﻿ Primulaxinningensis (Primulaceae), a new species from karst caves in Hunan, China

﻿Abstract Primulaxinningensis Wei Zhang bis & J.W.Shao, a new species from Hunan Province, China, is described. Its leaf morphology is similar to the P.merrilliana complex and flower morphology similar to P.cicutariifolia, but it can be distinguished from the former by the black pollen sac, corolla lobes apex obviously emarginate and can be differed from the latter by cotyledon triangular obovate, plants densely covered with glandular hairs and special habitat (karst caves). The whole plastid genome of this new species is 151, 601–151, 630 bp in length. Based on the whole plastid genome sequences, phylogenetic trees revealed that the new species did not genetically relate to the above two mentioned morphologically similar species, but it was closely related to P.hubeiensis. Currently, only three populations were discovered within a small distribution area, thus, it is preliminarily considered as Vulnerable (VU) according to criteria of the IUCN Red List.


Introduction
Primula L. is the largest genus in Primulaceae and comprises about 500 species worldwide. The genus is mainly distributed in temperate and alpine regions of the Northern Hemisphere, with only a few species in the Southern Hemisphere, i.e. Africa, tropical Asia and South America (Richards 2002). There are approximately 300 native Primula species in China and the modern distribution centres of this genus are located along both sides of the Himalayas to Yunnan and western Sichuan (Hu and Kelso 1996).
The sect. Ranunculoides C.M.Hu is a unique group in Primula, characterised by pinnately compound leaves and calyx not inflated at the base (Hu 1990;Hu and Kelso 1996). This section now includes P. ranunculoides F.H.Chen, P. cicutariifolia Pax, P. jiugongshanensis J.W.Shao, P. hubeiensis X.W. Li and P. merrilliana complex (Shao et al. 2012;He et al. 2017;Li et al. 2018;He et al. 2021). They are all endemic to central or eastern China and often grow at the waterside or the edge of broadleaf deciduous forests between 50 and 1600 m (Shao et al. 2012;He et al. 2017;Zhang et al. 2021).
In March 2016, during our field expeditions in Shimen Village, Xinning County, Hunan Province, China, we encountered a suspicious species of sect. Ranunculoides. The plants were restricted to growing on the walls and ground near the entrance to karst caves and are quite different from other known related species. After careful morphological observations, together with evidence from molecular phylogenetic analyses, based on the chloroplast genome, this suspicious species was confirmed as a new species. Here, the investigation results are reported, the new species is named as Primula xinningensis Wei Zhang bis & J.W.Shao and is described.

Sampling and morphological analyses
The studied specimens were collected in Shimen Village (26°30'27.22"N, 110°40'56.82"E, altitude: 468 m), Xinning County, Hunan Province, China. Voucher specimens were deposited at the Herbarium of Anhui Normal University (ANUB). The morphological description of the new species was based on examination of fresh material and herbarium specimens. A total of 10 diagnostic characteristics of the new species were identified and compared to related species in the Primula sect. Ranunculoides (Shao et al. 2012;He et al. 2017;Li et al. 2018;He et al. 2021).

Genome sequencing, assembly and annotation
Genomic DNA was extracted from dried leaves using a modified CTAB protocol (Doyle and Doyle 1987). The quality and concentration of DNA products were assessed via agarose gel electrophoresis and spectrophotometry and the qualified DNA sample was sent to BGI-Shenzhen (Shenzhen, China) for library construction and next-generation sequencing. Finally, we obtained ca. 2 Gb of high-quality clean data, the complete chloroplast genome was assembled using GetOrganelle described in Jin et al. (2019) and the annotation was conducted with Plastid Genome Annotator (Qu et al. 2019), coupled with manual correction using Geneious v. 9.1.4 (Kearse et al. 2012). The plastome of P. hubeiensis (Genbank accession number: MT268976) was used as the reference genomes for annotation. The cp genome maps were drawn using OGDRAW (Greiner et al. 2019). All sequences generated in this study were submitted to the NCBI database, the accession numbers are ON208991 (P. xinningensis), ON208990 (P. xinningensis) and ON221323 (P. hubeiensis), respectively.

Phylogenetic analyses
In order to determine the phylogenetic relationship of the new species, we downloaded 28 accessions cp genome sequences of primula from the NCBI (Fig. 2). All sequences were aligned with MAFFT v.7 (Katoh and Standley 2013) using the default settings and adjusted manually where necessary using MEGA 7.0.14 (Kumar et al. 2016). Phylogenetic analyses were conducted using Maximum Likelihood (ML) and Bayesian Inference (BI) methods with Androsace paxiana and Lysimachia congestiflora as outgroups (Xu et al. 2020). The ML analysis was conducted using RAxML-HPC BlackBox v.8.1.24 at the CIPRES Science Gateway website (Miller et al. 2010;Stamatakis 2014) with 1000 bootstrap replicates, the (GTR) + G + I model being used in ML analyses. For the BI analysis, the best substitution model was determined according to Bayesian Information Criterion (BIC) with ModelFinder (Kalyaanamoorthy et al. 2017). The BI analysis was performed using MrBayes v.3.2 (Ronquist et al. 2012). The Markov Chain Monte Carlo (MCMC) algorithm was run for 10 million generations and the trees were sampled every 1000 generations. Convergence was determined by examining the average standard deviation of the split frequencies (< 0.01). The first 25% of the trees were discarded as burn-in and the remaining trees were used to generate the consensus tree.

Characteristics of the complete plastid genome
The length of complete plastid genome of P. xinningensis comprised 151,601-151,630 bp ( Fig. 1). It possessed typical quadripartite structure: IRa, IRb, LSC and SSC; the characteristics and statistics of the plastid genome are summarised in Table 1.

Molecular phylogenetic relationship
Phylogenetic relationships of the new species and related species were constructed, based on the whole plastid genome using ML and BI analyses. The results showed that P. xinningensis affiliate to sect. Ranunculoides. In sect. Ranunculoides, Primula merrilliana complex, P. cicutariifolia and P. jiugongshanensis clustered in one clade and the other three species (P. xinningensis, P. hubeiensis and P. ranunculoides) clustered in another clade (Fig. 2). Primula xinningensis is a sister species of P. hubeiensis, and their individuals were respectively grouped into a monophyly with high support (posterior probability (PP) = 1, bootstrap support (BS) = 100%) (Fig. 2).

Morphological comparison
In morphology, this new species is very similar to P. merrilliana complex in leaf pinnae shape and degree of division and similar to P. cicutariifolia in floral characters, but can be easily distinguished from the former by the black pollen sac, corolla lobes apex obviously emarginate and can be differed from the latter by cotyledon triangular obovate, pinna margin usually pinnatipartite and plants densely covered with glandular hairs (Table 2, Figs 3,4). Although, in the phylogenetic relationship, P. xinningensis is closely related to P. hubeiensis, there were obvious morphological differences between them in pinna division pattern (segments margin entire vs. segments margin serrate), the length of glandular hair (0.07-0.42 mm vs. 0.76-0.88 mm) and flowers size and type (homostylous and corolla diameter 8-12 mm vs. distyly and corolla diameter 13-18 mm) (  (Fig. 5). Diagnosis. Cotyledon triangular obovate, leaves and scape densely covered with short glandular hairs, flowers long homostyled, corolla lobe apices conspicuously emarginate, pollen sac black.
Phenology all of them near the entrance to the karst caves and each with about 100-250 individuals. The surrounding area is cultivated field with strong human activities. The Extent of Occurrence (EOO) is less than 10 km 2 and the known Area of Occupancy (AOO) is less than 0.5 km 2 . Therefore, the conservation status of this new species is evaluated as 'Vulnerable' (VU) as it meets criterion D1 and D2, according to the IUCN Red List Categories and Criteria (IUCN 2019). In addition, P. shimemensis is a homostylous  species. Although Primula containing ca. 500 species, there are only ca. 45 species having monomorphic populations (Mast et al. 2006). Therefore, the recognition of this new species increases the homostylous species diversity in Primula and can provide valuable material for studying the evolution and maintenance mechanism of distylous flowers.