Phylogeny and diversification of genus Sanicula L. (Apiaceae): novel insights from plastid phylogenomic analyses.

BACKGROUND: The genus Sanicula L. is a unique perennial herb that holds important medicinal values. Although the previous studies on Sanicula provided us with a good research basis, its taxonomic system and interspecific relationships have not been satisfactorily resolved, especially for those endemic to China. Moreover, the evolutionary history of this genus also remains inadequately understood. The plastid genomes possessing highly conserved structure and limited evolutionary rate have proved to be an effective tool for studying plant phylogeny and evolution. RESULTS: In the current study, we newly sequenced and assembled fifteen Sanicula complete plastomes. Combined with two previously reported plastomes, we performed comprehensively plastid phylogenomics analyses to gain novel insights into the evolutionary history of this genus. The comparative results indicated that the seventeen plastomes exhibited a high degree of conservation and similarity in terms of their structure, size, GC content, gene order, IR borders, codon bias patterns and SSRs profiles. Such as all of them displayed a typical quadripartite structure, including a large single copy region (LSC: 85,074-86,197 bp), a small single copy region (SSC: 17,047-17,132 bp) separated by a pair of inverted repeat regions (IRs: 26,176-26,334 bp). And the seventeen plastomes had similar IR boundaries and the adjacent genes were identical. The rps19 gene was located at the junction of the LSC/IRa, the IRa/SSC junction region was located between the trnN gene and ndhF gene, the ycf1 gene appeared in the SSC/IRb junction and the IRb/LSC boundary was located between rpl12 gene and trnH gene. Twelve specific mutation hotspots (atpF, cemA, accD, rpl22, rbcL, matK, ycf1, trnH-psbA, ycf4-cemA, rbcL-accD, trnE-trnT and trnG-trnR) were identified that can serve as potential DNA barcodes for species identification within the genus Sanicula. Furthermore, the plastomes data and Internal Transcribed Spacer (ITS) sequences were performed to reconstruct the phylogeny of Sanicula. Although the tree topologies of them were incongruent, both provided strong evidence supporting the monophyly of Saniculoideae and Apioideae. In addition, the sister groups between Saniculoideae and Apioideae were strongly suggested. The Sanicula species involved in this study were clustered into a clade, and the Eryngium species were also clustered together. However, it was clearly observed that the sections of Sanicula involved in the current study were not respectively recovered as monophyletic group. Molecular dating analysis explored that the origin of this genus was occurred during the late Eocene period, approximately 37.84 Ma (95% HPD: 20.33-52.21 Ma) years ago and the diversification of the genus was occurred in early Miocene 18.38 Ma (95% HPD: 10.68-25.28 Ma). CONCLUSION: The plastome-based tree and ITS-based tree generated incongruences, which may be attributed to the event of hybridization/introgression, incomplete lineage sorting (ILS) and chloroplast capture. Our study highlighted the power of plastome data to significantly improve the phylogenetic supports and resolutions, and to efficiently explore the evolutionary history of this genus. Molecular dating analysis explored that the diversification of the genus occurred in the early Miocene, which was largely influenced by the prevalence of the East Asian monsoon and the uplift of the Hengduan Mountains (HDM). In summary, our study provides novel insights into the plastome evolution, phylogenetic relationships, taxonomic framework and evolution of genus Sanicula.

Plastid Phylogenomic Analyses Reveal a Cryptic Species of Ligusticopsis (Apiaceae, Angiosperms).

Ligusticopsis litangensis is identified and described as a cryptic species from Sichuan Province, China. Although the distribution of this cryptic species overlaps with that of Ligusticopsis capillacea and Ligusticopsis dielsiana, the morphological boundaries between them are explicit and have obviously distinguishable characters. The main distinguishing features of the cryptic species are as follows: long conical multi-branched roots, very short pedicels in compound umbels, unequal rays, oblong-globose fruits, 1-2 vittae per furrow and 3-4 vittae on the commissure. The above-mentioned features differ somewhat from other species within the genus Ligusticopsis, but generally coincide with the morphological boundaries defined for the genus Ligusticopsis. To determine the taxonomic position of L. litangensis, we sequenced and assembled the plastomes of L. litangensis and compared them with the plastomes of 11 other species of the genus Ligusticopsis. Notably, both phylogenetic analyses based on ITS sequences and the complete chloroplast genome robustly supported that three accessions of L. litangensis are monophyletic clade and then nested in Ligusticopsis genus. Moreover, the plastid genomes of 12 Ligusticopsis species, including the new species, were highly conserved in terms of gene order, gene content, codon bias, IR boundaries and SSR content. Overall, the integration of morphological, comparative genomic and phylogenetic evidence indicates that Ligusticopsis litangensis actually represents a new species.

First Report of Colletotrichum siamense causing anthracnose on Podocarpus macrophyllus in Sichuan Province of China.

Podocarpus macrophyllus (Thunb.) D. Don is a perennial evergreen tree of the Podocarpaceae family, which is widely used in landscape, medicine and forest interplanting (Qin et al. 2021). In August 2020, approximately 10% of the leaves have expressed symptoms of anthracnose in the campus of Sichuan Agricultural University (E103°51'35.88″, N30°42'30.41″). The lesions were light brown small sunken spots on the leaf tip in the early stage, then spread along the petiole to expanded into larger, irregular gray-white lesions in the late stage, with sparse black dots arranged above. The edge of the lesion was obvious with a fine smooth dark brown line. Samples taken from the lesions were surface disinfected for 3 min in 4% sodium hypochlorite, rinsed in sterile water and plated on potato dextrose agar (PDA), Eight single-spore cultures isolates from 10 samples were obtained and subcultured. After five days at 25°C in the dark, the mycelium of a representative culture LJS1 covered the entire plate surface (9 cm diameter). Hyphae were initially white at first, and turned pale grayish in the later stage. After about 10 days, a large number of pink conidial mass were formed around the center. Conidia 14.7 - 18.6 µm (mean 16.2 µm) in length and 4.4 -7.1 µm (mean 5.8 µm) in width (n = 100), nonseptate, cylindrical, two ends round or one end slightly acute. Conidial appressoria 5.7 - 9.3 µm (mean 7.8 µm) in length and 4.4 - 7.9 µm (mean 6.2 µm) in width (n = 50), clavate, ovoid to slightly irregular. Based on these characteristics, isolates were tentatively identified as Colletotrichum siamense complex (Sharma et al. 2013). Pathogenicity tests were conducted by spraying a conidial suspension of LJS1 (1 × 107conidia/ml) to 10 wounded and 10 non-wounded leaves from P. macrophyllus plants. Two areas of cuticle on either side of the midrib of each leaf were wounded by lightly scratching with a needle prior to inoculation (Du et al. 2020). As a control, distilled water was sprayed onto an equal number of wounded and non-wounded leaves. All inoculated and control plants were incubated in greenhouse (about 25 ± 2°C). Lesions similar to those observed in the field appeared on all wounded inoculated leaves within eight days after inoculation, whereas the non-wounded inoculated leaves and the controls remained symptomless. Reisolations of the putative pathogen were confirmed through morphological characteristics and the representative culture LJS1 were confirmed to be the pathogenic agents. The internal transcribed spaces (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), gene spacer region between Apn2 and Mat1-2(ApMat) genes were sequenced (Sharma et al. 2013) and deposited in GenBank (accession numbers OK036793, OK067325 and OK086086 respectively). These sequences were highly identical to those of C. siamense Prihastuti, L. Cai & K.D. Hyde (culture LF 139): accession numbers KJ955087.1 (99%), KJ954788.1 (99%), KJ954503.1 (99%), respectively. Based on the morphology and our multi-locus approach, the pathogen was convincingly identified for the first time as C. siamense. However, there are no reports of C. siamense causing anthracnose on P. macrophyllus worldwide. The identification of the causal agent of the disease made clear the pathogen causing anthracnose on P. macrophyllus, and provide theoretical basis for the diagnosis and treatment of disease.

Plastid Phylogenomic Analyses Reveal the Taxonomic Position of Peucedanum franchetii.

Peucedanum franchetii is a famous folk medicinal plant in China. However, the taxonomy of the P. franchetii has not been sufficiently resolved. Due to similar morphological features between P. franchetii and Ligusticopsis members, the World Flora Online (WFO) Plant List suggested that this species transformed into the genus Ligusticopsis and merged with Ligusticopsis likiangensis. However, both species are obviously diverse in leaf shape, bracts, and bracteoles. To check the taxonomic position of P. franchetii, we newly sequenced and assembled the plastome of P. franchetii and compared it with nine other plastomes of the genus Ligusticopsis. Ten plastomes were highly conserved and similar in gene order, codon bias, RNA editing sites, IR borders, and SSRs. Nevertheless, 10 mutation hotspot regions (infA, rps8, matK, ndhF, rps15, psbA-trnH, rps2-rpoC2, psbA-trnK, ycf2-trnL, and ccsA-ndhD) were still detected. In addition, both phylogenetic analyses based on plastome data and ITS sequences robustly supported that P. franchetii was not clustered with members of Peucedanum but nested in Ligusticopsis. P. franchetii was sister to L. likiangensis in the ITS topology but clustered with L. capillacea in the plastome tree. These findings implied that P. franchetii should be transferred to genus Ligusticopsis and not merged with L. likiangensis, but as an independent species, which was further verified by morphological evidences. Therefore, transferring P. franchetii under the genus Ligusticopsis as an independent species was reasonable, and a new combination was presented.