Small Single-copy Region Research Articles

The first chloroplast sequence of Rosa davurica Pall. var. Davurica

Rosa davurica Pall. var. davurica is a member of the plant family Rosaceae. Although R. davurica has high application value, its chloroplast genome sequence has not been reported. This study aims to reveal the genetic characteristics of the chloroplast genome of Rosa roxburghii. The length of its total chloroplast DNA is 156,971 bp, with 37.22% G/C content. Its chloroplast genome has two inverted repeat (IRa and IRb) regions totaling 26,051 bp which are separated by a large single copy (LSC) region of 86,032 bp and a small single copy (SSC) region of 18,837 bp. The genome contains 131 independent genes (86 protein-coding, 37 tRNA, and 8 rRNA), and there are 18 repeated genes within the IR region. Among these genes, 17 genes contained one or two introns. The phylogenetic analysis showed that R. davurica was relatively close to other Rosa species, such as the Rosa hybrid.

The complete chloroplast genome sequence of Aconitum tschangbaischanense (Ranunculaceae)

The perennial herbal medicine species Aconitum tschangbaischanense, is endemic to Changhai Mountain, Jilin province. In this study, we attempted to uncover the complete chloroplast (cp) genome of A. tschangbaischanense based on sequencing data using the Illumina sequencing technology. As per the results: (1) the length of its complete cp genome is 155,881 bp with a typical tetrad structure; (2) the structure of its cp genome contains large single-copy and small single-copy (LSC and SSC) regions of 86,351 and 16,9444 bp, respectively, isolated by two inverted repeat regions (IRs) of 26,293 bp; (3) we annotated a total 131 genes, consisting of 86 protein-coding genes, eight rRNA genes, and 37 tRNA genes. According to the maximum-likelihood phylogenetic tree based on complete cp genomes, A. tschangbaischanense, showed close association with A. carmichaelii, which belongs to clade I. Finally, this study provides the characteristics of the cp genome of A. tschangbaischanense, and its phylogenetic position.

Characterization of the complete chloroplast genome of Gypsophila huashanensis Y. W. Tsui & D. Q. Lu, an endemic herb species in China

Gypsophila huashanensis Y. W. Tsui & D. Q. Lu (Caryophyllaceae) is an endemic herb species to the Qinling Mountains in China. In this study, we characterized its whole plastid genome using the Illumina sequencing platform. The complete plastid genome of G. huashanensis is 152,457 bp in length, including a large single-copy DNA region of 83,476 bp, a small single-copy DNA region of 17,345 bp, and a pair of inverted repeat DNA sequences of 25,818 bp. The genome contains 130 genes comprising 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Evolutionary analysis showed that the non-coding regions of Caryophyllaceae exhibit a higher level of divergence than the exon regions. Gene site selection analysis suggested that 11 coding protein genes (accD, atpF, ndhA, ndhB, petB, petD, rpoCl, rpoC2, rps16, ycfl, and ycf2) have some sites under protein sequence evolution. Phylogenetic analysis showed that G. huashanensis is most closely related to the congeneric species G. oldhamiana. These results are very useful for studying phylogenetic evolution and species divergence in the family Caryophyllaceae.

The complete chloroplast genome sequence of Phyllostachys incarnata Wen, 1982

Phyllostachys incarnata Wen, 1982 is one of the important material and edible bamboo specie of high quality in China. We reported the complete chloroplast(cp) genome of P. incarnata in this study. The cp genome of P. incarnata (GenBank accession number: OL457160) was a typical tetrad structure with a full length of 139,689 bp, comprising a pair of inverted repeated (IR) regions (21,798 bp) separated by a large single-copy (LSC) region (83,221 bp) and a small single-copy (SSC) region (12,872 bp). And the cp genome contained 136 genes, including 90 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Phylogenetic analysis based on 19 cp genomes suggested that P. incarnata was relatively close to P. glauca among the species analyzed.

The complete chloroplast genome of Eurya rubiginosa var. attenuata H. T. Chang (Pentaphylacaceae)

Eurya rubiginosa var. attenuata is a valuable multiuse tree with a long history of use in China. It has great economic and ecological importance and is used for landscape and urban planting, soil improvement, and raw materials for food production. However, genomic studies of E. rubiginosa var. attenuata are limited. Meanwhile, the classification of this taxon is controversial. In this study, the complete plastome of E. rubiginosa var. attenuata was successfully sequenced and assembled. The chloroplast genome is 157,215 bp in length with a 37.3% GC content. The chloroplast genome structure includes a quadripartite structure comprising a pair of inverted repeat (IR) sequences of 25,872 bp, a small single-copy (SSC) region of 18,216 bp, and a large single-copy (LSC) region of 87,255 bp. The genome contains 128 genes, including 83 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic inference based on complete plastome analysis showed that E. rubiginosa var. attenuata is closely related to E. alata and belongs to the family Pentaphylacaceae, which differs from the results of the traditional Engler system. The chloroplast genome sequence assembly and phylogenetic analysis enrich the genetic resources of Pentaphylacaceae and provide a molecular basis for further studies on the phylogeny of the family.

A complete chloroplast genome sequence of Viola albida Palibin 1899 (Violaceae), a member of VIOLA ALBIDA complex

The VIOLA ALBIDA complex is a complicated group with taxonomic problems having continuous leaf variations and composed of taxa related to the following names: Viola albida, V. albida var. takahashii, and V. chaerophylloides. As a first step to understanding the genomic nature of this complex, this study identified the whole chloroplast genome of V. albida. The genome is 157,692 bp in length (36.3% of GC content) and contains four subregions: a large single copy region of 86,220 bp, a small single copy region of 17,248 bp, and a pair of inverted regions of 27,112 bp each. An annotation of the gene identifies 111 unique genes, including 77 protein-coding genes, four rRNA genes, and 30 tRNA genes. The phylogenetic analysis of this genome with selected cp genomes from Viola identifies the close relationship between V. albida and V. ulleungdoensis. It is noteworthy that V. chaerophylloides, traditionally recognized as a member of the VIOLA ALBIDA complex, is genetically distant from V. albida and forms a sister group of all other members of the subsection Patellares. Our genome report is expected to serve as a basis for understanding the identity of the VIOLA ALBIDA complex.

Chloroplast genome characteristics and phylogeny of the sinodielsia clade (apiaceae: apioideae)

BackgroundThe Sinodielsia clade of the subfamily Apioideae (Apiacieae) was established in 2008, and it is composed of 37 species from 17 genera. Its circumscription is still poorly delimited and unstable, and interspecific relationships in the clade lack comprehensive analysis. Chloroplast (cp.) genomes provide valuable and informative data sources for evolutionary biology and have been widely used in studies on plant phylogeny. To infer the phylogenetic history of the Sinodielsia clade, we assembled complete cp. genomes of 39 species and then performed phylogenetic analysis based on these cp. genome sequence data combined with 66 published cp. genomes from 16 genera relative to the Sinodielsia clade.ResultsThese 39 newly assembled genomes had a typical quadripartite structure with two inverted repeat regions (IRs: 17,599–31,486 bp) separated by a large single-copy region (LSC: 82,048–94,046 bp) and a small single-copy region (SSC: 16,343–17,917 bp). The phylogenetic analysis showed that 19 species were clustered into the Sinodielsia clade, and they were divided into two subclades. Six mutation hotspot regions were detected from the whole cp. genomes among the Sinodielsia clade, namely, rbcL–accD, ycf4–cemA, petA–psbJ, ycf1–ndhF, ndhF–rpl32 and ycf1, and it was found that ndhF–rpl32 and ycf1 were highly variable in the 105 sampled cp. genomes.ConclusionThe Sinodielsia clade was subdivided into two subclades relevant to geographical distributions, except for cultivated and introduced species. Six mutation hotspot regions, especially ndhF–rpl32 and ycf1, could be used as potential DNA markers in the identification and phylogenetic analyses of the Sinodielsia clade and Apioideae. Our study provided new insights into the phylogeny of the Sinodielsia clade and valuable information on cp. genome evolution in Apioideae.

Complete Chloroplast Genome Determination of Ranunculus sceleratus from Republic of Korea (Ranunculaceae) and Comparative Chloroplast Genomes of the Members of the Ranunculus Genus.

Ranunculus sceleratus (family: Ranunculaceae) is a medicinally and economically important plant; however, gaps in taxonomic and species identification limit its practical applicability. This study aimed to sequence the chloroplast genome of R. sceleratus from Republic of Korea. Chloroplast sequences were compared and analyzed among Ranunculus species. The chloroplast genome was assembled from Illumina HiSeq 2500 sequencing raw data. The genome was 156,329 bp and had a typical quadripartite structure comprising a small single-copy region, a large single-copy region, and two inverted repeats. Fifty-three simple sequence repeats were identified in the four quadrant structural regions. The region between the ndhC and trnV-UAC genes could be useful as a genetic marker to distinguish between R. sceleratus populations from Republic of Korea and China. The Ranunculus species formed a single lineage. To differentiate between Ranunculus species, we identified 16 hotspot regions and confirmed their potential using specific barcodes based on phylogenetic tree and BLAST-based analyses. The ndhE, ndhF, rpl23, atpF, rps4, and rpoA genes had a high posterior probability of codon sites in positive selection, while the amino acid site varied between Ranunculus species and other genera. Comparison of the Ranunculus genomes provides useful information regarding species identification and evolution that could guide future phylogenetic analyses.

Complete Chloroplast Genome of Bamboo Species Pleioblastus ovatoauritus and Comparative Analysis of Pleioblastus from China and Japan

Pleioblastus ovatoauritus T.H.Wen ex W.Y.Zhang is bamboo species published in 2018, originated from and existing in southeastern China. The chloroplast genome of Pl. ovatoauritus was obtained using a high-throughput sequencing platform. The chloroplast genome is up to 139,708 bp in length and displays a typical quadripartite structure with one large single-copy region, one small single-copy region, and two inverted repeat regions. There are 82 protein-coding genes, 8 rRNA genes, and 39 tRNA genes in the plastome genome. However, the interspecific relationship of Pleioblastus species originated from China and Japan has not been revealed explicitly. To understand their relationship, data from four Chinese species and four Japanese species were selected to investigate the distinctions between their genome structures, codon usage patterns, and SSR sites. We moved forward to examine the sequence divergence and polymorphic sites between the eight species. Phylogenetic trees were then plotted using the maximum likelihood method based on different parts of the sequences. Obvious difference found in the JLB boundary and a split in the phylograms contributed to our decision to split Pleioblastus species of China and Japan into different clades. Moreover, taxonomy using the subgenera concept in Flora Reipublicae Popularis Sinicae proved untenable. Nine SSR primers for Pleioblastus genus were then developed from cp genomes, aimed at facilitating identification and germplasm investigation.

CPJSdraw: analysis and visualization of junction sites of chloroplast genomes.

Chloroplast genomes are usually circular molecules, and most of them are tetrad structures with two inverted repeat (IR) regions, a large single-copy region, and a small single-copy region. IR contraction and expansion are among the genetic diversities during the evolution of plant chloroplast genomes. The only previously released tool for the visualization of junction sites of the regions does not consider the diversity of the starting point of genomes, which leads to incorrect results or even no results for the examination of IR contraction and expansion. In this work, a new tool named CPJSdraw was developed for visualizing the junction sites of chloroplast genomes. CPJSdraw can format the starting point of the irregular linearized genome, correct the junction sites of IR and single-copy regions, display the tetrad structure, visualize the junction sites of any number (≥1) of chloroplast genomes, show the transcription direction of genes adjacent to junction sites, and indicate the IR expansion or contraction of chloroplast genomes. CPJSdraw is a software that is universal and reliable in analysis and visualization of IR expansion or contraction of chloroplast genomes. CPJSdraw has more accurate analysis and more complete functions when compared with previously released tool. CPJSdraw as a perl package and tested data are available at http://dx.doi.org/10.5281/zenodo.7669480 for English users. In addition, an online version with a Chinese interface is available at http://cloud.genepioneer.com:9929/#/tool/alltool/detail/335.

Comparison of the chloroplast genomes and phylogenomic analysis of Elaeocarpaceae.

Elaeocarpaceae is a vital family in tropical and subtropical forests. Compared with the important position of Elaeocarpaceae species in forest ecosystem and the concern of medicinal value, the most research on Elaeocarpaceae are classification and taxonomy. Molecular systematics has corrected the morphological misjudgment, and it belongs to Oxalidales. Phylogenetic and divergence time estimates of Elaeocarpaceae is mostly constructed by using chloroplast gene fragments. At present, although there are reports on the chloroplast structure of Elaeocarpaceae, a comprehensive analysis of the chloroplast structure of Elaeocarpaceae is lacking. To understand the variation in chloroplast sequence size and structure in Elaeocarpaceae, the chloroplast genomes of nine species were sequenced using the Illumina HiSeq 2500 platform and further assembled and annotated with Elaeocarpus japonicus and Sloanea sinensis (family Elaeocarpaceae) as references. A phylogenomic tree was constructed based on the complete chloroplast genomes of the 11 species representing five genera of Elaeocarpaceae. Chloroplast genome characteristics were examined by using Circoletto and IRscope software. The results revealed the following: (a) The 11 sequenced chloroplast genomes ranged in size from 157,546 to 159,400 bp. (b) The chloroplast genomes of Elaeocarpus, Sloanea, Crinodendron and Vallea lacked the rpl32 gene in the small single-copy (SSC) region. The large single-copy (LSC) region of the chloroplast genomes lacked the ndhK gene in Elaeocarpus, Vallea stipularis, and Aristotelia fruticosa. The LSC region of the chloroplast genomes lacked the infA gene in genus Elaeocarpus and Crinodendron patagua. (c) Through inverted repeat (IR) expansion and contraction analysis, a significant difference was found between the LSC/IRB and IRA/LSC boundaries among these species. Rps3 was detected in the neighboring regions of the LSC and IRb regions in Elaeocarpus. (d) Phylogenomic analysis revealed that the genus Elaeocarpus is closely related to Crinodendron patagua on an independent branch and Aristotelia fruticosa is closely related to Vallea stipularis, forming a clade with the genus Sloanea. Structural comparisons showed that Elaeocarpaceae diverged at 60 Mya, the genus Elaeocarpus diverged 53 Mya and that the genus Sloanea diverged 0.44 Mya. These results provide new insight into the evolution of the Elaeocarpaceae.

Comparative plastome genomics, taxonomic delimitation and evolutionary divergences of Tetraena hamiensis var. qatarensis and Tetraena simplex (Zygophyllaceae)

The Zygophyllum and Tetraena genera are intriguingly important ecologically and medicinally. Based on morphological characteristics, T. hamiensis var. qatarensis, and T. simplex were transferred from Zygophyllum to Tetraena with the least genomic datasets available. Hence, we sequenced the T. hamiensis and T. simplex and performed in-depth comparative genomics, phylogenetic analysis, and estimated time divergences. The complete plastomes ranged between 106,720 and 106,446 bp—typically smaller than angiosperms plastomes. The plastome circular genomes are divided into large single-copy regions (~ 80,964 bp), small single-copy regions (~ 17,416 bp), and two inverted repeats regions (~ 4170 bp) in both Tetraena species. An unusual shrinkage of IR regions 16–24 kb was identified. This resulted in the loss of 16 genes, including 11 ndh genes which encode the NADH dehydrogenase subunits, and a significant size reduction of Tetraena plastomes compared to other angiosperms. The inter-species variations and similarities were identified using genome-wide comparisons. Phylogenetic trees generated by analyzing the whole plastomes, protein-coding genes, matK, rbcL, and cssA genes exhibited identical topologies, indicating that both species are sisters to the genus Tetraena and may not belong to Zygophyllum. Similarly, based on the entire plastome and proteins coding genes datasets, the time divergence of Zygophyllum and Tetraena was 36.6 Ma and 34.4 Ma, respectively. Tetraena stem ages were 31.7 and 18.2 Ma based on full plastome and protein-coding genes. The current study presents the plastome as a distinguishing and identification feature among the closely related Tetraena and Zygophyllum species. It can be potentially used as a universal super-barcode for identifying plants.

Complete chloroplast genome sequence of Eranthis byunsanensis B.Y. Sun (Ranunculaceae), an endemic species in Korea

The Korean endemic Eranthis byunsanensis B.Y. Sun, 1993 (Ranunculaceae) is a rare plant distributed in the southwestern part of the Korean Peninsula. The complete chloroplast (cp) genome of E. byunsanensis was sequenced by next-generation sequencing (NGS) using an Illumina HiSeq X platform. The cp genome of E. byunsanensis is 160,324 bp in length with 37.9% GC content. It showed a typical quadripartite structure consisting of a pair of inverted repeats (IRs; 28,356 bp), a large single-copy region (LSC; 87,671 bp), and a small single-copy region (SSC; 15,941 bp). The cp genome comprises 130 genes including 85 protein-coding genes (PCGs), 37 tRNA genes, and eight rRNA genes. The molecular phylogenetic analysis indicates that E. byunsanensis is closely related to Eranthis stellata, both of which belong to the genus Eranthis.

Analysis of the complete plastomes of Bidens pilosa L. 1753 (Asteraceae, Coreopsideae) from Beijing, China reveals high genetic diversity and possible misidentifications

Bidens pilosa L. 1753 is a perennial herbaceous flowering plant, traditionally used in foods and medicines. In this study, we sequenced, assembled, and characterized the complete plastome of B. pilosa from Beijing, China. The plastome (MN385242) is circularized with a conservative quadripartite structure. Its length is 150,524 bp, including a large single-copy region (83,535 bp), a small single-copy region (17,627 bp), and a pair of inverted repeat regions (each 24,681 bp). The plastome consists of 128 genes, including 78 unique protein-coding, 28 unique tRNA, and 4 unique rRNA genes. Phylogenetic analyses showed all five B. pilosa plants couldn’t form a monophyletic clade and were separated into three clades. The results of K2P distance and molecular markers were all consistent with those of phylogenetic analysis, revealing high genetic diversity and even possible misidentifications of the B. pilosa. Our results highlighted the importance of correct species identification of materials in medicinal products.

First record of the complete chloroplast genome of Polygonatum infundiflorum (Asparagaceae), a Korean endemic species

Polygonatum infundiflorum Y.S. Kim, B.U. Oh & C.G. Jang et al. 1998 is a Korean endemic species. This is first report on the complete chloroplast genome sequence of P. infundiflorum. The complete chloroplast genome length was 154,578 bp with a GC content of 37.7%. The large single-copy (LSC) region was 83,527 bp long, and the small single-copy (SSC) region was 18,457 bp long. The paired inverted repeats (IRs) were 26,297 bp and separated the LCS and SSC regions. There were 113 genes, comprising 78 protein-coding genes, four rRNA genes, 30 tRNA genes, and one pseudogene (infA). In total, 16 genes contained one intron, and two genes contained two introns. Phylogenetic analysis suggested that Polygonatum was divided into three sections, each forming a monophyletic group. P. infundiflorum was sister to P. macropodum and formed a monophyletic group with P. inflatum. This study provides basic information for future research and contributes to taxonomic and genetic studies on Polygonatum.

The complete chloroplast genome of Primula vialii (Primulaceae), an ornamental plant

Primula vialii Delavay ex Franch. (1905) is an alpine species with an ornamental value. In this study, we sequenced, assembled, and annotated the chloroplast genome of P. vialii. The results showed that it was a double-stranded, closed circular DNA with 154,897 bp in length, comprising a small single-copy (SSC) region of 17,766 bp, a large single-copy (LSC) region of 85,379 bp and a pair of inverted repeat (IR) regions of 25,876 bp. A total of 113 unique genes were annotated, including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. The phylogenetic analysis revealed that P. vialii is closely related to Primula flaccida. The cp genomic data will be useful for systematics and evolutionary studies of Primula.

The complete chloroplast genome sequence of Sedum bulbiferum (Crassulaceae)

Sedum bulbiferum is a traditional medicinal plant in China, with few reports on its chloroplast genome. In this study, the chloroplast genome of Sedum bulbiferum was characterized, and its phylogenetic position among other closely related species was studied. The results showed that the full length of the chloroplast genome was 150,074 bp, containing a large single-copy (LSC) region and a small single-copy (SSC) region of 81,730 and 16,726 bp, respectively, as well as two inverted repeat regions (IRs) of 25,809 bp like other plants. A total of 128 genes were found, including 83 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis showed that Sedum bulbiferum is closely related to Sedum emarginatum, Sedum alfredii, Sedum tricarpum, Sedum plumbizincicola, and Sedum sarmentosum.

Characterization of the complete chloroplast genome sequence of Limnophila sessiliflora Blume 1826 (Plantaginaceae)

Limnophila sessiliflora Blume 1826 is a perennial amphibious herb with ornamental and water purification value that is widespread in temperate and tropical Asia. In the present study, we sequenced, assembled, and annotated the complete chloroplast (cp) genome of L. sessiliflora. It is 152,395 bp in length, with a typical quadripartite structure, comprising a pair of inverted repeat regions (IRs; 25,545 bp), a large single-copy region (LSC; 83,163 bp), and a small single-copy (SSC; 18,142 bp) region. The whole cp genome contained 135 genes, including 89 protein-coding genes (PCGs), 38 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. The maximum-likelihood (ML) phylogenetic analysis indicated that L. sessiliflora was closely related to the genera Bacopa and Scoparia in the tribe Gratioleae of Plantaginaceae. This cp genome provides a valuable genetic resource for phylogenetic study.

The pan-plastome of tartary buckwheat (fagopyrum tataricum): key insights into genetic diversity and the history of lineage divergence

BackgroundTartary buckwheat (Fagopyrum tataricum) is an important food and medicine crop plant, which has been cultivated for 4000 years. A nuclear genome has been generated for this species, while an intraspecific pan-plastome has yet to be produced. As such a detailed understanding of the maternal genealogy of Tartary buckwheat has not been thoroughly investigated.ResultsIn this study, we de novo assembled 513 complete plastomes of Fagopyrum and compared with 8 complete plastomes of Fagopyrum downloaded from the NCBI database to construct a pan-plastome for F. tartaricum and resolve genomic variation. The complete plastomes of the 513 newly assembled Fagopyrum plastome sizes ranged from 159,253 bp to 159,576 bp with total GC contents ranged from 37.76 to 37.97%. These plastomes all maintained the typical quadripartite structure, consisting of a pair of inverted repeat regions (IRA and IRB) separated by a large single copy region (LSC) and a small single copy region (SSC). Although the structure and gene content of the Fagopyrum plastomes are conserved, numerous nucleotide variations were detected from which population structure could be resolved. The nucleotide variants were most abundant in the non-coding regions of the genome and of those the intergenic regions had the most. Mutational hotspots were primarily found in the LSC regions. The complete 521 Fagopyrum plastomes were divided into five genetic clusters, among which 509 Tartary buckwheat plastomes were divided into three genetic clusters (Ft-I/Ft-II/Ft-III). The genetic diversity in the Tartary buckwheat genetic clusters was the greatest in Ft-III, and the genetic distance between Ft-I and Ft-II was the largest. Based on the results of population structure and genetic diversity analysis, Ft-III was further subdivided into three subgroups Ft-IIIa, Ft-IIIb, and Ft-IIIc. Divergence time estimation indicated that the genera Fagopyrum and Rheum (rhubarb) shared a common ancestor about 48 million years ago (mya) and that intraspecies divergence in Tartary buckwheat began around 0.42 mya.ConclusionsThe resolution of pan-plastome diversity in Tartary buckwheat provides an important resource for future projects such as marker-assisted breeding and germplasm preservation.

Characterization of the complete chloroplast genome of Dalbergia congesta (Fabaceae), an endangered legume endemic to the Nilgiri Hills of Tamil Nadu, India

Reference-guided de novo assembly of the Dalbergia congesta chloroplast genome was carried out using whole-genome sequencing data. The newly generated chloroplast genome size had a total length of 156,048 bp and a GC content of 36.1%. The plastome showed the classical quadripartite structure with two inverted repeats regions (IRs; each 25,715 bp) separating the large single-copy region (LSC; 85,456 bp) from the small single-copy region (SSC; 19,162 bp). The plastid genome contained 111 unique genes, including 77 protein-coding genes (CDS), 30 tRNAs, and 4 rRNAs. The phylogenomic analyses based on whole chloroplast genome sequences recovered Dalbergia as a distinct clade of the Papilionoideae, with Dalbergia congesta having a sister relationship to a clade comprising D. fusca and D. cultrata. The newly available plastome sequence will facilitate future genetic and conservational research aiming to protect this economically important but highly threatened legume species.