Balanophora xinfeniae (Balanophoraceae), a new species from Xizang, China

241 Balanophora xinfeniae (Balanophoraceae), a new species from Xizang, China Chen-Long Fu1, Jia-Ning Zhou1, Wei-Hua Liao1, Tong Zhang2, Bo Xu3, Meng Li1 1 Co-Innovation Center for the Sustainable Forestry in Southern China, College of life sciences, Nanjing Forestry University, Nanjing 210037, Jiangsu, China 2 Central South Inventory and Planning Institute of National Forestry and Grassland Administration, Changsha, 410014, China 3 CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610213, Sichuan, China Corresponding authors: Bo Xu ([email protected]); Meng Li ([email protected]) Copyright: © Chen-Long Fu et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Balanophora xinfeniae C.L.Fu, M.Li & B.Xu, a new species discovered in Xizang, China, is described and illustrated here. Molecular phylogenetic analyses and morphological comparisons strongly support B. xinfeniae as a new species within the genus Balanophora. The most distinctive characteristic of the new species is its dioecious sexual system, with male flowers having a 3-lobed perianth, opposite leaves with serrated tips, and yellow scapes. Although it shares morphological similarities with B. henryi, B. xinfeniae can be distinguished by its flat spheroid tubers without stellate lenticels, yellow scape, prominently serrated apical leaf margins, and nearly spherical or ovoid-ellipsoid female inflorescences. Key words: Balanophora xinfeniae, China, holoparasitic plants, phylogeny, taxonomy Introduction Balanophora J. R. Forst. & G. Forst. is a genus of holoparasitic plants consisting of about 25 species distributed mainly in tropical Southeast and East Asia (Hansen 1972; Mutinda et al. 2024). In traditional medicine, species of Balanophora are often used to treat hemorrhoids, stomach aches, and liver ailments and can also be used as antipyretics (Teng et al. 2000; Li et al. 2006; Trakulsomboon et al. 2006; Mutinda et al. 2024). Regarding their lifestyle, Balanophora comprises highly specialized root holoparasites characterized by underground connections with their hosts. Over an extended period of adaptive evolution, these plants have experienced significant gene loss from their genomes (Su et al. 2019; Chen et al. 2023) and extensive morphological reduction, leaving only a limited number of traits available for taxonomic classification (Job and Bertel 2015; Yu et al. 2022). Morphologically, Balanophora can be readily divided into two subgenera: subgenus Balanophora (4to 6-merous male flowers) and subgenus Balania (3-merous male flowers), a result that is also supported by molecular systematics (Yu et al. 2022). However, resolving relationships among these subgenera at the species level continues to be a challenging task. DNA regions with Academic editor: Marcos A. Caraballo-Ortiz Received: 1 January 2025 Accepted: 5 November 2025 Published: 19 November 2025 Citation: Fu C-L, Zhou J-N, Liao W-H, Zhang T, Xu B, Li M (2025) Balanophora xinfeniae (Balanophoraceae), a new species from Xizang, China. PhytoKeys 266: 241–252. https://doi.org/10.3897/ phytokeys.266.147400 PhytoKeys 266: 241–252 (2025) DOI: 10.3897/phytokeys.266.147400 242 PhytoKeys 266: 241–252 (2025), DOI: 10.3897/phytokeys.266.147400 Chen-Long Fu et al.: Balanophora xinfeniae (Balanophoraceae) from China high substitution rates provide effective tools to evaluate the monophyly of Balanophora species defined by morphological traits. Recent studies have improved our understanding of species boundaries within this genus by comparing classifications based on morphological characters with results from molecular phylogenetic analyses (Hsiao et al. 2010; Su et al. 2012; Yu et al. 2021). For instance, Hansen (1972) considered the morphological differences among several species as intraspecific variation, synonymizing them under B. harlandii. In contrast, a more recent study based primarily on molecular phylogenetic analysis provided support for recognizing three distinct taxa—B. harlandii, B. henryi, and B. kawakamii—with clades corresponding to these taxa being strongly supported by molecular data (Yu et al. 2022). During our fieldwork from April to May 2024 in the forests near Gelin Village, Motuo County, Xizang (China), we discovered a Balanophora that differed from all previously described species reported in China, India, Nepal, and other countries. Through morphological observations and the construction of a phylogenetic tree using nuclear ribosomal internal transcribed spacer (ITS) sequences, we concluded that this taxon represents an undescribed species of Balanophora. Materials and methods Morphological study Morphological observations were conducted based on living plants in the field. Measurements were performed manually with rulers in the field or with the software ImageJ in the laboratory using photographs. We restricted morphological comparisons to three closely related species. At the same time, all images of specimens potentially related to the new species from China, India, Nepal, and other countries deposited in the herbaria A, CDBI, CDCM, GAC, HHBG, IBSC, IBK, LBG, K, KUN, NAS, P, PE, SHM, and ZM were observed and studied (acronyms follow Thiers 2024). The terminology used in the morphological description follows the Flora of China (Huang and Murata 2003). Observations of flowers and claviform bodies were carried out using an Olympus SZX10 stereomicroscope. Phylogenetic study DNA extractions, PCR procedures, and primers followed Yu et al. (2021). The ITS region of the new species was sequenced using the Sanger method. In addition, we downloaded ITS sequences of other Balanophora species available in the Nucleotide database of NCBI (https://www.ncbi.nlm.nih.gov). Langsdorffia hypogaea was selected as the outgroup for this study based on the results presented by Sanchez-Puerta et al. (2023). A total of 54 samples were used in this study, including multiple accessions for some species (Table 1). All sequences were aligned using MAFFT v7.526 following default parameters (Katoh et al. 2002; Katoh and Standley 2013), and the ends were manually trimmed. The model TPM3u+F+I+R2 for ML analysis and TPM3uf+I+G for BI analysis was selected based on the Bayesian Information Criterion (BIC) using jModelTest v2.1.10 (Darriba et al. 2012). Maximum likelihood (ML) analyses were performed using IQ-TREE v2.3.6 with 10,000 ultrafast bootstrap approximations (Minh et al. 2020). A Bayesian inference (BI) analysis was performed using 243 PhytoKeys 266: 241–252 (2025), DOI: 10.3897/phytokeys.266.147400 Chen-Long Fu et al.: Balanophora xinfeniae (Balanophoraceae) from China MrBayes v3.2.7 (Ronquist et al. 2012). A Markov chain Monte Carlo analysis was conducted for 20,000,000 generations with one cold and three hot chains, each starting from a random tree and sampled every 1,000 generations. The first 25% of the trees were discarded as burn-in, and the remaining trees were used to construct majority-rule consensus trees. The final trees obtained from the ML and BI analyses were visualized using FigTree v1.4.2 (Rambaut 2009). Results and discussion The circumscription of B. flava, B. fargesii, and B. involucrata remains a persistent taxonomic contention. Geng (2016) proposed that B. fargesii and B. involucrata are sister groups with distinct clades based on ITS sequences. In contrast, Lidén (2019) suggested that B. fargesii is synonymous with B. involucrata, while Yu et al. (2022) posited that B. flava and B. fargesii are synonymous with B. involucrata. In this study, these species are considered part of the B. involucrata complex, and we are not addressing taxonomic divisions within this group here. Instead, for clarity and to facilitate morphological comparisons, we refer to the dioecious individuals within the complex as B. flava and the monoecious individuals as B. involucrata. Table 1. Taxa used in the phylogenetic analyses with their GenBank accession numbers. Species Accession number Species Accession number B. appressa MW414662 B. laxiflora JN392888 B. appressa MW414670 B. laxiflora JN392889 B. flava OM103570 B. laxiflora MW414661 B. flava OM103567 B. laxiflora MW414659 B. fungosa JN392881 B. laxiflora JN392890 B. harlandii OM103582 B. laxiflora JN392891 B. harlandii OM103583 B. laxiflora JN392892 B. harlandii OM103585 B. laxiflora JN392893 B. henryi OM103573 B. parajaponica MT482093 B. henryi OM103574 B. parajaponica MT498081 B. henryi OM103575 B. parajaponica MW414666 B. henryi OM103576 B. parajaponica MW414667 B. henryi OM103577 B. reflexa EU598798 B. indica JN392880 B. reflexa KT763380 B. involucrata OM103568 B. subcupularis OM103584 B. involucrata OM103569 B. subcupularis OM418783 B. involucrata OM103571 B. subcupularis OM418784 B. involucrata OM103572 B. tobiracola JN392901 B. japonica JN392882 B. tobiracola OM103578 B. japonica MW414664 B. xinfeniae PQ640032 B. japonica MW414665 B. xinfeniae PQ640033 B. kawakamii JN392900 B. yakushimensis JN392894 B. kawakamii OM103579 B. yakushimensis MW414660 B. kawakamii OM103580 B. yakushimensis MW414663 B. kawakamii OM103581 B. yakushimensis MW414668 B. latisepala OM418779 B. yakushimensis MW414669 B. latisepala OP120928 Langsdorffia hypogaea KT709672 244 PhytoKeys 266: 241–252 (2025), DOI: 10.3897/phytokeys.266.147400 Chen-Long Fu et al.: Balanophora xinfeniae (Balanophoraceae) from China The topologies of the phylogenetic trees constructed by the ML and BI methods are highly consistent (Suppl. materials 1, 2). Our results showed that two samples of the new species were well resolved as a distinct clade, which is the sister group to the B. involucrata complex (BS = 1; PP = 98%) (Fig. 1). Figure 1. The cladogram of Balanophora constructed using maximum likelihood (left) and Bayesian inference (right) based on ITS sequences. Numbers at nodes indicate posterior probabilities (PP) or bootstrap percentages (BS). Branches highlighted in red indicate topological incongruence between the two trees. Labels for concordant topologies are not displayed on the BI tree. B. laxiflora JN392891 B. henryi OM103573 B. japonica MW414664 B. laxiflora JN392889 B. latisepala OP120928 B. japonica MW414665 B. flava OM103567 B. subcupularis OM103584 B. kawakamii OM103579 B. latisepala OM418779 B. laxiflora JN392892 B. japonica JN392882 B. flava OM103570 B. yakushimensis JN392894 B. laxiflora JN392893 B. harlandii OM103585 B. parajaponica MT482093 B. tobiracola JN392901 B. appressa MW414670 B. appressa MW414662 B. kawakamii OM103580 B. henryi OM103577 B. involucrata OM103569 B. henryi OM103574 B. laxiflora MW414659 B. involucrata OM103568 B. yakushimensis MW414668 B. involucrata OM103571 B. yakushimensis MW414660 B. parajaponica MW414666 Langsdorffia hypogaea KT709672 B. involucrata OM103572. B. laxiflora MW414661 B. parajaponica MW414667 B. harlandii OM103583 B. subcupularis OM418784 B. subcupularis OM418783 B. yakushimensis MW414669 B. reflexa KT763380 B. kawakamii OM103581 B. kawakamii JN392900 B. yakushimensis MW414663 B. xinfeniae PQ640032 B. henryi OM103576 B. laxiflora JN392888 B. reflexa EU598798 B. xinfeniae PQ640033 B. henryi OM103575 B. parajaponica MT498081 B. fungosa JN392881 B. indica JN392880 B. tobiracola OM103578 B. laxiflora JN392890 B. harlandii OM103582 99 98 100 97 92 97 100 19 36 100 84 71 98 49 100 100 100 100 100 100 100 67 95 80 100 53 100 59 100 99 57 100 100 100 100 91 100 100 100 56 58 100 98 100 0.98 0.34 1 0.87 0.35 1 OM418783 OM418784 OM103584 JN392889 JN392893 JN392892 JN392890 JN392891 0.38 0.38 1 1 0.32 1 1 0.92 1 0.45 0.78 0.81 1 0.39 0.08 0.12 0.12 1 1 1 1 1 1 1 0.94 0.89 0.33 1 1 0.97 0.73 1 1 1 0.69 0.18 0.18 OM103574 OM103577 OM103575 OM103576 1 0.40 0.87 0.43 1 0.98 0.86 0.45 ML tree BI tree 245 PhytoKeys 266: 241–252 (2025), DOI: 10.3897/phytokeys.266.147400 Chen-Long Fu et al.: Balanophora xinfeniae (Balanophoraceae) from China The new species described in this study is morphologically most similar to B. henryi, along with the closely related species B. involucrata and B. flava. In the phylogeny, the new species was placed in a clade containing these three species (Fig. 1). We therefore focused our comparison of morphological characters on these four species (Table 2; Fig. 2). The most distinctive characteristics of the new species are its dioecious sexual system (vs. monoecious in B. involucrata), opposite leaves (vs. connate into a single sheath-like whorl in B. flava and B. involucrata) with serrated margins (vs. crenate in B. flava and B. involucrata and entire in B. henryi), and yellow scapes (vs. red in B. involucrata and red or red to yellow in B. henryi). Based on the results of molecular and morphological analyses, we believe that there is sufficient evidence to support the validity of this taxon as distinctly different from B. henryi, B. involucrata, and B. flava. Therefore, we recommend that it be treated as a new species. Figure 2. Morphology of Balanophora xinfeniae (A–C), B. henryi (D–F), B. involucrata (G–I), and B. flava (J–M). A, D, G, J, K. Male and female individuals; B, E, H, L. Leaves; C, F, I, M. Tubers. Photos by Meng Li and Wei-Hua Liao. 246 PhytoKeys 266: 241–252 (2025), DOI: 10.3897/phytokeys.266.147400 Chen-Long Fu et al.: Balanophora xinfeniae (Balanophoraceae) from China Table 2. Comparison of morphological features among four related species of Balanophora. B. xinfeniae B. henryi B. involucrata B. flava Reproduction Dioecious Dioecious Monoecious Dioecious Tuber branching shape Flat spheroid or subglobose Irregularly spherical or oblate Urceolate, slightly elongated Urceolate Tuber surface Densely covered with granular warts Densely covered with granular warts Densely covered with granular warts and yellowish stellate lenticels Densely covered with granular warts and yellowish stellate lenticels Color of scapes Yellow Red or red to yellow Red Yellow Leaf arrangement Opposite Opposite Connate into a single sheath-like whorl Connate into a single sheath-like whorl Leaves apex Serrated Entire Crenate Crenate Shape of female inflorescence Nearly spherical or ovoid-ellipsoid Broadly ovoid Ovoid or short ellipsoid Ovoid or short ellipsoid Flowering period April to May September to November July to August July to August Taxonomic treatment Balanophora xinfeniae C.L.Fu, M.Li & B.Xu, sp. nov. urn:lsid:ipni.org:names:77372106-1 Figs 3, 4 Type. China • Xizang Autonomous Region: Motuo County (Mêdog), Gelin Village, 29.210161, 95.191856, alt. 1650 m, 29 Apr 2024 (fl.), Meng Li, C.L. Fu & J.N. Zhou YLZB11691-A (holotype: CDBI! Barcode number: CDBI0298364, ♀; isotypes: NAS! Barcode number: NAS00708465; NF!). Diagnosis. Balanophora xinfeniae is morphologically similar to B. henryi, but the two species can be distinguished by several features. The tuber branching shape of B. xinfeniae is characterized as flat spheroid or subglobose, whereas that of B. henryi is irregularly spherical or oblate. The scapes of B. xinfeniae are yellow, in contrast to the red or red-to-yellow scapes of B. henryi. In B. xinfeniae, the apical margins of the leaves are prominently serrate, whereas those of B. henryi are entire. The female inflorescence of B. xinfeniae is nearly spherical or ovoid-ellipsoid, while that of B. henryi is broadly ovoid. The flowering period of B. xinfeniae is from April to May, compared to September to November for B. henryi (Table 2; Fig. 2). Description. Plants dioecious. Tuber yellowish brown, branched or unbranched, surface scabrous, densely covered with granular warts; tuber branches flat spheroid or subglobose, 0.8–2.0 cm in diam. Scapes yellow, 2–12 × 0.5–1.2 cm. Leaves 6–10, yellow to reddish, opposite, scaly, oblong-ovate, apical margins prominently and irregularly serrate, with serrations arranged approximately parallel to the leaf margin, 1.2–3.5 × 0.3–1.8 cm. Male inflorescences subspheroid to ovoid-ellipsoid, 1.8–2.5 × 1.5–2.0 cm. Bracts truncate with expanded liplike margin, fused side by side into a hexagonal alveolus. Male flowers: pedicellate, inserted basally in alveolus, 3-merous, 2–4 mm in diam. Perianth lobes broadly deltoid. Synandria subdiscoid; anthers 3, transversely dehiscent. Female inflorescence nearly spherical or ovoid-ellipsoid. Claviform bodies obovoid, shortly stiped, apically truncate; cuticular ridges of apical cells labyrinth-like. Female flowers: both in the lower parts of the claviform bodies and on the main axis of the inflorescence. 247 PhytoKeys 266: 241–252 (2025), DOI: 10.3897/phytokeys.266.147400 Chen-Long Fu et al.: Balanophora xinfeniae (Balanophoraceae) from China Distribution and habitat. The new species is currently known only from the type locality in Gelin Village, Motuo County, Xizang Autonomous Region (Fig. 5). It grows in shaded forest habitats on moist mountain slopes. The hosts are usually species of Fagaceae, such as Quercus lamellosa and Castanopsis indica. Figure 3. Balanophora xinfeniae C.L. Fu, M. Li & B. Xu. A, B. Habitats (both male and female; arrows point to female individuals); C. Female individual (upper) and male individual (lower); D. Male individual; E. Female individual; F. Leaves and scape; G. Leaves; H. Tuber; I. Male inflorescence; J. Female inflorescence; K. Female flower and claviform body. Photos by Meng Li and Chen-Long Fu. 248 PhytoKeys 266: 241–252 (2025), DOI: 10.3897/phytokeys.266.147400 Chen-Long Fu et al.: Balanophora xinfeniae (Balanophoraceae) from China Figure 4. Line illustration of Balanophora xinfeniae. A. Male individual; B. Male flower; C. Female individual; D. Female flowers surrounding one claviform body. Drawn by Zi-Heng Yu based on YLZB11691-A and YLZB11691-B specimens stored in CDBI. Figure 5. Distribution of Balanophora xinfeniae (Balanophoraceae). 249 PhytoKeys 266: 241–252 (2025), DOI: 10.3897/phytokeys.266.147400 Chen-Long Fu et al.: Balanophora xinfeniae (Balanophoraceae) from China Phenology. The species has been observed flowering from April to May. Etymology. The species epithet xinfeniae honors Prof. Xin-Fen Gao (former curator of the CDBI Herbarium), a distinguished botanist who made significant contributions to the taxonomy of seed plants, lycopods, and pteridophytes. Vernacular name. Chinese Mandarin: Xìn fēn shé gū (信芬蛇菰). Additional specimens examined. China • Xizang Autonomous Region: Motuo County, Gelin Village, 29.210161°N, 95.191856°E, alt. 1650 m, 29 Apr 2024 (fl.), Meng Li, C.L. Fu & J.N. Zhou YLZB11691-B (paratype: CDBI! Barcode number: CDBI0298363, ♂; NAS! Barcode number: NAS00708510, ♂; NF!, ♂); • Gelin Village, 29.220615°N, 95.189094°E, alt. 1602 m, 1 May 2024 (fl.), Meng Li MD2405013-B (paratype: CDBI! Barcode number: CDBI0298362, ♂); • Gelin Village, alt. 1700 m, 24 May 1983 (fl.), B.S. Li, S.Z. Cheng & Z.C. Nie 03761 (PE! Barcode number: PE01491372; PE01801408); • Beibeng Township, alt. 1650 m, 11 May 1983 (fl.), S.Z. Cheng, Z.C. Nie & B.S. Li 04844 (PE! Barcode number: PE01491371). Key to the new species of Balanophora and three morphologically similar taxa 1 Leaves connate into a single sheath-like whorl ...........................................2 – Leaves opposite ...........................................................................................3 2 Plants monoecious, male flowers clustered basally on inflorescence ........ ................................................................................. Balanophora involucrata – Plants dioecious ................................................................Balanophora flava 3 Scapes yellow, apical leaf margins prominently and irregularly serrate, female inflorescence nearly spherical or ovoid-ellipsoid .......... Balanophora xinfeniae – Scapes red or red to yellow, leaf margins entire, female inflorescence broadly ovoid .................................................................. Balanophora henryi Acknowledgements We sincerely thank the reviewers and editors for their valuable comments. We are grateful to Prof. Yong Yang for his guidance on specimen preparation and to Xiao-Xia Ding and Xiao-Yu Dong for preparing the type specimens. We also thank Yan-Feng Song for assistance in examining the specimens stored in PE and extend special thanks to Zi-Heng Yu for drawing the line illustrations. We appreciate the support of the Forestry and Grassland Bureau of Linzhi City and the Forestry and Grassland Bureau of Motuo County for issuing the collection permits. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Use of AI No use of AI was reported.