ISSN 1346-7565 Acta Phytotax. Geobot. 68 (2): 83–92 (2017) doi: 10.18942/apg.201703 Molecular Phylogenetic Analysis of Japanese Miscanthus (Poaceae) 1 2 3,† 2 HIDENORI NAKAMORI , MIKI TOMITA , HIROSHI AZUMA , TAKEHIRO MASUZAWA 2,* AND TORU TOKUOKA 1Graduate School of Integrated Science and Technology, Shizuoka University, Ohya, Suruga-ku, Shizuoka, 422-8529, Japan; 2Department of Biological Science, Faculty of Science, Shizuoka University, Ohya, Suruga-ku, Shizuoka, 422-8529, Japan. *[email protected] (author for corresponding); 3Department of Botany, Graduate School of Science, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan; †Present Name & address: HIROSHI SUZUKI; Liberal arts and Sciences, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu-shi, Toyama 939-0398, Japan Miscanthus (Poaceae) comprises about 20 species, of which seven species and two forms occur in Japan. There is controversy whether M. condensatus is a separate species or a variety or subspecies of M. sinen- sis. To determine its taxonomic status, we conducted a molecular phylogenetic analysis using DNA se- quences of the atpB-rbcL, psbC-trnS(UGA), rpl20-rps12, trnL(UAA)-trnF(GAA), trnS(GGA)- trnT(UGU), and nuclear ITS regions, and the Adh1 gene from 31 samples of the seven Japanese species of Miscanthus. The neighbor-joining (NJ) tree based on the cpDNA sequences shows that M. condensa- tus and M. sinensis share two haplotypes, and that the nuclear ITS and Adh1 sequences of the two species are identical, making it difficult to distinguishM. condensatus from M. sinensis based on DNA sequenc- es. The evidence indicates that hybridization between the two species has proceeded rapidly, or that M. condensatus is derived from a diverging lineage of M. sinensis. The status of M. condensatus remains controversial, but our findings support provisionally treating M. condensatus as the infraspecific taxon, M. sinensis var. condensatus. Key words: Miscanthus, Miscanthus condensatus, Miscanthus sinensis, molecular phylogeny, Poaceae Miscanthus Andersson (Poaceae), which some count for five Miscanthus( condensatus, M. comprises about 20 species, occurs mainly in floridulus, M. oligostachyus, M. sinensis, and M. Southeast Asia (Clayton & Renvoize 1986). Sev- tinctorius) is 2n = 38. A chromosome count of 2n en species, as well as two forms, of Miscanthus = 57 has also been reported for M. condensatus occur in Japan (Osada 1989). It has been proposed (Adati 1958), but Hirayoshi et al. (1959) reported that Miscanthus be separated into three sections never having observed such a count. For M. inter- (for review, see Nishiwaki & Nadir 2014). With medius, counts of 2n = 76 and 114 have been re- respect to the Japanese species of Miscanthus, M. ported, and for M. sacchariflorus, 2n = 76 (Hi- sacchariflorus was assigned to section Triarrhe- rayoshi et al. 1956, 1959, 1960, 1964, for review na; M. floridulus, M. sinensis, and M. condensat- see Nishiwaki & Nadir 2014). Based on these us were assigned to section Eumiscanthus, and studies, it has been inferred that M. intermedius M. tinctorius, M. oligostachyus, and M. interme- and M. sacchariflorus formed via interspecific dius were assigned to section Kariyasua. hybridization (Hirayoshi et al. 1956, 1959, 1960, The cytology of the genus has also been stud- 1964). ied to determine the relationships among the spe- The biological and agricultural aspects of the cies. Of the seven Japanese species, the chromo- three Japanese species of Miscanthus in section 84 Acta Phytotax. Geobot. Vol. 68 Eumiscanthus have also been well studied. Of the tus accessions from the 40 M. sinensis acces- three, Miscanhus sinensis is a dominant perenni- sions. However, that study did not include sam- al grass and its ecology has been thoroughly ex- ples from mainland Japan. To understand the amined (e.g. Ministry of the Environment et al. phylogeography of M. sinensis and to clarify the 2007, Stewart et al. 2009, Quinn et al. 2012, Clark taxonomic status of M. condensatus, it is neces- et al. 2014, 2015). Miscanthus floridulus was in- sary to combine the approaches of the previous vestigated as a potential biofuel energy crop studies to produce an integrated dataset that gives (Bullard 1996, Bullard et al. 1997, Christian et al. a clearer picture of the situation. 1997), and in other studies the genetic variation Here, we present molecular phylogenetic among the Japanese populations was found to be analyses, based on sequencing of the chloroplast minimal (Ibaragi et al. 2013). The taxonomy of and nuclear DNA regions, of the seven Japanese the third species, M. condensatus, has been con- species of Miscanthus. They represent all three troversial. Many authors treat M. condensatus as sections of Miscanthus that occur on mainland a distinct species that is specialized to grow near Japan. In addition, we discuss the taxonomic seashores (Ohwi 1965, 1975, 1982, Osada 1989). treatment of M. condensatus. Others have treated it as a variant or subspecies of M. sinensis, because of the morphological simi- larity between the two (Ohwi 1953, Makino 1961, Materials and Methods Clayton & Renvoize 1986, Koyama 1987). More- over, Osada (1989) reported intermediate forms Taxon sampling, DNA extraction, amplification, between the two species occurring with low fre- and sequencing quency in transitional habitats between the coast DNA sequences of the five cpDNA regions and inland areas. For clarity, in this study we use atpB- rbcL, psbC- trnS(UGA), rpl20- rps12, the name M. condensatus throughout, following trnL(UAA)-trnF(GAA), trnS(GGA)-trnT(UGU), the convention of Osada (1989). the nuclear ITS regions, and Adh1 gene of 31 sam- Some phylogeographic studies of Miscanthus ples from the seven Japanese species of Miscan- sinensis have been conducted based on DNA thus were used for the molecular phylogenetic markers (Chiang et al. 2003, Iwata et al. 2005a, b, analyses (Table 1). Fresh leaves collected from Slavov et al. 2012, Shimono et al. 2013, Clark et wild plants were dried and preserved in silica gel. al. 2014, 2015). Shimono et al. (2013) reported DNA samples were extracted from the dried that nine haplotypes occur in Japan, based on 636 leaves. Voucher specimens for this study were samples from 30 populations, and using four re- deposited at Kyoto University (KYO). gions of chloroplast DNA (cpDNA). Two of the Total genomic DNA was extracted using the nine haplotypes were widely distributed across modified cetyltrimethylammonium bromide mainland Japan and within most populations. In (CTAB) method of Doyle & Doyle (1987). The contrast, Hayakawa et al. (2014) reported that the DNA regions used in this study were amplified populations on mainland Japan comprise a mono- using polymerase chain reaction (PCR). The PCR phyletic group, based on analyses using nuclear mixture (20 μL) contained 1 μL template DNA, internal transcribed spacer (ITS) sequences. 1.6 μL dNTPs (2.5 mM each), 0.2 μL of each However, the report included no mention of the primer (10 μM), 2 μL of 10× Taq buffer (contain- taxonomic treatment of M. condensatus. It is un- ing 20 mM MgCl2), 0.5 U Taq polymerase (Ex- clear whether it was included in the analyses, as Taq; Takara Bio, Japan). The PCR was performed an infraspecies, or excluded as an independent with a T100 Thermal Cycler (Bio-Rad Laborato- species. Chiang et al. (2003) estimated the DNA ries), using 35 cycles, each with 1 min denatur- sequence variation of the nuclear Adh1 locus in ation at 94°C, 1 min annealing at 55°C , an exten- both M. sinensis and M. condensatus and found sion of 1 min at 72°C, and a final extension of 5 strong support for separating the 10 M. condensa- min at 72°C. After checking for a single band us- June 2017 NAKAMORI & AL.–Phylogeny of Japanese Miscanthus 85 ing electrophoresis on 0.5% agarose TAE gel comprised 597 characters; 22 sites were variable stained with Midori Green DNA Stain (NIPPON among all samples (Table 4). Genetics Co, Ltd), the PCR products were puri- In the data matrix for the five cpDNA regions, fied via enzyme treatment. A 2-μL mixture con- six of the Miscanthus condensatus samples taining 0.3 U Exonuclease I (Takara Bio) and 0.3 (CO_1 to 6), two of the M. sinensis samples (SI_7 U calf intestine alkaline phosphatase (Toyobo, & 8), and two of the M. tinctorius samples (TI_1 Japan) was added to each PCR tube to degrade & 2) (hereafter, cpDNA group 1) were identical. remaining primers and dephosphorylate any re- Other groups that were identical in these regions maining dNTPs. The tubes were incubated at were: i) four of the M. floridulus samples (FL_1 37°C for 30 min, and then enzymes were dena- to 4; cpDNA group 2); ii) six M. sinensis samples tured at 80°C for 15 min. (SI_1 to 6) and three M. condensatus samples Direct sequencing of both strands was con- (CO_7 to 9) (cpDNA group 3); iii) three M. oli- ducted on an ABI 3100 Genetic Analyzer (Ap- gostachyus samples (OL_1 to 3; cpDNA group 4); plied Biosystems) using the BigDye Terminator and iv) two M. intermedius samples (IN_1 & 2; version 3.1 Cycle Sequencing Ready Reaction Kit cpDNA group 5). In the data matrix for the nucle- (Applied Biosystems Japan) following the manu- ar ITS sequences, all species except M. oli- facturer’s protocol. Primers for amplification gostachyus showed no variation. There was also and/or sequencing are listed in Table 2. We man- no variation between M. sinensis and M. conden- ually aligned the DNA sequences obtained using satus. With respect to the nuclear Adh1 gene se- MEGA v. 6.06 (Tamura et al. 2013). quences, four samples of M. sinensis samples and five of M. condensatus were identical (see acces- Phylogenetic analysis sion nos. in Table 1). The phylogenetic tree of cpDNA was con- We compared the sequence data with data structed by the neighbor-joining method (NJ) im- from previous studies (Shimono et al.