a& PhytoKeys 237: 231-243 (2024) eel hyto Keys DOI: 10.3897/phytokeys.237.111977 Research Article The identity of Bupleurum jeholense (Apiaceae) Li-Hua Wang’, Xue-Min Xu2, Yi He’®, Quan-Ru Liu'® 1 Key Laboratory of Biodiversity Science and Ecological Engineering, Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China 2 School of Life Sciences, Zhengzhou University, Zhengzhou 450001, Henan China Corresponding author: Quan-Ru Liu (liuquanru@bnu.edu.cn) Abstract Bupleurum jeholense Nakai (Apioideae, Apiaceae), originally found in the Wuling Mountain of China, was initially described as a species but later treated as a variety of B. sibiricum Vest ex Spreng. However, researchers have recently found that it is more closely related to B. chinense DC. In this study, we conducted morphological and phylogenetic analyses as well as chromosome counting to determine the taxonomic status of B. sibiricum var. jeholense (Nakai) Chu. Our results showed that B. sibiricum var. jeholense and B. chinense share common features (i.e., bracteoles 5 and stem solid) that distinguish both from B. sibiricum var. sibiricum. The chromosome number of B. sibiricum var. jeholense was found to be the same as in B. chinense (i.e., 2n = 12), whereas the chromosome number of B. sibiricum var. sibiricum was 64. A phylogenetic tree based on complete chloroplast genome data revealed a close relationship between B. sibiricum var. jeholense and B. chinense. Finally, B. sibiricum var. jeholense and B. chinense were mainly found to differ in plant height, number of stems, and middle stem leaves. Based on this evidence, we propose a new combination: Bupleurum chinense var. jeholense (Nakai) Q.R.Liu & L.H.Wang. Key words: Apiaceae, Bup/eurum chinense DC. var. jeholense (Nakai) Q.R.Liu & L.H.Wang, new combination OPEN Qaccess Academic editor: Alexander Sennikov Received: 31 August 2023 Introduction Bupleurum is a monophyletic genus in the tribe Bupleureae (Apioideae, Apia- Accepted: 9 January 2024 ceae) (Downie et al. 2000). It contains around 180-195 species, is distributed Published: 31 January 2024 throughout Eurasia and North Africa, with one species each in North America and South Africa, while being adventive in Australasia (Plunkett et al. 2019). Citation: Wang L-H, Xu XM, He Y, Liu China is a major diversity center of Bupleurum, containing 42 species and 16 QR (2024) The identity of Bupleurum Varieties (Sheh and Watson 2005). This genus can be easily recognized based jeholense (Apiaceae). PhytoKeys 237: . ‘ : : ; . 231-243, https://doi.org/10.3897/ on its entire and single leaves, which usually show parallel venation and dis- phytokeys.237.111977 tinct bracts and bracteoles. However, the morphological characteristics used for species delimitation of this genus remain limited, thereby rendering species Convright Oui Huwangat al identification difficult. To address this problem, it is necessary to perform de- This is an open access article distributed under ~—s tailed examinations and observations in field populations. terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Zl Li-Hua Wang et al.: Bupleurum jeholense Bupleurum jeholense Nakai was initially described on the basis of specimens collected from the Wuling Mountain (China). Nakai stated that the characters of this species are as follows: middle stem leaves oblanceolate, base tapering, apex obtuse or acute, apiculate, bracteoles 5, exceeding flowers (Nakai 1937). Later, Chu (Shan and Li 1974) treated B. jeholense as a variety of B. sibiricum Vest ex Spreng, i.e., B. sibiricum var. jeholense (Nakai) Chu. Chu reported that B. jeholense was similar to B. sibiricum, with the main difference being in brac- teole number. For example, B. jeholense commonly has five bracteoles, where- as B. sibiricum usually has 7-12 bracteoles. This difference was thought to be associated with a geographic distribution, i.e., the transition from the main dis- tribution area in Siberia to the limited populations found in the Wuling Mountain (Liaoning Forestry Soil Research Institute 1977). During recent fieldwork on Dongling Mountain (Beijing, China), we found that the distribution of Bupleurum species was closely linked to altitude. Nor- mally, B. chinense is distributed at low altitudes (<1600 m). However, with the increasing altitude, we found a continuous variation in B. chinense; for example, plants became shorter; the number of branches decreased; the num- ber of bracteoles gradually changed from 3 to 5 and varied in length from obviously shorter than the umbel to almost equal. Furthermore, as the alti- tude increased to 1600 m, B. chinense was gradually replaced by B. sibiricum var. jeholense. After checking the specimens, we also found that there were misidentifications between B. chinense and B. sibiricum var. jeholense. Sub- sequent phylogenetic analyses of Chinese Bup/leurum spp. based on nrDNA ITS and chloroplast markers (i.e., trnH-psbA and matk) indicated that B. si- biricum var. jeholense was more closely related to B. chinense, whereas B. sibiricum var. sibiricum was closely related to B. smithii Wolff (Wang et al. 2011b). Hence, we doubt whether B. sibiricum var. jeholense is a morpholog- ical variation of B. chinense that is adapted to high-altitude locations in the Yan Mountains. We therefore collected and checked several specimens from the type locality of B. jeholense. We conducted morphological observations, statistical compar- isons, cytological studies, and a phylogenetic analysis based on the complete chloroplast genome to clarify the relationship among B. sibiricum var. jeho- lense, B. sibiricum var. sibiricum, and B. chinense. Materials and methods Morphological observations Using the existing scientific literature and the relevant type specimens, we col- lected new specimens from the type locality of B. sibiricum var. jeholense and compared these with images of the type specimens. Specimens from field col- lections, CVH (https://www.cvh.ac.cn/), and online herbarium collections (MW, LE) were used to count bracteoles. In total, we obtained bracteole count data for 129 specimens of B. sibiricum var. jeholense, 183 specimens of B. sibiricum var. sibiricum, and 183 specimens of B. chinense. We analyzed these data us- ing R (beanplot package) to produce boxplots (Kampstra 2008). Morphological terminology was used according to Kljuykov et al. (2004). Voucher specimens were deposited to the BNU herbarium. PhytoKeys 237: 231-243 (2024), DOI: 10.3897/phytokeys.237.111977 999 Li-Hua Wang et al.: Bupleurum jeholense Cytology All materials used for cytological studies were obtained from the Wuling Moun- tain (B. sibiricum var. jeholense), Wutai Mountain (B. chinense), and Daging Mountain (B. sibiricum var. sibiricum). All voucher specimens are listed in Table 1. Chromosome preparations were produced using acid digestion and hypoton- ic wall removal and photographed. Three technical replicates were obtained for each taxon. This procedure was adapted from Li et al. (2021). Sampling and molecular analysis Fresh plant leaves were collected from the field and quickly dried with silica gel for DNA extraction. Specimen voucher information is shown in Table 1. DNA was extracted using an HP Plant DNA hypotonic (D2485-02; Omega Bio- Tek). DNA samples were then sent to Beijing Novogene Corporation for qual- ity testing and resequencing. An Illumina HiSeq X sequencing platform was used to generate approximately 10 GB of data for each sample. The chloroplast genome was then assembled from clean data using GetOrganelle (Jin et al. 2020). PGA (Qu et al. 2019) was used to annotate the resulting chloroplast genome. Sequences for B. yinchowense Shan et Y.Li (MT075711) and B. sikan- gense X.J.He & C.B.Wang (NC056803) were downloaded from NCBI (https:// www.ncbi.nim.nih.gov/nucleotide/) to be used as references. All chloroplast genome sequences generated here were deposited in the NCBI GenBank data- base (accession numbers listed in Table 1). Finally, twenty-one plastid genome sequences were downloaded from NCBI (see Appendix) for phylogenetic com- parisons. This included 19 species of Bup/eurum and two species of Pleuros- permum Hoffm., which were used as outgroups. In total, 25 sequences were imported into PhyloSuite (Zhang et al. 2020). The mafft module (Katoh et al. 2019) was used for sequence alignment, and the ModelFinder module (Kalyaanamoorthy et al. 2017) was used to calculate the nucleotide substitution model for all aligned sequences. A maximum likeli- hood (ML) tree was then constructed using IQ-TREE (Minh et al. 2020), with the nucleotide substitution model set to TVM+F+R2 and a standard bootstrap val- ue of 1000. Results were considered reliable when the bootstrap support value (BS) was 270% (Kress et al. 2002). A Bayesian (BI) tree was constructed using MrBayes (Huelsenbeck and Ronquist 2001), with the GTR+F+I+G4 model using the following settings: mcmcp ngen = 2,000,000, printfregq = 10,000, nchains = 4, and burninfrac = 25%. Results were considered reliable when the posterior probability (PP) was 20.95. The effective sample size (>200) was determined using Tracer version 1.7 (Rambaut et al. 2018). Table 1. Voucher information and GenBank accession numbers for newly sequenced plastome sequences. Taxon B. chinense B. smithii Location Voucher information Accession Dongling Mountain, Beijing, China BNU2021HBO002 (BNU) OR387523 Xiaowutai Mountain, Hebei, China BNU2020DT007(BNU) OR387522 B. sibiricum var. sibiricum | Daqing Mountain, Inner Mongolia, China | BNU2021NMG017(BNU) OR387525 B. sibiricum var. jeholense Wuling Mountain, Hebei, China BNU2021HB025(BNU) OR387524 PhytoKeys 237: 231-243 (2024), DOI: 10.3897/phytokeys.237.111977 933 Li-Hua Wang et al.: Bupleurum jeholense Results Morphological observations The bean plot indicated that the number of bracteoles of B. sibiricum var. je- holense was mostly 5 and occasionally 6, whereas that of B. sibiricum var. sibiricum was (6)7-8(9). Bupleurum chinense had 5, sometimes 4 or even 3 bracteoles at lower elevations or in an understory (Figs 1, 2). The stems of B. chinense and B. sibiricum var. jeholense were solid, lacking a cavity, where- as those of B. sibiricum var. sibiricum were hollow at all internodes, which led to the formation of a cavity (Fig. 3). Bupleurum sibiricum var. jeholense and B. chinense were found to mainly differ in height, number of stems, and pres- ence of middle stem leaves. The morphological characteristics of B. sibiricum var. jeholense plants are as follows: height below 40 cm, several stems, with 1-2 branches per stem, middle stem leaves narrower, and middle leaf length to width ratio 10-16. In contrast, B. chinense plants had single, occasionally several stem 40-90 cm high, with 2-4 branches per stem, and middle stem leaf length to width ratio 6-10. A comparison of morphological characters is shown in Table 2. Chromosome numbers Cytological analysis revealed that the chromosome number of B. sibiricum var. jeholense was 2n = 12 (x = 6), i.e., the same as B. chinense but different from B. sibiricum var. sibiricum (2n = 64, x = 8) (Fig. 4). We report the chromosome number of B. sibiricum var. jeholense for the first time, and the chromosome numbers of B. chinense and B. sibiricum var. sibiricum determined here are con- sistent with previous reports (Pan et al. 1995; Qin et al. 1989). Number of bracteoles B. chinense _ B. sibiricum var. jeholense B. sibiricum var. sibiricum Species Figure 1. Comparison of the number of bracteoles found in Bupleurum chinense, B. sibir- icum var. jeholense and B. sibiricum var. sibiricum. PhytoKeys 237: 231-243 (2024), DOI: 10.3897/phytokeys.237.111977 934 Li-Hua Wang et al.: Bupleurum jeholense Imm Figure 2. Umbel and bracteoles of the taxa under study A Bupleurum chinense B B. sibiricum var. jeholense C B. sibiricum var. sibiricum. Figure 3. Stem transections of the taxa under study A Bupleurum chinense B B. sibiricum var. jeholense C B. sibiricum var. sibiricum. Table 2. Comparison of the morphological characteristics and geographic distributions of three taxa under study. Character Height Stem (number) Stem (branching) Stem (presence of cavity) Basal leaves Basal leaf size Middle stem leaves Middle stem leaf length to width ratio Upper stem leaves Bracteole relative length Bracteoles Distribution B. chinense 40-90 cm Single, occasionally several 2-4-branched Solid Withering early 4-7 x 0.6-0.8 cm 4-12 x 0.6-1.8 cm 6-10 Not embracing Shorter than flowers (3-4) 5, lanceolate Northeast and Central China PhytoKeys 237: 231-243 (2024), DOI: 10.3897/phytokeys.237.111977 B. sibiricum var. jeholense 20-40 cm Many, clustered 1-2-branched Solid Withering early 9-10 x 0.3-0.8 cm 6=12%0551.2-cm 10-16 Not embracing Shorter than or equal to flowers 5 (6-7), lanceolate Yan mountains (Hebei and Beijing) B. sibiricum var. sibiricum 30-70 cm Many, clustered 1-2-branched Hollow in all internodes Persistent, many 1:2=25.%:0:7-1.6:em 6-14 x 0.5-1.6 cm 10-14 Rounded-cuneate, embracing Exceeding flowers (6) 7-12, elliptic-lanceolate Heilongjiang, Liaoning, Inner Mongolia, Mongolia, and Russia 235 Li-Hua Wang et al.: Bupleurum jeholense is "a Figure 4. Metaphase chromosomes of the taxa under study A Bupleurum chinense BB. sibiricum var. jeholense C B. sibiri- cum var. sibiricum. Phylogenetic analysis The size of the four Bupleurum chloroplast genomes ranged from 155,706 to 155,858 bp. The complete chloroplast genome had a typical circular quadri- partite structure and consisted of a pair of inverted repeat regions separated by the large single copy and small single copy regions. The topologies of the ML and BI trees constructed using the cp genome sequences were consistent. These results showed that the genus Bupleurum can be divided into two clades, with all Chinese Bup/eurum plants belonging to B. subg. Bupleurum. Bupleurum sibiricum var. sibiricum was found to cluster with B. smithii and B. bicaule Helm (BS = 100% PP = 1), whereas B. chinense was found to cluster with B. sibiricum var. jeholense (BS = 100% PP = 1) and was more distantly related to B. longira- diatum Turcz., B. falcatum L. and B. boissieuanum H. Wolff (Fig. 5). Discussion Comparative morphology Botanists have closely monitored the number of bracteoles because this charac- ter is essential in the taxonomy of Bupleurum. Moreover, bracteole number is an important indicator of the taxonomic position of B. sibiricum var. jeholense in the genus. Five bracteoles are generally present in both B. chinense and B. sibiricum var. jeholense. However, in environments such as the understory, B. chinense may have only three or four bracteoles. Furthermore, 6-7 bracteoles have occa- sionally been observed in B. sibiricum var. jeholense. We observed that the num- ber of bracteoles in B. sibiricum samples collected from the Daging Mountain, which were collected at the same latitude and altitude as the samples collected from Wuling Mountain, was not 5, as would be expected for B. sibiricum var. jeholense. Thus, we speculated that the 5-bracteoled B. sibiricum var. jeholense may not have evolved from the 12-bracteoled B. sibiricum var. sibiricum. In the classification of the genus Bup/eurum, stem structural characteristics have rarely been examined. After observing numerous specimens, we found PhytoKeys 237: 231-243 (2024), DOI: 10.3897/phytokeys.237.111977 036 Li-Hua Wang et al.: Bupleurum jeholense Pleurospermum szechenyii NCO71808 P. astrantioideum NCO71798 Bupleurum gibraltaricum NC060427 700 -“ L_____ B. fruticosum NC060426 B. shanianum NC058629 hamiltonii NC056298 candollei MT261183 rockii MT261189 marginatum MN968501 thianschanicum MT261192 densiflorum MT261184 euphorbioides NC053888 sibiricum MT261190 sibiricum Outgroup Subg. Penninervia 100, 1 100, 1 Subg. Bupleurum smithii bicaule NC057641 angustissimum NC057639 sikangense NC056803 . commelynoideum NC056802 . boissieuanum NC036017 . falcatum NC027834 . longiradiatum MT261186 . chinense MN854379 . sibiricum var. jeholense & chinense MNBS4379 Jehatense r LB ohinense by tt ty oh to oo bb iS] . chinense Figure 5. Phylogenetic tree of some Chinese Bupleurum species as inferred from chloroplast genomes using ML analy- ses (numbers below branches represent ML bootstrap values and BI posterior probabilities). that the stem of B. sibiricum var. sibiricum was hollow and contained a sub- stantial cavity. In contrast, the stems of B. chinense and B. sibiricum var. jeho- lense lacked this cavity. The presence or absence of a stem cavity is a stable character and does not change with the period of growth. For example, B. ko- marovianum was once treated as a variety of B. chinense (Liaoning Forestry Soil Research Institute 1977) until Wang et al. (2011b) found that the stems of B. komarovianum were hollow. The authors combined morphological and chromosomal evidence to argue that B. komarovianum should be reinstated as a separate species. Finally, the basal leaves of B. sibiricum var. sibiricum were found to be persistent, whereas those of B. sibiricum var. jeholense were found to wither at the flowering and fruiting stages, as in B. chinense. Bupleurum chinense is widely distributed throughout East Asia and is often cultivated as a medicinal plant. The morphology of this species varies with the environment. Bup/eurum sibiricum var. jeholense may be a variant of B. chinense that has adapted to the cold environments found in high-altitude mountains. In particular, the bracteole number may have increased to protect flowers at higher altitudes (Kofidis et al. 2007). Compared with B. chinense plants, B. sibir- icum var. jeholense plants are shorter, with several stems, fewer stem branches, and narrower middle stem leaves. Overall, the above evidence suggests that B. sibiricum var. jeholense should be treated as a variety of B. chinense. Cytological analysis Chromosomal variation plays a vital role in species formation, and the di- versity of chromosome size and number is therefore an important character that can be used to track Bupleurum species that have adapted to differ- ent habitats (Wang 2011a; Weiss-Schneeweiss and Schneeweiss 2013). PhytoKeys 237: 231-243 (2024), DOI: 10.3897/phytokeys.237.111977 997 Li-Hua Wang et al.: Bupleurum jeholense Bupleurum sibiricum var. jeholense differs from B. sibiricum var. sibiricum in both chromosome number and basal number, but these values are iden- tical to those of B. chinense. In this study, B. chinense and B. sibiricum var. jeholense were both found to have a chromosome number of 12 (diploid), which is the common basic number reported for this genus (Wang 2011a). Regarding the reported chromosome number of 64 in B. sibiricum from a population collected in Inner Mongolia, Qin et al. (1989) and Jiang et al. (2002) assumed that B. sibiricum was octoploid (i.e., with a basal number of 8), marking the highest level of polyploidy found in this genus. This ob- servation may be due to chromosomal polyploidy, a type common in colder climates (Jiang et al. 2002). Thus, in terms of both chromosome number and basal number, B. sibiricum var. jeholense appears to be only distantly related to B. sibiricum var. sibiricum. Phylogenetic analysis In this study, a phylogenetic tree was reconstructed using chloroplast genome data. Our findings were consistent with those of Wang et al. (2011b). In particular, we found that Bupleurum sibiricum var. jeholense was embedded in B. chinense, which forms a sister clade with B. yinchowense. In addition, B. sibiricum var. sibir- icum is sister to B. smithii and B. bicaule. In contrast to B. chinense, the distribu- tions of B. sibiricum and B. bicaule are ranging from Siberia to northeastern China. Distribution Bupleurum sibiricum is distributed widely throughout temperate Asia. It often co-occurs with B. scorzonerifolium in arid meadows in Inner Mongolia and Sibe- ria at elevations of 700-2000 m. In the field, B. sibiricum var. jeholense is often found to co-occur with B. chinense at different altitudes in the same mountain. At present, B. sibiricum var. jeholense is found only in high-altitude areas of the Yan Mountains. Moreover, it has a narrow distribution area and is confined to altitudes of 1500-2000 m. In contrast, B. chinense is widely distributed and is found at altitudes ranging from 200 to 1600 m in Northeast China; however, it has also been found isolated at an altitude of 2100 m in Northwest China. Given these findings, we speculate that B. sibiricum var. jeholense is a special- ized morphological variant of B. chinense that has specifically adapted to high altitudes. Overall, in terms of distribution and habitat, B. sibiricum var. jeholense differs considerably from B. sibiricum and is more similar to B. chinense. Taxonomic treatment Bupleurum chinense var. jeholense (Nakai) Q.R.Liu & L.H.Wang, comb. nov. urn:lsid:ipni.org:names:77335467-1 — Bupleurum jeholense Nakai in J. Jap. Bot. 13: 482 (1937) — Bupleurum sibiricum var. jeholense (Nakai) Chu in Shan & Li, Acta Phytotax. Sin. 12 (3): 272 (1974). = Bupleurum jeholense var. latifolium Nakai in J. Jap. Bot. 13: 482 (1937). Type. CHINA. Hebei: Wuling Mountain, 1800 m, 2 Sept 1933, Nakai, Honda et Kitagawa s. n. (holotype: T1I0082957!). PhytoKeys 237: 231-243 (2024), DOI: 10.3897/phytokeys.237.111977 038 Li-Hua Wang et al.: Bupleurum jeholense ayague> @ TR TET ET CT DISSV19 49499Y910]09 PRP EeMORPMTTTTONTTOTTONTTTT (UC CTTO ee ree enna recor eer) Plants of Jehol collected by The First Scientific Expedition to Jehol in 1933. Nodew av 444 2 Locality IE. BRS ai Date sti. 7 leg ane Lat Collectors: Prof. T. Naxar, Dr. M. Honpa, Mr, M. Kiracawa, Figure 6. lsotype of Bupleurum chinense var. jeholense (Photo from TI Herbarium). PhytoKeys 237: 231-243 (2024), DOI: 10.3897/phytokeys.237.111977 ee | 239 Li-Hua Wang et al.: Bupleurum jeholense Figure 7. Bupleurum chinense var. jeholense A plant B umbel and bract C flowers D umbel (side view) E pollen F trans- verse section of mericarp. 240 PhytoKeys 237: 231-243 (2024), DOI: 10.3897/phytokeys.237.111977 Li-Hua Wang et al.: Bupleurum jeholense Type. CHINA. Hebei: Wuling Mountain, 1500-2000 m, 2 Sept 1933, Nakai, Hon- da et Kitagawa, s. n. (holotype: not barcode, fig in protologue pp 481!:; isotypes: T10082958! T10082959! (Fig. 6) TI0082960! T10082961)], Diagnosis. Bupleurum chinense var. jeholense is morphologically similar to var. chinense and can be distinguished from the latter by plant size (>40 cm), the presence of multiple stems, with 1-2 branches per stem, thinner middle stem leaves, leaf length to width ratio 10-16, and the presence of 5 bracteoles. Description. Plants 20-40 cm, perennial. Root stout, brown, woody. Stem solitary or several, solid, petioles often purplish-red, clasping base without fi- brous remnant sheaths. Basal leaves oblanceolate, 5-10 x 0.3-0.8 cm, base petioles, apex acuminate. Middle leaves sessile, oblanceolate, 4.5-12 x 0.4- 1.4 cm, 7-9-nerved, base tapering, apex obtuse or acute, apiculate. Apical leaves small. Umbels 5-12, nearly equal or unequal rays 0.4—4 cm long; bracts of 1-5 unequal leaflets, often obsolete or deciduous, 3-15 x 0.6-11 mm, 4-7-nerved; bracteoles 5, lanceolate, 3-7 x 0.6-0.8 mm, exceeding flowers; umbellule 4-12 mm across, 8-14-flowered. Petals bright yellow. Stylopodium low-conic, discoid, dark yellow. Fruit oblong, brown, ca. 2.2-3.5 x 0.9-1.5 mm; ribs prominent, narrowly winged, wings pale brown; vittae 3(—4) in each furrow, 4 on commissure (Fig. 7). Fl. July-August and Fr. August-October. 2n = 12. Phenology. Flowering and fruiting from August to October. Distribution and habit. Hebei, Beijing. It grows in mountains at elevations of 1500-2000 m. Additional specimens examined. CHINA. Beijing: Fangshan county, Jin-Wu Wang s.n. (PEY!); Mentougou county, Xiao-Liu QS-186 (BJFC!); Quan-Ru Liu 200609009 (BNU!); Xue Lin 05 05-4-114 (BJFC!); Gang-Min Zhang 201008036 (BJFC!); Xian-Yun Mu 1821 (BJFC!); Duan-Zheng Lu s.n. (BUFC!); Quan-Ru Liu DLO25-2 (BNU!); Quan-Ru Liu DLO26-2 (BNU!); Quan-Ru Liu DLO27-1(BNU!); Mi- yun county, Xian-Yun Mu 1924 (BJFC!). Hebei: Xinglong county, Li-Hua Wang BNU2021HB029 (BNU!); Li-Hua Wang BNU2021HB025 (BNU!); Jia-Yi Liu 0845 2190 (TIE!); Jin-Wu Wang s.n. (PEY!); Zhen-Fu Fang 825 (NAS!); Ze-Hui Pan 83940 (NAS!); Ze-Hui Pan 83939 (NAS!); Shen-E Liu 4833 (IFP!); Zhen-Fu Fang 826 (IFP!); Quan-Ru Liu WLS068 (BNU!); Xin-Yuan Liu 1652 (KUN!); Jia-Yi Liu 08450 (TIE!); Jia-Yi Liu 00485 (TIE!); Biaobenshi 2190 (PE!); Wu-Xiu Zhang 917 (PE!). Acknowledgements We gratefully acknowledge the collaboration of the curators and staff of the herbaria mentioned in the text for their assistance upon visits, as well as for ac- cess to digital specimen images. Special thanks are due to Professor Akiko of the University of Tokyo for kindly providing photographs of the type specimens. We thank Dr. Dan-Hui Liu for the analyses of molecular sequence data and Xun Sun for the field assistance. Additional information Conflict of interest The authors have declared that no competing interests exist. PhytoKeys 237: 231-243 (2024), DOI: 10.3897/phytokeys.237.111977 51 Li-Hua Wang et al.: Bupleurum jeholense Ethical statement No ethical statement was reported. Funding This research was funded by the Biodiversity survey and assessment project of the Min- istry of Ecological Environment (2019HJ2096001006). Author contributions Funding acquisition: QRL. Investigation: LHW. Software: XMX, LHW. Supervision: QRL. Writing — review and editing: YH, XMX, LHW. Author ORCIDs Yi He © https://orcid.org/0000-0002-6925-7299 Quan-Ru Liu © https://orcid.org/0000-0003-4270-4746 Data availability All of the data that support the findings of this study are available in the main text. References Downie SR, Katz-Downie DS, Watson MF (2000) A phylogeny of the flowering plant fam- ily Apiaceae based on chloroplast DNA rpl16 and rpoC1 intron sequences: Towards a suprageneric classification of subfamily Apioideae. American Journal of Botany 87(2): 273-292. https://doi.org/10.2307/2656915 Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. 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