Primula sunhangii (Primulaceae): a new species from Hubei, Central China

Abstract This report provides a description of Primula sunhangii from the Shennongjia Forestry District, Hubei Province in Central China, which is categorized as a new species of the primrose family. Primula sunhangii is morphologically similar to P. involucrata Wall. ex Duby in terms of its simple umbel, efarinose, and prolonged bracts. However, P. sunhangii is distinguished by its glabrous sepal, short petiole (compare with blade) and cylindrical calyx and capsule. Molecular phylogenetic analysis based on nuclear and cpDNA genes demonstrates that P. sunhangii and P. involucrata are closely related. Combining genetic and morphological data, the recognition of P. sunhangii as a unique new species is supported.


Introduction
The well-known horticultural genus Primula is the largest in the Primulaceae. There are approximately 500 Primula species worldwide, with the majority distributed in the North Temperate Zone and a small number of outlying species located in the mountainous regions of Africa (e.g. Ethiopia), tropical Asia (e.g. Java and Sumatra), and South America (Richards 2002). In general, Primula is distinguished from other genera by being multi-scapose with a long corolla and heterostylous flowers. There are approximately 300 native Primula species in China, for which the preferred habitat is the relatively warm and humid regions of the Himalayas and Hengduan mountainous regions, in particular the Yunnan and Sichuan provinces of Southwest China that represent Primula biodiversity hotspots (Hu and Kelso 1996).
Currently, many Primula species are poorly characterized, with a number of descriptions based on a single sample, which potentially results in undocumented details of characteristics such as pin or thrum morphology, fruiting, and ecology (Hu and Kelso 1996). Taxonomic study of Primula in China remains insufficient and requires continued efforts, as demonstrated by the large number of reports describing new species, including P. pengzhouensis C. M. Hu, G. Hao & Y. Xu. (Xu et al. 2017), P. anthemifolia G. Hao, C. M. Hu & Yuan Xu (Xu et al. 2015), and P. mianyangensis G. Hao & C. M. Hu (Wu et al. 2013) collected from Sichuan; P. hubeiensis X. W. Li (Li et al. 2017) collected from Hubei; and P. undulifolia G. Hao, C. M. Hu & Y. Xu (Xu et al. 2016) collected from Hunan.
The Shennongjia Forestry District, located in western Hubei, Central China, adjacent to eastern Sichuan, is well known for its rich biodiversity, in particular the rare and endangered golden monkey. The Shennongjia mountain range is a large geographical area (ca. 3,250 km 2 ) known as the roof of central China, which represents the second geographical step of China in a west-east direction and is characterized by the majority of its mountain peaks being over 3,000 m. The flora of this area is mainly influenced by the monsoon from the Pacific Ocean, which differs from the weather systems that affect Yunnan or western Sichuan in Southwest China. Moreover, its environment is moist from March to November, which is maintained by the local atmosphere, influenced by both the Yangtze and Hanjiang rivers that surround the Shennongjia area. (Deng et al. 2018) In 2011, during several field expeditions in Shennongjia, an unusual Primula population was discovered, comprised of plants similar to P. involucrata, belonging to Sect. Aleuritia Duby, with comparable glabrous and efarinose leaves, denticulate leaf margins, umbels, cylindrical calyx, funnel-form corolla and bracts base prolonged below into auriculate appendage. However, these individuals were clearly distinguished by their glabrous sepal, cylindrical calyx and capsule, truncate or acute leaves base, and shorter scape. We thus hypothesized this population represents a new species, which was verified by subsequent morphological and molecular phylogenetic comparisons and thus described as Primula sunhangii.

Morphological analysis
Morphological analysis was performed using recently collected specimens and those sampled in 2011 from Shennongjia. A vernier caliper and a dissecting microscope were used in the measurement. We referred to the keys to sections and species in Flora Reipublicae Popularis Sinicae (Hu 1990) and Flora of China (Hu and Kelso 1996). We also examined all species in Sect. Aleuritia. Specimens are deposited in the herbaria of Kunming Institute of Botany (KUN) and Jishou University (JIU).

Taxon sampling and outgroup selection
The phylogenetic analysis was mainly based on the recently published framework of Primula (Yan et al. 2015), with Sect. Aleuritia as the focal group. We performed sequencing of nuclear ITS as well as rbcL and matK of P. sunhangii, and downloaded 84 sequences of 28 species from GenBank. Omphalogramma delavayi (Franch.) Franch. was selected as the outgroup. All GenBank accession numbers of studied taxa are listed in Suppl. material 1.

DNA extraction and sequencing
Molecular materials were collected from Shennongjia in 2011. A Tiangen DNA extraction kit was used. PCR was performed according to standard protocols (Kusukawa et al. 1990). The primers used were those described by Yan et al. (2015) ( Table 2). DNA sequencing was performed by Tsingke Biological Technology in Kunming.

Phylogenetic Analysis
The raw sequences were manually edited and aligned using BioEdit 7.0.4.1 (Hall 2001). In the ITS, rbcL, matK and combined (including all three markers above) phylogenetic analysis, maximum likelihood (ML) analysis was conducted using RAxML 8.2.10 (Stamatakis 2014) and run for 1,000 bootstrap iterations under a GTRGAMMA model. Bayesian inference (BI) analysis was carried out using Mr-Bayes 3.2.6 (Ronquist et al. 2012) and run for 2,000,0000 generations under a GTR + I + G substitution model, which was selected using jModelTest2 2.1.6 (Darriba et al. 2012). Diagnosis. Primula sunhangii is similar to P. involucrata in glabrous, efarinose, ovate leaves, and lanceolate bracts base prolonged below into auriculate appendage and its length. But it differs from P. involucrata in the following characters: glabrous sepal, cylindrical calyx and capsule, truncate or acute leaves base, and shorter scape (at most 3 times the leaves).
Pin flowers: corolla tube ca. 8.5 mm; stamens ca. 3.5 mm above base of corolla tube; the style is not or slightly exerted.
Thrum flowers: corolla tube ca. 1 cm; stamens toward apex of corolla tube; the style is slightly shorter than calyx.
Conservation status and IUCN preliminary assessment. The new species has only two known populations described thus far. The populations are both in subareas of Shennongjia, one in Shennongding and another in Laojunshan. Each population has ca. 40 individuals. The habitats are situated in a tourist attraction zone, with an average of 20,000 tourists visiting daily. According to IUCN red list categories and criteria, conservation status of this species should be Critically Endangered (CR) (B2abiii).
Relationship with related species. Based on its glabrous, efarinose, ovate leaves, lanceolate bracts base prolonged below into auriculate appendage, and its length, P. sunhangii is most morphologically similar to P. involucrata (Hu 1990;Hu and Kelso 1996). But P. sunhangii differs from P. involucrata in glabrous sepal, cylindrical calyx and capsule, truncate or acute leaves base, and shorter scape (at most 3 times the leaves). (Table 1).
We performed phylogenetic analyses using nuclear ITS as well as rbcL and matK of P. sunhangii and related species. Separate analyses for all three markers get six trees (ML and Bayesian) with similar structures. Combined molecular phylogenetic analyses shows that P. sunhangii and P. involucrata are sister taxa, with high support (Fig. 4). They have 19 different sites in the 2020 bp nucleotide sequence.
These genetic results and morphological data clearly support that Primula sunhangii be recognized as a distinct new species.