Confirmation of the Systematic Position of Biondia and Merrillanthus (Apocynaceae) Based on Molecular Evidence

Miao Liao¹, Sijin Zeng², Guangda Tang¹,³*

¹College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou 510642, China
²Key Laboratory of Plant Resources Conservation and Sustainable Utilization & Guangdong Provincial Key Laboratory of Digital Botanical Garden, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China
³Henry Fok College of Biology and Agriculture, Shaoguan University, Shaoguan 512005, Guangdong, China

Abstract

Biondia Schltr. is a genus endemic to China comprising approximately 13 species, while Merrillanthus Chun & Tsiang is a monotypic genus distributed only in China and Cambodia. Both genera have been subsumed into Vincetoxicum Wolf, but their phylogenetic positions and taxonomic status require further investigation due to limited sampling and incomplete phylogenetic analysis. We reconstructed phylogenetic trees of subtribe Tylophorinae using two nuclear ribosomal DNA regions (ITS, ETS) and five plastid DNA regions (psbA–trnH, trnG, trnL, trnL–F, trnT–L), analyzed both separately and in combination. Our sampling included the type species of each genus: Biondia chinensis Schltr. (= V. shaanxiense (Schltr.) Meve & Liede) and Merrillanthus hainanensis Chun & Tsiang (= V. hainanense (Chun & Tsiang) Meve, H.H. Kong & Liede). The results demonstrate that both Biondia and Merrillanthus are nested within Vincetoxicum. Biondia chinensis is sister to B. henryi (Warb.) Tsiang & Li (= V. henryi (Warb.) Meve & Liede), and this clade groups with V. kawaroense Meve & Liede, whereas B. insignis Tsiang (= V. insigne (Tsiang) Meve, H.H. Kong & Liede) falls within a separate lineage (the Subtropical Clade). Merrillanthus forms a clade with V. cissoides (Blume) Kuntze and V. philippicum Meve, Omlor & Liede. Both nuclear and plastid datasets support the inclusion of Biondia and Merrillanthus within Vincetoxicum, but Biondia is polyphyletic. Additional species sampling and combined data analysis are needed to further elucidate interspecific relationships within the expanded Vincetoxicum.

Keywords: Apocynaceae, Vincetoxicum, Biondia, Merrillanthus, phylogeny, China

Introduction

Apocynaceae, as circumscribed in the Angiosperm Phylogeny Group IV (APG IV) system, belongs to Gentianales along with Gelsemiaceae, Gentianaceae, Loganiaceae, and Rubiaceae (Chase et al., 2016). The family encompasses the traditionally recognized Apocynaceae s.s. and Asclepiadaceae, comprising approximately 5,350 species primarily distributed in tropical and subtropical regions (Endress et al., 2018). A relatively stable molecular phylogenetic framework for Apocynaceae has been established (Fishbein et al., 2018), leading to the consolidation or repositioning of several revised genera and reducing the total number from 422 (Endress & Bruyns 2000) to 378 (Endress et al., 2018).

Vincetoxicum Wolf belongs to tribe Asclepiadeae, subtribe Tylophorinae, and has undergone substantial taxonomic revision (Endress et al., 2018). Traditionally, Vincetoxicum is characterized by erect stems (occasionally twining at the apex), fascicled fibrous roots, rotate corollas, five fleshy corona lobes, and the presence of translucent latex (Qiu et al., 1989; Liede, 1996). The genus shares similar corona, gynostegium, and pollinarium structures with Cynanchum L., leading some researchers to advocate for its inclusion within Cynanchum (Jiang and Li, 1977; Forster, 1991; Li et al., 1995). However, others have argued for its recognition as a distinct genus (Markgraf, 1972; Ali & Khatoon, 1982). Qiu et al. (1989) proposed the independence of Vincetoxicum from Cynanchum based on evidence from chemical constituents, morphology, chromosome numbers, and distribution patterns, a view supported by Liede (1996) using morphological and chemical data, who further suggested its closest relationship with Tylophora R. Br.

Molecular phylogenetic studies have revealed that Vincetoxicum is distantly related to Cynanchum (Liede, 2001; Rapini et al., 2007), instead showing closer affinity with Tylophora, although both genera are non-monophyletic. Six genera—Biondia Schltr., Blyttia Arn., Diplostigma K. Schum., Goydera Liede, Pleurostelma Baill., and Rhyncharrhena F. Muell.—are nested within the Vincetoxicum–Tylophora clade (Liede-Schumann et al., 2012, 2016). Consequently, Tylophora and these six morphologically similar genera have been subsumed into Vincetoxicum, resulting in numerous new combinations and names (Kidyoo & Kidyoo, 2018; Liede-Schumann & Meve, 2018; Hsu et al., 2021; Shah et al., 2021). Three genera originally distributed primarily in China—Merrillanthus Chun & Tsiang, Pentastelma Tsiang & Li, and Belostemma Wall. ex Wight—have also been incorporated into Vincetoxicum. The newly defined Vincetoxicum comprises over 150 species, typically with translucent latex, small flowers with rotate corollas (occasionally campanulate, urceolate, or long-conical), coronas composed of fleshy, separate lobes on the stamens or forming rings from fused stamens and interstaminal parts, erect, horizontal, or ascending caudicles, and a distribution spanning tropical and subtropical Africa, Asia, and Eurasia (Endress et al., 2018).

Biondia comprises approximately 13 species endemic to eastern and southwestern China, while Merrillanthus is monotypic, containing only M. hainanensis, distributed in Guangdong, Hainan (China), and Cambodia (Jiang and Li, 1977; Li et al., 1995). In the phylogenetic analyses by Liede-Schumann et al. (2012, 2016), Biondia was represented by only two species—B. insignis and B. henryi—lacking the type species B. chinensis, and no Merrillanthus samples were included. Some researchers have reported chloroplast genome data for B. chinensis and M. hainanensis with preliminary phylogenetic analyses: B. chinensis appeared sister to V. rossicum, but the tree included only a single Vincetoxicum species (Rao et al., 2018), while M. hainanensis appeared sister to B. insignis, again with limited sampling (Xiong et al., 2019). Thus, the placement of these two species within Vincetoxicum and their relationships to specific lineages remain unclear.

In this study, we incorporated data for B. chinensis and M. hainanensis, with newly generated sequences for M. hainanensis and published shallow-sequencing data for B. chinensis from Rao et al. (2018), combined with existing molecular data for other Vincetoxicum species (Liede-Schumann et al., 2016), to conduct phylogenetic analyses aimed at clarifying the systematic positions and taxonomic归属 of these two genera.

1.1 Taxon Sampling and Molecular Data Sources

Data for Biondia chinensis (voucher: ZJB-2017-152-1, deposited at Shaanxi Normal University) were obtained from shallow-sequencing genomic data in Rao et al. (2018). For Merrillanthus hainanensis (voucher: LHB-AP17, deposited at South China Agricultural University), fresh leaves were sampled, dried in silica gel, and stored on dry ice before being shipped to Novogene Bioinformatics Technology Co., Ltd. (Wuhan) for total genomic DNA extraction, library construction, and sequencing. Following quality control of the DNA, random fragmentation produced ~350 bp libraries for paired-end 150 bp sequencing, yielding 10 Gb of clean data after quality filtering. Both the existing shallow-sequencing data for B. chinensis and the newly generated data for M. hainanensis were assembled using GetOrganelle v1.7 (Jin et al., 2020) with default parameters. The resulting fastg files were visualized and extracted using Bandage 0.8 (Wick et al., 2015) and Geneious Prime 2019 (https://www.geneious.com/) to obtain the final ribosomal genome sequence for B. chinensis and both chloroplast and ribosomal genome sequences for M. hainanensis. The chloroplast genome of M. hainanensis was annotated using Plastid Genome Annotator (PGA) (Qu et al., 2019) with Amborella trichopoda Baill. (AJ506156) and Apocynum venetum L. (MT313688) as references, followed by manual correction in Geneious Prime 2019 based on log files. Ribosomal genomes for both species were annotated in Geneious Prime 2019 using the nrDNA continuous fragment (18S+ITS1+5.8S+ITS2+26S) from Asclepias coulteri A.Gray (JN665084) and the ETS sequence from V. biglandulosum (Endl.) Kuntze (LN880610) as references. Required ribosomal and chloroplast gene fragments were extracted using the Extract function, and all data were submitted to GenBank (https://www.ncbi.nlm.nih.gov/).

Based on previously published molecular phylogenetic data for subtribe Tylophorinae (Liede-Schumann et al., 2016), we supplemented DNA data for M. hainanensis and B. chinensis, selecting three species from subtribe Cynanchinae as outgroups. Phylogenetic trees were constructed using: (1) five chloroplast gene regions (psbA–trnH, trnG, trnL, trnL–F, trnT–L) for 139 species; (2) two ribosomal gene regions (ETS, ITS) for 136 species; and (3) combined datasets for 139 species. Sequence details are provided in Table 1, and voucher specimen information is available in Liede-Schumann et al. (2016). Missing sequence data for some samples were coded as gaps in the alignment matrices.

1.2 Sequence Alignment and Concatenation

Individual gene regions were aligned using MAFFT (Katoh & Standley, 2013). Multiple alignments were concatenated in Geneious Prime 2019 to produce combined matrices.

1.3 Phylogenetic Analysis

Maximum likelihood (ML) phylogenetic analyses were performed using IQ-TREE (Nguyen et al., 2015). ModelFinder within IQ-TREE was used to automatically test and select the best-fit substitution models according to the Bayesian Information Criterion (BIC): TVM+F+R3 for the combined ribosomal dataset, K3Pu+F+R3 for the combined plastid dataset, and TVM+F+R3 for the combined nuclear and plastid dataset. Branch support was assessed using 1,000 SH-aLRT replicates (Guindon et al., 2010) and ultrafast bootstrap (UFBoot) analyses (Minh et al., 2013). Branches with SH-aLRT ≥ 80% and UFBoot ≥ 95% were considered well-supported and reliable. Resulting trees were visualized in FigTree 1.4.2 (Rambaut, 2012).

2 Results and Analysis

Phylogenetic trees constructed from the ribosomal gene regions (ETS, ITS) revealed that Vincetoxicum is non-monophyletic within subtribe Tylophorinae, with Pentatropis R. Br. ex Wight & Arn. nested within it and forming a sister clade to V. apiculatum + V. sylvaticum + V. tenuipedunculatum (SH-aLRT = 82.8%, UFBoot = 82%) (Fig. 1). Trees based on plastid gene regions and the combined dataset both resolved Tylophorinae into two major clades, with Pentatropis as sister to Vincetoxicum (support values: 99.8/100 and 99.9/100, respectively).

All three datasets strongly supported Merrillanthus grouping with V. cissoides (T. cissoides) and V. philippicum (T. parviflora) (support values: 100/100, 97.9/100, and 100/100 in Figs. 1, 2, and 3, respectively). The three Biondia species were nested within the Vincetoxicum clade but were non-monophyletic. In the ribosomal and combined datasets, B. chinensis was sister to B. henryi (= V. henryi) with strong support (97.7/100 and 97.8/100, respectively; Figs. 1 and 3), and this clade grouped with V. kawaroense (T. japonica) (97.1/100 and 97.8/100). In the plastid dataset tree, B. chinensis and B. henryi formed a clade but with low support (31.3/67) (Fig. 2). Biondia insignis (= V. insigne) consistently fell within the Subtropical Clade across all three trees (support values: 100/100, 93.8/100, and 100/100 in Figs. 1, 2, and 3, respectively), where it grouped with V. villosum (T. villosa) (support values: 74.3/100, 0/96, and 72.6/100).

3 Discussion

3.1 Phylogenetic Position and Generic Status of Biondia

When Schlechter (1905) established Biondia, he distinguished it from Marsdenia R. Br. by its pendulous pollinia (vs. erect) and from Tylophora by its campanulate or sub-campanulate corolla (vs. deeply 5-lobed rotate or broadly rotate corollas), placing it in subtribe Asclepiadinae. Gilbert et al. (1995) noted that Biondia is characterized by narrow leaves with minute pale spots on the adaxial surface, small flowers with reduced, often annular corollas and well-developed corolla tubes, and observed similar pale spots in some Tylophora species, indicating morphological overlap between the genera. Since Tylophora has been subsumed into Vincetoxicum (Liede-Schumann et al., 2012; Liede-Schumann & Meve, 2018; Endress et al., 2018), our molecular results support the inclusion of Biondia within Vincetoxicum.

Phylogenetic analyses show that all three Biondia species are nested within Vincetoxicum. Biondia chinensis and B. henryi form one clade, while B. insignis groups with V. villosum (T. villosa) within the Subtropical Clade. However, these species differ markedly in morphology: B. insignis has branches and petioles covered with uniseriate short hairs, linear-lanceolate leaves that are glabrous except for the midvein, and a sub-campanulate, glabrous corolla, whereas V. villosum has densely pubescent branches and petioles, ovate or ovate-oblong leaves that are hairy on both surfaces, and a rotate corolla with long soft hairs. Geographically, B. insignis is distributed in Guizhou, Hunan, Sichuan, Tibet, and Yunnan, China (Li et al., 1995), while V. villosum occurs on Java (Backer & Bakhuizen, 1965). The close phylogenetic relationship between B. insignis and V. villosum despite their substantial morphological and geographic differences warrants further investigation with expanded sampling.

3.2 Systematic Position and Generic Status of Merrillanthus

When Merrillanthus was described, it was considered most similar to Pycnostelma Bunge ex Decne. (now subsumed in Vincetoxicum; Liede-Schumann & Meve, 2018). Both genera have coronas adnate to the stamens but differ in growth habit, corona lobe shape, corolla lobe fusion patterns, and fruit morphology. Merrillanthus is a woody liana with dorsally raised, basally thickened corona lobes, connate corolla lobes that are fused at the apex before anthesis, and solitary, fusiform, large follicles (9–12 cm long, 3.5–4 cm in diameter). In contrast, Pycnostelma is an erect herb with coronas thickened toward the apex, deeply lobed corollas, and smaller, lanceolate fruits (Chun & Tsiang, 1941). The corolla lobe fusion in Merrillanthus resembles that of Heterostemma Wight & Arn. and Hoya R. Br., while its membranous, cordate leaves are similar to Telosma Coville. However, Merrillanthus can be distinguished by its pendulous pollinia (vs. erect in the other three genera) (Chun & Tsiang, 1941). Jiang and Li (1977) noted similarities between Merrillanthus and the Indian genus Iphisia Wight & Arn. (= Vincetoxicum Wolf), differing in corolla lobe adhesion at the bud stage and pollinium orientation. The floral structure of Merrillanthus is also similar to Tylophora augustiniana (Hemsl.) Craib (= V. augustinianum (Hemsl.) Meve & Liede), though with larger flowers. The large follicles with thick fibrous mesocarp in Merrillanthus contrast with the thin-walled, papery follicles typical of Tylophora, supporting its recognition as a distinct genus (Gilbert et al., 1995).

Although we did not include V. augustinianum in our analysis, our results show Merrillanthus grouping with two former Tylophora species, V. cissoides (T. cissoides) and V. philippicum (T. parviflora). These species share large fruits (10–12 cm × 3–5 cm and 13–15 cm × 3–4 cm, respectively) and similar leaf sizes and shapes. Geographically, V. cissoides is widespread in eastern Indonesia and New Guinea (Forster, 1994), V. philippicum is restricted to the Philippines (Meve et al., 2002), and Merrillanthus occurs in Guangdong, Hainan (China), and Cambodia (Li et al., 1995). These findings suggest that fruit size, caudicle orientation, and leaf morphology may be insufficient characters for generic delimitation within Tylophorinae but could serve as useful species-level diagnostic traits within Vincetoxicum. Initially placed in subtribe Asclepiadinae based on its pendulous pollinia (Chun & Tsiang, 1941), Merrillanthus was subsequently assigned to subtribe Cynanchinae (Endress et al., 2007) and then to Tylophorinae (Endress et al., 2014, 2018) following the merger of Apocynaceae and Asclepiadaceae (Endress & Bruyns, 2000). Our study supports the current classification of Merrillanthus within Vincetoxicum and its placement in subtribe Tylophorinae.

3.3 Phylogeny of Vincetoxicum

Although the combined nuclear and plastid dataset resolves Vincetoxicum as monophyletic, the dataset contains relatively few informative sites, resulting in low support for many interspecific relationships within the genus. High-throughput sequencing-based phylogenetic analyses will be necessary to reconstruct robust relationships within Vincetoxicum. The genus has experienced considerable taxonomic changes, making a comprehensive revision based on genomic data and morphological character analysis essential.

Acknowledgments

We thank Dr. Zou Jiabin from Shaanxi Normal University for providing molecular data and Dr. Zhang Caifei from Wuhan Botanical Garden for assistance with data acquisition.

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