﻿Molecular, morphological, and morphometric evidence reveal a new, critically endangered rattlepod (Crotalaria, Fabaceae/Leguminosae, Papilionoideae) from tropical China

﻿Abstract Here, we describe a new species of Crotalaria L. discovered in Mengla County, Xishuangbanna Dai Autonomous Prefecture, Yunnan, China. The new species, Crotalariamenglaensis S.A.Rather, was confirmed by identifying diagnostic morphological characteristics, performing principal component analyses of phenotypic traits, and phylogenetic analyses based on nuclear ITS and plastid matK sequences. Phylogenetic analyses recovered the two accessions of the new species to be sister to C.bracteata Roxb. ex DC. In turn, these two species formed the sister clade to the two accessions of C.incana L. The morphometric analyses revealed that all three species were distinct, while the analyses of distinctive characters enabled unambiguous distinction of the new species by its growth habit, leaflets, flower structure and pod morphology. In contrast to the two related species, the new species is currently known only from ca. 100 mature individuals. Thus, this species is considered to be critically endangered.


Introduction
Xishuangbanna, located in the most southwestern part of Yunnan Province and sharing borders with Myanmar and Laos, is well recognized for its rich biodiversity.Its tropical forests play a vital role in global terrestrial biodiversity conservation efforts (Feng et al. 2018).Unfortunately, many plants in Xishuangbanna have recently faced significant threats from deforestation and the establishment of artificial plantations, especially rubber plantations (Brinck et al. 2017;Liu et al. 2017;Yang et al. 2021;Yang et al. 2023).To enable effective protection of the unique and rich diversity of Xishuangbanna, efforts are needed to record species diversity, including that of many species still unknown to science (Chang et al. 2018;Chang et al. 2022;Chen et al. 2022).PhytoKeys 242: 333-348 (2024), DOI: 10.3897/phytokeys.242.122407Shabir A. Rather et al.: New endangered rattlepod in tropical China Here, we focus on accessions belonging to the legume genus Crotalaria L., which comprises approximately 702 species worldwide (Rockinger et al. 2017;Rather et al. 2018).Its highest species diversity is found in Africa and Madagascar, with an estimated 540 species.It has also expanded to South America and North America, with 64 and 34 species, respectively (Flores et al. 2006;Pandey et al. 2010;Le Roux et al. 2013;Rather et al. 2018).India hosts the largest number of species in Asia (ca.80 species), followed by Southeast Asian countries, which collectively host 105 species (Lock and Simpson 1991;Rather et al. 2018).Approximately 45 species have been recorded to occur in China, predominantly in Southwest China, including nine endemics and six introduced species (Li et al. 2010).The genus exhibits both annual and perennial life forms and various growth forms (prostrate to erect herbs, undershrubs, robust shrubs, and occasionally small trees) and occupies various habitats, such as open grasslands, roadsides, and forest edges (Polhill 1982;Rather et al. 2018).Crotalaria is characterized by papilionoid flowers, the presence of paired callosities on the standard petal, a rostrate keel, 5 + 5 dimorphic anthers, a hairy style, inflated pods and the presence of pyrrolizidine alkaloids (Le Roux et al. 2013;Rather et al. 2018).
In the present study, several interesting specimens of Crotalaria were collected during field trips to Mengla County in Yunnan Province, China.Initially, some plants observed in the Mengpengzhen area of the Xishuangbanna Dai Autonomous Prefecture could not be assigned to any known taxa.Thus, we considered three priority explanations.The first explanation considered interpreted that these accessions are natural hybrids formed between two sympatrically occurring Crotalaria species, namely, C. bracteata Roxb.ex DC. and C. incana L. However, upon closer examination, the newly discovered species did not match either of these taxa.The plants exhibited differences in numerous characteristics, including plant height, leaflet shape, inflorescence, flower, pod shape, indumentum, and number of seeds per pod, among others.The subsequent discovery of numerous plants during further surveys, which included nearly 50 mature individuals and several immature plants spread over an area of 0.1 km 2 , eliminated the possibility that these plants were hybrids.The second explanation interpreted these accessions as a new distributional record of a known species within the genus Crotalaria L. However, there have been no documented new records for the genus Crotalaria L. This possibility was ruled out after unsuccessful attempts to identify the plants using existing identification keys (Brach and Song 2006;Li et al. 2010).Additionally, we consulted taxonomists at various institutes in China who were unable to recognize the taxa collected.Furthermore, comparisons with verified images of other Crotalaria L. taxa available in the Plant Image Library of China (PPBC; https://ppbc.iplant.cn/)also failed to yield any proper matches.The final explanation considered these plants to represent a new, previously undescribed taxon.This study was designed to confirm this hypothesis by focusing on three lines of evidence, namely, traditional diagnostic morphological character identification, morphometric studies using principal component analyses, and phylogenetic analyses using both plastid matK and nrITS DNA sequences.Finally, we present a comprehensive taxonomic description of this newly discovered Crotalaria L. species, supplemented with taxonomic comments and accompanying photographs.

Ethics statement
The geographic sites where the newly identified species was found do not coincide with any designated natural conservation areas.Therefore, specific permission for access to these locations was not needed.

Morphological observations
The morphological analysis and description of the newly discovered species were prepared using freshly collected samples.The flowers were preserved in FAA solution (formaldehyde-glacial acetic acid-alcohol) for further studies.They were rehydrated using a mixture of water and detergent to observe the corolla in detail, followed by dissection.Minute corolla features were examined using a Stemi 305 binocular microscope.Morphological terminology adhered to the standards set by Harris and Harris (2001), Hickey and King (2007) for vegetative characters, Hewson (1990) for indumentum description, and Endress (2010) for inflorescence morphology.A comparison of the significant morphological features of the new species with those of its allied species C. incana L. and C. bracteata Roxb.ex DC. was performed (Table 1).
The identification of the allied species C. incana L and C. bracteata Roxb.ex DC. was established through previous revisionary and systematic studies (Brach and Song 2006;Ansari 2008;Le Roux et al. 2013) and examinations of their types and other authentic specimens housed in large herbaria, such as PE, KIB, WUK, HITBC, CAL, DUH, FRLH, M, MH, SJC, and SKU.Additionally, virtual images of these species sourced from repositories such as the JSTOR Global Plants (JSTOR 2024), China Virtual Herbarium (Chinese Virtual Herbarium 2024), Flora of Pakistan (Eflora of Pakistan 2024), and several other prominent online herbaria (B, BM, BR, E, FI, FOB, G-DC, K, L, LINN, NYBG, P, TUB) were also analysed.
A distribution map was constructed to visualize the geographical distribution of the newly identified species.This map was developed with a foundational base map constructed from Natural Earth (www.naturalearthdata.com) and generated using QGIS version 3.28.2(QGIS 2021) (Fig. 1).

Taxa sampling for molecular study
Fresh and disease-free leaves were collected from specimens in the field and promptly dried using silica gel to facilitate subsequent DNA extraction.The voucher specimens were preserved at the Herbarium of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences (HITBC), and detailed information about each sample is provided in Suppl.material 1.The analysis included a total of 81 accessions, covering both the ITS region and the plastid marker matK.Additionally, this dataset included two outgroup sequences from Bolusia amboensis and Euchlora hirsuta, as well as nine publicly available sequences of African Crotalaria sourced from the NCBI databases (https://www.ncbi.nlm.nih.gov).Overall, our dataset comprised 81 individuals, representing 56 distinct Crotalaria species (Suppl.material 1).

DNA Extraction, PCR Amplification and Sequencing
Genomic DNA was isolated using a DNeasy Plant Mini Kit (Qiagen, Amsterdam, The Netherlands) following the manufacturer's protocol.The DNA quantity was confirmed via 0.8% agarose gel electrophoresis, and its concentration was determined using a SmartSpec TM Plus Spectrophotometer (Bio-Rad, Hercules, CA, United States).Before amplification, the DNA samples were stored at -20 °C.
Polymerase chain reactions (PCR) were performed in a 25 µL reaction volume comprising 2.5 µL of 10× buffer with 2 mM MgCl2, 1 U of Taq DNA polymerase, 1 µL of dNTPs (0.125 mM), 1 µL of each primer (5 pM), and 30-50 ng of total DNA.Nuclease-free water was added to reach the final volume.The optimal PCR conditions and detailed primer information are listed in Suppl.material 2. PCR products were visualized by electrophoresis on 0.8% agarose gels, followed by purification using BioMed multifunctional DNA fragment purification recovery kits (Beijing, China).The purified products were sequenced using the same primers used for PCR amplification.Sequencing was conducted on an ABI 3730 automated sequencer at Sangon Biotech, Shanghai, China.

Sequence alignment and data analysis
To ensure the accuracy and authenticity of the sequences, the original trace files were subjected to rigorous validation through web-based BLASTn searches on the NCBI platform.We conducted sequence alignment in Geneious version 8.1.7,which included trimming, visual inspection, and manual adjustments (Kearse et al. 2012).The trimming parameters were set to an error probability of 0.1 per base and a quality threshold of 20, allowing the removal of any low-quality base calls at the 5' and 3' ends of the sequenced PCR products.Each gene was aligned separately using MUSCLE (Edgar 2004) within Geneious.To improve alignment quality and accuracy, ambiguous regions were trimmed using Gblocks v0.91b (Castresana 2000).Individual alignments were then concatenated to create a two-gene alignment for all 81 samples.Microsatellite repeats were excluded, and gaps were considered as missing data.Phylogenetic analyses were performed using the standard-maximum-likelihood (ML) method with IQ-TREE (Nguyen et

Morphometric analyses
To assess potential differences between the new species and their closest relatives and to determine which traits were most relevant for their identification, we conducted a principal component analysis (PCA) using the "factoextra" package in R version 4.3.0(Kassambara and Mundt 2020; R Core Team 2023) with a significance level set at 5%.We examined three to five specimens of C. menglaensis S.A.Rather, C. incana L., and C. bracteata Roxb.ex DC.The length and width of leaflets, flowers, standards, wings, keels, seeds and pods were measured (Suppl.material 3).Correlation analysis was performed to eliminate highly correlated traits (r > 0.71) using the "corrplot" package in R version 4.3.0(Wei and Simko 2021; R Core Team 2023).In total, four traits were retained for the PCA biplot analysis: keel length (KL), standard width (SW), seed width (SEW), and seed length (SEL) (Suppl.material 3).

Results and discussion
The proposed new species, Crotalaria menglaensis S.A.Rather, resembles C. incana L. and C. bracteata Roxb.Ex DC.However, it differs from the former in several aspects.It has an ovate to oblanceolate leaflet shape with a pubescent leaf surface, an obovate-orbicular standard shape, a straight keel beak, and an elliptical to oblong pod shape.It differs from the latter in having a stem surface covered with white hairs, a pilose bract surface, a notched standard apex, planar callosity, an angled keel shape, and a tomentose pod indumentum.A comprehensive morphological comparison is presented in Table 1 to elucidate the distinctions between the new taxon and its closest relatives.
Phenology.The plants were observed to bear flowers and fruits from October to January.
Etymology.The specific epithet of the new species "menglaensis" is derived from the type locality of this species.
Distribution and habitat.Crotalaria menglaensis S.A. Rather is found in grasslands and exposed areas of Mengpeng, Mengla County, within the Xishuangbanna Dai Autonomous Prefecture, Yunnan, China.
Uses.Locals use the pods of this species as a food source.Additionally, its roots and seeds are utilized in traditional medicine to treat various digestive disorders.
IUCN Red List Category.This species is exclusively documented in a single location where clustered populations of fewer than 100 mature individuals have been observed.Its habitat is adjacent to roads and agricultural land and is consistently affected by anthropogenic activities such as grazing, deforestation, cultivation, and landscape management.The potential degradation of its natural habitat and restricted geographical range significantly threatens its survival.Therefore, according to the IUCN Standards and Petitions Committee (2019), this species should be considered critically endangered under criteria A4, B2a, C2a, and D1.These criteria denote species facing a very high risk of extinction in the wild.

Figure 1 .
Figure 1.Map visualizing the only known occurrences of Crotalaria menglaensis S.A.Rather in Mengpeng village of Xishuangbanna Dai Autonomous Prefecture, Yunnan, China (red dot).

Figure 2 .
Figure2.Phylogenetic hypothesis of the genus Crotalaria based on the concatenated matrix including matK and nrITS sequences constructed via maximum likelihood as implemented in IQ Tree.Bootstrap values are printed above the branches.Since Bayesian analyses resulted in almost the same topology, only the ML tree made is presented here.The new species Crotalaria menglaensis S.A.Rather was marked in red.The names on the right side of the phylogeny correspond to the infrageneric classification of the genus Crotalaria by LeRoux et al. (2013).

Figure 3 .
Figure 3. Scatter plot visualizing Dim1 and Dim2 from the principal component analyses based on the assembled morphological trait variables and accessions of the three species nested in the Incanae clade (see Fig. 2), namely, C. incana L., C. bracteata Roxb.ex DC. and the new species Crotalaria menglaensis S.A.Rather.Dim1 explained 44.3% of the variation, whereas DIM2 explained 40.2%.The vectors corresponded to KL = keel length, SW -standard width, SEW -seed width, and SEL -seed length.

Figure 4 .
Figure 4. Crotalaria menglaensis S.A.Rather A habit B plant twigs with leaves and flowers C inflorescence with flowers D inflorescences with flowers and fruits E flower in dorsal, lateral, and ventral views F calyx showing the dorsal and ventral surfaces G standard adaxial surface H standard abaxial surface with paired planar callosity pairs at the base with white silky pubescence I wing petals with prominent cavae and a distinct claw J adaxial and abaxial surfaces of keel petals, beak not twisted, pubescence along the margins from the middle to the base of the keel petal K anthers monodelphous, 10 dimorphic anthers (common to all the species within the genus) L gynoecium showing the ovary, style, and stigma M pod in ventral, dorsal, and lateral views N pod splitted longitudinally with young seeds.

Table 1 .
Comparisons among Crotalaria menglaensis S.A.Rather, C. incana L. and C. bracteata Roxb.ex DC.The bold font represents the main distinguishing features of the new species.

Table 2 .
Variance in the contributions of morphological trait variables as determined by principal component analysis.