J. Jpn. Bot. 93(4): 240–252 (2018)

A New Record of the Critically Endangered Pondweed, Potamogeton lucens (Potamogetonaceae) from Aomori Prefecture, Japan

Kohtaroh S a,*, Mitsuru Ub, Hiroki Yc, Yushi Fd, Shiori Ha, Osamu Te, f, Yoshiko If, Masamitsu Kf, Naoko Kf, Akio Mf, Toshikazu Nf, Akiko Yf, Shingo Kd and Takashi Sa

aFaculty of Education, Niigata University, 2-8050, Ikarashi, Nishi-ku, Niigata, 950-2181 JAPAN; bIwaki, Fukushima, 970-8002 JAPAN; cThe Shirakami Institute for Environmental Sciences, Hirosaki University, 3, Bunkyo-cho, Hirosaki, Aomori, 036-8561 JAPAN; dFaculty of Symbiotic Systems Science, Fukushima University, 1, Kanayagawa, Fukushima, 960-1296 JAPAN; eAomori City Forestry Museum, 2-4, Yanakawa, Aomori, 038-0012 JAPAN; fThe Botanical Society of Tsugaru, 1-2-9, Wakaba, Goshogawara, Aomori, 037-0069 JAPAN *Corresponding author: [email protected]

(Accepted on February 14, 2018)

A new locality for Potamogeton lucens L. (Potamogetonaceae) was recorded from a pond in Aomori Pref., Tohoku District, Japan. In Japan, this species is critically endangered, and the sole remaining naturally established population was previously known only from one other locality. In June 2017, we found fragments of a submerged plant species that resembled P. lucens. We conducted fi eld surveys and identifi ed it as P. lucens based on morphological measurements, nuclear and chloroplast molecular analyses, a herbarium specimen survey, and pollen fertility. Although we could not reveal its population size and distribution in the pond, at least an established population exists because many fragments and a plant with developed roots were collected. This new locality of P. lucens represents the new northern limit of the species in Japan, which is over 500 km away from its nearest distributional record. As developments are now in progress around the pond area, urgent protection by a conservation program is needed.

Key words: Aomori Prefecture, aquatic plants, critically-endangered, fl ora, hybridization, Potamogeton gramineus, Potamogeton lucens, Potamogetonaceae, Red List.

Shining pondweed, Potamogeton lucens L. veins (Guo et al. 2010, Kadono 2014). It is (Potamogetonaceae; Japanese name, Gasha- widely distributed in the eastern and northern moku), is a rare aquatic macrophyte in Japan. hemisphere (Wiegleb and Kaplan 1998). This species is an obligate submerged plant with Although the IUCN Red List ver. 3.1 categorizes large and herbaceous stipules and subsessile to P. lucens as a least concern species (IUCN Red shortly petiolate leaves with distinctive netted List, http://www.iucnredlist.org/, confirmed in

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Fig. 1. Habit and habitat of Potamogeton lucens. a. Southern shore of the pond where detached segments (18 June 2017). b. Floating fragment (18 June 2017). c. Fragment of an individual (18 June 2017). d. Submerged leaves and an infl orescence (4 July 2017). e. Pollen grains stained with lactophenol cotton blue (10 July 2017). Bar: 1.0 cm (d), 0.1 mm (e).

3 Oct. 2017), the Ministry of the Environment, manure (Makino 1905). However, almost all Government of Japan (2015) categorized the populations have disappeared due to water species (as P. lucens var. teganumensis) as a pollution (Kadono 2014). Currently, the species critically endangered species in the Japanese only occurs naturally in a pond in Fukuoka Red List. In Japan, over 100 years ago, the Prefecture (Uehara et al. 2006, Chiba Prefectural species was so abundant in some lakes in Government 2017). In several artificial ponds Kanto District that farmers used the plants as in Chiba Prefecture, plants of the species had 242 植物研究雑誌 第 93 巻 第 4 号 2018 年 8 月

Fig. 2. Specimen suspected to be a Potamogeton lucens hybrid collected by M. Usuba on 25 July 1989 (M. Usuba 14025, NGU4567). The details of the locality was concealed to prevent excessive collection. August 2018 The Journal of Japanese Botany Vol. 93 No. 4 243 grown until recently, but all of them disappeared to the fragments, possibly hybridizing with a few years after sprouting from their soil P. lucens. The larger leaves of the fragments seed banks (Tanaka 2012, Chiba Prefectural might be caused by any hybridization. In order Government 2017, K. Hayashi pers. comm.). to identify the fragments, it was necessary to Therefore, conservation efforts are now being examine their morphological and molecular conducted in these localities (e.g., Amano et al. status. Molecular analyses based on chloroplast 2008, Tanaka 2012). and nuclear DNA have often been used to In June 2017, we found fragments of a identify parents of Potamogeton hybrids (e.g., submerged plant similar to Potamogeton lucens Ito et al. 2007, Zalewska-Gałosz et al. 2010, on the southern shore of a pond in Tsugaru- Yang et al. 2016). shi, Aomori Prefecture (Fig. 1a, b). Their The aim of this study was to identify the submerged leaves possessed relatively short fragments of the submerged plant similar to petioles, long stipules, and had distinctive Potamogeton lucens based on morphological netted veins (Fig. 1c). However, it seemed that measurements, nuclear and chloroplast its blades were larger than the description of molecular analyses, a herbarium specimen P. lucens subsp. sinicus (Migo) H. Hara var. survey, and pollen fertility. The specimens teganumensis Makino (Kadono 2014). Because collected by M. Usuba in the 1980s, which were the Japanese or Asian P. lucens is characterized suspected to be hybrids, were also subjected by smaller leaves, spikes, flowers, fruits, and to molecular analyses for identification. We achenes and more slender branches and stalks discuss the growing conditions and conservation compared with the European species, it is often requirements of the plant similar to P. lucens. treated as a subspecies or variety of the species (Makino 1905, Hara 1985). Furthermore, in Materials and Methods the 1980s, one of the authors (M. Usuba) had Field survey collected two Potamogeton specimens that were The unnamed pond from which fragments morphologically similar to P. lucens hybrids were found is located in Tsugaru-shi, Aomori from an artificial irrigation ditch near the pond Prefecture, Tohoku District, Japan (Fig. 3). We (Fig. 2). These had an uneven leaf size (2.5–7.0 concealed the details of the locality to prevent cm), well branched stems in the upper part, excessive collection, as Potamogeton lucens as well as subsessile leaves with distinctive is occasionally sold as an aquarium plant in netted veins, resembling the description of P. Japan. Its area and perimeter is approximately ×angustifolius (Zalewska-Gałosz 2002; Fig. 2). 14 ha and 1.76 km, respectively. The pond was As with other Potamogeton species, P. formed 20–30 years ago by construction. Based lucens often crosses with other related species. on three aerial photographs, there were no ponds Among them, P. ×angustifolius J. Presl (with at this site in 1985, a construction site and water P. gramineus L.), P. ×salicifolius Wolfg. (with body at the east and west shores of the current P. perfoliatus L.), and P. ×inbaensis Kadono pond were observed in 1990 respectively, and (with P. wrightii Morong) have a considerably the pond was fully formed in 1995 (Geospatial variable morphology and these hybrids Information Authority of Japan, http://mapps. sometimes bear quite a resemblance to P. lucens gsi.go.jp/maplibSearch.do#1, confirmed in (Kadono 1983, Zalewska-Gałosz 2002, Ito et al. 28 Aug. 2017). In 1986, before the artificial 2007). Additionally, five Potamogeton species, formation of this pond, it had been wet bog (M. P. natans L., P. distinctus A. Benn., P. crispus Usuba personal observation). Water quality L., P. maackianus A. Benn., and P. berchtoldii measurements were taken at 11:50 a.m. on Fieber., were found from the pond in addition 26 July 2017 at the shore where we found the 244 植物研究雑誌 第 93 巻 第 4 号 2018 年 8 月

Fig. 3. Revised distribution map of Potamogeton lucens in Japan presented by Kadono (1994). White and black circles indicate disappeared and established populations, respectively. The underlined population indicates the new locality. fragments using the LAQUAtwin B-712 meter northern shores of the pond (ca. 0.93 km; 53% of (HORIBA Advanced Techno, Kyoto, Japan). the pond shore) by walking; we could not survey Water temperature was 27.5 °C, pH was 7.80– the other shores because they were difficult to 7.90, and electrical conductivity was 0.25–0.26 approach. In addition to walking, we collected ms/cm. The aquatic macrophyte flora in this aquatic macrophytes using an aquatic plant pond had not been surveyed before, although sampler (F–K octopus sampler; Kato et al. in a comprehensive aquatic floristic research has prep.). Fragments of submerged plants similar to been conducted near the pond in 2005–2006 P. lucens were collected for molecular analyses (Higuchi and Fukuhara unpublished data). and morphological observations each time they In the fi eld survey, we collected fragments of were observed. Potamogeton gramineous, which submerged plants similar to Potamogeton lucens. is the most closely related species to P. lucens Surveys were conducted on 18 June, 4 July, and (Iida et al. 2004, Lindqvist et al. 2006, Ito et al. 26 July 2017. We surveyed the southern and 2016) and often hybridizes with P. lucens, was August 2018 The Journal of Japanese Botany Vol. 93 No. 4 245 also collected to compare their molecular status randomly counted more than 499 pollen grains with the fragments. We recorded all submerged, from each anther, and viability was calculated as floating-leaved, and free-floating macrophyte the number of stained pollen grains divided by species to elucidate the aquatic macrophyte fl ora the total number of pollen grains examined. of the pond. Molecular analyses Morphological observations To identify the collected fragments by The morphology of the collected fragments molecular analyses, we sequenced the nuclear similar to Potamogeton lucens was observed. internal transcribed spacer (ITS) and the For qualitative characters, the presence chloroplast rpl20-rps12 intergenic spacer. We or absence of heterophylly, leaf shape or sampled leaf materials from 10 fragments of characteristics, leaf apex, and leaf veins were submerged plants similar to Potamogeton lucens observed. For quantitative characters, petiole and two P. gramineus plants collected from the length, blade length and width, and stipule length same pond. Two specimens collected by M. were measured and the number of carpels were Usuba (M. Usuba 12841, NGU4566 and M. counted. Twenty leaves from fi ve non-blooming Usuba 14025, NGU4567) were also sampled. fragments and two inflorescences from two The samples were dried with silica gel until blooming fragments were used to examine DNA extraction. vegetative and reproductive organs, respectively. Genomic DNA was extracted using the Their morphology was compared to the Gentra Puregene Tissue Kit (Qiagen, Hilden, descriptions of P. lucens reported in Japanese, Germany). According to the protocol of Kaplan Chinese, and European literature (Preston and Fehrer (2004, 2006, 2011) and Ito et al. 1995, Guo et al. 2010, Kadono 2014) and to (2007), we selected the amplified regions, descriptions of three hybrids, P. ×angustifolius, nuclear ITS (ITS1, 5.8S, ITS2), and chloroplast P. ×salicifolius, and P. ×inbaensis, which rpl20-rps12 intergenic spacer. Primers ITS4 are sometimes difficult to distinguish and ITS5 (White et al. 1990) and rpl20 and morphologically from P. lucens (Zalewska- 5ʹ-rps12 (Hamilton et al. 1999) were used for Gałosz 2002, Kadono 2014). In addition, we amplifi cation of ITS and the rpl20-rps12 spacer, preliminarily surveyed herbarium specimens respectively. of P. lucens at the Osaka Museum of Natural Polymerase chain reaction (PCR) was History (OSA) to compare the morphology of performed following the protocol of the Qiagen specimens with the description of the Japanese Multiplex PCR Kit (Qiagen) with purified literature (e.g., Makino 1905, Kadono 2014). DNA, 4 μl of 2× Multiplex PCR Master Mix, The seven examined fragments were donated as and 0.2 μM of each primer at a final volume voucher specimens to the herbarium of Niigata of 8 μl using a T100 thermal cycler (Bio-Rad, University (NGU; Appendix 1). Hercules, California, USA) under the following Pollen viability of the fragments was conditions: initial denaturation at 95 °C for 15 estimated based on the stainability of the pollen min; 28 cycles of denaturation at 94 °C for 30 s, grains with a cotton blue staining solution for 10 annealing at 55 °C for 1 min and 30 s, extension min. The staining solution was a 20% phenol, at 72 °C for 1 min; and a final extension at 60 20% lactic acid, and 40% glycerin aqueous °C for 30 min. PCR products were purified solution to which 1% lactophenol cotton blue using Illustra ExoStar (GE Healthcare UK, Little aqueous solution was added. We removed six Chalfont, UK) and sequenced directly using anthers from the above two inflorescences and an ABI BigDye Terminator Cycle Sequencing collected the pollen grains on a glass slide. We Kit version 3.1 (Applied Biosystems, Foster 246 植物研究雑誌 第 93 巻 第 4 号 2018 年 8 月

Table 1. List of Potamogeton samples used for a comparison of internal transcribed spacer (ITS) and rpl20-5ʹrps12 spacer sequences ITS1, 5.8s, ITS2 rpl20-5ʹrps12 Locality 1 2 GHtype Accession no. Htype Accession no. P. lucens a a Japan: Aomori Pref., Tsugaru LNR-1 LC316763 LCP-1 LC316765 3 b 4 c Japan: Chiba Pref., Tega-gawa river basin 1 LNR-1 AB206990 LCP-1 AB871496 d d Japan: Chiba Pref., Tega-gawa river basin 2 LNR-2 HQ263509 LCP-1 HQ263453 3 b Japan: Fukuoka Pref., Kitakyushu 1 LNR-1 AB206990 d d Japan: Fukuoka Pref., Kitakyushu 2 LNR-1 HQ263508 LCP-1 HQ263452 e e Czech Republic: Distr. Pardubice, Hrobice LNR-3 EF174584 LCP-2 EF174595 d d Finland: Päijät-Häme, Hollola LNR-1 HQ263510 LCP-2 HQ263454 d d Germany: Mecklenburg-Vorpommern, Feldberg LNR-1 HQ263512 LCP-2 HQ263456 e e The Netherlands: Limburg, Arcen LNR-4 EF174583 LCP-2 EF174594 f f Poland: NE Poland, Rospuda River LNR-1 FJ883591 LCP-2 FJ883609 f f Poland: NW Poland, Drawa River LNR-1 FJ883592 LCP-2 FJ883610 d d Russia: Irkutsk, Ziminskiy, Ignay LNR-1 HQ263511 LCP-2 HQ263455 g China: Yunnan, Heqing LNR-1 KX062124–5 g China: Yunnan, Dali LNR-1 KX062126–7 P. gramineus a a Japan: Aomori Pref., Tsugaru GNR LC316764 GCP LC316766 Superscripts on accession nos. indicate references: aPresent study; bIto et al. (2007); cIto et al. (2014); dKaplan and Fehrer (2011); eKaplan and Fehrer (2007); fZalewska-Gałosz et al. (2010); gYang et al. (2016). 1Genotype or haplotype of nuclear ribosomal DNA. 2Haplotype of chloroplast DNA. 3Treated as “Potamogeton lucens L. subsp. sinicus (Migo) H. Hara var. teganumensis Makino”. 4The locality was confi rmed based on a personal communication with the author, Y. Ito, because it was not indicated in the reference.

City, California, USA) on an ABI PRISM 3130 because they included suspicious sequences, Genetic Analyzer (Applied Biosystems). The possibly derived from hybridization with other individual nucleotide sequences were read species. The consensus sequence of each locus using FinchTV version 1.4.0 (Geospiza, Seattle, was aligned using the program MAFFT version Washington, USA). The resulting sequences 7 (Katoh and Standley 2013) with default were deposited in the DNA Data Bank of Japan settings. The aligned sequences were compared (DDBJ), GenBank, and the European Molecular with the program MEGA 6.06 (Tamura et Biology Laboratory database (EMBL) (Table 1). al. 2013) and a plant similar to P. lucens was We compared the sequences of our samples identifi ed based on these sequence comparisons. with those of Potamogeton lucens and P. We did not treat one-base gaps yielded by single gramineus from DDBJ, GenBank, and EMBL sequence repeats as variations because most of (Table 1). Only sequences that included the gaps were indicated by unclear peak patterns published reference studies were used for the that were diffi cult to read. comparison (i.e., Ito et al. 2007, 2014, Kaplan and Fehrer 2007, 2011, Zalewska-Gałosz et Results al. 2010, Yang et al. 2016). Although Wang et Field survey and morphological identifi cation al. (2007) also sequenced ITS of P. lucens, the We collected about 30 fragments that sequences were not included in the comparison had drifted ashore at the southern shore near August 2018 The Journal of Japanese Botany Vol. 93 No. 4 247

Table 2. Measurements of Potamogeton lucens in the present study and as cited in species descriptions in the literature Locality Aomori Pref.1 Japan2 China2 UK2 References Present study Kadono (2014)3 Guo et al. (2010) Preston (1995) Blade length 11.2 ± 3.8 (5–17.4) cm 5–12 cm 2–18 cm (5.5–)7.5–20.0(–26.0) cm Blade width 2.0 ± 0.4 (1.3–2.7) cm 1.2–2.5 cm 0.8–5.0 cm (1.7–)2.5–6.5 cm Petiole 0.5 ± 0.3 (0.2–1.6) cm < 1 cm 0.2–1.5 cm 0.1–1.2(–2.5) cm Stipules 3.8 ± 1.2 (1.3–6) cm 2–4 cm 2–8(–11) cm 3.5–8.0(–11.0) cm Carpels 4 4 4 4–5(–6) 1Measured in the present study: values excluding carpel numbers were indicated as “mean ± standard deviation (range)”. 2Cited from species descriptions in the literature: values show general range and those in parentheses show minimum or maximum values. 3Treated as “Potamogeton lucens L. subsp. sinicus (Migo) H. Hara var. teganumensis Makino”. the location in which the first fragment was are shown in Table 2. We found 17 Potamogeton collected. No other fragments were found along lucens specimens at OSA that had been collected the remaining shore investigated. Recorded from Gunma, Ibaraki, Chiba, Shiga, and aquatic macrophytes, except for emergent Fukuoka Prefectures. Among them, specimens macrophytes, from the ponds in addition to the with large leaves (ca. 16 cm blade length) were fragments are as follows: Hydrilla verticillata (L. sometimes observed in those collected from f.) Royle, Najas marina L., N. oguraensis Miki, the Fukuoka Prefecture (e.g., M. Ohno 7446-4, N. graminea Delile, Vallisneria natans (Lour.) OSA270713). H. Hara, Potamogeton natans, P. distinctus, P. crispus, P. maackianus, P. berchtoldii, Molecular identifi cation Sparganium sp., Trapa japonica Flerow, We sequenced 610 and 725 bp in the and Utricularia sp. During the 26 July 2017 nuclear ITS and chloroplast rpl20-rps12 observation, a plant similar to P. lucens with spacer, respectively, among fragments similar developed roots was collected by the aquatic- to Potamogeton lucens and P. gramineus. plant sampler at the southern shore, although all The sequences of all fragments similar to P. fragments collected did not possess such roots. lucens were identical to the one haplotype of The fragments exhibited only submerged P. lucens in the two loci (haplotypes LNR-1 and leaves with an elliptic to lanceolate blade LCP-1; Table 1). Potamogeton gramineus also and distinctive netted veins, a mucronate or exhibited haplotypes GNR and GCP in the two cuspidate apex, and relatively short petioles (Fig. loci. These haplotypes were distinguished from 1c). Blade length, blade width, petiole length, LNR-1 (by four substitutions) and LCP-1 (by two and stipule length of the fragments were 11.2 substitutions), respectively (Tables 3, 4). No ± 3.8 cm (5–17.4), 2.0 ± 0.4 cm (1.3–2.7), 0.5 heterozygosity was detected in the nuclear DNA ± 0.3 cm (0.2–1.6), and 3.8 ± 1.2 cm (1.3–6), of any of our samples. We could not amplify and respectively (Table 2). Most fragments were > 1 sequence Usuba’s specimens collected in 1986 m in length (the longest segments reached 2 m) and 1989. and their lower internodes were generally long Nuclear and chloroplast DNA haplotypes (< 25 cm). The fragments had four carpels (Fig. of the fragments were identical to the DNA 1d) and pollen viability of two infl orescences of database sequences of Potamogeton lucens blooming fragments was 87.4 ± 0.5% and 92.9 (Tables 1, 3, 4). The sequences listed in the ± 1.0% (Fig. 1e). Descriptions from the literature database, which were collected from localities 248 植物研究雑誌 第 93 巻 第 4 号 2018 年 8 月

Table 3. Variable sites among genotypes or haplotypes of Table 4. Variable sites among haplotypes of Potamogeton lucens and P. gramineus in nuclear ITS Potamogeton lucens and P. gramineus in sequences chloroplast rpl20-5ʹrps12 spacers Variable sites variable sites GH 1 H 1 type 91 164 181 188 501 502 529 555 type 18 213 467–473 P. lucens P. lucens LNR-1 AGGATTTC LCP-1 TG - LNR-2 AGGRTTTC LCP-2 T G TTCAAGA LNR-3 RGKATTTC P. gramineus LNR-4 AGGAWTTC GCP-1 AT - P. gramineus 1Haplotype of chloroplast DNA. GNR ATGATCGT 1Genotype or haplotype of nuclear ribosomal DNA. all over the world, exhibited four nrDNA On the other hand, their morphology did not haplotypes (LNR-1, LNR-2, LNR-3, LNR-4) and two agree the descriptions of P. ×angustifolius, cpDNA haplotypes (LCP-1, LCP-2). LNR-2, LNR-3, P. ×salicifolius, or P. ×inbaensis in terms of and LNR-4 were only distinguished from LNR-1 by conspicuous herbaceous stipules, relatively nucleotide ambiguity codes, and our fragments short petioles that were well differentiated had LNR-1 that did not include any nucleotide from the blades, absence of fl oating leaves, and ambiguity codes (Table 3). In cpDNA, LCP-1 little branched stems (Zalewska-Gałosz 2002, was from all Japanese samples, including the Kadono 2014). The high pollen viability of the fragments, and LCP-2 was from other countries. fragments can indicate low possibility that they These two haplotypes were distinguished by one represent hybrids. indel (Table 4). We identified the fragments collected from The sequences of Potamogeton gramineus the unnamed pond as Potamogeton lucens based from the same pond were also identical to on its morphology, nuclear and chloroplast one of the sequences in the DNA database DNA sequences, and high pollen viability. We in both nuclear and chloroplast DNAs (e.g., refrain from identification of the fragments to FJ883587, HQ263490, DQ468866). Although intraspecifi c level: the fragments showed slightly various ITS and chloroplast sequences of P. larger morphology than the descriptions of the gramineus were submitted in the database (e.g., known variety in Japan, although the sequences EF174589, KX062106, DQ468864), the P. from the fragments were identical to those of the lucens sequences of LNR-1–LNR-4 and LCP-1–LCP- variety from the DNA database (Kadono 2014; 2 differed from them by at least four and one Table 2). We also found specimens with larger substitutions, respectively. leaves collected from the already known locality. Further studies are needed to reconsider the Discussion intraspecifi c taxonomic status of this species. Identifi cation of collected fragments In the fragments collected from the pond, we The morphological characteristics of the did not detect any evidence of hybridization such collected fragments were consistent with the as heterozygosity in nuclear DNA, inconsistency description of Potamogeton lucens from the between nuclear and chloroplast DNA, sharing UK and China (Table 2): they were likely to haplotypes with closely related species, or have a slightly larger blade and stipule length relatively low pollen viability. Therefore, the compared to the description of P. lucens subsp. fragments were not identified as a hybrid of sinicus var. teganumensis (Kadono 2014). Potamogeton lucens. We could not identify August 2018 The Journal of Japanese Botany Vol. 93 No. 4 249 genetically the specimens collected by M. is widely distributed in China (Guo et al. 2010) Usuba that were suspected to be hybrids (Fig. and has limited distribution in Russian Far East 2). The amplification difficulty in both nuclear (Tzvelev 1987). Because of its wide distribution and chloroplast DNAs was possibly caused by including high latitude areas like Finland the age of the specimens. Since any P. lucens (Wiegleb and Kaplan 1998; Table 1), additional hybrids may be discovered from near the pond, new localities may be discovered by surveying continual studies that focus on P. lucens hybrids freshwater macrophyte flora in the Hokkaido are needed. Prefecture and Tohoku District areas. This population is important in Japan as the Conditions of the Potamogeton lucens growing conditions of other existing populations population have worsened. In a native population in The population size and distribution of Fukuoka Prefecture, the population size had Potamogeton lucens in the pond were not drastically declined during the 2000s (Amano revealed in this study. Whereas, we conclude et al. 2008, Shimizu 2011, Manabe et al. 2015), that P. lucens is at least established in the pond although it has been restored gradually after the because many fragments and a plant with declining (Shimizu 2016). In Chiba Prefecture, developed roots were collected. Potamogeton plants of the species sometimes sprouted from lucens vegetation patches were probably soil seed banks, but the plants soon disappeared distributed in deeper water (at least > 2 m depth) despite efforts of re-establishment (Saito 1991, because many long fragments with mostly Tanaka 2012, Chiba Prefectural Government long internodes were collected. It is necessary 2017, K. Hayashi personal communication). Ex to reveal their population size and distribution situ conservation and investigations of soil seed for a conservation program by investigating banks in these two localities are being conducted deep water habitats. Although P. lucens is (Momohara et al. 2001, Kushiro et al. 2009, occasionally sold as an aquarium plant in Japan, Shimizu et al. 2011, Tanaka 2012, Manabe et al. the possibility of population establishment 2016). Therefore, it is preferable to immediately caused by discarded aquarium plants is likely to conserve this newly discovered population be relatively low because human activity was as with the other existing Potamogeton hardly observed there, except for fi shing. lucens populations in Japan. In the Tsugaru Quasi-National Park, including the whole Significances of the discovery of the neighborhood of the pond, land developments Potamogeton lucens population in Japan are now in progress. Furthermore, the aquatic In Japan, the discovery of Potamogeton vegetation of several ponds in this area has been lucens in a new locality is the first in these 30 deeply impacted by human activities since the years since Ohno (1987, 1988). Based on our 1970s (Higuchi and Fukuhara unpublished data, identification, we revised the distribution map Inoue 2005). If stable populations are confi rmed of P. lucens presented by Kadono (1994; Fig. by continuous investigations, their presence may 3). This locality represents the second extant play an important role in the conservation of naturally established one subsequent to the Oito- Japanese P. lucens in the near future. ike pond, Fukuoka Prefecture, Kyushu District. Furthermore, this locality represents a new The authors thank three anonymous northern limit of the species in Japan, which is reviewers for their constructive comments; over 500 km away from its nearest distributional Dr. Satoko Iida and Dr. Yasuro Kadono record, Gunma Prefecture, Kanto District (Fig. (Kobe University) for the identification of the 3; Kadono 1994). Surrounding Japan, P. lucens fragments of Potamogeton lucens; Mr. Masashi 250 植物研究雑誌 第 93 巻 第 4 号 2018 年 8 月

Yokogawa (Osaka Museum of Natural History) Ito Y., Tanaka Nr. and Uehara K. 2007. Inferring the origin for facilitating the examination of herbarium of Potamogeton ×inbaensis (Potamogetonaceae) using nuclear and chloroplast DNA sequences. J. Jpn. Bot. specimens and literature; Mr. Kozi Hayashi 82: 20–28. (Natural History Museum and Institute, Chiba) Ito Y., Robledo G. L., Iharlegui L. and Tanaka Nr. 2016. for providing information of the species in Chiba Phylogeny of Potamogeton (Potamogetonaceae) Prefecture; Mr. Ivan Ryzhii (Niigata University) revisited: Implications for hybridization and for translation of a Russian literature; and Dr. introgression in Argentina. Bull. Natl. Mus. Nat. Sci., Ser. B 42: 131–141. Tohru Manabe (Kitakyushu Museum of Natural Kadono Y. 1983. New hybrids of Potamogeton from Lake History and Human History) for providing Inba-numa, Chiba Prefecture. Acta Phytotax. Geobot. literature. This research was supported by 34: 51–54 (in Japanese with English abstract). the Environment Research and Technology Kadono Y. 1994. Aquatic plants of Japan. Bun-ichi Sogo Shuppan, Tokyo (in Japanese). Development Fund (4-1705) of Environmental Kadono Y. 2014. A Field Guide of Aquatic Plants of Japan. Restoration and Conservation Agency. Bun-ichi Sogo Shuppan, Tokyo (in Japanese). Kaplan Z. and Fehrer J. 2004. Evidence for the hybrid References origin of Potamogeton ×cooperi (Potamogetonaceae): Amano M., Ohno M., Suda R., Iida S., Kadono Y. Traditional morphology-based taxonomy and and Kosuge K. 2008. Origin and present status of molecular techniques in concert. Folia Geobot. 39: Potamogeton ×inbaensis in a natural population of 431–453. Oitoike pond, Kitakyushu-City, Japan. Bunrui 8: 129– Kaplan Z. and Fehrer J. 2006. Comparison of natural and 139 (in Japanese with English abstract). artificial hybridization in Potamogeton. Preslia 78: Chiba Prefectural Government (Chiba-ken Kankyou- 303–316. Seikatsu-bu Shizen-hogo-ka). 2017. The Red List in Kaplan Z. and Fehrer J. 2007. Molecular evidence for a Chiba Prefecture, plants and fungi, 2017 revised edition natural primary triple hybrid in plants revealed from (Chiba-ken Red List, Shokubutsu-Kinrui hen, 2017 direct sequencing. Ann. Bot. 99: 1213–1222. Kaitei-ban). Chiba Prefectural Government (Chiba- Kaplan Z. and Fehrer J. 2011. Erroneous identities of ken Kankyou-Seikatsu-bu Shizen-hogo-ka), Chiba (in Potamogeton hybrids corrected by molecular analysis Japanese; http://www.bdcchiba.jp/endangered/2017/ of plants from type clones. Taxon 60: 758–766. redlist2017.pdf confi rmed in Aug. 25, 2017). Katoh K. and Standley D. M. 2013. MAFFT multiple Guo Y., Haynes R. R., Hellquist C. B. and Kaplan Z. sequence alignment software version 7: Improvements 2010. Potamogetonaceae. In: Wu Z. Y., Raven P. in performance and usability. Mol. Biol. Evol. 30: H. and Hong D. Y. (eds.), Flora of China Volume 772–780. 23 (Acoraceae through Cyperaceae). pp. 108–115. Kushiro K., Hayashi N. and Nishihiro J. 2009. A restoration Science Press, Beijing & Missouri Botanical Garden of submerged vegetations from propagule banks by Press, St. Louis. making small ponds at the shore of Lake Imba-numa, Hamilton M. B. 1999. Four primer pairs for the Japan. Ecol. Civil Eng. 12: 141–147 (in Japanese). amplification of chloroplast intergenic regions with Lindqvist C., Laet J. D., Haynes R. R., Aagesen L., Keener intraspecifi c variations. Mol. Ecol. 8: 521–523. B. R. and Albert V. A. 2006. Molecular phylogenetics Hara H. 1985. Comments on the East Asiatic plants (17). J. of an aquatic plant lineage, Potamogetonaceae. Jap. Bot. 60: 230–238. Cladistics 22: 568–588. Iida S., Kosuge K. and Kadono Y. 2004. Molecular Makino T. 1905. Observations on the flora of Japan phylogeny of Japanese Potamogeton species in light (continued from p. 110). Bot. Mag. (Tokyo) 19: 131– of noncoding chloroplast sequences. Aquat. Bot. 80: 158. 115–127. Manabe T., Suda R., and Shimizu K. 2015. Report on the Inoue M. 2005. Plants in Tsugaru Peninsula (Tsugaru-Hanto factors aff ecting the decline in Potamogeton dentatus, no Shokubutsu). Aomori-Bungei-Kyokai Syuppan-bu, an endangered water plant, in Kitakyushu City. Bull. Goshogawara (in Japanese). Kitakyushu Mus. Nat. Hist. Hum., Ser. A 13: 7–18. Ito Y., Tanaka Nr., Pooma R. and Tanaka Nb. 2014. DNA Manabe T., Suda R., and Shimizu K. 2016. Emergence barcoding reveals a new record of Potamogeton of submerged plant Potamogeton dentatus from the distinctus (Potamogetonaceae) and its natural hybrids, sediment propagule bank. Landsc. Ecol. Manag. P. distinctus × P. nodosus and P. distinctus × P. wrightii (Keikan-Seitaigaku) 21: 121–125 (in Japanese). (P. ×malainoides) from Myanmar. Biodivers. Data J. 2: Ministry of the Environment, Government of Japan. 2015. e1073. Red Data Book 2014, 8: plant I. Gyousei, Tokyo (in August 2018 The Journal of Japanese Botany Vol. 93 No. 4 251

Japanese). Uehara K., Tanaka Nr., Momohara A. and Zhou Z.-K. Momohara A., Uehara K., Fujiki T. and Tanaka Nr. 2001. 2006. Genetic diversity of an endangered aquatic plant, Distribution and preservation of seed bank in lake Potamogeton lucens subspecies sinicus. Aquat. Bot. sediment of Teganuma, Chiba, Central Japan. Ann. 85: 350–354. Tsukuba Bot. Gard. 20: 1–9 (in Japanese with English Wang Q. D., Zhang T. and Wang J. B. 2007. Phylogenetic abstract). relationships and hybrid origin of Potamogeton species Ohno M. 1987. Plants in Kitakyushu 11: Potamogeton (Potamogetonaceae) distributed in China: insights x inbaensis Kadono (Kitakyushu no shokubutsu 11: from the nuclear ribosomal internal transcribed spacer “Inba-mo”). Natural History of Kitakyushu Natural sequence (ITS). Pl. Syst. Evol. 267: 65–78. History Association (Watashi-tachi no shizen-shi, Wiegleb G. and Kaplan Z. 1998. An account of the species Kitakyushu-shizenshi tomo-no-kai) no. 26: 19 (in of Potamogeton L. (Potamogetonaceae). Folia Geobot. Japanese). 33: 241–316. Ohno M. 1988. Plants in Kitakyushu 12: Potamogeton White T. J., Bruns T., Lee S. and Taylor J. 1990. dentatus Hagstr. (Kitakyushu no shokubutsu 12: Amplification and direct sequencing of fungal “Gasya-moku”). Natural History of Kitakyushu ribosomal RNA genes for phylogenetics. In: Innis M. Natural History Association (Watashi-tachi no shizen- A., Gelfand D. H., Sninsky J. J. and White T. J. (eds.), shi, Kitakyushu-shizenshi tomo-no-kai) no. 27: 22 (in PCR Protocols: A guide to methods and applications. Japanese). pp. 315–322. Academic Press, New York. Preston C. D. 1995. Pondweeds of Great Britain and Ireland Yang T., Zhang T., Guo Y. and Liu X. 2016. Identifi cation (BSBI Handbooks Volume: 8). BSBI Publications, of hybrids in Potamogeton: incongruence between Bristol. plastid and ITS regions solved by a novel barcoding Saito Y. 1991. The appearance of Gasha-moku of a marker PHYB. PLoS One 11: e0166177. phantom (Maboroshi no Gasya-moku no Shutsugen). Zalewska-Gałosz J. 2002. Occurrence and distribution of Bull. Water Plant Soc. Japan no. 43: 24–26 (in Potamogeton hybrids (Potamogetonaceae) in Poland. Japanese). Feddes Repert. 113: 380–393. Shimizu K. 2011. Activities for revival of Potamogeton Zalewska-Gałosz J., Ronikier M. and Kaplan Z. 2010. dentatus 1: investigating the cause of its deterioration Discovery of a new, recurrently formed Potamogeton (Gasha-moku fukkatsu ni mukete 1: suitai-youin wo hybrid in Europe and Africa: Molecular evidence and saguru). Natural History of Kitakyushu Natural History morphological comparison of different clones. Taxon Association (Watashi-tachi no shizen-shi, Kitakyushu- 59: 559–566. shizenshi tomo-no-kai) no. 116: 5–8 (in Japanese). Shimizu K. 2016. Activities for revival of Potamogeton Appendix 1 dentatus 3: draining of the pond to restore the Herbarium specimens examined. population (Gasha-moku fukkatsu ni mukete 3: saisei Potamogeton lucens L. no kirifuda wa mizu-otoshi). Natural History of JAPAN. Aomori Pref. Tsugaru-shi. K.Shutoh & Kitakyushu Natural History Association (Watashi-tachi T.Shiga 1–5, 18 June 2017 (NGU4568–4572, voucher for no shizen-shi, Kitakyushu-shizenshi tomo-no-kai) no. examination of vegetative organs); H.Yamagishi & al. 13, 134: 1–6 (in Japanese). 14, 4 July 2017 (NGU4681, 4682, voucher for examination Shimizu K., Yamamoto T. and Ichu T. 2011. Activities for of reproductive organs). Gunma Pref. Tatebayashi-shi. revival of Potamogeton dentatus 2: ex situ conservation Y.Kuniya s.n., 9 Sep. 1953 (OSA174712, two sheets were of the species (Gasha-moku fukkatsu ni mukete 2: stored); T.Hiroi 7083, 22 Aug. 1954, (OSA); T.Satomi Gasya-moku wo sodate, fuyasu). Natural History of s.n., 22 Aug. 1954 (OSA). Ibaraki Pref. Itako-shi. Kitakyushu Natural History Association (Watashi-tachi H.Tsurumachi s.n., 2 Dec. 1928 (OSA). Chiba Pref. Tega- no shizen-shi, Kitakyushu-shizenshi tomo-no-kai) no. numa lake. S.Miki s.n., 14 July 1926 (OSA); H.Nakano 117: 1–6 (in Japanese). s.n., 14 July 1926 (OSA). Inba-numa lake. I.Ikushima s.n., Tamura K., Stecher G., Peterson D., Filipski A. and Kumar 30 June 1965 (OSA). Shiga Pref. Ohtsu-shi. S.Miki s.n., 17 S. 2013. MEGA6: Molecular Evolutionary Genetics May 1927 (OSA); S.Miki s.n., 1 Sep. 1928 (OSA); S.Miki Analysis version 6.0. Mol. Biol. Evol. 30: 2725–2729. s.n., 10 July 1932 (OSA); S.Kitamura s.n., 18 Aug. 1928 Tanaka Nr. 2012. The science of aquatic plants, which is (OSA); S.Kitamura s.n., Oct. 1928 (OSA8817); S.Kitamura heterodox one (Itan no syokubutsu, Mizu-kusa wo s.n., 16 Nov. 1932 (OSA). Fukuoka Pref. Kitakyushu-shi. Kagaku suru). Bere Shuppan, Tokyo (in Japanese). M.Ohno 7173, 1 May 2000 (OSA270722); M.Ohno 7175- Tzvelev N. N. 1987. Potamogetonaceae Dumort. In: 1, 7175-2, 24 May 2000 (OSA270723, 270724); M.Ohno Charkevicz S. S. (ed.), Plantae Vasculares Orientis 7446-1–9, 18 June 2000 (OSA270713). Extremi Sovietici. 2: 317–335. Nauka, Leningrad (in Specimens suspected to be a hybrid produced by P. lucens Russian). JAPAN. Aomori Pref. Tsugaru-shi. M.Usuba 12841, 252 植物研究雑誌 第 93 巻 第 4 号 2018 年 8 月

23 Aug. 1986 (NGU4566); M.Usuba 14025, 25 July 1989 Potamogeton gramineus L. (NGU4567). JAPAN. Aomori Pref. Tsugaru-shi. K.Shutoh & T.Shiga 2401, 2402, 18 June 2017 (NGU4690, 4691).

首藤光太郎a，薄葉 満b，山岸洋貴c，藤田優志d，平松 栞a， 辻村 収e, f，石戸谷芳子f，葛西政光f，葛西直子f，松本 明男f，乗田利一f，横山昭子f，兼子伸吾d，志賀 隆a：青 森県でガシャモク（ヒルムシロ科）の新産地を発見 青森県つがる市の一湖沼から，ガシャモク ら，少なくとも湖内に生育しているものと考えられる． Potamogeton lucens L.（ヒルムシロ科）を報告した． 本集団は，ガシャモクの国内の自然集団としては 2 ヶ 本種は，かつては北関東などで多産したものの国内の既 所目で，国内における既知産地の北限を 500 km 以上更 知産地のほとんどで消滅し，環境省レッドデータブッ 新するものである．湖沼周辺では開発が進んでおり，ガ ク 2014 で絶滅危惧 IA 類に指定されている．筆者らは， シャモクの国内からの絶滅を防ぐためにも，本集団の早 2017 年 6 月および 7 月に当湖沼で類似した切れ藻を発 急な保全が望まれる． a 見した．発見した切れ藻は国内のガシャモクの記載より （ 新潟大学教育学部， b もわずかに大きな葉の長さ，幅，托葉をもっていたもの 福島県いわき市， c の，形態測定，塩基配列，花粉稔性，標本調査に基づき 弘前大学白神自然環境研究所， d 本種と同定した．現地では本種の生育状況を確認するこ 福島大学共生システム理工学類， e とはできなかったが，30 以上の切れ藻が湖沼の南岸の 青森市森林博物館， f みに漂着し，南岸付近で根をもつ株が採集されたことか 津軽植物の会）