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Research Article
Two new species of Paraphlomis (Lamiales, Lamiaceae) from limestone karsts in Guangdong Province, China
expand article infoWan-Yi Zhao‡, Qin-Dai Xiong‡, Rang-Min Wu§, You-Hong Zeng|, Zhi-Bin Xie, Ya-Ping Chen#, Qiang Fan‡
‡ Sun Yat-sen University, Guangzhou, China
§ Qingyuan Forestry Bureau, Qingyuan, China
| Qingyuan Jinji Forestry Farm, Qingyuan, China
¶ Yingde Forestry Bureau, Qingyuan, China
# Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China

Corresponding author: Qiang Fan ( fanqiang@mail.sysu.edu.cn )

Academic editor: Alan Paton
© 2024 Wan-Yi Zhao, Qin-Dai Xiong, Rang-Min Wu, You-Hong Zeng, Zhi-Bin Xie, Ya-Ping Chen, Qiang Fan.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Zhao W-Y, Xiong Q-D, Wu R-M, Zeng Y-H, Xie Z-B, Chen Y-P, Fan Q (2024) Two new species of Paraphlomis (Lamiales, Lamiaceae) from limestone karsts in Guangdong Province, China. PhytoKeys 243: 231-248. https://doi.org/10.3897/phytokeys.243.114415
Open Access

Abstract

Paraphlomis qingyuanensis and P. baiwanensis (Lamiaceae), two new species from the limestone area in Guangdong Province, China, are described. Morphologically, both species belong to P. ser. Subcoriaceae C.Y. Wu & H.W. Li. A close relationship between the two new and P. subcoriacea was revealed by molecular phylogenetic analyses based on ETS and ITS. Further morphological and population genetic evidence indicated that they are distinct species in Paraphlomis. According to the IUCN Red List Categories and Criteria, P. qingyuanensis and P. baiwanensis were assessed as Endangered (EN) and Deficient (DD), respectively.

Key words

IUCN, limestone, new taxon, Paraphlomideae, phylogeny, Qingyuan, RAD-seq

Introduction

Paraphlomis (Prain) Prain is a member of the tribe Paraphlomideae Bendiksby (Lamiaceae, Lamioideae) (Bendiksby et al. 2011; Li et al. 2016; Zhao et al. 2021). Most species of Paraphlomis are distributed in southern China, with several species occurring in the Himalayas, Korea and Southeast Asia (Li and Hedge 1994; Wu and Li 1997; Ko et al. 2014; Chen et al. 2021). Previous molecular phylogenetic studies of Paraphlomis revealed that the genus was not monophyletic, because species of Matsumurella were recovered within it (Chen et al. 2021; Chen et al. 2022b; Guo et al. 2023). In general, Paraphlomis is characterized 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; Ko et al. 2014; Chen et al. 2021).

As currently circumscribed, a total of 37 species and seven varieties are recognised within Paraphlomis (Li and Hedge 1994; Chen et al. 2022a, 2022b; Yuan et al. 2022; Guo et al. 2023; Yan et al. 2023). China is the distribution center of Paraphlomis, with 23 species documented in the Flora of China (Li and Hedge 1994). In recent years, a number of new species and infraspecies of Paraphlomis were reported in China, including P. javanica var. pteropoda D. Fang & K.J. Yan and P. javanica var. angustifolia f. albinervia D. Fang & K.J. Yan (Yan and Fang 2009); P. breviflora B.Y. Ding, Y.L. Xu & Z.H. Chen (Ding et al. 2019); P. kuankuoshuiensis R.B. Zhang, D. Tan & C.B. Ma (Zhang et al. 2020); P. jiangyongensis X.L. Yu & A. Liu and P. coronata (Vaniot) Y.P. Chen & C.L. Xiang (Chen et al. 2021); P. nana Y.P. Chen, C. Xiong & C.L. Xiang (Chen et al. 2022c); P. longicalyx Y.P. Chen & C.L. Xiang (Chen et al. 2022a); P. hsiwenii Y.P.Chen & XiongLi (Chen et al. 2022b); P. strictiflora J.C.Yuan, B.Chen & C.L.Xiang (Yuan et al. 2022); P. jinggangshanensis Boufford, W.B. Liao & W.Y. Zhao (Zhao et al. 2022); P. yingdeensis W.Y.Zhao, Y.Q.Li & Q.Fan (Guo et al. 2023), P. caloneura K.J.Yan, Y.P.Chen & Y.Feng Huang (Yan et al. 2023).

During a botanical expedition of the limestone area in Qingyuan city, Guangdong Province in 2023, we discovered two unknown species of Paraphlomis. The calyx teeth of the two unknown species extended into wings from veins, which are consistent with the characteristics of Paraphlomis ser. Subcoriaceae C.Y. Wu et H.W. Li (Li 1965; Wu and Li 1977). However, their morphological characters differ from the two known two species of the series, P. subcoriacea C. Y. Wu ex H. W. Li and P. brevifolia C. Y. Wu & H. W. Li. Thus, we suspected both of them were undescribed species. After careful field observations, morphological comparisons with other species of Paraphlomis, and molecular phylogenetic studies, we confirmed that they were new species and named them as P. qingyuanensis W.Y. Zhao, R.M. Wu & Q. Fan and P. baiwanensis W.Y. Zhao, Y.P. Chen & Q. Fan.

Materials and methods

Morphological study

The flowering and fruiting plants of the two new species were examined in the field from August to December in 2023 and compared with herbarium specimens deposited in IBSC, KUN, GCMI and SYS (herbarium acronyms follow Thiers 2023). The two putative new species were most similar to Paraphlomis subcoriacea and P. brevifolia. We have carried out several field work trips to the collection site of the type specimens of these two species from December 2023 to April 2024. Unfortunately, we did not find P. brevifolia in the field due to a lack of detailed collection site information (Li 1965). Therefore, the morphological features contrasting with those of P. brevifolia were based on its type specimens (IBSC0005124). All morphological characteristics were measured using dissecting microscopes.

Phylogenetic analyses

The nuclear ribosomal internal and external transcribed spacers (ITS and ETS) were used for reconstructing the phylogeny of the suspected new species and related taxa based on previous study (Chen et al. 2021; Zhao et al. 2022). Most sequences were downloaded from GenBank, except for the two nuclear ribosomal DNA (nrDNA) sequences of the two new species and Paraphlomis subcoriacea, which were newly sequenced in the present study. Genomic DNA of the suspected new species was extracted from silica-gel-dried leaves using the modified 2× CTAB procedure of Doyle and Doyle (1987). The ITS and ETS sequences were amplified with primer pairs 17SE/26SE (Sun et al. 1994) and ETSB/IGS (Beardsley and Olmstead 2002), respectively, with PCR amplification and sequencing following Chen et al. (2016). A total of 49 accessions representing 30 species and four varieties/subspecies of Paraphlomis and two species of Matsumurella were sampled in the phylogenetic study. Phlomoides bracteosa (Royle ex Benth.) Kamelin & Makhm. and Phlomis fruticosa Sieber ex C. Presl were selected as the outgroups. The GenBank accession numbers are listed in Appendix 1.

Nucleotide sequences were aligned using MAFFT 7 (Katoh and Standley 2013). After removing aligned columns with more than 70% missing data using Phyx (Brown et al. 2017), the two nrDNA regions were concatenated for phylogenetic reconstruction. The phylogenetic relationships were assessed using the Bayesian inference (BI) and maximum likelihood (ML) methods, and both were implemented on the online server Cyberinfrastructure for Phylogenetic Research Science (CIPRES) Gateway (http://www.phylo.org/; Miller et al. 2010). The BI analysis was carried out using MrBayes 3.2.7a (Ronquist et al. 2012) and the ML analysis using RAxML-HPC2 (Stamatakis 2014), with detailed settings following that of Chen et al. (2021). The resulting BI tree with posterior probabilities (PP) and best-scoring ML tree with bootstrap support (BS) values were visualized and annotated using TreeGraph 2 (Stover and Müller 2010).

Population genetic structure analyses

To investigate the phylogenetic relationship between the two newly discovered species and their close relatives, we further conducted an analysis of their population genetic structure. A total of 84 individuals were sampled from seven populations of the two putative Paraphlomis new species and its close relative P. subcoriacea (Appendix 2). The fresh leaves were dried and stored with silica gel, and then sent to JieRui BioScience Co. Ltd. (Guangzhou, China) for DNA extraction, ddRAD-seq library preparation, and Illumina sequencing. The produced raw data was processed with the software Stacks 2.55 (Catchen et al. 2013). The procedure “process_radtags” was used to demultiplex RAD tags, “denovo_map.pl” to process all the fastq files, and “populations” to filter the data by setting “--min-maf 0.05 --max-obs-het 0.7 -R 0.8 –write-random-snp –vcf”. The produced vcf file was transformed to ped file using bed file using software vcftools 0.1.16 (Danecek et al. 2011), and to bed file using the software Plink v1.90 (Chang et al. 2015). The produced bed file was used to perform Bayesian cluster analysis with the software ADMIXTURE v1.3.0 (Alexander et al. 2009), in which the number of groups (K) was set from 1–6 and the optimal K was determined by the minimum value of cross-validation error (CV). Principal coordinate analysis (PCA) was performed with Plink, and python script were used to draw the scatter diagram.

Results and discussion

The combined nuclear data set was 1211 bp (773 bp for ITS, 438 bp for ETS) in length, including 347 variable sites (165 for ITS, 182 for ETS) and 200 parsimony-informative characters (87 for ITS, 113 for ETS). The resulting phylogenetic tree of Paraphlomis in this study was similar to that in previous studies (Chen et al. 2021; Zhao et al. 2022). The two accessions of P. baiwanensis grouped together (Fig. 1: BS = 100%/PP < 0.50) and this new species was sister to P. subcoriacea (Fig. 1: BS = 100%/PP = 1.00). The P. baiwanensis-P. subcoriacea clade was further sister to another new species, P. qingyuanensis (Fig. 1: BS = 99%/PP = 1.00). All three species were nested within the previously suggested “Clade III” by Chen et al. (2021) (Fig. 1: BS = 78%/PP = 1.00). Species of “Clade III” (marked in blue in Fig. 1) are characterized by hairy nutlets/ovaries (Chen et al. 2021; 2022b). The nutlets of both new species are sparsely hispid and densely glandular at apex (Figs 2M, 5I, 6H), which confirmed the significance of nutlet morphology for the infrageneric classification of Paraphlomis (Chen et al. 2021).

Figure 1. 

The best-scoring maximum likelihood tree of Paraphlomis inferred from concatenated nrDNA (ETS and ITS) dataset. Support values ≥ 50% BS or 0.50 PP are displayed above and below the branches, respectively. Multiple accessions of the same species are numbered according to Appendix 1.

Figure 2. 

Paraphlomis qingyuanensis A habitat B plants C individual D inflorescences with unopened flowers E opening flowers F leaves G stolons and roots H lateral view of flower I upper lip of flower J dissected corolla and stamens K infructescence L frontal and lateral view of calyx M dissected calyx and seeds. (Photographs: A, B by Qin-Dai Xiong; C–G by Wan-Yi Zhao).

The close relationships among the two new species and P. subcoriacea are also supported by morphological evidence. In morphology, the two putative new species are most similar to Paraphlomis brevifolia C. Y. Wu et H. W. Li and P. subcoriacea C. Y. Wu ex H. W. Li. They share the features such as conspicuously extended calyx teeth, and the fact that they are also growing in a limestone habitat. A comparison of their morphological features is presented in Table 1 and Fig. 4.

Table 1.
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Morphological comparisons among Paraphlomis baiwanensis, P. brevifolia, P. qingyuanensis, and P. subcoriacea.

Characters P. qingyuanensis P. baiwanensis P. subcoriacea P. brevifolia
Stem 50–90 cm, slender, 1.4–3.1 mm in diam 50–80 cm tall, erect, stout, 3.6–5.3 mm in diam ca. 60 cm, ca. 3.6 mm in diam ca. 40 cm, ca. 2.5 mm in diam
Stem branching much branched unbranched or 2–3 branched unbranched or 2–3 branched unbranched
Length of petiole 0.4–1.6 cm 0.6–1.3 cm 1.0–1.3 cm 0.3–0.6 cm
Leaf size 2.7–9.5 cm long, 1.2–2.5 cm wide 12.5–18.7 cm long, 2.6–4.8 cm wide 7–15 cm long, 1.5–3.2 cm wide 5–8 cm long, 2.4–3.4 cm wide
Leaf base cuneate, not decurrent cuneate or abruptly obtuse, not decurrent attenuate or abruptly obtuse, not decurrent obtuse to rounded, not decurrent
Leaf texture papery leathery thin leathery thin leathery
Leaf vein 2–4 (-5) pairs 5–7 (-8) pairs 5–6 pairs 4–5 pairs
Calyx teeth subtruncate, extended into wings from veins subtruncate, extended into wings from veins subtruncate, extended into wings from veins triangular, conspicuously extended into wings from veins
Corolla upper lip yellow, lower lip red with purple spots white white or purple-white unknown

The putative new species Paraphlomis qingyuanensis differs from P. brevifolia by its slender and much branched stem (vs. unbranched) (Figs 2C, 4A), leaves without glandular (vs. abaxially golden glandular) (Table 1). Furthermore, the reticulate veins and pilose indumentum on leaves of P. brevifolia are more obvious (Fig. 4B). P. qingyuanensis could also be easily distinguished from P. subcoriacea by its papery leaves (vs. thin leathery), fewer leaf veins, 2–4 pairs (vs. 5–6 pairs), and smaller leaf size (Table 1, Fig. 4A, D).

Paraphlomis baiwanensis is most similar to P. subcoriacea. They share such features as leaves lanceolate, 5–7 lateral vein pairs, and leaf base shape (Table 1). However, P. baiwanensis could be easily distinguished from the latter by its stout stem (diam. 3.6–5.3 mm vs. ca. 3.6 mm), leathery leaves (vs. thin leathery), and larger leaves size (12.5–18.7 cm × 2.6–4.8 cm vs. 7–15 cm × 1.5–3.2 cm) (Table 1, Fig. 4C, D). Furthermore, the distribution areas of the two species are separated by a distance of approximately 45 km, exhibiting complete non-overlap (Fig. 7A). The results of population genetic analysis also confirmed significant differences in their genetic structure (Fig. 7B).

Bayesian cluster analysis showed lowest CV value as K = 3, each of the three species P. baiwanensis (P1), P. subcoriacea (P2–4), and P. qingyuanensis (P5–7) possesses a unique gene pool, and no gene admixture is observed in any individuals (Fig. 7B). PCA analysis reveals similar results in which the 84 individuals are assigned into three groups and individuals of the same species are clustered into the same group (Fig. 7C). These results showed that individuals of the three species can be separated clearly from each other based on genomic data, strongly supported their species status and no obvious gene flow was observed among the three species.

Taxonomic treatment

Paraphlomis qingyuanensis W.Y.Zhao, R.M.Wu & Q.Fan, sp. nov.

urn:lsid:ipni.org:names:77344362-1
Figs 2, 3, 4A 清远假糙苏

Type

China. Guangdong Province: Yingde City, Huanghua Town, near Hegushi, on the limestone valley, 24°13'N, 112°56'E, alt. 135 m, 7 September 2023, Xiong Qin-Dai ZWY-3793 (holotype: SYS00236954! isotypes: KUN, SYS00236955!, SYS00236968!, SYS00236969!).

Diagnosis

Paraphlomis qingyuanensis is morphologically similar to P. brevifolia and P. subcoriacea, but differs from the latter two species by its slender and much branched stem, papery leaves, inconspicuous reticulate veining, and fewer leaf veins.

Description

Herbs perennial, 50–90 cm tall; stoloniferous, with sparsely villous. Stems erect or decumbent, slender and much branched, 4-angled, diameter 1.4–3.1 mm, densely retrorse pilose. Leaves opposite; lamina ovate to oblong, papery, 2.7–9.5 cm long, 1.2–2.5 cm wide, apex obtuse, base cuneate, margin crenate-serrate; adaxially green, abaxially light green, sparsely pilose on both sides, more densely on viens; lateral veins 2–4 (-5)-paired; petiole 0.4–1.6 cm long, densely pilose. Verticillasters born in upper leaf axils, cymes 1–4-flowered; bracteoles inconspicuous, ca. 0.5 mm long, early deciduous; pedicels ca. 1 mm long, sparsely pilose. Calyx light green, obconical, 4.5–5 mm long, outside with sparsely pilose and white glandular, glabrous inside, conspicuously 10-veined; teeth 5, inconspicuous so that calyx mouth appears subtruncate, extended into wings from veins as 1.5–2.0 mm long. Corolla 1.0–1.2 cm long; tube ca. 0.3 cm long, ca. 1 mm in diam., straight, inside of throat dark purple, with pubescent annulate; 2-lipped, villous outside, upper lip oblong, erect and galeate, apex undulate or bilobate, ca. 8 mm long, ca. 4 mm wide; lower lip reflexed, ca. 4.5 mm long, 3-lobed, medium lobe largest, suborbicular, ca. 5 mm long, ca. 4.5 mm wide, apex emarginate, white, dotted with purplish-red spots, lateral lobes ovate, ca. 3.5 mm long, ca. 3 mm wide, apex obtuse, yellow. Stamens 4, inserted above middle and upper of corolla tube, straight, included, filaments flat, sparsely puberulent-villous, anther cells 2, ovoid, glabrous. Style filiform, included, glabrous, apex subequally 2-lobed, ca. 1 cm long. Ovary 4-loculed, truncate at apex. Nutlets triquetrous-oblong, ca. 2.5 mm long, apex sparsely hispid and glandular.

Figure 3. 

Line drawing of Paraphlomis qingyuanensis A plant B lateral view of flower C upper lip of flower D lateral view of calyx E inner view of calyx F style G dissected corolla and stamens H front view of calyx (Drawn by Zhong-Jin Wang).

Distribution, habitat and conservation status

Currently, only three populations of Paraphlomis qingyuanensis were found in Huanghua Town of Yingde City, Qingyuan City in Guangdong Province (Fig. 7A). These populations were located in the subtropical monsoon climate region, in a large area of karst landform. The distribution area of P. qingyuanensis is extremely fragmented, and it is not within a protected area. Human activity, such as forestry production and tourism, have a negative effect on population regeneration. Thus, P. qingyuanensis is here suggested to be endangered (EN) according to IUCN categories guidelines B2(a, b(iii)) (AOO < 500 km2, number of locations <5, and habitat affected by human activities) (IUCN Standards and Petitions Subcommittee 2022).

Figure 4. 

Morphological comparison of Paraphlomis qingyuanensis, P. brevifolia, P. baiwanensis, and P. subcoriacea. Paraphlomis qingyuanensis A plant of P. qingyuanensis B plant of P. brevifolia C plant of P. baiwanensis D plant of P. subcoriacea (Photographs by Wan-Yi Zhao; photographs of P. brevifolia from isotype W. Chen 84411, IBSC0005124).

Phenology

Flowering was observed from August to December, and fruiting from September to December.

Etymology

The specific epithet “qingyuanensis” is derived from the type locality of the new species, i.e. Qingyaun City in Guangdong Province, China.

Additional specimens examined

Paraphlomis qingyuanensis (paratypes): China. Guangdong Province: Qingyuan City, Yingde City, Huanghua Town, near Hegushi, on the limestone valley, 24°13'N, 112°56'E, alt. 132 m, 6 September 2023, Qin-Dai Xiong QYK-HH-156 (SYS); Yingde city, Huanghua town, Hegushi, 24°13'35.7"N, 112°56'08.97"E, alt. 114 m, 16 December 2023, Qiang Fan 20255 (SYS); Yingde city, Huanghua town, near Hegushi, 24°13'22.71"N, 112°56'05.71"E, alt. 159 m, 26 December 2023, Qiang Fan & Qin-Dai Xiong QYK-HH-1904 (SYS); Yingde city, Huanghua town, Huanghua park, 24°12'03.46"N, 112°54'10.05"E, alt. 205 m, 25 December 2023, Qiang Fan & Qin-Dai Xiong QYK-HH-1882 (SYS).

Paraphlomis baiwanensis W.Y.Zhao, Y.P.Chen & Q.Fan, sp. nov.

urn:lsid:ipni.org:names:77344363-1
Figs 4C, 5, 6 白湾假糙苏

Type

China. Guangdong Province: Qingyuan city, Qingxin district, Baiwan town, Hecang village, on the limestone cliff, 24°15'09.40"N, 112°46'47.32"E, alt. 310 m, 15 December 2023, Qiang Fan 20251 (holotype: SYS00236952!; isotypes: KUN, SYS00236953!).

Diagnosis

Paraphlomis baiwanensis is morphologically similar to P. subcoriacea, but differs from the latter by its stout stem, larger and leathery leaves, and larger flower.

Description

Herbs perennial, 50–80 cm tall; stoloniferous, with villous indumentum. Stems erect, stout, unbranched or 2–3 branched, 4-angled, diameter 3.6–5.3 mm, with densely retrorse pilose hairs. Leaves opposite; lamina long ovate to lanceolate, leathery, 12.5–18.7 cm long, 2.6–4.8 cm wide, apex acuminate, base cuneate or abruptly obtuse, margin serrulate; adaxially dark green, with densely pilose, abaxially light green with densely brown glandular, sparsely pilose, more densely on veins; lateral veins 5–7 (-8)-paired, raised abaxially and deeply impressed adaxially, anastomosing at leaf margin; petiole 0.6–1.3 cm long, densely pilose. Verticillasters borne in upper leaf axils, cymes (2-) 5–9-flowered; bracteoles inconspicuous, early deciduous; pedicels ca. 1.2–2.0 mm long, densely pilose. Calyx light green, obconical, 5.2–5.7 mm long, glabrous inside, outside with densely retrorse pilose, conspicuously 10-veined; calyx teeth 5, inconspicuous, throat appearing subtruncate, calyx veins extended into wings as 2.5–3.0 mm long. Corolla 1.5–2.1 cm long, white; tube ca. 7 mm long, ca. 2 mm in diam., straight, inside of throat with pubescent annulate; 2-lipped, villous outside, upper lip oblong, galeate, apex undulate, ca. 8–10 mm long, ca. 3.8–5.5 mm wide; lower lip reflexed, ca. 7 mm long, 3-lobed, medium lobe largest, suborbicular, ca. 6.5 mm long, ca. 6 mm wide, apex emarginate, lateral lobes ovate, ca. 4.5 mm long, ca. 3.2 mm wide, apex obtuse. Stamens 4, inserted above middle and upper of corolla tube, straight, included, filaments flat, 7–9 mm long, sparsely puberulent-villous, anther cells 2, ovoid, glabrous. Style filiform, included, glabrous, apex subequally 2-lobed, ca. 1 cm long. Ovary 4-loculed, truncate at apex. Nutlets triquetrous-oblong, ca. 3.8 mm long, apex sparsely hispid.

Figure 5. 

Paraphlomis baiwanensis A plant B young branch and leaves C flowering branch D leaves E lateral view of flower F front view of flower G lateral view of calyx H front view of calyx I seeds (Photographs: A, B, D, G, I by Wan-Yi Zhao; C by Qin-Dai Xiong; E-F, H by Qiang Fan).

Distribution, habitat and conservation status

Paraphlomis baiwanensis is currently known to occur only in Baiwan town, Guangdong in one population numbering less than one hundred individuals. It was observed to grow on limestone cliffs at altitudes about 300 m. Its known population was located in Qingxin Baiwan Provincial Nature Reserve of Guangdong which is well-protected. More field investigations are needed to determine its wild distribution. Therefore, the Protection level of Paraphlomis baiwanensis was suggested as Data Deficient (DD) based on the IUCN Red List Criteria (IUCN Standards and Petitions Subcommittee 2022).

Figure 6. 

Line drawing of Paraphlomis baiwanensis A plant B lateral view of a flower C frontal view of corolla D stamen E style F front view of calyx G lateral view of calyx tube H seed (Drawn by Rong-En Wu).

Phenology

Flowering was observed from June to September, and fruiting from August to December.

Etymology

The specific epithet “baiwanensis” is derived from the type locality of the new species, i.e. Qingxin Baiwan Provincial Nature Reserve of Guangdong, Qingyuan, China.

Figure 7. 

Geographic distribution and population genetic structure of Paraphlomis baiwanensis (P1), P. subcoriacea (P2–4), and P. qingyuanensis (P5–7) based on Admixture analysis A geographic distribution of the seven populations used for analysis B two-dimensional clustering of genetic variation from seven populations obtained via principal component analysis C Populations genetic structure result based on Admixture analysis.

Additional specimens examined

Paraphlomis baiwanensis (paratypes): China. Guangdong Province: Qingyuan city, Qingxin district, Baiwan town, Hecang village, on the limestone cliff, 24°15'09.40"N, 112°46'47.32"E, alt. 310 m, 23 August 2023,Yi-Wen Liu QYK-BW-1470 (SYS).

Paraphlomis brevifolia: China. Guangxi Province: Luocheng City, 1939, Wen Chen 84411 (IBSC0005124, isotype).

Paraphlomis subcoriacea: China. Guangdong Province: Qingyuan City, Yangshan City, Chengjia country, Wuyuan village, Baizhushan, 20 June 1956, Liang Deng 1572 (IBSC0005130, isotype); Yangshan City, Dalang Town, Chakeng village, 7 August 1936, Liang Deng 263 (IBK00059958; IBSC0585118); Yangshan City, Lingbei Town, Xiatianxia, 24°39'48.99"N, 112°45'41.73"E, alt. 295 m, 21 December 2023, Qiang Fan 20257 (SYS); Yangshan City, Lingbei Town, Yanzidong, 24°39'16.92"N, 112°46'59.95"E, alt. 156 m, 27 December 2023, Qiang Fan & Qin-Dai Xiong QYK-LB-1920 (SYS); Yangshan City, Chengjia Town, Baizhuling (collection site of type specimen), 24°39'39.56"N, 112°49'45.69"E, alt. 387 m, 27 December 2023, Qiang Fan & Qin-Dai Xiong QYK-CJ-1911 (SYS).

Acknowledgements

We thank Fu-Gang Li, Fu-Zhen Liu from Yingde Forestry Bureau and Xin-Yu Jia from Guangdong Qingxin Baiwan Provincial Nature Reserve for their help in the field work. We are deeply grateful to Zhong-Jin Wang, Rong-En Wu for their excellent illustrations in the manuscript.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by the Foundation of Qingyuan Forestry Bureau (HT-99982024-0030), Guangdong Provincial Ecological Forestry Development Project (2020141), the Yunnan Fundamental Research Project (202301AT070303), and Nanling Ecosystem Monitoring Project (GDEEMC-2023-19).

Author contributions

Q.F conceptualized and led the project. Field investigation and sample collection were conducted by Q.D.X, Q.F, R.M.W, Y.H.Z, and Z.B.X. W.YZ, and Q.D.X examined and detailed the fine structure of the species represented in the study. The phylogenetic analysis and population genetic analysis were carried out by Y.P.C, and Q.F. The initial draft of the manuscript was prepared by W.YZ and Y.P.C. All the authors approved the final manuscript.

Author ORCIDs

Wan-Yi Zhao https://orcid.org/0000-0002-6103-1236

Qin-Dai Xiong https://orcid.org/0009-0006-3645-7362

Rang-Min Wu https://orcid.org/0009-0004-3545-1933

You-Hong Zeng https://orcid.org/0009-0004-1171-5762

Zhi-Bin Xie https://orcid.org/0009-0004-9382-8073

Ya-Ping Chen https://orcid.org/0000-0002-7502-1848

Qiang Fan https://orcid.org/0000-0003-4254-6936

Data availability

The newly-obtained ITS and ETS sequences have been submitted to the NCBI (accession number: PP897029–PP897034; PP897950–PP897955). RAD raw data was deposited to the NCBI SRA database with BioProject PRJNA1107389. All of the other data that support the findings of this study are available in the main text.

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Appendix 1

Table A1.
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Sequence information for all samples used in present study. A “/” indicates a missing sequence. Herbarium abbreviations are listed after the vouchers. The accession numbers marked in bold represent sequences newly generated.

Taxon Voucher Country ITS ETS
Matsumurella chinensis (Benth.) Bendiksby 1 Y. Yang OYY00316 (KUN) Pingxiang, Jiangxi, China MW602147 MW602117
Matsumurella chinensis (Benth.) Bendiksby 2 Y. Yang OYY00131 (KUN) Guilin, Guangxi, China MW602148 MW602118
Matsumurella yangsoensis (Y.Z. Sun) Bendiksby L. Wu & W.B. Xu 10965 (IBK) Yangshuo, Guangxi, China MW602142 MW602112
Paraphlomis albida Hand.-Mazz. var. albida A. Liu et al. LK0841 (CSFI) Ningyuan, Hunan, China MW602124 MW602091
Paraphlomis albida var. brevidens Hand.-Mazz. Y.P. Chen EM312 (KUN) Hezhou, Guangxi, China MW602130 MW602098
Paraphlomis albiflora (Hemsl.) Hand.-Mazz. C.M. Tan et al. 1806393 (JJF) Jiujiang, Jiangxi, China / MW602101
Paraphlomis baiwanensis 1 Y.S. Chen et al. QY20230302 (IBSC) Qingyuan, Guangdong, China PP897029 PP897950
Paraphlomis baiwanensis 2 Q. Fan et al. 20251 (SYS) Qingyuan, Guangdong, China PP897030 PP897951
Paraphlomis caloneura K.J. Yan, Y.P. Chen & Y.F. Huang W.H. Wu et al. LHT1841 (KUN) Napo, Guangxi, China OQ627454 OQ628080
Paraphlomis coronata (Vaniot) Y.P. Chen & C.L. Xiang 1 E.D. Liu et al. 3043 (KUN) Emeishan, Sichuan, China MW602137 MW602107
Paraphlomis coronata (Vaniot) Y.P. Chen & C.L. Xiang 2 C.L. Xiang 358 (KUN) Jiangkou, Guizhou, China MW602123 MW602090
Paraphlomis foliata (Dunn) C.Y. Wu & H.W. Li subsp. foliata S.P. Chen s.n. (KUN) Jiangle, Fujian, China / MW602097
Paraphlomis foliata subsp. montigena X.H. Guo & S.B. Zhou Y.C. Dai s.n. (KUN) Hangzhou, Zhejiang, China OM836064 OM884453
Paraphlomis gracilis (Hemsl.) Kudô var. gracilis 1 A. Liu LK0931 (CSFI) Changsha, Hunan, China MW602134 MW602104
Paraphlomis gracilis (Hemsl.) Kudô var. gracilis 2 C.L. Xiang XCL1315 (KUN) Chongqing, China MW602141 MW602111
Paraphlomis gracilis var. lutienensis (Y.Z. Sun) C.Y. Wu C.L. Xiang XCL881 (KUN) Shibing, Guizhou, China MW602131 MW602099
Paraphlomis hirsutissima C.Y. Wu & H.W. Li Zhao & G. Chen XCL2115 (KUN) Malipo, Yunnan, China OQ627453 OQ628079
Paraphlomis hispida C.Y. Wu X. Li LX200702 (GXF) Napo, Guangxi, China MW602132 MW602102
Paraphlomis hsiwenii Y.P. Chen & Xiong Li 1 W.H. Wu et al. DD426 (KUN) Jingxi, Guangxi, China OP605346 OP609841
Paraphlomis hsiwenii Y.P. Chen & Xiong Li 2 W.H. Wu et al. DD426 (KUN) Jingxi, Guangxi, China OP605347 OP609842
Paraphlomis intermedia C.Y. Wu & H.W. Li X. Zhong et al. ZX16823 (CSH) Suichang, Zhejiang, China MW602135 MW602105
Paraphlomis javanica (Blume) Prain var. javanica 1 Y.P. Chen s.n. (KUN) Kunming, Yunnan, China MW602121 MW602088
Paraphlomis javanica (Blume) Prain var. javanica 2 L.B. Jia et al. JLB0029 (KUN) Maguan, Yunnan, China MW602143 MW602113
Paraphlomis javanica var. pteropoda D. Fang & K.J. Yan X. Li 2020090501 (GXF) Jingxi, Guangxi, China MW602140 MW602110
Paraphlomis jiangyongensis X.L. Yu & A. Liu 1 A. Liu et al. LK1104 (CSFI) Jiangyong, Hunan, China MW602128 MW602095
Paraphlomis jiangyongensis X.L. Yu & A. Liu 2 A. Liu et al. LK1104 (CSFI) Jiangyong, Hunan, China MW602129 MW602096
Paraphlomis kwangtungensis C.Y. Wu & H.W. Li Q. Fan et al. 19738 (SYS) Qujiang, Guangdong, China PP713070 PP706067
Paraphlomis lanceolata Hand.-Mazz. 1 C.Z. Huang s.n. (KUN) Guidong, Hunan, China MW602145 MW602115
Paraphlomis lanceolata Hand.-Mazz. 2 A. Liu et al. LK0825 (CSFI) Ningyuan, Hunan, China MW602146 MW602116
Paraphlomis lancidentata Y.Z. Sun X. Zhong et al. ZX16824 (CSH) Suichang, Zhejiang, China MW602136 MW602106
Paraphlomis longicalyx Y.P. Chen & C.L. Xiang Y.P. Chen et al. EM583 (KUN) Huanjiang, Guangxi, China OK104771 OK104774
Paraphlomis membranacea C.Y. Wu & H.W. Li M.S. Nuraliev 1057 (MW) Thanh Son, Phu Tho, Vietnam / MW602100
Paraphlomis nana Y.P. Chen, C. Xiong & C.L. Xiang 1 C. Xiong XC21097 (KUN) Chengkou, Chongqing, China OM836062 OM884451
Paraphlomis nana Y.P. Chen, C. Xiong & C.L. Xiang 2 C. Xiong & H.L. Zhou XC21126 (KUN) Wushan, Chongqing, China OM836063 OM884452
Paraphlomis octopus Q. Fan, Y.P. Chen & Ying Liu 1 Y.P. Chen & Y. Zhao EM1391 (KUN) Huaiji, Guangdong, China MW602126 MW602093
Paraphlomis octopus Q. Fan, Y.P. Chen & Ying Liu 2 Q. Fan et al. 19752 (SYS) Fengkai, Guangdong, China PP713071 PP706068
Paraphlomis octopus Q. Fan, Y.P. Chen & Ying Liu 3 Q. Fan et al. 19760 (SYS) Pingle, Guangxi, China PP713072 PP706069
Paraphlomis pagantha Dunn 1 L.X. Yuan et al. s.n. (KUN) Qionghai, Hainan, China OP605345 OP609840
Paraphlomis pagantha Dunn 2 X.Y. Jiang et al. HN001 (SYS) Wenchang, Hainan, China PP713073 PP706070
Paraphlomis patentisetulosa C.Y. Wu C.L. Su et al. XY015 (KUN) Xinyi, Guangdong, China OQ627455 OQ628081
Paraphlomis paucisetosa C.Y. Wu 1 X.X. Zhu s.n. (KUN) Malipo, Yunnan, China MW602125 MW602092
Paraphlomis paucisetosa C.Y. Wu 2 X. Li LX200704 (GXF) Napo, Guangxi, China MW602133 MW602103
Paraphlomis qingyuanensis 1 Q. Fan et al. QYK-HH-1904 (SYS) Yingde, Guangdong, China PP897031 PP897952
Paraphlomis qingyuanensis 2 Q. Fan et al. 20255 (SYS) Yingde, Guangdong, China PP897032 PP897953
Paraphlomis reflexa C.Y. Wu & H.W. Li Z.Z. Yang et al. s.n. (HIB) Tongshan, Hubei, China MW602122 MW602089
Paraphlomis strictiflora J.C. Yuan, B. Chen & C.L. Xiang B. Chen et al. CB05956 (CSH) Yinjiang, Guizhou, China / OP609839
Paraphlomis subcoriacea 1 Q. Fan et al. QYK-CJ-1911 (SYS) Yangshan, Guangdong, China PP897033 PP897954
Paraphlomis subcoriacea 2 Q. Fan et al. QYK-LB-1920 (SYS) Yangshan, Guangdong, China PP897034 PP897955
Paraphlomis yingdeensis W.Y. Zhao, Y.Q. Li & Q. Fan Q. Fan et al. 19013 (SYS) Yingde, Guangdong, China OP605348 OP609843
Phlomis fruticosa L. Y. Tong s.n. (KUN) Shanghai, China (cultivated) MW602119 MW602086
Phlomoides dentosa var. glabrescens (Danguy) C.L. Xiang & H. Peng Y.P. Chen EM360 (KUN) Beijing, China (cultivated) MW602120 MW602087

Appendix 2

Table A2.
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Populations information for PCA and Admixture analysis used in this study.

Population ID Species Voucher GPS Number of individual
P1 Paraphlomis baiwanensis Q. Fan 20251 (SYS); Hecang village, Baiwan town, Qingyuan city, Guangdong, China 24°15'09.40"N, 112°46'47.32"E 10
P2 Paraphlomis subcoriacea Q. Fan 20257 (SYS); Xiatianxia, Lingbei town, Yangshan city, Guangdong, China 24°39'48.99"N, 112°45'41.73"E 15
P3 Paraphlomis subcoriacea QYK-LB-1920 (SYS); Yanzidong, Lingbei town, Yangshan city, Guangdong, China 24°39'16.92"N, 112°46'59.95"E 6
P4 Paraphlomis subcoriacea QYK-CJ-1911 (SYS); Baizhuling, Chengjia country, Yangshan city, Guangdong, China 24°39'39.56"N, 112°49'45.69"E 15
P5 Paraphlomis qingyuanensis Q. Fan 20255 (SYS); Hegushi, Huanghua town, Yingde city, Guangdong, China 24°13'35.7"N, 112°56'08.97"E 14
P6 Paraphlomis qingyuanensis QYK-HH-1882 (SYS); Huanghua park, Huanghua town, Yingde city, Guangdong, China 24°12'03.46"N, 112°54'10.05"E 15
P7 Paraphlomis qingyuanensis QYK-HH-1904 (SYS); near Hegushi, Huanghua town, Yingde city, Guangdong, China 24°13'22.71"N, 112°56'05.71"E 10
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