The complete chloroplast genome sequences of two ornamental Epimedium species (Berberidaceae).

Both Epimedium sutchuenense Franch. 1894 and E. fargesii Franch. 1894 are perennial herbs with excellent ornamental values due to their showy flowers. However, little molecular research has been done on these species. Here, the complete chloroplast (cp) genome of E. sutchuenense and E. fargesii was analyzed and reported. The cp genome sizes of E. sutchuenense and E. fargesii were 157, 263 and 157, 133 bp, respectively. Both the two cp genomes contained a pair of inverted repeat regions (IRs) (25,782 and 25,796 bp), separated by the small single-copy (SSC) region (17,106 and 17,071 bp) and a large single-copy (LSC) region (88,593 and 88,470 bp). A total of 113 unique genes were annotated in each of the two cp genomes, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Phylogenetic analysis supported a close relationship between E. sutchuenense and E. wushanense, while E. fargesii had no clear clustering branch.

Background

Epimedium L., the largest genus of Berberidaceae, is a perennial herb discontinuously distributed in eastern Asia and the Mediterranean regions (Stearn 2002; Ying et al. 2011). The genus contains approximately 68 species/taxa, among which 58 species were distributed in China (Xu et al. 2020). It was noted that 27 Chinese Epimedium species were listed in the Red List of China Higher Plants (Qin et al. 2017). Some Epimedium species are of outstanding ornamental values (Probst 1998; Ren et al. 2008; Avent 2010). One of such species, E. sutchuenense Franch. 1894 is an excellent ornamental groundcover plant because of its rapidly growing rhizomes and attractive foliage and flowers. The species is mainly distributed in Sichuan, Hubei, Shaanxi provinces, and Chongqing city of China (Stearn 2002; Ying et al. 2011). Another species, E. fargesii Franch. 1894 is endemic to mountainous areas of Chongqing City, Sichuan, and Hubei Provinces (Stearn 2002; Ying et al. 2011; Xu et al. 2020). Due to its excellent ornamental values, the plant has been over-exploited and endangered (Qin et al. 2017).

Epimedium is one of the most taxonomically difficult representatives of Berberidaceae. Although the complete chloroplast (cp) genomes of same species have been presented recently (Liu et al. 2021a, 2021b), more molecular evidences are indispensable for further investigation of Epimedium. In this study, the sampling sites of the current two species were at a distance of about 40 km from those of the two published ones. Given the complex terrains of mountains, the distances would cause the genetic differences within the same species. Meanwhile, the phylogenetic tree with more species would more accurately explain the evolutionary status of the two species. Here, the complete cp genome of E. sutchuenense and E. fargesii was sequenced, and the phylogenetic relationship within Epimedium was analyzed. The baseline information would be beneficial to the conservation of the horticultural germplasm resources.

Methods

An individual of E. sutchuenense was collected from Shennongjia Forest District, Hubei Province, China (latitude 31.4734, longitude 110.3910); the sample of E. fargesii was collected from Chengkou County, Chongqing, China (latitude 31.8391, longitude 108.6277). The specimens of the two species were deposited at the Herbaria of Jiangxi University of Chinese Medicine (JXCM) (https://www.jxutcm.edu.cn/, Yanqin Xu, xuyanqin927@163.com) under the voucher numbers Y. Q. Xu and S. Y. Tian 2021006 and Y. Q. Xu and S. X. Liu 2016024, respectively.

Genome DNA was isolated from 0.5 g fresh leaves using the CTAB method (Doyle and Doyle 1987). The purified DNA was used to build a sequencing library with the Illumina NovaSeq 6000 platform. The complete cp genomes of two species were assembled using GetOrgnelle v1.71 (Jin et al. 2020). The resultant genomes were annotated by the genome annotator GeSeq (Tillich et al. 2017) with E. wushanense (NC_044891) as the reference, and the results were manually adjusted by Geneious (Kearse et al. 2012). The complete annotation cp genomes were deposited in GenBank with accession numbers MZ645926 (E. sutchuenense) and MZ603801 (E. fargesii).

A maximum-likelihood (ML) phylogenetic tree was constructed based on the 19 published complete cp genomes of Epimedium, with Vancouveria hexandra as an out-group. The ML tree was computed by PhyML v.3.0 (Stéphane et al. 2010) under the best model (F81) evaluated by Jmodeltest (Darriba et al. 2012).

Results

The lengths of the complete cp genomes of E. sutchuenense and E. fargesii were 157,263 and 157,133 bp, respectively. Both the two cp genomes exhibited typical quadripartite structures, including two inverted repeat regions (IRs), one large single-copy (LSC) region, and one small single-copy (SSC) region. For E. sutchuenense, the lengths of the IR, LSC, and SSC regions were 25,782 bp, 88,593 bp, and 17,106 bp, respectively; for E. fargesii, the lengths of the corresponding regions were 25,796 bp, 88,470 bp, and 17,071 bp, respectively. Both the two cp genomes showed overall GC content of 38.78%. The GC contents of E. sutchuenense were 37.37, 32.77, and 43.20% in the LSC, SSC, and IR regions, respectively, while these of E. fargesii were 37.38, 32.74, and 43.18%, respectively. Each of the two cp genomes was comprised of 113 unique genes, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Among them, 19 genes were duplicated, including seven protein-coding genes, eight tRNA genes, and four rRNA genes.

The phylogenetic analysis showed a close relationship between E. sutchuenense and E. wushanense, although the two species belong to different series with distinct morphological traits (Figure 1). The similar phylogenetic pattern was also reported in recent studies within the genus Epimedium (Huang et al. 2020; Guo et al. 2021). Contrastingly, E. fargesii had no clear cluster with other species of Epimedium, which might be associated with the short history of speciation of the species (Zhang et al. 2007; De Smet et al. 2012; Guo et al. 2021).

Figure 1.

The maximum-likelihood (ML) phylogenetic tree based on 19 complete chloroplast genomes, with Vancouveria hexandra as an out-group. Numbers at the right of nodes are bootstrap support values (≥50).