﻿Primulawolongensis (Primulaceae), a new species of the primrose from Sichuan, China

﻿Abstract This paper describes and illustrates a new species of Primulaceae, Primulawolongensissp. nov. from Wolong National Nature Reserve in Sichuan Province, China. It is very rare and currently only known from its type locality. The new species belongs to subsection Chartacea of the section Petiolares on account of lacking bud scales at flowering, being efarinose and having distinct petiolate leaves with more or less rounded lamina. The new species can be differentiated from other members of the subsection by leaf blade margin dentate, and leaf veins which are not raised, scape shorter than or equal to pedicels, yellow flowers and location of stamens of the corolla tube at thrum flower. Molecular phylogenetic analysis based on nuclear ribosome internal transcribed spacer (nrITS) demonstrated that P.wolongensis was sister to subgen. Auriculastrum. Primulawolongensis is currently known from a single location in Wolong Town, and its conservation status is assessed as Data Deficient (DD).


Introduction
Primula L. is the most widespread genus in Primulaceae. It contains about 500 species mainly distributed throughout the moister and cooler regions of the northern hemisphere (Hu 1990;Hu and Kelso 1996;APG 2016). The modern distribution center of the genus is the great mountain chain of the East Himalayan and Hengduan Mountains (Hu 1994;Hu and Kelso 1996). More than 300 species were found in this region, accounting for 78% of the total number of species (Richards 2003). The establishment of Primula petiolaris Wall. and its allies as representing a distinct section of the genus was the work of Pax (1889). The sect. Petiolares has nearly 60 species, mainly distributed in the Himalayan range and the alpine regions of Western China (Hu 1994;Hu and Kelso 1996;Richards 2003). Its members are easily recognized by the capsule globose, included within the calyx-tube lacking valves and with an apical membrane crumbling at maturity (Hu and Kelso 1996). The sect. Petiolares was divided into 5 subsections according to bud-scales conspicuous and persisting at flowering time, length of scapes at flowering, and whether there is a honeycomb-reticulate below the blade, namely subsect.  (Smith and Fletcher 1944). In recent years, many new species have been reported in this section (Hu and Geng 2003;Li and Hu 2009;Rankin 2010;Hu and Hao 2011;Xu et al. 2014Xu et al. , 2015Xu et al. , 2016Ju et al. 2018;Yuan et al. 2018) and it's likely more undescribed taxon will be discovered.
In May 2021, an unusual population of Primula was discovered in moss-covered crevices of wet cliffs above the tree-line, in Sichuan Province. After consulting relevant literature (Smith and Fletcher 1944;Hu 1986;Hu 1990;Fang 1994Fang , 2003Hu and Kelso 1996;Wu 1999;Richards 2003) and herbarium specimens (BM, CDBI, E, FI, HNWP, IBSC, K, KUN, P, PE, US, and WU), we concluded that it is an undescribed taxon, belonging to Primula sect. Petiolares in morphology. Based on the morphological data of fresh materials and herbarium specimens, we describe this new species as follows.

Morphological analyses
Morphological description and measurements of Primula wolongensis were based on living plants and dried herbarium specimens. The taxonomic description follows the terminology used by Beentje (2012). Voucher specimens and additional silica-gel dried leaves are stored at CDBI Herbarium (herbarium follows Thiers 2021).

DNA extraction, amplification and sequencing
Except for the newly generated sequence of Primula wolongensis in this study, the sequences of the remaining 62 samples representing nine subgenera of Primula and two outgroups (Androsace integra and A. paxiana) in molecular phylogenetic analysis were retrieved from GenBank. Total DNA was extracted exclusively from silica-gel dried leaves via a Plant DNA Isolation Kit (Cat.No.DE-06111). We used the same primers as Xu et al. (2012) to amplify the nuclear ribosome internal transcribed spacer (nrITS) through polymerase chain reaction (PCR). All DNA samples were sent to TSINGKE Biotech Co. Ltd (Chengdu, China) for sequencing and then deposited to GenBank under the accession number OP901719.

Results
The molecular phylogenetic tree showed that Primula wolongensis was sister to subgen. Auriculastrum (ML = 95, Fig. 1) which included sect. Auricula Duby, sect. Cuneifolia Balf.f., sect. Dodecatheon (L.) A.R.Mast & Reveal, sect. Parryi W.W.Sm. ex Wendelbo and sect. Suffrutescens A.J.Richards. Apart from the sect. Amethystina, which occurs in the Himalayan-Transverse Mountains, species of all other sections of this subgenus are found in Europe, North America, and northern Japan through to British Columbia. The position of P. wolongensis in the genus Primula is clearly divergent between morphological and molecular evidence, and more evidence is needed to understand the evolutionary history of the species. Diagnosis. Amongst the Chinese members of subsect. Chartacea, the new species is easily recognized by the following combination of characters: leaf blade margin dentate, leaf veins which are not raised, scape which is shorter or equal with pedicel, corolla yellow and location of stamens of the corolla tube at thrum flower.
The new species is morphologically similar to P. arunachalensis Basak & Maiti and P. fenghwaiana C.M.Hu & G.Hao, but can be easily distinguished from P. arunachalensis by its shorter rootstocks, petioles 3-5× as long as leaf blade (versus 1-2× as long as leaf blade), reticulation of veins obscure on both surfaces (versus veins slightly impressed adaxially and conspicuous abaxially), corolla lobes apex emarginate (versus corolla lobes margin denticulate to lacerate), heterostylous (versus homostylous). Compared with P. fenghwaiana, the difference of the new species is the petioles 3-5× as long as the leaf blade (versus ca.2/3 the length of the leaf blade), leaf blade base cordate (versus base broadly cuneate to almost rounded), leaf blade margin irregular dentate (versus margin remotely denticulate), scapes and pedicels sparsely short-stalked glandular (versus densely covered with minute glandular hairs), corollas yellow (versus pink to white), the position of stamens at thrum flower tube on the middle (versus on the apex).
Distribution and habitat. The species has so far only been found at its type locality in Wolong Town, Wenchuan county, Sichuan Province. It grows in the cracks of steep wet cliffs covered with moss above the tree-line.
Conservation status. Data Deficient (DD). Currently, only one population with more than 100 individuals has been found in the type locality. According to the guidelines for using the IUCN Red List categories and criteria (IUCN Standards and Petitions Subcommittee 2022), the conservation status of the new species is 'Data Deficient (DD)'. Further explorations in the adjacent mountainous tracts are necessary for an adequate assessment.
Discussion. Following Hu's (1990) taxonomic treatment of this genus, this new species belongs to Primula section Petiolares on account of globose capsule included within the calyx-tube that does not open by valves but apically crumbling at membrane apex at maturity (Figs 2-4).
Further morphological analysis shows that the new species is allied with subsect. Chartacea by having glabrous and efarinose plants, at flowering time devoid of basal bud-scales, more or less rounded blades and slender petioles. Including the newly described here, there are nine species reported so far for this subsection (Smith and Fletcher 1944;Hu 1990;Basak and Maiti 2000;Rankin 2010;Hu and Hao 2011;Xu et al. 2015;Wei et al. 2022). Amongst the Chinese members of subsect. Chartacea, the new species, is morphologically most similar to P. arunachalensis and P. fenghwaiana in its bud-scales lacking at anthesis, slender petioles, rounded blades, and scape shorter than or equal to pedicels, but can be recognized by almost absent rhizomes, morphological features of leaves, inflorescences, and flowers. Further morphological comparisons among P. wolongensis, P. arunachalensis and P. fenghwaiana are shown in Table 1.