PRIMULINA WENII ( GESNERIACEAE ) , A NEW SPECIES FROM CHINA J

Primulina wenii (Gesneriaceae) is described and illustrated here as a new species. Morphologically, this new species resembles P. juliae (Hance) Mich. Möller & A. Weber, but it is distinct from the latter by some obvious features in leaf blade, bract, indumentum, corolla, filament, stigma and pistil. Nuclear ribosomal internal transcribed spacer (ITS) region and plastid trnL-F intronspacer (trnL-F) DNA sequence data from the new species and its 22 relatives are used to determine the systematic position of the new species within Primulina. Molecular evidence suggests that P. wenii is strongly supported as a sister to a clade in which P. juliae is included. The conservation status of P. wenii is assessed as "Critically Endangered" (CR) according to IUCN Red List categories and criteria. Introduction A great number of new species of Primulina have been described since 2011 at present the number has rapidly reached more than 150 species in China (Möller et al., 2016). Undoubtedly, the limestone areas of South-west and South China and North Vietnam are considered to be of the highest biodiversity and differentiation for Primulina (Gesneriaceae) (Wei et al., 2010; Wen et al., 2015). It is easily understood that very few species of Primulina in Fujian province have been recorded. Before 1992, there were two species and one variety, namely P. pinnatifida (Hand.Mazz.) Y.Z. Wang [former Chirita pinnatifida (Hand.-Mazz.) Burtt], Primulina fimbrisepala (Hand.-Mazz.) Y.Z. Wang (former Chirita fimbrisepala Hand.-Mazz.) and Chirita gueilinensis W.T. Wang var. brachycarpa W.T. Wang (Zhang, 1993). However, Chirita gueilinensis var. brachycarpa was brought into Chirita juliae Hance (Wang et al., 1998; Li and Wang, 2004) based on the similar morphological characters of the two congeners. But soon afterwards, Chirita along with several other associated genera, namely almost all the species and Chirita sect. Gibbosaccus C.B. Clarke, two species of Wentsaiboea D. Fang & D.H. Qin (W. renifolia D. Fang & D.H. Qin (2004) and W. luochengensis Yan Liu & W.B. Xu (2010)) and all the species of Chiritopsis W.T. Wang (1981) were shifted to Primulina Hance (Li and Wang, 2007; Wang et al., 2011; Weber et al., 2011). It was originally the monotypic genus (Hance, 1883). Subsequently, a new species of Primulina, viz., P. xiuningensis (X.L. Liu & X.H. Guo) Mich. Möller & A. Weber (former Chiritopsis xiuningensis X.L. Liu & X.H. Guo) (Liu and Guo, 1989; Weber et al., 2011), from Fujian, China was discovered and reported formally in recent years (Geng et al., 2014). To sum it up, Fujian province in East China cannot still be considered to have high biodiversity of Primulina because only three species were found in this area. Gesneriaceae Conservation Center of China (GCCC), Guilin Botanical Garden, Guangxi Inst. of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, CN-541006 Guilin, P.R. China. Corresponding author.Email: yanljzju@163.com


Introduction
A great number of new species of Primulina have been described since 2011 at present the number has rapidly reached more than 150 species in China (Möller et al., 2016).Undoubtedly, the limestone areas of South-west and South China and North Vietnam are considered to be of the highest biodiversity and differentiation for Primulina (Gesneriaceae) (Wei et al., 2010;Wen et al., 2015).It is easily understood that very few species of Primulina in Fujian province have been recorded.Before 1992, there were two species and one variety, namely P. pinnatifida (Hand.-Mazz.)Y.Z.Wang [former Chirita pinnatifida (Hand.-Mazz.)Burtt], Primulina fimbrisepala (Hand.-Mazz.)Y.Z.Wang (former Chirita fimbrisepala Hand.-Mazz.)and Chirita gueilinensis W.T. Wang var.brachycarpa W.T. Wang (Zhang, 1993).However, Chirita gueilinensis var.brachycarpa was brought into Chirita juliae Hance (Wang et al., 1998;Li and Wang, 2004) based on the similar morphological characters of the two congeners.But soon afterwards, Chirita along with several other associated genera, namely almost all the species and Chirita sect.Gibbosaccus C.B. Clarke, two species of Wentsaiboea D. Fang & D.H. Qin (W. renifolia D. Fang & D.H. Qin (2004) and W. luochengensis Yan Liu & W.B. Xu (2010)) and all the species of Chiritopsis W.T. Wang (1981) were shifted to Primulina Hance (Li and Wang, 2007;Wang et al., 2011;Weber et al., 2011).It was originally the monotypic genus (Hance, 1883) (Liu and Guo, 1989;Weber et al., 2011), from Fujian, China was discovered and reported formally in recent years (Geng et al., 2014).To sum it up, Fujian province in East China cannot still be considered to have high biodiversity of Primulina because only three species were found in this area.
In 2012, one of the authors found a population of the peculiar Gesneriaceae plants growing on a rocky slope near a stream in a limestone gorge close to Fuzhou city, Fujian province, China.Firstly, we considered it to be Prmulina juliae because it is recorded in Ninghua County and Songxi County of Fujian.The next year we re-visited the same location to collect specimens with flowers, and we found that the two similar congeners are actually different.We sent some specimens with flowers and fruits to IBK (Dr.Fang Wen, FW) for identification.He thought they should be an unknown species and have never been seen before.After consulting relevant literature (Wang, 1985;Wang et al., 1990Wang et al., , 1998;;Li and Wang, 2004;Wei et al., 2010;Wang et al., 2011;Weber et al., 2011;Möller et al., 2011Möller et al., , 2016;;Xu et al., 2012;Möller and Clark, 2013) and checking herbarium specimens of Primulina (Some former Chirita), 'World checklist of Gesneriaceae' (Skog and Boggan, 2007) and 'The genera of Gesneriaceae' (Weber and Skog, 2007), and consulting Dr. Fang Wen based on his rich experience and knowledge about the identification and taxonomy of Primulina, we found the morphology of these newly discovered plants to be similar to P. juliae (Hance) Mich.Mich.& A. Weber (Wei et al., 2010).However, they differ significantly in some characters.We have determined that they belong to a new species of Primulina viz.P. wenii, sp.nov.The new species Primulina wenii is described and illustrated below and compared with the morphologically similar P. juliae and their conservation status is evaluated.

Material and Methods
An overview of the genus Primulina from South, South-west and East China was made.All the available specimens of Primulina stored in the following herbaria in China (ANU, BJFC, CDBI, CTC, HGAS, HIB, IBK, IBSC, KUN, PE, SZ) were used as material of Primulina from recent fieldwork by the re-searching team of authors in South, South-west and East China (Thiers, 2015).All the morphological characters were studied under dissecting microscopes, and described using the terminology presented by Wang et al. (1990Wang et al. ( , 1998)).
Leaf material of the new species was collected in the field and dried by silica gel for DNA extraction (Chase and Hills, 1991).The nuclear ribosomal internal transcribed spacer (ITS) region and the plastid trnL-F intron spacer (trnL-F) were used as molecular markers.The molecular methods and protocols followed Möller et al. (2009Möller et al. ( , 2011)).GenBank accession numbers for ITS and trnL-F of the new species are KX985576 and KX985577, respectively (Table 1).To elucidate the phylogenetic affinities of the new species, ingroups (22 species of Primulina) and outgroups [Ornithoboea wildeana Craib, Paraboea rufescens (Franch.)B.L.Burtt] selection were chosen based on recent phylogenetic analyses (Li et al., 2007;Mölleret al., 2011;Weber et al., 2011;Kang et al., 2014), and from which sequences were available from GenBank (Table 1).
Etymology: The species epithet 'wenii' is used to commemorate a Chinese botanist, Fang Wen, who studied Gesneriaceae of China for many years, and is devoted to the conservation and taxonomy of Chinese Gesneriaceae plants.
Habitat: Primulina wenii grows on shaded moist turf of limestone cliff in subtropical limestone evergreen broad-leaf forest on a North-facing slope of a limestone hill at an altitude of about 130 meters.The climate of Fuzhou, Fujian province is the monsoon of subtropical moist marine climate zone.The average annual temperature of Fuzhou is 20.1°C, the average annual precipitation is c. 1718.1 mm.

Molecular analysis
The combined matrix had a length of 1,670 characters, 672 for ITS and 998 for trnL-F.Of the 254 (15.2%) varied, 257 (15.4%) were parsimoniously informative, including the indels.The maximum parsimony analysis on the combined matrix resulted in two trees of 937 steps in length, a consistency index (CI) of 0.7311, retention index (RI) of 0.6644 and homoplasy index (HI) of 0.2689.The strict consensus tree (Fig. 3) was highly resolved and tree topology was consistent with the previous phylogenetic study by Möller et al. (2011) and Kang et al. (2014).
DNA analyses of sequence data show that the new species is sister to a clade (BS = 100) comprising of Primulina tabacum, P. danxiaensis, P. juliae and P. xiuningensis.Of these species the undescribed species is morphologically most similar to P. juliae from which it can be distinguished by certain qualitative and quantitative characters in leaf blade, bract, indumentum, corolla, filament, stigma and pistil.For instance, it is obviously different that the peduncles of P. wenii are densely covered with erectly spreading white eglandular pubescent and villous hairs, the number of bracts is three, filaments are glabrous, and pistil is densely covered with white glandular puberulent hairs.The morphological differences between the two species (P.wenii and P. juliae) are depicted in Table 2.

Fig. 2 .
Fig. 2. Primulina juliae.A. Habit; B. Leaf blade -adaxial surface; C. Front view of corolla; D. Lateral view of corolla; E. Cyme, calyx lobes and indumentum; F. Young straight capsules.A-B and E-F: photographed in type location by J. Li; C-D: photographed in nursery of Gesneriad Conservation Center of China by F. Wen.