Leaf and flower characters mapped on to supertree for Rutaceae.

<p>Tree topology and clade labels match those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072493#pone-0072493-g003" target="_blank">Fig. 3</a>.</p

Major Clades of Australasian Rutoideae (Rutaceae) Based on <i>rbc</i>L and <i>atp</i>B Sequences

<div><p>Background</p><p>Rutaceae subfamily Rutoideae (46 genera, c. 660 species) is diverse in both rainforests and sclerophyll vegetation of Australasia. Australia and New Caledonia are centres of endemism with a number of genera and species distributed disjunctly between the two regions. Our aim was to generate a high-level molecular phylogeny for the Australasian Rutoideae and identify major clades as a framework for assessing morphological and biogeographic patterns and taxonomy.</p><p>Methodology/Principal Findings</p><p>Phylogenetic analyses were based on chloroplast genes, <i>rbc</i>L and <i>atp</i>B, for 108 samples (78 new here), including 38 of 46 Australasian genera. Results were integrated with those from other molecular studies to produce a supertree for Rutaceae worldwide, including 115 of 154 genera. Australasian clades are poorly matched with existing tribal classifications, and genera <i>Philotheca</i> and <i>Boronia</i> are not monophyletic. Major sclerophyll lineages in Australia belong to two separate clades, each with an early divergence between rainforest and sclerophyll taxa. Dehiscent fruits with seeds ejected at maturity (often associated with myrmecochory) are inferred as ancestral; derived states include woody capsules with winged seeds, samaras, fleshy drupes, and retention and display of seeds in dehisced fruits (the last two states adaptations to bird dispersal, with multiple origins among rainforest genera). Patterns of relationship and levels of sequence divergence in some taxa, mostly species, with bird-dispersed (<i>Acronychia</i>, <i>Sarcomelicope</i>, <i>Halfordia</i> and <i>Melicope</i>) or winged (<i>Flindersia</i>) seeds are consistent with recent long-distance dispersal between Australia and New Caledonia. Other deeper Australian/New Caledonian divergences, some involving ant-dispersed taxa (e.g., <i>Neoschmidia</i>), suggest older vicariance.</p><p>Conclusions/Significance</p><p>This comprehensive molecular phylogeny of the Australasian Rutoideae gives a broad overview of the group’s evolutionary and biogeographic history. Deficiencies of infrafamilial classifications of Rutoideae have long been recognised, and our results provide a basis for taxonomic revision and a necessary framework for more focused studies of genera and species.</p></div

Supertree summarising relationships in Rutaceae, combining results of the present study with those of other molecular phylogenies (see Methods for details).

<p>The tree includes 115 of the 154 genera currently recognised <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072493#pone.0072493-Kubitzki1" target="_blank">[<b>2</b>]</a>. The distribution of fruit types is optimised on the tree (colour coding of branches). Also shown are the limits of subfamilies and some tribes, including circumscriptions of the tribe Boronieae by: a, Engler <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072493#pone.0072493-Engler1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072493#pone.0072493-Engler2" target="_blank">[4]</a>; b, Armstrong <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072493#pone.0072493-Armstrong1" target="_blank">[5]</a>; c, Hartley <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072493#pone.0072493-Hartley6" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072493#pone.0072493-Hartley7" target="_blank">[32]</a>. Clades R1 and R2 match those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072493#pone-0072493-g001" target="_blank">Fig. 1</a>; clades A-E match those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072493#pone-0072493-g002" target="_blank">Fig. 2</a>. Letter codes following taxon names in clade R2 indicate other tribal placements in the classification of Engler <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072493#pone.0072493-Engler1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072493#pone.0072493-Engler2" target="_blank">[4]</a> (Z, Zanthoxyleae; R, Ruteae; T, Toddalieae; F, Flinderseae). Asterisks denote clades with <50% bootstrap support in analyses of the primary data.</p

Examples of variation in Australasian Rutoideae in leaves, fruits and seeds.

<p><b>A</b>, opposite, compound leaves of rainforest tree <i>Melicope polybotrya</i>. <b>B</b>, opposite, compound leaves of xeromorphic shrub <i>Boronia ternata</i>. <b>C</b>, alternate, simple and terete leaves of <i>Philotheca tubiflora</i>. <b>D</b>, dehiscent fruit (follicles) of <i>Myrtopsis</i>, from which the seeds and endocarp are forcibly ejected at maturity. <b>E</b>, fruit and seeds of <i>Melicope glaberrima</i>. The seeds are adapted for bird dispersal. At maturity they remain attached to the follicles (“displayed” in the canopy). They have a shiny outer layer (pellicle), below which there is a spongy layer (sarcotesta) surrounding a strong inner layer of the seed coat (sclerotesta) capable of withstanding endozoochory. <b>F</b>, syncarpous drupes of <i>Halfordia kendack</i> (i.e., each drupe is the product of a single flower). <b>G</b>, apocarpous drupes of <i>Comptonella microcarpa</i> (i.e., each drupe is the product of a single carpel, with multiple drupes per flower). <b>H</b>, syncarpous drupe of <i>Acronychia laevis</i>. <b>I</b>, woody capsule of <i>Flindersia australis,</i> with winged seeds. <b>J</b>, winged samara of <i>Pentaceras australis</i>. <b>K</b>, seed of <i>Philotheca difformis</i>. An adaxial portion of endocarp remains attached to dehisced seeds and is assumed to function as an elaiosome for ant dispersal. [Credits: A, P. Ladiges; H, Peter Woodward; I, N. Turland; J, USDA-ARS, U.S. National Arboretum, U.S. National Seed Herbarium, Washington, DC, prepared by Robert J. Gibbons for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072493#pone.0072493-Kirkbride1" target="_blank">[126]</a>; B, C, D, E, F, G, K, M. Bayly.]</p

Genera of Australasian Rutaceae: geographic distributions and species numbers.

<p>Genera of Australasian Rutaceae: geographic distributions and species numbers.</p

Age estimates for selected New Caledonian/Australian divergences based on Bayesian relaxed clock molecular dating.

<p>Estimates include means plus upper and lower bounds of 95% highest posterior density intervals. Numbers of nucleotide differences associated with each divergence are also shown; these are the total number of base differences observed in <i>rbc</i>L and <i>atp</i>B combined, ignoring missing data and ambiguous base calls. Dispersal vectors are inferred from fruit and seed morphology.</p>*<p>Seeds are forcibly ejected from the fruits, but have no obvious features for secondary dispersal by ants or other animals (i.e., there is no elaiosome, obvious sarcotesta or pellicle).</p

Strict consensus of trees produced by MP analysis of combined <i>rbc</i>L and <i>atp</i>B sequences.

<p>Details have been simplified for some clades (indicated by terminal triangles). The most parsimonious trees were 1277 steps long (958 steps excluding autapomorphies), with CI  =  0.59 and RI  =  0.81. Numbers below branches are MP bootstrap values, followed by posterior probability values from BI analysis of the same dataset. Distribution information is shown to the right of taxon names; areas shown in brackets are not represented by samples in this study. Taxa in green occur chiefly in rainforests. Asterisks denote taxa that occur in a range of habitats, sometimes including rainforests. Abbreviations: Aus, Australia; LHI, Lord Howe Island; Mal, Malesian region; NC, New Caledonia; NG, Indonesian Papua and Papua New Guinea.</p

Chloroplast variation is incongruent with classification of the Australian bloodwood eucalypts (genus <i>Corymbia</i>, family Myrtaceae)

<div><p>Previous molecular phylogenetic analyses have resolved the Australian bloodwood eucalypt genus <i>Corymbia</i> (~100 species) as either monophyletic or paraphyletic with respect to <i>Angophora</i> (9–10 species). Here we assess relationships of <i>Corymbia</i> and <i>Angophora</i> using a large dataset of chloroplast DNA sequences (121,016 base pairs; from 90 accessions representing 55 <i>Corymbia</i> and 8 <i>Angophora</i> species, plus 33 accessions of related genera), skimmed from high throughput sequencing of genomic DNA, and compare results with new analyses of nuclear ITS sequences (119 accessions) from previous studies. Maximum likelihood and maximum parsimony analyses of cpDNA resolve well supported trees with most nodes having >95% bootstrap support. These trees strongly reject monophyly of <i>Corymbia</i>, its two subgenera (<i>Corymbia</i> and <i>Blakella</i>), most taxonomic sections (<i>Abbreviatae</i>, <i>Maculatae</i>, <i>Naviculares</i>, <i>Septentrionales</i>), and several species. ITS trees weakly indicate paraphyly of <i>Corymbia</i> (bootstrap support <50% for maximum likelihood, and 71% for parsimony), but are highly incongruent with the cpDNA analyses, in that they support monophyly of both subgenera and some taxonomic sections of <i>Corymbia</i>. The striking incongruence between cpDNA trees and both morphological taxonomy and ITS trees is attributed largely to chloroplast introgression between taxa, because of geographic sharing of chloroplast clades across taxonomic groups. Such introgression has been widely inferred in studies of the related genus <i>Eucalyptus</i>. This is the first report of its likely prevalence in <i>Corymbia</i> and <i>Angophora</i>, but this is consistent with previous morphological inferences of hybridisation between species. Our findings (based on continent-wide sampling) highlight a need for more focussed studies to assess the extent of hybridisation and introgression in the evolutionary history of these genera, and that critical testing of the classification of <i>Corymbia</i> and <i>Angophora</i> requires additional sequence data from nuclear genomes.</p></div

Phylogeny resulting from nrDNA analyses.

<p>Best-scoring maximum likelihood tree from a RAxML analysis (final ML optimization likelihood of -4709.258464) of a nrITS dataset (663 base pairs, 337 distinct alignment patterns, 119 accessions) of eucalypts. The outgroup (OG), major groups in <i>Eucalyptus</i> (‘Eudesmids’, ‘Monocalypts’ [Mono], and ‘Symphyomyrts’ [Symph]), subgenera and infrageneric groups of <i>Corymbia</i> are indicated with bars or colouring scheme (see textbox) on the tree. Species names are followed by GenBank accession numbers. Bootstrap support values are shown as percent maximum likelihood/maximum parsimony (MP mapped onto ML tree) with weighted edges indicating 100% support for both ML and MP. Support values <50% are omitted or dashed when the alternate analysis method had ≥50% support. ⁱ Identified in GenBank (GB) as <i>Angophora exul</i> K.D.Hill, a name that is synonymised with <i>A</i>. <i>bakeri</i> in the current Australian Plant Census (APC) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195034#pone.0195034.ref001" target="_blank">1</a>]. ⁱⁱ Identified in GB as <i>Angophora euryphylla</i> (G.J.Leach) L.A.S.Johnson & K.D.Hill, a name that is synonymised with <i>A</i>. <i>costata</i> subsp. <i>euryphylla</i> in the current APC. ⁱⁱⁱ Identified in GB as <i>Corymbia variegata</i> (F.Muell.) K.D.Hill & L.A.S.Johnson, a name that is synonymised with <i>C</i>. <i>citriodora</i> in the current APC. ^iv Identified in GB as <i>Corymbia dolichocarpa</i> (D.J.Carr & S.G.M.Carr) K.D.Hill & L.A.S.Johnson, a name that is synonymised with <i>C</i>. <i>clarksoniana</i> in the current APC. ^v Identified in GB as <i>Corymbia dampieri</i> (D.J.Carr & S.G.M.Carr) K.D.Hill & L.A.S.Johnson, a name that is synonymised with <i>C</i>. <i>greeniana</i> in the current APC. ^vi Identified in GB as <i>Corymbia dimorpha</i> (Brooker & A.R.Bean) K.D.Hill & L.A.S.Johnson, a name that is synonymised with <i>C</i>. <i>peltata</i> in the current APC. ^vii Identified in GB as <i>Corymbia catenaria</i> K.D.Hill & L.A.S.Johnson, a name that is synonymised with <i>C</i>. <i>watsoniana</i> subsp. <i>capillata</i> in the current APC.</p

Phylogeny resulting from nrDNA analyses.

<p>Best-scoring maximum likelihood tree from a RAxML analysis (final ML optimization likelihood of -4709.258464) of a nrITS dataset (663 base pairs, 337 distinct alignment patterns, 119 accessions) of eucalypts. The outgroup (OG), major groups in <i>Eucalyptus</i> (‘Eudesmids’, ‘Monocalypts’ [Mono], and ‘Symphyomyrts’ [Symph]), subgenera and infrageneric groups of <i>Corymbia</i> are indicated with bars or colouring scheme (see textbox) on the tree. Species names are followed by GenBank accession numbers. Bootstrap support values are shown as percent maximum likelihood/maximum parsimony (MP mapped onto ML tree) with weighted edges indicating 100% support for both ML and MP. Support values <50% are omitted or dashed when the alternate analysis method had ≥50% support. ⁱ Identified in GenBank (GB) as <i>Angophora exul</i> K.D.Hill, a name that is synonymised with <i>A</i>. <i>bakeri</i> in the current Australian Plant Census (APC) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195034#pone.0195034.ref001" target="_blank">1</a>]. ⁱⁱ Identified in GB as <i>Angophora euryphylla</i> (G.J.Leach) L.A.S.Johnson & K.D.Hill, a name that is synonymised with <i>A</i>. <i>costata</i> subsp. <i>euryphylla</i> in the current APC. ⁱⁱⁱ Identified in GB as <i>Corymbia variegata</i> (F.Muell.) K.D.Hill & L.A.S.Johnson, a name that is synonymised with <i>C</i>. <i>citriodora</i> in the current APC. ^iv Identified in GB as <i>Corymbia dolichocarpa</i> (D.J.Carr & S.G.M.Carr) K.D.Hill & L.A.S.Johnson, a name that is synonymised with <i>C</i>. <i>clarksoniana</i> in the current APC. ^v Identified in GB as <i>Corymbia dampieri</i> (D.J.Carr & S.G.M.Carr) K.D.Hill & L.A.S.Johnson, a name that is synonymised with <i>C</i>. <i>greeniana</i> in the current APC. ^vi Identified in GB as <i>Corymbia dimorpha</i> (Brooker & A.R.Bean) K.D.Hill & L.A.S.Johnson, a name that is synonymised with <i>C</i>. <i>peltata</i> in the current APC. ^vii Identified in GB as <i>Corymbia catenaria</i> K.D.Hill & L.A.S.Johnson, a name that is synonymised with <i>C</i>. <i>watsoniana</i> subsp. <i>capillata</i> in the current APC.</p