High genetic diversity and low differentiation present challenges in taxonomy and systematics of Salix. Chloroplast (cp) genome sequencing is efficient for providing new genomic information and elucidating phylogenetic relationships. Salix spathulifolia Seemen, S. cupularis Rehder, and S. annulifera C.Marquand & Airy Shaw are three shrubby willows spread in high-altitude regions in western China. In this study, the integrated circular cp genomes were sequenced and analyzed, and a phylogeny of Salix was constructed on the basis of the cp genomes. The results of chloroplast assembly and annotation information were used to characterize genome feature and interspecific variation. The phylogenetic position of the three willows was evaluated using phylogenetic analysis. Full-length cp genomes were 155,566–155,680 bp with a typical double-stranded circular quadripartite structure, containing one large single-copy region (LSC, 84,431–4552 bp), one small single-copy region (SSC: 16,206–16,221 bp), and two inverted repeats (IR: 27,453–27,461 bp). The cp genomes encoded 130 genes, including 8 rRNA genes, 37 tRNA genes, and 85 protein-coding genes. The guanine-cytosine (GC) content of the overall genome was 36.7%. Comparison among the three willows’ cp genomes revealed high similarity. Phylogenetic analysis indicated that S. spathulifolia was a basal taxon of clade I, while S. annulifera formed a monophyletic group with S. rorida Laksch.; S. cupularis was sister to S. suchowensis W.C. Cheng and S. psammophila Z. Wang & Chang Y. Yang. The complete chloroplast genomes of the three willows provides an additional sequence-based resource for studying the phylogeny and evolutionary history of Salicaceae.

The utility of transcriptome data in plant phylogenetics has gained popularity in recent years. However, because RNA degrades much more easily than DNA, the logistics of obtaining fresh tissues has become a major limiting factor for widely applying this method. Here, we used Ranunculaceae to test whether silica-dried plant tissues could be used for RNA extraction and subsequent phylogenomic studies. We sequenced 27 transcriptomes, 21 from silica gel-dried (SD-samples) and six from liquid nitrogen-preserved (LN-samples) leaf tissues, and downloaded 27 additional transcriptomes from GenBank. Our results showed that although the LN-samples produced slightly better reads than the SD-samples, there were no significant differences in RNA quality and quantity, assembled contig lengths and numbers, and BUSCO comparisons between two treatments. Using this data, we conducted phylogenomic analyses, including concatenated- and coalescent-based phylogenetic reconstruction, molecular dating, coalescent simulation, phylogenetic network estimation, and whole genome duplication (WGD) inference. The resulting phylogeny was consistent with previous studies with higher resolution and statistical support. The 11 core Ranunculaceae tribes grouped into two chromosome type clades (T- and R-types), with high support. Discordance among gene trees is likely due to hybridization and introgression, ancient genetic polymorphism and incomplete lineage sorting. Our results strongly support one ancient hybridization event within the R-type clade and three WGD events in Ranunculales. Evolution of the three Ranunculaceae chromosome types is likely not directly related to WGD events. By clearly resolving the Ranunculaceae phylogeny, we demonstrated that SD-samples can be used for RNA-seq and phylotranscriptomic studies of angiosperms.

Introduction: Accurate phylogenetic reconstruction is crucial for understanding evolutionary relationships and biodiversity. Despite advances in molecular systematics, the relationships within Pandanales—which include Cyclanthaceae, Pandanaceae, Stemonaceae, Triuridaceae, and Velloziaceae—remain unresolved. This study aims to clarify these relationships by analyzing transcriptomic and genomic data from these families.Methods: We analyzed transcriptomic and genomic data from 20 samples representing all five families of Pandanales. Our approach involved assembling 2,668 single-copy orthologous genes (SCOGs) and conducting phylogenetic analyses using both coalescent- and concatenation-based methods, alongside plastid genome data. Additionally, we employed HyDe for gene flow analysis and conducted coalescent simulations and QuIBL analyses to explore sources of phylogenetic conflict. We also investigated whole-genome duplication (WGD) events within Pandanales.Results: The phylogenetic analyses produced strongly supported but topologically incongruent trees. Our gene flow analysis suggested that the concatenation-based topology likely reflects the true evolutionary history of Pandanales. We identified two significant ancient gene flow events: one between Velloziaceae and Triuridaceae, and another between Triuridaceae and the C-P clade (Cyclanthaceae + Pandanaceae). Furthermore, we detected five whole-genome duplication (WGD) events, including two that occurred before the Cretaceous–Paleogene boundary in Stemonaceae and Pandanaceae, one in Triuridaceae during the mid-Paleogene, and two within Velloziaceae near the Paleogene–Neogene boundary.Discussion: Our findings indicate that gene flow, rather than incomplete lineage sorting, is the primary source of phylogenetic conflict at certain nodes within Pandanales. The identified WGD events likely played a significant role in facilitating adaptation and diversification under changing environmental conditions. These results not only resolve long-standing phylogenetic conflicts but also enhance our understanding of the mechanisms driving plant diversification within this order.

The tribe Delphinieae (Ranunculaceae) has long posed challenges in terms of both its phylogeny and its taxonomy. This study integrates 60 newly sequenced p