Crassula Peduncularis and C. Saginoides (Crassulaceae), Newly Naturalized in Japan, and Their Genetic Differences from C

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Crassula Peduncularis and C. Saginoides (Crassulaceae), Newly Naturalized in Japan, and Their Genetic Differences from C ISSN 1346-7565 Acta Phytotax. Geobot. 70 (2): 119–127 (2019) doi: 10.18942/apg.201818 Crassula peduncularis and C. saginoides (Crassulaceae), Newly Naturalized in Japan, and their Genetic Differences from C. aquatica 1,* 2 3 3 Shinji Fujii , TadaShi YamaShiro , Sachiko horie and maSaYuki maki 1Department of Environmental Science, University of Human Environments, Okazaki, Aichi, 444-3505, Japan. * [email protected] (author for correspondence); 2Division of Bioscience and Bioindustry, Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima 770-8513, Japan; 3Botanical Gardens, Tohoku University, Sendai, Miyagi 980-0862, Japan Recently, we found two species of Crassula newly introduced into central Japan. Based on their mor- phology, we identified them asCrassula peduncularis and C. saginoides, native to the New World. They closely resemble C. aquatica, which is indigenous to Japan. Here, we describe the morphological distinc- tions among the three species in detail. In addition, we examined genetic differences among the three species based on sequence variations in the nuclear ribosomal ITS region and three chloroplast inter- genic regions. Although C. saginoides was considered to be an inland form of C. aquatica and had been treated as a synonym of C. aquatica, the degree of genetic differentiation was relatively large, suggesting that these two taxa should be considered to be independent species. Key words: Crassula aquatica, Crassula peduncularis, Crassula saginoides, genetic differentiation, morphology, naturalized Crassula L. (Crassulaceae) is a large genus of guished from C. aquatica by the pedicels of the mostly terrestrial succulent plants, but also con- former that tend to elongate in fruit while those of tains annual, aquatic or semi-aquatic species the latter remain less than 1 mm long. Crassula with a worldwide distribution. In Japan, only C. solieri differs from C. aquatica also has elongat- aquatica is indigenous (Ohba 2001), but Crassula ed pedicels in fruit, as in C. saginoides, but is tillaea Lester-Garland (Tillaea muscosa L.), unique in the smooth, shiny surface cells of the which is native to Europe, was recently recorded seeds. Moran (1994, 2009), however, considered (Katsuyama et al. 2005). Although some species C. saginoides to be only an inland phenotype of of Crassula, such as C. aquatica and C. tillaea, C. aquatica, under which he treated it as a syn- have been treated as species of the genus Tillaea onym. (e.g., Borisova 1939, Kunjun 2001, Ohba 2001, In 2015, one of us (Fujii) discovered plants of Katsuyama et al. 2005), a recent molecular phylo- Crassula on Honshu that had not been reported genetic study indicated that Tillaea was polyphy- previously from Japan. Although they were simi- letically embedded within Crassula and was in- lar to C. aquatica, we found differences between cluded within Crassula by Mort et al. (2009). In them in certain morphological traits. Hereafter, revising Crassula in the New World, Bywater & we refer to the unknowns as “C. aquatica-like Wickens (1984) pointed out that C. saginoides plants.” In this study, we examined the morphol- (Maxim.) Bywater & Wickens and C. solieri ogy of “C. aquatica-like plant,” and sequenced (Gay) Meigen are morphologically similar to C. the nuclear ribosomal ITS (nrITS) region and aquatica. Crassula saginoides can be distin- three chloroplast intergenic regions to compare 120 Acta Phytotax. Geobot. Vol. 70 with Crassula aquatica. Based on morphological μM of each of the two primers, 0.1 mM of each and genetic studies we clarified the identity of the dNTP, 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2 C. aquatica-like plants. mM MgCl2, and 0.25 U of Taq polymerase (Am- plicon Inc., Irvine, CA). The reactions for all four regions were initiated with initial denaturation at Materials and Methods 95 °C for 5 minutes followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s, and Morphological observations a final extension at 72 °C for 10 minutes on an Plants collected in the field and deposited in ABI GeneAmp 9700 thermal cycler (Applied herbaria were examined for morphological char- Biosystems, Foster City, CA). The purified PCR acteristics and for details of their collection (loca- products were sequenced using a BigDye termi- tion and year of collection). Field surveys to lo- nator v.3.1 Cycle Sequencing Kit (Applied Bio- cate additional plants were conducted throughout systems) on an ABI 3100 Genetic Analyzer (Ap- Japan: Hokkaido, Honshu, Shikoku and Kyushu, plied Biosystems). The sequences were aligned and specimens in the herbaria: INM, KYO, with BioEdit v.7.0.5 (Hall 1999). A haplotype net- MAK, OSA, TI, TNS, TUS, Gunma Museum of work based on mutational steps in three chloro- Natural History, the herbarium of Niigata City. plast gene regions was created using TCS 1.21 (Clement et al. 2000) to evaluate possible genetic Seed morphology using scanning electron mi- relationships among haplotypes under the 95% croscopy statistical parsimony criterion. All sequences The seed coats of the Crassula aquatica-like newly determined here were deposited in Gen- plants (populations ZYO and KDZ in Table 1) Bank (DDBJ). and of C. aquatica (populations SZK and TBS in Table 1) were examined using a JSM-6010PLUS/ LA scanning electron microscope (SEM; JEOL, Results and Discussion Tokyo, Japan) in low vacuum mode (50 Pa) at 10 kV. Morphological observations Crassula aquatica and the C. aquatica-like DNA sequencing plants were erect, up to 7 cm tall, had branched For DNA sequencing, one individual per pop- stems (unbranched in small plants); leaves 1.5–7 ulation from three populations of the Crassula mm long, solitary flowers, four petals and acute, aquatica-like plants and nine populations of the many-seeded carpels. We distinguished three native C. aquatica (Table 1) were examined. types (I, II and III) based on morphological fea- Whole plants were collected and placed in plastic tures. Type I plants, which occurred throughout bags with a zip fastener and refrigerated until Japan (Hokkaido, Honshu, Shikoku and Kyushu, DNA extraction. Fig. 3A), were green in the spring fruiting season, We sequenced the nrITS regions and three had obtuse (or sometimes acute) leaves, fruiting chloroplast gene regions: trnHGUG-psbA, trnCGCA- capsules sessile or subsessile, carpels ascending rpoB, and 3’trnVUAC-ndhC. The primer pairs fol- or almost patent (or sometimes erect) at maturity lowed White et al. (1990) for nrITS, Shaw et al. (Fig. 1A) and rugulose-striate seed surface (Fig. (2005) for trnHGUG-psbA and trnCGCA-rpoB, and 2A). These features are characteristic of C. aquat- Shaw et al. (2007) for 3’trnVUAC-ndhC. Total DNA ica. was extracted from a shoot of each sample ac- The type II plants, found only in the Kinki cording to the cetyltrimethylammonium bromide District, Honshu (Fig. 3B), were often purple dur- (CTAB) method of Doyle & Doyle (1990). PCR ing the spring fruiting season, the leaves were amplifications were conducted in a final volume acute, the capsules were sessile or on pedicels to of 10 μl using 10–20 ng of template DNA, 0.15 15 mm long (varying even within the same plant, June 2019 Fujii & al. – Taxonomy of Exotic Crassula in Japan 121 Table 1. Populations examined in molecular phylogenetic analyses. Population Geographic 1) 2) Locality Voucher (herbarium acronym) Ribotype Chlorotype code coordinate TYPE I (Crassula aquatica) Fujii 17198 BHR Onsui pond, Bihoro, Hokkaido 43.787, 144.163 B B (KYO, MAK, TNS, TUS) Uchi-numa, Kurihara, Sugiyama, Kasai & Emi 8558 UCN 38.713, 141.082 A A Miyagi Pref. (TUS) Morinji, Tatebayashi, 3) TBS 36.226, 139.529 Aoki 17337 (GMNH ) B B Gunma Pref. Otama pond, Hakone, HKN 35.206, 139.041 Fujii 16999 (KYO, TUS) B B Kanagawa Pref. Nishikan-ku, Niigata, NGT 37.748, 138.890 Fujii 13969 (KYO, OSA) B B Niigata Pref. Aoi-ku, Shizuoka, SZK 35.014, 138.394 Fujii 17354 (KYO, OSA, TUS) B B Shizuoka Pref. HMJ Shigo, Himeji, Hyogo Pref. 34.815, 134.711 Fujii 16991 (KYO) A A MIE Sugari, Owase, Mie Pref. 34.094, 136.295 Fujii & Yamamoto 13206 (OSA) B B Fujii 16735 TOK Ohtsu, Naruto, Tokushima Pref. 34.161, 134.580 B B (KYO, MAK, TNS, TUS) TYPE II (Crassula peduncularis) Uji (Yodo) River, Yawata, Fujii 16952 YOD 34.894, 135.700 C C Kyoto Pref. (KYO, OSA, TNS, TUS) Fujii 16951 ZYO Tomino, Zyoyo, Kyoto Pref. 34.859, 135.774 C C (KYO, OSA, TNS, TUS) TYPE III (Crassula saginoides) Fujii & Maki 17313 KDZ Kiso River, Kaidzu, Gifu Pref. 35.233, 136.675 C D (KYO, OSA, TNS, TUS) 1) See Table 3. 2) See Table 4. 3) Gunma Museum of Natural History. with pedicel usually longest on the lower portion roplast gene regions, while type I had an indel of the shoot), carpels erect at maturity (Fig. 1B), variation among populations in 224–281 bp (Ta- each cell of the striate seed surface with 2–5 pa- bles 1 & 3). Figure 4 shows the TCS haplotype pillae (Fig. 2B). network based on mutations in the chloroplast The type III plants, found only at one locality gene regions, indicating a genetic gap between (KDZ) along the Kiso River in Honshu (Fig. 3B), types I, II and III, and also between types II and were similar to the type II plants in color, leaves III. The genetic evidence supports the recogni- and carpels, but differed in that the fruiting cap- tion of the three taxa in spite of their similar mor- sules were usually sessile or sometimes on pedi- phology. Because of a general lack of good mor- cels to 15 mm long (Fig. 1C) and the seeds were phological characters for distinguishing among striate-rugulose but lacked the papillae (Fig.
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