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).

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.

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.

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.

Figure 1. 

Plastid genome map of P. xinningensis sp. nov.

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).

Figure 2. 

Phylogenetic relationships of P. xinningensis sp. nov. and related species inferred from ML and BI analyses, based on the whole plastid genome. Numbers on the branches indicate the bootstrap support of the ML and the posterior probability of BI analyses. NCBI accession numbers were shown in the parentheses.

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) (Table 2, Figs 3, 4). Detailed morphological comparisons between the new species and other related species in sect. Ranunculoides are summarised in the following key:

Figure 3. 

Living images of P. xinningensis sp. nov. A, D habitat B, C plant in seedling E plant in flowering.

Figure 4. 

Morphological characters of P. xinningensis sp. nov. A, B longitudinally dissected of floral tube C infructescence D, F leaves morphology E opened capsule and seeds G leaf surface glandular hairs H leaf margin glandular hairs I rachis glandular hairs.