Petrocodon Longitubus (Gesneriaceae), a New Species from Guizhou, China

Phytotaxa 408 (4): 267–275 ISSN 1179-3155 (print edition) https://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2019 Magnolia Press Article ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.408.4.3

Petrocodon longitubus (Gesneriaceae), a new species from Guizhou, China

CONG-RUI LI1, #, FANG-PU LIU2, 3, #, SUDHINDRA R. GADAGKAR4, 5 & YANG LUO1, * 1Guizhou Academy of Forestry, Guiyang 550005, Guizhou, China 2State key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China 3University of Chinese Academy of Sciences, Beijing 100049, China 4College of Graduate Studies, Midwestern University Glendale, AZ 85308, USA 5College of Veterinary Medicine, Midwestern University, Glendale, AZ 85308, USA #These authors contributed equally to this work *Author for correspondence: [email protected]

Abstract

Petrocodon longitubus, a new species of Gesneriaceae from Guizhou province, China, is described and illustrated. It is similar to P. hunanensis in having four stamens, but readily differs in being rosulate and stemless and having a narrowly infundibuliform floral tube with the adaxial lip bending upward. Molecular evidence indicates that it is sister to P. hunanensis, but the two species differ markedly in morphology.

Keywords: ITS, limestone flora, molecular phylogeny, taxonomy, trnL-F

Introduction

Molecular phylogenetic research on Gesneriaceae over the last two decades has greatly advanced our understanding of species relationships and generic delimitations (Möller et al. 2011). Petrocodon Hance (1883: 167) was once treated as a genus of three species and one variety (Jiang et al. 2011, Wei 2006), but several genera have been merged within it based on recent molecular studies (Wang et al. 2010, Möller et al. 2011, Wang et al. 2011, Weber et al. 2011a, 2011b, Xu et al. 2014). This circumscription of Petrocodon has resulted in increased morphological variation within the genus (Weber et al. 2011a). For example, Petrocodon scopulorus (W.Y.Chun 1946: 34) Y.Z.Wang (2011: 60) has actinomorphic corolla and four or five stamens, while other species have zygomorphic corolla, two stamens and two or three staminodes. Moreover, the color and shape of the corolla now include more variation other than the white, urceolate to campanulate corollas as originally ascribed (Yu et al. 2015). During a routine field investigation in Guizhou Province in 2018, we collected plants similar to P. hunanensis X.L.Yu & Ming Li in Yu et al. (2015: 65) in having four stamens. However, all the plants were rosulate and stemless, different from the caulescent P. hunanensis. We concluded that these plants represent an undescribed species in Petrocodon based on morphological and molecular data and a search through relevant literature (Wang 1984, Wei 2006, Wei et al. 2010, Jiang et al. 2011, Weber et al. 2011a, Wen et al. 2012, Chen et al. 2014, Hong et al. 2014, Xu et al. 2014, Yu et al. 2015). Molecular evidence indicates that this new species, hereafter named P. longitubus Cong R.Li & Yang Luo, is sister to P. hunanensis.

Material and Methods

Plant materials ITS and trnL-F DNA sequences from 22 species of Petrocodon (except for P. hunanensis and P. longitubus) were acquired from GenBank (Appendix 1) and compared with leaf material obtained from the unidentified plants from Guizhou, which were dried using silica gel. Voucher specimens were deposited in GF and PE.

Accepted by Hai He: 12 May 2019; published: 4 Jul. 2019 267 The ingroup, representing the diversity in distribution in Petrocodon, comprised 19 species and one variety. Several species of Petrocodon were excluded from our analysis because of significant differences in morphology from P. longitubus. The outgroup comprised two species of Didymocarpus Wall. (1819: 378), a close relative of Petrocodon (Li & Wang 2007, Möller et al. 2009, Wang et al. 2011, Weber et al. 2011a, 2011b). Vouchers and sequence information are given in Appendix 1.

DNA extraction and PCR Total DNA was extracted from silica-gel dried leaves using the Tian quick DNAsecure Plant kit (Tiangen Biotech, Beijing, China) following the manufacturer’s protocol and was used as a template in the polymerase chain reaction (PCR). The markers ITS and trnL-F were amplified using ITS primers ITS1 and ITS4 (White et al. 1990) and trnL-F primers c and f (Taberlet et al. 1991), respectively. PCR was performed in reactions containing 30–50 ng genomic –1 DNA, 1 μl of each primer (5 pmol μl ), 10 μl 2 × Taq PCR MasterMix, and ddH2O to make up 20 μl. PCR amplification was done at 94°C for 3 min followed by 35 cycles at 94°C for 30 s, 30 s at the annealing temperature specific for each primer pair (ITS: 55°C; trnL-F: 52°C), 72°C for 50 s, and a final extension at 72°C for 5 min.

Sequence alignment and Phylogenetic analysis Sequences were aligned and adjusted manually using the software Geneious version 11.0.3. ITS and trnL-F alignments were analyzed separately using Maximum Parsimony in PAUP*4.0B10 (Swofford 2002). The incongruence length difference test (Farris et al. 1994) was used as implemented in PAUP*4.0B10 to assess potential incongruence between ITS and trnL-F phylogenies, and a conflict was detected between the two datasets (p <0.01). Incongruences with greater than 75% bootstrap support and/or 0.95 posterior probabilities were regarded as significant (Nishii et al. 2015). However, since no hard conflicts (MP-BS > 75% or BI-PP > 0.95) were found between the two single gene trees, we concatenated them to carry out the following analysis. The combined dataset was analyzed using Maximum Parsimony as implemented in PAUP*4.0B10. All characters were unordered and had equal weight. Gaps were treated as missing data. Starting trees were obtained via stepwise addition. Heuristic searches were carried out with 1000 replicates of random addition, one tree being held at each step during stepwise addition, with tree-bisection-reconnection (TBR) branch-swapping strategy. The robustness of different clades was assessed by means of nonparametric bootstrapping (Felsenstein 1985) using 1000 replicates and a heuristic search with 1000 replicates of random sequence addition and TBR branch swapping. The combined dataset was also analyzed using Bayesian inference in MrBayes version 3.2.2 (Ronquist & Huelsenbeck 2003). For this purpose, the model of sequence evolution was first determined to be K3Pu+F+G4 using IQ-Tree version 1.6.6 (Nylander 2004). 1,000,000 generations were run in two independent analyses, each with four Markov chain Monte Carlo (MCMC) chains. One tree was sampled every 500 generations (=2000 trees). The first 400 trees were discarded as burn-in. Posterior probabilities (PP) obtained from the analysis were used to indicate the support for each branch.

Results and Discussion

Parsimony analyses resulted in eight trees of equal length (L=635; CI=0.764; RI=0.611). The topology derived from Bayesian analyses was congruent with the MP trees. While both phylogenies showed that Petrocodon longitubus were sister to P. hunanensis with mixed support values (MP-BS <50%; BI-PP =0.9; Fig. 1).

Taxonomic treatment

Petrocodon longitubus Cong R.Li & Yang Luo, sp. nov. (Figs. 2 & 3). Diagnosis:—Petrocodon longitubus is morphologically similar to P. hunanensis, but readily differs by being rosulate and stemless, having a narrowly infundibuliform corolla tube with the adaxial lip bent upward and geniculate filaments. Type:—CHINA. Guizhou: Wangmo County, Mashan Town, Shanhua Village, elevation 1100–1300 m, on rock surface, 22 July 2018, Cong R. Li Wangmo1073 (holotype PE-02309204!; isotypes PE-02309201, PE-02309202, PE-02309203, GF!).

268 • Phytotaxa 408 (4) © 2019 Magnolia Press li et al. FIGURE 1. Majority rule consensus tree based on Bayesian inference analysis of Petrocodon based on ITS and trnL-F region. MP- BS/BI-PP values (> 50%) are shown above branches. Asterisk represents MP-BS support values below 50%. Petrocodon longitubus is highlighted in bold font. a new species of Petrocodon longitubus Phytotaxa 408 (4) © 2019 Magnolia Press • 269 FIGURE 2. Petrocodon longitubus Cong R.Li & Yang Luo (Drawn by Hua Xie). (A) mature plant, (B) opened corolla showing stamens, (C) pistil, (D) fruit, (E) abaxial leaf blade, (F) calyx and disc, (G) anther, (H) mature stigma.

270 • Phytotaxa 408 (4) © 2019 Magnolia Press li et al. FIGURE 3. Petrocodon longitubus Cong R.Li & Yang Luo (Photo by Hua Xie). (A) habitat, (B) mature plant, (C) adaxial surface of leaf blade, (D) abaxial surface of leaf blade, (E) calyx and disc, (F) front view of flower, (G) lateral view of flower, (H) young fruit, (I) pistil with calyx and disc, (J) opened corolla showing stamens and staminodes. a new species of Petrocodon longitubus Phytotaxa 408 (4) © 2019 Magnolia Press • 271 Herbs, perennial. Rhizomes 5–15 cm long, 4–6 mm in diameter; with many long fibrous roots. Leaves alternate or crowded at apex of rhizome; petiole 2–4.5 cm long, densely reddish purple pubescent; blade thickly papery, ovate, 2.5–5 × 1.4–2.5 cm, base broadly cuneate to round, margin crenate, apex slightly acuminate to obtuse, adaxial surface green, abaxial surface reddish purple, both surfaces with dense reddish purple bristles; lateral veins 2–4 pairs, adaxially pale, abaxially prominent. Inflorescence cymes 2–6, 1-(rarely 2) flowered; peduncle 10–25 cm long, densely reddish- purple pubescent. Bracts 2, opposite, ovate-lanceolate, 3–5 × ca. 1.5 mm, margin entire, outside densely villous, inside with sparse pubescent. Pedicels 0.5–1 cm long, with dense reddish-purple bristles. Calyx 5-parted to base, lobes triangular-lanceolate, 5–7 × 1–1.5 mm, outside densely puberulent, inside glabrous, margin entire. Corolla purple or reddish, 3.5–4 cm long, corolla tube narrowly infundibuliform, outside densely puberulent, inside glabrous, 0.8–1 cm in diameter at mouth, tube 2–2.8 cm long, inside with three pale purple stripes; limb 2-lipped, adaxial lip ca. 4 mm long, 2–lobed, lobes ovate, abaxial lip ca. 4 mm long, 3-lobed, lobes ovate, 8–10 mm long, all lobes were bending upward. Fertile stamens 4, abaxial stamens adnate to ca. 1.8 cm above base of corolla tube, filaments ca. 0.8 cm long, adaxial stamens adnate to ca. 1.5 cm above base of corolla tube, 0.5 cm long, twisted; all filaments geniculate and with dense short glandular hairs; anthers ellipsoid, coherent in pairs; staminode obscure. Pistil 3.5–4 cm long, densely puberulent, ovary 2–2.3 cm long, ca. 1 mm in diam., style 0.5–2 cm long, ca. 0.3 mm in diam., pubescent; stigma 2- lobed, ovate, ca. 4 mm long, densely pubescent. Disc annular, yellow, ca. 1 mm tall, margin entire. Capsule virgate, 4-valved, 4–6 cm × 1–2 mm, densely puberulent. Distribution and habitat:—Petrocodon longitubus is known only from the type locality in Shanhua Village, Mashan Town, Wangmo County, Guizhou Province, China. It grows on moist shady rock surfaces at elevation range of 1100–1300 m. Phenology:—Flowering from June to August, and fruiting from July to September. Etymology:—The specific epithet is derived from the narrowly infundibuliform corolla tube. Conservation Status:—Petrocodon longitubus is currently known only from the type locality in karst region by one population, scattered over an area of 10,000 m2. It may be susceptible to human activities such as collecting, defores tation and other land use. It is therefore suggested to be classified as ‘Vulnerable’ (VU) using the IUCN categories and criteria (IUCN 2017). Similar species:—Petrocodon longitubus is morphologically similar to P. hunanensis in having four fertile stamens, zygomorphic flowers and similarly shaped leaf blades. The morphological comparison of the two species is also summarized in Table 1.

TABLE 1. Morphological comparison between P. longitubus and P. hunanensis. Character P. longitubus P. hunanensis

Leaf blade with dense reddish-purple bristles with dense white pubescence Stems stemless caulescent Cymes 1-flowered, rarely 2-flowered 1–4-flowered Petiole 2–4.5 cm long 0.5–2.5 cm long Peduncle 10–25 cm long 2–7 cm long Pedicel 0.5–1.2 cm long, densely reddish-purple pubescent 0.3–2 (–2.8) cm long, densely pubescent Corolla tube narrowly infundibuliform infundibuliform Adaxial lip bent upward projecting Staminode 1, obscure 1, ca. 1 mm long Filaments geniculate nearly straight Ovary 1.6–2.3 cm long ca. 1 cm long

Acknowledgements

This study was supported by the Study on Plant Resources Investigation, Collection and Cultivation Techniques of Gesneriaceae in Guizhou, Guizhou Science and Technology Department ([2017]2523) and Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain (17-259-23). We are grateful to Dr. David E. Boufford of Harvard University Herbaria and Prof. Zhiyun Zhang of Institute of Botany, the Chinese Academy of Sciences for

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Taxa and GenBank accession numbers of ITS and trnL-F sequences for phylogenetic analysis (sequences of Petrocodon longitubus Cong R.Li & Yang Luo and Petrocodon hunanensis X.L.Yu & Ming Li were unpublished). Taxon ITS trnL-F

Petrocodon ainsliifolius W.H.Chen & Y.M.Shui HQ633038 HQ632941

Petrocodon coccineus (C.Y.Wu) Yin Z.Wang KF202292 KF202299

Petrocodon coriaceifolius (Y.G.Wei) Y.G.Wei & A.Weber KY796058 KY796060

Petrocodon dealbatus Hance KR337020 KR476565

Petrocodon dealbatus var. denticulatus (W.T.Wang) W.T.Wang JF697578 JF697590

Petrocodon ferrugineus Y.G.Wei HQ633043 HQ632946

Petrocodon hancei (Hemsl.) Mich.Möller & A.Weber KY796057 KY796059

Petrocodon hechiensis (Y.G.Wei, Yan Liu & F.Wen) Y.G.Wei & Mich.Möller KR337018 KR476563

Petrocodon hispidus (W.T.Wang) A.Weber & Mich.Möller KF202294 KF202301

Petrocodon integrifolius (D.Fang & L.Zeng) A.Weber & Mich.Möller HQ633037 HQ632940

Petrocodon lithophilus Y.M.Shui, W.H.Chen & Mich.Möller KF202296 KF202303

Petrocodon lui (Yan Liu & W.B.Xu) A.Weber & Mich.Möller HQ633035 HQ632938

Petrocodon multiflorus F.Wen & Y.S.Jiang KJ475411 KM232660

Petrocodon pulchriflorus Y.B.Lu & Q.Zhang KX579058 KX579059

Petrocodon retroflexus Qiang Zhang & J.Guo KX579060 KX579061

Petrocodon scopulorus (Chun) Yin Z.Wang KR337023 KR476567

Petrocodon tiandengensis (Yan Liu & B.Pan) A.Weber & Mich.Möller JX506960 JX506850

Petrocodon viridescens W.H.Chen, M.Möller HQ633036 HQ632939

Didymocarpus cortusifolius H.Lév. HQ632995 HQ632898

Didymocarpus yuenlingensis W.T.Wang KR337011 KR476555