ORIGINAL ARTICLE. Stephen A. Smith 1,2 *

Transcription

1 Journal of Biogeography (J. Biogeogr.) (2009) 36, ORIGINAL ARTICLE Taking into account phylogenetic and divergence-time uncertainty in a parametric biogeographical analysis of the Northern Hemisphere plant clade Caprifolieae Stephen A. Smith 1,2 * 1 Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, USA and 2 National Evolutionary Synthesis Center, Durham, NC, USA ABSTRACT Aim To examine the biogeographical history of the angiosperm clade Caprifolieae (Caprifoliaceae) using parametric biogeographical reconstruction methods. The existing parametric method was extended to evaluate biogeographical reconstructions over the distribution of dated phylogenies. This method provides a framework for reconstructing large-scale biogeography with parametric methods, while accounting for uncertainty in the phylogenetic relationships and divergence time. Location Asia, Europe and North America. Methods The biogeographical history of the major lineages of Caprifolieae was reconstructed over the posterior distribution of dated trees generated from Bayesian divergence-time analyses. Results from a model with no geological constraints were compared with those from one where movement is disallowed across the North Atlantic after the Eocene. The most plausible scenarios were segregated at each node to test whether particular scenarios were reconstructed for particular divergence times. The parametric biogeographical method was also extended to estimate connectivity between areas so that the probability of dispersal between the major areas of the Northern Hemisphere could be explored. Results Phylogenetic results for Caprifolieae agreed with previous estimates using smaller sampling, but uncertainty remained despite efforts to resolve the relationships of the four genera within this clade using multiple markers. In addition to topological uncertainty, there were few fossils available for calibrations, resulting in large confidence intervals for divergence times. Divergence-time analyses put the diversification of Caprifolieae at between 36 and 51 Ma and showed that Caprifolieae probably originated in Asia, with multiple movements into Europe and western and eastern North America. *Correspondence: Stephen A. Smith, National Evolutionary Synthesis Center, 2024 W. Main Street, Suite A200, Durham, NC 27705, USA. samsmith@nescent.org Main conclusions Newly developed parametric methods for biogeographical reconstruction incorporate more data and better models. Here, the parametric biogeographical reconstruction method has been extended to allow for topological and divergence-time uncertainty. The analyses of Caprifolieae demonstrated that biogeographical hypotheses can be explored even where there are large confidence intervals on divergence times and uncertainty in topology. These results add to the growing evidence that Asia was an important source of Northern Hemisphere diversity throughout the Cenozoic. Keywords Bayesian, biogeography, Caprifolieae, divergence times, Lonicera, parametric methods, phylogenetics doi: /j x

2 Uncertainty and parametric biogeographical reconstruction INTRODUCTION Recent synthetic analyses have identified and explored major patterns of plant and animal movements around the Northern Hemisphere (Wen, 1999; Sanmartín et al., 2001; Donoghue & Smith, 2004). For plants, these analyses have supported a general pattern of movement from Asia to North America that was facilitated by asymmetric dispersal routes. For example, movements across the Atlantic were less frequent after the Oligocene when connections were less viable (i.e. the North Atlantic Land Bridge was no longer accessible), while movements across Beringia were possible and common throughout the Tertiary. Geological evidence suggests that a complex interplay of a variety of factors was involved (e.g. light, temperature, moisture), including the physical connections that influenced the movement of plants in the Northern Hemisphere (Tiffney, 1985a,b; Tiffney & Manchester, 2001), but traditional biogeographical reconstruction methods and analyses have not emphasized times of events (Ronquist, 1997). Donoghue & Moore (2003) pointed out the possible pitfalls of inferring biogeographical scenarios without temporal information (i.e. pseudo-congruence), which is especially problematic for the Northern Hemisphere where connections varied over time (Tiffney & Manchester, 2001). While many phylogenetic and biogeographical studies have included some analysis of divergence times (e.g. Xiang et al., 2000; Donoghue et al., 2001a; Milne, 2004; Renner & Zhang, 2004; Eckert & Hall, 2006; Ickert-Bond & Wen, 2006; Zhou et al., 2006; Nie et al., 2008), these have not been incorporated explicitly into biogeographical analyses. Many studies have relied on fossil information (e.g. Xiang et al., 2004) but these were often hindered by the poor temporal resolution obtained from fossil evidence for estimating how ancestral species evolved and dispersed. There are few comprehensive biogeographical analyses that incorporate divergence times for Northern Hemisphere plant clades (but see Bell, 2007; Drummond, 2008; Gussarova et al., 2008; Xiang & Thomas, 2008). Recently developed parametric biogeographical methods (Ree et al., 2005; Ree & Smith, 2008) provide a powerful framework for testing biogeographical hypotheses. Unlike dispersal vicariance analyses (DIVA; Ronquist, 1997), these methods make use of divergence-time information. Moreover, the parametric method [as implemented in Lagrange (Ree & Smith, 2008)], allows comprehensive geographical connectivity models to be explored and tested (see the example in Ree & Smith, 2008). For example, geographical connectivity models can be fixed or estimated to evaluate different land bridge hypotheses. These new biogeographical methods require a fixed bifurcating phylogenetic tree with branch lengths proportional to time. Few species-level phylogenies are fully resolved (for Northern Hemisphere examples see Wen et al., 1998; Manos & Stanford, 2001; Winkworth & Donoghue, 2005; Xiang et al., 2004) and there are many sources of error inherent in divergence-time estimation, such as uncertainty in the placement of fossils and stratigraphic ranges (Marshall, 1997, 2008; Smith & Peterson, 2002). Recent advances in divergence-time estimation employ Bayesian methodology that allows for simultaneous incorporation of uncertainty in topology, rates of evolution and fossil age (Drummond et al., 2006; Drummond & Rambaut, 2007). A full Bayesian approach has yet to be developed for biogeographical reconstruction. Ideally, this would integrate over not only tree space but also dispersal and extinction parameters and uncertainty in the connectivity model. Nevertheless, maximum-likelihood biogeographical estimates can be conducted across the posterior distribution of Bayesian dated trees allowing for the incorporation of two major sources of uncertainty: topology and divergence times. Nylander et al. (2008) explored a similar approach with DIVA (Ronquist, 1997), but it does not utilize divergence-time estimates. As mentioned above, divergence times for many biogeographical questions are essential for correlating directions of movement with possible geological connections and events (Sanmartín et al., 2001; Donoghue & Moore, 2003; Donoghue & Smith, 2004; Sanmartín & Ronquist, 2004), making the parametric method of biogeographical reconstruction in some ways more appealing than DIVA. The parametric method also has the advantage of incorporating more speciation models, allowing for within-area speciation as well as between-area speciation (Ree et al., 2005). For this study, I extended the parametric biogeographical method to estimate: (1) reconstructions over the posterior distribution of dated phylogenies, and (2) probabilities of dispersal between areas. I focused on Caprifolieae (Caprifoliaceae; as in Donoghue et al., 2003), a clade consisting of four genera: Leycesteria (c. 8 species), Lonicera (c. 180 species), Symphoricarpos (c. 16 species) and Triosteum (c. 6 species), with Heptacodium (1 species) typically reconstructed as sister to these four (Bell et al., 2001; Donoghue et al., 2001a,b; Zhang et al., 2003; Theis et al., 2008). Caprifolieae is distributed throughout the Northern Hemisphere including western and eastern North America, Europe and Asia. This clade has been included in many phylogenetic studies using a sparse, representative sample of species (i.e. Bell et al., 2001; Donoghue et al., 2001a,b; Zhang et al., 2003); Lonicera (Theis et al., 2008) and Triosteum (Gould & Donoghue, 2000) have been examined with denser sampling. Previous studies have found the relationships of these genera hard to resolve, possibly reflecting an ancient rapid radiation (Bell & Donoghue, 2005). The biogeography of Caprifolieae has yet to be explored; nevertheless the clade exhibits interesting biogeographical problems. With the exception of Leycesteria, members of Caprifolieae contain classic biogeographical patterns including Asia North America disjunctions (Triosteum, Symphoricarpos) and western North America Europe disjunctions (e.g. Lonicera subgenus Caprifolium) with sister species distributed in non-adjacent areas. The group broadly demonstrates the typical Tertiary Northern Hemisphere plant pattern (Wen, 1999; Donoghue et al., 2001a; Donoghue & Smith, 2004). Biogeographical analysis of Caprifolieae would not only help to resolve the historical movements of this clade throughout the Cenozoic but would also demonstrate how Journal of Biogeography 36,

3 S. A. Smith other problematic clades, with poorly resolved relationships, might be analysed with reference to biogeography. Of particular interest in Caprifolieae are the routes of movement, which have been examined for Triosteum (Donoghue et al., 2001a); sampling has been insufficient to examine these questions in the other genera, especially Lonicera (Donoghue et al., 2003). In order properly to address these biogeographical problems in Caprifolieae, topological and divergence-time uncertainty must be incorporated. This may be achieved by extending the parametric biogeographical reconstruction method both (1) to allow for topological and temporal uncertainty, and (2) to examine connectivity scenarios by estimating probability of dispersal between areas. MATERIALS AND METHODS Sampling and sequences Caprifolieae (Dipsacales) consists of c. 210 species divided into four genera. For this study, 79 species of Lonicera, four species of Symphoricarpos, six species of Triosteum and two species of Leycesteria were sampled, and except for Symphoricarpos all major biogeographical regions have been represented with this sampling. For a previous study, the ndhf and trnl trnf chloroplast regions were sampled for an Asian species of Symphoricarpos (Symphoricarpos sinensis); however, this gene region is poorly represented in the other taxa and was therefore excluded (Zhang et al., 2003). Heptacodium, consisting of a single species, has frequently been estimated as sister to Caprifolieae (Bell et al., 2001; Smith & Donoghue, 2008; Winkworth et al., 2008) and all analyses here have been rooted along that branch. The largest group, Lonicera, is typically separated into two subgenera (Caprifolium, Lonicera), the largest of which, Lonicera subgenus Lonicera, is typically separated into four sections, Coeloxylosteum, Isoxylosteum, Nintooa and Isika (Rehder, 1903; Hara, 1983; Hsu & Wang, 1988), and a number of subsections. Each major group of Lonicera has been represented by the sampling strategy in this study and 32 Lonicera species were added to the species previously sampled by Theis et al. (2008). The DNA sequences for Caprifolieae are listed in Table 1. Fifty internal transcribed spacer (ITS) sequences and 56 trns trng sequences were added for Lonicera and Symphoricarpos to the sequences from the Theis et al. (2008) study (see Tables S1, S2). Genomic DNA was isolated from silica dried leaf tissue or herbarium samples using the Qiagen DNeasy plant kit (Qiagen, La Jolla, CA, USA). Standard primers and polymerase chain reaction (PCR) protocols were used for ITS and trns trng; procedures for trns trng are detailed in Shaw et al. (2005). Amplification products were purified using the QIAquick PCR purification kit (Qiagen). Automated sequencing reactions used the ABI PRISM BigDye terminator cycle sequencing ready reaction kit (ABI, Foster City, CA, USA) and were analysed using an ABI 3730 Genetic Analyzer (ABI). The new sequences were combined with sequences of atpb rbcl, matk, petn psbm, psbm trnd, rpob trnc, trns trng and ITS from previous studies (Gould & Donoghue, 2000; Theis et al., 2008). Phylogenetic analyses Data matrices were aligned using dialign (version 2.2; Morgenstern, 1999), and sites with at least 50% missing data were trimmed. The cleaned data matrices were then concatenated. All matrix manipulation employed Phyutility (Smith & Dunn, 2008). All phylogenetic analyses were conducted using Metropolis-coupled Markov chain Monte Carlo (MCMC) chains as implemented in MrBayes and all analyses employed a mixed model (version 3.1.2; Ronquist & Huelsenbeck, 2003; Altekar et al., 2004) with all gene regions partitioned and parameters between partitions unlinked. In each analysis, four chains were run for 10 7 generations, sampling every 10 3 generations. MrBayes analyses were replicated (n = 3) to verify convergence to the same topology. Convergence was also determined by examining effective sample size (ESS) and trace plots in Tracer (version 1.4; Rambaut & Drummond, 2007). Consensus trees (all compatible groups represented and 50% majority rule) were calculated on samples generated after generations. To examine the relationships between genera, additional analyses were conducted with MrBayes, while constraining each possible resolution of the four genera (30 total analyses, two for each of the 15 possible resolutions). This is in addition to the unconstrained MrBayes analyses discussed above. The Bayes factor was then computed between the harmonic mean of each run reported by MrBayes. The Bayes factor calculation comparing model one (M 1 ; in this case topology 1) and model two (M 2 ; in this case topology 2) is BF ¼ f ðxjm 1Þ f ðxjm 2 Þ where the data are denoted with x and f(x M i ) is the marginal likelihood of the model i (Kass & Raftery, 1995). The alternative models in this case are the alternative topological constraints, and the marginal likelihoods of all alternative topologies were compared with the marginal likelihood of the highest scoring topology. Typically, the significance of the Table 1 Statistics for aligned gene regions of Caprifolieae used for estimating the phylogeny and divergence times. atpb rbcl ITS petn psbm psbm trnd rpob trnc trns trng No. of sequences Sequence length (bp) Aligned length (bp) Journal of Biogeography 36,

4 Uncertainty and parametric biogeographical reconstruction difference between two models, in this case two trees, is determined using twice the log difference between two models. Values greater than 10 are considered highly significant and values less than 6 are considered of low significance. Fossil calibrations and divergence-time analyses Two macrofossils of Lonicera have been identified. Lonicera protojaponica is a fossilized leaf from the late Miocene ( Ma) Tatsumitoge Flora of Honshu, Japan. This fossil is considered similar to extant Lonicera gracilipes based on leaf shape and venation characters (Ozaki, 1980, 1991). Fossil seeds identified as Lonicera alpigena were found in Germany and are considered to be from the Messinian to Zanclean ( Ma; Mai, 2001). Lonicera pollen is known from throughout the Eocene (Graham, 1999); Symphoricarpos pollen is known from the late Oligocene ( Ma; Graham, 1999). Given the inability to place the microfossils to species within Lonicera or Symphoricarpos, I considered the split between Symphoricarpos and Lonicera to be distributed during the Eocene (lognormal prior with mean = 2.0 and SD = 0.6 set at 33.8 Ma; Graham, 1999). Lognormal priors are chosen to favour younger ages closer to the fossil age, but allowing for older ages if supported by the data. The split between L. gracilipes and its sister was considered to have occurred by 5.3 Ma (lognormal prior with mean = 0 and SD = 1 starting at 5.3 Ma; Ozaki, 1991). The split between L. alpigena and its sister was considered to have occurred by 3.6 Ma (lognormal prior with mean = 0 and SD = 1 starting at 3.6 Ma; Mai, 2001). To allow for uncertainty in divergence-time estimates because of topological and fossil uncertainty, the Bayesian divergence-time method implemented in beast (version 1.4.7; Drummond & Rambaut, 2007) was employed. The uncorrelated lognormal (UCLN; Drummond et al., 2006) model of rate evolution was chosen, as it does not require rates to be heritable. That is, molecular rates are not necessarily inherited from parent node to child node through the phylogeny. Most previous divergence-time estimates have assumed an autocorrelated model of evolution, including those for Dipsacales (Bell & Donoghue, 2005). The assumption of autocorrelation is problematic due to lineage-specific rate heterogeneity, making fast-evolving branches older and slowly-evolving branches younger. For plants, lineage-specific rate heterogeneity linked to life history may be widespread (Kay et al., 2006; Smith & Donoghue, 2008). These patterns of rate heterogeneity are especially apparent in clades that contain both woody (Lonicera, Symphoricarpos, Leycesteria) and herbaceous species (Triosteum). The Bayesian divergence-time analyses were conducted specifying prior distributions for the fossil nodes discussed above. beast does not employ a coupled MCMC (as in MrBayes), potentially making it more prone to getting stuck in local optima. Therefore, based on strong support in the MrBayes analyses (see Fig. 1b), each genus was constrained to be monophyletic. Convergence to the same posterior distribution was determined by replicating the beast analysis (n = 3), and then comparing posterior distributions of divergence times and parameters of the analyses. Each analysis was run for 10 7 generations, sampling every 10 3 generations. Resulting posterior distributions for parameter estimates were examined in Tracer (version 1.4; Rambaut & Drummond, 2007) and maximum credibility trees, representing the maximum a posteriori topology, were calculated after removing burn-in with TreeAnnotator (version 1.4.7). Biogeographical reconstructions In order to determine the broad scale geographical evolution of Caprifolieae, parametric likelihood analyses were conducted as implemented in Lagrange (version 2.0.1; Ree & Smith, 2008). For these analyses, each species was assigned to one or more of four areas: eastern North America (ena), western North America (wna), Europe (Eu) and Asia (As). Instead of reconstructing geographical ranges at every internal node, I focused on 12 nodes of particular interest, chosen because of the biogeographical pattern of extant species in the clade. Maximum-likelihood analyses were conducted on target nodes as in Ree & Smith (2008). With Lagrange, the connectivity between areas can be modelled extensively; here, two connectivity scenarios are considered. The first does not constrain movement between areas at any time. The second disallows movement across the North Atlantic after the Eocene (34 Ma; Tiffney & Manchester, 2001), making movements from western North America and eastern North America to Europe impossible after the Eocene. Although the parametric biogeographical reconstruction method can impose extremely complex biogeographical scenarios, the use of these constraints makes large simplifying assumptions that may be unnecessary. For example, how the development of grasslands in central North America influenced movements of North American Lonicera is unclear. Some Lonicera are adapted to drier climates and therefore may be more amenable to xeric areas, while others are more mesic, making the grasslands a biogeographical barrier. For the purposes of this study, only a simple scenario other than an unconstrained model was explored where the interruption of the North Atlantic Land Bridge would disallow easy movement across the Atlantic. At least 1500 reconstructions for each scenario were calculated over the posterior distribution of dated trees estimated from the beast analyses. For each of the more than 3000 analyses both dispersal and extinction were estimated to maximize the likelihood of the biogeographical scenario (as described in Ree & Smith, 2008). Estimating connectivity between areas Ree et al. (2005) and Ree & Smith (2008) describe models of biogeographical connectivity in which movement between areas, or the probability of dispersal between particular areas, can be modelled extensively. There may be differential rates of dispersal and movement between areas due to physical Journal of Biogeography 36,

5 S. A. Smith (a) (b) Figure 1 (a) Phylogenetic tree for Caprifolieae using atpb rbcl, ITS, petn psbm, psbm trnd, rpob trnc and trns trng gene regions produced from unconstrained Bayesian analyses showing all compatible groups. (b) Majority rule consensus (50%) tree from Bayesian phylogenetic analyses. Major clades are labelled. Sympho. = Symphoricarpos; Leycest. = Leycesteria. connectivity, wind patterns and other biotic and abiotic factors (Ree et al., 2005). For example, one of the scenarios tested above disallowed dispersal (i.e. making the probability of dispersal zero) between North America and Europe after the Eocene. In addition to setting these parameters, we may also estimate these parameters. Sanmartín et al. (2008) explore the estimation of the probability of dispersal between areas with extended nucleotide models on species in the Canary Islands Journal of Biogeography 36,

6 Uncertainty and parametric biogeographical reconstruction Their implementation, however, does not allow for the multitude of speciation models explored in Ree et al. (2005). Here, I extend the parametric biogeographical reconstruction method to estimate the probability of dispersal between areas. Therefore, instead of setting the parameter for probability of dispersal between areas, we can estimate the probability of dispersal between areas based on the data. As described in Ree & Smith (2008), these dispersal rates can be asymmetric. As with the reconstructions, this analysis was run over the posterior distribution of dated trees. RESULTS Phylogenetic analyses Statistics for the aligned gene regions can be found in Table 1. Phylogenetic analyses largely confirm previous analyses with different sampling (i.e. Gould & Donoghue, 2000; Bell et al., 2001; Donoghue et al., 2001b; Theis et al., 2008; see Fig. 1). All genera are found to be monophyletic, with the monophyly of Triosteum weakly supported (69% posterior probability). This may be at least partly due to Triosteum sinuatum having only an ITS sequence; data for all other gene regions are missing. Triosteum sinuatum is found to be sister to the other five species of Triosteum when it is placed within Triosteum. Uncertainty remains for relationships within Lonicera, and the Caprifolium subgenus of Lonicera is particularly poorly resolved. Bayes factors were calculated for all alternative placements of the four genera (Kass & Raftery, 1995; see Fig. 2). The bestsupported topology from the constrained analyses places Symphoricarpos as sister to Lonicera, then Triosteum as sister to that clade, then Leycesteria. However, three other topologies are not significantly different (as defined in Materials and Methods): (1) Symphoricarpos as sister to Lonicera, then Leycesteria as sister to that clade, then Triosteum; (2) Symphoricarpos as sister to Leycesteria then Lonicera as sister to that clade, then Triosteum; and (3) Symphoricarpos as sister to Leycesteria then Triosteum as sister to that clade, then Lonicera. The topology with Symphoricarpos as sister to Leycesteria then Lonicera as sister to that clade, then Triosteum as sister to that clade is the topology supported by the unconstrained MrBayes analyses (Fig. 1), and is not significantly different from the other topologies listed above. Divergence-time estimation The estimate of the maximum a posteriori topology calculated from the Bayesian divergence-time analyses is the same as that estimated by the unconstrained MrBayes analyses (see Fig. 2). The crown group of Lonicera is estimated to be Myr old and the Lonicera Caprifolium crown clade originated between 7 and 17 Ma. The crown group of Symphoricarpos is estimated to be 4 17 Ma, with the Leycesteria crown clade originating between 4 and 22 Ma. The split between Symphoricarpos + Leycesteria and Lonicera originated between 35 and 44 Ma. The crown clade Triosteum is estimated to be Ma old. The Caprifolieae crown originated between 36 and 51 Ma, and the Caprifolieae + Heptacodium clade originated between 37 and 77 Ma. I used two metrics to evaluate the appropriateness of assuming a model of uncorrelated rates of molecular evolution when estimating divergence times. First, I examined the covariance statistic, which measures the amount of autocorrelation. The distribution for these analyses centres on zero [P = ) with a 95% highest probability density (HPD) of )0.353 to 0.352], implying that there is little support for molecular rates to be inherited from parent to child nodes throughout the phylogeny. However, it is worth noting that this measure may be a poor indicator of autocorrelation of rates through the tree and has not yet been extensively examined. Second, I examined the coefficient of variation, which measures the proportion of the variation in rates surrounding the mean. The distribution of possible coefficients of variation is centred far from zero (0.637 with 95% HPD ), suggesting that rates vary more than 60% from the mean a result indicative of extreme rate heterogeneity that was specifically accounted for in the model. Biogeographical analyses Maximum-likelihood reconstructions for the more than 1500 analyses on the 12 major nodes estimated across the posterior distribution of dated phylogenies are presented in Fig. 3 (see the figure for speciation scenarios). Only results that were represented in at least 5% of the analyses are reported. Because ancestral geographical ranges were calculated over the posterior distribution of dated phylogenies with varying divergence times at each node, results are segregated by time to determine whether particular biogeographical scenarios were reconstructed only during certain times. Results for both models, unconstrained and disallowing dispersal across the North Atlantic after the Eocene, are presented (Fig. 3; for unconstrained results see Fig. S1 in Supporting Information). The root, the node separating Triosteum from Symphoricarpos + Leycesteria + Lonicera, and the node separating Symporicarpos + Leycesteria from Lonicera are all reconstructed with ancestral distributions in Asia under both geographical models. The crown Triosteum clade is reconstructed to be in eastern North America + Asia most frequently (Fig. 3, node 6; purple histogram), in Asia with slightly lower frequency (green histogram), and widespread between eastern North America + Asia with a within-area speciation in eastern North America (yellow histogram). There are differences in the divergence times for which each scenario is reconstructed. For example, the within-area speciation scenario is only reconstructed for more recent times (c Ma). The node separating Symphoricarpos and Leycesteria is reconstructed to be in Asia most frequently (Fig. 3, node 5; green histogram) and less frequently widespread between Asia and eastern North America (blue histogram) or widespread between Asia and western North America (purple histogram). Journal of Biogeography 36,

7 S. A. Smith Figure 2 Maximum clade credibility tree of Caprifolieae produced from Bayesian divergence-time analyses. Below the tree are the posterior distributions of heights for the root and crown clades of interest. Lonicera + Symph. is the crown height estimate for the most recent common ancestor of Lonicera and Symphoricarpos. Figure 3 Biogeographical reconstructions of Caprifolieae produced from reconstructing over the posterior distribution of dated phylogenies. Individual maximum-likelihood biogeographical reconstructions were conducted on individual trees sampled from the posterior distribution of dated trees. Uncertainty in reconstructions (i.e. different estimated maximum-likelihood ranges based on different samples pulled from the posterior distribution of dated phylogenies) at major nodes is shown on the left with histograms. Although the consensus topology is shown, reconstructions were conducted over the posterior distribution of trees, allowing for alternative topologies to be included. Individual histograms represent the frequency of recovering particular scenarios in individual maximum-likelihood analyses. The x-axis for histograms represents time (in Ma) and the globes show the biogeographical scenario reconstructed. Keys for histograms are shown on the left, coloured by the unique colour in the map. Lines also connect the reconstructed range with the relevant histogram. Within-area speciation scenarios are shown with two colours (only one of which is used to colour the relevant histogram). Lines above the histograms show the extent of the date range for reconstructions. This figure shows the results using the land bridge constraint: see Fig. S1 for unconstrained results. The results do not differ dramatically; for more information see text Journal of Biogeography 36,

8 Uncertainty and parametric biogeographical reconstruction Journal of Biogeography 36,

9 S. A. Smith (a) (b) Figure 4 (a) A graphical representation of the estimated probability of dispersal success between areas. (b) Estimates were conducted over the posterior distribution of dated phylogenies. Boxplots showing the uncertainty in estimates of the probability of dispersal success between areas. Light grey lines represent the extent of the outliers. The y-axis represents the estimated connectivity from the area on the x-axis to the area designated above the boxplot. Geographical reconstructions differ at different divergence times. All reconstructions overlap between 30 and 40 Ma, while Asia is reconstructed to older times and widespread reconstructions occur at younger times. The land bridge model, where the movement across the Atlantic is disallowed after the Eocene, reconstructs an ancestor between Asia and eastern North America with much lower frequency than the unrestricted model (Fig. S1). The crown clade of Symphoricarpos is reconstructed almost equally to be widespread between western North America and eastern North America (Fig. 3, node 4), with within-area speciation events occurring in eastern North America (orange histogram) or in western North America (yellow histogram). A narrower ancestral range is also reconstructed in just western North America with lower frequency (red histogram). There is little difference between the land bridge model and the unrestricted model. There is no discernible difference between the timing of the two widespread scenarios. However, the narrower ancestral range scenario occurs only at older (c Ma) times while the widespread scenario occurs at more recent times (c Ma). It should be noted that the analyses are missing the one Asian species (S. sinensis), which is likely to change the hypothesized ancestral range for crown Symphoricarpos. The crown clade of Lonicera is reconstructed, most frequently, to be distributed in Asia (Fig. 3, node 3; green histogram) and less frequently as widespread between Asia, Europe and western North America (blue histogram). The widespread ancestor is only reconstructed at more recent times (c Ma). The land bridge model reconstructs the widespread ancestor with much higher frequency than the unrestricted model (Fig. S1). Within Lonicera, the ancestor of subgenus Lonicera is reconstructed to be in Asia. Node 1, representing a well-supported clade distributed in western North America, Europe and Asia is reconstructed to be widespread in Europe and Asia (brown histogram) and with less frequency just in Asia (green histogram). The narrower range is only reconstructed at older times (c Ma). The crown clade of Lonicera subgenus Caprifolium has highly variable reconstructions (Fig. 3, node 2). Western North America is present in every reconstruction. A widespread ancestor between western North America and Europe and a widespread ancestor between western North America, Asia and Europe are the most frequently reconstructed scenarios. The land bridge model shows less variability, with only four reconstructions represented in at least 5% of analyses as compared to six in the unconstrained reconstructions. For the land bridge model, reconstructions in order from highest to lowest frequency are: (1) widespread Europe and western North America (red histogram); (2) widespread Europe, western North America, and Asia (blue histogram); (3) widespread Europe, Asia, western North America, with a within-area speciation in western North America (orange histogram); and (4) widespread across all areas (yellow histogram). The more widespread ancestral scenarios using the land bridge model are reconstructed only at more recent time intervals, c Ma as opposed to 5 25 Ma for the other scenarios; there is little discernible difference in the timing of the unconstrained reconstructions. Estimating connectivity between areas Estimates of the probabilities of dispersal between areas calculated over the posterior distribution of dated phylogenies are presented in Fig. 4. The highest probability of movement is found between adjacent areas in both directions: Eu + As, ena + wna. Movement probabilities between non-adjacent regions are asymmetric and only from the Old World to the New World: Eu to wna, As to ena. All estimates have high variance, with outliers falling far outside the mean values. DISCUSSION Phylogenetic results The phylogenetic results presented here do not differ dramatically from those presented in previous studies of Caprifolieae (see Bell et al., 2001; Theis et al., 2008). Specifically, there is uncertainty in many of the deeper nodes, corresponding to the 2332 Journal of Biogeography 36,

10 Uncertainty and parametric biogeographical reconstruction taxonomic genera, as well as internal nodes, especially within Lonicera. The two subgenera of Lonicera Lonicera and Lonicera Caprifolium, are well supported, but the four sections of Lonicera subgenus Lonicera are poorly supported. Theis et al. (2008) found support for the monophyly of sections Coeloxylosteum and Nintooa, but due either to increased sampling or the effects of missing data the monophyly of these sections is not supported here. Sections Isoxylosteum and Isika are not monophyletic in either study. Although there is still uncertainty as to the specific relationships of the genera, topology tests using the Bayes factor significantly lowered the range of possibilities with this dataset from 15 topologies to four. Specifically, four topologies have very similar harmonic means and insignificant Bayes factors (2 difference > 6; Kass & Raftery, 1995). In two topologies, Symphoricarpos is sister to Leycesteria and Triosteum is not sister to Lonicera. In the other two topologies, Symphoricarpos is sister to Lonicera and Triosteum is not sister to Leycesteria. There is at least one apparent geographical consequence of the alternative topologies. In all but one of these topologies where Lonicera is sister to the other genera the dominant geographical range of the early diverging clades is reconstructed as Asia. However, if Lonicera was supported with more data to be the sister group of the rest, a more widespread ancestor could be supported. It should be noted that the best-supported topology is different from the topology recovered by unconstrained MrBayes and beast analyses. However, the topology recovered by the unconstrained analyses was found to be insignificantly different from the highest scoring topology from Bayes factor analyses. Analyses conducted on the placement of the genera suggest that certain scenarios cannot be ruled out until more data are brought to bear on this problem. Despite topological uncertainty, interesting patterns are apparent with the resulting phylogeny. Specifically, it is evident that perfoliate leaves with tightly clustered terminal inflorescences probably evolved at least twice: once in Triosteum and once in Lonicera. The Lonicera subgenus Caprifolium has perfoliate leaves subtending the terminal inflorescences while the Lonicera subgenus has petiolate (non-perfoliate) leaves with paired axillary flowers. In parallel, T. sinuatum, Triosteum pinnatifidum, Triosteum himalayanum and Triosteum sp. (as identified by Gould & Donoghue, 2000) have perfoliate leaves with terminal inflorescence, while Triosteum perfoliatum, Triosteum angustifolium and Triosteum aurantiacum all have petiolate leaves with axillary flowers. The phylogenetic analyses illustrate an interesting molecular evolution pattern in the Caprifolium clade. Not only is there particularly poor resolution within the clade, suggesting a rapid radiation or lower mutation rate, but there are also numerous short branches within the clade and a long stem branch separating it from its common ancestor with subgenus Lonicera. Also, as noted above, it exhibits a unique leaf and inflorescence morphology for Lonicera; however, there is no reason to think that this morphology should be associated with different rates of molecular evolution leading to the extreme uncertainty and shorter branch lengths. Differences in population size and major fluctuations have also been suggested to affect rates of molecular evolution (Ohta, 1992; Weinreich, 2001) but the Caprifolium clade does not stand out as being exceptionally different in this regard. Further studies are required to resolve and explore the early evolution of the Caprifolium clade of Lonicera. Divergence-time estimates Divergence times for Caprifolieae have been estimated with a variety of methods with much more limited sampling by Bell & Donoghue (2005), who used Diplodipelta as a fossil calibration (Manchester & Donoghue, 1995). The results presented here differ only slightly from those presented by Bell & Donoghue (2005), despite the fact that the topology of Caprifolieae used by Bell & Donoghue (2005) differed from those described here. Bell & Donoghue (2005) found the clade Caprifolieae + Heptacodium to originate between 70 and 84 Ma with penalized likelihood. The results presented here differ only in including more recent dates (37 77 Ma) that do not fall outside the Bayesian estimates in Bell & Donoghue (2005; Ma). Crown Caprifolieae is estimated to be between 36 and 51 Ma as compared with 17 and 67 Ma in Bell & Donoghue (2005). Without more internal calibrations for Caprifolieae, it is unlikely that better divergence times will be obtained. The crown clade of Triosteum was estimated to be 32 Myr old (with 95% HPD Ma), and other than the root, has the highest interval for divergence-time uncertainty. At least two factors may be affecting the divergence-time estimation of this node. Across Caprifolieae, I detected significant rate heterogeneity, evidenced by both the UCLN and estimates for covariance statistic. Triosteum is the only herbaceous clade in Caprifolieae, and recent analyses have found high rate heterogeneity in herbs versus trees/shrubs (Kay et al., 2006; Smith & Donoghue, 2008). Smith & Donoghue (2008) found lineage-specific rate heterogeneity between Triosteum and Lonicera, which were sister clades in their maximum-likelihood analysis of Dipsacales. Not properly accounting for this rate difference could bias herbaceous, faster-evolving, clades to be older than more slowly evolving tree/shrub clades. The uncorrelated lognormal model used here (Drummond et al., 2006) should accommodate for lineage specific rate differences, but how well it does needs to be explored further. Another possible factor affecting the dating of this node is the long branch separating T. sinuatum from the remaining Triosteum species. Species sampling for this clade in these analyses is very good, but ITS is only available for T. sinuatum. Biogeographical reconstructions The backbone nodes of Caprifolieae + Heptacodium were reconstructed, with few exceptions, to be distributed in Asia, suggesting Asia as the origin for diversification of Caprifolieae during the Cenozoic. This general pattern has been well documented in other cases (summarized and see references within Wen, 1999; Qian & Ricklefs, 2000; Xiang et al., 2000; Journal of Biogeography 36,

11 S. A. Smith Xiang & Soltis, 2001; Donoghue & Smith, 2004; Xiang et al., 2004). Asia has been considered a source of diversity throughout the Tertiary for northern temperate groups, possibly due to it being less susceptible to extinction events or possibly due to increased diversification rates (Tiffney, 1985a; Graham, 1999; Wen, 1999; Xiang et al., 2004). By examining the timing of the maximum likelihood biogeographical reconstruction results at each node, we find that certain scenarios are reconstructed only during particular times. For Symphoricarpos, the widespread ranges are reconstructed only during more recent times (4 14 Ma) a particularly interesting pattern for a group of western and eastern North American species. The Rocky Mountains continued to uplift during this time, with resulting climate changes perhaps promoting speciation (Graham, 1999). Presumably, this geological event affected the speciation and diversification of this North American clade, while these reconstructions suggest that it was widespread across the barrier. An interesting pattern regarding the size of the reconstructed range is seen by temporally segregating the times in which reconstructions occur. Smaller ranges are often reconstructed in early times. This is seen in Symphoricarpos, Symphoricarpos + Leycesteria, Lonicera and Node 1 within Lonicera. This is probably due to the fact that when the node of interest has a longer branch there is more time for biogeographical events (dispersal and extinction) to occur. Specifically, nodes followed by long branches can have more restricted ranges as dispersal can occur along the long branch. Unlike parsimony event-based methods (DIVA), the parametric method can integrate the timing of nodes in biogeographical analyses, which allows for the identification of such patterns. Segregating the reconstructions at each node by divergence time also provides predictions for reconstructions should more accurate divergence times be estimated in future analyses. For example, in the case of Symphoricarpos, if the crown is eventually reconstructed to originate before 20 Ma then instead of a widespread ancestor, a narrow western North American ancestral reconstruction should be favoured. Of course, inclusion of the single Asian species of Symphoricarpos will also help resolve the ancestral distribution of Symphoricarpos. Numerous dispersals throughout the Tertiary are supported here, as has been observed in many other Northern Hemisphere plant clades (Wen, 1999; Donoghue et al., 2001a; Milne & Abbott, 2002; Donoghue & Smith, 2004; Xiang et al., 2004; Winkworth & Donoghue, 2005). Because the backbone of the phylogeny is generally reconstructed to be distributed in Asia, descendant nodes distributed either outside Asia or widespread require dispersals (and extinctions) along the stems. The earliest dispersals occur by the end of the Eocene with crown Triosteum and Symphoricarpos + Leycesteria, from Asia into North America in both cases. Dispersals that are more recent have occurred within Symphoricarpos within North America and the two major clades of Lonicera throughout the Northern Hemisphere. This suggests not a single dispersal around the Northern Hemisphere but multiple events influencing biogeographical patterns. Many of these dispersals resulted in intercontinental disjuncts; at least six of these are found in the Caprifolieae, including three Asia/ eastern North America and two western North America/Europe disjuncts. Disjunctions between North America and Europe, as in Lonicera subgenus Caprifolium and Lonicera subgenus Lonicera, suggest dispersal across the North Atlantic Land Bridge (Tiffney, 1985b). However, the divergence-time estimates post-date the availability of the land bridge (Fig. 2). By the Oligocene, the North Atlantic Land Bridge is considered mostly inaccessible and the relevant dispersals in the Caprifolieae appear to have occurred after the Oligocene Miocene boundary (Tiffney, 1985b; Tiffney & Manchester, 2001). Therefore, dispersal through Beringia and subsequent extinction in Asia may be more likely. A particularly interesting geographical pattern is seen in Lonicera subgenus Caprifolium. This clade is distributed throughout the Northern Hemisphere and is estimated to be of relatively recent origin (7 17 Ma) as compared with crown Lonicera subgenus Lonicera. The stem leading to the crown is mostly distributed within Asia, while the extant species live in Europe, Asia and eastern and western North America. This suggests either dispersal around the Northern Hemisphere, resulting in a widespread species that was subsequently broken up by a number of vicariance events, or multiple dispersal events around the Northern Hemisphere, leading the reconstruction method to support a widespread ancestor. Estimating connectivity between areas The parameter estimates reflect results found in other broadscale analyses (see Donoghue & Smith, 2004). The probability of dispersal in Caprifolieae is found to be high between adjacent areas (wna + ena and Eu + As) and between disjunct areas (ena + As and wna + Eu), but not between adjacent areas spanning the oceans (As + wna and Eu + ena). As mentioned above, there are extant European and western North American disjuncts in at least Lonicera subgenus Caprifolium and North American and Asian disjuncts in at least Triosteum. These and other disjunct distributions are probably driving the parameter estimates of movements between unconnected areas. In general, it is likely that the more recent patterns, found in Caprifolium and Triosteum, are driving the parameter estimates. These results are found to have high variance, an expected result considering the uncertainty in topology and divergence times and the large number of free parameters (n = 14). Another interesting pattern emerged from these analyses where disjunct dispersals are estimated to be asymmetrical in both cases from the Old World to the New World. Asymmetrical dispersal patterns between Asia and North America may result from the estimation of ancestral distributions within Asia reconstructed along the deeper nodes of the phylogeny, making dispersal out of Asia more likely than dispersal into Asia. It is unclear why dispersal between Europe and western North America did not occur equally in both directions. The results concerning parameter estimates should be considered preliminary for many reasons. First, the 2334 Journal of Biogeography 36,

12 Uncertainty and parametric biogeographical reconstruction biogeographical data can be thought of as a single observation for each species, as compared with thousands of nucleotide observations for phylogenetic analyses. The number of free parameters for these estimates is relatively high considering the level of data. Therefore these results can be considered to have a high error rate. Additionally, this type of analysis has not been extensively explored. Despite these possible shortcomings, the results are compelling and other datasets should be explored in this way. between areas. In the example of Caprifolieae, it is shown how even relatively unresolved phylogenies can be examined with parametric biogeographical methods to produce interpretable and meaningful results. In addition, it is demonstrated how parameter estimates of connectivity can help inform our understanding of routes of movements. The biogeographical reconstructions support origins within Asia with multiple dispersals around the Northern Hemisphere during the Cenozoic, many through Beringia. Phylogenetic uncertainty and reconstructions Uncertainties in the relationships of clades and divergencetime estimates are paramount in ancestral reconstructions, whether biogeographical, ecological or morphological. Here, despite large confidence intervals in topology and divergence time, there are robust results for a number of biogeographical reconstructions in Caprifolieae. Hypotheses can be focused by reconstructing particular nodes of interest instead of reconstructing every, possibly poorly resolved, node. For example, there is little uncertainty in the biogeographical results for the backbone of Caprifolieae, despite high uncertainty in the divergence times and topology. Parameter estimates, and the associated variance, provide interpretable results concerning movement between areas. Most importantly, the results presented here more accurately reflect our confidence (or lack thereof) in particular scenarios and results. Future analyses and developments Although these analyses of Caprifolieae demonstrate how to incorporate uncertainty into biogeographical analyses, a full Bayesian approach for biogeographical reconstruction should be pursued. This should allow for integrating over uncertainty in parameter estimates specific to biogeographical reconstructions (i.e. dispersal and extinction). Another exciting prospect is to conduct analyses estimating the probability of dispersal between areas on other clades with similar distribution patterns. As noted above, the parameter estimates here are likely to have high error rates due to the low data content. However, estimating dispersal across multiple clades will increase the number of data (Sanmartín et al., 2008), providing more accuracy in the estimates. In addition to tallying disjunction types, as has been done in previous analyses (see Sanmartín et al., 2001; Donoghue & Smith, 2004; Sanmartín & Ronquist, 2004), these estimates could be instrumental in identifying corridors and the timing of dispersals between areas. CONCLUSIONS In this study, a recently developed biogeographical reconstruction method is extended to perform reconstructions over the posterior distribution of dated phylogenies. The method is also extended to allow for estimating probabilities of dispersal ACKNOWLEDGEMENTS Valuable feedback was gained from the working group on Phytogeography of the Northern Hemisphere sponsored by NESCent (NSF EF ) and from M. Donoghue, J. Beaulieu, R. Ree, T. Near, M. Smith, J. Wen, A. Greenberg and two anonymous referees. John Lambshead was especially helpful in improving the manuscript. S.A.S. was partially supported by the Cyberinfrastructure for Phylogenetic Research (CIPRES) programme (NSF no. EF ) and by the National Evolutionary Synthesis Center (NESCent; NSF no. EF ). REFERENCES Altekar, G., Dwarkadas, S., Huelsenbeck, J.P. & Ronquist, F. (2004) Parallel Metropolis coupled Markov chain Monte Carlo for Bayesian phylogenetic inference. Bioinformatics, 20, Bell, C.D. (2007) Phylogenetic placement and biogeography of the North American species of Valerianella (Valerianaceae: Dipsacales) based on chloroplast and nuclear DNA. Molecular Phylogenetics and Evolution, 44, Bell, C.D. & Donoghue, M.J. (2005) Dating the Dipsacales: comparing models, genes, and evolutionary implications. American Journal of Botany, 92, Bell, C.D., Edwards, E.J., Kim, S. & Donoghue, M.J. (2001) Dipsacales phylogeny based on chloroplast DNA sequences. Harvard Papers in Botany, 6, Donoghue, M.J. & Moore, B.R. (2003) Toward an integrative historical biogeography. Integrative and Comparative Biology, 43, Donoghue, M.J. & Smith, S.A. (2004) Patterns in the assembly of temperate forests around the Northern Hemisphere. Philosophical Transactions of the Royal Society B: Biological Sciences, 359, Donoghue, M.J., Bell, C.D. & Li, J.H. (2001a) Phylogenetic patterns in Northern Hemisphere plant geography. International Journal of Plant Sciences, 162, S41 S52. Donoghue, M.J., Eriksson, T., Reeves, P.A. & Olmstead, R.G. (2001b) Phylogeny and phylogenetic taxonomy of Dipsacales, with special reference to Sinadoxa and Tetradoxa (Adoxaceae). Harvard Papers in Botany, 6, Donoghue, M.J., Bell, C.D. & Winkworth, R.C. (2003) The evolution of reproductive characters in Dipsacales. International Journal of Plant Sciences, 164, S453 S464. Journal of Biogeography 36,