Resolving complicated relationships of the Panax bipinnatifidus complex in southwestern China by RAD-seq data

Highlights

• ddRAD data resolve the phylogeny of P. binpinnatifidus complex.

• Phylogenetic relationships largely agreed with morphologically described species.

• The complex may have undergone a complicated evolutionary history with localized admixture.

Abstract

The P. binpinnatifidus complex included most of the Panax species distributed in Sino-Himalaya regions except for P. pseudoginseng, P. stipuleanatus and P. notoginseng. However, the delimitation and identification of these taxa within the species complex are very difficult due to the existence of morphological intermediates, and their evolutionary relationships remain unresolved despite several studies have been carried out based on traditional DNA markers. The taxonomic uncertainty hinders the identification, conservation and exploration of these wild populations of Panax. To study this species complex, we employed ddRAD-seq data of these taxa from 18 different localities of southwestern China, using two RAD analysis pipelines, STACKS and pyRAD. Based on the results of phylogenetic analysis, the species complex was divided into four clades with high supports, which largely agreed with morphologically described species. Two clades, corresponding to P. vietnamensis and P. zingiberensis, respectively, were sister groups, indicating that these two species had a closer genetic relationship; the third clade was consisted of samples with bamboo-like rhizomes named as P. wangianus clade, and the fourth one with moniliform rhizomes was named as P. bipinnatifidus clade. The population genetic structure analysis and D-statistics test showed the localized admixture among these species, which indicated that introgression had occurred among the related lineages continuously distributed in southeastern Yunnan and adjacent regions.

Introduction

Panax L., the ginseng genus, is a small genus of family Araliaceae, and almost every species is regarded as an important medicinal herb. Geographically, this genus disjunctly distributes in eastern Asia and eastern North America (Wen and Nowicke, 1999). In detail, P. trifolius L. and P. quinquefolius L. occur in North America, and P. ginseng C. A. Meyer could be found in northeastern Asia, including northeastern China, Korean peninsula and eastern Russia, while P. japonicus C. A. Meyer distributes in Japan (Wen, 2001). Himalayas and central and southwestern China is considered as the current center of evolution and diversification of Panax, and seven species and one varieties distribute in these areas (Wen, 2001), which are P. pseudoginseng Wallich, P. stipuleanatus H. T. Tsai & K. M. Feng, P. notoginseng (Burkill) F. H. Chen, P. zingiberensis C. Y. Wu & K. M. Feng, P. vietnamensis Ha & Grushv, P. wangianus S. C. Sun, P. bipinnatifidus Seemann, and P. bipinnatifidus Seemann var. angustifolius (Burkill). Only three species among these taxa, P. pseudoginseng, P. stipuleanatus, and P. notoginseng were relatively easy to be distinguished morphologically based on the difference of stipule-like appendages at petiole base. However, the delimitation and identification of the remaining taxa from this region are challenging, due to the high similarity in flora and fruits as well as the existence of morphological intermediates in rhizomes and leaflets (Wen, 2001, Xiang and Lowry, 2007). P. vietnamensis and P. wangianus, rhizomes are bamboo-like with short and thick internodes, while leaflets of the former are obovate-elliptic to oblong, the latter are narrowly lanceolate to broadly linear. P. zingiberensis is morphologically characterized by its fleshy and ginger-like rootstocks and sessile leaflets. P. bipinnatifidus and P. bipinnatifidus var. angustifolius are characterized by their moniliform rhizomes with slender internodes and subglobose nodes (Wen, 2001).

As to the phylogeny and evolutionary history of Panax, several studies have been carried out based on nuclear genes and chloroplast markers (Wen and Zimmer, 1996, Choi and Wen, 2000, Zhu et al., 2003b, Lee and Wen, 2004), however, the phylogenetic relationships within many clades were poorly resolved, especially for those morphologically resembled taxa. The P. bipinnatifidus species complex described by Zuo et al. (2011) for the first time because of the taxonomical difficulty included most of the Panax species distributed in Sino-Himalaya regions except for the well-defined ones (P. pseudoginseng, P. stipuleanatus and P. notoginseng). In the study of Zuo et al. (2015), this species complex exhibited a pattern of evolutionary radiation. And the genetic relationships among the taxa within this species complex were not resolved in the later studies using few DNA markers (Shi et al., 2015, Zuo et al., 2017) The clustering analysis using three cpDNA fragments and four nDNA fragments conducted by Zhou et al. (2018) suggested that these morphologically described species or varieties as described by Xiang and Lowry (2007) formed several paraphyletic subclades, and some populations still exhibited an admixture in genetic components. A poor understanding of the relationships among these taxa have largely caused the taxonomic disagreements. To elucidate evolutionary patterns of the species complex, more informative markers are needed for more extensive investigations.

Next-generation sequencing (NGS) techniques have changed the research in the field of molecular systematics from phylogenetics analysed with a few SNPs to phylogenomics with hundreds or even thousands of SNPs (Posada, 2016). The RAD-seq approach has been regarded as a cost-effective method for generating large-scale sequence data from non-model organisms through high-throughput NGS technology for conducting genome-wide evolutionary studies (Davey and Blaxter, 2010). This approach has proven attractive in species delimitation and phylogeny within and among closely related species, and it often offers even greater potential than complete chloroplast genomes sequencing (Wagner et al., 2013, Taranto et al., 2016, Wang et al., 2017). In addition, because of the ability to generate thousands of loci (Baird et al., 2008), RAD-seq has made it possible to infer the historical introgression patterns by using D-statistic test (Durand et al., 2011) in some taxa, such as Heliconius butterflies (Dasmahapatra et al., 2012), Pedicularis (Eaton and Ree, 2013) and Catostomus fishes (Bangs et al., 2018).

Therefore, the objectives of this study are to (1) reconstruct phylogenetic relationships of the P. bipinnatifidus complex distributed in southwestern China by using genome-wide SNPs generated by RAD sequencing and, (2) access their genetic structure and the possible gene flow so as to resolve the complicated relationships among these taxa.