Edwards & al. • Melanthera alliance biogeography and relationships (Compositae) TAXON 67 (3) • June 2018: 552–564 Biogeography and relationships within the Melanthera alliance: A pan-tropical lineage (Compositae: Heliantheae: Ecliptinae) Robert D. Edwards,1 Jason T. Cantley,2 Marian M. Chau,3 Sterling C. Keeley3 & Vicki A. Funk1 1 Department of Botany, National Museum of Natural History, Smithsonian Institution, Washington, 10th and Constitution Ave., Washington, D.C. 20560-0166, U.S.A. 2 Department of Biology, San Francisco State University, 1600 Holloway Ave., San Francisco, California 94132, U.S.A. 3 Department of Botany, University of Hawai‘i, Mānoa, 3190 Maile Way, Honolulu, Hawaii 96822, U.S.A. Author for correspondence: Robert D. Edwards, bortedwards@gmail.com DOI https://doi.org/10.12705/673.6 Abstract The taxonomic history of the Melanthera alliance is long and convoluted with many generic name changes and requires a robust phylogeny to clarify taxonomic concepts within the group and to begin asking questions of its evolutionary history. For a time, prevailing classifications placed all species in the genus Melanthera except for a handful of tetraploids from the Hawaiian Islands being recognized as a distinct genus: Lipochaeta. Recent morphological revision has reopened debate by proposing six genera: Apowollastonia, Echinocephalum, Lipotriche, and Melanthera, and two Pacific Island genera representing diploids (Wollastonia) and tetraploids (Lipochaeta), plus four closely related genera expected to fall outside the alliance (Acunniana, Indocypraea, Lipoblepharis, Quadribractea). Here, we present the most comprehensive molecular phylogeny to date of the taxa variously associated with Melanthera in order to test these competing generic limits and explore the biogeographic history of this pan-tropical lineage. The data are consistent with six segregate genera, including the sinking of Hawaiian Islands members of Wollastonia (Melanthera) back into a broader concept of Lipochaeta, although there is currently no recognized morphological synapomorphy to distinguish Lipochaeta s.l. from Wollastonia. Our results suggest that the Melanthera alliance originated some time during the Pliocene or Pleistocene and a strong contemporary presence of the alliance and closely related Ecliptinae outgroups in the Americas suggests that this region may have been the center of origin with subsequent dispersal. We illustrate the difficulty of reconstructing the dispersal history of the remaining genera and present the most parsimonious colonization hypotheses. Keywords Apowollastonia; Asteraceae; Compositae; Echinocephalum; Ecliptinae; Hawaiian Islands; Lipochaeta; Lipotriche; Pacific biogeography; Wedelia; Wollastonia Supplementary Material The Electronic Supplement (Figs. S1 & S2) is available from https://doi.org/10.12705/673.6.S; files for MCC tree and alignment matrix were deposited at TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S22771) INTRODUCTION Although the integrity of the Compositae Giseke (= Asteraceae Bercht. & J.Presl.) has not been called into question since its initial circumscription (Adanson, 1763, see Funk & al., 2009 chapter 1 for details), there have been numerous disagreements about generic limits within the family. While some have been resolved, many remain unclear or in dispute, including relationships within the subtribe Ecliptinae Less.—a monophyletic group of 48 genera and more than 420 species within the tribe Heliantheae Cass. (Panero, 2007). The monophyly of the Ecliptinae is robust, but relationships within and among genera remain poorly resolved (Panero & al., 1999), notably the limits and relationships among taxa representing the informal Melanthera Rohr alliance and their relationship to the closely related genera Blainvillea Cass., Lipotriche R.Br., and Wedelia Jacq. (including taxa previously recognized as Aspilia Thouars). A review of taxonomic concepts within the alliance can be found in both Wagner & Robinson (2002) and Orchard (2013), and as it currently stands these two studies represent the two primary competing morphologically based taxonomic hypotheses for the group: H1: Two sister genera sensu Wagner & Robinson (2002) (Lipochaeta DC. + Melanthera). H2: Five smaller genera sensu Ochard (2013) (Apowollastonia Orchard + Echinocephalum Gardner + Lipochaeta + Lipotriche + Melanthera + Wollastonia DC. ex Decne.). The first hypothesis (Wagner & Robinson, 2002) takes a simple approach in sinking all but the tetraploid Hawaiian Islands species (Lipochaeta s.str.) into one large genus, Melanthera. This amalgamates species from South, Central, and North America, as well as Asia and the Pacific in an unwieldy and rather uninformative framework. The second hypothesis is the result of more recent work of Orchard (2013) that, although primarily focused on Australian taxa, resulted in broader generic implications and divisions across the alliance: Melanthera was reduced to a few white-flowered species in southeastern U.S.A., the Caribbean Islands, and the east coast Article history: Received: 29 Jan 2018 | returned for (first) revision: 10 Mar 2018 | (last) revision received: 10 Apr 2018 | accepted: 11 Apr 2018 | published: online fast track, 4 Jun 2018 ; in print and online issues, 6 Jul 2018 || Associate Editor: Alfonso Susanna || Published online “open-access” under the terms of the Creative Commons Attribution 4.0 (CC-BY 4.0) License || © International Association for Plant Taxonomy (IAPT) 2018, all rights reserved 552 Version of Record TAXON 67 (3) • June 2018: 552–564 Edwards & al. • Melanthera alliance biogeography and relationships (Compositae) of Mesoamerica and South America, while several small genera were resurrected, being distributed widely from India through the Indo-Pacific Islands to Japan. The tetraploid Hawaiian Islands taxa were maintained as Lipochaeta per Wagner & Robinson (2002), while all other Pacific taxa were placed in Wollastonia. The novel genus Apowollastonia was created for eight Australian taxa, and Wedelia, a sister genus to the alliance, was restricted to only taxa from the Americas and Africa. In this paper we test both of these hypotheses, but for clarity use the five-genus nomenclature of Orchard (2013) for the remainder of the introduction as it allows greater resolution for discussion. Work addressing these taxa is understandably difficult with widespread dispersal across the tropics and many recent local and endemic radiations. The taxonomic concepts outlined above have been based on traditional morphological data and address a number of species that are opportunistic colonizers and pioneers with high phenotypic plasticity yet poorly divergent general morphologies (Panero & al., 1999) (Fig. 1). These are all factors that have proven challenging to traditional taxonomy and there is an expectation of non-monophyly or recircumscription when tested with molecular data. Some genetic work has attempted to resolve regional subsets of these genera (e.g., Panero & al., 1999) but species from the Pacific and the Americas have been largely neglected as the work was based on a limited taxonomic sampling (approximately one representative species from each genus) and several chloroplast loci. While these data suggest a well-supported sister relationship between Lipochaeta and Wollastonia, there is only weak support for Melanthera as sister to these, and for the placement of these three genera in relation to the rest of the subtribe (nested between a well-supported clade including Wedelia plus five other genera, and the rest of the group). Poor resolution has restricted our ability to adequately address generic limits across the entire subtribe or understand the pan-tropical biogeographical connections within the group. Hawaiian Islands Lipochaeta. — Of particular interest beyond the lump-or-split alternatives posed by the two competing hypotheses outlined above is one commonality: the restriction of the genus Lipochaeta to only tetraploid Hawaiian Islands taxa. As currently understood, there are 16 endemic taxa with representatives on all eight of the main islands and the Northwest Islands. They occupy a range of habitats from coastal strand through dry shrubland/forests to mesic forests or hanging valleys up to 1800 m elevation. Seven are listed as endangered and an additional four species are believed to have become extinct since the mid-twentieth century (L. degeneri Sherff, Wollastonia bryanii (Sherff) Orchard, W. perdita (Sherff) Orchard, W. populafolia (Sherff) Orchard—Wagner & al., 1999). Traditionally all 16 species have been placed in the genus Lipochaeta based both on morphological (Gardner, 1979) and secondary plant chemistry data (Gardner & LaDuke, 1978). However, cytology, morphology, and biochemistry has been also used to separate the taxa into two groups: L. sect. Lipochaeta being tetraploids (n = 26), with four corolla lobes, flavonols plus flavones; and sect. Aphanopappus (Endl.) Benth. & Hook.f. being diploids (n = 15), with five corolla lobes and only flavonols. The combinations of these characters along with successful hybridization experiments led to the hypothesis (H3) that tetraploid taxa (L. sect. Lipochaeta) arose from hybridization between an unknown Wollastonia-like (n = 15) ancestor and an unknown Wedelia-like (n = 11) ancestor (Rabakonandrianina & Carr, 1981). Citing this suspected recent shared ancestry between the tetraploid Lipochaeta and Wollastonia Wagner & Robinson (2002) combined them, and in the process merged them into a larger pan-tropical Melanthera genus. The competing concept of Orchard (2013) retains the merging of diploid Hawaiian taxa into Wollastonia s.str., but recognizes this as a genus distinct from Melanthera. There is still uncertainty as to whether the two Hawaiian Islands cytological groups should be represented as distinct genera or not, and until now this has not been tested using molecular data. Other taxa. — Orchard (2013) not only placed the diploid Hawaiian Islands taxa in Wollastonia but also included four narrowly distributed species of the Pacific and Pacific Rim (Orchard, 2013), plus Wollastonia biflora (L.) DC. which is widespread across islands and coastal areas in the Indo-Pacific and Asia-Pacific regions. As relationships within the group are unclear, these taxa may be closely related to any of a number of genera, most likely Apowollastonia, Echinocephalum, Lipoblepharis Orchard, Lipotriche, or Melanthera. Of these, two genera (previously considered Melanthera by Wagner & Robinson, 2002) were described as distinct by Orchard (2013): Apowollastonia with eight taxa restricted to Australia, and Lipoblepharis with five species ranging from India to Indonesia, China and Japan. Of the remaining taxa, Echinocephalum is a monospecific genus (sensu Orchard, 2013) with E. latifolium Gardner found in open damp savannas in Paraguay and Brazil, Lipotriche includes 12 species found in mostly tropical areas throughout Africa (Hind, 2014), and Melanthera s.str. contains only three species all found in the southeastern United States, one of which also occurs in the Caribbean and east coasts of Central and South America. Orchard (2013) also detailed three new monotypic Ecliptinae genera with close affinities to the Melanthera alliance: Quadribractea Orchard from Malesia, Acunniana Orchard from Australia, and Indocypraea Orchard from Southeast Asia and India. Of these only Indocypraea is included in this study due to a lack of material for the other genera. Dispersal. — Distributional patterns within the Ecliptinae vary from narrowly endemic to widespread, and conspecifics or populations within species can be separated by large distances. Achenes in some species of Wollastonia and Lipochaeta are clearly water-dispersed and are associated with beach and sea-cliff habitats across the Hawaiian Islands. For example, Fig. 1J shows a dissected achene of L. integrifolia (Nutt.) A.Gray with a well-developed light brown periderm consisting of layers of cells enclosing large gas filled vacuoles that facilitate buoyancy. Experiments conducted on the widespread W. biflora show that similarly buoyant achenes allow for flotation for at least 90 days in seawater while retaining 30% viability (Nakanishi, 1988). Such viability suggests that W. biflora (and perhaps other species in the alliance, e.g., L. integrifolia) could disperse over considerable distances Version of Record 553 Edwards & al. • Melanthera alliance biogeography and relationships (Compositae) and establish new populations. This ability has the potential to allow repeated dispersals, with divergence followed by re-dispersal of taxa confounding simple reconstruction of biogeographic origins and making the treatment of local insular floras in isolation difficult. An unstable grasp of phylogeny within the Ecliptinae, coupled with the diasporic TAXON 67 (3) • June 2018: 552–564 distribution of the group has made inferring biogeographical patterns difficult, especially within the Pacific where relationships are unclear (notably Lipochaeta and Wollastonia). As currently understood, the closest relatives of these taxa are North American (Panero & al., 1999), rather than from the Australasian region. Fig. 1. Photos of Pacific Ecliptinae taxa. A, Coastal limestone habitat in Guam of Wollastonia biflora showing B, Typical opposite-leaved phyllotaxy and ribbed stem; C, W. uniflora of New Caledonia of sand and lithified sand dune habitats; D, Lipochaeta integrifolia and E, L. rockii of coastal sea cliffs on the Hawaiian Island of Moloka‘i; F, L. remyi of O‘ahu Island showing red involucral bracts; G, L. succulenta of Kaua‘i Island; H, L. lobata of O‘ahu Island; I, L. kamolensis of Maui Island; J, A fruit cross-section of L. integrifolia, widespread across many Hawaiian Islands, highlighting the outermost buoyant light-brown fruit tissue; K, An infructescence with prominent receptacular bracts each surrounding a developing fruit/flower, and L, Typical coastal habitat, here from O‘ahu Island; M, Sphagneticola trilobata, is naturalized across the Hawaiian archipelago and distantly related within the subtribe Ecliptinae. — Photos by J.T. Cantley, except C, which is used with permission from Gildas Gâteblé. Scale bars: A, D & E: 10 cm; B, I & L: 3 cm; C, F–H, K & M: 2 cm; J = 0.5. 554 Version of Record TAXON 67 (3) • June 2018: 552–564 Edwards & al. • Melanthera alliance biogeography and relationships (Compositae) Aims. — In this study, we seek to address the generic boundaries of the Pacific and North American taxa by closing the gaps from previous studies and testing the two prevailing taxonomic hypotheses (Hypotheses 1 and 2). This will allow us to answer questions central to the origin of the Hawaiian Islands and Pacific Ecliptinae Lipochaeta, Melanthera, and Wollastonia including the hybrid origin of the tetraploid Lipochaeta (Hypothesis 3) and the biogeographic origins of Pacific taxa. MATERIALS AND METHODS Sampling and sequencing. — Plant material was obtained from field collections in the early 1990s or between 2011 and 2013, and herbarium specimens (US, PTBG). Voucher specimens, when collected, were deposited at US, PTBG, and/or HAW. DNA was extracted from 33 specimens representing all 16 extant Hawaiian Islands species (including coverage across some varieties), with a further 25 specimens representing all Asia, Pacific, and Neotropical species and some African species of the alliance. Only two of eight Australian Apowollastonia taxa were included. In addition, 28 specimens representing 14 outgroup Ecliptinae genera were included to better understand the position of the alliance within the Ecliptinae. Two species of Montanoa Cerv. (Montanoinae) were used as outgroups beyond the subtribe. Details of the tissue collections are provided in Appendix 1. Vouchers for all samples are indicated, but for many of the Hawaiian material no “direct voucher” was collected at the time of tissue sampling. In some cases, these are rare or endangered species where there is insufficient material to permit the collection of a full herbarium sheet. Similar circumstances can occur when full vouchers are destroyed during sampling, lost, or are logistically impossible to obtain or transport. In these instances, we have retrospectively designated “indirect vouchers” (Funk & al., 2018). Here, if an herbarium voucher specimen has been previously collected from the same locality and authoritatively identified as the species corresponding to the tissue sample then it is nominated as an indirect voucher. Of the 33 Hawaiian Islands samples, 22 have been indirectly vouchered, with 4 unable to be even indirectly validated. The identification and designation of many of these is convoluted (details are presented in Appendix 1) and the process behind establishing indirect vouchers for this study and the general need for nominating vouchers even when primary material is unavailable is presented in Funk & al. (2018). DNA samples were prepared by hand with leaves ground in a mortar and pestle with liquid nitrogen, followed by extraction using a DNeasy Plant Mini Kit (Qiagen, Valencia, California, U.S.A.). Samples older than 1990 and specimens for which DNA preservation protocol was poor at the time of collection were extracted using the QiaAmp DNA Stool Mini Kit (Qiagen), following the manufacturer’s protocols with modified volumes and a lengthened initial incubation period of 2 hours at 70°C. Samples processed immediately after their collection in the 1990s were extracted using a standard CTAB protocol (Doyle & Dickson, 1987). DNA was sequenced for two nuclear (ITS, ETS) and two chloroplast (trnH-psbA, trnQrps16) non-coding regions. The ITS and ETS markers have relatively rapid rates of evolution and have been found to be useful for phylogenetic studies in general, and in particular for Asteraceae (Baldwin, 1992; Baldwin & Markos, 1998). The full internal transcribed spacers 1 and 2 and the 5.8S nuclear ribosomal DNA gene region (ITS) were amplified using the primers ITS4 and ITS5 (after Markos & Baldwin, 2001; White & al., 1990). The external transcribed spacer nuclear ribosomal DNA gene region (ETS) was amplified using the primers ETShel-1 and 18S-ETS, which were specifically designed for the ETS region in the Heliantheae (Baldwin & Markos, 1998). The plastid psbA intron plus spacer was amplified using primers psbA and trnH, after Oxelman & al. (1997). trnQ-rps16 intergenic spacer was amplified using primers trnQ and rps16, after Shaw & al. (2007). All PCR reactions were performed with 25 µl of reaction cocktail containing 12.75 µl of sterilized H 2O, 2.0 µl of 20 mM dNTPs (Pharmacia Biotech, Piscataway, New Jersey, U.S.A.) in an equimolar solution, 2.5 µl of 10× PCR reaction Buffer A (Promega, Madison, Wisconsin, U.S.A.), 1.25 µl of 25 mM MgCl2, 0.5 µl 10 mg/ml Bovine Serum Albumin (Sigma, St. Louis, Missouri, U.S.A.), 1 µl of 10 mM of each of the two primers, 0.5 µl Biolase Red Taq DNA polymerase enzyme (Bioline, Taunton, Massachusetts, U.S.A.) and 4 µl of DNA template. The final amount of DNA template and PCR reaction cocktail and Taq was adjusted as necessary to generate sufficient PCR products for DNA sequencing. ITS amplifications were performed on a Bio-Rad thermal cycler c1000 (Bio-Rad, Hercules, California, U.S.A.) using an initial denaturing step of 94°C, 2 min; followed by 35 cycles of (94°C, 1 min; 50°C, 1 min; 72°C, 2 min), then a final 72°C, 7 min elongation step. Amplification of the ETS region was achieved using the same PCR cycling program as the ITS region with a raised annealing temperature of 62°C. For the plastid trnQ-rps16 intergenic spacer and psbA-trnH spacer the amplification program was an initial denaturing step of 95°C, 2 min, followed by 35 cycles of (95°C, 0.5 min; 57°C, 1 min; 72°C, 2 min) and a final elongation step of 72˚C, 7 min. Samples were purified prior to sequencing using an Exo-Sap enzymatic PCR product pre-sequencing protocol (USB) for 45 min. A final volume of 8.2 µl was used for sequencing reactions. This consisted of 2.0 µl of sterilized H 2O, 3.2 µl of 1 mM primer and 3.0 µl of purified DNA template. Sequencing was conducted at the Advanced Studies of Genomics, Proteomics and Bioinformatics facility at the University of Hawai‘i at Mānoa. All four loci were sequenced for all taxa except Apowollastonia hirbernica (Kel930) which failed to amplify for ITS. Sequences were edited using Sequencher v.3.1.10 (Gene Codes, 1999) and aligned by MUSCLE (Edgar, 2004) using default parameters as implemented in MEGA v.7 (Tamura & al., 2011). Alignments were then manually inspected by eye and adjusted where necessary. Several indels appeared potentially informative, but due to a lack of understanding of indel evolution and concerns about potential bias in coding, all were left uncoded. Version of Record 555 Edwards & al. • Melanthera alliance biogeography and relationships (Compositae) Phylogenetic analyses. — A best-fit model (GTR + I + Γ) was identified for all individual gene regions as well as the combined nuclear dataset using the Akaike information criterion (AIC) in jModelTest v.2 (Posada & Crandall, 1998; Posada & Buckley, 2004). To test for any significant conflict between genes, trees for each individual gene as well as separate analyses for nuclear-only and chloroplast-only markers were constructed under a Bayesian model (BI) implemented in MrBayes (Hueselenbeck & Ronquist, 2001) via the Cyber Infrastructure for Phylogenetic Research (CIPRES) online portal (http://www. phylo.org/). Comparison of topologies (Electr. Suppl.: Fig. S1A–F) showed some unsupported incongruence between chloroplast loci. The concatenated ncDNA (faster evolving) is most strongly congruent with the full combined analysis, recovering monophyly of all ingroup genera (but with Apowollastonia placed sister to Lipochaeta). Concatenated cpDNA shows less resolution, and the unlikely placement of several outgroup taxa (e.g., Blainvillea cunninghamii (DC.) Orchard, B. tenuicaulis (Hook.f.) Benth & Hook.f. and Wedelia goyazensis Gardner) within the ingroup, as well as the pushing of Echinocephalum + Lipotriche outside the expected ingroup. These differences are reflective of the individual loci, with both ncDNA markers returning similar relationships to the full dataset (with differences between the two markers being the placement of Wollastonia inside or outside Echinocephalum + Lipotriche and Melanthera). Individual cpDNA loci lack much resolution within Lipochaeta, and the shuffling of clades and non-monophyly is especially apparent in trnQ when compared to the other markers. Constant across all partitions is the monophyly of Lipochaeta s.l., and the two sections within it are recovered as monophyletic by both ncDNA loci, with the exception of the placement of L. fauriei H.Lév. This taxon (diploid, and sister to the polyploidy group in the combined analysis) is conflicted between nuclear markers, being nested deeply inside the diploid clade in ITS but within the polyploid group for ETS, while sister to diploid taxon for both ncDNA markers. Overall, recovery of five monophyletic ingroup clades was generally consistent across all markers, and with the concatenated dataset representing shared patterns from across these loci, it was used for further analyses. Estimation of phylogeny under a maximum likelihood (ML) model was implemented in RAxML v.7.0.4 (Stamatakis, 2006) with nonparametric bootstrap replicates (1000) calculated with the thorough bootstrap replicate option selected and allowing all free model parameters to be estimated. For BI analysis in MrBayes the concatenated dataset was subjected to Markov Chain Monte Carlo (MCMC) sampling performed with two replicates of four chains (one hot, three cold) each with a heating temperature of 0.2. Twenty-five million generations were completed with sampling occurring every 1000 generations. A discarded burn-in period was estimated by visually inspecting plotted log likelihood values versus generation time to determine the point at which convergence had been reached using Tracer v.1.5 (Rambaut & Drummond, 2009). The remaining trees were combined in FigTree v.1.3.1 (Rambaut, 2009) to construct a consensus tree where Bayesian posterior probabilities (PP) were calculated for internal node support of the resulting phylogenetic reconstruction. 556 TAXON 67 (3) • June 2018: 552–564 Molecular clock analyses. — Divergence times were estimated using BEAST v.2.4.5 (Drummond & al., 2006; Bouckaert & al., 2014) under two scenarios: (i) an analysis with all taxa, and (ii) an analysis without the tetraploid Hawaiian Islands Lipochaeta taxa. Tetraploid taxa were removed to assess the potential effect of reticulate evolution on the inferred phylogenetic reconstructions and divergence times under a hypothesis of a hybrid origin of these taxa. Two African taxa Lipotriche richardsiae (Wild) D.J.N.Hind and L. scandens (Schumach.) Orchard were removed from the BEAST analyses as when they were included the topologies recovered were highly discordant from those using MrBayes and RAxML. Reasons for this are unclear, but removal of these two taxa rectified the issue, returning a topology consistent with the other methods. Analyses both with and without Hawaiian polyploid taxa used a fossil constraint and information on the ages of island in the Hawaiian Archipelago for time calibration. The Compositae generally lack trustworthy macrofossils that are useful for dating the family and its subdivisions, but an appropriate dated macrofossil is available that dates the root of our tree to younger than 0.5–45.0 Ma (Macphail & Hill, 1994; Martínez-Millán, 2010). The node representing all Hawaiian Islands taxa was loosely constrained between 0.0 and 29.0 Ma based on the estimated age of continuous emergent land suitable for colonization (Clague & al., 2010). For both analyses an .xml file was generated with BEAUti v.2.4.5 (Bouckaert & al., 2014) and edited allowing parameters to be estimated for each partition. All sequence evolution priors were set as default except for the tree shape, which was set to follow a birth-death speciation process, and the parameters relating to a relaxed clock. The substitution model was the same as used in MrBayes (GTR + I + Γ) and an uncorrelated lognormal molecular clock with a Yule prior was used for branch lengths. Several short runs were performed to examine the optimal performance of the prior and a final run of 30 million Markov Chain Monte Carlo (MCMC) generations (sampled every 1000) was completed for each analysis. Convergence of the stationary distribution and effective sample sizes were checked by visual inspection of plotted posterior estimates using the software Tracer v.1.5 (Rambaut & Drummond, 2009). After discarding the first 7500 trees as burn-in, the samples were summarized as a maximum clade credibility tree using TreeAnnotator v.1.6.1 (Rambaut & Drummond, 2009) with the posterior probability limit set to 0.5 and summarizing mean node heights. The results for both runs were visualized using FigTree v.1.3.1 (Rambaut, 2009). RESULTS The phylogenetic reconstructions estimated under BI and ML analyses showed significant topological congruence for nodes with posterior probability (PP) > 0.5 and bootstrap (BS) values > 50% so we present just the BI topology and indicate PP values > 0.90 and BS values > 70 (Fig. 2A). Similarly, the two BEAST molecular clock analyses (all taxa or with tetraploid Hawaiian Islands taxa removed) were largely congruent in topology and estimated node ages, and showed only minor topological Version of Record TAXON 67 (3) • June 2018: 552–564 Edwards & al. • Melanthera alliance biogeography and relationships (Compositae) differences from the BI and ML analyses. The two dated analyses did not differ from each other in topology, but there were minor differences in HPD node estimates. Differences between the BEAST (dated) and BI/ML reconstructions include the positioning of the outgroup Eclipta L., which is nested considerably deeper into the phylogeny in the dated analysis. Broadly, the data show strong support for the monophyly of subtribe Ecliptinae, and for much of the backbone of the phylogeny. Some of the relationships between the outgroup genera have not previously been suggested, and there is also evidence that Wedelia is polyphyletic as currently recognized: Wedelia calycina Rich. falls into a clade that is the sister group of its congeners along with species from a number of other genera, and W. reticulata DC. nested even further away from these (Fig. 2A). Given the tortured history of Wedelia (Orchard, 2013), this non-monophyly is not surprising. It should be noted that four taxa previously considered Aspilia but currently considered Wedelia sensu Orchard (W. subpetiolata (Baker) B.L.Turner, W. kotschyi (Sch.Bip.) Soldano, and two unidentifiable to species level), form a distinct clade suggesting further investigation of these relationships may support previous taxonomies. The two other outgroup genera with multiple accessions form wellsupported monophyletic clades (Blainvillea, Lundellianthus H.Rob.). The Melanthera alliance as a whole is sister clade to the genus Perymenium Schrad. of western North America, similar to the relationship recovered by Panero & al. (1999) who found Perymenium + Lundellianthus to be the sister group, but Lundellianthus is more distantly placed in our phylogeny. Four main clades were recovered within the alliance: (a) Echinocephalum + Lipotriche, (b) Lipochaeta + Hawaiian Islands Wollastonia, (c) non-Hawaiian Islands Wollastonia, and (d) Melanthera, with all but the relationship between Lipochaeta and Wollastonia (BS of 64) well supported. These results are not favourable for the ongoing recognition of a twogenus taxonomy (Hypothesis 1; Wagner & Robinson, 2002) being more (but not entirely) consistent with the alternative five-genus hypothesis of Orchard (2013). Contrary to both competing taxonomies, all Hawaiian Islands taxa are recovered as a single well-supported lineage with tetraploids (Lipochaeta) nested within the diploid clade (Wollastonia). The remaining Pacific & Indian Ocean Wollastonia form a clade that is the sister group of the Hawaiian Islands species, with these two clades sharing a most recent common ancestor with the white-flowered North American Melanthera (Fig. 2A). African Lipotriche and South American Echinocephalum form a well-supported clade that is the sister group of the rest of the alliance. Of note is that Indocypraea, a newly erected monotypic genus is nested well within Wollastonia, the genus within which this taxon was previously recognized. The molecular clock analyses recovered the stem age of all taxa from the Hawaiian Island as 0.73–1.79 million years, Wollastonia s.l. (including Lipochaeta) as 1.19–1.79 million years (Electr. Suppl: Fig. S2). The divergences that established African Lipotriche + South American Echinocephalum and southeast North American Melanthera are estimated to have occurred 0.97–2.01 Ma and 0.58–2.01 Ma, and indicate that Pacific lineages arose during the Pliocene or Pleistocene. Dates for deeper divergences have considerably wider error margins, with the emergence of the Ecliptinae estimated as sometime during the mid to late Miocene. DISCUSSION Taxonomy within the alliance. — Our results do not favour Hypothesis 1 and the broader generic concepts of Wagner & Robinson (2002), with well-supported and well-defined clades corresponding to previously recognized genera within their concept of Melanthera s.l. (Fig. 2B). Instead, relationships are more closely congruent with those of Orchard (2013; Hypothesis 2) with the exception that Hawaiian Islands members of the complex, currently recognized as belonging to two distinct genera (Lipochaeta, Wollastonia), form a well-supported monophyletic group, but with one (Lipochaeta) rendering the other (Wollastonia) paraphyletic (see below). Otherwise, members of the alliance fall into five clearly recognisable and wellsupported clades: Hawaiian Wollastonia + Lipochaeta are the sister group of the Pacific and Asian members of Wollastonia— if all the Hawaiian members of Wollastonia are returned to Lipochaeta then both genera are monophyletic. These clades are diagnosable molecularly and geographically, with the Hawaiian Island clade following a distinct evolutionary trajectory in isolation. Morphological diagnostics for Lipochaeta and Wollastonia under this concept are not currently available, and further work addressing this would be useful for field identification. The alternative—a sinking of Lipochaeta into Wollastonia—results in a more readily diagnosable taxon morphologically, however obscures molecular and geographic resolution. While non-monophyletic relationships are acknowledged to be a property of early cladogenesis, decisions on assigning taxon rank above species are ultimately subjective. As genera are an artificial construct, decisions on at what point a lineage is sufficiently distinct are largely philosophical, and the argument that morphological differences between the diploid and polyploidy taxa here (4-merous vs. 5-merous florets) indicate a sufficient degree of differentiation for generic status is hard to support. Our data show little genetic difference between the two groups, with separation clearly very recent, and while isolation can be predicted by degree of molecular divergence (Orr, 2005), no such rule exists for morphological differences. Similarly, while karyotypic differentiation has certainly greatly reduced fertility between the two ploidy groups, crosses are still possible (Rabakonandrianina, 1980), and similar instances of recent isolation between other species of different ploidy within the tribe (e.g., the Helianthus annuus L. complex; Kantar & al. 2014) are not considered worthy of generic status. The molecular data presented here support the recognition of a combined genus of diploid plus polyploid species, equivalent in divergence to their sister genera. We take this, in combination with geographic unity and minimal internal divergence, as evidence to refer to all the Hawaiian Island taxa as Lipochaeta from here on. If distinction between diploid and polyploid groups is desired, informal infra-generic names already exist (diploid = sect. Aphanopappus; polyploid Version of Record 557 Edwards & al. • Melanthera alliance biogeography and relationships (Compositae) monophyletic group, it renders Orchard’s broad concept of Wollastonia s.l. paraphyletic with little advantage in erecting additional genera in order to retain recognition. Even attempting to recognize the diploid Hawaiian Islands taxa alone as a novel genus is problematic as our 4-locus and combined ncDNA data suggest that Lipochaeta fauriei is more closely related to the tetraploid taxa than the rest of the diploid group (although there is conflict in this relationship between individual loci). A return to a more inclusive classification and rejection of the ploidy-based separation of Hawaiian Islands taxa requires no nomenclatural changes and best represents the evolutionary origins of the lineage as currently understood. These results do not confirm a hybrid Lipochaeta (n = 15)-Wollastonia (n = 11) origin for the tetraploid Lipochaeta (n = 26), with no obvious phylogenetic conflict that would be consistent with divergent parental origins. Any allopolyploid event would appear to have likely involved members from within the Hawaiian Islands Lipochaeta and their immediate ancestors, or be the result of an autopolyploidization event with subsequent chromosome loss. Polyploidy followed by descending dysploidy is welldocumented in the Compositae, including Heliantheae (Baldwin & al., 2002; Watanabe & al., 2007; Semple & Watanabe, 2009). Our data are based on a relatively modest set of loci and signatures of hybrid origin may well have been largely ameliorated through multigenerational backcrossing to a Lipochaeta-like lineage or concerted evolution of the markers sampled (Fuertes Aguilar & al., 1999). Further investigation of hybrid origins would benefit from high-throughput deep-sequencing methods. The placement of diploid Lipochaeta fauriei as the sister taxon to the tetraploid clade is intriguing and exploring the ancestry of this relationship could prove fruitful. It should be noted that L. fauriei is represented by an “indirect voucher” (Funk & al., 2018) in our dataset and there is no way to definitively check its identity. At a finer scale, sampling of multiple individuals per species reveals what appears to be non-monophyly in three Hawaiian taxa, Lipochaeta lavarum (Gaudich) DC., L. lobata (Gaudich.) DC., and L. succulenta (Hook. & Arn.) DC., with each accession having a sister-relationship to a different species. Similarly, Wollastonia biflora is represented by four lineages that do not share a most recent common ancestor, and geographic variation has previously been indicated in each of these species with the recognition of distinct varieties. It is possible that each lineage represents a parallel evolution of a common morphotype in response to similar environmental conditions resulting in non-sister cryptic species. This would not be unlike the evolution of analogous morphotypes in response to similar niches during the process of adaptive radiation inferred in the related Silversword group, also found on Fig. 2. A, Bayesian phylogenetic reconstruction for the informal Melanthera alliance and Ecliptinae outgroups. Circles above nodes represent BI posterior probability (PP) and those below nodes are ML bootstrap support (BS), values are: black circles ≥ 0.95(PP)/95%(BS); dark grey circles ≥ 0.85/85%; light grey circles ≥ 0.75/75%; white circles ≥ 0.70/70%. Taxonomy follows Orchard (2013) except for diploid Hawaiian Island taxa which are recognized as Lipochaeta. Colored circles indicate polyphyletic taxa. B, Diagrammatic representations of the phylogeny under the two competing taxonomic hypotheses tested are also presented. C, Map of the distribution of species colored according to the phylogeny. D, Simplified phylogeny for Lipochaeta, Wollastonia, Apowollastonia, and Melanthera showing the four equally parsimonious dispersal hypotheses (H1–4 in text) with 3 dispersal events each denoted by red stars. Color of branches indicates region of occupation per the map in B. H = Hawaiian Islands; A/P = Asia-Pacific; AU = Australia; AM = the Americas. 558 Version of Record ◄ = sect. Lipochaeta). As such, Lipochaeta (all Hawaiian Islands taxa) is the sister taxon of Wollastonia, and the two are in turn the sister clade to the Australian genus Apowollastonia, thus forming a large well-defined Asia-Pacific clade. This AsiaPacific clade is the sister group of the white-flowered North and Central American Melanthera clade. The earliest-diverging clade of the alliance contains two monophyletic genera, the monospecific Echinocephalum (South America) and Lipotriche (Africa) and is the sister group of the rest of the Melanthera alliance. Despite geographic separation, the close relationship between the South American and African genera is perhaps not surprising given a number of shared similarities in habitat preference (open savannas) and close association with fresh water. The placement of Indocypraea well within Wollastonia is also not entirely surprising given that it has traditionally been recognized in this genus, but is at odds with the findings of Ren (2016) where, while other relationships are broadly congruent with ours (e.g., a Wollastonia /Lipochaeta /Melanthera clade), representatives of Indocypraea fall well outside the ingroup as considered here. The use of chloroplast loci only in the previous study makes it hard to lend much weight to this less intuitive relationship, especially given the confused and possibly reticulate origins of these genera, which would require nuclear data to unravel. Further work on this taxon may help resolve this conflict, but our results, combining both nuclear and plastid loci, indicate that Wollastonia montana is best treated as a morphologically anomalous species not warranting generic status. Although we were unable to sample the two remaining genera described by Orchard (2013), a strong signal of geographic regionalization predicts Acunniana to be closely related to Apowollastonia with which it is partly sympatric, while Lipoblepharis and Quadribractea could be expected to align with Asian members of Wollastonia, the genus from which they were recently separated. Other relationships across outgroups representing the Ecliptinae subtribe are mostly straightforward except for Wedelia where taxa fall into four distinct clades rendering the genus highly paraphyletic. That Wedelia may represent another complex taxonomic knot is not unexpected (Wagner & Robinson, 2002; Orchard, 2012) and it is hoped that increased sampling and representation of taxa in future studies will be able to properly address relationships within this group. Pacific and Hawaiian islands taxa. — The recovery of a well-supported monophyletic clade representing the Hawaiian Islands members of the Ecliptinae (see above) is consistent with a traditionally recognized single genus (Lipochaeta sensu Gardner, 1979). While the tetraploid Lipochaeta appear to have a single origin and form a moderately well supported TAXON 67 (3) • June 2018: 552–564 TAXON 67 (3) • June 2018: 552–564 C. D. Edwards & al. • Melanthera alliance biogeography and relationships (Compositae) (M08) Lipochaeta integrifolia Kaua‘i (M11) Lipochaeta integrifolia Lana‘i (M01) Lipochaeta integrifolia O‘ahu (M02) Lipochaeta lavarum Lana‘i (M09) Lipochaeta integrifolia Moloka‘i (M15) Lipochaeta lavarum var. ovata Maui (M100) Lipochaeta lavarum Kaho‘olawe Hawaiian Islands (M05) Lipochaeta subcordata Hawai‘i (diploid) (M13) Lipochaeta lavarum Maui (M14) Lipochaeta lavaraum var. hillebrandiana Maui (M21) Lipochaeta venosa Hawai‘i (M12) Lipochaeta kamolensis East Maui (M26) Lipochaeta subcordata Hawai‘i (M10) Lipochaeta integrifolia Hawai‘i (M22) Lipochaeta waimeaensis Kaua‘i (M03) Lipochaeta micrantha var. exigua Kaua‘i Lipochaeta (M06) Lipochaeta tenuis O‘ahu (M19) Lipochaeta tenuifolia O‘ahu (M20) Lipochaeta tenuis O‘ahu (M04) Lipochaeta remyi O‘ahu (M39) Lipochaeta succulenta Hawai‘i (M40) Lipochaeta succulenta Maui (M31) Lipochaeta heterophylla Lana‘i (M33) Lipochaeta lobata Maui (M27) Lipochaeta connata var. acris Maui (M29) Lipochaeta connata var. connata Kaua‘i (M30) Lipochaeta heterophylla Moloka‘i (M37) Lipochaeta rockii Moloka‘i Hawaiian Islands (M28) Lipochaeta connata var. acris Kaua‘i (polyploid) (M36) Lipochaeta rockii var. dissecta Maui (M41) Lipochaeta succulenta Kaua‘i (M32) Lipochaeta lobata O‘ahu (M44) Lipochaeta fauriei Kaua‘i (M23) Wollastonia biflora Japan (M48) Wollastonia biflora Taiwan Asia (M82) Wollastonia dentata Taiwan (M83) Wollastonia (Indocypraea) montana China (M102) Wollastonia biflora Rapa Iti Wollastonia (M81) Wollastonia biflora PNG (M103) Wollastonia biflora Marshall Islands (M104) Wollastonia biflora Pohnpei South Pacific (M24) Wollastonia biflora var. canescens Guam (M51) Wollastonia lifuana New Caledonia (M101) Wollastonia lifuana New Caledonia (Kel931) Apowollastonia stirlingii subsp. fontaliciana Apowollastonia (Kel930) Apowollastonia hirbernica Australia (M47) Melanthra angustifolia Florida (M54) Melanthera parvifolia Florida Melanthera (M53) Melanthera nivea Georgia North America & Caribbean (M52) Melanthera nivea Florida (M69) Lipotriche scandens Uganda (M73) Lipotriche scandens Madagascar (M70) Lipotriche scandens Ghana Lipotriche (M72) Lipotriche triternata Namibia Africa (M79) Lipotriche rhombifolia Mali (M80) Lipotriche scandens subsp. dregei Guinea (M49) Echinocephalum latifolium Uruguay South America (M50) Echinocephalum latifolium Brazil Echinocephalum A. H A/P H1 AU AM H A/P H2 AU AM H A/P H3 AU AM H A/P H4 AU AM sensu Orchard (2013) (M59) Perymenium berlandieri Mexico (M66) Wedelia reticulata Puerto Rico (M110) Wedelia reticulata Puerto Rico (M67) Tilesia macrocephala Ecuador (M60) Perymeniopsis ovalifolia Mexico (M61) Riencourtia latifolia Brazil (M87) Blainvillea gayana Botswana (M88) Blainvillea acmella India (M84) Blainvillea rhomboidea Africa (M85) Blainvillea tenuicaulis Ecuador (M86) Blainvillea cunninghamii Australia  (M55) Wedelia subpetiolata Brazil (M71) Wedelia kotschyi Namibia  ex-Aspilia (M75) Wedelia sp. Tanzania (M76) Wedelia sp. Tanzania  (M58) Oyedaea boliviana Bolivia (M63) Steiractinia sodiroi Ecuador (M65) Wedelia calycina DR (M109) Elaphandra patentipilis Colombia (M68) Zexmenia virgulta Costa Rica (M90) Wedelia brachylepis Brazil (M91) Wedelia buphtalnifolia Argentina (M89) Wedelia goyazensis Brazil (M64) Wedelia acapulcensis Mexico (M106) Lundellianthus guatemalensis Guatemala (M107) Lundellianthus salvinii Guatemala (M62) Sphagneticola trilobata O‘ahu B. sensu Wagner & Robinson (2002) Ecliptinae outgroups (naturalized from Central America) (M57) Eclipta prostrata Virginia M94 Montanoa hibiscifolia Kaua‘i (naturalized from Mexico) M95 Montanoa karwinskii Mexico Montanoinae outgroups Version of Record 0.20 559 Edwards & al. • Melanthera alliance biogeography and relationships (Compositae) the Hawaiian Islands (Baldwin, 2006). Alternatively, recent island colonisation could lead to the nesting of distinct peripatric species in a phylogenetic reconstruction where gene trees are incompletely sorted, or in a third scenario, where there is gene exchange between a wide-ranging species and local taxa via hybridization and potentially speciation with gene-flow. All three possibilities would not be unexpected given the nature of these species, but plausibility for the local hybridization hypothesis is particularly strong in W. biflora, a cosmopolitan and highly variable species with multiple accessions from across its geographic range clustering more closely with other geographically proximal species than with each other (Fig. 2C). Considerably more sampling and data are required for any implications to be drawn here and one must keep in mind that a number of the samples used in this study do not have “direct vouchers” thus their species designation could be questioned (Funk & al., 2018). Exploring which of these processes may be at play in the Hawaiian Island taxa and whether inter-species introgression explains regional paraphyly in an otherwise widely distributed species such as W. biflora would be of considerable interest from an evolutionary perspective while assisting in refining species level taxonomy within the group, and the treatment of Indocyprea. Biogeographic implications. — Our phylogenetic data supports a strong biogeographic pattern of dispersal events followed by localized radiations, with each genus within the Melanthera alliance largely confined to a discrete region without overlapping distributions. Our data also shows that dates for divergences between clades fall within the ages of all of the land-masses under consideration, including currently emergent Hawaiian Islands (< 6 Ma; Lim & Marshall, 2017) making any region a plausible sink or source. It should be noted that one of the two calibration points for the analysis was derived from the age of the islands, although very loosely constrained. The large number of Ecliptinae outgroups from the Americas gives weight to the hypothesis that this was the centre of origin from which the alliance arose, however resolving dispersal patterns is problematic in any group with high long-distance dispersal potential, without a good fossil record, and with no relevant biogeographic calibration points (e.g., verifiable vicariance events) especially when coupled with the possibility of extinction confounding correct ancestral reconstruction, or high asymmetrical dispersal probability (Cook & Crisp, 2005). Assuming an American origin for the group, the most parsimonious reconstruction for the history of Lipotriche, nested between American clades, is the dispersal of an ancestor from the Americas to Africa 0.72–0.97 Ma. While it is impossible to rule out two separate instances of dispersal from Africa to the Americas, or concurrent dispersal to Africa and South America from another location where the group has subsequently become extinct, these scenarios require additional steps (although see Crisp & al., 2011 for problems in assumption of parsimony in biogeographic reconstruction). For the remaining colonization events, there are four most phylogenetically parsimonious hypotheses each requiring three dispersal events (Fig. 2D), and each with greater or lesser plausibility given what we know about biogeography of the regions from other groups: 560 TAXON 67 (3) • June 2018: 552–564 ● H1 reconstructs dispersal from the Americas to Australia, back through the Asia-Pacific and ultimately to the Hawaiian Islands. Direct east-west dispersal from the Americas to Australia is highly unusual (Cook & Crisp, 2005) requiring the traversal of long distances despite the direction being generally favourable for equatorial ocean currents. Movement from Australia into the Asia-Pacific is relatively frequent (Crisp & al., 2009), and dispersal from Asia-Pacific to the Hawaiian Islands is also consistent with the majority of Hawaiian Island plant colonisations (Wagner & al., 1999; Keely & Funk, 2011). ● H2 also requires an initial dispersal to Australia and then a subsequent colonization of the Hawaiian Islands followed by movement out into the Asia-Pacific region. This scenario suffers both from the low probability of a dispersal from Australia directly to the Hawaiian Islands (approximately 5% of studied dispersals—Wagner in prep.) and an extremely rare dispersal event out of the Hawaiian Islands (fewer than a dozen lineages—Baldwin & Wagner, 2010; Keeley & Funk, 2011). Poor dispersal of endemic Hawaiian Island taxa may often be the result of a loss of dispersal mechanisms (common to island species), something that does not seem to have happened to many members of the Melanthera alliance, including some Hawaiian Island species (Lipochaeta) which have retained their seed buoyancy chambers. ● H3 proposes an initial Americas to the Asia-Pacific dispersal with subsequent colonisation of both Australia and the Hawaiian Islands from there. This scenario benefits from individual movements of relatively short distances (South America to Rapa Iti is almost half the distance of South America to Australia), including the potential for island hopping through the Pacific as a means of distribution to other regions, and is the more common pathway in to the Hawaiian Islands as noted in H2 above. ● H4 reconstructs dispersal directly from the Americas to the Hawaiian Islands with two subsequent dispersal events from there to Australia and to Asia-Pacific. This pathway requires shorter dispersal distances, and an initial movement from the Americas to the Hawaiian Islands is consistent with several other Compositae lineages most notably Bidens L. (Ganders & al., 2000; Knope & al., 2012) and the Silversword alliance (Baldwin & Wagner, 2010) where ancestry and dates of divergence unequivocally support direct colonization from the continent. But, the overall proportions of dispersal events from the Americas to the Hawaiian Islands is relatively low (North Temperate ~9%; Neotropical ~13%) (Wagner, in prep.) and as with H2 this scenario suffers from the rarity of dispersal events out of the Hawaiian Islands. Even with the addition of supporting information and what is known from other plant groups, any of the four hypotheses outlined above are plausible. The authors are divided on whether to favour H1 or H3, but in agreement that H4 is unlikely and H2 is least probable. Clearly long-distance dispersal has happened multiple times, but without knowing the probability of each hypothesized event, determining the most likely scenario can be subjective. Developing probabilistic mathematical models for dispersal events from data across plant groups, taking into account direction, time-frame, size of sinks and sources, Version of Record TAXON 67 (3) • June 2018: 552–564 Edwards & al. • Melanthera alliance biogeography and relationships (Compositae) and hypothesized dispersal mechanisms would be valuable for predicting or reconstructing movement across regions for these taxa and other lineages. Additionally, it should be noted that recently described taxa (Orchard, 2013) not sampled in this study—namely Acunniana (Australia), Lipoblepharis (India to Japan) and Quadribractea (Malesia) plus the seven nonsampled taxa of Apowollastonia (Australia) could further confuse or help clarify any current interpretation of the dispersal patterns if included in future studies. CONCLUSIONS A stable taxonomy is beginning to emerge for the Melanthera alliance at a generic level, with progress toward a better understanding of the Ecliptinae as a whole. We support the recognition of five genera: Lipochaeta as a genus of all species endemic to the Hawaiian Islands, Wollastonia including the Asia, Indian Ocean and southern Pacific taxa, Melanthera from southeastern U.S.A. and coastal areas of the Caribbean, southern and meso-America, Lipotriche from Africa, and Echinocephalum from South America. We can neither support nor reject a hybrid origin for the tetraploid Lipochaeta from any ancestor outside the Hawaiian Islands Lipochaeta group. Even with good phylogenetic resolution we encounter difficulty in reconstructing biogeographic origins for highly dispersed taxa with few lines of complimentary evidence, although an American origin for the alliance as a whole appears most likely. Numerous non-monophyletic species suggest that establishing a completely stable taxonomy may continue to prove problematic, but further research into these species has the potential to derive many interesting questions about gene flow and species identity in a highly mobile group spread across geographic regions. The framework we provide is important both for identifying areas that require improved sampling, and for refining and testing hypotheses of the biogeography and evolution of this complex and dynamic group of plants. AUTHOR CONTRIBUTIONS VAF conceived, designed and funded the project. Fieldwork was conducted by VAF, SCK, JTC, and MMC, lab work by SCK, and data alignment and analysis by JTC and RDE. The manuscript was written by RDE and JTC with contributions from VAF. — RDE, http://orcid. org/0000-0002-4993-2453: SCK, http://orcid.org/0000-0003-1581-6478; VAF, http://orcid.org/0000-0002-7975-1450 ACKNOWLEDGEMENTS The authors thank Gildas Gâteblé (Jardin de IAC St.-Louis, New Caledonia), Jun Wen (Smithsonian Institution) and Kuo-Fang Chung (National Taiwan University, Taiwan) for providing field collections, Peter C. Jobson (Northern Territory Herbarium [NT], Alice Springs, Australia) and Christopher T. Martine (Bucknell University, Lewisburg, Pennsylvania, U.S.A.) for assisting in and support of Apowollastonia collections, Raymund Chan and Carol Kelloff (Smithsonian Institution) for the Apowollastonia sequencing work, Warren Wagner (Smithsonian Institution) for insightful comments and discussion of the manuscript, and two anonymous reviewers for their time. 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Manual of the Flowering Plants of Hawai‘i, ed. 2, 2 vols. Honolulu: University of Hawai‘i and Bishop Museum Press. Watanabe, K., Yahara, T., Hashimoto, G., Nagatani, Y., Soejima, A., Kawahara, T. & Nakazawa, M. 2007. Chromosome numbers and karyotypes in Asteraceae. Ann. Missouri Bot. Gard. 94: 643—654. https://doi.org/10.3417/0026-6493(2007)94[643:CNAKIA]2.0.CO;2 White, T.J., Bruns, T., Lee, S. & Taylor, J.W. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315–322 in: Innis, M.A, Gelfand D.H., Sninsky J.J. & White, T.J. (eds.), PCR protocols: A guide to methods and applications. San Diego: Academic Press. https://doi.org/10.1016/ B978-0-12-372180-8.50042-1 Version of Record TAXON 67 (3) • June 2018: 552–564 Edwards & al. • Melanthera alliance biogeography and relationships (Compositae) Appendix 1. Taxa and GenBank accession numbers for specimens studied. Voucher data are in the following format: taxon name, extraction number (for reference across studies), country, locality, collector and collection number (with asterisk if indirect voucher), original collection information if indirect voucher was required, ITS, ETS, trnH-psbA, trnQ-rps16 GenBank accession number. All sequences were newly obtained for this study. Apowollastonia hirbernica Orchard, Kel930, Australia, Northern Territory: Limmen National Park, J. Cantley & al. 1117 (US), N/A, –, MH151956, MH152044, MH152219; Apowollastonia stirlingii subsp. fontaliciana Orchard, Kel931, Australia, Northern Territory: Standley Chasm Trail, J. Cantley & al. 1118 (US), N/A, MH152132, MH151957, MH152045, MH152220; Blainvillea acmella (L.) Philipson, M88, India, Punjab: Topi Park, R.R. Stewart 15114 (US), N/A, MH152213, MH152038, MH152126, MH152301; Blainvillea cunninghamii (DC.) Orchard, M86, Australia, Queensland: Roko Island, B.M. Waterhouse 6351 (US), N/A, MH152211, MH152036, MH152124, MH152299; Blainvillea gayana Cass., M87, Botswana, Ngamiland, P.A. Smith 5696 (US), N/A, MH152212, MH152037, MH152125, MH152300; Blainvillea rhomboidea Cass., M84, Brazil, Espirito Santo Reserva Florestal de Linhares ex Africa, D.A. Folli 2611 (US), N/A, MH152209, MH152034, MH152122, MH152297; Blainvillea tenuicaulis (Hook.f.) Benth & Hook.f., M85, Ecuador, Galapagos: Albemarle Island, J.T. Howell 9487 (US), N/A, MH152210, MH152035, MH152123, MH152298; Echinocephalum latifolium Gardner, M49, Uruguay, Paysandu: near Uruguay River, M. Bonifacino & al. 4291 (US), N/A, MH152178, MH152003, MH152091, MH152266; Echinocephalum latifolium Gardner, M50, Brazil, Bahia: Salvador, M.B.B. Alves 27 (US), N/A, MH152179, MH152004, MH152092, MH152267; Eclipta prostrata (L.) L., M57, U.S.A., Virginia: Falls Church, V.A. Funk 12780 (US), N/A, MH152185, MH152010, MH152098, MH152273; Elaphandra patentipilis (S.F.Blake) Pruski & G.P. Mèndez, M109, Colombia, Narino Ricuarte Mun, T.B. Croat 71511 (US), N/A, MH152149, MH151974, MH152062, MH152237; Indocypraea montana (Blume) Orchard, M83, China, Guangxi: Guilin, E.S. Chow & Wan 79087 (US), N/A, MH152208, MH152033, MH152121, MH152296; Lipochaeta connata var. acris (Sherff) W.L.Wagner & H.Rob., M27, U.S.A., Hawaii (Maui) Canyon E of Iao Needle, R.W. Hobdy 2083* (BISH), R. Hobdy & S. Keeley s.n.: near Iao Needle, Mar 1993, MH152163, MH151988, MH152076, MH152251; Lipochaeta connata var. acris (Sherff) W.L.Wagner & H.Rob., M28, U.S.A., Hawaii (Kaua‘i) Kalalau Valley: Kalalau Trail from Hanakapiai to Hoolulu, T. Flynn 424* (PTBG), S. Keeley s.n.: Kauai Kalalau trail past Hanakapiai, Apr 1993, MH152164, MH151989, MH152077, MH152252; Lipochaeta connata var. connata (Gaudich.) DC., M29, U.S.A., Hawaii (Kaua‘i) Waimea Canyon: Koaiae River 1–2 miles upstream from Lonomea Shelter, Lorence & al. 6774* (PTBG), S. Keeley s.n.: Waimea Canyon, Jul 1992, MH152165, MH151990, MH152078, MH152253; Lipochaeta fauriei H.Lév., M44, U.S.A., Hawaii (Kaua‘i) Haeleele Ridge, Wood & Lau & Perlman 271* (PTBG), N/A, Puu ka Pele Forest Reserve Haeleele Ridge, MH152175, MH152000, MH152088, MH152263; Lipochaeta heterophylla A.Gray, M30, U.S.A., Hawaii (Moloka‘i) Moomomi Beach, Hobdy & Keeley & Baker 3577 (US), N/A, MH152166, MH151991, MH152079, MH152254; Lipochaeta heterophylla A.Gray, M31, U.S.A., Hawaii (Lana‘i) Keamoku Rd, Hobdy & Keeley & Baker 3578 (US), N/A, MH152167, MH151992, MH152080, MH152255; Lipochaeta integrifolia (Nutt.) A.Gray, M01, U.S.A., Hawaii (O‘ahu) Kaena Point, V.A. Funk, M.M. Chow, J. Cantley & B. Gagne 12784* (US), S. Keeley & J. Obata 4567: Kaena Point 31, Mar 1992, MH152133, MH151958, MH152046, MH152221; Lipochaeta integrifolia (Nutt.) A.Gray, M08, U.S.A., Hawaii (Kaua‘i – collected O‘ahu) Bishop Museum: Atherton Halau, J. Lau 1807* (BISH), S. Keeley s.n.: Waimea Falls Park from cutting #74c2094A, 27 Apr 1992, MH152139, MH151964, MH152052, MH152227; Lipochaeta integrifolia (Nutt.) A.Gray, M09, U.S.A., Hawaii (Moloka‘i) Moomomi Beach, E. Rabakonandrianina RB107* (BISH), R. Hobdy & S. Keeley s.n.: Moomomi Beach, 2 Mar 1993, MH152140, MH151965, MH152053, MH152228; Lipochaeta integrifolia (Nutt.) A.Gray, M10, U.S.A., Hawaii (Hawai‘i) South Point, C. Corn ESP224* (BISH), S. Keeley s.n.: Hawaii Isl. South Point, Jul 1992, MH152141, MH151966, MH152054, MH152229; Lipochaeta integrifolia (Nutt.) A.Gray, M11, U.S.A., Hawaii (Lana‘i) Poaiwa, O. Degner 28377* (US), R. Hobdy, S. Keeley & Baker s.n.: Kukui Point, Mar 1993, MH152150, MH151975, MH152063, MH152238; Lipochaeta kamolensis O.Deg. & Sherff, M12, U.S.A., Hawaii (Maui) Kahikinui: West slope of Kamole Gulch, R.W. Hobdy 2293* (BISH), R. Hobdy & S. Keeley s.n.: Maui Kamole Gulch, 26 Mar 1993, MH152152, MH151977, MH152065, MH152240; Lipochaeta lavarum (Gaudich.) DC., M02, U.S.A., Hawaii (Lana‘i) Kahea Gulch, D. Orr s.n.* (BISH), S. Keeley s.n.: Waimea Falls Park from seedlot #89s47, 27 Apr 1992; original collection from Kahia Lanai, MH152134, MH151959, MH152047, MH152222; Lipochaeta lavarum (Gaudich.) DC., M100, U.S.A., Hawaii (Kaho‘olawe) Makawao District, K.R. Wood, S. Perlman, J. Lau & C. Rowland 1719 (PTBG), N/A, MH152142, MH151967, MH152055, MH152230; Lipochaeta lavarum (Gaudich.) DC., M13, U.S.A., Hawaii (Maui) Lihua Peak lower slopes above Puuhipa, R.W. Hobdy 878* (BISH), W. Char s.n.: West Maui, May 1993, MH152153, MH151978, MH152066, MH152241; Lipochaeta lavarum var. hillebrandiana Sherff, M14, U.S.A., Hawaii (Maui) Hanaula Rd, R.W. Hobdy s.n.* (US), R. Hobdy & S. Keeley s.n.: Kings Trail at Hanaula Rd, 31 Mar 1993, MH152154, MH151979, MH152067, MH152242; Lipochaeta lavarum var. ovata Sherff, M15, U.S.A., Hawaii (Maui) South Coast: West end of Kanaio Beach, R.W. Hobdy 1285* (BISH), R. Hobdy & S. Keeley s.n.: Maui Kanaio pop2, Apr 1993, MH152155, MH151980, MH152068, MH152243; Lipochaeta lobata (Gaudich.) DC., M32, U.S.A., Hawaii (O‘ahu) Kaena Point, W.N. Takeuchi 2007* (BISH), S. Keeley & J. Obata 4555: Kaena Point, 31 Mar 1992, MH152168, MH151993, MH152081, MH152256; Lipochaeta lobata (Gaudich.) DC., M33, U.S.A., Hawaii (Maui) Hanaula Rd, R.W. Hobdy s.n.* (BISH), R.W. Hobdy & S. Keeley: Hanaula Rd Jct Hawaiian Trail, Mar 1993, MH152169, MH151994, MH152082, MH152257; Lipochaeta micrantha var. exigua (O.Deg. & Sherff) R.C.Gardner, M03, U.S.A., Hawaii (Kaua‘i) Hoary Head Range, T. Flynn 735* (PTBG), S. Keeley s.n.: Waimea Falls Park from cutting #90531, 27 Apr 1992; original collection by Perlman: Kauai, 5 Jul 1990, MH152135, MH151960, MH152048, MH152223; Lipochaeta remyi A.Gray, M04, U.S.A., Hawaii (O‘ahu) Kealia Trail above Dillingham Airfield, T. Flynn 783* (PTBG), S. Keeley & J. Obata 4573: Kealia Trail, 31 Mar 1992, MH152136, MH151961, MH152049, MH152224; Lipochaeta rockii Sherff, M37, U.S.A., Hawaii (Moloka‘i) Kamiloloa, R.W. Hobdy 892* (BISH), R.W. Hobdy & S. Keeley s.n.: Kamiloloa Rd 210m, Mar 1993, MH152171, MH151996, MH152084, MH152259; Lipochaeta rockii var. dissecta Sherff, M36, U.S.A., Hawaii (Maui) Makena District: Puu o Kali near Poolenalena, R.W. Hobdy & S. Keeley 3584 (US), N/A, MH152170, MH151995, MH152083, MH152258; Lipochaeta subcordata A.Gray, M05, U.S.A., Hawaii (Hawai‘i) Pohakuloa Training Area between Mauna Loa and Mauna Kea, J. Davis 299* (BISH), S. Keeley s.n.: from Waimea Falls Park cutting #79c562, 27 Apr 1992; original collection from Pohakuloa, MH152137, MH151962, MH152050, MH152225; Lipochaeta subcordata A.Gray, M26, U.S.A., Hawaii (Hawai‘i) Pohakuloa Training Area Bobcat Trail, S. Garner 1a* (US), S. Garner & S. Keeley s.n.: Pohakuloa Bobcat Trail pop2, Apr 1993, MH152162, MH151987, MH152075, MH152250; Lipochaeta succulenta (Hook. & Arn.) DC., M39, U.S.A., Hawaii (Hawai‘i) Hilo: margins of Lokoaka pond, R.L. Stemmermann 7190* (BISH), S. Keeley s.n.: Hilo fishpond, Apr 1993, MH152172, MH151997, MH152085, MH152260; Lipochaeta succulenta (Hook. & Arn.) DC., M40, U.S.A., Hawaii (Moloka‘i) Wailau Valley, F.R. Fosberg 9663* (BISH), P. Welton s.n.: Wailau Valley, Feb 1993, MH152173, MH151998, MH152086, MH152261; Lipochaeta succulenta (Hook. & Arn.) DC., M41, U.S.A., Hawaii (Kaua‘i) Kipu Kai on sand above beach, A.M. Alexander & L. Kelloff 5335* (BISH), S. Keeley s.n.: NTBG from cutting #89085, Apr 1993; original collection by K. Lilleeng-Rosenberge: Kipukai, 18 Oct 1989, MH152174, MH151999, MH152087, MH152262; Lipochaeta tenuifolia A.Gray, M19, U.S.A., Hawaii (O‘ahu) Waianae Kai, J. Obata s.n.* (PTBG), J. Obata & Fenstenacher s.n.: Waimea Falls Park from cutting #79c561; original collection: Waianae Kai with no voucher, 8 May 1992, MH152156, MH151981, MH152069, MH152244; Lipochaeta tenuis O.Deg. & Sherff, M06, U.S.A., Hawaii (O‘ahu) Waianae Mts, S. Perlman 5647* (PTBG), S. Keeley s.n.: Waimea Falls Park from seedlot #905466, 22 Apr 1992; original collection by Perlman s.n., 21 Jul 1992, MH152138, MH151963, MH152051, MH152226; Lipochaeta tenuis O.Deg. & Sherff, M20, U.S.A., Hawaii (O‘ahu) Waianae Kai: slope of Kaala, J. Obata s.n.* (PTBG), J. Obata & Fenstenacher s.n.: Waianae Kai near Kolekole, Mar 1992., MH152157, MH151982, MH152070, MH152245; Lipochaeta venosa Sherff, M21, U.S.A., Hawaii (Hawai‘i) Sth Kohala District: Cinder cone across hwy from Nohonaohe Nth of old Aalii pumping station, J. Davis 712* (BISH), S. Garner & S. Keeley s.n.: Nohonohae, Apr 1993, MH152158, MH151983, MH152071, MH152246; Lipochaeta waimeaensis H.St.John, M22, U.S.A., Hawaii (Kaua‘i) Waimea Canyon: 3 miles below junction of Waimea and Kekaha roads below old canyon rim, S. Perlman 11790* (PTBG), T. Flynn & S. Keeley s.n.: Waimea Canyon Aug 1992, MH152159, MH151984, MH152072, MH152247; Lipotriche rhombifolia (O.Hoffm. & Muschl.) D.J.N.Hind, M79, Mali, Fodobougou, B. Carre & al. ML311 (US), N/A, MH152204, MH152029, MH152117, MH152292; Lipotriche scandens (Schumach.) Orchard, M70, Ghana, Kibi, M. Merello & al. 1222 (US), N/A, MH152198, MH152023, MH152111, MH152286; Lipotriche scandens subsp. dregei (DC.) Orchard, M80, Guinea, Nzerekore, A. Haba & F. Soropogui s.n. (US), N/A, MH152205, MH152030, MH152118, MH152293; Version of Record 563 Edwards & al. • Melanthera alliance biogeography and relationships (Compositae) TAXON 67 (3) • June 2018: 552–564 Appendix 1. Continued. Lipotriche scandens (Schumach.) Orchard, M69, Uganda, Kyadondo County: West Mengo Kanyanya Valley, P.K. Rwaburindore 1136 (US), N/A, MH152197, MH152022, MH152110, MH152285; Lipotriche scandens (Schumach.) Orchard, M73, Tanzania, Kigoma, Y.S. Abeid 3545 (US), N/A, MH152201, MH152026, MH152114, MH152289; Lipotriche triternata (Klatt) Orchard, M72, Namibia, Kunene, E.S. Klassen & al. 1950 (US), N/A, MH152200, MH152025, MH152113, MH152288; Lundellianthus guatemalensis (Donn.Sm.) Strother, M106, Guatemala, Peten Dept, E. Contreras 10555 (US), N/A, MH152147, MH151972, MH152060, MH152235; Lundellianthus salvinii (Hemsl.) Strother, M107, Guatemala, Sacatepequez Dept, A.L. Norrbom 90G-6 (US), N/A, MH152148, MH151973, MH152061, MH152236; Melanthera angustifolia A.Rich., M47, U.S.A., Florida: Marathon Key, W.C. Allen 86 (US), N/A, MH152176, MH152001, MH152089, MH152264; Melanthera nivea (L.) Small, M52, U.S.A., Florida: Key Largo, W.C. Allen 72 (US), N/A, MH152181, MH152006, MH152094, MH152269; Melanthera nivea (L.) Small, M53, U.S.A., Georgia: Jekyll Island, M.T. Strong 4106 (US), N/A, MH152182, MH152007, MH152095, MH152270; Melanthera parvifolia Small, M54, U.S.A., Florida: Porter Russell Pinelands, W.C. Allen 50 (US), N/A, MH152183, MH152008, MH152096, MH152271; Montanoa hibiscifolia Benth., M94, Hawaii, ex Mexico (naturalized in Hawaii), D.H. Lorence 6676 (US), N/A, MH152217, MH152042, MH152130, MH152305; Montanoa karwinskii DC., M95, Mexico, Jalisco, V.A. Funk & A. Delgado 12602 (US), N/A, MH152218, MH152043, MH152131, MH152306; Oyedaea boliviana Britton, M58, Bolivia, La Paz Vilaque, S.G. Beck 28256 (US), N/A, MH152186, MH152011, MH152099, MH152274; Perymeniopsis ovalifolia (A.Gray) H.Rob., M60, Mexico, Puebla, J. Amith 1725 (US), N/A, MH152188, MH152013, MH152101, MH152276; Perymenium berlandieri DC., M59, Mexico, Puebla, J.B. Fay & A. Cronquist 120 (US), N/A, MH152187, MH152012, MH152100, MH152275; Riencourtia latifolia Gardner, M61, Brazil, Tocantins: Ilhado Bananal Parque Nacional do Araguaina, M. Aparecida de Silva & al. 3983 (US) N/A, MH152189, MH152014, MH152102, MH152277; Sphagneticola trilobata (L.) Pruski, M62, U.S.A., Univ. of Hawaii Manoa, M.M. Chau 50 (HAW), N/A, MH152190, MH152015, MH152103, MH152278; Steiractinia sodiroi (Hieron.) S.F.Blake, M63, Ecuador, Pinchincha, G. Webster & al. 31302 (US), N/A, MH152191, MH152016, MH152104, MH152279; Tilesia macrocephala (H.Rob.) Pruski, M67, Ecuador, Loja, J.J. Pipoly 6374 (US), N/A, MH152195, MH152020, MH152108, MH152283; Wedelia acapulcensis Kunth, M64, Mexico, Co’ahuila, Henrickson, Riskind & al. 22571 (US), N/A, MH152192, MH152017, MH152105, MH152280; Wedelia brachylepis Griseb., M90, Brazil, Mato Grosso do Sul: Puerto Murtinho, Hatschbach & al. 76562 (US), N/A, MH152215, MH152040, MH152128, MH152303; Wedelia buphtalnifolia Lorentz, M91, Argentina, Cordoba, Villafane 198 (US), N/A, MH152216, MH152041, MH152129, MH152304; Wedelia calycina Rich., M65, Dominican Rep, Altagracia, P. Acevedo & al. 14117 (US), N/A, MH152193, MH152018, MH152106, MH152281; Wedelia goyazensis Gardner, M89, Brazil, Bahia: Fazendo do Conde, Hatschbach & al. 75647 (US), N/A, MH152214, MH152039, MH152127, MH152302; Wedelia kotschyi, (Sch.Bip.) Soldano, M71, Namibia, Caprivi, G.L. Maggs GM-723 (US), N/A, MH152199, MH152024, MH152112, MH152287; Wedelia reticulata DC., M110, Puerto Rico, Aguadilla Bo. Caimital Bajo, P. Acevedo 13441 (US), N/A, MH152151, MH151976, MH152064, MH152239; Wedelia reticulata DC., M66, Puerto Rico, Aguadilla, P. Acevedo & al. 13441 (US), N/A, MH152194, MH152019, MH152107, MH152282; Wedelia sp.1, M75, Tanzania, Kigoma, Y.S. Abeid 3553 (US), N/A, MH152202, MH152027, MH152115, MH152290; Wedelia sp.2, M76, Tanzania, Kigoma, Y.S. Abeid 3554 (US), N/A, MH152203, MH152028, MH152116, MH152291; Wedelia subpetiolata (Baker) B.L.Turner, M55, Brazil, Minas Gerais, J.R. Pirani 4102 (US), N/A, MH152184, MH152009, MH152097, MH152272; Wollastonia biflora (L.) DC., M102, French Polynesia, Austral Islands: Rapa, S. Perlman 18031 (PTBG), N/A, MH152144, MH151969, MH152057, MH152232; Wollastonia biflora (L.) DC., M103, Marshall Islands, Kwajalein, A. Whistler & O. Steele 11210 (PTBG), N/A, MH152145, MH151970, MH152058, MH152233; Wollastonia biflora (L.) DC., M104, Federated States of Micronesia, Pohnpei: U Municipality Nanisou, A. Dores 184 (PTBG), N/A, MH152146, MH151971, MH152059, MH152234; Wollastonia biflora (L.) DC., M23, U.S.A., PTBG Garden ex Okinawa, K. Woolliams 165* (PTBG), S. Keeley s.n.: NTBG, 27 Apr 1992; original collection: Okinawa Japan, MH152160, MH151985, MH152073, MH152248; Wollastonia biflora (L.) DC., M48, Taiwan, Taitung Hsien, C-H. Liu 680 (US), N/A, MH152177, MH152002, MH152090, MH152265; Wollastonia biflora (L.) DC., M81, Papua New Guinea, Morobe Province: Salamaua Peninsula, J. Wen 12270 (US), N/A, MH152206, MH152031, MH152119, MH152294; Wollastonia biflora var. canescens (Gaudich.) Fosberg, M24, Guam, Anderson Air Force Base, S. Perlman & K. Wood 14282* (PTBG), W. Char s.n.: NTBG Mar 1993; original collection: Agat Bay Guam, MH152161, MH151986, MH152074, MH152249; Wollastonia dentata (H.Lév. & Vaniot) Orchard, M82, Taiwan, New Taipei City, K-F. Chung 2024 (US), N/A, MH152207, MH152032, MH152120, MH152295; Wollastonia lifuana (Hochr.) Fosb., M101, New Caledonia, Mont-Dore: Jardin de IAC St-Louis (native to Lifou), G. Gateble 92 (US), N/A, MH152143, MH151968, MH152056, MH152231; Wollastonia lifuana (Hochr.) Fosb., M51, New Caledonia, Isle Brosse: 1 km SE of Isle des Pins, D. Mueller-Dombois 81081308 (US), N/A, MH152180, MH152005, MH152093, MH152268; Zexmenia virgulta Klatt, M68, Costa Rica, Alajuela, V.A. Funk & al. 10745 (US), N/A, MH152196, MH152021, MH152109, MH152284; 564 Version of Record