Research Article
Research Article
 Primula xinningensis (Primulaceae), a new species from karst caves in Hunan, China
expand article infoWei Zhang§, Yu Zhang, Jian Wen Shao§
‡ Anhui Normal University, Wuhu, China
§ Provincial Key Laboratory of Conservation and Utilization of Biological Resources, Wuhu, China
Open Access


Primula xinningensis 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.


Homostyly, P. cicutariifolia, P. hubeiensis, P. merrilliana complex, section Ranunculoides


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.

Materials and methods

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.

Table 1.

Basic characteristics of cp genomes of Primula xinningensis sp. nov.

Characteristic Primula xinningensis
Total length (bp) 151,601–151,630
GC% 36.8%–36.8%
LSC length (bp) 83,421–83,466
SSC length (bp) 17,583–17,599
IR length (bp) 25,292
Total genes 113
Protein-coding genes 80
rRNA genes 4
tRNA genes 29
Figure 1. 

Plastid genome map of P. xinningensis sp. nov.

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).

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.

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) (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:

Table 2.

Morphological and ecological features comparison between P. xinningensis sp. nov. and its related species.

Features P. xinningensis P. hubeiensis P. cicutariifolia P. merrilliana complex
Floral morph Homostylous Distylous Homostylous Distylous or homostylous
Umbel layers 1 1–2 1 1–3
Corolla diameter 8–12 mm 13–18 mm 6–10 mm 9–19 mm
Corolla lobes Apex conspicuously emarginate Apex conspicuously emarginate Apex conspicuously emarginate Apex rounded
Scape length 0.8–2 cm 3.5–9.6 cm 1–3 cm 1.3–9 cm
Pollens Pantoporate Pantoporate Pantoporate Pantoporate or stephanocolpate
Pollen sac Black Yellow Yellow Yellow
Cotyledon Triangular obovate Ovate Ovate Ovate
Older Leaves Pinnatisect, with 11–19 pinnae, the terminal pinna similar to others, 3-lobed or parted Pinnatisect, with 13–19 pinnae, the terminal pinna similar to others, 3-lobed or parted, margin coarsely dentate Pinnatisect, with 7–17 pinnae, the terminal pinna similar to others, 3-lobed Pinnatisect, with 11–21 pinnae, the terminal pinna similar to others, 3-lobed or parted
Glandular hairs Leaves and scape densely covered with glandular hairs (0.07–0.42 mm) Leaves and scape densely covered with glandular hairs (0.76–0.88 mm) without without
Distribution Hunan Hubei Anhui, Zhejiang Anhui, Zhejiang
Habitat Karst caves Shady damp rock crevices Stream sides or under broadleaf deciduous forests of northern slopes Stream sides or under broadleaf deciduous forests of northern slopes
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.

Key to the species of sect. Ranunculoides

1 Corolla lobe apices rounded P. merrilliana complex
Corolla lobe apices obviously emarginated 2
2 Compound leaves with 3–9 pinnae; scape apices differentiating to bulblets late in flowering P. ranunculoides
Compound leaves with 7–21 pinnae; scape apices lacking bulblets 3
3 Plants densely covered with glandular hairs 4
Plants glabrous 5
4 Cotyledon triangular obovate, pinna segments margin entire, flower homostylous, corolla diameter 8–12 mm P. xinningensis
Cotyledon ovate, pinna segments margin serrate, flower distylous, corolla diameter 13–18 mm P. hubeiensis
5 Flower homostylous, umbels solitary, limb ca. 6–10 mm across P. cicutariifolia
Flower distylous, umbels usually 2, limb ca.11–19 mm across P. jiugongshanensis

Taxonomic treatment

Primula xinningensis Wei Zhang bis & J.W.Shao, sp. nov.

Figs 3, 4, 5


China. Hunan, Xinning County, Shimen Village, 26°30'27.22"N, 110°40'56.82"E, alt. 458 m, 28 Mar 2016, Wei.Zhang & Jian.Wen.Shao ZW20160328 (holotype: ANUB!; isotypes: ANUB!, PE!) (Fig. 5).

Figure 5. 

Holotype of P. xinningensis Wei Zhang bis & J.W.Shao, sp. nov.


Cotyledon triangular obovate, leaves and scape densely covered with short glandular hairs, flowers long homostyled, corolla lobe apices conspicuously emarginate, pollen sac black.


Herb biennial, dwarf, densely covered with short glandular hairs. Leaves 9–25 in an open rosette; petiole 1.0–2.0 cm long; leaf blade pinnatisect, 4.0–8.0 cm long, 1.0–2.0 cm wide; pinnae 5–9 pairs, elliptic, margin usually pinnatifid, segments 3–5, apex mucronulate. Scapes 1–8 in each plant, 0.8–2.0 (–2.5) cm tall, carrying one umbel, usually 3 flowers per umbel; bracts linear lanceolate, 2–3 mm long. Pedicel slender, 0.3–0.8 cm. Flowers long homostylous. Calyx narrowly campanulate, 1.0–3.0 mm long, split to the middle; lobes lanceolate, apex acuminate, not outward curvature. Corolla pale red, flowers’ limb 8.0–12.0 mm across, lobes obovate, ca. 2.5 mm wide, apex conspicuously emarginate, corolla tube 5.8–7.0 mm, both stamens and stigma at the mouth of the corolla tube, pollen sac black. Capsule subglobose, 1.0–3.0 mm in diam., dehiscing by valves.


Flowering from March to April, fruiting from April to May.

Chinese name

Xīn níng yǔ yè bào chūn (新宁羽叶报春).


The specific epithet ‘xinningensis’ refers to the locality, Xinning County, Hunan, China.

Distribution and ecology

P. xinningensis is known only from Shimen Village, Xinning County, Hunan, China. Growing on the walls or ground of karst caves, at an altitude of 385–487 m. The main accompanied species were Primulina latinervis (W.T.Wang) Mich. Möller & A. Weber. (Gesneriaceae), Cyrtomium fortunei J.Sm. (Dryopteridaceae) and Pteris multifida Poir. (Pteridaceae).

Conservation status

Vulnerable (VU D1 and D2). This species is endemic to China, Hunan Province, Xinning County, Shimen Village. We only found three populations, 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 km2 and the known Area of Occupancy (AOO) is less than 0.5 km2. 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.

Additional specimen examined

China. Hunan: Xinning County, Wanfeng Forest Farm, alt. 450 m, 22 Apr 1995, Lin Bo Luo 00205565 (PE); Xinning County, Wanfeng Forest Farm, alt. 450 m, 22 Apr 1995, Lin Bo Luo 00353811 (IBK); Xinning County, Shuimiao Town, Jiangmu Village, alt. 347 m, 19 Feb 2014, Xun Lin Yu & Hui Zhou 028303 (CSFI); Xinning County, Shuimiao Town, Jiangmu Village, alt. 347 m, 19 Feb 2014, Xun Lin Yu & Hui Zhou 028304 (CSFI).


This work was supported by National Natural Science Foundation of China (No.32070370) and China’s National Basic Science and Technology Program (2019FY101810). The authors also thank the reviewers for their constructive comments to improve this manuscript.


  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19(1): 11–15.
  • Greiner S, Lehwark P, Bock R (2019) OrganellarGenomeDRAW (OGDRAW) version 1.3.1: Expanded toolkit for the graphical visualization of organellar genomes. Nucleic Acids Research 47(1): 59–64.
  • He X, Song LY, Wu YF, Liu J, Shao JW (2017) Primula jiugongshanensis sp. nov. (Primulaceae) from China, based on morphological and molecular evidence. Nordic Journal of Botany 35(3): 328–333.
  • He X, Cao JJ, Zhang W, Li YQ, Zhang C, Li XH, Shao JW (2021) Integrative taxonomy of herbaceous plants with narrow fragmented distributions: A case study on Primula merrilliana species complex. Journal of Systematics and Evolution (early view).
  • Hu CM (1990) Primula. In: Chen FH, Hu CM (Eds) Flora Reipublicae Popularis Sinicae, Chapter 59. Science Press, Beijing, 1–245.
  • Hu CM, Kelso S (1996) Primulaceae. In: Wu ZY, Raven PH (Eds) Flora of China. Science Press & Missouri Botanical Garden Press, Beijing & St. Louis.
  • Jin JJ, Yu WB, Yang JB, Song Y, Li DZ (2019) GetOrganelle: A fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biology 21(1): e241.
  • Kalyaanamoorthy S, Minh BQ, Wong T, von Haeseler A, Jermiin LS (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14(6): 587–589.
  • Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30(4): 772–780.
  • Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Tierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics (Oxford, England) 28(12): 1647–1649.
  • Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7): 1870–1874.
  • Li XW, Bao DC, Huang HD, Xie JF (2018) Primula hubeiensis (primulaceae), a new species from central china. Novon A Journal for Botanical Nomenclature 25(2): 162–165.
  • Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. 2010 Gateway Computing Environments Workshop (GCE), 1–8.
  • Richards A (2002) Primula (2nd edn.). B.T. Batsford, London.
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542.
  • Shao JW, Wu YF, Kan XZ, Liang TJ, Zhang XP (2012) Reappraisal of Primula ranunculoides, (Primulaceae), an endangered species endemic to China, based on morphological, molecular genetic and reproductive characters. Botanical Journal of the Linnean Society 169(2): 338–349.
  • Xu WB, Xia BS, Li XW (2020) The complete chloroplast genome sequences of five pinnate-leaved Primula species and phylogenetic analyses. Scientific Reports 10(1): e20782.
  • Zhang W, Hu YF, He X, Zhou W, Shao JW (2021) Evolution of autonomous selfing in marginal habitats: Spatiotemporal variation in the floral traits of the distylous Primula wannanensis. Frontiers in Plant Science 12: e781281.