1 Podocarp Evolution: A Molecular Phylogenetic Perspective Edward Biffin, John G. Conran, and Andrew J. Lowe ABSTRACT. Phylogenetic reconstructions of the relationships among extant taxa can be used to infer the nature of the processes that have generated contemporary patterns of biotic diversity. In this study, we present a molecular phylogenetic hypothesis for the conifer family Podocarpaceae based upon three DNA fragments that have been sampled for approximately 90 taxa. We use Bayesian relaxed- clock methods and four fossil con- straints to estimate divergence times among the lineages of Podocarpaceae. Our dating analyses suggest that although the family is old (Triassic–Jurassic), the extant species groups are of recent evolutionary origin (mid- to late Cenozoic), a pattern that could reflect a temporal increase in the rate lineage accumulation or, alternatively, a high and constant rate of extinction. Our data do not support the hypothesis that Podocarpaceae have diversified at a homogeneous rate, instead providing strong evidence for a three- to eightfold increase in diversification associated with the Podocarpoid– Dacrydioid clade, which radiated in the mid- to late Cretaceous to the earliest Cenozoic, around 60–94 mya. This group includes a predominance of taxa that develop broad leaves and/or leaf- like shoots and are distributed predominantly throughout the tropics. Tropical podocarps with broad leaves may have experienced reduced extinction and/or increased speciation coincident with the radiation of the angiosperms, the expansion of megathermal forests, and relatively stable tropical climates that were widespread through the Tertiary. Edward Biffin, John G. Conran, and Andrew J. INTRODUCTION Lowe, Australian Centre for Evolutionary Biol- ogy and Biodiversity, School of Earth and Envi- Patterns of species diversity reflect the balance of speciation and extinction ronmental Science, The University of Adelaide, Adelaide, South Australia 5005, Australia. Cor- over the evolutionary history of life. These, in turn, are parameters influenced respondence: E. Biffin, edward.biffin@adelaide by extrinsic factors, such as environmental condition and long- term processes .edu.au. of geological and climatic change, and intrinsic attributes of organisms, such Manuscript received 13 April 2010; accepted as morphological innovations that increase the propensity for speciation or 9 July 2010. reduce the risk of extinction. The key aims of evolutionary biologists are to 2 • SMITHSONIAN CONTRIBUTIONS TO botany explain patterns of diversity and, foremost, to determine phylogenetic data with null expectations (e.g., Rabosky whether there is evidence for significant heterogeneity in et al., 2007; Rabosky and Lovette, 2008). the “per lineage” patterns of diversity that require expla- The conifer family Podocarpaceae comprises approxi- nation (Sanderson and Wojciechowski, 1996; Magallón mately 173 species and 18 genera (Farjon, 1998) distributed and Sanderson, 2001; Davies et al., 2004; Moore and primarily in the Southern Hemisphere, although extending Donoghue, 2007; Rabosky et al., 2007). Phylogenetic northward as far as subtropical China and Japan and to reconstructions of evolutionary relationships provide an Mexico and the Caribbean (see Enright and Jaffré, this vol- indirect record of the speciation events that have led to ex- ume; Dalling et al., this volume). The podocarps have a rich tant species. Because evolutionary rates can be estimated fossil record that suggests an origin in the Triassic and a dis- from phylogenetic data, such reconstructions can help to tribution in both the Northern and Southern hemispheres elucidate the significance and drivers of biotic diversity through the Mesozoic, although by the Cenozoic the fossil patterns (Moore and Donoghue, 2007; Ricklefs, 2007). record of the family is overwhelmingly southern (Hill and Molecular phylogenetics has revolutionized the field Brodribb, 1999). Currently, the Podocarpaceae comprise of evolutionary biology. For instance, the molecular clock a majority of species-poor (Figure 1.1), range-restricted hypothesis predicts that the level of genetic divergence be- genera (e.g., Acmopyle, Lagarostrobos, Microcachrys, tween any two lineages will be proportional to the time Microstrobos, Saxegothaea) that are presumably relictual, since divergence from a most recent common ancestor. as evidenced by the fossil record, which indicates broader Therefore, using external calibrations (e.g., timing of vi- distributions and greater species diversities in the past (Hill cariance events, fossils of known age, and phylogenetic and Brodribb, 1999). Relatively few genera are species rich affinity) or known mutation rates, it is possible to esti- and widely distributed, although Podocarpus comprises ap- mate the age of all of the splits in a molecular phyloge- proximately 105 species (Figure 1.1) and occurs on all con- netic tree (which often comprise a majority of splits with tinents except Antarctica and Europe. no associated fossil data to directly estimate the age). “Nearest living relative” comparisons of Podocar- There has been justified criticism of the molecular clock paceae suggest the conservation of morphology, commu- hypothesis. In particular, there is strong evidence that in nity associations, and ecological response over evolutionary most lineages, the constancy of mutation rates in proteins time (Brodribb and Hill, 2004). From this perspective, the or DNA sequences cannot be assumed (e.g., Ayala, 1997; Podocarpaceae provide an outstanding opportunity to ex- Bromham and Penny, 2003). Recently developed meth- plore the influences of organism–environment interactions ods, which attempt to incorporate heterogeneity into phy- in the context of large-scale geological and climatic change logenetic analysis by specifying a model of rate variation in shaping patterns of extant distribution and diversity. For among lineages (referred to as relaxed- clock methods), example, the majority of podocarp species presently occur are believed to provide more realistic estimates of diver- within angiosperm- dominated humid forests. This pattern gence times in the absence of rate constancy (for recent is of considerable interest to paleobotanists, ecologists, and reviews see Bromham and Penny, 2003; Rutschmann, biogeographers (e.g., Brodribb and Hill, 2004; Brodribb 2006). Furthermore, there have been promising devel- and Feild, 2008), given that conifers in general have been opments in methods to account for uncertainty inherent considered less competitive than angiosperms in produc- in the fossil record; Bayesian methods, in particular, can tive environments (Bond, 1989). Using the ecophysiologi- incorporate fossil calibrations because parametric prior cal tolerances of extant species as representative of closely probability distributions make fewer assumptions (rela- allied extinct fossil taxa, it has been argued that charac- tive to “fixed”-point calibrations) concerning the nodal teristics such as leaf flattening and associated physiologies placement of a given fossil datum on a phylogenetic tree have promoted the persistence of Podocarpaceae in the face (Yang and Rannala, 2006; Sanders and Lee, 2007). With of angiosperm competition (Brodribb and Hill, 1997; Bro- improved confidence in hypotheses, there has been a di- dribb and Feild, 2008). However, within Australia, these versification of questions and associated methodologies characteristics may also be associated with range contrac- developed around molecular clock phylogenies. These tion and at least local extinction of several lineages as a include the examination of vicariance versus dispersal ex- consequence of increasing aridity in the Miocene– Pliocene planations for diversity patterns (e.g., Crisp and Cook, (Hill and Brodribb, 1999; Brodribb and Hill, 2004). 2007), the timing of evolutionary radiations and/or ex- Here we use molecular (DNA sequence) data, first, to tinctions and coincidence with environmental change assess phylogenetic relationships among Podocarpaceae (e.g., Davis et al., 2005), and estimation of the tempo of and, second, using a relaxed molecular clock approach, to diversification using a statistical framework to contrast estimate the timing of diversification events for the major number 95 • 3 FIGURE 1.1. Frequency distribution of species/genus for the Podocarpaceae (estimates according to Farjon, 1998). lineages. From this perspective we explore macroevolu- morphology (Hart, 1987; Kelch, 1997, 1998) and mo- tionary patterns within the family and specifically use the lecular (DNA sequence) data (Kelch, 1998, 2002; Conran dated molecular phylogeny to test whether it is necessary et al., 2000; Sinclair et al., 2002). A key focus of these to invoke among-lineage variation in diversification rates studies has been the assessment of relationships for clas- to explain the disparities in extant diversity among major sification; for instance, the status of Phyllocladus has been groups of Podocarpaceae. Our results indicate a highly controversial, although the elevation of this taxon to fam- significant shift in diversification rates corresponding to ily level as Phyllocladaceae (e.g., Page, 1990a; Bobrov et approximately the Cretaceous–Tertiary boundary. The al., 1999) is not supported by phylogenetic analyses to significance of this diversification rate shift is briefly ex- date (Conran et al., 2000; Quinn et al., 2002; Sinclair et plored in the context of the angiosperm radiation, bioge- al., 2002; Wagstaff,