Abstract
Background and Aims Cypripedium is the most widespread and morphologically diverse genus of slipper orchids. Despite several published phylogenies, the topology and monophyly of its infrageneric taxa remained uncertain. Here, we aimed to reconstruct a robust section-level phylogeny of Cypripedium and explore its evolutionary history using target capture data for the first time.
Methods We used the orchid-specific bait set Orchidaceae963 in combination with transcriptomic data to reconstruct the phylogeny of Cypripedium based on 913 nuclear loci, covering all 13 sections. Subsequently, we investigated discordance among nuclear and chloroplast trees, estimated divergence times and ancestral ranges, searched for anomaly zones, polytomies, and diversification rate shifts, and identified potential gene (genome) duplication and hybridization events.
Key Results All sections were recovered as monophyletic, contrary to the two subsections within sect. Cypripedium. The two subclades within this section did not correspond to its subsections but matched the geographic distribution of their species. Additionally, we discovered high levels of discordance in the short backbone branches of the genus and within sect. Cypripedium, which can be attributed to hybridization events detected based on phylogenetic network analyses, and incomplete lineage sorting caused by rapid radiation. Our biogeographic analysis suggested a Neotropical origin of the genus during the Oligocene (∼30 Ma), with a lineage of potentially hybrid origin spreading to the Old World in the Early Miocene (∼22 Ma). The rapid radiation at the backbone likely occurred in Southeast Asia around the Middle Miocene Climatic Transition (∼15–13 Ma), followed by several independent dispersals back to the New World. Moreover, the Pliocene-Quaternary glacial cycles may have contributed to further speciation and reticulate evolution within Cypripedium.
Conclusions Our study provided novel insights into the evolutionary history of Cypripedium based on high-throughput molecular data, shedding light on the dynamics of its distribution and diversity patterns from its origin to the present.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
We carried out additional analyses to make our results more robust. Specifically, we added 14 more samples to our nuclear target enrichment dataset, including representative taxa from two previously missing sections, therefore covering all 13 sections of Cypripedium. For this reason, all analyses were re-performed from the beginning, with most yielding similar results, except the results regarding the whole genome duplication (WGD). To cross-examine whether the increased gene duplication percentage (>20%) indicated by our target enrichment data could represent a WGD, we sequenced 10 new transcriptomes across Cypripedium and conducted a Ks plots analysis using transcriptomic data for most sections of the genus. The results of the Ks plots analysis did not support a WGD within Cypripedium. Thus, the elevated gene duplications detected using the target enrichment data could be caused by different factors, such as the increased sequencing of multi-copy genes using the Orchidaceae963 bait set in Cypripedium or assembly artifacts of the target enrichment pipeline that we used. Therefore, we have adjusted the title, results, and discussion according to these new data and results. Additionally, in the revised manuscript we focused on the conflict at the backbone of the phylogeny. We also assembled and inferred a phylogeny from cpDNA and investigated the discordance between nuclear and chloroplast data, showing that their section-level topologies are incongruent. We have also focused our phylogenetic network analysis on the backbone, finding potential ancestral hybridization events between sections, while the analyses at the intra-sectional level were treated as secondary and moved to the supplementary material.
