Isoëtes L. (1753: 1100) is the only extant genus of the family Isoëtaceae, with an extensive fossil record dating from the Devonian Period (Pigg, 2001; Wang et al., 2019) to rapid diversification in the Cenozoic Era (Pereira et al., 2017b, 2021; Wood et al., 2020) . The genus contains about 250 species, widely distributed from tropical to subarctic regions, and grows in various habitats in most parts of the world (PPGI, 2016; Troia et al., 2016) . However, many Isoëtes species have restricted habitats and small populations, putting them at risk of extinction (Kang et al., 2005; Liu X et al., 2005; Gentili et al., 2010; Troia et al., 2016; Brunton & Troia, 2018; Singh et al., 2021) . Interestingly, hybridization and polyploidization frequently occur in Isoëtes, and more than half of the known species are polyploid ranging from tetraploid to dodecaploid (Hickey, 1984; Taylor & Hickey, 1992; Liu et al., 2004; Kim et al., 2010; Pereira, 2015; Troia et al., 2016; Grigoryan et al., 2020) . Although this ancient genus is widely distributed in various habitats and has a complex evolutionary history, its simple morphology — a tuft of linear sporophylls on the lobed rootstock — are remarkably similar among species, making interspecific identification difficult (Taylor & Hickey, 1992; Troia et al., 2016) . Isoëtes species are mostly distributed in America, southern Europe, Africa and Australia, with relatively few species in Asia (Troia et al., 2016; Pereira et al., 2017b, 2021; Brunton & Troia, 2018; Choi et al., 2018; Singh et al., 2021) . The Isoëtes species diversity in Asia is probably underestimated, and many cryptic species may not be identified (Schafran, 2019) .

Characteristics such as habitat, velum, ligule, megaspore size and texture, chromosome count, and DNA sequences are generally employed to distinguish species of Isoëtes (Hickey, 1986; Pereira, 2015; Troia et al., 2016) ; however, the characteristics of leaf morphology and anatomy are influenced by local environmental conditions (Liu et al., 2006; Cavalheiro-Filho et al., 2021; Singh et al., 2021) . Several species have recently been described and illustrated around the world (Mora-Olivo et al., 2016; Schafran et al., 2016; Pereira et al., 2016, 2017a, 2019; Li et al., 2019; Lu et al., 2021) . In China, seven species — I. yunguiensis Q. F. Wang & W. C. Taylor, I. hypsophila Hand.-Mazz., I. taiwanensis De Vol, I. shangrilaensis X. Li & Y.Q. Huang, I. baodongii Y. F. Gu, Y. H. Yan & Yi J. Lu, I. sinensis T. C. Palmer and I. orientalis H. Liu & Q.F. Wang — have been reported (Handel-Mazzetti, 1923; Palmer, 1927; De Vol, 1972; Wang et al., 2002; Liu H et al., 2005; Zhang & Taylor, 2013; Li et al., 2019; Lu et al., 2021) .

We conducted several field surveys of the genus in China and collected some tetraploid samples (2n=4x=44) previously considered as I. sinensis (2n=4x=44) with morphological and anatomical variation (Liu et al., 2004; Liu et al., 2006; Dai et al., 2020, 2021) . By comparing morphological features and spore textures, these samples from Ningxiang City and Tongdao County in Hunan Province were found to be different from I. sinensis. In addition, our plastid phylogenomic analysis indicated that these samples formed monophyletic clades, which did not cluster together with I. sinensis. The sample from Ningxiang City, here named I. longpingii, is similar to I. orientalis (2n=6x=66) on megaspore ornamentation and that from Tongdao County, here named I. xiangfei, is similar to I. yunguiensis (2n=2x=22) on megaspore ornamen-tation. These two new species are described and illustrated in the present study.

1 Materials and Methods

Isoëtes species possess prominent spore morpholo-gical characteristics, and the spore ornamentation terminology established by Hickey (1986) was used in the present study. Megaspores and microspores of samples were examined under a scanning electron microscope. Spores were mounted on a double-sided adhesive tape attached to metal stubs, sputter-coated with platinum, and observed under a field emission scanning electron microscope (Quanta250; FEI, Hillsboro, Oregon, U.S.A.) at 30 kV. Megaspore and microspore sizes from 3-5 individuals were measured using Photoshop CS5 (Adobe Systems Inc., USA) .

To determine the chromosome number, young root tips of the sporophytes were pretreated in a saturated aqueous solution of p-dichlorobenzene for 3-5 h and subsequently fixed in Carnoy’s solution (1∶3, glacial acetic acid∶95% ethanol) for 1 h at 4℃. Subsequently, the samples were hydrolyzed with a mixture of 3% cellulase and 2.5% pectinase for 10 min at room temperature. They were then stained with carbol fuchsin. The chromosomes of the samples were counted and photographed using a Carl Zeiss Axio Scope A1 photomicroscope (Jena, Germany) .

2 Taxonomic Treatment

2.1 Isoëtes longpingii Y.H. Yan, Y.F. Gu & J.P. Shu (Fig.1, Fig.3: A-D)

Diagnosis: — Isoëtes longpingii is similar to I. sinensis, but differs in its small megaspores (310-410 μm, mean=350 μm, n=20) , sparsely tuberculate-cristate [vs. larger (mean 410 μm) , densely tuberculate-cristate]and bilobed rhizome corms (vs. trilobed in I. sinensis) . It is also similar to hexaploid I. orientalis, but differs in its megaspores tuberculate-cristate, and 44 chromosomes (vs.66 in I. orientalis) (Fig.3, Fig.4, Table1) .

Type: — CHINA. Hunan Province, Changsha City, Ningxiang City, Yujia’ao Village, 28°14′27.91″ N, 112°17′44.86″ E, alt.131 m, June15, 2019. Zhiguo Ou YYH15160 (holotype, PE! barcode 02347221; isotype: NOCC!, IBSC!) .

Description: — Plants aquatic. Rhizome corms: 2-lobed. Sporophylls: widely spreading, 40-60 in a tuft, white at base, green above, spirally arranged, 20-60 cm long, ca.1 mm wide, flattened on the adaxial side, rounded on the abaxial side, base flat and alate, peripheral fibrous bundles present, central intrastelar canal4. Sporangia: basal, obovate, 3.5-4.5×2.5-3.0 mm. Ligule: cuniform, 2.3-2.6×1.1-1.3 mm. Mega-spores: white when dry, earthy yellow when wet, 310-410 μm (mean=350 μm, n =20) in diameter, proximalhemisphere tuberculate, distal hemisphere echinate-cristate. Microspores: gray en masse, elliptic, vertical axis length 27-30 μm (mean=29 μm, n =20) , surface echinate. Chromosome numbers 2n=4x=44.

Note:* Cited from Liu et al. (2008) . Datas of I. shangrilaensis and I. baodongii are cited from Li et al. (2019) and Lu et al. (2021) , respectively.

Distribution: — Ningxiang City, Changsha City, Hunan Province.

Ecology: — Submerged in a small pond; alt.131 m.

Etymology: — The specific epithet “longpingii” is derived from the name of the late Prof. YUAN Longping, who devoted his entire life to hybrid rice research in Changsha City (where the species was discovered) and greatly contributed to global food security and poverty alleviation (Wang, 2021) .

IUCN Red List category: — A single population of about 10 individuals is found in only one location in Yujia’ao Village, Ningxiang City, Hunan Province. Isoëtes longpingii is critically endangered (CR) [A1a; B2ab (iv) c; C2a (ii) ; D (2) ] according to the Red List Categories and Criteria (IUCN, 2022) .

2.2 Isoëtes xiangfei Y.H. Yan, Y.F. Gu & J.P. Shu (Fig.2, Fig.4: A-D)

Diagnosis: — Isoëtes xiangfei is a tetraploid, morphologically similar to the diploid I. yunguiensis and the tetraploid I. sinensis in megaspore ornamentation, but differs in its less reticulate, lower megaspore ornamentation, and densely low-tuberculate (vs. sparsely tuberculate-echinate) microspores (Fig.3, Fig.4, Table1) .

Type: — CHINA. Hunan Province: Huaihua City, Tongdao County, 26°14′56.82″ N, 109°50′0.33″ E, alt.298 m, December 28, 2019. Juan Yang Fern08928 (holotype: PE! barcode 02347220; isotype: NOCC!, IBSC!) .

Description: — Plants aquatic. Rhizome corms 3-lobed. Sporophylls 20-60 in a tuft, widely spreading, white at the base, green above, spirally arranged, 15-35 cm long, 2-3 mm wide, flattened on the adaxial side, rounded on the abaxial side, base flat and alate, peripheral fibrous bundles present, central intrastelar canal4. Sporangia: basal, oblong, 4.4-5.0×2.9-3.3 mm. Ligule: oval triangle, 2.5-2.7×1.4-1.6 mm. Megaspores: white when dry, earthy yellow when wet, 390-450 μm (mean=430 μm, n =20) in diameter; proximal hemisphere cristate, distal hemisphere reticulate. Microspores: gray en masse, elliptic, monolete, vertical axis 26-28 μm (mean=27 μm, n =20) , surface echinate. Chromosome numbers 2n=4x=44.

Distribution: — Tongdao and Huitong counties, Huaihua City, Hunan Province.

Ecology: — Wetlands and small gullies; alt.298 m.

Etymology: — The specific epithet “xiangfei” is derived from the name of Xiangfei, who are two respectable goddesses of the Xiangjiang River in Hunan Province, where the aquatic plant was discovered. “Xiang” is the name of the longest river in Hunan Province, and “fei” is the wife of the king in Chinese. Xiangfei, two sisters, married the legendary King Shun at the same time.

IUCN Red List category: — Isoëtes xiangfei is found in Tongdao and Huitong counties, Hunan Province. There are about 200 individuals, which are well conserved by the local forestry department. It is vulnerable (VU) [B2ab (iii) ; C2a (i) ] according to the Red List Categories and Criteria (IUCN, 2022) .

3 Key to Isoëtes of China

(1) Individual diploid. ······ (2)

(1) Individual polyploid. ······ (6)

(2) Megaspore levigate or just tuberculate-regulate. ······ (3)

(2) Megaspore with various types of ornamentation. ······ (4)

(3) Leaves 2-5 cm long, microspores echinate. ······I. hypsophila

(3) Leaves 3-18 cm long, microspores echinate to cristate. ······I. shangrilaensis

(4) Megaspore tuberculate-cristate, just distributing in Taiwan. ······I. taiwanensis

(4) Megaspore echinate or cristate-reticulate. ······ (5)

(5) Microspore echinate, distributing in Zhejiang. ······I. baodongii

(5) Microspore levigate-granulate, distributing in Yunnan and Guizhou. ······I. yunguiensis

(6) Leaves ca.1 mm wide at mid-length. ······ (7)

(6) Leaves 2-3 mm wide at mid-length. ······I. xiangfei

(7) Megaspore ornamentation cristate-reticulate, chromosome numbers 66. ······I. orientalis

(7) Megaspore without reticulate ornamentation, chromosome numbers 44. ······ (8)

(8) Rhizome2-lobed, megaspore310-410 μm (mean=350 μm) . ······I. longpingii

(8) Rhizome3-lobed, megaspore340-450 μm (mean=409 μm) . ······I. sinensis

Acknowledgments   The authors thank Prof. YU Xunlin from Central South University of Forestry & Technology for assisting in the field surveys, Prof. YANG Qiner and Prof. DENG Yunfei from South China Botanical Garden, Chinese Academy of Sciences for their assisting in the correction of Latin name.

参考文献