﻿Paraphlomisyingdeensis (Lamiaceae), a new species from Guangdong (China)

﻿Abstract Paraphlomisyingdeensis (Lamiaceae), a new species from the limestone area in northern Guangdong Province, China, is described and illustrated. Phylogenetic analyses, based on two nuclear DNA regions (ITS and ETS) and three plastid DNA regions (rpl32-trnL, rps16 and trnL-trnF), suggest that P.yingdeensis represents a distinct species in Paraphlomis. Morphologically, P.yingdeensis is similar to P.foliatasubsp.montigena and P.nana, but can be distinguished from the former by its densely villous lamina and calyx, not decurrent base of lamina and bristle-like-acuminate apex of calyx teeth, and distinguished from the latter by its significantly taller plant (15–20 cm vs. 1–5 cm) and larger lamina (6.2–16.5 × 4–11.5 vs. 2–7 × 1.5–4 cm), densely villous stem, lamina and calyx and yellow corolla.


Introduction
As a member of tribe Paraphlomideae (Lamiaceae, Lamioideae) (Bendiksby et al. 2011;Zhao et al. 2021), the genus Paraphlomis Prain is characterised by its herbaceous habit, actinomorphic calyx with five lobes less than half as long as the tube, corolla 2-lipped (1/3) with hairy upper lip, but hardly bearded along the margin, included stamens and an apically truncate ovary (Wu and Li 1977;Bendiksby et al. 2011;Chen et al. 2021). A total of 36 species and seven varieties are recognised within Paraphlomis, most of which are distributed in southern China (Chen et al. 2022b;Yuan et al. 2022), with several species occurring in the Himalayas, Korea and Southeast Asia (Li and Hedge 1994;Ko et al. 2014;Chen et al. 2021). Many species of Paraphlomis are endemics of limestone soils, including the recently described P. kuankuoshuiensis R.B. Zhang, D. Tan & C.B. Ma (Zhang et al. 2020), P. longicalyx Y.P. Chen & C.L. Xiang (Chen et al. 2022a) and P. hsiwenii Y.P. Chen & X. Li (Chen et al. 2022b). This shows species richness of Paraphlomis has been quite underrated and more field investigations are needed to infer its diversity in limestone areas.
The botanical expedition to the Shimentai National Nature Reserve in Guangdong Province, China in October 2021, showed an unknown species of Paraphlomis. Based on other field observations (from May to August in 2022), morphological comparisons with congeneric species, as well as molecular phylogenetic studies, we confirmed that it represented a new species, here described and illustrated.

Morphological study
Field observations and collections of the new species were carried out from May to August in 2022 in Boluo Town of Yingde City in northern Guangdong Province, China. Morphological comparisons of the putative new species with other Paraphlomis species were conducted firstly by consulting relevant taxonomic literature, included "Flora of China" (Li and Hedge 1994), "Flora of Guangdong" (Luo 1995) and other recently described species and infraspecies of Paraphlomis (Yan and Fang 2009;Ding et al. 2019;Zhang et al. 2020;Chen et al. 2021Chen et al. , 2022aZhao et al. 2022). We also carried out a check of herbarium specimens deposited in LBG, AU, IBK, FJFC, PE, ANUB, KUN, FJSI and SYS (herbarium acronyms following Thiers 2022). All morphological characters were measured using dissecting microscopes.

Phylogenetic analyses
Previous molecular phylogenetic study revealed genus Paraphlomis is not monophyletic, because species of Matsumurella were recovered within it Chen et al. 2022b). Thus, Matsumurella was also included in our phylogenetic analyses. A total of 37 accessions, representing 20 species and four varieties/subspecies of Para-phlomis and two Matsumurella species were selected as ingroups. One species each of Phlomis L. and Phlomoides Moench were included as outgroups following Chen et al. (2022a, b). Except for the three accessions of the new species that were newly sampled here, sequences of the remaining accessions were all retrieved from our previous studies (Chen et al. , 2022a. Genomic DNA of the potential new species was extracted from silica-gel-dried leaves using the modified 2× CTAB procedure of Doyle and Doyle (1987). We selected five DNA markers for the phylogenetic reconstruction, including two nuclear ribosomal regions (internal and external transcribed spacers, i.e. ITS and ETS) and three plastid DNA regions (rpl32-trnL, rps16 and trnL-trnF). Primers used for the polymerase chain reaction (PCR) amplification and sequencing were the same as those of Chen et al. (2021), while PCR procedures followed those described in Chen et al. (2016). The specimen information of samples and GenBank accession numbers for all sequences are listed in Appendix 1.
Raw sequences were assembled and edited using Sequencher 4.1.4 (Gene Codes, Ann Arbor, MI, USA) and then aligned using MUSCLE (Edgar 2004) and manually adjusted in MEGA 6.0 (Tamura et al. 2013). Bayesian Inference (BI) (Ronquist et al. 2012) and Maximum Likelihood (ML) (Stamatakis 2014) analyses were used for phylogenetic reconstruction and detailed settings for the two analyses followed those described in Chen et al. (2021). The resulting trees with posterior probabilities (PP) and Bootstrap support (BS) values were visualised and annotated in TreeGraph 2 (Stöver and Müller 2010). The combined nuclear dataset and the combined plastid dataset were initially analysed separately. Topological incongruence between the two reconstructions was visually inspected, based on the thresholds of PP ≥ 0.95 and/or BS ≥ 70%. After excluding the taxa that exhibited strong conflicts between the nuclear tree and the plastid tree, the combined nuclear dataset and the combined plastid dataset were then concatenated for phylogenetic analyses.

Results and discussion
The aligned length of the combined nuclear dataset was 1254 bp (810 bp for ITS, 444 bp for ETS) and that of the combined plastid dataset was 2479 bp (850 bp for rpl32-trnL, 812 bp for rps16, 817 bp for trnL-trnF). Since the placements of three taxa, Paraphlomis albiflora (Hemsl.) Hand.-Mazz., P. nana Y.P. Chen, C. Xiong & C.L. Xiang and P. javanica var. pteropoda D. Fang & K.J. Yan, showed hard incongruences in the nuclear tree (Appendix 2) and the plastid tree (Appendix 3), these taxa were excluded prior to the combination of the nuclear and plastid datasets. All the resulting trees ( Fig. 1; Appendices 2-3) were topologically consistent with those in previous study . With the two species of Matsumurella deeply nested within Paraphlomis, both genera were shown to be non-monophyletic. The three individuals of the putative new species formed a strongly supported clade ( Fig. 1: PP = 1.00 / BS = 100%), but its relationship with other species of Matsumurella-Paraphlomis was not resolved.
Our  (Chen et al. 2022b, c) and our present analyses ( Fig. 1; Appendices 2-3) indicated that P. foliata subsp. montigena might represent an independent species within the genus as it is distantly related to P. foliata subsp. foliata. The new species can be distinguished from P. foliata subsp. montigena in the morphology and indumentum of laminae and calyces. Both the laminae and calyces are densely villous in P. yingdeensis, but are sparsely strigose in P. foliata subsp. montigena; the base of lamina is broadly cuneate and not decurrent in the new species, but is cuneate and decurrent in P. foliata subsp. montigena; P. yingdeensis has bristle-like-acuminate apex of calyx teeth, in contrast, the apex of calyx teeth of P. foliata subsp. montigena is acuminate. The phylogenetic placement of P. nana was conflicting in the nuclear tree and plastid tree, but it was consistently sister to P. albiflora (Appendices 2-3). Both P. nana and P. yingdeensis have translucent and membranous calyces with bristle-like-acuminate apex of calyx teeth. The two species mainly differ in the height of plants, size and indumentum of laminae, as well as colour of corollae. Specifically, plants of P. nana are 1-5 cm tall, whereas those of P. yingdeensis are 10-20 cm tall. The stems and laminae are densely villous in P. nana, but are densely strigose in P. yingdeensis. Moreover, P. yingdeensis has significantly larger laminae than P. nana (6.2-16.5 × 4-11.5 cm vs. 2-7 × 1.5-4 cm) and the corollae of P. yingdeensis are yellow, differing from the white corollae of P. nana. Detailed morphological comparisons amongst the three taxa were summarised in Table 1.