Two New Tetraploid Quillwort Species, Isoëtes longpingii and I. xiangfei from China (Isoëtaceae)

Jiangping Shu¹², Yufeng Gu¹³, Zhiguo Ou⁴, Wen Shao⁵, Juan Yang⁶, Qiyong Lu⁶, Xianchun Zhang⁷, Baodong Liu³, Ruijiang Wang², Yuehong Yan¹

¹ Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization, the Orchid Conservation & Research Center of Shenzhen, Shenzhen 518114, Guangdong, China
² Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China
³ Life Science and Technology College, Harbin Normal University, Key Laboratory of Plant Biology in Colleges of Heilongjiang Province, Harbin 150025, China
⁴ Hunan Shangjie Instrument Co., Ltd., Changsha 410000, China
⁵ Shanghai Chenshan Botanical Garden, Shanghai Chenshan Plant Research Center, Chinese Academy of Sciences, Shanghai 201602, China
⁶ Tongdao Dong Autonomous County Forestry Bureau, Huaihua 418500, Hunan, China
⁷ State Key Laboratory of Systematic and Evolution Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

Abstract

Two new tetraploid quillwort species, Isoëtes longpingii and I. xiangfei, from China are described and illustrated. I. longpingii is a fully submerged plant found in a small pond in Hunan Province. It is morphologically similar to I. sinensis but differs in its small, tuberculate-cristate megaspores and soft, slender leaves that grow up to 60 cm. It is also similar to the hexaploid I. orientalis but differs in having 44 chromosomes and tuberculate-cristate megaspores. I. xiangfei is most similar to the diploid I. yunguiensis in megaspore ornamentation but differs in its microspore ornamentation, oblong sporangium, and 44 chromosomes. Only a few individuals of I. longpingii occur in Ningxiang County, Hunan Province, while I. xiangfei is distributed in the wetlands of Tongdao and Huitong counties in Hunan Province. Due to their limited geographic ranges, small populations, low individual numbers, and disturbed habitats, I. longpingii and I. xiangfei are evaluated as Critically Endangered (CR) and Vulnerable (VU), respectively, according to IUCN Red List criteria. A key to all currently known Chinese quillworts is also provided to facilitate further taxonomic identification and conservation of these rare and endangered plants in China.

Keywords: new taxon, Isoëtes, lycophytes, polyploid, heterosporous, aquatic plants

Funding: This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA19050404), the National Natural Science Foundation of China (3217020964), the Harbin Normal University Postgraduate Innovation Project (HSDBSCX2021-01), and the Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization (OC202103).

Author Information: Jiangping Shu (1991–), Ph.D. candidate, research focus on fern phylogenomics, (E-mail) jpshu@scbg.ac.cn. *Corresponding authors: Yuehong Yan, Ph.D., Professor, research focus on fern diversity and evolution, (E-mail) yhyan@sibs.ac.cn; Ruijiang Wang, Ph.D., Professor, research focus on plant molecular systematics and evolution, (E-mail) wangrj@scbg.ac.cn

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) through rapid diversification in the Cenozoic Era (Pereira et al., 2017b, 2021; Wood et al., 2020). The genus contains approximately 250 species widely distributed from tropical to subarctic regions, inhabiting various habitats across most parts of the world (PPGI, 2016; Troia et al., 2016). However, most Isoëtes species have restricted habitats and small populations, placing 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 occur frequently in Isoëtes, with more than half of known species being polyploids 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 across various habitats and has a complex evolutionary history, its simple morphology—a tuft of linear sporophylls on a lobed rootstock—is remarkably similar among species, making interspecific identification difficult (Taylor & Hickey, 1992; Troia et al., 2016). Isoëtes species are predominantly distributed in the Americas, 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 diversity of Isoëtes species in Asia is probably underestimated, and many cryptic species may remain unidentified (Schafran, 2019).

Characteristics such as habitat, velum, ligule, megaspore size and texture, chromosome count, and DNA sequences are generally employed to distinguish Isoëtes species (Hickey, 1986; Pereira, 2015; Troia et al., 2016). However, 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 worldwide (Mora-Olivo et al., 2016; Pereira et al., 2016, 2017a, 2019; Schafran et al., 2016; Li et al., 2019; Lu et al., 2021). In China, seven species have been reported: 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 (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 extensive field surveys of the genus across China and collected tetraploid samples (2n=4x=44) previously identified as I. sinensis (2n=4x=44) that exhibited morphological and anatomical variation (Liu et al., 2004; Liu et al., 2006; Dai et al., 2020, 2021). By comparing morphological features and spore texture, samples from Ningxiang and Tongdao counties in Hunan Province were found to differ from I. sinensis. Additionally, our plastid phylogenomic analysis indicated that these samples formed monophyletic clades that did not cluster with I. sinensis (Gu et al., unpublished). The sample from Ningxiang County, here named I. longpingii, resembles I. orientalis (2n=6x=66) in megaspore ornamentation, while the sample from Tongdao County, here named I. xiangfei, resembles I. yunguiensis (2n=2x=22) in megaspore ornamentation. These two new species are described and illustrated in the present study.

Materials and Methods

Isoëtes species possess prominent spore morphological characteristics, and the spore ornamentation terminology established by Hickey (1986) was used in this study. Megaspores and microspores were examined under a scanning electron microscope. Spores were mounted on 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 chromosome number, young root tips of 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 °C. The samples were then hydrolyzed with a mixture of 3% cellulase and 2.5% pectinase for 10 min at room temperature and stained with carbol fuchsin. Chromosomes were counted and photographed using a Carl Zeiss Axio Scope A1 photomicroscope (Jena, Germany).

Taxonomic Treatment

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) that are sparsely tuberculate-cristate [vs. larger (mean 410 µm) and densely tuberculate-cristate] and bilobed rhizome corms (vs. trilobed in I. sinensis). It is also similar to the hexaploid I. orientalis but differs in having tuberculate-cristate megaspores and 44 chromosomes (vs. 66 in I. orientalis) (Fig. 3, Fig. 4, Table 1).

Type: CHINA. Hunan Province, Changsha City, Ningxiang County, Yujia'ao Village, 28°14'27.91'' N, 112°17'44.86'' E, alt. 131 m, June 15, 2019. Zhiguo Ou YYH15160 (holotype, PE!; 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 canal 4. Sporangia basal, obovate, 3.5–4.5 × 2.5–3.0 mm. Ligule cuneiform, 2.3–2.6 × 1.1–1.3 mm. Megaspores white when dry, earthy yellow when wet, 310–410 µm (mean = 350 µm, n = 20) in diameter, proximal hemisphere tuberculate, distal hemisphere echinate-cristate. Microspores gray en masse, elliptic, vertical axis length 27–30 µm (mean = 29 µm, n = 20), surface echinate. Chromosome number 2n = 4x = 44.

Distribution: Ningxiang County, Changsha City, Hunan Province.

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

Etymology: The specific epithet "longpingii" honors the late Prof. Long-Ping Yuan, who devoted his 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 ten individuals is found at only one location in Yujia'ao Village, Ningxiang County, Hunan Province. Isoëtes longpingii is Critically Endangered (CR) [A1a; B2ab(iv)c; C2a(ii); D(2)] according to the Red List Categories and Criteria (Subcommittee, 2019).

Fig. 1 Morphological and anatomic characteristics of Isoëtes longpingii Y.H. Yan, Y.F. Gu & J.P. Shu. A. Habit; B. Microsporangia; C. Megasporangia; D. Chromosomes (2n = 4x = 44); E. Transection of leaf; F. Ligule; G. Transection of rhizome (2-lobed).

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 microspores (vs. sparsely tuberculate-echinate) (Fig. 3, Fig. 4, Table 1).

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!; 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 canal 4. Sporangia basal, oblong, 4.4–5.0 × 2.9–3.3 mm. Ligule oval-triangular, 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 number 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" derives from the name of the Xiangfei, two revered goddesses of the Xiangjiang River in Hunan Province where this aquatic plant was discovered. "Xiang" is the longest river in Hunan Province, and "fei" means wife in Chinese. According to legend, the Xiangfei sisters married King Shun simultaneously.

IUCN Red List category: Isoëtes xiangfei occurs in Tongdao and Huitong counties, Hunan Province, with approximately 200 individuals that are well conserved by the local forestry department. It is Vulnerable (VU) [B2ab(iii); C2a(i)] according to the Red List Categories and Criteria (Subcommittee, 2019).

Fig. 2 Morphological and anatomic characteristics of Isoëtes xiangfei Y.H. Yan, Y.F. Gu & J.P. Shu. A. Habit; B. Plants with wide wing at the base of leaf; C. Microsporangia; D. Megasporangia; E. Ligule; F. Transection of rhizome (3-lobed); G. Transection of leaf; H. Chromosomes.

Fig. 3 Palynological comparison between Isoëtes longpingii and I. sinensis. A–D. Isoëtes longpingii (YYH15160). A. Proximal view of megaspore; B. Distal view of megaspore; C. Proximal view of microspore; D. Distal view of microspore. E–H. I. sinensis from China (cited from Liu et al. 2008). E. Proximal view of megaspore; F. Distal view of megaspore; G. Proximal view of microspore; H. Distal view of microspore. I–L. I. sinensis from Japan (cited from Watanabe et al. 1996). I. Proximal view of megaspore; J. Distal view of megaspore; K. Proximal view of microspore; L. Distal view of microspore. M–P. I. sinensis from Korea (cited from Takamiya 2001). M. Proximal view of megaspore; N. Distal view of megaspore; O. Proximal view of microspore; P. Distal view of microspore.

Fig. 4 Palynological comparison between Isoëtes xiangfei, I. yunguiensis and I. orientalis. A–D. Isoëtes xiangfei (Fern08928). A. Proximal view of megaspore; B. Distal view of megaspore; C. Proximal view of microspore; D. Distal view of microspore. E–H. I. yunguiensis (YYH15163). E. Proximal view of megaspore; F. Distal view of megaspore; G. Proximal view of microspore; H. Distal view of microspore. I–L. I. orientalis (Fern08748). I. Proximal view of megaspore; J. Distal view of megaspore; K. Proximal view of microspore; L. Distal view of microspore.

Table 1 Spore features of Isoëtes from China

Note: Data for I. hypsophila, I. yunguiensis, I. taiwanensis, I. sinensis, and I. orientalis were cited from Liu et al. (2008). Data for I. shangrilaensis and I. baodongii were cited from Li et al. (2019) and Lu et al. (2021), respectively.

Key to Isoëtes of China

1. Individual diploid → 2
2. Individual polyploid → 6
3. Megaspore levigate or just tuberculate-regulate → 3
4. Megaspore with various types of ornamentation → 4
5. Leaves 2–5 cm long, microspores echinate → I. hypsophila
6. Leaves 3–18 cm long, microspores echinate to cristate → I. shangrilaensis
7. Megaspore tuberculate-cristate, just distributing in Taiwan → I. taiwanensis
8. Megaspore echinate or cristate-reticulate → 5
9. Microspore echinate, distributing in Zhejiang → I. baodongii
10. Microspore levigate-granulate, distributing in Yunnan and Guizhou → I. yunguiensis
11. Leaves ca. 1 mm wide at mid-length → 7
12. Leaves 2–3 mm wide at mid-length → I. xiangfei
13. Megaspore ornamentation cristate-reticulate, chromosome number 66 → I. orientalis
14. Megaspore without reticulate ornamentation, chromosome number 44 → 8
15. Rhizome 2-lobed, megaspore 310–410 µm (mean = 350 µm) → I. longpingii
16. Rhizome 3-lobed, megaspore 340–450 µm (mean = 409 µm) → I. sinensis

Acknowledgments

The authors thank Prof. Xun-Lin Yu from Central South University of Forestry & Technology for assistance with field surveys, and Prof. Qin-Er Yang and Prof. Yun-Fei Deng from South China Botanical Garden, Chinese Academy of Sciences for assistance with correction of Latin names. We also thank TopEdit (www.topeditsci.com) for linguistic assistance during manuscript preparation.

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