Many recent studies have addressed the mechanisms operating during the early stages of speciation, but surprisingly few studies have tested theoretical predictions on the evolution of strong reproductive isolation (RI). To help address this gap, we first undertook a quantitative review of the hybrid zone literature for flowering plants in relation to reproductive barriers. Then, using Populus as an exemplary model group, we analysed genome-wide variation for phylogenetic tree topologies in both early- and late-stage speciation taxa to determine how these patterns may be related to the genomic architecture of RI. Our plant literature survey revealed variation in barrier complexity and an association between barrier number and introgressive gene flow. Focusing on Populus, our genome-wide analysis of tree topologies in speciating poplar taxa points to unusually complex genomic architectures of RI, consistent with earlier genome-wide association studies. These architectures appear to facilitate the ‘escape’ of introgressed genome segments from polygenic barriers even with strong RI, thus affecting their relationships with recombination rates. Placed within the context of the broader literature, our data illustrate how phylogenomic approaches hold great promise for addressing the evolution and temporary breakdown of RI during late stages of speciation. This article is part of the theme issue ‘Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

Speciation, the continuous process by which new species form, is often investigated by looking at the variation of nucleotide diversity and differentiation across the genome (hereafter genomic landscapes). A key challenge lies in how to determine the main evolutionary forces at play shaping these patterns. One promising strategy, albeit little used to date, is to comparatively investigate these genomic landscapes as a progression through time by using a series of species pairs along a divergence gradient. Here, we resequenced 201 whole-genomes from eight closely related Populus species, with pairs of species at different stages along the divergence gradient to learn more about speciation processes. Using population structure and ancestry analyses, we document extensive introgression between some species pairs, especially those with parapatric distributions. We further investigate genomic landscapes, focusing on within-species (nucleotide diversity and recombination rate) and among-species (relative and absolute divergence) summary statistics of diversity and divergence. We observe highly conserved patterns of genomic divergence across species pairs. Independent of the stage across the divergence gradient, we find support for signatures of linked selection (i.e., the interaction between natural selection and genetic linkage) in shaping these genomic landscapes, along with gene flow and standing genetic variation. We highlight the importance of investigating genomic patterns on multiple species across a divergence gradient and discuss prospects to better understand the evolutionary forces shaping the genomic landscapes of diversity and differentiation.

Plant restoration strategies are ubiquitously employed for the purposes of soil and water conservation and ecological improvement in forest ecosystems. Despite N and temperature being acknowledged as pivotal factors affecting plant restoration outcomes, their effects on soil fertility, microbial communities, and shrub biomass remain underexplored, particularly in the loess hilly regions of China. Here, we examined the growth patterns and nutrient acquisition abilities of three shrub species, Periploca sepium, Amorpha fruticosa, and Vitex negundo, along with the attendant alterations in soil properties and microbial community composition under controlled greenhouse conditions. Specifically, we imposed three levels of N fertilization (200, 400, and 600 kg ha⁻¹; designated as N1, N2, and N3, respectively) and temperature regimes (18–23, 25–30, and 32–37 °C; labeled T1, T2, and T3, respectively). The results indicated a significant interplay between the combination of N fertilization and temperature significantly affecting shrub growth. Optimal growth conditions, as evidenced by the highest dry biomass accumulation, were identified as N3T1 for A. fruticosa, N1T1 for P. sepium, and N2T2 for V. negundo, with these conditions differentially influencing roots, stems, and leaves. Furthermore, soil microorganisms also responded significantly to the N fertilization and temperature. However, this was largely dependent on shrub species and soil nutrients. For A. fruticosa under N3T1 conditions, Actinobacteria and Basidiomycota abundances correlated strongly with soil C, N, and P contents, while leaf N uptake significantly correlated with the structure of both bacterial and fungal communities. For P. sepium at N1T1, Acidobacteriota was dominant in response to soil N and C, while leaf C uptake and leaf and stem N uptake positively correlated with bacterial and fungal communities, respectively. For V. negundo at N2T2, Chloroflexi had the greatest abundance, responding to the greatest variation in soil N and C, while its stem N uptake was significantly related to the structure of the fungal communities. Thus, our findings underscored the intricate interplay between abiotic factors, shrub growth, soil fertility, and microbial community dynamics, providing insights into the optimization of plant restoration efforts in ecologically sensitive regions.

The size of leaves is a vital factor in the development and overall biomass of a plant, serving as a key indicator of how a plant adapts to its environment. Rhamnus heterophylla, a species known for its heteromorphic leaves of varying sizes, presents an intriguing case for studying leaf development at the molecular level. To gain insights for further studies on the underlying mechanisms, we constructed a comprehensive reference transcriptome database using both SMART sequencing and Illumina RNA-seq technologies. Our analysis of the transcriptome data identified 88,546 isoforms, featuring an N50 size of 2386 base pairs. Furthermore, we identified 2932 transcription factors from 55 gene families, along with 14,947 unigenes that underwent alternative splicing. By comparing the gene expression patterns between large and small leaves, we pinpointed 982 differentially expressed genes (DEGs). Among these DEGs, 116 genes exhibit significantly greater activity in small leaves, while 866 genes display significantly greater activity in large leaves. Functional enrichment analyses revealed the significant involvement of these DEGs in various hormone signaling pathways. Notably, we detected a significant decrease in the expression of several genes associated with auxin synthesis, such as ARFs, GRF8, and IAA27, in small leaves. This finding sheds light on their potential role in leaf size regulation in R. heterophylla, providing valuable insights into the genes underlying this mechanism.

Many Cypripedium orchids are threatened and legally protected, restricting destructive or large-scale wild sampling and leaving population-scale plastome resources scarce. Using small leaf samples collected with permission and assistance from local reserve staff, we assembled and annotated 62 complete chloroplast genomes from total genomic DNA sequencing data, representing 53 Cypripedium taibaiense and 9 C. flavum individuals. The plastomes were 191,252 to 204,050 bp long and retained the typical quadripartite structure. In the final GenBank annotations, unique intact CDS content was consistent within each species (79 genes in C. taibaiense and 75 in C. flavum), and the four canonical rRNA genes were each present in two copies. A phylogeny based on concatenated nucleotide alignments of protein-coding sequences recovered the two species as reciprocal monophyletic, whereas whole-plastome analyses including non-coding regions captured population-level variation. Across the dataset, we detected 5,574 SNPs and 60,941 indels, mainly in the large and small single-copy regions. This population-scale plastome dataset provides a practical reference for phylogeographic analysis, marker development, and conservation genomic studies in Cypripedium.