長興水韭，中國水韭屬一個四倍體新種

Iso?tes changxingensis （Iso?taceae）， a new tetraploid quillwort species from China

TONG Ling1， SHEN Jiahao2，3，4， ZHAO Xiao1， YAN Yuehong5， ZHU Yufen6，

WU Jianyong1， GU Yufeng5， LIU Xingjian2，3，4

（ 1. Nanjing Institute of Environmental Sciences， Ministry of Ecology and Environment of the People’s Republic of China， Nanjing 210042， China;

2. Institute of Botany， Jiangsu Province and Chinese Academy of Sciences， Nanjing 210014， China; 3. Nanjing Botanical Garden Mem. Sun Yat-Sen， Nanjing 210014， China; 4. Jiangsu Key Laboratory for the Research and Utilization of Plant Resources， Nanjing 210014， China; 5. KeyLaboratory"of National Forestry and Grassland Administration for Orchid Conservation and Utilization， National Orchid Conservation amp; Research Center of Shenzhen，Shenzhen 518114， Guangdong， China; 6. Huzhou Ecological Environment Bureau， Changxing Branch， Huzhou 313100， Zhejiang， China ）

Abstract： "Iso?tes changxingensis， a new species from Zhejiang Province， China， is illustrated and described here. The new species morphologically resembles I. baodongii， but it differs from the latter by 44 chromosomes， megaspore 317 - 411 μm （mean=360 μm） ［vs. 22， 390 - 510 μm （mean=450 μm）］. It is also extremely similar to I. longpingii in terms of megaspore size， with the difference that I. changxingensis rhizome corms 3-lobed， leaves 2 - 3 mm wide at middle， megaspore echinate-cristate （vs. 2-lobed， 1 mm， tuberculate-cristate）. This species is distinguished from I. sinensis by its smaller megaspore， different surface ornamentation， and leaf blade 20 - 60 cm ［vs. 340 - 450 μm （mean=409 μm）， cristate， 15 - 30 cm］. According to Guidlines for Using the IUCN Red List Criteria， the conservation status of I. changxingensis should be regarded as endangered （EN）.

Key words： new taxon， Iso?tes， lycophytes， endangered， spore ornamentation， key to Iso?tes

CLC number： "Q949

Document code： "A

Article ID： "1000-3142（2024）04-0611-08

摘"要： "水韭屬是一類具有異形孢子的古老石松類植物，全屬物種均被列入國家重點保護野生植物。該文報道了在浙江省長興縣新發現的一個四倍體水韭屬居群。基于形態學、孢粉學和細胞學證據，將該物種命名為長興水韭（Iso?tes changxingensis），并詳細描述了其形態特征。長興水韭與保東水韭（I. baodongii）在植株形態及孢子紋飾方面都較為相似，不同之處在于其染色體為44條，大孢子極面直徑為317～411 μm（平均為360 μm）（vs. 染色體22條，大孢子極面直徑為390～510 μm，平均為450 μm）。與同為四倍體的隆平水韭（I. longpingii）在孢子大小方面極為接近，不同之處在于其根狀莖3裂，葉片中間寬為2～3 mm，大孢子表面具棘刺-脊條狀紋飾（vs. 根狀莖2裂，葉片中間寬度為1 mm，大孢子表面具瘤狀-脊條狀紋飾）。該種與中華水韭（I. sinensis）的區別在于其大孢子較小，表面紋飾不同，葉片長為20～60 cm（vs. 大孢子極面直徑為340～450 μm，平均為409 μm，具脊條狀突起紋飾，葉片長為15～30 cm）。長興水韭目前僅分布于其模式產地的一處溝渠，由于其分布區狹窄，野生居群數量較少，棲息地環境受到人為干擾，因此根據IUCN紅色名錄評估標準可將長興水韭暫定為瀕危（EN）等級。由于該種面臨生境破壞和野外人為刈割的風險，因此很有必要開展遷地保護及野外回歸等相關工作。該文編制了中國已報道的水韭屬物種的分種檢索表，為該屬物種的鑒定和保護工作提供了參考。

關鍵詞： 新分類群， 水韭屬， 石松類植物， 瀕危， 孢子紋飾， 水韭屬檢索表

Iso?tes L. （Iso?taceae， 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 Iso?tes 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 amp; Hickey， 1992; Choi et al.， 2008; Bagella et al.， 2011; Freund et al.， 2018; Gu et al.， 2023a）. The identification characteristics of Iso?tes mainly include habitat， leaf vein， ligule， megaspore size and texture， chromosome count and DNA sequence （Kott amp; Britton， 1983; Hickey， 1986， Taylor et al.， 1993; Troia et al.， 2016; Bolin et al.， 2017; Brunton amp; 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 amp; Troia， 2018）.

At present， ten species of Iso?tes 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 amp; W. C. Taylor （Wang et al.， 2002）， I. orientalis H. Liu amp; Q. F. Wang （Liu et al.， 2005）， I. shangrilaensis X. Li， Y. Huang， X. Dai， amp; X. Liu （Li et al.， 2019）， I. baodongii Y. F. Gu， Y. H. Yan， amp; Yi J. Lu （Lu et al.， 2021）， I. longpingii Y. H. Yan， Y. F. Gu， amp; J. P. Shu， and I. xiangfei Y. H. Yan， Y. F. Gu， amp; J. P. Shu （Shu et al.， 2022）， and newly reported species I. fengii Y. F. Gu amp; Y. H. Yan （Gu et al.， 2023b）. According to the List of National Key Protected Wild Plants released in September 2021， all species of Iso?tes are list as first-level protection （http：//www.forestry.gov.cn/main/5461/20210908/162515850572900.html）. The distribu-tion scopes of Iso?tes species are wide and the altitude spans are very large. Iso?tes 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 Iso?tes species in the United States， while only ten accepted Iso?tes species in China （Troia et al.， 2016）. The relatively sparse number of Iso?tes 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 Iso?tes 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

Iso?tes changxingensis[HTSS][STBZ] Y. F. Gu amp; J. H. Shen， sp. nov. （長興水韭， 新擬， Fig. 1-Fig. 3）

Type： CHINA， Zhejiang Province， Changxing （長興） County， Xiaopu （小浦） Town， in an artificial ditch， 31.014349° N， 119.779106° E， alt. 50 m， 27 August 2023. Jiahao Shen amp; Ling Tong 2515 （holotype： NAS！; isotype： NOCC！）.

Diagnosis： Iso?tes 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 blade 20-60 cm "［vs. megaspore 340 -450 μm， mean=409 μm， cristate， 15-30 cm in I. sinensis］. （Fig. 1-Fig. 3， Table 1）

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 canal 4. 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： Iso?tes changxingensis is known only from Changxing County， Huzhou City of Zhejiang Province， China.

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

Cytology： Iso?tes 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： Iso?tes 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 Iso?tes Species in China

Key to Iso?tes species from China

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

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

2. Megaspore laevigate， microspore rugulateI. hypsophila

2. Megaspore tuberculate-rugulate， microspore echinate to cristateI. shangrilaensis

3. Leaves 1 - 2 mm wide at middle4

3. Leaves 2 - 10 mm wide at middle7

4. Megaspore cristate-reticulate， microspore echinate-tuberculateI. orientalis

4. Megaspore cristate or tuberculate-cristate， microspore echinate5

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

5. Megaspore tuberculate-cristate， 280 - 410 μm in diameter on the proximal face6

6. Individual diploid （2n=22）， just distributing in TaiwanI. taiwanensis

6. Individual tetraploid （2n=44）， just distributing in HunanI. longpingii

7. Microspore tuberculate or levigate-granulate8

7. Microspore echinate9

8. Individual tetraploid （2n=44）， just distributing in HunanI. xiangfei

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

9. Megaspore echinate-cristate10

9. Megaspore reticulateI. fengii

10.Individual diploid （2n=22）， megaspore 390 - 510 μm （mean=450 μm） in diameter on the proximal faceI. baodongii

10.Individual tetraploid （2n=44）， megaspore 317-411 μm （mean=360 μm） in diameter on the proximal faceI. changxingensis

中國水韭屬檢索表

1. 植株較矮小，多分布于高海拔地區（H gt; 2 000 m）2

1. 植株較高大，多分布于中低海拔地區（H lt; 2 000 m）3

2. 大孢子表面光滑，小孢子表面具突起紋飾高寒水韭I. hypsophila

2. 大孢子表面具瘤狀-突起紋飾，小孢子表面具棘刺至脊條狀突起紋飾香格里拉水韭I. shangrilaensis

3. 葉片中部較窄，寬度1～2 mm4

3. 葉片中部較寬，寬度2～10 mm7

4. 大孢子表面具網絡狀紋飾; 小孢子表面具棘刺狀瘤狀突起紋飾東方水韭I. orientalis

4. 大孢子表面具脊條狀或瘤狀突起紋飾；小孢子表面具棘刺狀突起紋飾5

5. 大孢子表面具脊條狀突起紋飾，平均極面直徑409 μm中華水韭I. sinensis

5. 大孢子表面具瘤狀-脊條狀突起紋飾，平均極面直徑小于400 μm6

6. 染色體數目為22，分布于臺灣臺灣水韭I. taiwanensis

6. 染色體數目為44，分布于湖南隆平水韭I. longpingii

7. 小孢子表面具瘤狀突起紋飾或具疣狀顆粒至近乎光滑8

7. 小孢子表面具棘刺狀突起紋飾9

8. 染色體數目為44，分布于湖南湘妃水韭I. xiangfei

8. 染色體數目為22，分布于云南和貴州云貴水韭I. yunguiensis

9. 大孢子表面具棘刺-脊條狀突起紋飾10

9. 大孢子表面具網絡狀紋飾青鋒水韭I. fengii

10. 染色體數目為22，大孢子平均極面直徑為450 μm保東水韭I. baodongii

10. 染色體數目為44，大孢子平均極面直徑為360 μm長興水韭I. changxingensis

Acknowledgments

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

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（責任編輯"李"莉"王登惠）

DOI： 10.11931/guihaia.gxzw202312022

童玲， 沈佳豪， 趙曉， 等， 2024.長興水韭，中國水韭屬一個四倍體新種 . 廣西植物， 44（4）： 611-618.

TONG L， SHEN JH， ZHAO X， et al.， 2024.

Iso?tes changxingensis （Iso?taceae）， a new tetraploid quillwort species from China .

Guihaia， 44（4）： 611-618.

收稿日期： "2024-01-18"接受日期： [HTSS]2024-02-19

基金項目： ""長興縣生物多樣性本底調查評估項目（202308160013）；生態環境部“生物多樣性調查、觀測和評估”項目（2019HJ2096001006）。

第一作者： 童玲（1996—），碩士，助理研究員，研究方向為植物多樣性保護與利用，（E-mail） 2896049234@qq.com。

通信作者： 顧鈺峰，博士，研究方向為蕨類植物系統分類學，（E-mail） shguyufeng@163.com; 劉興劍，高級實驗師，研究方向為植物分類、活植物引種和遷地保育技術，（E-mail） liux-ingjian1974@163.com。