Research Article
Print
Research Article
Molecular and morphological evidence supports the resurrection of Chrysosplenium guangxiense H.G.Ye & Gui C.Zhang (Saxifragaceae)
expand article infoLong-Fei Fu‡, Tian-Ge Yang§, De-Qing Lan§, Xi-Tang Chen|, Hong Liu§
‡ Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin, China
§ South-Central Minzu University, Wuhan, China
| Hubei Jiugongshan National Nature Reserve Administration, Xianning, China

Corresponding author: Hong Liu ( liuhong@mail.scuec.edu.cn )

Academic editor: Hugo de Boer
© 2024 Long-Fei Fu, Tian-Ge Yang, De-Qing Lan, Xi-Tang Chen, Hong Liu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Fu L-F, Yang T-G, Lan D-Q, Chen X-T, Liu H (2024) Molecular and morphological evidence supports the resurrection of Chrysosplenium guangxiense H.G.Ye & Gui C.Zhang (Saxifragaceae). PhytoKeys 243: 185-198. https://doi.org/10.3897/phytokeys.243.125742
Open Access

Abstract

Chrysosplenium guangxiense H.G.Ye & Gui C.Zhang was first described as a new species in 1994 but later synonymized in the Flora of China treatment with C. glossophyllum H.Hara. Plastid genomes and nrDNA sequences were used to infer the phylogenetic relationships of selected taxa in Chrysosplenium. Our phylogenetic analyses revealed that C. guangxiense belongs to sect. Alternifolia, is closely related to Chrysosplenium hydrocotylifolium H.Lév. & Vaniot but distant from C. glossophyllum. Morphologically, C. guangxiense could be easily distinguished from C. glossophyllum by having robust rhizomes, basal leaves with a long cuneate base and fewer teeth in the margin, curled sepal margins, and red, larger seeds. It could also be easily distinguished from C. hydrocotylifolium by possessing long elliptic leaves and a long cuneate leaf base. Along with the phylogenetic studies, the complete plastid genome of C. guangxiense was also reported. The plastid genome was 154,004 bp in length and comprised two inverted repeats (IRs) of 28,120 bp, separated by a large single-copy of 80,646 bp and a small single-copy of 17,118 bp. A total of 111 functional genes were discovered, comprising 78 protein-coding genes, 29 tRNA genes, and four rRNA genes. Based on assessment of morphological and molecular data Chrysosplenium guangxiense H.G.Ye & Gui C.Zhang is resurrected from C. glossophyllum H.Hara at species level. A global conservation assessment classifies C. guangxiense as Vulnerable (VU).

Key words

Chrysosplenium, phylogeny, plastid genome, Saxifragaceae, taxonomy

Introduction

Chrysosplenium L. (Saxifragaceae) comprises more than 70 species of perennial herbs (Kim et al. 2019; Fu et al. 2020, 2021). Chrysosplenium is distributed throughout Asia, America and Europe (Pan and Ohba 2001; Soltis 2007). The latest checklist of Chinese Chrysosplenium included 35 species (Pan and Ohba 2001). Their earlier revisions classified the genus into two subgenera (subg. Chrysosplenium and subg. Gamosplenium) based on leaf arrangement (Pan 1986a, b). This character was also considered by Hara, who divided Chrysosplenium into two sections, namely sect. Alternifolia and sect. Oppositifolia (Hara 1957). The following molecular analyses (Soltis et al. 2001; Fu et al. 2021) demonstrated that these two subgenera/sections are monophyletic and sister to each other, further confirming that leaf arrangement is a good indicator of the relationships within the genus. However, a recent systematic study based on a complete chloroplast genome and nrDNA data challenged this relationship as their results recovered an additional clade composed of two species with alternate leaves (as members of sect. Alternifolia). The newly defined clade was recognized as a basal clade sister to the rest of the species of Chrysosplenium (Yang et al. 2023).

Chrysosplenium guangxiense H.G.Ye & Gui C.Zhang was first described as a new species in 1994 by having ovate-elliptic leaves, an acuminate apex, a cuneate base, fewer dentate margins, and a depressed sepal apex, enabling it to be distinguished from its similar species, C. glossophyllum H.Hara (Ye and Zhang 1994). Subsequently, it appeared as a synonym of the latter in Flora of China without additional explanation (Pan and Ohba 2001). We assumed that the authors considered these differences were insufficient to distinguish them. It is possible that the scarcity of C. glossophyllum species in China, with its only population in Sichuan Province, may have led to this misinterpretation. Molecular data, however, could provide a means to confirm the systematic position of morphological similarities and to evaluate the phylogenetic informativeness of morphological characters (Scotland et al. 2003).

In 2019, we conducted an extensive investigation in Tianlin County, Baise City, Guangxi, China, the type locality of Chrysosplenium guangxiense. We collected a plant of Chrysosplenium, which was then confirmed as C. guangxiense. Following a thorough literature survey (Hara 1957; Pan 1992; Pan and Ohba 2001; Liu et al. 2016; Wei 2018; Kim et al. 2019; Fu et al. 2020, 2021; Wei et al. 2022), along with the molecular evidence, it was confirmed that C. guangxiense is a different species from C. glossophyllum.

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 microscope (SEM) observations; seeds were collected from the field and dried with silica gel. The pre-treatments, including impurity removal, air-drying, and gold-coating, were performed following Fu et al. (2020). Observations and photographs were taken under a Hitachi SU8010 scanning electron micrograph. At least 15 seeds were used to determine their size and ornamentation. A conservation assessment was undertaken following the IUCN (2019).

Genomic DNA extraction, sequencing, plastid genome and nrDNA assembly and annotation

The genomic DNA was extracted using the modified CTAB method (Doyle and Doyle 1987). The short-insertion library (300 bp) was constructed and then sequenced to obtain 2×150 bp paired-end data using the Illumina NovaSeq platform at Majorbio Company (Shanghai, China). The raw data was filtered through Trimmomatic v. 0.39 (Bolger et al. 2014) to obtain clean data, and then the clean data were quality-controlled using FastQC v. 0.11.9 (Simon 2020). The complete plastid genome and nrDNA sequence were assembled using GetOrganelle v. 1.7.5 (Jin et al. 2020), and annotation was performed using CPGAVAS2 (Shi et al. 2019) and PGA (Qu et al. 2019).

Phylogenetic analysis

To confirm the phylogenetic placement of Chrysosplenium guangxiense, we undertook phylogenetic studies using the chloroplast (CP) genomes and nrDNA sequences obtained in a previous study (Yang et al. 2023). Forty-seven species of Chrysosplenium as in-group, and two species from other genera in Saxifragaceae and Itea chinensis Hook. & Arn. from Iteaceae as an out-group were sampled. The species names and GenBank accession numbers are listed in Table 1.

Table 1.
Download as
CSV
XLSX

Species names and GenBank accession numbers of plastid genomes and nrDNA sequence used in this study (* newly generated sequences).

Species Location Voucher specimens Herbarium Plastid GenBank number nrDNA GenBank number
Chrysosplenium album Maxim. Nikkou-shi, Japan HSN09815 HSN OK336556 OP154009
Chrysosplenium aureobracteatum Y.I.Kim & Y.D.Kim Gangwon Province, South Korea KYI-2009032 (Kim et al. 2018) MG878089 MK989509
Chrysosplenium biondianum Engl. Shanxi, China HZ2017050107362 HSN OK336542 OP154015
Chrysosplenium carnosum Hook.f. et Thoms. Sichuan, China HSN013113 HSN OK336564 OP154016
Chrysosplenium davidianum Decne. ex Maxim. Sichuan, China HSN06442 HSN OK336537 OP154017
Chrysosplenium delavayi Franch. Sangzhi, Hunan, China SZ2016080907105 HSN OK336539 OP154018
Chrysosplenium dubium J. Gayex DC. Georgia P03_WF11 (Folk et al. 2019) – OP154019
Chrysosplenium echinus Maxim. Nikkou-shi, Japan HSN09817 HSN OK336557 OP154020
Chrysosplenium fauriae Franch. Nikkou-shi, Japan HSN09823 HSN OK336561 OP154021
Chrysosplenium flagelliferum Fr. Schmidt. Nikkou-shi, Japan HSN09816 HSN OK336541 OP154022
Chrysosplenium forrestii Diels Nikkou-shi, Japan HSN7797 HSN OK336565 OP154024
Chrysosplenium giraldianum Engl. Sichuan, China JZ2018042507981 HSN OK336548 OP154025
Chrysosplenium glossophyllum H. Hara Sichuan, China QCS2017102608035 HSN OK336544 OP154026
Chrysosplenium grayanum Maxim. Nikkou-shi, Japan HSN09810 HSN OK336555 OP154027
Chrysosplenium griffithii Hook.f. et Thoms. Shanxi, China HSN7760 HSN OK336547 OP154028
Chrysosplenium guangxiense H.G.Ye & Gui C.Zhang Guangxi, China HSN13356 HSN OP093635 * OR941245 *
Chrysosplenium henryi Franch. Sangzhi, Hunan, China HSN7505 HSN OK336532 OP154030
Chrysosplenium hydrocotylifolium H. Lév. & Vaniot Hubei, China HSN09188 HSN OK336540 OP154031
Chrysosplenium japonicum (Maxim.) Makino Zhejiang, China HSN7909 HSN OK336554 OP154032
Chrysosplenium kamtschaticum Fisch. ex Seringe Shimane-ken, Japan DG2019032310004 HSN MT371065 OP154033
Chrysosplenium kiotense Ohwi. Nikkou-shi, Japan HSN09818 HSN OK336558 OP154034
Chrysosplenium lanuginosum Hook.f. et Thoms. Anhui, China BD2017030507343 HSN OK336534 OP154035
Chrysosplenium lectus-cochleae Kitagawa Jilin, China HSN7379 HSN OK336550 OP154036
Chrysosplenium macrophyllum Oliv. Hubei, China BD2017030507344 HSN MK973001 OP154037
Chrysosplenium macrospermum Y.I.Kim & Y.D.Kim Jilin, China CBS2016062406656 HSN OK336562 OP154038
Chrysosplenium macrostemon Maxim. ex Franch. et Sav. Nikkou-shi, Japan HSN09820 HSN OK336560 OP154039
Chrysosplenium microspermum Franch. Jinfo Mountain, Chongqing, China HSNTG025 HSN OK336546 OP154040
Chrysosplenium nepalense D.Don Tengchong, Yunnan, China GLGH20170607375 HSN OK336535 OP154043
Chrysosplenium nudicaule Bunge Gansu, China HSN07772 HSN MZ424445 OP154044
Chrysosplenium oppositifolium L. Wales, UK BGN_RN_W (Folk et al. 2019) OR397749 OP154057
Chrysosplenium pilosum Maxim. Nikkou-shi, Japan HSN09819 HSN OK336559 OP154045
Chrysosplenium valdepilosum (Ohwi) S.H.Kang & J.W.Han Jilin, China HSN09819 HSN OR397753 OP154046
Chrysosplenium qinlingense Z.P.Jien ex J.T.Pan Sichuan, China HSN7980 HSN OK336549 OP154047
Chrysosplenium ramosum Maxim. Jilin, China SJH2017052107372 HSN MK973002 OP154048
Chrysosplenium sedakowii Turcz. Irkutsk, Russia P02_WC8 (Folk et al. 2019) – OP154049
Chrysosplenium serreanum Hand.-Mazz. Jilin, China SJH2017052107371 HSN OK336538 OP154050
Chrysosplenium sinicum Maxim. Hunan, China TPS2017042407504 HSN MT362051 OP154051
Chrysosplenium taibaishanense J.T.Pan Shanxi, China HSN7761 HSN OK336552 OP154052
Chrysosplenium uniflorum Maxim. Tibet, China HSN7380 HSN OK336533 OP154053
Chrysosplenium zhouzhiense Hong Liu Shanxi, China HSN13356 HSN OK336551 OP154055
Chrysosplenium alternifolium L. Shimane-ken, Japan DG2019032310003 HSN OK336545 OP154010
Chrysosplenium tetrandrum (N. Lund) Th. Fries Nunavut, Canada Brysting_01-065_CAN CAN OR397750 OP154052
Chrysosplenium wrightii Franch. & Sav. Yukon, Canada Bennett_08-125_CAN CAN OR397751 OP154059
Chrysosplenium valdivicum Hook. Chile P04_WG8 HSN OR397752 OP154060
Chrysosplenium zhangjiajieense X.L.Yu, Hui Zhou & D.S.Zhou Hunan, China ZJ2016031506369 HSN OK336563 OP154054
Peltoboykinia tellimoides (Maxim.) Hara Henan, China PT210814 (Yang et al. 2022) MZ779205 JQ895246
Saxifraga stolonifera Curt. Hubei, China S313 (Chen et al. 2022) NC_037882 MK092506
Itea chinensis C.K.Schneider Hunan, China S371 – NC_037884 MG730867

The chloroplast protein-coding genes (cpPCGs) were extracted from the CP genome using PhyloSuite v.1.2.3 (Zhang et al. 2020). These cpPCGs and nrDNA sequences were aligned by MAFFT v. 7.4 (Katoh and Standley 2013), and concatenated using PhyloSuite v.1.2.3 (Zhang et al. 2020) to form the cpPCGs+nrDNA matrix. The phylogenetic analyses of Chrysosplenium based on cpPCGs, nrDNA and cpPCGs+nrDNA matrices were performed using maximum likelihood (ML) and Bayesian inference (BI), respectively. The ML analyses were conducted using IQ-TREE v. 2.1.2 (Nguyen et al. 2015) with 1,000 bootstrap replicates and the default ModelFinder (Kalyaanamoorthy et al. 2017) to find GTR+F+I+G4 as the best-fit substitution model. Tree visualization was achieved in Figtree v. 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). For BI analysis, MrBayes v. 3.2.6 (Ronquist et al. 2012) was employed to obtain a maximum clade credibility (MCC) tree. BI analysis was performed using one million generations, two runs, 25% trees discarded as burn-in, and trees sampled every 1,000 generations (1,000 trees sampled in total) with the GTR model.

Results

Characteristics of the complete chloroplast genome

The CP genome of Chrysosplenium guangxiense comprised 154,004 bp (Fig. 1). The characteristics and statistics of the CP genome are summarized in Tables 4, 5.

Figure 1. 

Plastid genome map of Chrysosplenium guangxiense. The thick lines on the outer complete circle identify the inverted repeat regions (IRa and IRb). The innermost track of the plastome shows the GC content. Genes on the outside and inside of the map are transcribed in clockwise and counter directions, respectively.

Molecular phylogenetic studies

The cpPCGs matrix length was 71,919 bp, including 6,392 parsimony informative sites, 13,645 variable sites, and 55,865 conserved sites. The nrDNA matrix was 6,738 bp in length, with 765 parsimony informative sites, 1,200 variable sites, and 5,231 conserved sites. The cpPCGs+nrDNA matrix was 78,657 bp in length, with 7,157 parsimony informative sites, 14,845 variable sites, and 61,096 conserved sites. The phylogenetic tree of the cpPCGs matrix exhibited high confidence, while the phylogenetic tree of the nrDNA matrix had some branches with low support, and was significantly different from the former (Suppl. materials 1, 2). However, Chrysosplenium guangxiense was consistently related to C. hydrocotylifolium H.Lév. & Vaniot in both chloroplast and nuclear gene trees (Suppl. materials 1, 2). The phylogenetic tree of the cpPCGs+nrDNA matrix received a higher confidence value compared to trees generated from subsets (cpPCGs and nrDNA). Topologies obtained from BI and ML methods were congruent and showed that Chrysosplenium species clustered in a strongly supported clade (BS = 100%, PP = 1) which was further divided into three well-supported clades (defined as A-C clades; Fig. 2). Chrysosplenium guangxiense was recognized as a member of clade B and fell in its basal clade (BS = 100%, PP = 1; Fig. 2), which also included C. macrophyllum Oliv., C. zhangjiajieense X.L.Yu, Hui Zhou & D.S.Zhou, C. hydrocotylifolium, C. flagelliferum F.Schmidt, and C. zhouzhiense Hong Liu. Of these, C. guangxiense was most closely related to C. hydrocotylifolium (BS = 100%, PP = 1; Fig. 2). Although C. glossophyllum was also a member of clade B, it fell into a much more distant clade from C. guangxiense (Fig. 2).

Figure 2. 

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

Morphological observations

A suite of morphological characters including rhizome size, leaf shape, leaf margin dentate, sepal apex shape, and seed size of Chrysosplenium guangxiense and C. glossophyllum was consulted or observed. Chrysosplenium guangxiense had a robust rhizome, basal leaves with a long cuneate base and fewer teeth in the margin, curled sepal margins, and red, larger seeds that make it easily distinguished from C. glossophyllum (Table 2). Considering the phylogenetic results, a morphological comparison between C. guangxiense and C. hydrocotylifolium was also conducted. Chrysosplenium guangxiense had long elliptic leaves and a long cuneate leaf base, which can be easily distinguished from C. hydrocotylifolium (Table 3).

Table 2.
Download as
CSV
XLSX

Diagnostic comparison of Chrysosplenium guangxiense and C. glossophyllum.

Characters C. guangxiense C. glossophyllum
Rhizome Rhizome thick, crossed and nodular absent
Basal leaves base long cuneate, margin 10–20-crenate base rounded to subcordate; margin 20–36-crenate
Sepals margin curl margin uncurl
Seed red, 0.59–0.85 × 0.48–0.63 mm black, 0.50 × 0.40 mm
Table 3.
Download as
CSV
XLSX

Diagnostic comparison of Chrysosplenium guangxiense and C. hydrocotylifolium.

Characters C. guangxiense C. hydrocotylifolium
Basal leaves and cauline leaves Isophyllous heterophyllous
Basal leaves long elliptic, margin 14–24-crenate; base long cuneate orbicular, margin 34–39-crenate; base reniform
Table 4.
Download as
CSV
XLSX

Summary of the complete plastid genome of Chrysosplenium guangxiense.

Characteristic Chrysosplenium guangxiense
Size (base pair, bp) 154,004
LSC length (bp) 80,646
SSC length (bp) 17,118
IR length (bp) 28,120
Number of genes 111
Protein-coding genes 78
rRNA genes 4
tRNA genes 29
GC content 37.51%
Table 5.
Download as
CSV
XLSX

The gene statistics of the plastid genome of Chrysosplenium guangxiense. [Genes with one or two introns are indicated by one (*) or two asterisks (**), respectively. Genes in the IR regions are followed by the (×2) symbol].

Group of Genes Gene Name Number
tRNA genes trnK-UUU, trnQ-UUG, trnS-GCU, trnG-GCC, trnR-UCU, trnC-GCA, trnD-GUC, trnY-GUA, trnE-UUC, trnT-GGU, trnS-UGA, trnS-CGA, trnfM-CAU, trnS-GGA, trnT-UGU, trnL-UAA*, trnF-GAA, trnV-UAC*, trnM-CAU, trnW-CCA, trnP-UGG, trnR-ACG(×2), trnN-GUU(×2), trnI-GAU*(×2), trnA-UGC*(×2), trnL-UAG, trnI-CAU(×2), trnL-CAA(×2), trnV-GAC(×2) 29
rRNA genes rrn16 (×2), rrn23 (×2), rrn4.5 (×2), rrn5 (×2) 4
Ribosomal small subunit rpsl6, rps2, rps14, rps4, rps18, rps12 (×2), rps11, rps8, rps3 (×2), rps19 (×2), rps7 (×2), rps15 12
Ribosomal Large subunit rpl33, rpl20, rpl36, rpl14, rpl16, rpl22 (×2), rpl2 (×2), rpl23 (×2) 8
DNA-dependent RNA polymerase rpoC2, rpoC1*, rpoB, rpoA 4
Photosystem Ⅰ psaB, psaA, psaI, psaJ, psaC 5
Large subunit of rubisco rbcL 1
Photosystem Ⅱ psbA, psbK, psbI, psbM, psbD, psbC, psbZ, psbJ, psbL, psbF, psbE, psbB, psbT, psbN, psbH 15
NADH dehydrogenase ndhJ, ndhK, ndhC, ndhB*(×2), ndhF, ndhD, ndhE, ndhG, ndhI, ndhA*, ndhH 11
Cytochrome b/f complex petN, petA, petL, petG, petB, petD 6
ATP synthase atpA, atpF*, atpH, atpI, atpE, atpB 6
Maturase matK 1
Subunit of acetyl-CoA carboxylase accD* 1
Envelope membrane protein cemA 1
Protease clpP** 1
Translational initiation factor infA 1
c-type cytochrome synthesis ccsA 1
Conserved open reading frames(ycf) ycf3**, ycf4, ycf2 (×2), ycf1 (×2) 4

Discussion

Our phylogenetic result supported the monophyly of Chrysosplenium (Soltis et al. 2001; Fu et al. 2021; Yang et al. 2023). Besides two well-defined clades (denoted as sect. Oppositifolia and sect. Alternifolia), our result also revealed a third clade comprising two species from sect. Alternifolia, the topology of which is consistent with the previous study (Yang et al. 2023). This phylogenetic relationship indicated a non-monophyletic status of sect. Alternifolia and suggested that a deeper morphological character evolution across this phylogenetic framework is needed to evaluate the phylogenetic informativeness of characters.

In our phylogenetic tree, Chrysosplenium guangxiense was recovered as a member of sect. Alternifolia, most closely related to Chrysosplenium hydrocotylifolium (BS = 100%, PP = 1) but had a distant relationship with C. glossophyllum. It was easy to distinguish C. guangxiense from C. hydrocotylifolium by the long elliptic leaves and long cuneate leaf bases (Table 3). Our morphological comparison between C. guangxiense and C. glossophyllum also showed a suite of characters, including having a robust rhizome, basal leaves with a long cuneate base and fewer teeth in margin, and larger seeds in C. guangxiense which helped distinguish it from C. glossophyllum (Table 2). Furthermore, there was a typical viviparous phenomenon of C. guangxiense; the mature seeds were able to germinate directly in the opening capsule (Figs 3E, 4G, H). This feature has not been reported in any other Chrysosplenium species so far. Therefore, our molecular and morphological evidence supports C. guangxiense as a distinct species that resurrected from C. glossophyllum. We presented the following detailed taxonomic treatment for C. guangxiense.

Figure 3. 

Illustration of Chrysosplenium guangxiense H.G.Ye & Gui C.Zhang A habit in flowering phase B flower C indehiscent capsule D dehiscent capsule and seeds E germinated seeds in capsule F seeds G caulline leaf H, I bracteal leaf.3

Figure 4. 

Plate of Chrysosplenium guangxiense H.G.Ye & Gui C.Zhang A habit B, C inflorescence with flowers D, E basal leaves F fruit and seed G, H germinated seeds and seedlings in capsule I, J SEM of seed (Photos by Hong Liu).

Taxonomic treatment

Chrysosplenium guangxiense H.G.Ye & Gui C.Zhang in Acta Bot. Austro Sin. 9: 57, f. 1 (1994)

Figs 3, 4

Type

Tian Lin, 11 Oct. 1989, South China Exped. 2458 (holotype: IBSC!; isotype: IBSC!).

Description

Perennial herbs, 5.5–17 cm high. Root fibrous and robust. Long creeping rhizome developed, thick, crossed and nodular, 1–2 cm between each node, without stolons and bulbs. Flowering stem(s) always 1, erect, branched, 10–17 cm high, sparsely pilose, green, squared. Sterile branches arise from all basal leaves. Isophyllous, Basal leaves 3–6, alternate and clustered; petiole 1–6.5 cm long, white pilose; leaf blade long elliptic, 2.2–10.3 × 1.8–3.3 cm, abaxially glabrous, light green, adaxially with sparse long hispid, dark green, apex rounded, margin 14–25-crenate, base long cuneate. Cauline leaves always 1, petiole 1.3–2.2 cm long; blade 2.2–4.0 × 1.2–1.9 cm, long elliptic, glabrous in the abaxial side and with sparse hispid in the adaxial side; apex obtuse; margin obtusely dentate (9–13 teeth); base broadly cuneate; veins obvious in adaxial. Pleiochasium 9–16 cm wide, 10–15 cm high, extremely diffused, with 5–20-flowered cyme, branches sparsely hispid, surrounded by bracts; bracteal leaves green, elliptic to broadly ovate or round, glabrous. Flowers tetramerous, actinomorphic; sepals 4 (2 pairs), flat, green, 0.9–1.2 × 2.1–4.3 mm, broadly ovate, apex acute, with margin curls outward in fruiting time; disk obvious; stamens 8, ca. 0.2 mm long, shorter than sepals; anthers orange, 2-locular, longitudinally dehiscent; ovary 2-locular, semi-inferior; stigma 2; styles erect, ca. 0.2 mm long. Fruit a capsule, 5–7 mm long, green, smooth, 2-lobed (horn-shaped), equal, dehiscent along the adaxial suture; seeds numerous, red or reddish brown, obovoid, a raphe on one side, 594.19–855.33 × 475.41–625.7 μm, long papillose. Viviparous.

Additional specimen examined

Chrysosplenium glossophyllum. China. Sichuan: Kuan County (Dujiangyan City), 19 April 1930, F. T. Wang 20553 (PE!, NAS!); same locality, 6 May 1987, Xintang Ma & Zhilong Zhao 87-0521 (WCSBG!); same locality, 15 April 2013, LiXJ 353 (KUN!); same locality, 24 May 2016, Hong Liu, HSN06644 (HSN!); same locality, 26 October 2017, Hong Liu, HSN08105 (HSN!). Chrysosplenium guangxiense. China. Guangxi: Lingyun County, Baise City, 6 March 2014, Lingyun team 451027140305005 (GXMG!); Tianlin County, Baise City, 27 November 2019, Hong Liu HSN13356 (HSN!).

Conservation status

Chrysosplenium guangxiense is only known from two localities (IUCN criterion D2). At these two localities, the populations included ca. 200 mature individuals (IUCN criterion D1) growing in several patches. Using the IUCN methodology, C. guangxiense is classified as Vulnerable (VU) based on criteria D1 and D2: population size and the number of locations, combined with a plausible future threat that could drive this taxon to Critically Endangered or Extinct in a very short time. However, the vivipary of C. guangxiense may strengthen its adaptability to cope with future climate and environmental changes. The future threat is mainly due to grazing.

Conclusions

The phylogenetic analyses using plastomes and nuclear gene sequences of Chrysosplenium guangxiense reveal that C. guangxiense belongs to the sect. Alternifolia, is closely related to Chrysosplenium hydrocotylifolium, but distant from C. glossophyllum based on leaf morphology and other traits. Our findings support the resurrection of C. guangxiense as a distinct species and provide a detailed taxonomic treatment for its identification. The phylogenetic analyses confirm the monophyly of Chrysosplenium and reveal a non-monophyletic status of sect. Alternifolia. Further systematic studies of Chrysosplenium should focus on finding additional morphological characters with phylogenetic informativeness to disentangle the non-monophyletic sect. Alternifolia, and propose a new infrageneric classification and provide a stable framework for answering broader questions in evolutionary biology.

Acknowledgements

We want to thank Stephen Maciejewski, the Gesneriad Society, and Michael LoFurno, Associate Professor, Temple University, Philadelphia, USA, for their editorial assistance.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by the National Natural Science Foundation of China (No. 32170207), the Fund for Scientific Research Platforms of South-Central Minzu University (No. PTZ24019 & PTZ24018), the Fund of Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain, and the Basic Research Fund of Guangxi Academy of Sciences (CQZ-C-1901).

Author contributions

Data curation: TGY. Funding acquisition: LH, LFF. Investigation: HL, DQL, XTC. Project administration: HL, LFF. Writing – original draft: LFF, TGY, HL. Writing – review and editing: LFF, TGY, HL.

Author ORCIDs

Long-Fei Fu https://orcid.org/0000-0001-8708-4718

Tian-Ge Yang https://orcid.org/0000-0003-1681-1767

De-Qing Lan https://orcid.org/0000-0003-3277-357X

Xi-Tang Chen https://orcid.org/0000-0002-5461-7183

Hong Liu https://orcid.org/0000-0001-7227-2476

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

Reference

  • Chen Z, Yu X, Yang Y, Wei P, Zhang W, Li X, Liu C, Zhao S, Li X, Liu X (2022) Comparative analysis of chloroplast genomes within Saxifraga (Saxifragaceae) takes insights into their genomic evolution and adaption to the high-elevation environment. Genes 13(9): 1673. https://doi.org/10.3390/genes13091673
  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissues. Phytochemical Bulletin 19: 11–15.
  • Folk RA, Stubbs RL, Mort ME, Cellinese N, Allen JM, Soltis PS, Soltis DE, Guralnick RP (2019) Rates of niche and phenotype evolution lag behind diversification in a temperate radiation. Proceedings of the National Academy of Sciences of the United States of America 116(22): 10874–10882. https://doi.org/10.1073/pnas.1817999116
  • Hara H (1957) Synopsis of genus Chrysosplenium L. Journal of the Faculty of Science, University of Tokyo, Section III. Botany 7: 1–90.
  • IUCN (2019) Guidelines for using the IUCN red list categories and criteria. Version 14. Prepared by the Standards and Petitions Committee.
  • Jin JJ, Yu WB, Yang JB, Song Y, de-Pamphilis CW, Yi TS, Li DZ (2020) GetOrganelle: A fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biology 21(1): 241. https://doi.org/10.1186/s13059-020-02154-5
  • Kalyaanamoorthy S, Minh BQ, Wong TK, von-Haeseler A, Jermiin LS (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14(6): 587–589. https://doi.org/10.1038/nmeth.4285
  • Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30(4): 772–780. https://doi.org/10.1093/molbev/mst010
  • Kim YI, Lee JH, Kim YD (2018) The complete chloroplast genome of a Korean endemic plant Chrysosplenium aureobracteatum Y.I. Kim & Y.D. Kim (Saxifragaceae). Mitochondrial DNA, Part B, Resources 3(1): 380–381. https://doi.org/10.1080/23802359.2018.1450668
  • Nguyen LT, Schmidt HA, von-Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268–274. https://doi.org/10.1093/molbev/msu300
  • Pan JT (1986a) A study on the genus Chrysosplenium L. from China. Zhiwu Fenlei Xuebao 24(2): 81–97.
  • Pan JT (1986b) A study of the genus Chrysosplenium L. from China (sequel). A study of the genus Chrysosplenium L. from China (Cont.). Zhiwu Fenlei Xuebao 24(3): 203–214.
  • Pan JT (1992) Chrysosplenium L. In: Pan JT (Ed.) Flora of Reipublicae Popularis Sinicae 34(2). Science Press, Beijing, 234–279.
  • Pan JT, Ohba H (2001) Chrysosplenium. In: Wu ZY, Raven PH (Eds) Flora of China, Vol. 8. Science Press, Beijing, and Missouri Botanical Garden Press, St. Louis, 346–358.
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029
  • Shi L, Chen H, Jiang M, Wang L, Wu X, Huang L, Liu C (2019) CPGAVAS2, an integrated plastome sequence annotator and analyzer. Nucleic Acids Research 47(W1): W65–W73. https://doi.org/10.1093/nar/gkz345
  • Soltis DE, Tago-Nakazawa M, Xiang Q, Kawano S, Murata J, Wakabayashi M, Hibsch-Jetter C (2001) Phylogenetic relationships and evolution in Chrysosplenium (Saxifragaceae) based on matK sequence data. American Journal of Botany 88(5): 883–893. https://doi.org/10.2307/2657040
  • Wei YG (2018) The distribution and conservation status of native plants in Guangxi, China. China Forestry Publishing House, Beijing, 876 pp.
  • Wei YG, Do VT, Wen F (2022) A checklist to the plants of northern Vietnam. China Forestry Publishing House, Beijing, 451 pp.
  • Yang R, Li R, Dong M, Liu L (2022) Characterization of the complete chloroplast genome of Peltoboykinia tellimoides (Saxifragaceae). Mitochondrial DNA, Part B, Resources 7(5): 741–743. https://doi.org/10.1080/23802359.2022.2070040
  • Yang T, Wu Z, Tie J, Qin R, Wang J, Liu H (2023) A comprehensive analysis of chloroplast genome provides new insights into the evolution of the genus Chrysosplenium. International Journal of Molecular Sciences 24(19): 14735. https://doi.org/10.3390/ijms241914735
  • Ye H, Zhang G (1994) A new species of Chrysosplenium from Guangxi. Acta Botanica Austro Sinica 9: 57.
  • Zhang D, Gao F, Jakovli’c I, Zou H, Zhang J, Li WX, Wang GT (2020) PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20(1): 348–355. https://doi.org/10.1111/1755-0998.13096

1Tian-Ge Yang and Long-Fei Fu contributed equally to this work as first authors.

Supplementary materials

Supplementary material 1 

Phylogenetic tree of Chrysosplenium generated from maximum likelihood (ML) of cpPCGs dataset

Long-Fei Fu, Tian-Ge Yang, De-Qing Lan, Xi-Tang Chen, Hong Liu

Data type: jpg

Explanation note: Numbers on the branches indicate bootstrap values (left) of the ML analyses and the posterior probability (right) of Bayesian analyses.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (836.40 kb)
Supplementary material 2 

Phylogenetic tree of Chrysosplenium generated from maximum likelihood (ML) of nrDNA dataset

Long-Fei Fu, Tian-Ge Yang, De-Qing Lan, Xi-Tang Chen, Hong Liu

Data type: jpg

Explanation note: Numbers on the branches indicate bootstrap values (left) of the ML analyses and the posterior probability (right) of Bayesian analyses.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (858.42 kb)
login to comment