Chrysosplenium sangzhiense (Saxifragaceae), a new species from Hunan, China

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).


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.
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.

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).

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.

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.

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)
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).

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;Tanaka 1988a, b, 1989;Funamoto et al. 1997Funamoto et al. , 1999Funamoto et al. , 2000Funamoto et al. , 2004Funamoto 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.