ISSN 1346-7565 Acta Phytotax. Geobot. 71 (2): 103–128 (2020) doi: 10.18942/apg.201923 A Large-scale Phylogenetic Analysis of Dioscorea (Dioscoreaceae), with Reference to Character Evolution and Subgeneric Recognition 1,* 2 1 3 HIROSHI NODA , JUN YAMASHITA , SHIZUKA FUSE , RACHUN POOMA , 3 1 1 MANOP POOPATH , HIROSHI TOBE AND MINORU N. TAMURA 1Department of Botany, Graduate School of Science, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. *[email protected] (author for correspondence); 2Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki-shi, Okayama 710-0046, Japan; 3The Forest Herbarium, Department of National Parks, Wildlife and Plant Conservation, Chatuchak, Bangkok 10900, Thailand Dioscorea (Dioscoreaceae) is a diverse genus of more than 600 species. To understand relationships and character evolution within the genus, 273 samples from 183 species (including 28 newly sequenced spe- cies) based on four cpDNA regions were analyzed phylogenetically. The phylogenetic tree obtained com- prised eleven well-supported major clades, most of which further consisted of more than two subclades. Comparisons with previously proposed infrageneric taxa (23 to 58 sections and associated ‘genera’) showed that some sections/‘genera’ are monophyletic and others polyphyletic. As in previous studies, ‘D. sect. Stenophora’ was sister to the rest of the genus. The present analyses of character state distribution on the tree confirmed that D‘ . sect. Stenophora’ is characterized by having rhizomes, monosulcate pollen and a diploid chromosome number based on x = 10 (plesiomorphies), whereas the rest of the genus has tubers and bisulcate pollen (apomorphies), but is diverse in regard to chromosome number, stem twining direction, fruit types and seed wing morphology. Based on molecular and morphological evidence, two subgenera, Dioscorea (= ‘D. sect. Stenophora’) and Helmia, are proposed. For subgenus Helmia, a revi- sion of the infrageneric classification, especially for the species in the Old World, is needed. Keywords: Dioscorea, Dioscoreaceae, evolution, Helmia, molecular phylogeny, monocots, Stenophora, subgeneric classification, taxonomy Dioscorea L., the yams, with about 630 spe- recognized 23 sections for the Old World species, cies worldwide, along with Stenomeris Planch., and Huber (1998) proposed five ‘genera’ and 24 Tacca J. R. Forst. & G. Forst., and Trichopus ‘genus-equivalent sections.’ Huber (1998) further Gaertn. constitute the Dioscoreaceae R. Br. recognized at least three more ‘genus-equivalent (WCSP 2019). Most species have twining stems sections’ that lack descriptions. In all of these (Fig. 1a) and are dioecious (Fig. 1b, c). Because classification systems, a few species ofDioscorea the large number of species represent consider- were assigned to the separate genera Borderea able morphological diversity, Dioscorea has of- Miégev., Epipetrum Phil., Rajania L., Tamus L., ten been divided into infrageneric taxa (for re- and Testudinaria Salisb. because of their unique view see Caddick et al. 2002a). Uline (1898) di- morphological features. As indicated below, how- vided Dioscorea into 51 sections in three subgen- ever, molecular phylogenetic analyses have con- era Eudioscorea Pax, Helmia (Kunth & Gatt.) sistently shown that all of those species are nested Gris. and Testudinaria (Salisb. & Gatt.) Uline. within a large clade composed of more typical Knuth (1924) recognized four subgenera Eu- species of Dioscorea (Caddick et al. 2002a, dioscorea, Helmia, Stenophora (Uline) R. Knuth. Wilkin et al. 2005, Viruel et al. 2016, 2018, Couto and Testudinaria and recognized 58 sections et al. 2018). Accordingly, Dioscorea is now cir- within the former three subgenera. Burkill (1960) cumscribed to contain much more morphological 104 Acta Phytotax. Geobot. Vol. 71 Fig. 1. Terrestrial stems and flowers of Dioscorea. a, Twining stem (D. bulbifera); b, Staminate flower D.( tokoro); c, Pistillate flower D.( tokoro) with a distinct inferior ovary (arrowhead). Scale bars = 10 cm in a; 5mm in b, c. diversity than had been previously considered. cluding Rajania-cordata and Dioscorea-tentacu- In previous infrageneric classifications of Di- ligera) in the phylogenetic tree, and suggested oscorea (including the aforementioned associat- that previous systems of infrageneric classifica- ed genera) (Uline 1898, Knuth 1924, Burkill tion could be greatly simplified. Gaoet al. (2008) 1960, Huber 1998), diverse morphological char- analyzed the phylogeny of 17 species assigned to acters were used to distinguish one section (sub- D. sect. Stenophora using sequences of three cp- genus/genus) from another. The characters in- DNA regions (matK, rbcL, and trnL-F). Hsu et al. cluded: fruit type (capsule, samara or berry), seed (2013) analyzed the relationships of 44 Asian spe- morphology (winged or unwinged, and if winged, cies of Dioscorea using sequences of four cpD- wing types), underground stem (rhizome or tu- NA regions (matK, rbcL, atpB-rbcL, and trnL-F), ber), the direction of stem twining (right or left), and discussed the relationships between several and phyllotaxis (alternate or opposite). Diagnos- sections consisting of Asian species. More re- tic keys to the sections (subgenera/genera) dif- cently, Viruel et al. (2016) analyzed the phyloge- fered greatly between authors, and no consensus ny of 135 species of Dioscorea using sequences has emerged with regard to the recognition and/or from four cpDNA regions (matK, rbcL, atpB, and circumscription of many of the infrageneric taxa. trnL-F) and discussed the geographic origin of For the past two decades, molecular phyloge- the genus and potential evolutionary processes netic analyses have been used to better under- leading to the current distribution of the species. stand relationships within Dioscoreaceae and Di- Maurin et al. (2016) provided phylogenetic stud- oscorea (Caddick et al. 2002a, Wilkin et al. 2005, ies of Dioscorea with a focus on the African spe- Gao et al. 2008, Hsu et al. 2013, Maurin et al. cies using six cpDNA regions, including matK, 2016, Viruel et al. 2018, Soto Gomez et al. 2019), rbcL, and trnL-F. They newly sequenced six Af- and to elucidate the biogeographic origin and rican species. Viruel et al. (2018) further obtained species migrations in Dioscorea (Viruel et al. phylogenetic trees of 53 species of Dioscorea 2016, Couto et al. 2018). Wilkin et al. (2005) ana- based on the low copy nuclear gene Xdh, and lyzed the phylogeny of 67 species of Dioscorea showed that the order of divergence of the main using sequences of two cpDNA regions (matK clades of Dioscorea are congruent with those of and rbcL). They identified 10 major groups (in- the plastid-based trees. Couto et al. (2018) also June 2020 NODA & AL. – Phylogeny and character evolution of Dioscorea 105 analyzed the phylogeny of 161 species of Di- only at base and apex. Schols et al. (2005) further oscorea, focusing on Neotropical species, and ob- discussed pollen (size, aperture number, sexine tained new sequences of two chloroplast genes ornamentation, perforation density) evolution us- (matK and rbcL) from 34 species. Besides dis- ing a tree identical to that obtained by Wilkin et cussing the biogeographic history of Dioscorea, al. (2005). After the character state analyses of Couto et al. (2018) discussed how phylogeny sup- Wilkin et al. (2005) and Schols et al. (2005), se- ported the infrageneric classification proposed by quence data of cpDNA regions from additional Uline (1897) and Knuth (1924). They also con- species of Dioscorea have become available (e.g., cluded that although some of the sections pro- Gao et al. 2008, Hsu et al. 2013, Maurin et al. posed by Uline (1897) may be supported, the in- 2016, Viruel et al. 2016, Couto et al. 2018). Since frageneric classifications of Uline (1897) and a phylogenetic tree based on more species of Di- Knuth (1924) did not generally represent natural oscorea than were available to Wilkin et al. lineages. (2005) can now be obtained, we can discuss char- The aforementioned molecular phylogenetic acter evolution (particularly in Old World species studies of Dioscorea (Wilkin et al. 2005, Viruel groups) more extensively and precisely. et al. 2016, 2018, Couto et al. 2018) obtained sim- In the present paper, we combined all existing ilar tree topologies consisting of ten major clades, cpDNA sequence data with our original data to which were referred to as Stenophora, New World undertake a large-scale phylogenetic analysis of I (NWI), New World II (NWII), African, Medi- 183 species of Dioscorea (about 29% of the cur- terranean, New World III (NWIII; including ‘Ra- rently known species of Dioscorea). We com- jania’), compound leaved, Malagasy, Shan- pared the major clades/subclades in the resulting nicorea and Enantiophyllum. While the first three tree with previously proposed infrageneric clas- clades successively diverged, the order of diver- sifications to delineate the evolution of morpho- gence of the remaining seven differed according logical characters and to map their diversification to each author. Soto Gomez et al. (2019) analyzed within the genus. The morphological characters the genomic variation in 34 species of Dioscorea we discuss include not only those used in previ- that represent all major clades obtained by cpD- ous infrageneric classifications (Uline 1897, NA and nuclear Xdh sequence analyses (Viruel et 1898, Knuth 1924, Burkill 1960, Huber 1998), but al. 2018). They showed robust relationships