Based on morphological and molecular data, a new species of Gesneriaceae, Primulina yingdeensis, from Guangdong, China, is described and illustrated. It is morphologically most similar to P. hochiensis and P. hochiensis var. rosulata, but the three taxa differ from each other in discrete characters. They share yellow honey guides, but in the new species these are strongly ridged and with glands. At the same time, also based on morphological and molecular data, P. hochiensis var. rosulata is raised to the rank of species, as P. rosulata.
Version of record first published online on 15 November 2016 ahead of inclusion in December 2016 issue.
The genus Primulina Hance (1883) formerly composed of the single species P. tabacum Hance, now includes all species of Chirita sect. Gibbosaccus C. B. Clarke (1883), Chiritopsis W. T. Wang (1981) and two species of Wentsaiboea D. Fang & D. H. Qin (2004), based on taxonomic transfers supported by recent phylogenetic analyses (Möller & al. 2009, 2011; Liu & al. 2010; Wang & al. 2011; Weber & al. 2011a). The newly defined Primulina is one of the largest genera of the Old World Gesneriaceae, comprising currently around 170 species distributed in S and SW China and N Vietnam (Weber & al. 2011a; Möller & Clark 2013) with more than 40 taxa described in the last few years (Möller & al. 2016). The karst regions of S and SW China and N Vietnam show the highest biodiversity and differentiation of Primulina species, and most are narrowly endemic species with small populations distributed at only one or a few localities (Wen & Zhong 1998; Li & Wang 2004; Wei & al. 2004; Kang & al. 2014).
In 2011, during field investigations in Yingde City, N Guangdong, we collected an unknown species of Gesneriaceae with stolons and white flowers with a slender funnelform corolla tube. Although in corolla shape and size it is similar to Primulina hochiensis (C. C. Huang & X. X. Chen) Mich. Möller & A. Weber (Huang & Chen 1992; Weber & al. 2011a) and P. hochiensis var. rosulata F. Wen & Y. G. Wei (Wen & al. 2012), it differs in several other morphological characters, summarized in Table 1. These differences mean that the new collection can easily be distinguished from the described species in Primulina.
Morphological comparison of Primulina yingdeensis, P. hochiensis and P rosulata.
To evaluate distinctness and phylogenetic affinities, we carried out a phylogenetic analysis on a set of Primulina taxa including the three above-mentioned, morphologically similar taxa.
Material and methods
Material — Material of the undescribed species was collected on a limestone hill in Yingde City, Guangdong Province, China. Voucher specimens were deposited in IBSC (herbarium code according to Thiers [continuously updated]). For molecular work, leaf material from three different localities was collected. In addition, leaf material of the two morphologically similar taxa, Primulina hochiensis and P. hochiensis var. rosulata, was collected from two localities and one locality, respectively (see Appendix 1). All leaves were dried in silica gel. Relevant literature was consulted to compare the new collection to other species in Primulina (Wang 1981; Liu & Guo 1989; Wang & al. 1990, 1998; Huang & Chen 1992; Wei & al. 2000; Fang & Qin 2004; Li & Wang 2004; Shen & al. 2010; Wei & al. 2010; Wen & al. 2012; Wu & al. 2012; Chung & al. 2013; Ning & al. 2013; Zhao & al. 2013; Zheng & Deng 2014; Zhou & al. 2014; Guo & al. 2015; Ning & al. 2015).
The ingroup comprised 95 Primulina samples, comprising 91 taxa and 87 species including the presumed new species. Two additional species, i.e. Petrocodon dealbatus Hance (1883) and Petrocodon hancei (Hemsl.) Mich. Möller & A. Weber (Weber & al. 2011b), were selected as outgroups based on previous phylogenetic analyses (Möller & al. 2009, 2011).
DNA sequencing and phylogenetic analyses — Sequence data for all but five samples used in this study were obtained from GenBank (Appendix 2). For the extraction of genomic DNA from the five samples, PCR procedure followed Kang & al. (2014). DNA sequences generated in this study were deposited in NCBI GenBank with accession numbers KU528868 to KU528887 (Appendix 2). A total of 95 samples were used to reconstruct the molecular phylogeny using the nuclear internal transcribed spacer (ITS) and three plastid markers (trnL-trnF, rpl32-trnL and atpB-rbcL). The reconstruction of the phylogenetic tree by Bayesian inference (BI) was carried out in MrBayes v.3.2.2 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003; Ronquist & al. 2012). The GTR + I + Γ model was obtained for the combined dataset with the Akaike Information Criterion (AIC) in MrModeltest v.2.3 (Nylander 2004). Eight million generations were run in two independent analyses each with four chains (one cold and three heated). One tree was saved every 1000 generations (= 8000 trees), and the first 800 trees (10%) were discarded as burn-in, determined after plotting the log likelihoods in the “.stat” output file. The remaining trees were used to estimate a majority rule consensus tree with the “sumt” command and the Bayesian posterior probabilities (PP) obtained in MrBayes.
Results and Discussion
The concatenated and aligned DNA sequences were 4034 base pairs long. A total of 825 polymorphic sites were found, of which 456 were parsimony informative. Bayesian analysis of the molecular data revealed that the three accessions of the newly collected taxon clustered into a clade with maximum support (Fig. 1). The BI analysis also showed that the new species was sister to Primulina hochiensis var. rosulata with maximum support. Additionally, the clade comprising these two taxa nested on a polytomy including P. hochiensis var. hochiensis and P. glandulosa var. yangshuoensis (F. Wen & al.) Mich. Möller & A. Weber (Fig. 1). This configuration rendered P. hochiensis paraphyletic. The genetic distance is 0.015 between P. hochiensis var. hochiensis and P. hochiensis var. rosulata; it is 0.011 between P. hochiensis var. hochiensis and P. yingdeensis; 0.015 between P. glandulosa (D. Fang & al.) Yin Z. Wang and P. fimbrisepala (Hand.-Mazz.) Yin Z. Wang; 0.014 between P. verecunda (Chun) Mich. Möller & A. Weber and P. villosissima (W. T. Wang) Mich. Möller & A. Weber; and 0.013 between P. fimbrisepala and P. villosissima. Thus the genetic distance between P. hochiensis var. hochiensis and P. hochiensis var. rosulata is the same or larger than that between the other taxa. Because the sister relationship between P. hochiensis var. rosulata and the new taxon is strongly supported, but the monophyly of P. hochiensis is not supported, we raise P. hochiensis var. rosulata to the rank of species.
After consulting the relevant literature and performing molecular phylogenetic analyses, we conclude that our collection from Yingde City represents a new species of Primulina, which is described and illustrated here. In addition, some key characters of P. hochiensis var. rosulata are obviously different from P. yingdeensis and P. hochiensis var. hochiensis, such as plants without stolons, leaf blades elliptical to slightly ovate, and anthers glabrous; hence P. hochiensis var. rosulata is raised to the rank of species.
Primulina rosulata (F. Wen & Y. G. Wei) Z. L. Ning & X. Y. Zhuang, comb. & stat. nov. ≡ Primulina hochiensis var. rosulata F. Wen & Y. G. Wei in Phytotaxa 54: 37. 2012. — Holotype: China, Guangxi Zhuang Autonomous Region, Pingle county, Tong'an town, 24°34′47″N, 110°55′34″E, altitude 150 m, 17 Aug 2008, B. Gao 08171 (IBK!; isotype: BJFC!).
Distribution — Pingle County, NE Guangxi Autonomous Region, S China.
Holotype: China, Guangdong Province, Yingde City, Huanghua Town, 24°09′36″N, 112°54′00″E, altitude 185 m, on limestone rock surface in a karst cave, 27 Aug 2011, M. Kang & al. YD03-3 (IBSC!; isotypes: IBSC! and others to be distributed).
Diagnosis — The new species is most similar to Primulina rosulata, but differs by having stolons present (vs absent), peduncle longer, 6–15 cm (vs 3–9.5 cm), bracts larger, 15-23 × 2-7 mm (vs 4-5 × c. 1 mm), corolla inside densely glandular pubescent below stamen insertion (vs glabrous), honey guides strongly ridged, covered with glands (vs gently ridged, without glands), and anthers light yellow (vs dark purple or purple). The new species differs from P. hochiensis by having cymes 1- or 2-branched with 4–16 flowers (vs unbranched with 1–4 flowers), peduncle longer, 6–15 cm (vs 4–7 cm), bracts larger, 15-23 × 2-7 mm (vs 3-4 × c. 1.2 mm), corolla white (vs purple), inside densely glandular pubescent below stamen insertion (vs glabrous), honey guides strongly ridged, covered with glands (vs gently ridged, without glands), and anthers light yellow (vs purple).
Description — Herbs perennial, with stolons. Stem rhizomatous, subterete, 1.5–2.5 cm long, c. 1 cm in diam.; internodes inconspicuous. Leaves 15–18, crowded at stem and stolon apex. Petiole 3.5–5 cm long, 0.8–1 cm in diam., densely white appressed pubescent. Leaf blade ovate or ovate-elliptic, slightly asymmetric, 6-11(-15) × 4-6.5(-10) cm, herbaceous, abaxially densely appressed white pubescent, adaxially densely long villous and shortly pubescent, base cuneate, margin serrate or rarely entire, apex acute or rounded; lateral veins 5–7 on each side of midrib. Cymes 3-8, 1- or 2-branched, 4-16-flowered; peduncle 6–15 cm long, c. 0.3 cm in diam., densely puberulent; bracts 2, opposite, oblong or linear-lanceolate, 15-23 × 2-7 mm, pubescent, margin entire. Pedicel 1–3.5 cm long, pubescent. Calyx green with purple apex, rarely all green, 5-lobed nearly to base; lobes linear-lanceolate, 4-8 × 1-1.5 mm, pubescent. Corolla white, 2.4–3 cm long, outside glandular puberulent, inside densely glandular pubescent below stamen insertion, throat with two strongly ridged yellow longitudinal honey guides covered with glands; tube narrowly funnel-form, 1.8–2 cm long, orifice 6–8 mm in diam.; upper lip 2-lobed, lobes suborbicular, c. 5 × 6 mm, apex rounded; lower lip 3-lobed, lateral lobes oblong, 4-6 × 4-5 mm, apex rounded, central lobe subrounded, 4-5 × 4-5 mm, apex rounded. Stamens 2, adnate to c. 1.2 cm above base of corolla tube; filaments geniculate near middle, c. 7 mm long, glabrous; anthers light yellow, reniform, c. 5 × 4 mm, abaxially densely white villous. Staminodes 3, c. 2 mm long, glabrous, adnate to 6–8 mm above base of corolla tube. Disc annular, 0.5–1 mm high, glabrous, margin repand. Pistil 2.2–2.6 cm long; ovary linear, 8–10 mm long, puberulent; style 1.3–1.5 cm long, glan-dular puberulent; stigma obtrapeziform, c. 1 mm long, 2-lobed. Capsule linear, 1.5–1.8 cm long.
Phenology — Flowering from August to October, and fruiting from September to November.
Distribution and ecology — Primulina yingdeensis is currently known from five populations in Yingde City, NW Guangdong, S China (24.13–24.38°N, 112.90–113.09°E). During field surveys in Yingde City, we found that it is locally abundant and grows mainly on moist rock surfaces at altitudes of 125–225 m.
Conservation status — The population occurs close to the edge of a provincial road and near a village without any protective measures. Its habitat is vulnerable to destruction from human activities. The plant is easy to propagate from stolon cuttings. At present, we have introduced some individuals from the wild population into cultivation in the South China Botanical Garden and Guilin Botanical Garden, China.
Etymology — The specific epithet yingdeensis is derived from the name of the type locality, Yingde City.
Additional specimens seen (paratypes) — china: guangdong: Yingde City, Xiniu Town, Xiazai Village, 24.13°N, 113.00°E, altitude 185 m, 27 Aug 2011, Kang Ming YD01 (IBSC); Huanghua Town, Mingjing Village, 24.17°N, 112.93°E, altitude 205 m, 27 Aug 2011, Kang Ming YD110802 (IBSC); Huanghua Town, Guanyinyan, 24.16°N, 112.90°E, altitude 225 m, 27 Jun 2012, Kang Ming & Ning Zu-Lin YD03 (IBSC); Dawan Town, Changshan Village, 24.38°N, 112.97°E, altitude 200 m, 6 Aug 2012, Kang Ming & Ning Zu-Lin YD120806 (IBSC); Shigutang Town, 24.20°N, 113.09°E, altitude 125 m, 4 Aug 2015, Kang Ming & Yang Li-Hua YD12 (IBSC).
We would like to thank Prof. James F. Smith (Department of Biological Sciences, Boise State University) for help with comments and correction on the manuscript, and two anonymous reviewers for their valuable comments, which helped to improve an earlier version of this paper. We are also grateful to Yun-Xiao Liu for drawing the illustration (Fig. 2). This work was supported by grants from the NSFC-Guangdong Natural Science Foundation Joint Project (U1501211), Natural Science Fund of Guangdong (2015A030313795) and the Science and Technology Planning Project of Guangdong (2014A030304003).
GenBank accession numbers for phylogenetic reconstruction of Primulina. Sequences generated in this study are indicated in bold; otherwise they were obtained from Kang & al. (2014).