J. Jpn. Bot. 91 Suppl.: 231–249 (2016) A Taxonomic Revision of Amphicarpaea (Leguminosae) Including a Pollen Morphological Comparison with Shuteria a, b Hiroyoshi OHASHI * and Kazuaki OHASHI aHerbarium TUS, Botanical Garden, Tohoku University, Sendai, 980-0862 JAPAN; bSchool of Pharmacy, Iwate Medical University, Yahaba, Iwate, 028-3694 JAPAN *Corresponding author: [email protected] (Accepted on January 9, 2016) Amphicarpaea is newly circumscribed based on the structure of the pollen apertures having linear grooves or slight colporus-like furrows instead of distinct colpori. Three species are recognized as the members of the genus: A. edgeworthii and A. ferruginea in Asia and A. bracteata in North America. Amphicarpaea africana having distinct tricolporate pollen grains is returned to Shuteria, and Amphicarpaea lineata in China is regarded as identical to Shuteria hirsuta. Amphicarpaea ferruginea Benth. is lectotypified. Key words: Amphicarpaea, colporus-like furrows, Fabaceae, Phaseoleae, pollen aperture, pollen colporus, pollen morphology, Shuteria, Shuteria africana, typification. Amphicarpaea is a papilionaceous genus flowers. belonging to the tribe Phaseoleae. It was Amphicarpaea was first placed in its named on the basis of the nature of A. bracteata taxonomic position between Apios and having different two kinds of fruits: aerial and Glycine (Nuttall 1818). It has been classified in subterranean fruits. In fact, the species has two Phaseoleae subtribe Glycininae (Bentham 1865, kinds of aerial pods and subterranean ones. Lackey 1981, Schrire 2005) or tribe Glycineae Amphicarpaea edgeworthii has the same habitat. (Hutchinson 1964). Evolutionary relationships The aerial pods are produced from the aerial between Amphicarpaea and other genera chasmogamous flowers and cleistogamous ones. among Glycininae have been problematic. The subterranean pods arise from cleistogamous Bentham (1865) classified Amphicarpaea flowers borne on the subterranean shoots between Cologania and Dumasia. Taubert elongating from the cotyledon axil and geocarpic (1894) united Amphicarpaea with Cologania. pods from cleistogamous flowers on terrestrial Hutchinson (1964) put the genus next to creeping shoots grown from the aerial axil of the Shuteria. Turner and Fearing (1965) supposed lower leaves. These two species, A. bracteata a probable evolutionary relationship between and A. edgeworthii, are annual. In contrast, A. Amphicarpaea and Shuteria. In fact, several ferruginea and A. africana are perennial, having species were transferred between the two two kinds of aerial pods produced from aerial genera, e.g., A. africana or S. africana, A. chasmogamous flowers or cleistogamous ones, ferruginea or S. ferruginea, or A. trisperma or but lacking subterranean or geocarpic pods. S. trisperma. Lackey (1981) considered that Amphicarpaea lineata has no cleistogamous Amphicarpaea is particularly closely allied to —231— 232 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue Shuteria, Dumasia and Cologania in its flowers. Pollen grains of Shuteria were treated by However, Lee and Hymowitz (2001) analyzed Thuan (1972) who excluded Shuteria anomala molecular phylogenetic relations in the subtribe Pamp. and S. longipes Franch. from this genus and showed that “Amphicarpaea was sister to based partly on pollen morphological characters. the clade comprising Glycine and Teramnus He transferred S. anomala to a synonym rather than to Cologania.” Cologania, Dumasia of Amphicarpaea edgeworthii, but did not and Shuteria showed remote relationships accommodate S. longipes to any genus. Now S. with Amphicarpaea in their analysis. In recent longipes is placed in Hylodesmum of the tribe molecular analyses of the phaseoloid groups Desmodieae, H. longipes (Franch.) H. Ohashi (Phaseoleae s.l.), Amphicarpaea belongs to & R. R. Mill (Ohashi and Mill 2000). Ferguson the “Core-Phaseoleae” clade, while Shuteria and Skvarla (1981) introduced the Shuteria is distant from the clade and grouped with a pollen grains as subprolate, margocolporate, and Kennediinae-Desmodieae clade (Schrire 2005, reticulate with granules in the lumina. Stefanović et al. 2009). This paper aims to revise Amphicarpaea, as Materials and Methods the genus needs to have a clear circumscription. Pollen materials were obtained from A comparative pollen morphological study herbarium specimens kept in E and TUS. is emphasized between Amphicarpaea The pollen grains for SEM observation were and Shuteria, because these are hardly acetolysed following the standard method distinguishable from each other in external (Erdtman 1960) and dehydrated in an ethanol morphology, but no discussion has been made series. The air-dried samples were coated with of their pollen morphological relationships. osmium with a Filgen NL-OPC 60A osmium Detailed morphological descriptions for A. plasma coater, and examined with a Hitachi edgeworthii and A. ferruginea are provided. SU-8010 scanning electron microscope at Amphicarpaea lineata is newly placed as a the Center for Electron Microscopy and Bio- synonym of S. hirsuta in this paper. Imaging Research, Iwate Medical University. Observations by SEM are performed at 2.0 kV. 1. Pollen morphology of Amphicarpaea and The polar and equatorial lengths were measured Shuteria on at least 20 grains of each specimen. Pollen Pollen grains of Amphicarpaea were terminology in our study generally follows recorded by Schively as early as in 1897 for A. Punt et al. (1994), Hoen (1999) and Hesse et al. comosa (= A. bracteata). Differences in pollen (2009). morphology in Amphicarpaea were first shown by Meeson (1974) based on observations in light Pollen descriptions microscopy among the three species recognized Amphicarpaea africana Harms (Fig. 1) by Turner and Fearing (1965): 3-colporate in A. Pollen grains tricolporate; polar axis 25.3 africana, 4-porate in A. bracteata, and 3-porate ± 2.0 µm (range: 21.2–27.7 µm) (n = 20), in A. edgeworthii. He stated that “the pollen of equatorial diameter 23.9 ± 1.9 µm (21.0–27.2 Amphicarpaea edgeworthii and A. bracteata µm) (n = 28), P/E = 1.07 ± 0.08 (0.88–1.20), appear to be modifications of the basic form of spheroidal, equatorial view elliptic, polar view A. africana by loss of the colpi and reticulated triangular to circular. Colpori 0.60 times as sexine and an increase of retention in the number long as the polar axis, ca. 1.8–3.6 µm wide at of pores.” Ohashi et al. (2005) accepted this equator, narrowing to pointed ends; colporus view and regarded that the pollen of the genus membrane almost smooth, margines with has a basically colporate structure. continuous tectum; endoaperture ca. 0.12 times December 2016 Ohashi & Ohashi: Revision of Amphicarpaea 233 Fig. 1. Pollen grain of Amphicarpaea africana. A. Mesocolpium in equatorial view. B. Aperture in equatorial view. C. Polar view. D. Sculpture of mesocolpium. Voucher specimen: Congo, Semliki R. forest. Kassner Expedition 1908, no. 3088 (E). Scale bar: A, B, C = 10 µm; D = 1 µm. Fig. 2. Pollen grain of Amphicarpaea bracteata. A. Mesocolpium in equatorial view. B. Aperture in equatorial view. C. Polar view. D. Sculpture of mesocolpium. Voucher specimen: U.S.A. Vermont. Franklin Co., Fletcher, alt. ca. 240 m. 19 Aug. 1983. Boufford & Wood 23201 (TUS). Scale bar: A, B, C = 10 µm; D = 1 µm. 234 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue as long as the polar axis. Sexine semitectate; not. Ohashi et al. (2005) supposed the structure sculpture of mesocolpium reticulate, perforate as rudimentary colpori and regarded the pollen around margines, lumina 0.5–3 µm in diameter as colporate. The grooves or colporus-like at mesocolpium, with columellae elements. furrows are, however, different from the typical Voucher specimen: Congo. Semliki R. forest. colporus, because they seem to have no obvious Kassner Expedition 1908, no. 3088 (E). ectoaperture. The pollen grains of Amphicarpaea africana differ from those of the other two species in Amphicarpaea edgeworthii Benth. (Fig. 3) having distinct colpi. We compared the pollen Pollen grains triporate usually with slight with that of Shuteria hirsuta, because A. africana colporus-like furrows; polar axis 22.5 ± 1.2 had been regarded as a member of Shuteria, µm (range: 20.6–25.5 µm) (n = 25), equatorial and S. hirsuta appears to be most similar to diameter 19.8 ± 1.0 µm (18.1–21.9 µm) (n = 26), Amphicarpaea among Shuteria. Pollen grains P/E = 1.13 ± 0.06 (1.08–1.27), prolate spheroidal of S. hirsuta and S. vestita were reported by to subspheroidal, equatorial view elliptic, polar Thuan (1972). We confirmed that pollen grains view circular. Aperture ca. 0.18 times as long of these species are distinctly tricolporate. The as the polar axis, margines continuous with colporus in the two Shuteria species has the mesocolpium. Sexine semitectate; sculpture of same structure as that of A. africana, but not of mesocolpium granulate to rugulate with fine A. bracteata and A. edgeworthii. fossula. Voucher specimen: Japan. Miyagi Pref., Sendai-city, Aobayama. 22 Sep. 1985. Amphicarpaea bracteata (L.) Fernald (Fig. 2) Nemoto 2881 (TUS). Pollen grains tetraporate sometimes with The pollen grains of this species (also as linear grooves or slight colporus-like furrows; Amphicarpaea trisperma, A. edgeworthii var. polar axis 28.4 ± 1.0 µm (range: 26.4–30.3 japonica or A. bracteata subsp. edgeworthii) µm) (n = 20), equatorial diameter 25.0 ± 1.4 have been described as 3-colporate, 3-porate µm (22.2–27.4 µm) (n = 20), P/E = 1.14 ± 0.06 or 4–5-porate (Ikuse 1956, Huang 1968, 1972, (1.06–1.22), prolate spheroidal to subspheroidal, Meeson 1974, Liu 1991, Guo et al. 2003, Ohashi equatorial view elliptic, polar view circular et al. 2005). Such divergent descriptions were to polygonal. Colporus-like furrows (when produced due to occurrence of the colporus-like observed) continuous with mesocolpium, structure which are often indistinguishable from lacking margines, 0.44 times as long as the the tectum (Ohashi et al. 2005). Variation of the polar axis and ca. 1.9–3.2 µm wide at equator; colpori was illustrated in Ohashi et al. (2005; fig. aperture ca. 0.16 times as long as the polar axis.