A new species of Primulaceae, Primula undulifolia, is described from the hilly area of Hunan province in south-central China. Its morphology and distributional range suggest that it is allied to P. kwangtungensis, both adapted to subtropical climate, having contiguous distribution and similar habitat, growing on shady and moist cliffs. Petioles, scapes and pedicels of them are densely covered with rusty multicellular hairs, but the new species can be easily distinguished by its smaller flowers and narrowly oblong leaves with undulate margins. Molecular phylogenetic analysis based on four DNA markers (ITS, matK, trnL-F and rps16) confirmed the new species as an independent lineage and constitutes a main clade together with P. kwangtungensis, P. kweichouensis, P. wangii and P. hunanensis of Primula sect. Carolinella.
Citation: Xu Y, Yu X-L, Hu C-M, Hao G (2016) Morphological and Molecular Phylogenetic Data Reveal a New Species of Primula (Primulaceae) from Hunan, China. PLoS ONE 11(8): e0161172. https://doi.org/10.1371/journal.pone.0161172
Editor: Zhong-Jian Liu, The National Orchid Conservation Center of China; The Orchid Conservation & Research Center of Shenzhen, CHINA
Received: January 11, 2016; Accepted: July 31, 2016; Published: August 31, 2016
Copyright: © 2016 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This study was supported by the National Natural Science Foundation of China (grant no. 31570193, 31570222, www.nsfc.gov.cn) to Gang Hao and Hai-Fei Yan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The genus Primula L. can be explicitly recognized by its perennial habitat, basal rosette of leaves, heterostylous flowers with obvious tube and capsular fruits, with rare exemptions (with homostylous flowers, which have only one flower type, with anthers and stigma at the same level) [1, 2]. The heterostyly is a distinctive feature, with species possessing short-styled thrum-type flowers with the style included in the corolla tube, and the long-styled pin-type flowers with long style and the stigma presented at the throat of the tube. Recent molecular phylogenetic studies indicated that Primula (with Dionysia Fenzl, Sredinskya (Stein) Fed., Dodecatheon L., and Cortusa L. nested) is a major monophyletic clade of Primulaceae, while several genera traditionally included within the Primulaceae (e.g. Samolus L. and Lysimachia L.) have been transferred to the Theophrastaceae or to the Myrsinaceae [3–7]. This genus consists of more than 500 species, mainly distributed in the temperate zone of the Northern Hemisphere [2, 8, 9]. The modern center of diversity of Primula is in southwestern China (particularly in western Sichuan, eastern Xizang, and northwestern Yunnan), including approximately 300 species [1, 8].
Pax  proposed a preliminary infrageneric system of Primula and 20 sections were recognized, but the demarcation was unclear. Smith et al.  divided this genus into 31 sections further, and Hu  revised Chinese species of Primula and followed this treatment. To summary the relationships among the 31 sections delimited by Smith et al. , Wendelbo  assigned them into seven subgenera (i.e. subgen. Sphondylia (Duby) Ruprecht, subgen. Auriculastrum Schott, subgen. Primula, subgen. Auganthus (Link) Wendelbo, subgen. Carolinella (Hemsl.) Wendelbo, subgen. Craibia Wendelbo, and subgen. Aleuritia (Link) Wendelbo). In the recent system of Richards , another redefined seven subgenera, subgen. Sphondylia (Duby) Ruprecht, subgen. Auriculastrum Schott, subgen. Primula, subgen. Auganthus (Duby) Wendelbo, subgen. Carolinella (Hemsl.) Wendelbo, subgen. Pinnatae Richards, and subgen. Aleuritia (Duby) Wendelbo, were delimited.
Recently, some molecular phylogenetic analyses focusing on Primula have found that most of the subgenera and sections in the traditional subgeneric classification of Primula were non-monophyletic [5, 7, 13, 14]. Some of the diagnostic characters, such as the calyptrate capsule, might have evolved more than once in Primula, even in Primulaceae [14, 15]. The subgen. Carolinella was nested within subgen. Auganthus and differentiates into four separate lineages [14, 15]. However, a formal taxonomic treatment of the genus Primula based on the latest molecular phylogenetic studies has not yet been proposed . The traditional morphological characters (including the calyptrate capsule) are still the main evidence to establish subgroups of Primula in floras and taxonomical studies .
Subgenus Carolinella, characterized by capsules opening by a lid (calyptrate), includes the sole sect. Carolinella (Hemsl.) Pax, which is a small group in Primula, with 12 species (P. henryi (Hemsl.) Pax, P. chapaensis Gagnep., P. partschiana Pax, P. rugosa N.P. Balakr., P. kweichouensis W.W. Smith, P. kwangtungensis W.W. Smith, P. wangii F.H. Chen & C.M. Hu, P. levicalyx C.M. Hu & Z.R. Xu, P. calyptrata X. Gong & R.C. Fang, P. hunanensis G. Hao, C.M. Hu & X.L. Yu, P. intanoensis T. Yamazaki, P. cardioeides W.W. Smith & H.R. Fletcher) endemic to southwestern China and adjacent Vietnam and Thailand [2, 9, 16–19].
At first, members of sect. Carolinella were described as a separate genus, named as Carolinella Hemsl., consisting of only three species, Carolinella henryi Hemsl. (≡Primula henryi (Hemsl.) Pax), Carolinella cordifolia Hemsl. (≡Primula partschiana Pax) and Carolinella obovata Hemsl. (≡Primula rugosa Balakr.) [20, 21]. This genus was distinguished from Primula by the large cordate leaves and calyptrate capsule . Pax & Knuth  considered that the calyptrate capsules of Carolinella are in fact not operculate but rather dehisce by irregular calyptra as occurring in other Primulas, and merged it into Primula as a section. Subsequent taxonomists all followed this treatment [1, 2, 9, 11]. Although the sect. Carolinella was been recognized for a long time, some members (i.e. P. kwangtungensis and P. kweichouensis) have ever been placed in sect. Obconicolisteri Balf. f. .
In the early 2014, during field expedition in southwest Hunan, China, one of the authors fund a distinct Primula species with undulant margin of leaves. At first sight, this species is similar to P. kwangtungensis, and is also easily confused with some variants of P. obconica Hance (belongs to sect. Obconicolisteri). Further morphologic investigation of both flowering and fruiting individuals turned out that it represents an undescribed taxon. Evidenced by the unique calyptrate capsule among sections in Primula, it is supposedly affiliated to the sect. Carolinella. Thus, here we not only make a morphological comparison with related species but also conduct a phylogenetic analysis to determine the systematic position of the new species.
Materials and Methods
All samples employed in this study are not endangered nor protected in the sampled area, and none of the sampled locations are privately owned or protected by any law. No specific permits are required for the present study.
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The present phylogenetic analysis was based on the framework of Primula, with subgen. Auganthus as focal group, with reference to recent molecular phylogenetic studies [4–6, 13–15]. We downloaded 311 sequences for 66 species of Primula from GenBank. In addition, we sequenced five species including the new species and the recently described P. hunanensis. Totally, the ingroup included 342 sequences (86 accessions) of 68 species, representing all main sections of the genus Primula and all nine sections of subgenus Auganthus established by Richards . Two species of the related genera to Primula, i.e. Androsace sublanata Hand.-Mazz. and Soldanella alpina L., were selected as outgroups following previous phylogenetic studies [5, 6, 13, 23]. Voucher information and GenBank accession numbers are listed in S1 Table.
DNA extraction and sequencing
Total genomic DNA was extracted from silica gel-dried plant leaves following a modified CTAB protocol of Doyle & Doyle . The nucleotide sequences of the nuclear ribosomal internal transcribed spacers (ITS, including ITS1-5.8S-ITS2) and three chloroplast regions (matK, rps16 and trnL-F), which were employed to infer phylogenetic relationships of Primula in previous studies, were analyzed in this study. For ITS, the primers, PCR amplification and sequencing conditions followed Liu et al. . For chloroplast regions, the primers, PCR amplification and sequencing conditions followed Yan et al. , except the matK region of two samples (see S1 Table for details) that followed Yan et al. .
Sequence alignment and phylogenetic analysis
SeqMan (DNASTAR Inc., Madison, WI, USA) was used to assemble sequences. Multiple-sequence alignment for each marker was made using Clustal X 1.83 , and then minor manually adjusted using Se-Al version 2.0a11 . Following the treatment of Liu et al. , combined phylogenetic analysis (including all the four datasets) was carried out using maximum parsimony (MP) and Bayesian inference (BI).
MP analysis was conducted using heuristic searches of random taxon addition for 100 replicates through PAUP* version 4.0b10 , and tree bisection–reconnection (TBR) branch swapping with no limit to the number of trees saved. All characters were weighted equally and gaps were considered as missing data. Relative support for individual clades was evaluated by Bootstrap analyses, which was conducted from 1000 replicates of heuristic search with TBR branch-swapping and saving multiple trees .
Bayesian analysis was carried out using MrBayes version 3.2.1 . Best-fitting nucleotide substitution model for the combined data matrix was selected by ModelTest version 3.7 . The Bayesian Markov Chain Monte Carlo (MCMC) algorithm was run for 2000000 generations under a GTR + I + G substitution model, with sampling of trees every 200th generation. The initial 25% of sampled trees were discarded as burn-in after checking for stationarity and convergence of the chains with TRACER v1.6 . The 50% majority-rule consensus tree was calculated from remaining trees with nodal support summarized as posterior probabilities (PP).
Bootstrap values ≥ 80 and Bayesian posterior probabilities ≥ 0.95 were considered as robust support.
We observed and measured both fresh and pressed specimens of the new species to obtain the accurate description. All the quantitative characters, like the diameter of corolla, which were easily changed from fresh to dry, were measured from forty fresh individuals (with half thrum flowers and half pin flowers) in the field, randomly. Other qualitative characteristics, like the texture of leaf, which were stable, were observed from ten pressed specimens. Specimens of the related species, i.e. P. henryi, P. chapaensis, P. partschiana, P. rugosa, P. kweichouensis, P. kwangtungensis, P. wangii, P. hunanensis, P. levicalyx and P. calyptrate, were checked and a morphological comparison was made with the new species. The occurrences of the main diagnostic characters (inflorescence types and calyx shape) of the members of P. sect. Carolinella were displayed on the resulting phylogenetic tree. The information of the examined specimens can be found in the S1 Text.
Aligned length of the combined dataset (ITS + matK + trnL-F + rps16) was 4655 bp, of which 1901 sites were variable and 1253 were maximum parsimony informative. The majority consensus tree of the Bayesian analysis of the combined dataset is shown in Fig 1, and the topology was congruent with that of MP analysis. The combined matrix and the majority consensus tree were deposited in TreeBASE; study number S19166 (http://purl.org/phylo/treebase/phylows/study/TB2:S19166).
Numbers before and after the slash on the branches are parsimony bootstrap values (≥ 50%) and Bayesian posterior probabilities (≥ 0.5), respectively. Clades A-D represent the monophyletic clade of P. sect. Carolinella. (A’) Inflorescence of P. chapaensis; (B’) Inflorescence of P. kwangtungensis; (C’) Inflorescence of P. calyptrate; (D’) Inflorescence of P. rugosa; (A”) Calyx of P. chapaensis; (B”) Calyx of P. kwangtungensis; (C”) Calyx of P. calyptrate; (D”) Calyx of P. rugosa.
Species of sect. Carolinella were divided into four separate clades (Fig 1; clades A, B, C, D). Two samples of P. chapaensis deviated and were deeply nested within subgen. Auganthus (Fig 1; clade A). Other examined species of sect. Carolinella fell into three phylogenetically independent lineages (Fig 1; clades B, C, D). The clade including all the samples of P. partischiana and P. rugosa was first diverged from the remaining group of subgen. Carolinella (Fig 1; clade D). Then the clade of P. calyptrata was differentiated as an independent lineage (Fig 1; clade C). The new species together with P. hunanensis, P. wangii, P. kweichouensis and P. kwangtungensis, formed a clade and became the sister to the majority members of subgen. Auganthus (Fig 1; clade B). All the three samples of the new species formed a robust monophyletic group (Fig 1; clade B).
Primula undulifolia G. Hao, C.M. Hu & Y. Xu, sp. nov. [urn:lsid:ipni.org:names:XXXXX] (Figs 2 and 3) Type: China. Hunan province: Yongzhou City, Dong’an Xian, Damiaokou town, Xiexi village, 348 m a.s.l., 4 Mar., 2015, Y. Xu & T. J. Liu, Xu150001 (holotype: IBSC, isotype: IBSC).
Primula undulifolia sp. nov. (A) Plant; (B) Short-styled Flower; (C) Long-styled Flower; (D) Calyx. Drawn by Yunxiao LIU, from the holotype.
At first sight the new species seems to be some variants of P. obconica Hance, but the calyptrate capsule clearly shows that its true affinity is with the species of section Carolinella, and the most closely related species is P. kwangtungensis, but the new species can be easily distinguished from P. kwangtungensis by its smaller flowers and narrowly oblong leaves with undulate margins.
Herbs perennial, efarinose; rhizome very short, with numerous fibrous roots. Leaves in a rosette; petiole slender, 2.5–5.5 cm long, half the length of the blade to almost as long as it, densely covered with rusty curled multicellular hairs; leaf blade oblong to oblong-elliptic, 2.5–6.5 × 1.2–4 cm, papery when dry, abaxially sparsely pilose along main veins and veinlets, adaxially sparsely pilose along midvein, otherwise glabrous, apex rounded, base subrounded, sometimes slightly cordate and asymmetrical, margin undulate, with irregular wavy undulations, ciliate; lateral veins 5–6 on each side of midvein, c. 45° angled to midvein. Scapes 2 to many per plant, 2.5–8.5 cm long, together with pedicels densely covered with rusty multicellular hairs; umbel 3–5-flowered, solitary or with a second whorl of flower; bracts linear, 3–5 mm long, less than 1 mm broad, apex acute, more or less pilose at base; pedicels slender, 1.5–2 cm long, lengthening to 2.5 cm in fruit. Flowers distylous. Calyx campanulate to tubular-campanulate, 5–5.5 mm, split to a third of its length; tube pilose outside; lobes ovate, c. 1.5 mm wide, apex acute, glabrous. Corolla pale violet-purple, annulate, limb c. 1.5 cm wide, lobes obovate, c. 7 × 4 mm, bilobed, lobules entire; corolla tubes c. 9 mm long, thrum flowers with stamens inserted towards apex, style slightly exceeding the calyx; pin flower with stamens inserted on the middle of the corolla tube, slightly exceeding the calyx, style equaling the corolla tube. Capsule c. as long as the persistent calyx, with a fragile wall, at first opening by a thicker lid.
Distribution, habitat and phenology
The new species is presently known only from the type locality in Dong’an Xian of Hunan Province (Fig 4). It grows on shady and moist cliffs of karst landform, alt. 348 m. Flowering in March to April.
The species epithet “undulifolia” refers to the leaf margin of the new species with wavy and shallow undulations.
The new species is presently known only from the type collection with two small populations. The habitat adjoins to the farmland, and become fragile due to deforestations and farming activities. Based on current information and according to IUCN red list criteria , its conservation status is evaluated as ‘Critically Endangered’ (CR) (B1abiii).
Taxonomy and phylogenetic relationships
Based on the similar habit and vegetative characters, sect. Carolinella was considered having affinity with sect. Obconicolisteri, but can be distinguished by its calyptrate capsule . The new species also seems as a variant of P. obconica by the leaf morphology at first sight. In this study, traits of calyptrate capsule, tubular-campanulate calyx and rusty indumentum, as well as the molecule phylogenetic result all reveal that P. undulifolia should be affiliated to sect. Carolinella defined by Hu , and are closely related to P. hunanensis and P. kwangtungensis. While species from sect. Obconicolisteri generally have a broadly campanulate calyx.
The non-monophyly of sect. Carolinella shown in this study has been verified by the recent phylogenetic analyses [14, 15]. All the examined species of sect. Carolinella are deeply nested within subgen. Auganthus and divided into four clades (Fig 1; clades A, B, C, D). We confirmed the systematic position of the new species P. undulifolia, which clusters with P. hunanensis, P. wangii, P. kweichouensis and P. kwangtungensis (Fig 1; clade B). Morphologically, this clade can be distinguished from other clades of sect. Carolinella (Fig 1; clades A, C and D) by the umbel with heterostylous flowers (Fig 1B’). In contrast, species of clade A and D have abbreviated raceme (Fig 1A’ and 1D’) and clade C has superimposed umbel with homostylous flowers (Fig 1C’). Additionally, calyx lobes of clade B are ovate-triangular (Fig 1B”), while that of clade A are ovate and veined (Fig 1A”), clade C are ovate (Fig 1C”), clade D are triangular and apex acuminate to caudate (Fig 1D”).
Although, the phylogenetic relationship of clade B (Fig 1) is unresolved, all the three accessions of the new species P. undulifolia constitute a robust monophyletic group. Morphologically, P kwangtungensis is closely related to P. undulifolia with the same traits of indumentum and calyx, and it is sister to all the other species (including P hunanensis, P. wangii, P kweichouensis and P. undulifolia) of clade B (Fig 1). Meanwhile, except for P kwangtungensis, the remaining species of clade B (Fig 1) are distinct for each other: pedicel and calyx of P hunanensis are glabrous, leaf blade of P wangii is more or less cordate at base, calyx of P. kweichouensis split to 2/3 of its length and leaf margin of P. undulifolia is undulate. Otherwise, the geographical distributions of all five species, included in clad B (Fig 1), are non-overlapping (Fig 4).
Diversification of Primula in subtropical mountains
Previous studies [2, 8, 16, 25] indicated that the geographical distribution of the genus Primula is an outstanding example of high diversity and endemism. As a whole the distribution of the genus is worldwide, but with most of the species concentrated in a narrow area, stretching from East Himalaya to the high mountains of western provinces Yunnan and Sichuan of China. Of the 300 species in China, more than 80% occur in this area.
Meanwhile the recognized clades comprising the new species (Fig 1; clades A-D) in the present study represents a few outliers that are found in the subtropical mountains, with a distributional range from SE Yunnan and W Guangxi along Nanling mountain extending to N Guangdong, in a narrow (c. 250 km wide) strip of area, between latitude 23°N to 26°N, longitude 103°E to 113°E (Fig 4). This region harbors almost all the species of sect. Caronlinella (except P. intanoensis distributed in Thailand and P. cardioeides distributed in Vietnam) and a few members of sect. Monocarpisae (i.e. P. malacoides Franch. and P. cavaleriei Petitm.) and sect. Obconicolisteri (i.e. P. barbicalyx Wright, P. obconica Hance and P. apicicallosa D. Fang). Compared with the alpine species, the subtropical species of Primula have wide blade and conspicuous petiole. These species also demonstrated highly endemism and abundant diversity in the special ecological niche of Danxia landform and karst landform of this stripe. The morphological and distributional range of the new species suggest that it is closely related to P. hunanensis and P. kwangtungensis, all adapted to subtropical climate, having adjacent ranges and similar habitat, growing on acidic soil of Danxia landform or karst landform.
S1 Table. Species, voucher information and GenBank accession numbers.
We thank IBSC, PE, KUN, GXMI, K, P and E for access to the specimens, and Ms. Yunxiao LIU for the line drawings of the new species.
- Conceived and designed the experiments: GH CMH.
- Performed the experiments: YX.
- Analyzed the data: YX GH CMH.
- Contributed reagents/materials/analysis tools: YX GH.
- Wrote the paper: YX GH CMH XLY.
- Conducted field expedition: YX XLY.
- 1. Hu CM. Primulaceae. In: Chen FH, Hu CM, editors. Flora Reipublicae Popularis Sinicae. 59. Beijing: Science Press; 1990. pp. 1–277.
- 2. Hu CM, Kelso S. Primulaceae. In: Wu ZY, Raven PH, editors. Flora of China. 15. Beijing: Science Press, St. Louis: Missouri Botanical Garden Press; 1996. pp. 99–185.
- 3. Bremer B, Bremer K, Chase MW, Fay MF, Reveal JL, Soltis DE, et al. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Bot J Linn Soc. 2009; 161(2): 105–121.
- 4. Martins L, Oberprieler C, Hellwig FH. A phylogenetic analysis of Primulaceae s.l. based on internal transcribed spacer (ITS) DNA sequence data. Plant Syst Evol. 2003; 237(1–2): 75–85.
- 5. Mast AR, Kelso S, Conti E. Are any primroses (Primula) primitively monomorphic? New Phytol. 2006; 171(3): 605–616. pmid:16866962
- 6. Mast AR, Kelso S, Richards AJ, Lang DJ, Feller DMS, Conti E. Phylogenetic relationships in Primula L. and related genera (Primulaceae) based on noncoding chloroplast DNA. Int J Plant Sci. 2001; 162(6): 1381–1400.
- 7. Trift I, Kallersjo M, Anderberg AA. The monophyly of Primula (Primulaceae) evaluated by analysis of sequences from the chloroplast gene rbcL. Syst Bot. 2002; 27(2): 396–407.
- 8. Hu CM. On the geographical distribution of the Primulaceae. Journal of Tropical and Subtropical Botany. 1994; 2: 1–14.
- 9. Richards AJ. Primula. 2nd ed. London: B.T. Batsford Ltd; 2002.
- 10. Pax F. Monographische übersicht über die Arten der Gattung Primula. Botanische Jahrbücher. 1889; 10: 75–241.
- 11. Smith WW, Forrest G, Fletcher HR. The genus Primula. Plant Monograph Reprints. 1941–1949 (facsimile reprint 1977); 11: 1–835.
- 12. Wendelbo P. Studies in Primulaceae II: An account of Primula subgenus Sphondylia with a review of the subdivisions of the genus. Årbok for University of Bergen Mat-Naturv Serie. 1961; 11: 1–49.
- 13. de Vos JM, Hughes CE, Schneeweiss GM, Moore BR, Conti E. Heterostyly accelerates diversification via reduced extinction in primroses. P Roy Soc B-Biol Sci. 2014; 281: 20140075.
- 14. Liu YJ, Liu J, Hu CM, Hao G. Non-monophyly of Primula subgenera Auganthus and Carolinella (Primlaceae) as confirmed by the nuclear DNA sequence variation. Plant Syst Evol. 2015; 301(8): 2057–2071.
- 15. Yan HF, He CH, Peng CI, Hu CM, Hao G. Circumscription of Primula subgenus Auganthus (Primulaceae) based on chloroplast DNA sequences. J Syst Evol. 2010; 48(2): 123–132.
- 16. Yu XL, Xu Y, Hu CM, Hao G. Primula hunanensis sp. nov. (Primulaceae) from Hunan, south-central China. Nord J Bot. 2015; 33(3): 381–384.
- 17. Anderberg AA, El-Ghazaly G. Pollen morphology in Primula sect. Carolinella (Primulaceae) and its taxonomic implications. Nord J Bot. 2000; 20(1): 5–14.
- 18. Gong X, Fang RC. Primula calyptrata, a new species in section Carolinella (Primulaceae) from Yunnan, China. Novon. 2003; 13(2): 193–195.
- 19. Hu CM. A new species of Primula from Thailand with critical Notes on the section Carolinella. Nord J Bot. 1990; 10(4): 399–401.
- 20. Hemsley WB. Figures with descriptive characters and remarks of new and rare plants. Hooker's Icones Plantarum. 1902; 28(2): Pl. 2726.
- 21. Hemsley WB. Figures with descriptive characters and remarks of new and rare plants. Hooker's Icones Plantarum. 1903; 28(3): Pl. 2775.
- 22. Pax F, Knuth R. Primulaceae. In: Engler A, editor. Das Pflanzenreich Regni vegetabilis conspectus. 22 (IV, 237). Leipzig: Wilhelm Engelmann; 1905. pp. 45–47.
- 23. Kallersjo M, Bergqvist G, Anderberg AA. Generic realignment in primuloid families of the Ericales s.l.: A phylogenetic analysis based on DNA sequences from three chloroplast genes and morphology. Am J Bot. 2000; 87(9): 1325–1341. pmid:10991903
- 24. Doyle JJ, Doyle JL. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin. 1987; 19: 11–15.
- 25. Yan HF, Liu YJ, Xie XF, Zhang CY, Hu CM, Hao G, et al. DNA barcoding evaluation and its taxonomic implications in the species-rich genus Primula L. in China. PloS ONE. 2015; 10(4): e0122903. pmid:25875620
- 26. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997; 25(24): 4876–4882. pmid:9396791
- 27. Rambaut A. Se-al version2.0a11. http://treebioedacuk/software/seal/. 2002.
- 28. Swofford DL. PAUP*: Phylogenetic Analysis using parsimony (*and other methods), version 4.0b10 [computer program]. Sunderland, Massachusetts (USA): Sinauer Associates. 2002.
- 29. Felsenstein J. Confidence-limits on phylogenies: an approach using the bootstrap. Evolution. 1985; 39(4): 783–791.
- 30. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, et al. MrBayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012; 61(3): 539–542. pmid:22357727
- 31. Posada D, Crandall KA. MODELTEST: testing the model of DNA substitution. Bioinformatics. 1998; 14(9): 817–8. pmid:9918953
- 32. Rambaut A, Suchard M, Drummond A. Tracer. Version 1.6. http://treebioedacuk/software/tracer/. 2013.
- 33. IUCN. IUCN red list categories and criteria, version 3.1. Gland: IUCN Species Survival Commission. 2001.