Silene sunhangii (Caryophyllaceae), a new species from China

Abstract Silene sunhangii, a new species of Caryophyllaceae known from only three populations in Hubei and Hunan provinces of central China, is described. Both morphological and molecular data were used to assess the taxonomic status and relationships of this species. Morphologically, S. sunhangii is most similar to S. platyphylla Franch. from which it differs most readily in having 3-veined elliptical leaves without pubescence, tasseled catacorolla, pale purple to red petals without a linear lobe or narrow tooth and lanceolate, bifid to one third. A phylogenetic analysis based on nuclear ITS region identified the new species as a well-supported, independent lineage. Our new species is nested within a grade that encompasses species representing a polyphyletic Silene sect. Physolychnis (Benth.) Bocquet. Both the genetic and morphological data support the recognition of Silene sunhangii as a distinct species, although there is inconsistency between these two datasets as to the relationships of the new species.


Introduction
Silene L. (Sileneae, Caryophyllaceae) is the largest genus of Caryophyllaceae Juss., containing over 700 species (Melzheimer 1988;Rautenberg et al. 2010;Oxelman et al. 2011). It is distributed mainly in the Northern Hemisphere, but some species also occur in Africa and South America . Morphologically, Silene is characterized by having a synsepalous calyx, 3-5 carpels and a campanulate, clavate or ovate calyx tube. de Candolle (1824) had recognized just eight sections, using several morphological features, including those of habit, inflorescences and stems. Using life form as the primary character, Boissier (1867) recognized 31 sections for the genus, 11 containing annual species and 20 containing perennial species. Previously, the sectional classification within Silene was subsequently revised by Chowdhuri (1957) who recognized 44 sections, and it is this scheme that remains in place today. That study was based on a comprehensive sampling of species and a re-assessment of morphological characters. Uncertainties exist as to the number of subgenera that should be recognized for the genus. Rohrbach (1868) recognized two subgenera (subg. Silene and subg. Behenantha (Otth) Endl., based on seed characters) while Williams (1896) recognized three subgenera (subg. Gastrosilene Williams, subg. Conosilene Williams and subg. Eusilene Williams, based on calyx characters). Recent molecular studies (Oxelman et al. 1997;Petri and Oxelman 2011) support the subdivision of Silene into two major clades which correspond to subg. Silene and subg. Behenantha. Notwithstanding the above, deficiencies still exist within current classifications involving the genus and a comprehensive phylogenetic study is needed, especially as there is a suggestion in the results of both Oxelman et al. (1997) and Petri and Oxelman (2011) that Silene may be polyphyletic.
The treatment of Silene by Zhou et al. (2001) in the Flora of China recognized 110 species, of which 67 are endemic and geographically restricted within the country. Within China, species of Silene are widely distributed and show a large range of morphological variation. Historically, these species have been accommodated in 22 sections that were defined mostly by characters of the stems, petals, calyx and seeds (Zhou et al. 2001).
Field investigations conducted during this study revealed the existence of a distinctive entity of Silene in Hubei and Hunan provinces. Morphologically, this entity is most similar to S. platyphylla Franch. which occurs in Yunnan, but it differs significantly from that species in the characters of its root, leaves, petals, catacorolla and lobes. These morphological differences are supported by molecular evidence that justify the recognition of the Hubei and Hunan entity as a new species of Silene for China. It is therefore described below as Silene sunhangii.

Morphology
Natural populations of the new species were collected from three populations in Hubei and Hunan province ( Fig. 1, these data were submitted to PANGAEA, accession number 10.1594/PANGAEA.906581). Morphological characters recorded for the new species were based on fresh flowering and fruiting material collected from those populations. S. platyphylla were from herbarium material (KUN). A comparison of the new species with similar species is provided in Table 1.

Molecular analyses
Fresh leaves of the new species were dried in silica gel and total genomic DNA was extracted from 10-20 mg dried leaf tissue. Molecular material of S. platyphylla was collected from herbarium specimens (Appendix 1). The nuclear ITS locus was used for phylogeny. The PCR protocol used the following conditions: 5 min at 94, followed by 35 cycles of 1 min at 94 °C, 1 min at 53 °C, 2 min at 72 °C and then ending with a final extension of 5 min at 72 °C. The ITS primers used were ITS1 and ITS4, as described by White et al. (1990) and Urbatsch et al. (2000). Voucher specimen and GenBank accession information for taxa are listed in Appendix 1. DNA sequences were aligned using MAFFT software and then manually checked (Katoh et al. 2002). A total of 301-taxon data sets, including two newly published  (Huelsenbeck and Ronquist 2001;Stamatakis 2006), respectively. The best-fitting substitution models GTR for Bayesian inference were selected using ModelTest v.3.8, and branch support was computed with 1, 000 bootstrap replicates (Posada and Crandall 1998). ML analyses were conducted using the GTRGAMMA model with 1, 000 nonparametric bootstrapping replicates.
Phenology. Flowering occurs from February to April, and fruiting from April to June.
Etymology. The new species is named in honor of Chinese botanist, Prof. Hang Sun, who has made significant contributions to the flora of China.
Distribution, habitat and conservation status. Silene sunhangii is presently known from only Hubei and Hunan provinces in central China (Fig. 1). It grows in humid and evergreen or deciduous mixed forest, from 1214-2227 m (Fig. 1). A total of three natural populations have been located, each comprising less than 100 individuals distributed over an area not exceeding 100 m 2 . These populations are located within the Shennongjia National Nature Reserve (Hubei province), Houhe Nature Reserve (Hubei Province) and Huping Mountains (Hunan Province), and are therefore well-protected; there are no known threats to these populations. Further field studies are needed to more authoritatively determine the geographic range and frequency of this species. In the meantime, current evidence indicates that Silene sunhangii should be assigned the conservation status of "Data Deficient (DD)", following the IUCN Red List Criteria and Categories (IUCN 2017).
Taxonomic notes. Silene sunhangii is a perennial with densely ciliate, tuberous roots and dichasial cymose inflorescences containing many flowers. These characters indicate that the new species should be assigned to Silene sect. Cucubaloideae subsect. Silene Chowdhuri. It can be distinguished from all other species of Silene that possess lilac to red petals through its root, stem, leaf and corolla characters as described above. Morphologically, Silene sunhangii shows greatest similarities with S. platyphylla. The diagnosis above enables the two species to be reliably distinguished. Silene platyphylla is distributed in western Yunnan.

Molecular phylogenetic analysis
The results of our initial phylogenetic analysis, which included over 300 species, are not shown here but they did confirm the position of the new species within Silene. In Fig. 4, we present only those clades (38 species from that original matrix) which are relatively close to the new species. Due to the vagueness of outgroup, we constructed unrooted phylogenetic tree based on 38-taxon of Silene (Fig. 4). Clades associated with Silene platyphylla are also included because morphological criteria indicate that this species has similarities with S. sunhangii.
The aligned matrix consisted of 676 characters from 38 species, of which 165 were variable and 82 were parsimony-informative. Our results based on ITS produced trees with identical topology between BI and ML, and only the tree with bootstrap support values from ML analyses was presented (Fig. 4). According to these results, Silene sunhangii is nested within a grade that incorporates a polyphyletic Sect. Physolychnis  (Benth.) Bocquet. S. sunhangii is shown to be separated from associated taxa with very high support (BS = 97, PP = 1), and is well-removed from S. platyphylla. These results differ from those of the morphological study which placed S. sunhangii in sect. Cucubaloideae and showed it to be morphologically most similar to S. platyphylla. As al- ready noted, Sect. Physolychnis was resolved as polyphyletic. This section was shown to include the 'S. ajanensis group', an Asian clade, an American clade, and miscellaneous other species. These results are consistent with those of a previous study by Petri and Oxelman (2011). An unexplainable result was that S. platyphylla was well-separated from S. sunhangii, and included within a clade containing species of Sect. Cucubaloideae Edgew. et Hook. f.. These genetic results do clearly support the morphological data in recognizing Silene sunhangii as a distinct species. However, relationships of the new species do require further investigation.