubi’ (sister to Tropicranus albocristatus). We also confirm that an Asiatic lineage (Berenicor- nis) is sister to a trio of Afrotropical genera (Tropicranus [including ‘Tockus hartlaubi’], Ceratogymna, Bycan- istes). We present a summary phylogeny as a robust basis for further studies of hornbill ecology, evolution and historical biogeography. Ó 2013 Published by Elsevier Inc. 1. Introduction Table A1), some of which (e.g. Anthracoceros montani, Aceros wal- deni) are close to extinction. Because of these attributes, hornbills The hornbills and ground-hornbills comprising the family Buc- are becoming increasingly prominent as study systems in ecology erotidae are charismatic land-birds that have long been the focus (e.g. Holbrook and Smith, 2000; Holbrook et al., 2002; Kitamura, of research attention. Amongst evolutionary biologists, they are 2011) and conservation biology (e.g. Sethi and Howe, 2009; Lenz well known for their elaborate bill casques, cooperative breeding et al., 2011). systems, and the remarkable strategy of self-incarceration, the fe- The evolutionary history of the family has received less atten- males of many species sealing themselves into tree-holes for sev- tion, although the basic outline of hornbill systematics is now well eral weeks by plastering the entrances of their nest cavities established. Several anatomical features—including fused upper (Moreau, 1934; Kemp, 2001). Amongst ecologists, the vital contri- vertebrae (atlas and axis), long flattened upper eyelashes, and bution of hornbills as long-distance seed dispersers has led to them bilobular kidneys (Kemp, 2001)—are unique to hornbills, suggest- being viewed as keystone species (Trail, 2007), and implicated in ing that they form a relatively distinct clade. Their apparent diver- the historical expansion of Palaeotropical forests (Viseshakul gence from related families has led to some authors separating et al., 2011). They also play an important role in tribal cultures them into their own order, Bucerotiformes (e.g. Kemp, 1995). from South Africa to East Asia (Bennett et al., 1997). Unfortunately, Relationships within the family have been estimated on the basis as a corollary of their large size and need for extensive foraging of a qualitative assessment of characters such as phenotype, vocal- areas, many hornbills are highly sensitive to hunting and habitat izations and breeding behavior (Kemp and Crowe, 1985; Kemp, fragmentation, making them one of the most threatened compo- 1988), culminating in the publication of a consensus cladogram nents of tropical ecosystems (Kinnaird and O’Brien, 2007). Over a built using 26 such characters (Kemp, 1995). This tree has proved third of hornbill species are considered to be of conservation con- to be a useful framework for hornbill systematics, particularly cern globally, including 62% (20/32) of Asiatic species (see because its coverage (54 of 61 taxa) is reasonably comprehensive (Kinnaird and O’Brien, 2007). ⇑ Corresponding author. Fax: +44 (0)1865 271168. Several quantitative assessments of hornbill relationships have E-mail address: [email protected] (J.A. Tobias). been undertaken using molecular techniques, but all have been 1055-7903/$ - see front matter Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ympev.2013.02.012 Please cite this article in press as: Gonzalez, J.-C.T., et al. A comprehensive molecular phylogeny for the hornbills (Aves: Bucerotidae). Mol. Phylogenet. Evol. (2013), http://dx.doi.org/10.1016/j.ympev.2013.02.012 2 J.-C.T. Gonzalez et al. / Molecular Phylogenetics and Evolution xxx (2013) xxx–xxx based on highly incomplete datasets. The first steps involved karyo- kempi and T. damarensis) were added by downloading sequences logical studies focused on seven species (Belterman and de Boer, from GenBank (see Table 1). Direct sampling involved the extrac- 1984, 1990), and a 17-taxa tree constructed using DNA–DNA tion of genomic DNA from contemporary material (i.e. captive hybridization (Sibley and Ahlquist, 1990). These were followed by and wild-trapped individuals) and historical material (i.e. museum phylogenetic approaches generating partial cytochrome b (cyt b) samples collected over the last 160 years). Types and sources of sequences (189 bp) for 11 taxa (Morin et al., 1994; Srikwan and material are given in Table B1. Woodruff, 1998). The results of these analyses agreed on the place- For contemporary material, DNA was extracted from molted ment of the genus Bucorvus (ground-hornbills) as a highly divergent flight feathers and plucked pin-feathers (the latter preserved in sister clade to all other hornbills, perhaps warranting designation as 70–90% ethanol) following Morin et al. (1994). For historical mate- a separate family (Kemp, 1995). They also suggested that Tockus rial, we extracted DNA from toe-pads following Mundy et al. hornbills were an ancient lineage sharing a common ancestor with (1997). Historical samples were processed in a separate laboratory the rest of the Bucerotidae. Further sequencing led to expanded following standard extraction and polymerase chain reaction (PCR) mitochondrial DNA (mtDNA) phylogenies for hornbills, first includ- controls, and using stringent protocols to avoid cross-contamina- ing 22 species (Hübner et al., 2003), then more recently all 34 spe- tion with modern avian DNA (Lerner and Mindell, 2005). In all cies for which molecular data are currently available, i.e. 56% of cases, short fragments of genes (200–500 bp) were amplified to species diversity in the family (Viseshakul et al., 2011). improve recovery of degraded DNA. Amplification was mainly con- The phylogeny published by Viseshakul et al. (2011) provided ducted using a set of 17 newly designed primers developed from the most informative assessment of the historical relationships be- existing GenBank sequences using the program PRIMER3 v.04.0 tween major clades of hornbills, particularly as it contained at least (Rozen and Skaletsky, 2000). We also used two previously pub- one member from each genus. However, many nodes had low con- lished primers (Shapiro and Dumbacher, 2001). For full details of fidence in terms of bootstrap values, presumably because tree primers see Table S1 (Supplementary material). topology was based on variation in one mitochondrial gene (cyt PCR amplification was performed using pre-optimized Qiagen b) across a limited set of species. Moreover, most species were only HotStarTaq Master Mix in ABI 2720 thermal cyclers (Applied Bio- represented by partial sequences (400–1043 bp), whereas com- systems, Foster City, CA) and purified using the Qiagen Mini-elute plete gene sequences (1143 bp) were only available for 15 species, kit. The PCR profile followed for AK1 intron 5 was a touchdown of i.e. 25% of species diversity in the family. Viseshakul et al. (2011) 15 min at 95 °C, followed by 45 cycles of 95 °C for 45 s, 54 °C for noted that a fuller understanding of phylogenetic relationships 60 s, and 72 °C for 60 s, and a final extension phase at 72 °C for within the clade, as well as a better grasp of the timing of evolu- 10 min. The equivalent profile for cyt b was a touchdown of tionary events, could only be resolved by more comprehensive 15 min at 95 °C, followed by 45 cycles of 94 °C for 30 s, 52 °C for sampling of lineages and loci. 45 s, and 72 °C for 60 s, and a final extension phase at 72 °C for To address this issue we conducted the first complete species- 10 min. Cycle sequencing