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Chrysosplenium sangzhiense (Saxifragaceae), a new species from Hunan, China
expand article infoLong-Fei Fu, Tian-Ge Yang§, De-qing Lan§, Fang Wen, Hong Liu§
‡ Guangxi Institute of Botany, Chinese Academy of Sciences, Guilin, China
§ South-Central University for Nationalities, Wuhan, China
Open Access

Abstract

Chrysosplenium sangzhiense Hong Liu, a new species from Hunan, China, is described and illustrated. The phylogenetic analysis revealed that the new species belongs to subgen. Chrysosplenium and is closely related to C. grayanum, C. nepalense and C. sinicum. The chromosome number of the new species is 2n = 46, indicating a novel basic number x = 23 in Chrysosplenium that is different from other species. This also suggests that C. sangzhiense is probably an allopolyploid derivative of a species with x = 11 and one with x = 12. Morphologically, C. sangzhiense can be easily distinguished from C. grayanum, C. nepalense, C. sinicum and C. cavaleriei, a species not included in our phylogenetic analysis by a suite of characters relating to the sterile shoots, basal leaves, cauline leaves, flowering stem, sepals, disc, capsule and seed. A global conservation assessment is performed, and classifies C. sangzhiense as Least Concern (LC).

Keywords

Chrysosplenium, cytology, phylogeny, Saxifragaceae, subgen, taxonomy

Introduction

Chrysosplenium L. (1753) is a perennial herbaceous genus in Saxifragaceae and comprises more than 70 species (Kim et al. 2019; Fu et al. 2020). Chrysosplenium is distributed in Asia, America and Europe (Pan and Ohba 2001; Soltis 2007).

The latest revision of Chinese Chrysosplenium included 35 species (Pan and Ohba 2001). Although no particular infra-generic classification was adopted in this revision, use of leaf arrangement as the primary character in the key to species reflected the recognition of two subgenera in previous taxonomic revisions (Pan 1986a, b). In addition, seed surface has been used as an important character to delimit sections (Pan 1986a, b). Soltis et al. (2001) showed that the two subgenera are both monophyletic and sister to each other using matK sequence data, thereby confirming that leaf arrangement is a phylogenetically informative morphological character. Subsequent taxonomic research on Chinese Chrysosplenium has been undertaken by Liu et al. (2016), Kim et al. (2019) and Fu et al. (2020), bringing the total diversity of the Chinese flora to 38 species, of which 23 (60%) are endemic.

Previous studies have demonstrated that Chrysosplenium has a diverse basic chromosome number with x = 7, 8, 9, 10, 11, 12 and 13 at species level indicating cytological data provides important evidence for the delimitation and evolution of Chrysosplenium (Hara and Kurosawa 1963; Funamoto and Tanaka 1988a, b, 1989; Funamoto et al. 1997, 1999, 2000, 2004; Funamoto and Zhou 2010).

As part of ongoing research into the diversity of Chinese Chrysosplenium, the authors undertook an extensive fieldtrip in Hunan, China. During the trip an unknown species of Chrysosplenium was collected. Following a thorough literature survey (Hara 1957; Pan 1992; Pan and Ohba 2001; Liu et al. 2016; Kim et al. 2019; Fu et al. 2020) along with the molecular and cytological evidence, we confirmed that it is a distinct and undescribed species.

Materials and methods

Morphology observations and conservation assessments

All morphological characters were studied based on the material from field and herbarium specimens using a dissecting microscope (SMZ171, Motic, China). For seed morphology, we also undertook scanning electron micrograph (SEM) observation; seeds were collected from the field and dried by silica gel. The pre-treatment including impurities removing, air-drying and gold-coating was performed, following Fu et al. (2020). Observations and photographs were taken under a Hitachi SU8010 scanning electron microscope. At least 15 seeds were used to determine the size and surface. Conservation assessment was undertaken following IUCN (2019).

Genomic DNA extraction, PCR amplification, and Sequencing

To confirm the systematic position of this unknown species, we conducted phylogenetic studies using matK sequence data. We chose this DNA region due to its highest species coverage within the genus (Soltis et al. 2001; De Vere 2012; Saarela et al. 2013; Ebersbach et al. 2017; Kim et al. 2018) so that we could trace the most closely related species. Forty-eight species of Chrysosplenium as in-group and three species of Saxifraga and Itea as out-group were sampled. Of these, 15 sequences were obtained from the Genbank (https://www.ncbi.nlm.nih.gov/), while 36 sequences were newly generated. Their species names and GenBank accession numbers are listed in Table 1. DNA extraction, PCR amplification, and sequencing were performed following Soltis et al. (2001).

Table 1.

Species names and GenBank accession numbers of matK DNA sequences used in this study (* newly generated sequences).

Species Location Voucher specimens Herbarium Genbank number
Chrysosplenium album Maxim. Nikkou-shi, Japan HSN09815 HSN MW402998 *
Chrysosplenium aureobracteatum Y.I.Kim & Y.D.Kim Gangwon, South Korea KYI-2009032 AXY64019
Chrysosplenium biondianum Engl. Shanxi, China HZ2017050107362 HSN MW402999 *
Chrysosplenium carnosum Hook.f. et Thoms. Sichuan, China HSN013113 HSN MW403000 *
Chrysosplenium davidianum Decne. ex Maxim. Sichuan, China HSN06442 HSN MW403001 *
Chrysosplenium delavayi Franch. Hunan, China SZ2016080907105 HSN MW403002 *
Chrysosplenium echinus Maxim. Nikkou-shi, Japan HSN09817 HSN MW403003 *
Chrysosplenium fauriae Franch. Nikkou-shi, Japan HSN09823 HSN MW403004 *
Chrysosplenium flagelliferum Fr. Schmidt. Nikkou-shi, Japan HSN09816 HSN MW403005 *
Chrysosplenium forrestii Diels Nikkou-shi, Japan HSN7797 HSN MW403006 *
Chrysosplenium giraldianum Engl. Sichuan, China JZ2018042507981 HSN MW403007 *
Chrysosplenium glossophyllum Hara Sichuan, China QCS2017102608035 HSN MW403008 *
Chrysosplenium grayanum Maxim. Nikkou-shi, Japan HSN09810 HSN MW403009 *
Chrysosplenium griffithii Hook.f. et Thoms. Shanxi, China HSN7760 HSN MW403010 *
Chrysosplenium henryi Franch. Hunan, China HSN7505 HSN MW403011 *
Chrysosplenium hydrocotylifolium Lévl. et Vant. Hubei, China HSN09188 HSN MW403012 *
Chrysosplenium japonicum (Maxim.) Makino Zhejiang, China HSN7909 HSN MW403013 *
Chrysosplenium kamtschaticum Fisch. ex Seringe Shimane-ken, Japan DG2019032310004 HSN MW403014 *
Chrysosplenium kiotense Ohwi. Nikkou-shi, Japan HSN09818 HSN MW403015 *
Chrysosplenium lanuginosum Hook.f. et Thoms. Anhui, China BD2017030507343 HSN MW403016 *
Chrysosplenium lectus-cochleae Kitagawa Jilin, China HSN7379 HSN MW403017 *
Chrysosplenium macrophyllum Oliv. Hubei, China BD2017030507344 HSN MW403018 *
Chrysosplenium macrospermum Y.I.Kim & Y.D.Kim Jilin, China CBS2016062406656 HSN MW403019 *
Chrysosplenium macrostemon Maxim. ex Franch. et Sav. Nikkou-shi, Japan HSN09820 HSN MW403020 *
Chrysosplenium nepalense D.Don Yunnan, China GLGH20170607375 HSN MW403021 *
Chrysosplenium nudicaule Bunge Gansu, China HSN07772 HSN MW403022 *
Chrysosplenium pilosum Maxim. Nikkou-shi, Japan HSN09819 HSN MW403023 *
Chrysosplenium qinlingense Z.P.Jien ex J.T.Pan Sichuan, China HSN7980 HSN MW403024 *
Chrysosplenium ramosum Maxim. Jilin, China SJH2017052107372 HSN MW403025 *
Chrysosplenium serreanum Hand.-Mazz. Jilin, China SJH2017052107371 HSN MW403026 *
Chrysosplenium sinicum Maxim. Hunan, China TPS2017042407504 HSN MW403027 *
Chrysosplenium taibaishanense J.T.Pan Shanxi, China HSN7761 HSN MW403028 *
Chrysosplenium uniflorum Maxim. Tibet, China HSN7380 HSN MW403029 *
Chrysosplenium zhouzhiense Hong Liu Shanxi, China HSN13356 HSN MW403030 *
Chrysosplenium sangzhiense Hong Liu sp. nov. Hunan, China TPS2017042307449 HSN MW403032 *
Chrysosplenium alternifolium L. Shimane-ken, Japan DG2019032310003 HSN MT362050
Chrysosplenium maximowiczii Franch. et Sav. Kanagawa, Japan AB003053
Chrysosplenium nagasei Wakab. & H.Ohba Gifu, Japan AB003054
Chrysosplenium rhabdospermum Maxim. Nagasaki, Japan AB003058
Chrysosplenium tosaense Makino Saitama, Japan AB003059
Chrysosplenium iowense Rydb. Iowa, USA L34120
Chrysosplenium oppositifolium L. Wales, UK JN894973
Chrysosplenium rosendahlii Packer Northwest Territories, Canada KC474470
Chrysosplenium tetrandrum (N. Lund) Th. Fries Nunavut, Canada Brysting_01-065_CAN CAN KC474473
Chrysosplenium wrightii Franch. & Sav. Yukon, Canada Bennett_08-125_CAN CAN KC474474
Chrysosplenium americanum Schwein. ex Hook. Hatfield, New Hampshire, USA KU524206
Chrysosplenium valdivicum Hook. Chile KU524208
Chrysosplenium zhangjiajieense X.L.Yu, Hui Zhou & D.S.Zhou Hunan, China ZJ2016031506369 HSN MW402997 *
Saxifraga stolonifera Curt. Anhui, China HSN07355 HSN MW403031*
Itea chinensis C.K.Schneider NC_037884
Itea virginica L. MF350096

Phylogenetic analysis

We performed phylogenetic analyses of Chrysosplenium based on matK sequence dataset using Bayesian inference (BI) and maximum likelihood (ML). For BI analysis, we employed MrBayes v.3.2.6 (Ronquist et al. 2012) to obtain a maximum clade credibility (MCC) tree. The matrix of matK sequence was aligned by MAFFT. Bayesian inference was performed using one million generations, four runs, four chains, a temperature of 0.001, 25% trees discarded as burn-in, and trees sampled every 1,000 generations (1,000 trees sampled in total) with GTR+F+G4 model.

We conducted the ML analysis using IQ-TREE v 2.0.6 (Nguyen et al. 2015) with 1,000 bootstrap replicates, and default ModelFinder (Kalyaanamoorthy et al. 2017) to find TVM+F+R3 as the best-fit substitution model. Tree visualization was achieved in FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

Chromosome preparations

Living plants of the new species were cultivated in the green house of South-Central University for Nationalities. Actively growing root tips were harvested after 1–2 weeks. Cytological examination was performed following Funamoto and Zhou (2010). The best metaphase plates were photographed using an imager microscope with a camera attachment. At least 3–5 cells from 3–5 root tips of five individuals of the new species at somatic metaphase were counted to determine the chromosome numbers.

Results

Molecular phylogenetic studies

The aligned matrix of matK sequence was 1,644 characters. Of the 154 variable characters, 90 (58.44%) were parsimony-informative, including indels. BI and ML analyses resulted in the same tree topology which showed the undescribed species as belonging to a strongly supported clade (BP = 89%, PP = 1) that included Chrysosplenium grayanum Maxim. (1877), C. nepalense D.Don (1825) and C. sinicum Maxim. (1877) (Fig. 1).

Figure 1. 

Phylogenetic tree of Chrysosplenium generated from maximum likelihood (ML) of matK dataset. Numbers on the branches indicate bootstrap values (≥50%) of the ML and the posterior probability (≥0.5) of Bayesian inference analyses.

Chromosome characteristics

The chromosome number of Chrysosplenium sangzhiense was observed to be 2n = 46 (Fig. 2). The chromosome size fell into the range 0.93–2.43 μm, suggesting slight size variation. A detailed karyotype analysis was not possible because the chromosomes are small, and the position of centromere could not be determined.

Figure 2. 

Somatic chromosomes at metaphase of C. sangzhiense Hong Liu, sp. nov. from three different individuals. Scale bar: 10 μm.

Taxonomic treatment

Chrysosplenium sangzhiense Hong Liu, sp. nov.

Figs 3, 4, 5

Remarks

Similar to Chrysosplenium grayanum, C. nepalense, C. sinicum and C. cavaleriei (Table 2). C. sangzhiense differs from C. grayanum in its usually fewer cauline leaves, a square flowering stem and red-brown seeds; from C. nepalense it differs in its usually fewer cauline leaves, a square flowering stem and conspicuously unequal capsule lobes; from C. sinicum it differs in producing sterile shoots from all leaf axils, an absence of basal leaves, larger cauline leaves, and red-brown seeds; and from C. cavaleriei it differs in its erect sepals and absent disc.

Figure 3. 

Illustration of Chrysosplenium sangzhiense Hong Liu, sp. nov. A habit in flowering phase B non-flowering stem C inflorescence with flowers D flower E capsule F seeds.

Table 2.

Morphological comparison of Chrysosplenium sangzhiense, C. cavaleriei, C. grayanum, C. nepalense and C. sinicum.

Characters C. sangzhiense C. cavaleriei C. grayanum C. nepalense C. sinicum
Sterile branch from all leaf axils from near stem base from all leaf axils from all leaf axils only from basal leaf axils
Basal leaves absent absent absent absent present
Cauline leaves 2–3 pairs, 10–30 × 10–25 mm 1–3 pairs, 9–13 × 10–14 mm 2–7 pairs, 4–17 × 4–17 mm 3–5 pairs, 3–18 × 5–18 mm 1–2 pairs, 6–10.5 × 7.5–11.5 mm
Flowering stem (upper part) square unknown rounded rounded square
Sepals erect spreading erect erect erect
Disc absent distinct somewhat inconspicuous absent absent
Capsule lobe lobes conspicuous unequal lobes conspicuous unequal lobes conspicuous unequal lobes subequal lobes conspicuous unequal
Seed red brown, papillose dark brown, papillose dark brown, papillose red brown, smooth dark brown, papillose

Type

China. Hunan: Badagongshan National Nature Reserve, Sangzhi County, 29°47'10"N, 110°5'33"E, under broadleaved forests and near the stream in a mountain area at ca 1,220 m altitude, 22 April 2017, Hong Liu HSN07449 (holotype HSN; isotypes HSN, IBK).

Description

Perennial herbs, 10–25 cm tall. Root fibrous and robust. Rhizome long creeping without stolons or bulbs. Basal leaves absent. Sterile shoots well developed, arising from all leaf axils, round in cross-section, 5–15 cm long at anthesis, later elongate and decumbent, up to 50 cm long, rooting at nodes, without forming a rosette. Leaves of sterile shoots opposite, isophyllous, always ca 8 at anthesis, dark purple, petiole 6–10 mm long, blade 10–30 × 10–25 mm, rounded, glabrous, apex obtuse, margin obtusely dentate (10–16 teeth), base broadly cuneate; post-anthesis 10–30 or more, green, petiole 6–10 mm long, blade 20–35 × 15–20 mm, rounded or ovate, glabrous, apex obtuse, margin obtusely dentate (12–20 teeth), base broadly cuneate. Cauline leaves 4–6 (2–3 pairs), opposite, petiole 6–10 mm long; blade 6–13 × 5–12 mm, rounded or broadly ovate, glabrous, apex obtuse, margin obtusely dentate (10–14 teeth), base broadly cuneate. Flowering stem(s) erect, branched, 10–23 cm tall, glabrous, purple, square in cross-section. Inflorescence 8–25-flowered cyme, dense, 1.4–9 cm long, 5–10 cm in diam.; bracteal leaves yellow-green, triangular arrangement and unequal, the middle one larger, petiole 2–8 mm long, blade 4–15 × 7–10 mm, subrounded, glabrous, apex obtuse, margin obtusely dentate (6–12 teeth), base broadly cuneate; Flowers tetramerous, actinomorphic; sepals 4 (2 pairs), erect, yellow in flowering phase but turn green in fruiting time, 2–3 × 2–3 mm, broadly ovate, apex obtuse; disk absent; stamens 8, homostylic, 1–2 mm long, shorter than sepals; filaments slender, ca 1 mm long; anther yellow, 2-locular, longitudinally dehiscent; ovary 2-locular, semi-inferior; stigma 2; styles erect, ca 1–2 mm long. Fruit a capsule, 5–7 mm long, green, smooth, 2-lobed (horn-shaped), conspicuous unequal, dehiscent along the adaxial suture; seeds numerous, reddish brown, sub-ovoid, a raphe on one side, 650–800 × 600–750 μm, papillose.

Figure 4. 

Plate of Chrysosplenium sangzhiense Hong Liu, sp. nov. A habitat B habit in flowering phase C habit in non-flowering phase D flowering stem E inflorescence with flowers (Photos by Hong Liu).

Etymology

Chrysosplenium sangzhiense is named after the type locality, Sangzhi County, Hunan Province, China.

Vernacular name

sāng zhí jīn yāo (Chinese pronunciation); 桑植金腰 (Chinese name).

Conservation status

At present, Chrysosplenium sangzhiense is only known from a single locality (IUCN criterion D2). At this locality, the population is ca 500 mature individuals (IUCN criterion D1) growing in at least ten patches within a nature reserve. Using the IUCN methodology, C. sangzhiense would be classed as Vulnerable (VU), however no plausible threat could be found to confirm its status as the population is located within a protected area and not under threat in the near future. In addition, considering that the surrounding area has not been completely explored, there may be hitherto undocumented additional populations. For these reasons the Global Species Conservation Assessment for C. sangzhiense is Least Concern (LC).

Figure 5. 

Macro- and micro-morphology of Chrysosplenium spp. A C. sangzhiense, habit (A1), inflorescence with flowers (A2), scanning electron micrograph (SEM) of seed 200× (A3), 1,000× (A4) B C. grayanum, habit (B1), inflorescence with flowers (B2), SEM of seed 150× (B3), 1,000× (B4) C C. nepalense, habit (C1), inflorescence with flowers (C2), SEM of seed 150× (C3), 1,000× (C4) D C. sinicum habit (D1), inflorescence with flowers (D2) SEM of seed 150× (D3), 1,000× (D4) (Photos by Hong Liu).

Discussion

Our phylogenetic analysis is consistent with previous studies (Soltis et al. 2001) that Chrysosplenium is monophyletic and comprises two strongly supported clades namely subgen. Gamosplenium (with alternate leaves) and subgen. Chrysosplenium (with opposite leaves). C. sangzhiense is recovered as a member of subgen. Chrysosplenium and falls into a strongly supported clade that includes C. grayanum, C. nepalense and C. sinicum. In addition, C. cavaleriei H.Lév. & Vaniot (1911) is also a morphologically similar species despite that it is not included in our phylogenetic analysis. All five species are close morphologically (Table 2), but nevertheless distinguishable. C. grayanum is likely the most closely related species despite the fact that it is endemic to Japan, while C. nepalense, C. sinicum and C. cavaleriei are widespread in China.

The basic chromosome number of Japanese Chrysosplenium species is x = 11 or x = 12, but in China there is more diversity with x = 7, 8, 9, 10, 12 and 13 (Hara and Kurosawa 1963; Funamoto and Tanaka 1988a, b, 1989; Funamoto et al. 1997, 1999, 2000, 2004; Funamoto and Zhou 2010). Our cytological studies support this. The chromosome number of C. sangzhiense is 2n = 46 indicating its basic number to be x = 23. Given the relationship of reported basic chromosome number of Chrysosplenium, it suggests that the new species is probably an allopolyploid derivative of a species with x = 11 and one with x = 12. Furthermore, this is a novel basic number for the genus, and different from the closely related species such as C. grayanum (x = 11), C. sinicum (x = 12) and C. nepalense (x = 12) (Hara and Kurosawa 1963; Funamoto and Tanaka 1989; Funamoto et al. 1999; Funamoto and Zhou 2010).

Conclusion

In this study, we confirm and describe a new species of Chrysosplenium based on morphological, molecular and cytological evidence. The newly generated molecular data contributes to reconstruct a robust phylogenetic framework for further studies on the aspects of biogeography and character evolution of Chrysosplenium. In addition, a novel basic chromosome number for Chrysosplenium reported here will be useful data to evaluate the evolutionary pattern of chromosome number change and to estimate the basic chromosome number of clades of the genus.

Acknowledgements

This work was supported by the construction plan of Hubei province science and technology basic conditions platform (No.2017BEC014), Fund for key laboratory construction of Hubei province (No.2018BFC360) and the major projects of technological innovation in Hubei province (2019ABA101).

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