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Biological Journal of the Linnean Society The evolution of Australasian agamid lizards based on nuclear and mitochondrial genes, and the affinities of the thorny devil (Moloch horridus). For Peer Review Journal: Biological Journal of the Linnean Society Manuscript ID: BJLS-0023 Manuscript Type: Original Manuscript Date Submitted by the 26-Jun-2006 Author: Complete List of Authors: Hugall, Andrew; University of Adelaide, Earth and Environmental Sciences Foster, Ralph; South Australian Museum Lee, Michael; South Australian Museum Hutchinson, Mark; South Australian Museum agamidae, phylogeny, partition support, congruence, convergence, Keywords: molecular clock, aridification Biological Journal of the Linnean Society Page 1 of 33 Biological Journal of the Linnean Society 1 2 3 4 The evolution of Australasian agamid lizards based on nuclear and 5 mitochondrial genes, and the affinities of the thorny devil (Moloch 6 horridus). 7 8 9 A.F. Hugall1*, R. Foster2, M. Hutchinson2 and M.S.Y. Lee1,2 10 11 12 13 1 School of Earth and Environmental Sciences, University of Adelaide, SA 5005 14 2 15 Natural Sciences Building, South Australian Museum, Adelaide, SA 5000, Australia 16 17 *Corresponding Author, E-mail [email protected], Fax +61 8 8303 4364 18 19 20 For Peer Review 21 Running title: Austral Agamid Phylogeny 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Biological Journal of the Linnean Society Biological Journal of the Linnean Society Page 2 of 33 Austral Agamid Phylogeny 2 1 2 3 ABSTRACT 4 5 6 7 Recent mtDNA phylogenies of Australasian agamid lizards are highly incongruent with 8 existing morphological views. To resolve this discrepancy we sequenced two nuclear gene 9 10 regions, c-mos and BDNF. These were highly concordant with each other and the mtDNA 11 phylogeny, but not morphology. A combined molecular analysis reveals substantial hidden 12 13 support (additional phylogenetic signal that emerges only when the data sets interact in a 14 15 combined analysis), and produces a well-resolved tree that indicates extensive 16 morphological homoplasy, with many genera emerging as non-monophyletic (Amphibolurus, 17 18 Rankinia, Ctenophorus, Physignathus, Diporiphora). The water and forest dragons 19 20 (Physignathus and HypsilurusFor) formPeer a paraphyletic Review basal assemblage to the more derived 21 Australian forms such as Amphibolurus and Ctenophorus, which include almost all the xeric 22 23 forms. However, thorny devil Moloch horridus is a basal lineage and not closely related to the 24 other arid taxa. Tree topology, inferred divergence dates, palaogeographic and 25 26 palaeoclimatic data are all consistent with Miocene immigration into Australia from the north 27 28 via mesic forest ecomorphs, followed by initial diversification in mesic habitats before 29 radiation into xeric habitats driven by increasing aridity. 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Biological Journal of the Linnean Society Page 3 of 33 Biological Journal of the Linnean Society Austral Agamid Phylogeny 3 1 2 3 INTRODUCTION 4 5 6 7 The seventy species of agamid lizards ("dragons") are one of the most diverse and 8 prominent components of the Australian reptile fauna. Their origins and phylogenetic 9 10 relationships have been investigated using information from anatomy (Moody 1980; Witten 11 1982), cytogenetics (Witten 1983) and molecules (Macey et al. 2000; Honda et al. 2000; 12 13 Schulte, Melville & Larson 2003; Hugall and Lee 2004). The most detailed phylogeny is 14 15 based on a parsimony analysis of a long (~2000bp) mitochondrial segment spanning the 16 ND1 to COI genes, sampled for most species (Schulte et al. 2003). This amount of sequence 17 18 data provided a well-resolved phylogeny, corroborating some nodes previously proposed 19 20 based on morphology Fore.g. monophyly Peer of Pogona Review and Tympanocryptis, mesic forms 21 (Hypsilurus and Physignathus) basal to the core xeric radiation. Nevertheless, many of the 22 23 nodes retrieved were surprising, conflicting strongly with previous morphological 24 assessments. The lack of support for some generic arrangements could have been 25 26 expected, given that the taxonomic framework used for Australian agamids has been based 27 28 largely on phenetic comparisons (e.g. Storr 1982) rather than on attempts to recover 29 phylogenetic relationships. Even so, it was notable how the phylogeny of Schulte et al. 30 31 (2003) separated some species that are phenotypically very similar, and grouped others that 32 33 are strongly divergent. Many morphologically cohesive genera emerged as paraphyletic 34 (Ctenophorus, Diporiphora) or polyphyletic (Rankinia, Amphibolurus, Physignathus). The 35 36 basal position of the ground-dwelling, xeric Moloch alongside arboreal rainforest forms 37 38 probably the most unexpected result; the morphological specialisations of this genus are 39 extreme and well documented, but no one had doubted that it was a member of the 40 41 Australian arid zone radiation (Moody 1980, Greer 1989). Finally, relationships within the 42 diverse genus Ctenophorus were surprising, with none of the three ecomorphs (burrowing, 43 44 rock and vegetation dwelling) emerging as monophyletic (though monophyly of the latter two 45 46 ecomorphs could not be rejected: Melville, Schulte & Larson 2001). The new data on 47 phylogenetic relationships implies considerable homoplasy of body form and ecology, which 48 49 would make Australian agamids an especially useful group in which to study adaptive 50 51 changes (Melville et al. 2001; 2006). 52 It would be desirable to test the mtDNA phylogeny using independent molecular data 53 54 sets, because 1) any single locus is susceptible to stochastic (e.g. lineage sorting) and 55 systematic errors, 2) high levels of mtDNA divergence may compromise phylogenetic signal, 56 57 (3) parsimony analysis has been argued to be inferior to model-based likelihood and 58 59 Bayesian methods, especially for fast-evolving sequences with multiple hits, (4) there were 60 many surprising mtDNA clades retrieved. Here, we investigate the phylogeny of Australian agamids based on the full coding sequence of two nuclear genes: brain-derived neurotrophic Biological Journal of the Linnean Society Biological Journal of the Linnean Society Page 4 of 33 Austral Agamid Phylogeny 4 1 2 3 factor (BDNF) and the oncogene c-mos. This information is combined with mtDNA 4 5 sequences and analysed using parsimony, maximum likelihood and Bayesian methods. The 6 7 new nuclear data should either confirm or challenge the surprising mtDNA results mentioned 8 above. Finally, given the controversy over the timing of the Australian radiation (Schulte et al. 9 10 2003; Hugall and Lee 2004), we attempt to put a molecular time frame on it, and investigate 11 differences in divergence date estimates based on mtDNA (previously used: Melville et al. 12 13 2001, Schulte et al. 2003), the new nuclear sequences, and the combined data. 14 15 16 MATERIALS AND METHODS 17 18 19 20 Taxon sampling For Peer Review 21 22 23 The mtDNA study (Schulte et al. 2003) sequenced nearly every species of Australian 24 agamid. A subset of these species, and overseas (outgroup) taxa, were chosen for nuclear 25 26 analysis based on the following criteria. All Australian genera (except for the rare 27 28 Cryptagama, for which no adequately preserved tissues are available) were sampled, with 29 multiple exemplars for diverse or problematic (potentially non-monophyletic) genera. For 30 31 instance, the mtDNA suggested that Diporiphora consisted of three clades, and exemplars 32 33 from all three clades were chosen. All unusual intrageneric relationships (see above) 34 retrieved by the mtDNA data were also tested by the species sampling. For example, within 35 36 Ctenophorus, the mtDNA suggested that burrowing, sand ecomorph (reticulatus group) 37 38 consisted of four separate lineages, and exemplars from each lineage (C. gibba, clayi, 39 nuchalis and pictus) were thus included. 40 41 For five species (Moloch horridus, Physignathus cocincinus, P. lesueurii, 42 Ctenophorus cristatus, Amphibolurus muricatus), two specimens each were sequenced for 43 44 the nuclear genes: all these species were monophyletic with respect to the other 47 taxa 45 46 considered here. Finally, at least nine taxa were almost certainly heterozygote for the c-mos, 47 judging by clear double peak signal in both strands. Similarly, five taxa appeared 48 49 heterozygote for BDNF. Nine of these heterozygotes involved a single polymorphic site, one 50 51 three sites, three two sites, and one (Diporiphora albilabris c-mos) with seven sites. 52 However, in all cases, arbitrarily resolved possible alleles all were monophyletic with respect 53 54 to the other 47 taxa. Only one sequence per species was used in the final analyses and 55 polymorphic sites scored as ambiguous using the IUPAC code. 56 57 Table 1 lists museum numbers and localities of specimens sampled as well as 58 59 GenBank accession numbers of new sequences. For consistency, whenever possible we 60 used the same specimens sampled in the mitochondrial studies (Macey et al. 2000; Schulte et al. 2003) and/or a previous more limited c-mos study (see Hugall and Lee 2004). For three Biological Journal of the Linnean Society Page 5 of 33 Biological Journal of the Linnean Society Austral Agamid Phylogeny 5 1 2 3 of the outgroups, the combined data is a composite of one species for the nuclear data and 4 5 another species for the mtDNA data, due to the lack of suitable specimens. These have been 6 7 labeled "spp" in the combined data trees (Gonocephalus spp = G. grandis for mtDNA and G. 8 kuhlii for nuclear DNA; Uromastyx spp = U.