﻿Sileneophioglossa (Caryophyllaceae, Sileneae), a new species from southwest China

﻿Abstract Sileneophioglossa Huan C. Wang & Feng Yang, a new species of Caryophyllaceae, is here described and illustrated based on morphological and molecular evidence. The new species was found in Sichuan and Yunnan provinces, southwest China. Phylogenetic analysis based on ITS sequences showed this new species belongs to section Cucubaloides. Morphologically, it resembles S.phoenicodonta and S.viscidula, which were also found in the southwest China, but clearly differs from the latter two species by having 5–7 mm long calyces with sparsely hirtellous and short glandular hairs, white petals, linear limbs and lobes, and absent or oblong-linear coronal scales. A distribution map and a table with morphological diagnostic characters of new species and its closest relatives are provided, as well as a preliminary conservation assessment of S.ophioglossa under the IUCN criteria.

The first comprehensive revision of the genus Silene in China was made by Tang (1996) who recognized 112 species, 2 subspecies and 17 varieties. In the most recent treatment by Zhou et al. (2001), 110 species were accepted, 67 of which are endemic to China (endemism ratio of Silene in China is about 61%). More recently, three additional species of Silene were described or recorded from southwest China by Lin et al. (2019) and Yang et al. (2022 a, b); these findings highlight the need for continued field exploration and taxonomical research in the region.
During our field surveys and the herbarium studies for a taxonomic revision of Silene in the Sino-Himalayan region, an interesting plant was repeatedly encountered, but one that does not fit with any previously described species. Comparison with related species demonstrates that this plant actually represents a distinct species hitherto not described. Therefore, it is described as a new species herein and named as Silene ophioglossa Huan C. Wang & Feng Yang.

Morphological analyses
The study followed the normal practice of plant taxonomic survey and herbarium taxonomy. Morphological studies of the new species were based on observation of living plants and specimens from Yunnan and Sichuan, southwest China. Digital images available at the JSTOR Global Plants (http://plants.jstor.org/) and at the Chinese Virtual Herbarium (http://www.cvh.ac.cn/), as well the collections housed at CDBI, KUN, PE, PYU, XTBG and YUKU were examined and compared with the new species. Pertinent taxonomic literature (e.g. Wu 1993;Zhuang 1995;Tang 1996;Zhou et al. 2001) was consulted. Morphological studies were carried out on dried material under a stereomicroscope (Olympus SZX2, Tokyo, Japan) and measurements were made using a ruler and a metric vernier caliper.

Seed micromorphology
Mature seed samples were directly adhered to carbon adhesive tape. Then they were coated with gold palladium using a BAL-TEC SCD 005 cool sputter coater (BAL-TEC AG., Liechtenstein) at Yunnan University, Kunming, China. Observations were conducted using a QUANTA 200 scanning electron microscope (SEM) (FEI Co., U. S. A.) at 20.0 KV.

Phylogenetic study
To determine the phylogenetic position of the putative new species, the internal transcribed spacer region (ITS) of the nuclear ribosomal DNA was used as the molecular marker. Total genomic DNA of this new species and S. phoenicodonta were extracted from silica-gel dried leaves using the DNA secure plant kit (TIANGEN, Beijing, China). The PCR protocols followed Lin et al. (2019). The ITS primers used were ITS1 and ITS4, as described by White et al. (1990) and Urbatsch et al. (2000). The PCR products were bidirectionally sequenced with the same primers used for PCR amplifications in an ABI 3730xL DNA Analyzer (Applied Biosystems) at Kunming Branch of Beijing Qingke Biotechnology Co., Ltd. (Yunnan, China).
We used a total of 70 taxa of Silene as ingroups, being representatives of most sections of Silene, Heliosperma oliverae Niketić et Stevan., Atocion armeria (Fedor.) Fedor. and Petrocoptis pyrenaica A. Braun as outgroups following the previous phylogenetic analyses (Jafari et al. 2020). A total of 73-taxon data sets, including six newly published sequences, were obtained. Voucher specimen and GenBank accession information for taxa are listed in Appendix 1.
All sequences were aligned with MAFFT (Katoh and Standley 2013) using 'auto' strategy and normal alignment mode. Gap sites were removed with trimAl (Capella-Gutiérrez et al. 2009) using "-automated1" command. The best-fitting substitution models SYM+I+G model for Bayesian inference were selected using ModelFinder (Kalyaanamoorthy et al. 2017) in BIC criterion. MrBayes 3.2.6 (Ronquist et al. 2012) was used to conduct Bayesian phylogenetic analyses. Runs were performed for 5 million generations with sampling of trees every 500 generations. The initial 25% of sampled data were discarded as burn-in. Diagnosis. Silene ophioglossa is morphologically similar to S. phoenicodonta (Fig. 3), but clearly differs from the latter in having 5-7 mm (vs. 6-8 mm) long calyces sparsely hirtellous and short glandular hairs (vs. densely hirtellous and with short glandular hairs), white (vs. dark violet) petals, linear (vs. obovate) limbs and linear (vs. ovate or nearly band-shaped) lobes, absent or oblong-linear (vs. orbicular-linear) coronal scales.
Molecular phylogenetics. The ITS sequence region of Silene ophioglossa comprises 687 and 821 base pairs with a GC content of 53.3% and 54.4%. The alignment of 73 ITS sequences resulted in a matrix of 582 total characters, 350 of which are constant, 58 of the variable characters are singleton sites and 174 characters are parsimonyinformative sites.
Phylogenetic analyses using ITS sequences uncovered that the new species, S. ophioglossa, belongs to a clade A representing S. sect. Cucubaloides Edgeworth & Hook. f. which was recircumscribed by Jafari et al. (2020) in their recent studies (Fig. 4). This placement is also supported by its morphological characters, such as the ascending to sprawling, multibranched stems, ovate-elliptic or obovate-elliptic leaves, lax dichasial cymes and tubular-campanulate calyces. In the phylogenetic tree (Fig. 4), two sequences from S. ophioglossa constituted a monophyletic lineage with maximum support, and it is sister to a small subclade B that includes S. phoenicodonta and S. viscidula. This close relationship is also supported by their morphological similarity.
Seed micromorphology. Seeds of Silene ophioglossa are dark brown when mature, globose-reniform in shape, 0.94-1.12 mm long, 0.79-0.94 mm wide. The lateral surface of seed is concave. The dorsal surface is flat, ca. 0.57 mm wide. Its seed coat is formed by elongate epidermis cells with S-undulate and V-undulate anticlinal walls. The periclinal walls are convex and have granulate-papillate ornamentation ( Fig. 5A-C).
Phenology. Flowering and fruiting from June to September. Distribution and habitat. Silene ophioglossa is endemic to southwest China, where it has been collected from western Sichuan and north Yunnan (Fig. 6). Currently, it seems to be restricted to the Jinsha River basin. Silene ophioglossa usually occurs at elevations  Conservation status. Silene ophioglossa is known from five localities and has been found in thickets or at forest margins. However, we actually only investigated two points, and didn't have enough information about its distribution, abundance, or threats to this species. More information is needed for assignment of its conservation status. Therefore, we choose to assign this new species to the category data deficient (DD) following the IUCN guidelines (IUCN 2012(IUCN , 2022. Taxonomic notes. Morphologically and seed micromorphologically, Silene ophioglossa is most similar to S. phoenicodonta (Fig. 3), a species also distributed in southwest China (Fig. 6). They share ascending to sprawling, multibranched stems, ovateelliptic or obovate-elliptic leaves, dichasial cymes, 3 styles and globose-reniform seeds (Fig. 5), but the new species is distinguishable from the latter by its calyces 5-7 mm (vs. 6-8 mm) long, sparsely (vs. densely) hirtellous and with short glandular hairs, petals white (vs. dark violet), limbs linear (vs. obovate), lobes linear (vs. ovate or nearly band-shaped) and coronal scales absent or oblong-linear (vs. orbicular-linear).
Additional specimens examined (Paratypes