Isoëtes L. (Isoëtaceae, Lycopodiopsida) is an ancient heterosporous genus of lycophytes. It originated between the Late Devonian and Early Carboniferous (Yang et al., 2022) , occupying a key position in the evolution of terrestrial plants (Cui et al., 2022) . The genus contains about 250 species which are widely distributed from the tropics to the sub-arctic and South America is considered a center of both morphological and taxonomical diversity with more than 60 cataloged species (Pereira et al., 2016; Troia et al., 2016) . They thrive in diverse aquatic or wet soils habitats, including oligotrophic lakes, higher-altitude wetlands, seasonal pools, and intermittent streams (Pereira et al., 2016, 2017) . The uncertainty in the assessment of species diversity of Isoëtes mainly comes from the difficulty in classification, which is related to a series of habitat adaptation, simple and conservative morphology, convergence and reticular evolution (Taylor &Hickey, 1992; Choi et al., 2008; Bagella et al., 2011; Freund et al., 2018; Gu et al., 2023a) . The identification characteristics of Isoëtes mainly include habitat, leaf vein, ligule, megaspore size and texture, chromosome count and DNA sequence (Kott &Britton, 1983; Hickey, 1986, Taylor et al., 1993; Troia et al., 2016; Bolin et al., 2017; Brunton &Troia, 2018) . The most reliable feature is megaspore morphology, but this is often difficult to achieve under field conditions (Hickey, 1986; Holmes et al., 2005; Brunton &Troia, 2018) .

At present, ten species of Isoëtes have been described from China, including I. hypsophila Hand.-Mazz. (Handel-Mazzetti, 1923) , I. sinensis T. C. Palmer (Palmer, 1927) , I. taiwanensis De Vol (De Vol, 1972) , I. yunguiensis Q. F. Wang &W. C. Taylor (Wang et al., 2002) , I. orientalis H. Liu &Q. F. Wang (Liu et al., 2005) , I. shangrilaensis X. Li, Y. Huang, X. Dai, &X. Liu (Li et al., 2019) , I. baodongii Y. F. Gu, Y. H. Yan, &Yi J. Lu (Lu et al., 2021) , I. longpingii Y. H. Yan, Y. F. Gu, &J. P. Shu, and I. xiangfei Y. H. Yan, Y. F. Gu, &J. P. Shu (Shu et al., 2022) , and newly reported species I. fengii Y. F. Gu &Y. H. Yan (Gu et al., 2023b) . According to the List of National Key Protected Wild Plants released in September 2021, all species of Isoëtes are list as first-level protection (http://www.forestry.gov.cn/main/5461/20210908/162515850572900.html) . The distribu-tion scopes of Isoëtes species are wide and the altitude spans are very large. Isoëtes hypsophila is located in the Qinghai-Tibet Plateau (QTP) while I. sinensis grows in the middle and low reaches of the Yangtze River (Li et al., 2019) . In contrast to the United States, which is geographically at about the same latitude as China and has similar climatic conditions, there are more than 50 Isoëtes species in the United States, while only ten accepted Isoëtes species in China (Troia et al., 2016) . The relatively sparse number of Isoëtes species reported from China may be due to increased water pollution and urbanization, similar appearance between species, less taxonomic research and insufficient field exploration (Troia et al., 2016; Lu et al., 2021) .

During field investigation in Changxing County, Huzhou City, Zhejiang Province, China, we discovered one population of Isoëtes similar to some populations found in eastern China, such as I. baodongii and I. sinensis. However, determination of different morphological traits, cytology and spore ornamentation provided convincing evidence that the Changxing population constituted a new species, as proposed below.

1 Materials and Methods

The megaspores and microspores of the samples were observed via scanning electron microscopy. Spores were mounted on doublesided adhesive tape attached to metal stubs, sputter-coated with platinum, and viewed under a scanning electron microscope FlexSEM 1000 Ⅱ (Hitachi, Japan) at 5-7 kV. In our study, a total of 20 megaspores and 30 microspores from three to five individuals were measured using ImageJ2 (National Institutes of Health, USA) . Terms for describing the ornamentation of megaspores and microspores were taken from Hickey (1986) .

Root tips of 1 cm in length were taken from the sporophytes and pretreated in a mixture of 0.1% colchicine and 0.002 mol·L^-1 8-hydroxyquinoline (1∶1) for 3.5 h at room temperature, rinsed with water for three times, and then fixed in Carnot’s fixative of ethanol and glacial acetic acid (3∶1) for 6 h at 4℃. The samples were rinsed with water for three times, then dissociated with a mixture of 1 mol·L^-1 HCl and 45% glacial acetic acid (1∶1) for 45 min in a thermostatic bath at 37℃. Finally, the samples were rinsed with water for three times, and then stained with modified carbonic acid magenta for 4 h, and then pressed into slices by the conventional tablet pressing method. The chromosomes of the samples were counted by Nikon ECLIPSE Ci-S Biomicroscope (Nikon, Japan) and photographed using a Digital Sight Ds-Fi2 Camera System (Nikon, Japan) .

2 Taxonomic Treatment

Isoëtes changxingensis Y. F. Gu &J. H. Shen，sp. nov.（长兴水韭，新拟，Fig.1-Fig.3）

Type: CHINA，Zhejiang Province，Changxing（长兴）County，Xiaopu（小浦）Town，in an artificial ditch，31.014349° N，119.779106° E，alt.50 m，27 August 2023. Jiahao Shen &Ling Tong2515（holotype: NAS!; isotype: NOCC!）.

Diagnosis: Isoëtes changxingensis is a tetraploid that morphologically resembles I. baodongii, but differs in its 44 chromosomes [vs.22 in I. baodongii]. It is also similar to I. longpingii in megaspore size, but differs in its trilobed rhizome corms, megaspore echinate-cristate [vs. bilobed, tuberculate-cristate in I. longpingii]. This species is distinguished from I. sinensis by its smaller megaspore (317-411 μm, mean=360 μm) , different surface ornamentation, and leaf blade20-60 cm [vs. megaspore340-450 μm, mean=409 μm, cristate, 15-30 cm in I. sinensis]. (Fig.1-Fig.3, Table1)

Description: Plants aquatic. Rhizome corms 3-lobed. Sporophylls white basally, green above, spirally arranged, widely spreading, 20-60 cm long, 2-3 mm wide at mid-length, in tufts of 50 to 90, flattened on the adaxial side, rounded on the abaxial side, base flat and alate, peripheral fibrous bundles present, central intrastelar canal4. Ligule ovate-subtriangular, (3.0-3.5) mm × (2.0-2.5) mm. Sporangia basal, oblong, triangular ovate, (7.5-10.5) mm × (3.5-4.5) mm, sporangium wall clear. Megaspores rugulate, gray when wet, white when dry, ca.317-411 μm (mean = 360 μm) , proximal hemisphere echinate-cristate, distal hemisphere cristate. Microspores gray in mass, elliptic, vertical axis length 24-31 μm (mean = 27 μm) , echinate. Megaspores and microspores in different sporangia.

Distribution: Isoëtes changxingensis is known only from Changxing County, Huzhou City of Zhejiang Province, China.

Note: * indicates cite from Liu et al. (2008) and Shu et al. (2022) . Data of I. shangrilaensis, I. baodongii and I. fengii are cited from Li et al. (2019) , Lu et al. (2021) and Gu et al. (2023a, b) , respectively.

Etymology: The specific epithet is derived from its type locality, Changxing County.

Cytology: Isoëtes changxingensis is a tetraploid species with a chromosome number of 2n=4x=44. (Fig.3)

Ecology: Ditches beside farmland at an altitude of 50 m, it has been submerged in the upstream ditch at a higher water level while standing up in the ditch at a lower water level. It often blends in with associated species such as Pteris multifida Poir., Oenanthe javanica (Blume) DC., Pouzolzia zeylanica (L.) Benn., Commelina benghalensis L. and Gynostemma pentaphyllum (Thunb.) Makino at the distribution site.

IUCN Red List category: Isoëtes changxingensis is only found in Changxing County, Huzhou City, Zhejiang Province. There are about 300 individuals, but the ditches in which they are distributed are close to villagers’ farmlands, which poses a risk of pesticide contamination, vine shading, and farmers’ cutting. Based on currently available information, we proposed that I. changxingensis should be considered as ‘Endangered (EN) ’ [A2a; B2ab (iii) ; C2a (i) ] according to the Red List Categories and Criteria (IUCN, 2022) . This species is confronted with habitat destruction and wild human mowing, therefore, it is necessary to carry out the related conservation work such as ex situ conservation and field return, etc.

Additional specimen examined (Paratype) : CHINA, Nanjing Botanical Garden Mem. Sun Yat-Sen, cultivated plant, collected from type locality, 12 January 2024, Jiahao Shen 2707 (NAS!) .

3 Updated Key to Isoëtes Species in China

Key to Isoëtes species from China

1. Small ferns, mostly found at high altitudes (H ＞ 2 000 m) ······ 2

1. Taller plants, mostly found at low to medium altitudes (H ＜ 2 000 m) ······ 3

2. Megaspore laevigate, microspore rugulate ······I. hypsophila

2. Megaspore tuberculate-rugulate, microspore echinate to cristate ······I. shangrilaensis

3 . Leaves 1-2 mm wide at middle······4

3 . Leaves 2-10 mm wide at middle······7

4. Megaspore cristate-reticulate, microspore echinate-tuberculate ······I. orientalis

4. Megaspore cristate or tuberculate-cristate, microspore echinate······5

5. Megaspore cristate, 340-450 μm (mean=409 μm) in diameter on the proximal face······ I. sinensis

5. Megaspore tuberculate-cristate, 280-410 μm in diameter on the proximal face······6

6. Individual diploid (2n=22) , just distributing in Taiwan ······I. taiwanensis

6. Individual tetraploid (2n=44) , just distributing in Hunan ······I. longpingii

7 . Microspore tuberculate or levigate-granulate······8

7 . Microspore echinate······9

8. Individual tetraploid (2n=44) , just distributing in Hunan ······I. xiangfei

8. Individual diploid (2n=22) , distributing in Yunnan and Guizhou ······I. yunguiensis

9 . Megaspore echinate-cristate······10

9 . Megaspore reticulate ······I. fengii

1 0. Individual diploid (2n=22) , megaspore390-510 μm (mean=450 μm) in diameter on the proximal face ······I. baodongii

1 0. Individual tetraploid (2n=44) , megaspore317-411 μm (mean=360 μm) in diameter on the proximal face ······I. changxingensis

中国水韭属检索表

1 . 植株较矮小，多分布于高海拔地区（H ＞ 2 000 m）······ 2

1 . 植株较高大，多分布于中低海拔地区（H ＜ 2 000 m）······ 3

2 . 大孢子表面光滑，小孢子表面具突起纹饰······高寒水韭I. hypsophila

2. 大孢子表面具瘤状-突起纹饰，小孢子表面具棘刺至脊条状突起纹饰······香格里拉水韭I. shangrilaensis

3 . 叶片中部较窄，宽度1~2 mm ······4

3 . 叶片中部较宽，宽度2~10 mm ······7

4. 大孢子表面具网络状纹饰; 小孢子表面具棘刺状瘤状突起纹饰······东方水韭I. orientalis

4 . 大孢子表面具脊条状或瘤状突起纹饰；小孢子表面具棘刺状突起纹饰······ 5

5 . 大孢子表面具脊条状突起纹饰，平均极面直径409 μm ······中华水韭I. sinensis

5 . 大孢子表面具瘤状-脊条状突起纹饰，平均极面直径小于400 μm ······6

6 . 染色体数目为22，分布于台湾······台湾水韭I. taiwanensis

6 . 染色体数目为44，分布于湖南······隆平水韭I. longpingii

7 . 小孢子表面具瘤状突起纹饰或具疣状颗粒至近乎光滑······ 8

7 . 小孢子表面具棘刺状突起纹饰 ······9

8 . 染色体数目为44，分布于湖南······湘妃水韭I. xiangfei

8 . 染色体数目为22，分布于云南和贵州······云贵水韭I. yunguiensis

9 . 大孢子表面具棘刺-脊条状突起纹饰 ······10

9 . 大孢子表面具网络状纹饰······青锋水韭I. fengii

1 0. 染色体数目为22，大孢子平均极面直径为450 μm ······保东水韭I. baodongii

1 0. 染色体数目为44，大孢子平均极面直径为360 μm ······长兴水韭I. changxingensis

Acknowledgments The authors thank Prof. WANG Yuhua from Nanjing Agricultural University for scanning the spores.

参考文献