Return Verdesmummenglaense to the genus Hylodesmum (Fabaceae) based on morphological and molecular evidence

Abstract Verdesmummenglaense (C. Chen & X. J. Cui) H. Ohashi & K. Ohashi is a rare species in the tribe Desmodieae (Fabaceae) from Southwest China. The morphological observation shows that the species has minute capitate stigma and ebracteolate calyces, which are entirely different from the funnel-shaped stigma and bracteolate calyces of the genus Verdesmum H. Ohashi & K. Ohashi, but are consistent with those of the genus Hylodesmum H. Ohashi & R. R. Mill. The generic placement of V.menglaense within Hylodesmum was further supported by molecular evidence. Therefore, this species should be returned to Hylodesmum as H.menglaense (C. Chen & X. J. Cui) H. Ohashi & R. R. Mill. A full description including floral characters, a colour plate and a distribution map are first provided here for this species. After excluding the solo representative in China, Verdesmum should be removed from the record in Flora of China.


Introduction
Verdesmum H. Ohashi & K. Ohashi is a newly established genus in the tribe Desmodieae (Fabaceae) (Ohashi and Ohashi 2012a;Lewis et al. 2013;Azani et al. 2017), based on only one species V. hentyi (Verdc.) H. Ohashi & K. Ohashi which is a shrub distributed in Papua New Guinea and Malaysia and was first described in the genus Desmodium Desv. as D. hentyi Verdc. (Verdcout 1977;Ohashi 2004). This genus was considered to be most similar to Hylodesmum H. Ohashi & R. R. Mill, but differs in having funnelshaped terminal stigma, bracteolate calyces, linear pods, very narrow obovate-elliptic articles and stipes longer than fruiting pedicels (Ohashi and Ohashi 2012a). Amongst these characters, the shape of the stigma was considered to be the most important trait of Verdesmum and to be unique amongst the whole tribe Desmodieae Ohashi 2012a, 2013).
Verdesmum menglaense (C. Chen & X. J. Cui) H. Ohashi & K. Ohashi, the second species recognised in Verdesmum (Ohashi and Ohashi 2013), was originally published in the genus Podocarpium (Benth.) Y. C. Yang & P. H. Huang as P. menglaense C. Chen & X. J. Cui, based on two fruiting gatherings from Yunnan, Southwest China (Cui et al. 1987). The species had been suggested for transfer to the genus Desmodium Desv. by Ohashi (1995), but was not accepted by other taxonomists. Later, Ohashi and Mill (2000) found that Podocarpium was an illegitimate generic name and thus proposed to replace it by Hylodesmum H. Ohashi & R. R. Mill. Correspondingly, P. menglaense was proposed as Hylodesmum menglaense (C. Chen & X. J. Cui) H. Ohashi & R. R. Mill (Ohashi and Mill 2000;Gao 2006;Zhu et al. 2007;Huang and Ohashi 2010). The species was further transferred to the genus Verdesmum as V. menglaense (C. Chen & X. J. Cui) H. Ohashi & K. Ohashi for the similarity in the linear pods and very narrow obovateelliptic articles (Ohashi and Ohashi 2013). This treatment was followed by subsequent research (Zhu 2015a, Zhu 2015bLiu et al. 2015;Ohashi et al. 2018b). However, Verdesmum menglaense is a rare species endemic to Yunnan, Southwest China. After being published, it was not re-discovered in the field and its flowers have not been described in any literature (e.g. Ohashi 1995;Gao 2006;Huang and Ohashi 2010). Ohashi and Ohashi (2013) considered that it is difficult to determine the correct generic position of this species with the absence of flowers. Fortunately, in a collecting trip to Yunnan Province in 2010, we found several living individuals without flowers or fruits of this species at a streamside in the forest. Subsequently, these plants successfully produced flowers and fruits under cultivated conditions in South China Botanical Garden. Our morphological observation showed this species has terminal minute capitate stigma and ebracteolate calyces (Fig. 1). In these important floral characters, Verdesmum menglaense is thus distinct from Verdesmum, but is consistent with Hylodesmum. Furthermore, the placement of this species within Hylodesmum was also strongly supported by molecular evidence from the trnL-F sequences (Fig. 2). Therefore, this species should be returned to Hylodesmum as H. menglaense. Currently, Verdesmum just includes a single species (V. hentyi) and its distribution in China should be excluded.

Morphological studies
The morphological characters were examined based on the living plants and specimens kept in the HITBC, IBSC and KUN herbaria. Acronyms for the herbaria follow the Index Herbariorum (Thiers 2018). The distribution map was made by the software ArcGIS 10.2.

Molecular analyses
In order to clarify the generic position of the species Verdesmum menglaense within the Desmodium group of the tribe Desmodieae, a phylogeny was reconstructed based on analyses of the noncoding plastid marker trnL-F, which was often used in phylogenetic studies of this tribe in single or combined analyses with other DNA sequences (e.g. Stefanovic et al. 2009;Nemoto et al. 2010;Xu et al. 2012;Ohashi et al. 2018b). DNA sequences were downloaded mostly from Genbank (www.ncbi.nlm.nih.gov/Genbank) and 14 taxa were newly sequenced in the present study. In total, 53 species were sampled in phylogenetic analyses, including 23 of the 28 genera in the Desmodium group (Ohashi et al. 2018a) and 11 of the 12 species in the genus Hylodesmum (if Verdesmum menglaense is not considered). Information about relevant samples and Genbank accessions are listed in Appendix 1. The phylogenetic trees were reconstructed using two approaches: Maximum Likelihood (ML) and Bayesian Inference (BI). Detailed information about the experiment operations (DNA extraction and PCR amplification), sequences of primer used, model selection of the sequence matrix constructed and methods in tree reconstruction can be accessed in Li et al. (2009) and Yao et al. (2016).

Results and discussion
Results from phylogenetic analyses revealed that three groups (clade A: Lespedeza group, clade B: Phylloddium group and clade C: Desmodium group) were well supported in the tribe Desmodieae, just as reported in most recent research (Jabbour et al. 2018;Zhang et al. 2018;Ohashi et al. 2018a;Ohashi et al. 2018b). Although the type species of the genus Verdesmum was not sampled and thereby its phylogenetic position could not be resolved, the species V. menglaense was deeply embedded within the genus Hylodesmum in both of the ML (BS=98%) and BI (PP=1.00) analyses (Fig. 2). Thus, the taxonomic status of V. menglaense within the genus Hylodesmum was strongly supported by this molecular evidence, despite the absence of a good specific relationship.
Currently, Hylodesmum comprises 13 species (including H. menglaense) and 4 subspecies (Ohashi and Mill 2000;Huang and Ohashi 2010) Song et al. 2013). The genus is disjunctly distributed in eastern North America (3 species) and eastern Asia (10 species), one of which extends from Asia to Africa (Ohashi and Mill 2000;Woods 2008). China has the highest species richness in the genus and includes 10 species and 4 subspecies (Huang and Ohashi 2010;Song et al. 2013 Cui et al. (1987) and Ohashi (1995), because both species have calyx lobes much shorter than tube, lateral veins of leaflets not reaching margin and abxial surfaces of leaflets scattered with white spots. Especially, the white spots appear on the abxial blades only for the two species in the whole genus. However, H. menglaense has very narrow obovate-elliptic articles and pods with central isthmi between the articles, which are unique amongst the genus (Ohashi and Ohashi 2012a). When without fruits, we found that H. menglaense can be distinguished from H. leptopus by slightly larger and thicker terminal leaflets.  Description. Perennial herbs or subshrubs, 30-100 cm high. Stem erect, simple, usually woody at base. Stipules striate, lanceolate, 3.5 mm × 1 mm in size, green to brown, uncinate-hairy. Stipels subulate, ca. 1.4 mm long. Leaves 3-foliolate, scattered along stem; petiole 8-12 cm including rachis 1-2.5 cm long, uncinate-hairy; leaflet blades thickly papery to subleathery; adaxial surfaces dark green, shiny, glabrous; abaxial surfaces pale green, scattered with white spots, very sparsely uncinate-hairy under the microscope; terminal leaflet ovate, 12-19 cm × 7-10 cm in size, entire along margin, rounded or broadly cuneate at base, acuminate or caudate at apex, 2-stipellate at base of pulvinule; lateral veins about 5 pairs, not reaching margin; lateral leaflets slightly smaller, narrowly ovate to lanceolate, base oblique, 7-12 cm × 3-5 cm in size, sessile but pulvinule distinct, 1-stipellate at base of pulvinule; pulvinule ca. 5 mm long. Inflorescences terminal or axillary, sometimes borne at leafless nodes or near the base of  Phylogenetic relationships amongst 53 species from 30 genera of the tribe Desmodieae based on the trnL-F sequence data using Maximum Likelihood analysis. Numbers at the nodes are posterior probabilities and bootstrap percentages (PP, BP) from Bayesian and Maximum Likelihood analysis, respectively. A dash (--) indicates PP < 0.5 or BP < 50%. The grey cover shows the representative of Hylodesmum within which Verdesmum menglaense (indicated by bold font) was deeply embedded. old stem, pseudoracemose, up to 15-50 cm long, laxly flowered, 2-flowered per node, with minute hooked hairs. Primary bracts subtending the secondary bracts, narrowly triangular, acute at the apex, 4.3 mm × 1.6 mm in size, with uncinate hairs. Secondary bracts triangular, 1 mm × 0.7 mm in size, with uncinate hairs. Bracteole absent at base of calyx. Pedicles 2.5-3 mm long, with minute uncinate hairs. Calyx 4-lobed; tube 2.5-2.6 mm long; lobes much shorter than the tube, upper lobe minutely 2-toothed at the apex, lateral and lower lobes shallowly triangular with minute hooked hairs, 1.3-1.5 mm × 0.4-0.6 mm in size; floral disc absent. Corolla pale reddish-pink, glabrous; standard blade orbiculate or suborbiculate, 7.3 mm × 6.5 mm in size, reflexed, emarginate at the apex, suddenly cuneate to the base, with ca. 1.8 mm long claw; wings narrowly elliptic, 7.3 mm × 1.8 mm in size, slightly twisted, obtuse at the apex, slightly auriculate at the base, with ca. 1.8 mm long claw; keel-petals connate, 6.6 mm × 2.3 mm in size, obtuse at the apex, auriculate at the base, with ca. 2.3 mm long claw. Stamens 10, monadelphous, filaments connate into a tube, ca. 9 mm long. Ovary linear, minute uncinate-hairy, about 7.5 mm long including style 1.5 mm long, usually 2-5-ovuled, with a very short stipe; style curved upwards, with a terminal minute capitate stigma. Pods 2-5-jointed, linear, densely minute hooked hairy, with central isthmi between articles; fruiting pedicles 5-7 mm long, fruiting stipes 9-15 mm long; articles very narrow obovate-elliptic, 3.2-5.4 cm × 3.5-6 mm in size, covered with prominent reticulate veins when mature. Seeds 1 in each locule, very narrow obovateelliptic, 2.5-3.5 cm × 3 mm in size, without rim-arillate around the hilum.
Phenology. Under natural conditions in the field, the species was recorded in fruit from August to November. Under cultivated conditions in Guangzhou City, plants were observed in flower in October and in fruit from November to December. Conservation status. Before our investigation, only five type specimens of two fruiting gatherings have been found in a single locality for this species. We explored the type locality of this species and found two additional localities, but individual numbers of each of the three populations were discovered to be less than 30. Therefore this species might be considered as 'Critically Endangered' (CR) under the IUCN (2001) categories criteria C2a(i).
Notes. Hylodesmum menglaense was described as having terminal and/or axillary inflorescences in some references (Cui et al. 1987;Gao 2006;Huang and Ohashi 2010). Through examining type specimens, however, Ohashi and Ohashi (2013) pointed out that inflorescences of this species arise from leafless nodes or from the base of old stem, but seem not to be terminal. Our observations show this species does produce terminal inflorescences as well.

Appendix 1
Scientific names, GenBank accession numbers and voucher information for trnL-F used in the molecular analyses *indicates the taxon was newly sequenced in the present study