﻿Molecular and morphological evidence supports the resurrection of Chrysospleniumguangxiense H.G.Ye & Gui C.Zhang (Saxifragaceae)

﻿Abstract Chrysospleniumguangxiense 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 Chrysospleniumhydrocotylifolium 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 Chrysospleniumguangxiense 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).


Introduction
Chrysosplenium L. (Saxifragaceae) comprises more than 70 species of perennial herbs (Kim et al. 2019;Fu et al. 2020Fu et al. , 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. 2020Fu et al. , 2021;;Wei et al. 2022), along with the molecular evidence, it was confirmed that C. guangxiense is a different species from C. glossophyllum.

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 Ge-tOrganelle v. 1.7.5 (Jin et al. 2020), and annotation was performed using CPGA-VAS2 (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.

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.

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;

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).

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.

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.

Figure 1 .
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.

Figure 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.

Figure 3 .
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 .
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).

Table 1 .
Species names and GenBank accession numbers of plastid genomes and nrDNA sequence used in this study (* newly generated sequences).

Table 4 .
Summary of the complete plastid genome of Chrysosplenium guangxiense.

Table 5 .
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].