ISSN 1346-7565 Acta Phytotax. Geobot. 70 (3): 173–182 (2019) doi: 10.18942/apg.201825

A New Variety of × leptocephalus Koidz., var. fujiensis, Endemic to the Fuji Five Lakes and Lake Ashi, Central Japan

* Yasuro Kadono and Satoko Iida

Department of Biology, Graduate School of Science, Kobe University, Nada, Kobe, Hyogo 657-8501, Japan. *[email protected] (author for correspondence)

DNA analysis and morphological observation were conducted for a putative Potamogeton hybrid endemic to the Fuji Five Lakes and Lake Ashi and Potamogeton × leptocephalus from Lake Biwa, Japan. Both proved to be hybrids between P. maackianus and P. perfoliatus. However, the hybrid from the Fuji Five Lakes and Lake Ashi was distinct from P. × leptocephalus in certain morphological characteristics. It is described as a new variety P. × leptocephalus var. fujiensis.

Key words: hybrid origin, Potamogeton, Potamogeton maackianus, , variety

The Fuji Five Lakes (Fuji-Go-ko) are located (Ashi-no-ko), Hakone, Kanagawa Prefecture, as at the northern foot of Mt. Fuji in Yamanashi “Potamogeton crispus L. × P. maackianus A. Prefecture and consist of Lake Yamanaka, Lake Benn. (?)”, with a brief description. This identifi- Kawaguchi, Lake Sai, Lake Shoji and Lake cation was accepted by those of Matsuyama-Ser- Motosu. The first comprehensive study of the izawa et al. (2009) and Serizawa et al. (2013, macrophytic flora of these lakes was conducted 2014). Occurrences of the putative hybrid were by Nobuhara et al. (1971), who enumerated eight reported in Lake Shoji and Lake Sai (Serizawa et species of Potamogeton () from al. 2015), as well as in Lake Yamanaka. the five lakes, namely P. perfoliatus L., P. malaia- Specimens that may also be the putative hy- nus Miq. (=P. wrightii Morong), P. maackianus brid were first collected from Lake Shoji by Bun- A. Benn., P. crispus L., P. compressus L., P. oxy- zo Hayata in 1924 and deposited in TI (identified phyllus Miq., P. distinctus A. Benn. and P. pecti- as “Potamogeton crispus L.”). Other specimens natus L.[=Stuckenia pectinata (L.) Börner]. were collected from Lake Kawaguchi in 1961 and Hayakawa (1986) reported the occurrence of deposited in MAK. Of the nine sheets in MAK, two additional taxa of Potamogeton, a hybrid seven were identified as P.“ maackianus × P. oxy- named P. anguillanus Koidz. and “P. crispus × phyllus” by Masami Mizushima; the remaining P. maackainus” from Lake Yamanaka. The iden- two were identified by Nobushige Kato as Pota“ - tification of the latter hybrid was made provision- mogeton × leptocephalus Koidzumi”. ally by Y. Kadono based on the morphology and Potamogeton × leptocephalus Koidz. was de- sterility of the specimen sent to him. The sheath- scribed as a species based on specimens collected ing leaf base indicated P. maackianus as one of from Lake Unagi, Kagoshima Pref., Kyushu. The the putative parents and three, conspicuous, red- putative hybrid from Lake Kawaguchi and P. × dish nerves reminded him of P. crispus as a can- leptocephalus share some morphological charac- didate for the other putative parent. Kadono teristics, as described later, which must have led (1988) recorded this hybrid from Lake Ashi to Kato’s identification. Miki (1937) hypothe- 174 Acta Phytotax. Geobot. Vol. 70

Table 1. Locality of Potamogeton × leptocephalus and the putative hybrid examined in the DNA analysis. Species Locality Voucher specimen Potamogeton × leptocephalus Koidz. Japan: Pref. Shiga, Lake Biwa, Sugaura, Nagahama City Y. Kadono 17098(OSA) The putative hybrid Japan: Pref. Yamanashi, Lake Sai, Kawaguchiko-machi Y. Kadono 16103 (OSA) The putative hybrid Japan: Pref. Yamanashi, Lake Yamanaka, Yamanakako-mura Y. Kadono 16098 (OSA) The putative hybrid Japan: Pref. Kanagawa, Lake Ashi, Hakone-machi H. Sakayama & S. Kato s.n. (OSA) sized Potamogeton × leptocephalus to be a hy- mine the taxonomic identity of the hybrids. Both brid between P. maackianus and P. perfoliatus DNA regions were chosen based on our GenBank without in-depth discussion. His hypothesis of searches that found species-specific nucleotide the parental combination of P. × leptocephalus substitutions among four candidate species (Pota- has not been confirmed. Since then, P. × lepto- mogeton maackianus, P. perfoliatus, P. crispus, cephalus has also been recorded from Lake Biwa, and P. oxyphyllus) (Appendices 1 & 2). Shiga Pref., Honshu (Hamabata & Kira 1987, Ka- Total DNA was extracted from dried leaves of dono 1991). the putative hybrids from the Fuji Five Lakes (n = To determine the origin of hybrids in Potamo- 1 from each lake) and Lake Ashi (n = 1) and Pota- geton, molecular techniques such as allozyme mogeton × leptocephalus (n = 1), P. maackianus electrophoresis and DNA-based methods are use- (n = 1) and P. perfoliatus (n = 1) from Lake Biwa ful tools (e.g., Kaplan & Fehrer 2007, Kaplan et using NucleoSpinPlant II (Macherey-Nagel al. 2018, Zalewska-Gałosz & Ronikier 2012, Iida GmbH & Co. KG, Germany) according to the et al. 2018). In this study, we aimed to reveal the manufacturer’s protocol. Both ITS and rbcL were origin and taxonomic status of P. × leptocephalus amplified using γ-Taq polymerase (Toyobo, Japan). as well as the putative hybrid from the Fuji Five For ITS, 35 PCR cycles were performed at 94 °C Lakes and Lake Ashi by DNA and morphological for 30 s, 60 °C for 40 s, and at 72 °C for 30 s, using analyses, with special reference to the four candi- the primers F18S (5’- CCTTATCATTTAGAG- date parental species, P. maackianus, P. perfolia- GAAGGAG -3’) and R26S (5’- CTCCGCTTATT- tus, P. crispus and P. oxyphyllus, hypothesized by GATATGCTTAAAC -3’). The amplified prod- Miki (1937) or suggested by Kadono (1988) and ucts were sequenced directly using amplification Mizushima as mentioned above. primers and the internal sequence primer F5.8S (5’- TGACTCTCGGCAACGGATATC -3’) (Iida et al. 2018). Due to a length mutation in the paren- Materials and Methods tal ITS sequences, a sequence of positions 1–305 was determined by an F18S rRNA primer and a Samples sequence of positions 306–625 was determined Putative hybrid of Potamogeton were by an F5.8S rRNA primer. The amplification of collected from Lake Yamanaka, Lake Shoji, and rbcL was performed using primers rbcL26F Lake Ashi, while Potamogeton × leptocephalus (5’- TGTC ACCACAAACAGAGACTAAAGC was obtained from Lake Biwa (Table 1). Voucher -3’) and rbcL1375R (5’- ATACGATCTCTTTC specimens were deposited at OSA (transferred CATACTTCAC -3’) and sequenced using rb- from the herbarium of Kobe University). The cL1375R (Iida et al. 2007). ABI 3130 Genetic An- samples of P. maackianus and P. perfoliatus were alyzer (Applied Biosystems, USA) was used for collected from Lake Biwa. sequencing with a BigDye Terminator v.3.1 Cycle Sequencing Kit (ThermoFisher Scientific, USA) DNA analysis at Kobe University Biosignal Research Center. The nuclear-encoded internal transcribed Determined sequences of the putative hybrid and spacer (ITS) and the maternally inherited chloro- Potamogeton × leptocephalus were searched for plast-encoded gene rbcL were analyzed to deter- species-specific nucleotide substitutions or indels October 2019 Kadono & Iida– A New Variety of Potamogeton × leptocephalus 175

Table 2. Diagnostic ITS sequences of Potamogeton × leptocephalus, the putative hybrid and their parental candidates. Data after position 533 were not listed (see Fig. 2). Positions in the alignment Species 1 1 1 2 2 2 2 3 4 2 2 3 4 4 5 6 7 8 9 1 6 8 1 1 1 1 6 1 0 3 5 6 9 2 0 8 5 5 4 7 7 2 4 5 6 8 3 P. × leptocephalus T C/T A A/T G/C T G/T A/T C/T C/T C/T G/T G G A C T C/T G/T Putative hybrids L. Sai ...... L. Yamanaka ...... L. Ashi ...... Parental candidates P. maackianus . C . A G . G A C T T* G . . . . . T G* P. perfoliatus . T* . T* C* . T* T* T* C C T* . . . . . C T* P. crispus C* C T* A G . G A C T C A* C* T* T* T* A* T A* P. oxyphyllus . C . A G A* G A C C C G . . . . . C C*

Positions in the alignment Species 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 1 2 2 2 2 3 4 4 6 7 7 8 8 9 9 9 9 3 8 3 4 6 9 0 2 4 0 8 9 5 8 3 6 8 9 2 P. × leptocephalus C A/T C A C/T C A/G C/T C/T C C C/T - C G/C A/G A/G T Putative hybrids L. Sai ...... - . . . . . L. Yamanaka ...... - . . . . . L. Ashi ...... - . . . . . Parental candidates P. maackianus . T . . C . A C C* . . C* - . G G* A . P. perfoliatus . A* . . T . G T* T . . T - . C A G* . P. crispus T* T T* T* C T* A C T T* T* T T* T* G A A -* P. oxyphyllus . T . . T . G C T . . T - m C A A . .: the same as P. × leptocephalus, -: deletion, *: The species-specific nucleotide substitutions or indels. among four candidate species. in P. oxyphyllus). The obtained ITS sequences were 500 bp long Morphological comparison in the putative hybrid from the Fuji Five Lakes The morphology of the putative hybrid and and Lake Ashi and in Potamogeton × leptocepha- Potamogeton × leptocephalus was analyzed on lus (excluding an undetermined 88 bp sequence herbarium specimens at TI, MAK, and OSA, as around the 5.8S rRNA primer region). Although well as on living materials collected from the similar electropherograms were obtained from abovementioned lakes. Comparison was made on both the putative hybrid and P. × leptocephalus plants growing in an aquarium. before aligning position 532 (Fig. 1), the electro- pherograms of the hybrid showed complex pat- terns after aligning position 532 (Fig. 2). Species-specific nucleotide substitutions or Results indels of ITS were found for each candidate pa- rental species (Potamogeton crispus: 18 sites; DNA analysis P. maackianus: 5 sites; P. perfoliatus: 11 sites; The obtained rbcL sequences were 821 bp P. oxyphyllus: 2 sites in alignment positions long. The sequences in both the putative hybrid 1–533, Table 2). The sequences of the putative hy- from the Fuji Five Lakes and Lake Ashi and Pota- brid from the Fuji Five Lakes and Lake Ashi and mogeton × leptocephalus from Lake Biwa and in P. × leptocephalus exhibited additive states with P. maackianus were identical, but differed from respect to those of P. maackianus and P. perfolia- sequences in the other candidate parental species tus (Table 2, Figs. 1 & 2). Species-specific nucle- (four nucleotide substitutions in P. crispus, two otide substitutions of P. crispus and P. oxyphyllus substitution in P. perfoliatus and six substitutions were not detected in the overall ITS sequences of 176 Acta Phytotax. Geobot. Vol. 70

Fig. 1. Alignment and electropherogram of internal transcribed spacer (ITS) sequences of Potamogeton at alignment positions 110–170. Arrows indicate species-specific positions (114, 167). Lower case letters indicate mixed bases. the putative hybrid or P. × leptocephalus (Table sketched a four-carpellate flower ofPotamogeton 2). × leptocephalus from Lake Unagi (cultivated at the Botanical Gardens of Kyoto University). Al- Morphology though the number of flowers available for obser- The putative hybrid from the Fuji Five Lakes vation was limited, we observed flowers with two and Lake Ashi and Potamogeton × leptocephalus or three carpels among the specimens of P. × lep- from Lake Unagi and Lake Biwa shared morpho- tocephalus from Lake Biwa deposited in OSA logical characteristics including narrowly lanceo- and in the putative hybrid from the Fuji Five late leaves sheathing at the base and dimorphic Lakes (Fig.3B). flowering and sterile shoots (Fig. 3A). Miki (1937) The most pronounced differences between October 2019 Kadono & Iida– A New Variety of Potamogeton × leptocephalus 177

Fig. 2. Alignment and electropherogram of internal transcribed spacer (ITS) sequences of Potamogeton at alignment positions 521–580. Arrows indicate species-specific positions (560, 567, 571). Lower case letters indicate mixed bases. the putative hybrid from the Fuji Five Lakes and and limited mainly to the apical half of the leaf Lake Ashi and Potamogeton × leptocephalus margin in the putative hybrid. were in leaf characteristics. Leaves of the puta- The stele of the stem was of the oblong type tive hybrid were longer (to 7.5 cm, compared with with two median bundles sensu Ogden (1943) in a maximum length of 5 cm in P. × leptocephalus) the putative hybrid and Potamogeton × lepto- and less curled (Fig. 3A). The leaf apex of the pu- cephalus (Fig. 3D). Subepidermal bundles of tative hybrid was more or less acute, whereas in sclerenchymatous cells were present in the for- P. × leptocephalus it was obtuse or round (Fig. mer but absent in the latter from Lake Biwa (Fig. 3C). The entire margin of the leaves of P. × lepto- 3E & F). cephalus were toothed, whereas teeth were sparse 178 Acta Phytotax. Geobot. Vol. 70

Fig. 3. (A) Comparison of shoots of Potamogeton × leptocephalus var. fujiensis and var. leptocephalus grown under the same aquarium conditions. Flowering (left) and sterile shoots (middle) of var. fujiensis, and var. leptocephalus (right two). Scale bar 5 cm; (B) Spike bearing flowers with two carpels. Flower with three carpels inserted beneath. Scale bar 2.5 mm; (C) leaf apex of var. fujiensis (left) and var. leptocephalus (right). Note the acute and obtuse apices, respectively; (D) Trans- verse section of stem of var. fujiensis with oblong type stele with two median bundles. Scale bar 0.5 mm; (E) & (F) Clo- seup of transverse sections of stems of var. fujiensis (E) and var. leptocephalus (F). Note presence of subepidermal bun- dles in var. fujiensis (E) indicated by arrows. Scale bar 0.2 mm. October 2019 Kadono & Iida– A New Variety of Potamogeton × leptocephalus 179

Discussion Taxonomic treatment

Although the putative hybrid from the Fuji Potamogeton × leptocephalus Koidz. in Florula Five Lakes and Lake Ashi was distinct from Satsumensis 2: 162. 1931. Miki in Water Phanero- Potamogeton × leptocephalus in leaf morpholo- gams Jap. 30. 1937; Ohwi in Fl. Jap. rev. ed., 67. gy, our DNA analysis confirmed both of them to 1965; Kadono in Aquat. Pl. Jap. 41. 1994. be hybrids between P. maackianus and P. perfo- liatus. The parental combination was the same, var. fujiensis Kadono & Iida, var. nov.—Figs. 3 i.e., the former was the maternal parent and the & 4. latter was the paternal parent. Molecular evi- Potamogeton crispus L. × P. maackianus A. dence did not suggest either P. crispus or P. oxy- Benn. (?) Kadono in Fl. Kanagawa 1988, Jap. phyllus to be involved in hybridizations we ana- 192. 1988. lyzed. Our findings agree with the hypothesis of Similar to Potamogeton × leptocephalus var. leptocepha- Miki (1937), who hypothesized that P. × lepto- lus but distinguished by less curly leaves with acute apex. cephalus was the result of hybridization between Holotype: Japan, Yamanashi Prefecture, Kawagu- P. maackianus and P. perfoliatus. Our findings chiko-machi, Minami-tsuru-gun, Lake Kawaguchi, Sept. 6, 1961. T. Ohfusa s.n. (MAK 19022). also confirmed the presence of P. × leptocephalus in Fuji Five Lakes and Lake Ashi, supporting the Nom Jap. Fuji-ebimo (Kadono 2014). identification by N. Kato. The putative hybrid from the Fuji Five Lakes Herbs, perennial, evergreen, submerged. Rhi- and Lake Ashi and Potamogeton × leptocephalus zomes creeping, branched, 1st lateral internode from Lake Biwa have been shown to differ mor- not elongated, seemingly with one internode be- phologically. The putative hybrid also differed tween erect shoots, 2–5.5 cm long; erect shoot up from P. × leptocephalus from Lake Biwa in the to 85 cm long; stems terete; leaves narrowly lan- sequence electropherograms (Fig. 2). Both P. ceolate, 2.5–7.5 cm long, 4–7 mm wide, three maackianus and P. perfoliatus have been record- reddish conspicuous nerves with two additional ed from Lake Yamanaka and Lake Kawaguchi fine veins, apex more or less acute, margin sparse- (specimens collected in 1924 by B. Hayata are de- ly serrulate with single-celled teeth, sheathed at posited in TI; Nobuhara et al. 1971, Nagasaka et base (1–4 mm); sheath of basal part of stem lon- al. 2002) and Lake Ashi (Kadono 1988). The hy- ger than that of cauline part in sterile shoots; brid may have originated in one of these three leaves of flowering shoots distinctly shorter, to lakes and spread to adjacent lakes. 2.5 cm long and with distinct sheath at base; pe- Subepidermal bundles were present in Pota- duncle 2–4 (–6) cm long; spike 5–12 mm long mogeton × leptocephalus from Lake Unagi (Miki, with (3–) 5–6 flowers; two to three carpels; fruit 1937), but absent in the hybrid from Lake Biwa. not seen; stele of the stem oblong type with two This observation might also indicate independent median bundles, subepidermal bundles present. origins of P. × leptocephalus in lakes Biwa and Unagi. Further genetic comparison is needed to Distribution. endemic to the Fuji Five Lakes determine the origin of the hybrids in different and Lake Ashi, central Japan. regions. Although Potamogeton × leptocephalus from the Kagoshima region and Lake Biwa are Other specimens examined. morphologically similar, the hybrids from the Potamogeton × leptocephalus var. fujiensis Pref. Yamanashi: Lake Shoji, Aug.16, 1924, B. Hayata Fuji Five Lakes and Lake Ashi are clearly differ- (TI); Lake Kawaguchi, Nov. 17, 1961. T. Ohfusa s.n. ent from both groups. Thus, we formally describe (MAK); Lake Kawaguchi, Y. Kadono 11082 (OSA); Lake a new variety of P. × leptocephalus. Yamanaka, Y. Kadono 16098 (OSA); Lake Sai, Y. Kadono 16103 (OSA); Lake Shoji, Y. Kadono 17048 (OSA); Pref. 180 Acta Phytotax. Geobot. Vol. 70

Fig. 4. Holotype of Potamogeton × leptocephalus var. fujiensis Kadono & Iida (Ohfusa s.n. MAK 19022) October 2019 Kadono & Iida– A New Variety of Potamogeton × leptocephalus 181

Kanagawa: Lake Ashi, Oct. 25, 2017, H. Sakayama & S. Kaplan, Z. & J. Fehrer. 2007. Molecular evidence for a Kato s.n. (OSA). natural primary triple hybrid in plants revealed from Potamogeton × leptocephalus var. leptocephalus (repre- direct sequencing. Ann. Bot. 99: 1213–1222. sentative specimens only) Kaplan, Z., J. Fehrer, V. Bambasová & C. B. Hellquist. Kagoshima Pref.: Lake Unagi, Yamakawa-cho, Oct. 8, 2018. The endangered Florida pondweed (Potamoge- 1926 Y. Doi s.n. (KYO, OSA); Lake Ikeda, Ibusuki City, ton floridanus) is a hybrid: Why we need to under- Date unshown H. Kasaki s.n. (MAK); Shiga Pref.: Lake stand biodiversity thoroughly. PLoS One 13: Biwa, E. Hamabata 86092 (OSA); K. Seto 33818 (OSA); e0195241. S. Fujii 3982, 3992, 4031, 4035 (all in OSA); S. Fujii, Y. Matsuyama-Serizawa, K., K. Yoshizawa, K. Takahashi, Kadono & S. Iida 7110 (OSA); S. Fujii & T. Shiga 12894 T. Nakano, T. Yasuda & Y. Serizawa. 2009. A note on (OSA); Y. Kadono 17098 (OSA). aquatic macrophytes and macroalgae of Lake Ya- manaka in 2007. Bull. Water Soc., Japan 92: 1–9 (in Japanese). We express our sincere thanks to the curators of TI, Miki, S. 1937. The water phanerogams in Japan, with spe- MAK, and OSA for allowing us to examine specimens. cial reference to those in Province Yamashiro. Stud- We also thank H. Sakayama and S. Kato for providing liv- ies of Historical and Natural Monuments in Kyoto-fu ing plants from Lake Ashi. 18: 1–127 (in Japanese). Nagasaka, M., K. Yoshizawa, K. Ariizumi & K. Hi- rabayashi. 2002. Temporal changes and vertical dis- tribution of macrophytes in Lake Kawaguchi. Lim- nology 3: 107–114. Nobuhara, H., Y. Iwata & I. Ikusima. 1971. Distribution References of aquatic macrophytes in Fuji-Goko. In: Mount Fuji: Reports of Integrated Scientific Researches on Mt. Hamabata, E. & T. Kira. 1987. Aquatic plants of Lake Fuji, pp.559–577. Fuji-Kyuko- Co, Ltd. Fuji-Yoshida Biwa. Nihon no Seibutsu 1: 24–29 (in Japanese). (in Japanese). Hayakawa, R. 1986. Present status of aquatic macro- Ogden, E. C. 1943. The broad-leaved species of Potamo- phytes of Lake Yamanaka. Bull. Water Plant Soc., Ja- geton of North America north of Mexico. Rhodora pan 26: 2–5 (in Japanese). 45: 57–214. Iida, S., A. Yamada, M. Amano, J. Ishii, Y. Kadono & K. Serizawa, Y., Y. Sato, M. Fukashiro, K. Tsuchiya & K. Kosuge. 2007. Inherited maternal effects on the Matsuyama-Serizawa, 2013. Annual and yearly drought tolerance of a natural hybrid , changes in species composition and biomass of aquat- Potamogeton anguillanus. Jour. Plant Res. 120: 473– ic plants of Lake Yamanaka, northern foot of Mt. Fuji 481. from 2008 to 2010. Bull. Water Plant Soc., Japan 100: Iida, S., M. Ashiya & Y. Kadono. 2018. The hybrid origin 61–71 (in Japanese). of Potamogeton biwaensis Miki, an endemic sub- Serizawa, Y., K. Yoshizawa, K. Takahashi, S. Kato, H. merged plant in Lake Biwa, Japan. Aquat. Bot. 150: Nozaki & K. Matsuyama-Serizawa. 2014. Horizontal 23–26. and vertical distribution of hydrophytes in Lake Ya- Kadono, Y. 1988. Potamogetonaceae. In: The Flora- manaka, at the northern foot of Mt. Fuji in 2008. Kanagawa Association (ed.) Flora of Kanagawa 1988, Mount Fuji Research 8: 7–14 (in Japanese). Japan, pp. 190–193. Kanagawa Prefectural Museum, Serizawa, Y., T. Uejima, S. Nakamura, H. Watanabe, N. Yokohama (in Japanese). Shirasawa & K. Matsuyama-Serizawa. 2015. Aquatic Kadono, Y. 1991. Aquatic macrophytes of Shiga Prefec- plants, macroalgae and light environment of Lake Sai ture. In: Editorial Committee of Landscape and Envi- and Lake Shoji at the northern foot of Mt. Fuji, central ronment of Shiga (ed.) Landscape and Environment Japan. Bull. Fac. Educ. Human Sci., Yamanashi Univ. of Shiga: Scientific Studies of Shiga Prefecture, Ja- 17: 201–210 (in Japanese). pan, pp. 1275–1294. The Foundation of Nature Con- Zalewska-Gałosz, J. & M. Ronikier. 2012. Molecular evi- servation in Shiga Prefecture, Ohtsu (in Japanese). dence for two rare Potamogeton natans hybrids with Kadono, Y. 2014. A Field Guide to Aquatic Plants of Ja- reassessment of Potamogeton hybrid diversity in Po- pan. Bun’ichi Sogo Shuppan, Tokyo (in Japanese). land. Aquat. Bot. 103: 15–22.

Received August 1, 2018; accepted December 13, 2018 182 Acta Phytotax. Geobot. Vol. 70

Appendix 1. Localities and GenBank accession numbers of Potamogeton ITS sequences used to identify species-spe- cific nucleotide substitutions and indels. The substitutions and indels were regarded to be species-specific when they were shared by all accessions. Potamogeton maackianus A. Benn. China: Heilongjiang HQ167661, USA: New York State - HQ263522, USA: Ver- Sheng - JF977905, China: Jiangsu - JF977903, China: mont - EU596953, Potamogeton crispus L. Australia - Yunnan - JF977901, China: Dongxiang - DQ840267, GU814245, China: Anhui - JF977881, China: Guizhou - DQ840268, China: Tongjiang - DQ840269, DQ840270, JF977880, China: Hebei - JF977879, China: Jiangsu - Japan: Shiga - LC349934, Potamogeton perfoliatus L. JF977878, China: Honghu - DQ840284, DQ8402845, Austria - HQ263520, Australia - AB937782, Bosnia and China: Mishan - DQ840286, DQ8402867, USA: Connect- Herzegovina - HQ263521, China: Wuhan - DQ840288, icut - GU814243, Czech Republic - GU814242, Czech Re- DQ840289, China: Ruoergai - DQ8402690, DQ8402691, public - AY529524, Germany - AY529523, India - China: Heilongjiang Sheng - JF977910, China: Weinan - GU814244, Turkey - KX273104, Turkey - HQ167656, FJ956770, China: Yunnan - JF977914, Czech Republic - USA: Connecticut - EF526368, USA: Connecticut - HQ263523, Germany - AY529525, Japan - AB744008, Ja- EF526372, USA: Connecticut - EF526369, Potamogeton pan: Shiga - LC349931, Poland: NW Poland, Kramsko oxyphyllus Miq. Japan - KF270913, Japan: Ishikawa - Lake - FJ883583, Switzerland - AY529527, Turkey - AB617753, Japan: Ishikawa - AB617755

Appendix 2. Localities and GenBank accession numbers of Potamogeton rbcL sequences used to identify species-spe- cific nucleotide substitutions. The substitutions were regarded to be species-specific when they were shared by all accessions.

Potamogeton maackianus A. Benn. Japan: Fukuoka - mogeton crispus L. Japan: Shiga - AB196847, China: AB196944, China: Chengjiang - KX059535, China: Jian- Qushui - KX059518, China: Luding - KX059519, China: chuan - KX059534, China: Lijang - KX059533, China: Xiangyun - KX059520, China: Lanping - KX059521, Xiaoxingkai Lake WDZY22 - FJ956835, China: Mishan China: Linbao - FJ956836, Potamogeton oxyphyllus WDZY21 - FJ956834, Myanmer - AB506769, Potamoge- Miq. Japan: Shiga - AB196949, China - KX024597, Chi- ton perfoliatus L. Japan: Shiga - AB196951, China: na - KX024598, China - KX527492, China: Quanken - Weinan - EU741051, China: Mishan - EU741052, Pota- FJ956853