The genus Paris in the broad concept is an economically important group in the monocotyledonous family Melanthiaceae (tribe Parideae). The phylogeny of Paris was controversial in previous morphology-based classification and molecular phylogeny. Here, the complete cp genomes of eleven Paris taxa were sequenced, to better understand the evolutionary relationships among these plants and the mutation patterns in their chloroplast (cp) genomes. Comparative analyses indicated that the overall cp genome structure among the Paris taxa is quite similar. The triplication of trnI-CAU was found only in the cp genomes of P. quadrifolia and P. verticillata. Phylogenetic analyses based on the complete cp genomes did not resolve Paris as a monophyletic group, instead providing evidence supporting division of the twelve taxa into two segregate genera: Paris sensu strict and Daiswa. The sister relationship between Daiswa and Trillium was well supported. We recovered two fully supported lineages with divergent distribution in Daiswa; however, none of the previously recognized sections in Daiswa was resolved as monophyletic using plastome data, suggesting that the infrageneric relationships and biogeography of Daiswa species require further investigation. Ten highly divergent DNA regions, suitable for species identification, were detected among the 12 cp genomes. This study is the first successful attempt to provide well-supported evolutionary relationships in Paris based on phylogenomic analyses. The findings highlight the potential of the whole cp genomes for improving resolution in phylogeny as well as species identification in phylogenetically and taxonomically difficult plant genera.

Ultra-barcoding is a technique using whole plastomes and nuclear ribosomal DNA (nrDNA) sequences for plant species identification. Paris yunnanensis is a medicinal plant of great economic importance for the pharmaceutical industry. However, the alpha taxonomy of P. yunnanensis is still uncertain, hindering effective conservation and management of the germplasm. To resolve long-standing taxonomic disputes regarding this species, we newly generated the complete plastomes and nrDNA sequences from 22 P. yunnanensis accessions. Ultra-barcoding analyses suggest that P. yunnanensis as currently circumscribed is made up of two distinct genetic lineages, corresponding to the two phenotypes (“typical” and “high stem” form) identified early in our study. With distinct morphologies and distribution, the “high stem” form should be recognized as a previously unrecognized species; here it is described as a new species, P. liiana sp. nov. Moreover, the ultra-barcoding data do not support treatment of P. yunnanensis as a conspecific variety under Paris polyphylla. Our study represents a guiding practical application of ultra-barcoding for discovery of cryptic species in taxonomically challenging plant taxa. The findings highlight the great potential of ultra-barcoding as an effective tool for resolving perplexing problems in plant taxonomy.

Background: Paris(Melanthiaceae) is an economically important but taxonomically difficult genus, which is unique in angiosperms because some species have extremely large nuclear genomes. Phylogenetic relationships withinParishave long been controversial. Based on complete plastomes and nuclear ribosomal DNA (nrDNA) sequences, this study aims to reconstruct a robust phylogenetic tree and explore historical biogeography and clade diversification in the genus.Results: All 29 species currently recognized inPariswere sampled. Whole plastomes and nrDNA sequences were generated by the genome skimming approach. Phylogenetic relationships were reconstructed using the maximum likelihood and Bayesian inference methods. Based on the phylogenetic framework and molecular dating, biogeographic scenarios and historical diversification ofPariswere explored. Significant conflicts between plastid and nuclear datasets were identified, and the plastome tree is highly congruent with past interpretations of the morphology. Ancestral area reconstruction indicated thatParismay have originated in northeastern Asia and northern China, and has experienced multiple dispersal and vicariance events during its diversification. The rate of clade diversification has sharply accelerated since the Miocene/Pliocene boundary.Conclusions: Our results provide important insights for clarifying some of the long-standing taxonomic debates inParis. Cytonuclear discordance may have been caused by ancient and recent hybridizations in the genus. The climatic and geological changes since the late Miocene, such as the intensification of Asian monsoon and the rapid uplift of Qinghai-Tibet Plateau, as well as the climatic fluctuations during the Pleistocene, played essential roles in driving range expansion and radiative diversification inParis. Our findings challenge the theoretical prediction that large genome sizes may limit speciation.