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Old-New World and trans-African disjunctions of Thamnosma (Rutaceae): Intercontinental long-distance dispersal and local differentiation in the succulent biome

2010; Wiley; Volume: 98; Issue: 1 Linguagem: Inglês

10.3732/ajb.1000339

1537-2197

Mike Thiv, Timotheüs van der Niet, Frank Rutschmann, Mats Thulin, Thomas Brune, H. P. Linder,

Botanical Studies and Applications

American Journal of BotanyVolume 98, Issue 1 p. 76-87 Evolution and PhylogenyFree Access Old–New World and trans-African disjunctions of Thamnosma (Rutaceae): Intercontinental long-distance dispersal and local differentiation in the succulent biome† Mike Thiv, Corresponding Author Mike Thiv mike.thiv@smns-bw.de Botany Department, State Museum of Natural History Stuttgart, Rosenstein 1, 70191 Stuttgart, GermanyAuthor for correspondence (e-mail: mike.thiv@smns-bw.de)Search for more papers by this authorTimotheüs van der Niet, Timotheüs van der Niet School of Biological and Conservation Sciences, University of KwaZulu-Natal, Private Bag X01, Scottsville 3209, South AfricaSearch for more papers by this authorFrank Rutschmann, Frank Rutschmann Institute for Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich, SwitzerlandSearch for more papers by this authorMats Thulin, Mats Thulin Department of Systematic Biology, Uppsala University, Norbyvägen 18D 75236 Uppsala, SwedenSearch for more papers by this authorThomas Brune, Thomas Brune Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 25, 70593 Stuttgart, GermanySearch for more papers by this authorHans Peter Linder, Hans Peter Linder Institute for Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich, SwitzerlandSearch for more papers by this author Mike Thiv, Corresponding Author Mike Thiv mike.thiv@smns-bw.de Botany Department, State Museum of Natural History Stuttgart, Rosenstein 1, 70191 Stuttgart, GermanyAuthor for correspondence (e-mail: mike.thiv@smns-bw.de)Search for more papers by this authorTimotheüs van der Niet, Timotheüs van der Niet School of Biological and Conservation Sciences, University of KwaZulu-Natal, Private Bag X01, Scottsville 3209, South AfricaSearch for more papers by this authorFrank Rutschmann, Frank Rutschmann Institute for Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich, SwitzerlandSearch for more papers by this authorMats Thulin, Mats Thulin Department of Systematic Biology, Uppsala University, Norbyvägen 18D 75236 Uppsala, SwedenSearch for more papers by this authorThomas Brune, Thomas Brune Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 25, 70593 Stuttgart, GermanySearch for more papers by this authorHans Peter Linder, Hans Peter Linder Institute for Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich, SwitzerlandSearch for more papers by this author First published: 01 January 2011 https://doi.org/10.3732/ajb.1000339Citations: 31 † The authors would like to thank Jon Rebman (SDSU, USA) and Michael Denslow (Rancho Santa Ana Botanic Garden, USA) for providing plant material, Joachim Kadereit (Mainz, Germany) for helpful comments on the manuscript, and Mohamed Ali Hubaishan (AREA Research Station Mukalla), Ahmed Said Sulaiman (EPA Socotra), Said Masood Awad Al-Gareiri (Dept. Agriculture Socotra), and Mohamed El-Mashjary (EPA Sanaa, all Yemen) for support of the fieldwork in Socotra. The fieldwork was conducted as part of the BIOTA Yemen Project funded by the German Ministry for Research and Education (BMBF). The work was supported by grants of the German Research Foundation (DFG, Th830/1-1) and the Claraz-Schenkung (Switzerland) to M.T. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract • Premise of the study: The succulent biome is highly fragmented throughout the Old and New World. The resulting disjunctions on global and regional scales have been explained by various hypotheses. To evaluate these, we used Thamnosma, which is restricted to the succulent biome and has trans-Atlantic and trans-African disjunctions. Its three main distribution centers are in southern North America, southern and eastern Africa including Socotra. • Methods: We conducted parsimony, maximum likelihood, and Bayesian phylogenetic analyses based on chloroplast and nuclear sequence data. We applied molecular clock calculations using the programs BEAST and MULTIDIVTIME and biogeographic reconstructions using S-DIVA and Lagrange. • Key results: Our data indicate a weakly supported paraphyly of the New World species with respect to a palaeotropical lineage, which is further subdivided into a southern African and a Horn of Africa group. The disjunctions in Thamnosma are mostly dated to the Miocene. • Conclusions: We conclude that the Old–New World disjunction of Thamnosma is likely the result of long-distance dispersal. The Miocene closure of the arid corridor between southern and eastern Africa may have caused the split within the Old World lineage, thus making a vicariance explanation feasible. The colonization of Socotra is also due to long-distance dispersal. All recent Thamnosma species are part of the succulent biome, and the North American species may have been members of the arid Neogene Madro-Tertiary Geoflora. Phylogenetic niche conservatism, rare long-distance dispersal, and local differentiation account for the diversity among species of Thamnosma. 64 accounted for the diversification of legumes with a model that incorporated phylogenetic niche conservatism with local differentiation. The tendency of lineages to retain their ancestral ecological niche rather than adapting to new environments (phylogenetic niche conservatism) has has been demonstrated in diverse clades and regions (86; 15; 12). On the basis of phylogenetic niche conservatism, widely distributed clades should occupy similar habitats in remote areas, a pattern neatly demonstrated in the legumes (39; 64). The succulent biome, which is characterized by a climate with a long dry season, is found in relatively small fragments and isolated regions in both South and North America, in a large region in SW Africa, and from Tanzania to Pakistan. The predominant growth forms in these climates are usually evergreen, sclerophyllous, nonfire-adapted shrubs and succulents (48; 63). This succulent biome has been the object of some studies (e.g., 39; 63, 64, 12). Its characteristic climate and highly fragmented distribution make it an excellent system in which to investigate the effects of niche conservatism. The succulent biome shows a disjunction between the New and the Old World. This pattern is matched by the distribution of legumes where, e.g., the genera Arcoa Urb., Diphysa Jaqu., Pictetia DC., and Chapmannia Torr. & Gray occur in North and Mesoamerica and Tetrapterocarpon Humbert, Zygocarpum Thulin & Lavin, Ormocarpopsis R.Vig., Ormocarpum P. Beauv., and Chapmannia occur in Africa. In most of these cases, the New World group finds its corresponding sister clade in Africa. Several hypotheses were assumed to explain this pattern. Inferring from physiognomical similarities, links have been proposed between succulent biome taxa and dry boreotropical and Madrean-Tethyan representatives (38). Based on palaeontological and geological evidence, 81 proposed an Eocene North Atlantic Land bridge (55.8–33.9 million years ago [Ma]) linking the continents of the Northern Hemisphere, resulting in a circumboreal, deciduous, and evergreen heterogenous flora (boreotropical hypothesis, 88). 38 suggested that vicariance between North/Mesoamerica and Africa was possible via this land bridge for legumes. Similarily, only younger in time, the Madrean-Tethyan hypothesis suggested a common occurrence of recent and fossil sclerophyllous taxa in both southwestern North America and the Old World by the late Eocene to the late Paleogene (37.2–23 Ma, 2, 3, 4). Another possible vicariant origin of this disjunction may have been caused by an early Miocene (23–16 Ma) migration route via the Bering land bridge, as suggested by 67 and 80. In contrast to these vicariance scenarios, the intercontinental disjunction of legumes in the succulent biome was recently interpreted as result of long distance because the ages of these lineages are much younger than the assumed vicariance events (39). The succulent biome is also disjunct on the African continent. Numerous plants (e.g., Aizoon L. [Aizoaceae], Trichoneura Anderss. [Poaceae]) and animals (e.g., ostriches) occupy an "arid corridor" between southwestern and northeastern Africa (5; 83; 14; 34), with an extension to Mauretania (NW Africa). The closure and widening of this arid track has been linked to Miocene uplift and rifting in Central Africa and climate changes in the Pleistocene (Caujape-Castells et al., 2001; 11), which have led to isolated populations in these areas characterized by a short rainy season. Evidence supporting the arid track came from phylogeographical studies of ostriches (21) and phylogenetic investigations of Androcymbium Willd. (Colchicaceae, Caujape-Castells et al., 2001), Senecio L. (11), and Zygophyllum L. (Zygophyllaceae, 6). A small transoceanic disjunction in the succulent biome is found in the Horn of Africa region. This area covers the three provinces of the Eritreo–Arabian subregion of 70: Somalo-Ethiopia, South Arabia, and the Socotran archipelago. Socotra is of continental origin and separated from the Arabian plate about 15–18 Ma (56; 20; 82). In all three regions, the succulent biome is prevalent, leading to two explanations for Socotran–continental disjunctions. They are either vicariant relicts preceeding the rifting (postulated for chameleons; 42), or were established by long-distance dispersal over water (e.g., Aerva Forssk. [Amaranthaceae], 73]; Echidnopsis Hook. f. [Apocynaceae-Asclepiadoideae], 71]; and Campylanthus Roth [Plantaginaceae], 72). To explore the evolution of these disjunctions, we used the rutaceous genus Thamnosma Torr. & Frém. With only 11 species, Thamnosma occurs in three widely disjunct centers of distribution. Five species grow in southwestern North America, these could well be relicts of the North American Madro-Tertiary geoflora (1), an assemblage of sclerophyllous and microphyllous taxa that were adapted to conditions of low annual precipitation, high summer temperatures, and long sunshine periods. The other species are mainly found in Africa, where three species occur in the southern continent from Angola to the northern regions of South Africa, and another three species grow around the Horn of Africa extending to southern Arabia and the island of Socotra (77) (Table 1, Fig. 1). This pattern with trans-Atlantic and trans-African disjunctions of the succulent biome makes Thamnosma a suitable case study (1) to test the hypotheses on the origin of Old and New World disjunctions in the succulent biome, (2) to investigate the "arid corridor" between southern Africa and the Horn of Africa region, and (3) to evaluate a vicariance origin for the Socotran taxon. Figure 1Open in figure viewerPowerPoint Distribution map (http://www.aquarius.ifm-geomar.de) of Thamnosma in North and Central America and in Africa. Table 1. Species of Thamnosma, their distribution and defined areas of endemism: H = Horn of Africa region, N = Northern/ Mesoamerica, S = Southern Africa. Taxon Species distribution Area of endemism Thamnosma africana Engl. S Angola, Namibia S Thamnosma crenata (Engl.) Thulin South Africa, Northern Province S Thamnosma hirschii Stapf Yemen, Oman, N Somalia, Socotra H Thamnosma montana Torr. & Frém. S USA, Mexico N Thamnosma pailensis M. C. Johnst. Mexico, SE Coahuila N Thamnosma rhodesica (Bak. f.) Mendonça S+W Zimbabwe, Botswana S Thamnosma socotrana Balf. f. Yemen, Socotra H Thamnosma somalensis Thulin NE Somalia H Thamnosma stanfordii I. M. Johnst. Mexico, S Coahuila N Thamnosma texana Torr. S USA, Mexico N Thamnosma trifoliata I. M. Johnst. Mexico, Baja California N MATERIALS AND METHODS Taxon sampling To resolve the interspecific phylogenetic relationships of Thamnosma, we included all 11 species of Thamnosma (33; 77) in a combined analysis of the ITS, matK, and the atpB-rbcL spacer (Table 2), rooted with the the closely related Ruta L. (59). On the basis of three chloroplast markers, Thamnosma is closely related to the eastern Asian, monotypic Boenninghausenia Reichb. ex Meissner, Ruta L., Haplophyllum A. Juss., and Cneoridium dumosum Hook. f. (59). Of those, only Cneoridium Hook. f. and Ruta could be included in our study due to availability of material. 59 found a sister-group relationship between Thamnosma and Boenninghausenia. Still, the monotypic Psilopeganum Hemsl. ex Forb. & Hemsl., restricted to a small area of central China (66), may also be closely related to Thamnosma, because these are the only bicarpellate genera in the Rutinae and share capitate stigmas and similar flower merosity and seed shapes (17, 18; 59). This relationship is also supported by phytochemical data (acridones of type H1; 13). Because Psilopeganum shares more morphological synapomorphies with Thamnosma rather than with Boenninghausenia (cf. 59), it seems more likely that Thamnosma and Psilopeganum are phylogenetic sisters. However, it is unlikely that Psilopeganum is embedded in Thamnosma because they differ in the degree of carpel fusion and the size of their disc (17, 18). No DNA is available for Psilopeganum; thus these morphologically based interpretations have not yet been tested. Material was mostly taken from herbarium specimens or, in some cases, silica-gel-dried leaves. Vouchers and EMBL accession numbers are given Table 2. Table 2. Taxon sampling, vouchers and new EMBL numbers for this study. Herbaria acronyms are according to Index Herbariorum (http://sciweb.nybg.org/science2/IndexHerbariorum.asp). Taxon Collector No. Origin voucher ITS1 ITS2 matK atpB-rbcL spacer rbcL Cneoridium dumosum Hook.f. Thibault et Denslow s.n. Cult. Rancho Santa Ana Botanic Garden RSA674337 RSA FN552678 Euodia hupehensis Dode Wagen s.n. Cult. Bot. Garden Zurich Z FN552679 Ruta chalepensis L. Wagen s.n. Cult. Bot. Garden Zurich Z FN552647 FN552663 FN552615 FN552631 Thamnosma africana Goldblatt et al. 8927 Namibia MO FN552649 FN552665 FN552617 FN552633 Thamnosma africana Lavranos 21900 Namibia MO FN552648 FN552664 FN552616 FN552632 Thamnosma crenata Venter 11224 South Africa MO FN552650 FN552666 FN552618 FN552634 Thamnosma hirschii Miller 10000 Yemen, Socotra B FN552651 FN552667 FN552619 FN552635 Thamnosma hirschii Kilian & Hein NK 6164 Yemen B FN552653 FN552669 FN552621 FN552637 Thamnosma hirschii Thiv 3187 Yemen, Socotra Z FN552652 FN552668 FN552620 FN552636 FN552680 Thamnosma montana Landrum et Landrum 9022 USA, Arizona NY FN552654 FN552670 FN552622 FN552638 FN552681 Thamnosma montana White 4252 USA, California BM FN552655 — FN552623 FN552639 FN552682 Thamnosma pailensis Woodruff 369 Mexico, Coahuila BM FN552656 FN552671 FN552624 FN552640 FN552683 Thamnosma rhodesica Blomberg et al. BMP 104 Botswana UPS FN552657 FN552672 FN552625 FN552641 Thamnosma socotrana Thiv 3176 Yemen, Socotra STU, Z FN552658 FN552673 FN552626 FN552642 Thamnosma socotrana Kilian 2495 Yemen, Socotra B FN552684 Thamnosma somalensis Thulin et al. 9489 Somalia UPS FN552659 FN552674 FN552627 FN552643 Thamnosma stanfordii Chiang et al. 9545 Mexico, Coahuila MO, NY FN552660 FN552675 FN552628 FN552644 Thamnosma texana McGolderick s.n. USA, Texas L FN552661 FN552676 FN552629 FN552645 FN552685 Thamnosma trifoliata Rebman 7577 Mexico, Baja California SD FN552662 FN552677 FN552630 FN552646 FN552686 To estimate the ages of biogeographically relevant nodes in the Thamnosma phylogeny, we assembled a set of species representing all major clades of Rutaceae and two taxa with reliable fossils for the rbcL analysis. Thamnosma was represented by six North American and Old World species: southern African Thamnosma species were not included because amplifications did not succeed, possibly from degradation of the DNA extracted from herbarium material. Due to the use of degraded DNA from herbarium material, phylogenetic reconstruction and molecular dating were based on only half of the gene length of rbcL (790–813 bp). Laboratory work The molecular work followed standard protocols. For DNA extraction, the DNeasy plant extraction kit (Qiagen) was used according to the manufacturer's protocol. Amplifications were performed using 1.5 mM buffer, 0.625 mM MgCl2, 0.2 mM dNTPs, 0.05 U/μL Taq DNA polymerase (Amersham Biosciences, Freiburg, Germany), 0.325 μM primer, and 5 ng/μL DNA template. PCR profiles included 33 cycles of 94°C for 1 min, 50–55°C for 1 min, and 72°C for 2–3 min. For amplifications and sequencing, the following primers were used. ITS nrDNA: ITS-A 5′-GGAAGGAGAAGTCGTAACAAGG-3′, ITS-B 5′-CTTTTCCTCCGCTTATTGATATG-3′, ITS-D 5′-CTCTCGGCAACGGATATCTCG-3′ (all 7), ITS-R2 5′-CGTTCAAAGACTCGATGGTTC-3′ (84). Multiple DNA extractions and ITS sequencing for several accessions of Thamnosma, yielded identical results indicating the absence of paralogous copies of ITS. The atpB-rbcL intergenic spacer: atpB-F2 5′-GAAGTAGTAGGATTGATTCTC-3′, atpB-R5 5′-GAAGTAGTAGGATTGATTCTC-3′ (both 44); matK (these primers amplify the 3′ end of the matK gene and the adjacent intron): matK-Th-F 5′-TTATTCATCTGATTGGATCAT-3′ (designed in the present study), matK-1R 5′-GAACTAGTCGGATGGAGTAG-3′ (61); rbcL: rbcL-F1 5′-TCACCACAAACAGARACTAAAGC-3′, rbcL-R2 5′-RCGRTGRATGTGAAGAAG-3′ (both Long-Yin Qiu, University of Michigan, personal communication). The use of herbarium material only allowed the amplification of a partition of rbcL. PCR products were cleaned using the PCR purification kit (Qiagen, Hilden, Germany). Cycle sequencing was conducted using ABI PRISM BigDye 2.1 to obtain double stranded sequences. Resulting products were analyzed using automated sequencing systems ABI PRISM 3100 (PE Biosystems, Darmstadt, Germany). Phylogenetic analysis Sequences of all Thamnosma species were aligned using the program Clustal X version 1.81 (74) and then manually adjusted. The alignments are available in TreeBase (S10787, http://treebase.org). First, parsimony (MP) bootstrap analyses (options given later) were conducted for both chloroplast and nuclear data. To test for congruence between these two data sets, we conducted a partition homogeneity test as implemented in the program PAUP* 4.0b10 (69). Because this test failed to reject the hypothesis of congruency, the two data sets were combined (30; 85). Maximum likelihood (ML; 19) and parsimony analyses for the combined data were performed using PAUP* 4.0b10. For the ML analysis, the GTR+I+Γ model was used as indicated by the Akaike information criterion (AIC) in the program Modeltest 3.06 (53). For parsimony analyses, characters were equally weighted, character states were treated as unordered, and indels were treated as missing data. Parsimony analyses were carried out using 103 random-addition-sequence replicates, with Multrees in effect and tree-bisection-reconnection (TBR) swapping. Parsimony bootstrap analyses (104 replicates) were calculated using the closest Multrees and TBR options. Additionally, Bayesian inference using the combined data set was explored using the program MrBayes v. 3.1.2 (29). The GTR+Γ model was selected by AIC in the program MrModeltest (50). Two runs using four parallel chains of 5 × 106 generations with heats of 1.00, 0.83, 0.71, and 0.62 were performed, with a sample frequency of 102. Trees from the first 104 generations were discarded. We tested alternative scenarios using a parametric bootstrap analysis. This test is shown to be a statistically sound method of evaluating different alternative topological hypotheses (28; 22; 68). This procedure included the determination of ML parameters for the described constrained topologies. Based on these parameters, 99 simulated data sets were created using the program Seq-Gen (54). The simulated data sets were analyzed using maximum parsimony with the closest sequence addition and TBR branch swapping, testing for significant differences in lengths between the constrained tree as null hypothesis and the optimal tree. Molecular dating We used a molecular clock to date the disjunction between New and Old World species of Thamnosma. Based on a likelihood ratio (LR) test (19; 60; 49), substitution rates of the rbcL sequences were near clock-like. Nonetheless, we employed Bayesian dating using a relaxed clock. This method (76; 75) uses a probabilistic model to describe the change in evolutionary rate over time and uses the Markov chain Monte Carlo (MCMC) procedure to derive the posterior distribution of rates and time. It allows multiple calibration points and provides direct credibility intervals for estimated divergence times and substitution rates. We used the programs BEAST 1.4.8 and Tracer (16), which do not assume autocorrelation, and MULTIDIVTIME (76; 36, 75), which does. For the BEAST analysis of the rbcL data set, the model parameters determined as optimal by AIC (see results) under the GTR+Γ+I model and suggested priors taken from a prerun analysis were used. A relaxed clock model with an uncorrelated log-normal rate change was chosen. We tuned the operators using BEAST's auto-optimization option. We then executed two runs of 107 generations each, sampling every 103 generations, using random starting trees, and setting the coalescent process and a speciation model following a Yule process as tree prior. For all BEAST analyses, resulting posterior distributions for parameter estimates were checked in Tracer 1.4.1 (16), and maximum credibility trees, representing the maximum a posteriori topology, were calculated after removing burn-in with the program TreeAnnotator version 1.4.7. The .xml files are available for the Rutaceae analysis as Appendix S1 and for the Thamnosma analysis as Appendix S2 (online at http://www.amjbot.org/cgi/content/full/ajb.1000339/DC1). For the MULTIDIVTIME analyses, we followed the procedure outlined by 73 and a step-by-step manual by 58. Model parameters for the F84+Γ model (35) were estimated using the module BASEML in the program PAML (89). We estimated the maximum likelihood of the branch lengths of the rooted evolutionary tree together with a variance–covariance matrix of the branch length estimates by using the program ESTBRANCHES (76). We used MULTIDIVTIME to approximate the posterior distributions of substitution rates and divergence times by using a multivariate normal distribution of estimated branch lengths (provided here by ESTBRANCHES) and running the MCMC procedure with the following settings for the prior distributions: 1.50 for both rttm and rttmsd, 0.07 for both rtrate and rtratesd, 0.4 for both brownmean and brownsd, and 84 million years ago (Ma) for bigtime, which is the maximum age of Sapindales according to 87. This maximum age means that this node cannot be estimated to be older than this date. We ran the Markov chain for at least 103 cycles and collected one sample every 102 cycles, after an unsampled burn-in of 104 cycles. We repeated the analyses in BEAST and MULTIDIVTIME twice using different random starting number to assure convergence of the Markov chain and combined the results. The rate-corrected tree was calibrated with two fossil taxa. A continuous macrofossil record starting form the Late Eocene/Early Oligocene (40–35 Ma) is available for Euodia J.R.Forst. & G.Forst. and Zanthoxylum L. (25). Because the macrofossils can be exclusively attributed to these genera (24, 25; 79; 43), both were used as calibration points for our age estimates. The oldest known records of Euodia and Zanthoxylum date from the Late Eocene (35 Ma) and were treated as minimum ages for the stem of the corresponding lineages in the rbcL analysis (Fig. 2). For the BEAST analysis, we modeled the clades including Euodia and Zanthoxylum, each, as an exponential distribution (26) with a mean of 16.4 and an offset of 35 Ma, which corresponds to the maximum age of these fossils. With this mean value, the 95% distribution covered 84 Ma, which was regarded as big time in the MULTIDIVTIME analysis. Figure 2Open in figure viewerPowerPoint Clock-like maximum likelihood (ML) tree of selected Rutaceae based on rbcL sequences. Capitals A and B indicate calibration points. Lowercase letters a–d refer to nodes in Table 3. ML bootstrap values (>50%) are on the branches. Numbers behind taxon names refer to EMBL accession numbers (Table 2). Because rbcL was availabile only for a limited number of species, we used the age estimates for some nodes of the rbcL tree for a BEAST analysis of the combined ITS/matK/atpB-rbcL spacer data set including all Thamnosma species. This procedure followed the BEAST analysis for rbcL with the following specifications. Mean age estimates with standard deviations of the rbcL BEAST analysis (nodes b and d in Table 3) served as calibration points under a normal distribution. The GTR+Γ+I model was chosen. We then executed two runs of 108 generations each and sampled every 104 generations. Table 3. Results of the Bayesian dating using BEAST and MULTIDIVTIME showing combined mean ages and the 95% highest posterior density (HPD) of two runs. Nodes in lowercase letters refer to Fig. 2 and those in capital letters to Figs. 3 and 4. BEAST MULTIDIVTIME Node Mean (Ma) 95% HPD (Ma) SE Mean (Ma) 95% HPD (Ma) a 36.75 14.66-61.55 0.38 22.25 9.21–34.92 b/B 14.56 4.78-26.35 0.16 13.11 3.33–26.83 c 10.52 2.80-20.43 1.12 8.60 1.22–20.96 d/D 5.15 0.36-11.38 0.06 4.09 0.13–13.65 E 8.53 5.28-12.11 0.02 — — F 12.63 8.37-14.94 0.03 — — Biogeographic analyses We defined the following areas of endemism for Thamnosma: N = North/Central America, H = Horn of Africa including Socotra and Somalia, and S = Southern Africa (Table 1). The outgroup, Ruta chalepensis, was coded as Mediterranean, and we did not take into account the potential Asian relatives (see Materials and Methods: Taxon sampling). To reconstruct the geographical evolution of Thamnosma, we conducted a dispersal—vicariance analysis (57) using the program S-DIVA 1.9 (90). This method calculates the optimized areas over a set of trees, thus taking into account topological uncertainty. We used the 9000 trees retained from the BEAST analysis of the combined data set. The number of maximum areas was set to three because this reflects the ingroup's number of defined areas of distribution. To take into account the estimated time between speciation events, we also used the program Lagrange 2.0.1 (55) for the reconstruction of ancestral areas, using an ultrametric tree combining the ML topology with internal node age estimates from a BEAST analysis based on the combined data set (not shown). All combinations of areas were allowed in the adjacency matrix, and baseline rates of dispersal and local extinction were estimated. RESULTS Rutaceae phylogeny The aligned sequence lengths of rbcL were 811 bp. The optimal model of sequence evolution for this data set was the transversion (TVM+I+Γ) model: unequal base frequencies (A = 0.2721, C = 0.2039, G = 0.2442, T = 0.2798), six substitution types (A/C: 1.5287, A/G: 2.7801, A/T: 0.5796, C/G: 0.9464, C/T: 2.7801), gamma distribution of rates among sites with alpha shape parameter = 0.7607, and the proportion of invariable sites = 0.6326. The analysis using these parameters yielded a ML tree with a log-likelihood score of –lnL = 2962.76. According to the ML tree (Fig. 2), Thamnosma is highly supported as monophyletic (99% bootstrap value), and the position as sister to the Mediterranean Ruta is supported by a 75% bootstrap value. In the rbcL study, both genera appear weakly supported as sister to the Californian Cneoridium. Interspecific relationships of Thamnosma inferred from a reduced taxon sample were partly resolved by rbcL data. Except for the position of T. trifoliata, they correspond to the results of the second, more detailed analysis reported next. Thamnosma phylogeny The aligned sequence lengths were 224 bp (ITS1), 231 bp (ITS2), 664 bp (matK), and 994 bp (atpB-rbcL spacer), resulting in a total length of 2113 bp, of which 0.89% were scored as missing data. Of these characters, 248 were variable and 62 parsimony informative. A partition homogeneity test explained incongruence between the nuclear and chloroplast data at P = 0.954. Therefore, we combined the data sets. The MP analysis yielded a single most parsimonious tree with a length of 306 steps, a CI of 0.92 and a RI of 0.80. The optimal model of sequence evolution for this combined data set was the general time reversible (GTR+I+Γ) model: unequal base frequencies (A = 0.2793, C = 0.2042, G = 0.2169, T = 0.2996) and six substitution types (A/C: 1.0607, A/G: 1.4094, A/T: 0.3057, C/G: 1.3575, C/T: 3.0006), gamma distribution of rates among sites with alpha shape parameter = 0.6826, and the proportion of invariable sites = 0.5533. The analysis using these parameters yielded a ML tree with a log-likelihood score of –lnL = 4667.27. The MP, ML, and Bayesian analyses, all indicate that the North American species are paraphyletic relative to the Old World taxa (Figs. 3, 4). All analyses recognize a North American clade consisting of T. stanfordii, T. pailensis, and T. texana and a palaeotropical lineage including T. africana, T. crenata, T. rhodesica, T. somalensis, T. hirschii, and T. socotrana. This latter clade is subdivided into the southern African T. africana-T. crenata-T. rhodesica, and the Eastern African-Arabian-Socotran T. socotrana-T. somalensis-T. hirschii. The results differ with reg