J. Jpn. Bot. 93(1): 9–17 (2018) A Phylogenetic Study of Amphicarpaea with a New Genus Afroamphica (Leguminosae Tribe Phaseoleae) a b, Kazuaki OHASHI and Hiroyoshi OHASHI * aSchool of Pharmacy, Iwate Medical University, Yahaba, Iwate, 028-3694 JAPAN; bHerbarium, Botanical Garden, Tohoku University, Sendai, 980-0862 JAPAN *Corresponding author: [email protected] (Accepted on September 13, 2017) The genus Amphicarpaea was revised by Ohashi and Ohashi (2016) based mainly on pollen morphology in which Shuteria africana was resurrected from Amphicarpaea africana and the taxonomic position of A. ferruginea was settled in Amphicarpaea. In the present study, those results were reexamined in molecular phylogenetic analyses using chloroplast DNA and nuclear DNA (ITS). Our molecular data show that Shuteria africana is sister to the genus Amphicarpaea, and that A. ferruginea is sister to A. edgeworthii and A. bracteata within the genus. For a consistent systematic treatment with the discrepancy between the results of the pollen morphology and the present molecular study, a new monotypic genus Afroamphica is proposed for Shuteria africana. Key words: Afroamphica, Amphicarpaea, Amphicarpaea africana, Amphicarpaea ferruginea, Fabaceae, Leguminosae, new genus, Phaseoleae, Shuteria, Shuteria africana. Amphicarpaea was revised by Ohashi (Baker 1876) was placed in Amphicarpaea and Ohashi (2016) based mainly on pollen based on such evidence from pollen morphology morphology. The genus was then circumscribed as above, because the former two genera have to consist of three species: A. edgeworthii Benth. typical 3-colporate pollen grains. To confirm the and A. ferruginea Benth., both distributed in classification of these species based on pollen Asia, and A. bracteata (L.) Fernald in North morphology from our previous work (Ohashi America. Amphicarpaea africana (Hook. f.) and Ohashi 2016), we undertook molecular Harms, which had been widely recognized as analyses of these species. a member of the genus in recent taxonomic This paper aims to provide the results of the works (Hauman 1954, Turner and Fearing phylogenetic findings for our treatment on the 1965, Verdcourt 1971), was transferred to taxonomic position for Shuteria africana and Shuteria africana Hook. f., because of the Amphicarpaea ferruginea. distinct tricolporate pollen grains observed in species of Shuteria in contrast to the triporate Phylogenetic confirmation or tetraporate grains with linear grooves or The material used and the voucher specimens slight colporus-like furrows in Amphicarpaea. for Shuteria africana is ‘Plants of Uganda, In contrast, A. ferruginea, which was once Mobuku Valley, Ruwenzori. Alt. 6200 ft. Vine, attributed to Pueraria (Kurz 1874) or Shuteria beside river. Flower purple. 2 January 1939. —9— 10 植物研究雑誌 第 93 巻 第 1 号 2018 年 2 月 Fig. 1. The 50% majority-rule consensus tree (Bayesian analysis) of Amphicarpaea and Shuteria with species of Phaseoleae, Psoraleeae (Bituminaria, Cullen and Otholobium) and Desmodieae (Campylotropis, Desmodium, Kummerowia and Lespedeza) based on cpDNA dataset (trnK/ matK, trnL-trnF). Support values on the branches are presented as follows: BI-PP/ML-BS. NS indicates the node not supported. Scale bar shows the expected number of substitutions per site in Bayesian analysis. Mrs. M. V. Loveridge 298 (A).’ The methods BS = 92) (Fig. 1). Shuteria africana was sister used in this study are described at the end*. to all three species of Amphicarpaea (BI-PP = The phylogenetic tree based on chloroplast 1; ML-BS = 100). We obtained the trees with DNA (trnL-trnF and trnK/matK) revealed similar topology in both the BI and ML analyses. a clade composed of Shuteria africana and These results show that Shuteria africana is Amphicarpaea (BI-PP = 1; ML-BS = 100), more closely related to Amphicarpaea than to which formed a sister to the clade consisting of other species of Shuteria: S. vestita Wight & Glycine max (L.) Merr. and species of the tribe Arn. and S. involucrata (Wall.) Wight & Arn. ex Psoraleeae: Bituminaria bituminosa Kuntze, Walp. Cullen australasicum (Schltdl.) Hanelt, and To confirm the relationship betweenShuteria Otholobium striatum (Thunb.) C. H. Stirt., and africana and the species of Amphicarpaea, a the clade consisting of Teramnus uncinatus Sw. phylogenetic analysis based on six chloroplast and T. labialis (L. f.) Spreng. (BI-PP = 1; ML- DNA regions (trnL-trnF, trnK/matK, trnT-trnL, February 2018 The Journal of Japanese Botany Vol. 93 No. 1 11 Fig. 2. The 50% majority-rule consensus tree (Bayesian analysis) of Amphicarpaea with closely related species of Phaseoleae based on six chloroplast DNA regions (trnL-trnF, trnK/matK, trnT-trnL, trnG intron, rpl16 itron and trnL-rpl32). Support values on the branches are presented as follows: BI-PP/ML-BS. Scale bar shows the expected number of substitutions per site in Bayesian analysis. Fig. 3. The 50% majority-rule consensus tree (Bayesian analysis) of Shuteria africana (Amphicarpaea africana) derived from two specimens with species of Phaseoleae based on ca. 600 bp matK fragment. Support values on the branches are presented as follows: BI-PP/ML-BS. Scale bar shows the expected number of substitutions per site in Bayesian analysis. trnG intron, rpl16 itron and trnL-rpl32) was (the other is a sequence analyzed in this report). performed. The deduced tree (Fig. 2) showed The obtained tree (Fig. 3) shows the close a monophyletic Amphicarpaea clade sister to relationship of these two samples. Shuteria africana. The analysis based on nuclear DNA A short fragment of matK sequence of (ITS) also showed the monophyly of the S. africana obtained from another sample is Amphicarpaea-Shuteria africana clade with accessible in GenBank (Accession number: well supported values (BI-PP = 1; ML-BS = KX213379). Therefore we analyzed the 92) and the first separation of Shuteria africana phylogenetic relationship of the two S. africana (Fig. 4). Analysis of ITS using other models samples with this ca. 600 bp matK fragment (e.g., HYK85+G) obtained a similar clade 12 植物研究雑誌 第 93 巻 第 1 号 2018 年 2 月 Fig. 4. The 50% majority-rule consensus tree (Bayesian analysis) of Amphicarpaea and Shuteria with species of Phaseoleae, Psoraleeae (Bituminaria, Cullen and Otholobium) and Desmodieae (Campylotropis, Desmodium, Kummerowia and Lespedeza) based on nuclear DNA (ITS). Support values on the branches are presented as follows: BI-PP/ML-BS. Scale bar shows the expected number of substitutions per site in Bayesian analysis. containing Amphicarpaea and S. africana (data and Ohashi (2016), but supports the inclusion of not shown). Our molecular phylogenetic data on A. ferruginea in Amphicarpaea as indicated by chloroplast and nuclear DNA shows the close morphological features of the pollen. relationships between S. africana and the species The taxonomic position of Shuteria africana of Amphicarpaea. Additionally, the phylogenetic is problematic. The species is distinct from analysis supported the inclusion of A. ferruginea Amphicarpaea in floral and pollen morphology, in Amphicarpaea. but is close to Amphicarpaea based on molecular evidence. The phylogenetic position is Taxonomic treatment far from other species of Shuteria. We consider Results of the present phylogenetic that creating a new genus for the species is more analyses revealed a Shuteria africana- reasonable than retaining it in Amphicarpaea or Amphicarpaea clade composed of S. africana in Shuteria. In the sense of Ohashi and Ohashi and Amphicarpaea. Shuteria africana is sister (2016), Amphicarpaea in the strict sense is to the genus Amphicarpaea. The results did not more natural in morphology and palynology. support the recognition of Shuteria africana as We, therefore, propose here the distinct genus distinct from Amphicarpaea as treated by Ohashi Afroamphica to accommodate Shuteria africana. February 2018 The Journal of Japanese Botany Vol. 93 No. 1 13 Afroamphica H. Ohashi & K. Ohashi, gen. We thank Dr. David E. Boufford of Harvard nov. University Herbaria for supplying the material of [Diagnosis] Afroamphica is morphologically Shuteria africana and for correction of English intermediate between Amphicarpaea and with valuable comments and Dr. T. Nemoto of Shuteria—similar to Amphicarpaea in Ishinomaki Shenshu University for review of the having chasmogamous and cleistogamous manuscript. flowers, but differs from Shuteria in this characteristic. Whereas Afroamphica differs *Methods for phylogenetic analyses from Amphicarpaea in flowers and pollen Genomic DNA was extracted from leaf tissue using the grains, it resembles Shuteria in these characters. DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Eight In the flowers of Afroamphica, the standard chloroplast markers and ITS were amplified by PCR: trnK intron including matK (trnK/matK), trnL-trnF (trnL intron has a broadly obovate-round blade, the claw of and trnF-trnL spacer), trnT-trnL spacer, rps16 intron, rpl16 the wings is as long as the blade and the acute intron and trnL-rpl32 spacer. The PCR primers used for keel petals; in Amphicarpaea the blade of the these markers are as follows: trnK1L and trnK2R (Hu et al. standard is oblong-obovate, the claw of the 2000) for trnK/matK, primer ‘c’ and Primer ‘f’ (Taberlet et al. 1991) for trnL-trnF (trnL intron and trnF-trnL spacer), wings is 2–3 times longer than blade, and the trnA2 and trnB (Cronn et al. 2002) for trnT-trnL, trnG(UUC)* keel petals are obtuse. (Shaw et al. 2007) and 5ʹtrnG2G (Shaw et al. 2005) for [Description] Herbs, climbing, perennial. trnG, primer 1 and primer 3 (Lee and Hymowitz 2001) for Stems slender, with reflexed ferruginous hairs. rps16 intron, trnL(UAG) and rpl32-F (Shaw et al. 2007) for Leaves pinnately 3-foliolate, stipulate, petiolate; trnL-rpl32, F71 and R1516 (Jordan et al. 1996) for rpl16 intron and ITS4 and ITS5 (White et al. 1990) for ITS. The leaflets elliptic or obovate; stipelate. Racemes PCR condition and DNA sequencing analysis basically with axillary chasmogamous flowers, many- followed Ohashi et al. (2017). The accession numbers of flowered; peduncle and rachis with appressed newly sequenced data in this analysis are shown in Table hairs; bracts narrowly ovate. Chasmogamous 1.