Molecular phylogenetic and morphological analyses support recognition of a new species of Vincetoxicum (Apocynaceae, Asclepiadoideae) from eastern Thailand

An unknown Vincetoxicum species has recently been discovered in eastern Thailand. It is a twiner that resembles in its morphology Vincetoxicum flexuosum s.l., a variable taxon widely occurring from tropical Asia to Australia. Morphological examination showed that despite similarities in growth habit, leaf shape, gross appearance of inflorescence structure and flower colour, these two elements exhibit substantial differences in both vegetative and floral characters that clearly distinguish one from the other, particularly shape of the flower bud and indumentum on the corolla lobes. In addition, we also evaluated phylogenetic relationships based on DNA sequence data for ITS, trnT-L, trnL and trnL-F markers of this new Vincetoxicum sp. with congeners (including, inter alia, new sequences of the two varieties recognized in Thailand of V. flexuosum s.l., i.e. var. flexuosum and var. tenue). The analyses demonstrated that the new Vincetoxicum sp. is not closely related to the taxa recognized in V. flexuosum s.l. Instead, it was retrieved as sister to a clade containing the African taxa, Vincetoxicum caffrum, Vincetoxicum lycioides and Vincetoxicum fleckii. Therefore, integrated analyses of morphology and molecular phylogeny revealed the new Vincetoxicum sp. to be a well-defined species clearly distinct from V. flexuosum s.l., as well as from all other known congeners. The morphological similarity between the new Vincetoxicum sp. and V. flexuosum s.l. likely resulted from convergence, leading to various taxonomic complications. We here describe it as a new species, Vincetoxicum sangyojarniae, sp. nov., and provide a detailed description, illustration and photographs. Moreover, as phylogenetic relationships revealed that V. flexuosum s.l. is not monophyletic in its actual circumscription, a taxonomic reconsideration of this taxon is suggested.

Vincetoxicum s.l., under its broadened circumscription, is largely characterized by clear latex (rarely yellowish or whitish in some species of the former Tylophora), twining or erect stems, small flowers with a gynostegial corona formed by five mostly separate staminal lobes, and small round pollinia (Forster 1991;Liede-Schumann et al. 2012, 2016. There are more than 200 species naturally distributed in Europe, Africa, Asia and Australia. Some invasive species also occur in North America (Yamashiro et al. 2004 and Chao 2011; Liede-Schumann et al. 2016;Jiang et al. 2018;Liede-Schumann et al. 2018;Shah et al. 2018). In Thailand, the 14 known species are found from sea level up to an elevation of ca. 1600 m a. s. l. (Thaithong et al. 2018;Kidyoo 2016Kidyoo , 2020. In 2021, an unknown climbing Vincetoxicum was found in the protected area within Rajamangala University of Technology Isan Surin Campus, eastern Thailand. At first glance, it looks similar to V. flexuosum (R. Br.) Kuntze (Brown 1810; Kuntze 1891), a variable species widespread from tropical and subtropical Asia to northern Australia (Liede-Schumann et al. 2012). Owing to its considerable morphological variation, V. flexuosum has been recognized as a species complex (Schneidt 1999;Liede et al. 2002;Liede-Schumann et al. 2012 (Thaithong et al. 2018). Our newly discovered Vincetoxicum species, although having several morphological traits especially overlapping with those of V. flexuosum s.l., there are consistent characters that can be used to distinguish one from the other. Detailed comparisons of the similarities and differences in morphology and ecology between these two taxa are provided. Moreover, to corroborate the separation of these two entities, their systematic positions based on ITS, trnT-L, trnL and trnL-F DNA sequence data were investigated.

Molecular phylogenetic analysis
To evaluate the taxonomic status of the new Vincetoxicum species and its phylogenetic relationships with congeners, we included new DNA sequences of one accession of this plant, eight of V. flexuosum var. flexuosum, and four of V. flexuosum var. tenue, all newly collected from different locations throughout Thailand ( Fig. 1 and see Table 1 for voucher numbers and sampling locations), in a phylogenetic analysis of the genus based on combined ITS (nuclear marker) and cpDNA datasets. All sequences have been deposited in Gen-Bank (Table 1). DNA extraction and sequencing protocols were performed as described in Kidyoo et al. (2021). Three accessions of Pentatropis, the other genus in the subtribe Tylophorinae, were downloaded from GenBank (Liede-Schumann et al. 2016) and used as outgroup taxa. In addition, the homologous sequences of 29 other Vincetoxicum spp. were downloaded from GenBank (Liede et al. 2002;Liede-Schumann et al. 2012, 2016Xiong et al. 2020; see Table S1) for comparative analysis.

Phylogenetic analyses
SCLE sequence alignments were made using Mesquite v.3.51 (Maddison and Maddison 2019). PartitionFinder v.2.0.0 (Lanfear et al. 2017) was used to select a partition scheme and the nucleotide substitution models, based on the Akaike information criterion. Prior data blocks were defined by marker and codon position. The best-fit model for the combined ITS and cpDNA markers was GTR + I + G. Phylogenetic reconstructions based on the concatenated ITS and cpDNA datasets were performed using Bayesian inference with MrBayes v. 3 (Ronquist and Huelsenbeck 2003;Ronquist et al. 2012). Two analyses of four chains were run for 1,000,000 generations (after which the split deviation frequency value was lower than 0.01), sampling trees every 500 generations with 25% burn-in samples discarded for each run. The remaining trees were used to build a majority-rule consensus tree and to calculate Bayesian posterior probabilities (PP). The consensus trees were visualized using Mesquite v.3.61 (Maddison and Maddison 2019).

Taxonomic study
Samples of the new Vincetoxicum species were collected from the plant's natural habitats in Surin Province during 2021 and 2022. Voucher specimens were prepared and deposited at BKF and BCU. Vegetative and reproductive structures of the newly collected specimens and of specimens preserved in herbaria (BK, BKF, QBG, BCU, K, L, BM and P) were examined and compared. Furthermore, the similar V. flexuosum s.l. was also included for comparison. Morphological characteristics were mainly observed under light microscopy (LM).

Molecular phylogenetic analysis
GenBank accession numbers for the new sequences generated for this study are OP921231 to OP921246 for ITS and OP923868 to OP923893 for cpDNA (Table 1). Bayesian analyses of combined ITS and cpDNA sequences show that the specimens of V. flexuosum var. flexuosum, V. flexuosum var. tenue and the new Vincetoxicum sp. sequenced from Thailand do not form a monophyletic clade (Fig. 2). With the exception of the new Vincetoxicum sp., all the other Thai accessions are grouped together with high support (PP = 1) in a sister-group position to the New Caledonian/SW Pacific species, Vincetoxicum biglandulosum (PP = 0.57). This clade consists of two well-supported subclades, one of which comprises all the eight samples of V. flexuosum var. flexuosum from Thailand (PP = 1). In the second subclade, all accessions (four from Thailand and one from Borneo) of V. flexuosum var. tenue are placed together with high support (PP = 1). The accessions of V. flexuosum var. flexuosum and V. flexuosum var. perrottetianum from the Philippines and Malaysia are retrieved together in another well-supported clade (PP = 0.99). As for the new Vincetoxicum sp., it is retrieved as sister to the group comprising the African taxa Vincetoxicum caffrum, Vincetoxicum lycioides and Vincetoxicum fleckii, with moderate support (PP = 0.85). This group of four species is in turn sister, with moderate support (PP = 0.79), to the subclade constituted of Vincetoxicum philippicum, Vincetoxicum cissoides and Vincetoxicum hainanense from Philippines, Papua New Guinea and China, respectively. Overall, this phylogenetic analysis shows that Vincetoxicum flexuosum s.l. is not monophyletic in its actual circumscription as its 16 accessions were separated into three well-supported terminal clades, and the new Vincetoxicum sp. is not closely related to any of these clades.

Taxonomic study
Phylogenetic analysis allowed us to refine the taxonomic treatment of the elements of V. flexuosum s.l. included in the present study. The fact that the accessions of V. flexuosum var. flexuosum from Thailand were not placed in the same clade as V. flexuosum var. flexuosum and V. flexuosum var. perrottetianum from Malesiana (Philippines and Malaysia) suggested that the elements from Thailand might not belong to any of these taxa. Henceforth, we treat the These three species have similar growth habit, leaf shape and flower colour. They possess a slender twining stem and branches, ovate to lanceolate leaves that are glabrous on both surfaces (or sometimes sparsely pubescent on the midrib), and small red or reddish brown flowers (5-6 mm in diameter) with a broadly conical gynostegium. They also resemble one another in having branched cymes with zigzag rachises. Despite high morphological similarity between the three species, they can be separated one from the other mainly by the details of inflorescence architecture and branching pattern. Vincetoxicum tenue, although looks quite similar to V. aff. flexuosum and V. flexuosum sensu Schneidt, Meve & Liede in the gross appearance of inflorescence, it can be discerned based on its laxly panicle-like, dichotomously branched inflorescence with 5-11 markedly pedunculate cymules, each cymule with 5-8 pedicellate flowers loosely arranged on an elongated axis (Fig. 3c). By contrast, both V. aff. flexuosum and V. flexuosum sensu Schneidt, Meve & Liede have the inflorescences with 2-5 sessile (to subsessile) cymules, each cymule with 3-8 flowers closely packed on a short inconspicuous axis (Fig. 3a, b).
Considering details of flowers, the flowers of V. aff. flexuosum are consistently characterized by the distally thickened inner corolla tube. This thickening forms a continuous ring at the corolla throat around the gynostegial corona (Fig. 4c). The flowers of V. flexuosum sensu Schneidt, Meve & Liede and V. tenue, on the other hand, lack the thickened ring on the inner surface around the corolla tube (Fig. 4e). Within V. flexuosum sensu Schneidt, Meve & Liede, the discernible morphological difference between the two varieties, i.e. var. flexuosum and var. perrottetianum, is related to quantitative floral characteristic. Vincetoxicum flexuosum var. flexuosum has pedicellate flowers with long pedicels (usually more than 5 mm), while V. flexuosum var. perrottetianum has sessile to subsessile flowers (pedicel length less than 5 mm).
The new Vincetoxicum sp. is morphologically distinct and apparently also genetically distinct from other known Vincetoxicum species. It looks superficially similar to V. flexuosum s.l., but is different in details of floral characters. The new Vincetoxicum sp. has broadly pyramidal flower bud with an obtuse apex (Fig. 4b) and adaxially densely pubescent corolla lobes (Fig. 4a), which set it apart from V. flexuosum s.l. By contrast, the elements of V. flexuosum s.l. have the corolla lobes that are entirely glabrous on both sides, and the flower buds that are horizontally bilaterally symmetrical in shape (against the pyramidal flower bud that is asymmetrical in the horizontal axis of the new Vincetoxicum sp.), e.g. oblate with a rounded apex in V. aff. flexuosum (Fig. 4d) and oval with an obtuse apex in V. tenue (Fig. 4f). In light of this fact, the new Vincetoxicum sp. is described here as a new species, named as V. sangyojarniae (Figs. 5, 6). Given that Vincetoxicum sangyojarniae produces a panicle-like inflorescence bearing cymules that have elongated axes, it is morphologically more similar to V. tenue than it is to V. flexuosum sensu Schneidt, Meve & Liede or to V. aff. flexuosum. Detailed morphological description of the new species with illustrations, photographs and comparison with the most similar V. tenue is provided in the section 'Taxonomic treatment'.

Molecular phylogeny and morphology support recognition of a new species
Our taxonomic investigation underlines a significant variability within elements and a high degree of overlap between elements of V. flexuosum s.l. in morphological traits that have in many cases led to various taxonomic complications. Effectively, the molecular phylogenetic analysis in the present study shows that V. flexuosum s.l. in its current circumscription is not monophyletic, but includes three distinct groups, each of which should be regarded as separate species, i.e. V. tenue, V. aff. flexuosum and V. flexuosum sensu Schneidt, Meve & Liede. Morphologically, these three species are distinguished one from the other by the architecture and branching pattern of inflorescence, and thickening of the distal part of the inner corolla tube (see in 'Taxonomic study' of the Results section; Figs. 3, 4).
Overall, combined phylogenetic and morphological evidence reveals Vincetoxicum aff. flexuosum, previously misidentified as V. flexuosum var. flexuosum, to be another cryptic species hidden within V. flexuosum s.l. This element is widespread in lowland, occurring from near sea level up to 800 m. Its taxonomic status will be further disentangled in a future paper. Moreover, the results of this study suggest that the taxonomic status as an infraspecific rank of V. flexuosum var. tenue (Schneidt 1999;Liede et al. 2002;Liede-Schumann et al. 2012) needs to be reconsidered. Investigation of samples of this taxon obtained throughout its distribution range in Thailand revealed its substantial differences from V. flexuosum s.s. (Brown 1810; Kuntze 1891; Natural History Museum 2023) in leaf texture, inflorescence architecture and branching pattern (Fig. 3), and also in habitat preference-V. tenue is the only element of V. flexuosum s.l. that grows in mangrove forests. These findings support the reinstatement of V. tenue (Blume) Kuntze (Kuntze 1891) as an independent species. Vincetoxicum flexuosum var. perrottetianum, for its part, is morphologically hardly discernible from V. flexuosum s.s. The only difference between these two elements is the quantitative variation in length of the pedicels. Therefore, the results of this study confirm the taxonomic status as an infraspecific rank of V. flexuosum var. perrottetianum (Schneidt 1999;Liede et al. 2002;Liede-Schumann et al. 2012).
Our results also showed that V. sangyojarniae is a welldefined entity and clearly distinct from V. tenue and from other known Vincetoxicum species. Morphologically, V. sangyojarniae and V. tenue are similar in several vegetative and reproductive aspects. In addition to the common characters typically shared by all elements of V. flexuosum s.l., these two species more closely resemble each other in having panicle-like inflorescences (see in 'Taxonomic study' of the Results section; Fig. 3). The new species, however, can be clearly distinguished from V. tenue by several consistent characters. It has broadly pyramidal flower bud with an obtuse apex, and corolla lobes that are pubescent on the adaxial surface, the distal half of which covered with dense long white trichomes. On the contrary, V. tenue has oblate flower bud with a rounded apex. Its corolla lobes are always Molecular phylogenetic relationships revealed that Vincetoxicum sangyojarniae is evolutionarily more closely related to several African species, i.e. the climber V. lycioides, and the non-twiners V. caffrum and V. fleckii, than to all elements of V. flexuosum s.l. Therefore, the similarity in morphology between V. sangyojarniae and V. tenue as well as the other elements of V. flexuosum s.l. is likely the result of convergence.

Taxonomic treatment
Vincetoxicum sangyojarniae A.Kidyoo, sp. nov.-TYPE: Thailand, Surin Province, Muang district, Rajamangala Diagnosis: Vincetoxicum sangyojarniae resembles V. tenue in having a panicle-like cyme with a slender peduncle and pedicellate flowers arising from elongated axis of cymule but clearly differs by inflorescences that branch dichotomously 1-2 times, membranous leaves, broadly pyramidal flower buds with an obtuse apex and corolla lobes that are adaxially densely pubescent. On the contrary, V. tenue has inflorescences that branch dichotomously 5-11 times, coriaceous to thick coriaceous leaves, oblate flower buds with a rounded apex and corolla lobes that are glabrous on both surfaces.
Phenology: Flowering from September to January.

Distribution and habitat:
This new species is known only from the type locality. It grows in loam soil along roads in shady areas of disturbed mixed deciduous forest (Fig. 5a).
Conservation status: Vincetoxicum sangyojarniae is apparently rare, known from a single locality in Thailand. About 20 to 30 mature individuals were found growing in an area of 1500 m 2 that is frequently disturbed by human activities. Following IUCN (2019), a provisional conservation assessment of Endangered: B1ab(iii) is assigned owing to its extent of occurrence much smaller than 5,000 km 2 , its restricted geographic distribution and the small number of locations, which are susceptible to threats in the near future.