Systematic Botany (2008), 33(1): pp. 125–132 © Copyright 2008 by the American Society of Plant Taxonomists Austrobryonia (Cucurbitaceae), a New Australian Endemic Genus, is the Closest Living Relative to the Eurasian and Mediterranean Bryonia and Ecballium Hanno Schaefer,1,3 Ian R. H. Telford,2 and Susanne S. Renner1 1Systematic Botany, Department of Biology, University of Munich (LMU), Menzinger Str. 67, D-80638 Munich, Germany 2N.C.W. Beadle Herbarium, University of New England, Armidale, NSW 2351, Australia 3Author for correspondence ([email protected]) Communicating Editor: Javier Francisco-Ortega Abstract—The Cucurbitaceae genus Austrobryonia, with four species endemic to Australia, is described, illustrated, and placed in a phylogenetic context based on molecular and morphological data. In the Flora of Australia (Telford 1982), all four species were provisionally included in Mukia, but not formally described. Austrobryonia argillicola, A. centralis, A. micrantha, and A. pilbarensis are adapted to arid central regions of Australia. All species are known from several (7–27) localities, and their distributional ranges are allopatric. A phylogenetic analysis of plastid and nuclear DNA sequences that includes all four species in a family-wide context revealed that Austrobryonia is the closest living relative to a Eurasian and Mediterranean clade consisting of Bryonia L. and Ecballium L. An rbcL molecular clock, calibrated with Cucurbitaceae fossils, dates this rare biogeographic disjunction to minimally 42 my ago (with an error of ca. ± 25%), while the crown group of Austrobryonia may be about 8 my old. Keywords—Australia-Eurasia disjunction, molecular clock, Mukia micrantha. The Australian continent is relatively poor in genera and Mukia sp. C, Mukia sp. D, and Mukia sp. E (Telford 1982, pp. species of Cucurbitaceae. The last comprehensive treatment 182–187). (Telford 1982) lists 44 species, then assigned to 17 genera. Over the past quarter of a century, additional flowering Twelve genera and perhaps 30 species appear indigenous to and fruiting collections have become available that now per- the Australian continent (Benincasa Savi, Cucumis L., Diplocy- mit complete descriptions, including information on phenol- clos (Endl.) Post & Kuntze, Luffa Miller, Momordica L., Muel- ogy and natural ranges. Molecular work also clarified the lerargia Cogn., Neoachmandra W. J. de Wilde & Duyfjes, Neo- affinities of Mukia, Zehneria, and the five new species from alsomitra Hutch., Nothoalsomitra I. Telford, Sicyos L., Tricho- Australia. Our molecular phylogenetic analyses indicate that santhes L., and the new genus Austrobryonia described below), M. maderaspatana, M. javanica (Miq.) C. Jeffrey, Mukia sp. A, but only one genus was thought endemic to Australia. This is and Mukia sp. B belong in the genus Cucumis (Renner et al. the monotypic Nothoalsomitra, a liana of SE Queensland’s rain 2007), while M. micrantha and Mukia sp. C, D, and E belong in forests, originally described in Benincaseae (Telford 1982), a new genus Austrobryonia that forms the sister clade to a but placing far from them in a molecular tree of Cucurbita- Eurasian/Mediterranean clade comprising Bryonia L. and Ec- ceae that samples 21% of the family’s 800 species and 94% of ballium L. We here formally describe the new genus and spe- its 130 genera, including species from all Australian clades cies, present the first molecular phylogenies that include (Kocyan et al. 2007). these new taxa, and, based on a molecular clock analysis, The most problematic group in the Flora of Australia treat- discuss the possible origin of this unexpected Australian/ ment of Cucurbitaceae was the genus Mukia Arnold. As pre- Mediterranean/Eurasian disjunction. viously conceived, Mukia was thought to include nine spe- cies, five that range from China through Indo-China south- MATERIALS AND METHODS east to Java, Borneo, and the Philippines, and west through Morphology—Specimens were borrowed from BRI, CANB and NSW. Measurements were taken from rehydrated dried herbarium specimens. India, Pakistan, and the Yemen into subSaharan Africa, and Data Generation and Retrieval—DNA extraction and sequencing fol- five that were left undescribed (Telford 1982; De Wilde and lowed standard procedures, using the rbcL, matK, rpl20–rps12, and trnL Duyfjes 2006). The type species of Mukia, M. scabrella (L.) Arn. and trnL–F primers listed in Kocyan et al. (2007). In addition to these (= M. maderaspatana (L.) M. Roem.), occurs from subSaharan plastid regions, we sequenced the nuclear internal transcribed spacer Africa, through tropical and subtropical Asia to Australia. region (347 nt of ITS 1, 162 nt of the 5.8S gene, and 318 nt of ITS 2) using the ITS primers of Balthazar et al. (2000). Direct PCR amplification of ITS The pollen of the Asian Mukia species is so different from that yielded single bands and unambiguous base calls. Thirty-six sequences of the Australian M. micrantha (F. Muell.) F. Muell. that Jef- were newly generated for this study (GenBank accession numbers EF frey (1969a) suspected they might not be congeneric. He 487543–EF 487578; Appendix 1). thought the affinities of M. micrantha might lie with Zehneria Sequence Alignment and Phylogenetic Analyses—Sequences were ed- ited with Sequencher (4.6; Gene Codes, Ann Arbor, Michigan, USA) and Endl., Kedrostis Medik., or Ibervillea Greene, but could not aligned by eye, using MacClade 4.06 (Maddison and Maddison 2003). The decide about its true taxonomic position. Earlier, Ferdinand aligned plastid matrix comprises 4692 nucleotides after exclusion of a von Mueller (1854) had first included M. micrantha in the poly-A run in the trnL intron and a TATATA microsatellite region in the otherwise neotropical genus Cucurbita, then in the African/ trnL–F intergenic spacer. The aligned ITS matrix comprises 829 nucleo- Asian Zehneria (Mueller 1858/59), and finally in Mukia tides. Data matrices and trees have been deposited in TreeBASE (study number S1817). (Mueller 1860/61), but without specifying the basis for these Equally weighted parsimony analyses for matrices of nucleotides were placements. Naudin (1859) redescribed the species as Cucu- conducted using PAUP 4.0b10 (Swofford 2002). The search strategy in- mis muelleri, but again without providing any reasons for this volved 100 random addition replicates with TBR branch swapping, sav- generic placement. Given the extremely few collections of ing all optimal trees. For the parsimony analyses, gaps were treated as missing data, for maximum likelihood (ML) searches gaps were not re- what appeared to be as many as five species, Telford (1982) moved. To assess node support, parsimony bootstrap analyses were per- refrained from erecting a new genus and instead provision- formed using 1000 replicate heuristic searches, each with 10 random ad- ally discussed the new species as Mukia sp. A, Mukia sp. B, dition replicates and TBR branch swapping, saving all optimal trees. 125 126 SYSTEMATIC BOTANY [Volume 33 Maximum likelihood analyses (and ML bootstrap searches) were per- between Austrobryonia and Ecballium/Bryonia, and of about 8 formed using GARLI 0.94 (D. J. Zwickl, University of Texas-Austin, avail- my for the crown group of Austrobryonia (i.e. divergence of A. able at http://www.bio.utexas.edu/grad/zwickl/web/garli.html). GARLI searches relied on the GTR+G+P-invar model, the only model centralis from the ancestor of the lineage leading to A. micran- implemented in this program. Model parameters were estimated in tha and its relatives; Figs. 1, 2). Compared to 191 other rbcL GARLI over the duration of specified runs. sequences of Cucurbitaceae and outgroups, the four species To identify the closest relatives of Austrobryonia, we added Austrobryo- of Austrobryonia share a synapomorphic silent substitution at nia sequences to the dataset of Kocyan et al. (2007), which includes 123 of position 417 of the rbcL alpha helix 8 (GCG instead of GCC, the 130 genera of Cucurbitaceae (representing all tribes and subtribes) and 171 of the ca. 800 species. Additional sequences were obtained for the both coding for the amino acid alanine), and A. centralis in genera Gomphogyne, Indomelothria, Pseudosicydium, Urceodiscus, and addition has an autapomorphic substitution (CGA instead of Zanonia, which augmented our sampling to 128 of 130 genera. Parsimony AGA at position 167). and ML analyses of this dataset showed that Austrobryonia always forms a highly supported clade with Bryonia and Ecballium. Austrobryonia H. Schaef., gen. nov.—TYPE: Cucurbita mi- The two genera of Cucurbitaceae that have not yet been sequenced are Khmeriosicyos De Wilde & Duyfjes, a monotypic genus from Cambodia crantha F. Muell., Trans. Philos. Soc. Victoria 1: 17, 1854. known only from the type, and Papuasicyos Duyfjes, a monotypic genus from Papua New Guinea. Judging from their floral morphology, both are A Cucumeris grege Mukia (quae floribus eiusdem sexus in expected to fall in Benincaseae, far from Bryonia, Ecballium, and Austro- unaquaque axilla numerosis (1–20) fasciculatis, fructibus ru- bryonia. In Fig. 1, we show a phylogeny based on a reduced dataset of 75 bris et seminibus plerumque turgidis foveolatis gaudet) dif- taxa, including all tribes and subtribes and all genera containing Austra- lian species. fert floribus paucis (2–8) amborum sexuum in unaquaque Molecular Clock Analyses—rbcL branch lengths for Cucurbitaceae axilla consociatis, fructibus flavis vel flavovirentibus nec non were calculated under a GTR+G+I+clock model on the preferred ML seminibus compressis laevibus. topology obtained with five chloroplast loci from a 184-taxon matrix that Monoecious scabrid herbs; stems annual, from a perennat- contained Bryonia alba, B. dioica, and Ecballium elaterium (Kocyan et al. 2007). For the present study, we added Austrobryonia argillicola, A. centra- ing rootstock. Tendrils simple. Leaves petiolate; lamina more lis, A. micrantha, A. pilbarensis, and Bryonia verrucosa (the latter so as to or less ovate, cordate, acute, unlobed or shallowly 3-, 5- or span the root of Bryonia). The tree was rooted on Corynocarpaceae, Be- 7-lobed, dentate or serrate. Inflorescences of solitary flowers goniaceae, and Datiscaceae based on Zhang et al.