Legume Research

  • Chief EditorJ. S. Sandhu

  • Print ISSN 0250-5371

  • Online ISSN 0976-0571

  • NAAS Rating 6.80

  • SJR 0.391

  • Impact Factor 0.8 (2024)

Frequency :
Monthly (January, February, March, April, May, June, July, August, September, October, November and December)
Indexing Services :
BIOSIS Preview, ISI Citation Index, Biological Abstracts, Elsevier (Scopus and Embase), AGRICOLA, Google Scholar, CrossRef, CAB Abstracting Journals, Chemical Abstracts, Indian Science Abstracts, EBSCO Indexing Services, Index Copernicus
Submit

Research Article

Legume Research, volume 44 issue 8 (august 2021) : 882-887

Phylogenetic Relationships in Vicia Subgenus Vicilla (Fabaceae) Based on Combined Evidence from DNA Sequences

Fei-Fei Wu1, Weihong Sun1, Fang Liu2, Qiu Gao2, Meiyan Jin1, Bowen Liu1, Xian-Guo Wang1,*
1College of Grassland Science and Technology, China Agricultural University, Beijing, China.
2National Herbage Germplasm Conservation Centre of China, Beijing 100125, China.

  • Submitted04-11-2020|

  • Accepted29-03-2021|

  • First Online 06-05-2021|

  • doi 10.18805/LR-596

Cite article:- Wu Fei-Fei, Sun Weihong, Liu Fang, Gao Qiu, Jin Meiyan, Liu Bowen, Wang Xian-Guo (2021). Phylogenetic Relationships in Vicia Subgenus Vicilla (Fabaceae) Based on Combined Evidence from DNA Sequences . Legume Research. 44(8): 882-887. doi: 10.18805/LR-596.

ABSTRACT

Background: Vicia L. is rich in protein and can be used as a good forage, green manure, etc., but its classification is chaotic, which is not conducive to breeding and utilization. The purpose of the study is sequencing the nrDNA ITS region the chloroplast region (matK, rbcL and trnL-F) of the Vicia species of subgenus Vicilla of Vicia to resolve taxonomic contradictions.

Methods: Collecting seeds from the National Herbage Germplasm Conservation Centre of China (Beijing, China) and the germplasm bank of wild species (Kunming, China). The seedlings were cultivated in greenhouse as experimental materials. The nrDNA ITS region and the chloroplast region (matK, rbcL and trnL-F) were sequenced in 17 Vicia species of subgenus Vicilla of Vicia. Maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) for all the data sets were used to estimate the individual and multilocus phylogenetic trees are used in this study and the result showed a highly-resolved phylogeny of the Vicia species. 

Result: V. amurensis, V. costata and V. tibetica belong to section Vicilla. Sections Cassubicae, Americanae, Variegatae and Lenticula were monophyletic groups. The sections Vicilla, Cracca and Amurense appeared polyphyletic. Treatment of sections Lenticula and Ervumin a separate subgenus Ervum was not supported. The research evaluated the phylogenetic position of the subgenus Vicilla species and provides a theoretical basis for the precise classification of the subgenus Vicilla and the genus Vicia.

INTRODUCTION

Vicia L. belongs to the legume tribe Fabaceae, which is widely distributed in the temperate zones in the northern hemisphere (Leht, 2009) and mainly centered in Europe, Caucasus and China (Zhao et al., 2001). The number of Vicia species has been estimated about 150 by Kupicha (1976), 210 by Hanelt and Mettin (1989) and the different results were mainly attributed to the confused circumscription of species (Maxted, 1993; Shiran et al., 2014). There are 140-160 species in subgenus Vicilla (Hanelt and Mettin, 1989) and the number is more than the subgenus Vicia, which made it difficult to circumscribe species precisely. The phylogenetic relationships of the subgenus Vicilla has been studied by morphological characters (Leht, 2005), isozyme (Jaaska, 2005) and ITS sequences (Choi et al., 2006). Most of these studies have clarified some confused relationships, V. amoena was placed in section Cassubicae instead of section Vicilla. However, some species were still controversial. For instance, V. hirsuta was belonged to section Cracca by the studies of Kupicha (1976) and Lersten and Gunn (1982). However, Steele and Wojciechowski (2003) did not support the placement of V. hirsuta in section Cracca, which was basically branched from other Fabaceae species based on matK dataset. V. hirsuta was also branched from other Vicia species based on styler types and ITS datasets (Choi et al., 2006).
        
More and more technical methods have been applied to solve better confused circumscription of species. Morphological traits (Abozeid et al., 2018; Binzat et al., 2014), biochemical methods (Leht and Jaaska, 2002; Pal et al., 2010) and molecular characteristics (Choi et al., 2006; Schaefer et al., 2012; Shiran et al., 2014; Katkar et al., 2015; Narula et al., 2013; Prasanthi et al., 2009) have been used to determine the inter specific relationships within the genus Vicia. Kupicha (1976) divided the world-wide species of genus Vicia into subgenus Vicia (5 sections) and subgenus Vicilla (17 sections) (Jaaska, 2005) by morphological traits. Previous studies have shown that DNA sequences is good diagnostic characters for distinguishing between species (Choi et al., 2006; Schaefer et al., 2012; Shiran et al., 2014; Wu et al., 2020).
        
This study described DNA variations of 17 species belonging to 8 traditional sections of the subgenus Vicilla. The nrDNA ITS region and the chloroplast region (matK, rbcL and trnL-F) were sequenced by DNA sequences, meanwhile the phylogenetic trees (MP, ML and Bayesian trees) were constructed based on individual and multi locus. Our goal was to evaluate the phylogenetic position of the subgenus Vicilla species and the results will contribute to the precise classification of the subgenus Vicilla and the genus Vicia.

MATERIALS AND METHODS

Materials
 
Seeds were collected from the National Herbage Germplasm Conservation Centre of China (Beijing, China) and the germplasm bank of wild species (Kunming, China) (Table 1). The seedlings were raise in greenhouse as experimental materials.
 

Table 1: List of the taxa and accession investigated.


 
DNA extraction and PCR amplification
 
Total genomic DNA was extracted from fresh or silica-gel-derived leaves using a Plant Genomic DNA Extraction Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions.
        
The ITS region was amplified using the primers ITS-p5 and ITS-u4 (Cheng et al., 2016), the matK region was amplified using the primers matK472F and matK1248R (Yu et al., 2011), the rbcL region was amplified using the primer scp063F andcp063R (Dong et al., 2013) and the trnL-trnF region was amplified using the primers trnL-c (Taberlet et al., 1991) and cp054R (Dong et al., 2013). Amplification reactions were performed in a total volume of 20 μL, containing 2.0 μL of 10× Taq buffer, 2.0 μL of dNTPs (2 mmol/L), 0.2 μL of Taq polymerase (2.5 U/μL), 1 μL of each primer (5 μmol/L), 1 μL of genomic DNA (30-40 ng) and water to adjust the volume. The PCR program consisted of 94°C for 3 min, followed by 40 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 1 min and a final extension at 72°C for 5 min. The PCR products were examined via 1% agarose gel electrophoresis with ethidium bromide and visualized using an ultraviolet trans illuminator.
 
Phylogenetic analysis
 
Phylogenetic trees were estimated using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) for all the datasets. Two species of the sister genus Lathyrus L., Lathyrus latifolius L. and Lathyrus sylvestris L. were used as out groups (Jaaska, 2005; Kenicer et al., 2005).
        
Maximum parsimony analyses were performed using PAUP* v4.0 b 10 (Swofford, 2003). Heuristic search was conducted with 1000 bootstrap replicates using random addition, with all characters unordered and equal weighted. Gaps were treated as missing and multistate taxa was interpreted as uncertainty. Branch-swapping algorithm, tree-bisection-reconnection (TBR) was conducted with zero-length branches collapsed and all minimal-length trees (MulTrees) saved on different datasets. To evaluate node support, bootstrap analysis (BS) (Felsenstein, 1985) was performed using 1000 pseudo replicates, each with 10 random taxon addition replicates and TBR branch swapping. The combined dataset was tested for incongruence using the partition homogeneity test (PHT) (Farris et al., 1994), this test was performed as implemented with 10,000 bootstrap replicates. The overall degree of homoplasy was estimated using consistency and retention indices (CI and RI). Descriptive statistics reflecting the amount of phylogenetic signal in the parsimony analysis were given by the consistency index (CI) (Kluge and Farris, 1969), retention index (RI) (Farris, 1989), and the resulting rescaled consistency index (RC) (Farris, 1989).We considered nodes with bootstrap support values of ³85% as strongly supported, 75%-84% as moderately and 50-74% as weakly supported.
        
Maximum likelihood (ML) trees were inferred using the fast and effective stochastic algorithm implemented in IQ-TREE v1.6.2 software (Nguyen et al., 2015). Best-fit substitution models were selected using the test function in IQ-TREE based on the Bayesian Information Criterion (BIC). We performed 1000 nonparametric bootstrap replicates to investigate nodal support across topologies.
        
Bayesian inference (BI) was performed on MrBayes 3.2.6 (Ronquist et al., 2012). The Akaike information criterion (AIC), which performed with MrModel test 2.3 (Nylander, 2004), was used as a selection criterion to evaluate non-nested models. The Bayesian analyses were run with four Markov Chains, starting from random trees, for ten million generations and sampled every 100th generation. After the first 20% generations were discarded as burn-in, a majority-rule consensus tree with posterior probabilities (PP) was constructed. Posterior probabilities were used to evaluate support for all nodes. We consider that clades with posterior probabilities above 0.95 are strongly supported.

RESULTS AND DISCUSSION

Characteristics of DNA regions
 
Based on alignment using MAFFT and manual adjustment in MEGA, the ITS sequences comprised 670 positions, among which 592 were invariant and 59 were parsimony-informative characters. The matK alignment comprised 648 positions, among which 577 were invariant and 59 were parsimony-informative characters. The rbcL sequences comprised 1268 positions, among which 1215 were invariant and 48 were parsimony-informative characters. The trnL-trnF alignment comprised 547 positions, among which 494 were invariant and 49 were parsimony-informative characters. The combined sequence of the ITS and chloroplast regions was 3144 bp, among which 2869 were invariant and 216 were parsimony-informative characters (Table 2).
 

Table 2: Nucleotide sequence characteristics of the amplified DNA regions in Vicia species.


 
Cladistic analysis of relationships between species
 
The MP and ML trees were similar to the Bayesian topologies. Most phylogenetic analysis of individual and combined datasets have supported that almost all the species can gather to a highly supported monophyletic (Fig 1). The Vicia species which formed a monophyletic clade were sperated in five hierarchical subclades. In both individual trees (ITS, matK and rbcL) and combined trees (ITS+matK+rbcL+trnL-trnF), V. tetrasperma belonging to section Ervum formed a basal sub clade with a high boot strap value (BS_ML=100; BS_MP=100; PP=1.00). The species V. hirsuta formed a strongly supported sub clade (BS_ML=100; BS_MP=100; PP=1.00) based on the combined DNA dataset and ITS. The section Cracca clade contained two well-supported species, V. cracca (BS_ML=100; BS_MP=95; PP=0.99) and V. villosa (BS_ML=100; BS_MP=99; PP=1.00). V. bungei (Sect. Americanae) formed a sub clade with strongly supported (BS_ML=100; BS_MP=100; PP=0.99) in rbcL and combined trees and was linked with V. megalotropis (BS_ML=81; BS_MP=72; PP=0.99) as sister-species couples with a high support value in ITS-tree. But V. bungei was linked with V. hirsuta (BS_ML=59; BS_MP=40; PP=0.64) with a low support value in matK-tree. In trnL-trnF tree, V. bungei, V. hirsuta and V. tetrasperma were linked with a variable support value (BS_ML=69; BS_MP=72; PP=0.98).
 

Fig 1: Phylogenetic analysis (MP, ML and Bayesian trees) based on gene sequence of nrDNA ITS region and the chloroplast region (matK, rbcL and trnL-F).


 
The species of section Vicilla, Amurense and Cassubicae formed a well-supported monophyletic sub clade (BS_ML=100; BS_MP=100; PP=1.00). However, the section Cracca was supported as monophyletic group, while the section Vicilla appeared polyphyletic based on isozyme analysis (Jaaska, 2005). Unexpectedly, V. costata of section Cracca was intermingled to the sub clade with low bootstrap support (BS_ML=59; BS_MP=51; PP=0.51). The section Amurense (V. amurensis and V. tibetica) (Endo and Ohashi, 1996) and section Vicilla (V. amoena, V. pseudorobus, V. unijuga, V. ramuliflora and V. japonica) (Kupicha, 1976) were formed a subclade with a relative high supported value (BS_ML=81; BS_MP=61; PP=0.95). The V. multicaulis (section Cassibicae) (Kupicha, 1976) was basically linked to the subclade with significant bootstrap support (BS_ML=100; BS_MP=100; PP=1.00).
        
In the present study, V. amurensis and V. tibetica formed a well-supported clade, which was consisted with Kupicha (1976). Our results supported the attribution of both two species to section Vicilla based on the DNA phylogenetic trees. In terms of other two species, V. dichroantha formed a clade with V. villosa, and V. nummularia formed a clade with V. tetrasperma, which were different from the results of Endo and Ohashi (1996) based on floral organ morphological structure and Schaefer et al., (2012) based on DNA sequences. This may be attributed to the low number of species used to construct phylogenetic trees.
        
In this study, V. tetrasperma appeared as a separate monophyletic clade apart from the Lathyrus clade and was clustered with V. nummularia. Therefore, the results argued against the placement of V. tetrasperma in a separate subgenus Ervum (Choi et al., 2006; Radzhi, 1970) and favored its position in the section Ervum of subgenus Vicilla (Jaaska, 2005; Kupicha, 1976).
        
V. costata was basally linked with the section Vicilla and formed sister clade with V. multicaulis of section Cassubicae in this study. The result was different from the view of placing V. costata in section Cracca (Kupicha, 1976; Leht et al., 2002), which was mainly based on the morphological traits. Our results favored to classify V. costata in section Vicilla, which was consistent with the results of Schaefer et al., (2012) based on DNA sequences.

CONCLUSION

In conclusion, the nuclear and chloroplast phylogenetic study yielded a highly-resolved phylogeny in 17 Vicia species of the subgenus Vicilla in this study. The results showed that sections Cassubicae, Americanae, Variegatae and Lenticula were monophyletic groups. The sections Vicilla, Cracca and Amurense appeared polyphyletic, V. amurensis, V. costata and V. tibetica were placed into section Vicilla. Treatment of sections Lenticula and Ervum in a separate subgenus Ervum was not supported. We have provided additional evidence to resolve the chaos of subgenus Vicilla. Further morphological and phylogenetic studies are needed to determine the precise taxonomic location of Vicia species.

ACKNOWLEDGEMENT

The present work was financially supported by the National Natural Science Foundation of China (No. 31772657).

REFERENCES


  1. Abozeid,A., Liu, Y., Liu, J. and Tang, Z. (2018). Taxonomic implication of embryo micromorphology in the genus Vicia L. (Fabaceae). Plant Systematics and Evolution. 304: 33-42.

  2. Binzat, O.K., Kahraman, A. and Doğan, M. (2014). Pollen morphology of some taxa of Vicia L. subgenus Vicilla (Schur) Rouy (Fabaceae) from Turkey. Plant Systematics and Evolution. 300: 1867-1876.

  3. Cheng, T., Xu, C., Lei, L., Li, C., Zhang, Y. and Zhou, S. (2016). Barcoding the kingdom Plantae: new PCR primers for ITS regions of plants with improved universality and specificity. Mol Ecol Resour. 16: 138-149.

  4. Choi, B.H., Seok, D.I., Endo, Y. and Ohashi, H. (2006). Phylogenetic significance of stylar features in genus Vicia (Leguminosae): an analysis with molecular phylogeny. Journal of Plant Research. 119: 513-523.

  5. Dong, W.P., Xu, C., Cheng, T., Lin, K. and Zhou, S.L. (2013). Sequencing angiosperm plastid genomes made easy: a complete set of universal primers and a case study on the phylogeny of Saxifragales. Genome Biol Evol. 5: 989-997.

  6. Endo, Y. and Ohashi, H. (1996). The Infragenetic Position of East Asian Species of Vicia (Leguminosae). Journal of Japanese Botany. 71: 254-262.

  7. Farris, J.S. (1989). The retention index and the rescaled consistency index. Cladistics. 5: 417-419.

  8. Farris, J.S., Källersjö, M., Kluge, A.G. and Bult, C. (1994). Testing significance of incongruence. Cladistics. 10: 315-319.

  9. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 39: 783-791.

  10. Fennell, S.R., Powell, W. and Wright, F. (1998). Phylogenetic relationships between Vicia faba (Fabaceae) and related species inferred from chloroplasttrnL sequences. Plant Systematics and Evolution. 212: 247-259.

  11. Hanelt, P. and Mettin, D. (1989). Biosystematics of the genus Vicia L. (Leguminosae). Annual Review of Ecology and Systematics. 20: 199-223.

  12. Jaaska, V. (1997). Isoenzyme diversity and phylogenetic affinities in Vicia subgenus Vicia (Fabaceae). Genetic Resources and Crop Evolution. 44: 557-574.

  13. Jaaska,V. (2005). Isozyme variation and phylogenetic relationships in Vicia subgenus Cracca (Fabaceae). Annals of Botany. 96: 1085-1096.

  14. Kenicer, G.J., Kajita, T., Pennington, R.T. and Murata, J. (2005). Systematics and biogeography of Lathyrus (Leguminosae) based on internal transcribed spacer and cpDNA sequence data. Am J Bot. 92: 1199-1209.

  15. Khalik, K.N.A. and Algohary, I.H. (2013). Taxonomic relationships in some Vicia species from Egypt, based on seed morphology and SDS-PAGE of seed proteins. Acta Scientiarum Biological Sciences. 35: 603-611.

  16. Kluge, A.G. and Farris, J.S. (1969). Quantitative phyletics and the evolution of anurans. Systematic Biology. 18: 1-32.

  17. Kupicha, F.K. (1976). The infrageneric structure of Vicia. Notes from the Royal Botanic Garden Edinburgh. 34: 287-326.

  18. Leht, M. (2005). Cladistic and phenetic analyses of relationships in Vicia subgenus Cracca (Fabaceae) based on morphological data. Taxon. 54: 1023-1032.

  19. Leht, M. (2009). Phylogenetics of Vicia (Fabaceae) based on morphological data. Feddes Repertorium. 120: 379-393.

  20. Leht, M. and Jaaska, V. (2002). Cladistic and phenetic analysis of relationships in Vicia subgenus Vicia (Fabaceae) by morphology and isozymes. Plant Systematics and Evolution. 232: 237-260.

  21. Lersten, N.R. and Gunn, C.R. (1982). Testa characters in tribe Vicieae, with notes about tribes Abreae, Cicereae and Trifolieae (Fabaceae).Technical Bulletins. 118: 274-285.

  22. Maxted, N. (1993). A phenetic investigation of Vicia L. subgenus Vicia (Leguminosae, Vicieae). Botanical Journal of the Linnean Society. 111: 155-182.

  23. Nylander, J. (2004). MrModeltest ver. 2. Evolutionary Biology Centre.

  24. Radzhi, A. (1970).Konspekt sistemy kavkazskikh vidov roda Vicia L. (Conspectus systematis specierum caucasicarum generis Vicia L.). Novosti Sist. Vyssh. Rast. 7: 228-240.

  25. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. and Huelsenbeck, J.P. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic biology. 61: 539-542.

  26. Schaefer, H., Hechenleitner, P., Santos-Guerra, A., Sequeira, M.M.D., Pennington, R.T., Kenicer, G. and Carine, M.A. (2012). Systematics, biogeography and character evolution of the legume tribe Fabeae with special focus on the middle-Atlantic island lineages. BMC Evolutionary Biology.12: 250-268.

  27. Shiran, B., Kiani, S., Sehgal, D., Hafizi, A., ul-Hassan, T., Chaudhary, M. and Raina, S.N. (2014). Internal transcribed spacer sequences of nuclear ribosomal DNA resolving complex taxonomic history in the genus Vicia L. Genetic Resources and Crop Evolution. 61: 909-925.

  28. Steele, K.P. and Wojciechowski, M.F. (2003). Phylogenetic analyses of tribes Trifolieae and Vicieae, based on sequences of the plastid gene matK (Papilionoideae: Leguminosae), in: Klitgaard, B.B., Bruneau, A. (Eds.). Advances in Legume Systematics. Royal Botanic Gardens, Kew. pp. 355-370.

  29. Swofford, D. (2003). PAUP*: phylogenetic analysis using parsimony, version 4.0 b10.

  30. Taberlet, P., Gielly, L., Pautou, G. and Bouvet, J. (1991). Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology. 17: 1105-1109.

  31. Van de Wouw, M., Maxted, N., Chabane, K. and Ford-Lloyd, B. (2001). Molecular taxonomy of Vicia ser. Vicia based on amplified fragment length polymorphisms. Plant Systematics and Evolution. 229: 91-105.

  32. Wu, F.-F., Gao, Q., Liu, F., Wang, Z., Wang, J. L. and Wang and X. G. (2020). DNA barcoding evaluation of Vicia (Fabaceae): Comparative efficacy of six universal barcode loci on abundant species. Journal of Systematics and Evolution.

  33. Yu, J., Xue, J. and Zhou, S. (2011). New universal matK primers for DNA barcoding angiosperms. Journal of Systematics and Evolution. 49: 176-181.

  34. Zhao, Y. and Liu, Y. (2001). Study on Floristic and Ecological Geogrophical Distribution of the Genus Vicia L. in the Mongolian Plateau. Acta Scientiarum Naturalium Universitatis Neimongol. 32: 207-210.

  35. Katkar, M., Mane, S.S. and Kadam, N. (2015). Molecular characterization races of Fusarium oxysporumf.sp.Ciceri using RAPD and ISSR markers. Legume Research. 38:246-252.

  36. Narula, S., Anand, R.C., Dudeja, S.S. and Pathak, D.V. (2013). Molecular diversity of root and nodule endophytic bacteria from field pea (Pisum sativum L.). Legume Research. 36: 344-350.

  37. Pal, M., Brahmachary, R. L. and Ghosh, M. (2010). Comparative studies on physicochemical and biochemical characteristics of scented and non-scented strains of mung beans (Vigna radiata) of Indian origin. Legume Research. 33: 1-9.

  38. Prasanthi, L. Reddy, B.V., Rani, K.R. and Naidu, P.H. (2009). Molecular marker for screening fusarium wilt resistance in pigeonpea [Cajanus cajan (L.) Millspaugh]. Legume Research. 32:212-216.
Reviewed By

Download Article
In this Article
    Contact us
    Follow us

    Editorial Board

    View all (0)