Biol. Lett. the deep divergences (Livezey & Zusi 2001; Cracraft doi:10.1098/rsbl.2006.0523 et al. 2004; Fain & Houde 2004). DNA sequence Published online data have begun to clarify interfamily relationships for a handful of higher level groups such as some aquatic birds (van Tuinen et al. 2001), ‘higher land birds’ Diversification of Neoaves: (Johansson et al. 2001; Mayr et al. 2003), shorebirds (Ericson et al. 2003; Paton et al. 2003) and passerines integration of molecular (Barker et al. 2004). Recent analyses of morphological sequence data and fossils and molecular data support a sister group relationship between Galloanseres (land- and waterfowl) and all Per G. P. Ericson1,*, Cajsa L. Anderson2, other neognathous birds, the Neoaves (Livezey & Tom Britton3, Andrzej Elzanowski4, Zusi 2001; Mayr & Clarke 2003; Cracraft et al. 2004; Ulf S. Johansson5, Mari Ka¨llersjo¨ 1, Fain & Houde 2004). However, there are no Jan I. Ohlson1,6, Thomas J. Parsons7, hypotheses concerning the most basal neoavian diver- Dario Zuccon1 and Gerald Mayr8 gences, except for the proposed division of the group into Metaves and Coronaves based on an analysis of 1Department of Vertebrate Zoology and Molecular Systematics Laboratory, Swedish Museum of Natural History, PO Box 50007, b-fibrinogen sequence data (Fain & Houde 2004). 10405 Stockholm, Sweden Molecular clock analyses have suggested that the 2Department of Systematic Botany, Evolutionary Biology Centre, earliest diversification of Neoaves had already University of Uppsala, Norbyva¨gen 18D, 752 36 Uppsala, Sweden 3Department of Mathematics, and 6Department of Zoology, occurred in the Cretaceous (Hedges et al. 1996; University of Stockholm, 106 91 Stockholm, Sweden Cooper & Penny 1997; Cracraft 2001; van Tuinen & 4Institute of Zoology, University of Wroclaw, 21 Sienkiewicz Street, Hedges 2001). However, there are few neoavian 50335 Wroclaw, Poland fossils from the Cretaceous (Hope 2002; Feduccia 5DST/NRF Centre of Excellence at the Percy Fitzpatrick Institute, Evolutionary Genomics Group, Department of Botany and Zoology, 2003) and instead the palaeontological record University of Stellenbosch, Private Bag XI, Maiteland 7602, suggests that only a few neoavian lineages existed at South Africa the end of the Cretaceous, 65 Myr ago (Feduccia 7 International Commission on Missing Persons, Alipasˇina 45 A, 2003). The considerable diversity of stem group 71000 Sarajevo, Bosnia 8Forschungsinstitut Senckenberg, Sektion fu¨r Ornithologie, representatives of modern neoavian taxa, which is Senckenberganlage 25, 60325 Frankfurt am Main, Germany evident in the Early Eocene 50 Myr ago (Mayr 2005), *Author for correspondence ( [email protected]). would thus result from a rapid diversification of taxa, Patterns of diversification and timing of which filled the many vacant ecological niches after evolution within Neoaves, which includes almost the K/T boundary (Feduccia 2003). There is an 95% of all bird species, are virtually unknown. apparent conflict between earlier molecular datings On the other hand, molecular data consistently and the palaeontological record—but is this conflict indicate a Cretaceous origin of many neoavian real? The molecular dating methods must be correctly lineages and the fossil record seems to support calibrated to yield reliable data, and this has not an Early Tertiary diversification. Here, we previously been done in studies including Neoaves. present the first well-resolved molecular phylo- Since all Cretaceous fossils of neornithine birds are geny for Neoaves, together with divergence time estimates calibrated with a large number of very fragmentary (Hope 2002) and their identification stratigraphically and phylogenetically well- is often uncertain (Hope 2002), most calibrations documented fossils. Our study defines several have so far used a calculated age for the split between well-supported clades within Neoaves. The cali- galliforms and anseriforms (90 Myr ago) which is in bration results suggest that Neoaves, after an turn based on the diapsid/synapsid split age at initial split from Galloanseres in Mid-Cretac- 310 Myr ago (Hedges et al. 1996). However, Graur & eous, diversified around or soon after the K/T Martin (2004) have argued convincingly that this boundary. Our results thus do not contradict estimate is not reliable, and nor are any of the palaeontological data and show that there is no calibration points that are based on it. Here, we solid molecular evidence for an extensive pre- employ an alternative strategy and use multiple fossils Tertiary radiation of Neoaves. of more recent neoavian groups as internal calibration Keywords: Neoaves; phylogeny; nuclear DNA; points in order to test the different diversification fossils; molecular clock; divergence times models suggested by Penny & Phillips (2004). 1. INTRODUCTION 2. MATERIAL AND METHODS Birds are used as model organisms in many fields Traditional classification recognizes 145 families in Neoaves of biology, and the lack of a thorough understanding (Morony et al. 1975). We obtained genomic DNA from blood or tissue samples of 87 neoavian species representing 75 families. of their systematics has often compromised interpre- Charadriiformes (shorebirds and allies, 19 families in total) and tations of experiments and observations. The Passeriformes (passerines, 57 families in total), which have been DNA–DNA hybridization studies of Sibley & Ahl- shown to be monophyletic (Ericson et al. 2002, 2003; Paton et al. 2003; Barker et al. 2004), are represented by four and two families, quist (1990) have repeatedly been criticized for respectively. At least one genus was sampled from the remaining methodological reasons (Harshman 1994; Cracraft neoavian families. Two palaeognaths (Rhea and Apteryx), one et al. 2004), and the few cladistic analyses of Neoaves megapode and one screamer, were used as outgroups following the with dense taxon sampling show poor resolution of well-established hypothesis that Palaeognathae are the sister taxon of Neognathae, and that Galloanseres, in turn, are the sisters of The electronic supplementary material is available at http://dx.doi. Neoaves (Groth & Barrowclough 1999). The sample information org/10.1098/rsbl.2006.0523 or via http://www.journals.royalsoc.ac. and GenBank accession numbers are given in the electronic uk. supplementary material. Received 30 May 2006 This journal is q 2006 The Royal Society Accepted 6 July 2006 2 P. G. P. Ericson and others Diversification of Neoaves Accipitridae-Accipiter Accipitridae-Heterospizias 4 Accipitridae-Leptodon G Pandionidae Sagittariidae Momotidae Alcedinidae Todidae C Coraciidae Brachypteraciidae Meropidae Galbulidae Bucconidae Picidae A Indicatoridae Ramphastidae terrestrial 3 Capitonidae and arboreal B Upupidae Phoeniculidae groups Bucerotidae D Trogonidae E Coliidae F Tytonidae 2 Strigidae Cathartidae-Cathartes Cathartidae-Coragyps Leptosomidae Tyrannidae Laniidae Cacatuidae Psittacidae Falconidae-Falco Falconidae-Polyborus H Cariamidae Anhingidae 8 I Phalacrocoracidae J Sulidae K Fregatidae Ciconiidae-Jabiru Ciconiidae-Mycteria Ardeidae-Ardea Ardeidae-Tigrisoma Threskiornithidae-Harpiprion Threskiornithidae-Theristicus M Balaenicipitidae Scopidae Pelecanidae aquatic and Diomedeidae-Diomedea semi-aquatic 7 Diomedeidae-Phoebetria ‘Coronaves’ Pelecanoididae groups Procellariidae-Fulmarus Procellariidae-Puffinus Hydrobatidae L Spheniscidae X Gaviidae Aramidae V Gruidae 5 6 Psophiidae Rallidae-Aramides Rallidae-Laterallus N Heliornithidae Musophagidae Cuculidae-Cuculus Neoaves Cuculidae-Guira Otididae Turnicidae 9 Rynchopidae shorebirds O Jacanidae Charadriidae Apodidae P Hemiprocnidae Q Trochilidae nightbirds (except 1 Aegothelidae Podargidae owls), swifts and Nyctibiidae hummingbirds Steatornithidae Caprimulgidae ‘Metaves’ Eurypygidae Rhynochetidae R Pteroclididae Columbidae S Phoenicopteridae Podicipedidae Mesitornithidae-Mesitornis Mesitornithidae-Monias Opisthocomidae T Phaethontidae U Megapodiidae Anhimidae Apterygidae Rheidae Figure1. (Caption Opposite.) Biol. Lett. Diversification of Neoaves P. G. P. Ericson and others 3 Figure 1. (Opposite.) Family-level relationships within Neoaves estimated by Bayesian analysis of five nuclear genes (5007 nucleotide positions). Nodes that received a posterior probability value of less than 95% have been collapsed. Note that the branch lengths are not proportional to the number of nucleotide substitutions along each branch. Neoavian families fall into a few reciprocally monophyletic clades (coloured) that roughly correspond to ecological adaptations of extant taxa. Nodal numbers correspond to clades discussed in the text. Letters in boxes, referring to table 3 in the electronic supplementary material, indicate fossil calibration points. The aligned dataset consists of 5007 bp obtained from five gene Strongly supported is a previously unrecognized regions: c-myc (exon 3), RAG-1, myoglobin (intron 2), b-fibrinogen major clade (Johansson et al. 2001; Mayr et al. 2003), (intron 7) and ornithine decarboxylase (introns 6 and 7, along with the intercepting exon 7). For laboratory procedures, alignments, which includes diurnal birds of prey, seriemas, parrots selection of models for nucleotide substitutions, parsimony analysis and the ‘higher landbird assemblage’ (figure 1, and Bayesian analyses of individual gene regions, see electronic node 2). For the majority of families, traditionally supplementary material. Divergence times were estimated using two rate-smoothing included in the orders Coraciiformes and Piciformes methods,