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Introduction: Epimedium L., the largest herbaceous genus of Berberidaceae, is one of the most taxonomically difficult representatives. The classification and phylogenetic relationships within Epimedium are controversial and unresolved., Objectives: For the first time, we systematically studied the phylogeny and evolution of Epimedium based on plastid genome (plastome) data for better understanding this enigmatic genus., Methods: We explored the molecular phylogeny, assessed the infrageneric classification, estimated the divergence times, and inferred the ancestral states for flower traits of Epimedium based on 45 plastomes from 32 species., Results: The Epimedium plastome length ranged from 156,635 bp to 159,956 bp. Four types of plastome organization with different inverted repeat boundary changes were identified. Phylogenetic analysis revealed a strong support for the sister relationship of sect. Macroceras and sect. Diphyllon but did not provide a distinct route for petal evolution in sect. Diphyllon . Disharmony between phylogenetic relationships and traditional classification of sect. Diphyllon was observed. Results from divergence time analysis showed that Epimedium diverged in the early Pleistocene (~2.11 Ma, 95% HPD = 1.88-2.35 Ma). Ancestral character state reconstructions indicated transitions from long spur (large-flowered group) to other petal types (small-flowered group) in Epimedium ., Conclusion: These findings provide new insights into the relationships among Epimedium species and pave the way for better elucidation of the classification and evolution of this genus., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V. on behalf of Cairo University.)

Epimedium wushanense (Berberidaceae) is recorded as the source plant of Epimedii Wushanensis Folium in the Chinese Pharmacopoeia. However, controversies exist on the classification of E. wushanense and its closely related species, namely, E . pseudowushanense , E. chlorandrum , E. mikinorii , E. ilicifolium , and E. borealiguizhouense . These species are often confused with one another because of their highly similar morphological characteristics. This confusion leads to misuse in the medicinal market threatening efficiency and safety. Here, we studied the plastid genomes of these Epimedium species. Results show that the plastid genomes of E. wushanense and its relative species are typical circular tetramerous structure, with lengths of 156,855-158,251 bp. A total of 112 genes were identified from the Epimedium plastid genomes, including 78 protein-coding, 30 tRNA, and 4 rRNA genes. A loss of rpl32 gene in E. chlorandrum was found for the first time in this study. The phylogenetic trees constructed indicated that E. wushanense can be distinguished from its closely related species. E. wushanense shows a closer relationship to species in ser. Dolichocerae . In conclusion, the use of plastid genomes contributes useful genetic information for identifying medicinally important species E. wushanense and provides new evidence for understanding phylogenetic relationships within the Epimedium genus., Competing Interests: The authors declare no conflict of interest.

[Display omitted] • The phylogeny and evolution of Epimedium based on 45 plastomes were first studied systematically. • Four types of plastomes were recognized according to the variation of inverted repeat boundary. • A strong support for the sister relationship of sect. Macroceras and sect. Diphyllon was revealed. • The diversification of Epimedium was estimated in the early Pleistocene (∼2.11 Ma). • Long spur (large-flowered group) constituted the plesiomorphic state of Epimedium. • Disharmony existed between molecular phylogeny and traditional classification of sect. Diphyllon. Epimedium L., the largest herbaceous genus of Berberidaceae, is one of the most taxonomically difficult representatives. The classification and phylogenetic relationships within Epimedium are controversial and unresolved. For the first time, we systematically studied the phylogeny and evolution of Epimedium based on plastid genome (plastome) data for better understanding this enigmatic genus. We explored the molecular phylogeny, assessed the infrageneric classification, estimated the divergence times, and inferred the ancestral states for flower traits of Epimedium based on 45 plastomes from 32 species. The Epimedium plastome length ranged from 156,635 bp to 159,956 bp. Four types of plastome organization with different inverted repeat boundary changes were identified. Phylogenetic analysis revealed a strong support for the sister relationship of sect. Macroceras and sect. Diphyllon but did not provide a distinct route for petal evolution in sect. Diphyllon. Disharmony between phylogenetic relationships and traditional classification of sect. Diphyllon was observed. Results from divergence time analysis showed that Epimedium diverged in the early Pleistocene (~2.11 Ma, 95% HPD = 1.88–2.35 Ma). Ancestral character state reconstructions indicated transitions from long spur (large-flowered group) to other petal types (small-flowered group) in Epimedium. These findings provide new insights into the relationships among Epimedium species and pave the way for better elucidation of the classification and evolution of this genus. [ABSTRACT FROM AUTHOR]

Genus Epimedium consists of approximately 50 species in China, and more than half of them possess medicinal properties. The high similarity of species' morphological characteristics complicates the identification accuracy, leading to potential risks in herbal efficacy and medical safety. In this study, we tested the applicability of four single loci, namely, rbcL, psbA-trnH, internal transcribed spacer (ITS), and ITS2, and their combinations as DNA barcodes to identify 37 Epimedium species on the basis of the analyses, including the success rates of PCR amplifications and sequencing, specific genetic divergence, distance-based method, and character-based method. Among them, character-based method showed the best applicability for identifying Epimedium species. As for the DNA barcodes, psbA-trnH showed the best performance among the four single loci with nine species being correctly differentiated. Moreover, psbA-trnH + ITS and psbA-trnH + ITS + rbcL exhibited the highest identification ability among all the multilocus combinations, and 17 species, of which 12 are medicinally used, could be efficiently discriminated. The DNA barcode data set developed in our study contributes valuable information to Chinese resources of Epimedium. It provides a new means for discrimination of the species within this medicinally important genus, thus guaranteeing correct and safe usage of Herba Epimedii. [ABSTRACT FROM AUTHOR]