Standard models of speciation assume strictly dichotomous genealogies in which a species, the ancestor, is replaced by two offspring species. The reality is more complex: plant species can evolve from other species via isolation when genetic drift exceeds gene flow; lineage mixing can give rise to new species (hybrid taxa such as nothospecies and allopolyploids). The multi-copy, potentially multi-locus 5S rDNA is one of few gene regions conserving signal from dichotomous and reticulate evolutionary processes down to the level of intra-genomic recombination. Here, we provide the first high-throughput sequencing (HTS) 5S intergenic spacer (5S-IGS) data for a lineage of wind-pollinated subtropical to temperate trees, the Fagus crenata – F. sylvatica s.l. lineage, and its distant relative F. japonica. The observed 4,963 unique 5S-IGS variants reflect a long history of repeated incomplete lineage sorting and lineage mixing since the early Cenozoic of two or more paralogous-homoeologous 5S rDNA lineages. Extant species of Fagus are genetic mosaics and, at least to some part, of hybrid origin.

Anthropogenic activities are leading to changes in the environment at global scales, and understanding these changes requires rapid, high-throughput methods of assessment. Pollen DNA metabarcoding and related methods provide advantages in throughput and efficiency over traditional methods, such as microscopic identification of pollen and visual observation of plant-pollinator interactions. Pollen DNA metabarcoding is currently being applied to assessments of plant-pollinator interactions and their responses to land-use change such as increased agricultural intensity and urbanisation, surveillance of ecosystem change, and monitoring of spatiotemporal distribution of allergenic pollen. In combination with historical specimens, pollen DNA metabarcoding can compare contemporary and past ecosystems. Current technical challenges with pollen DNA metabarcoding include the need to understand the relationship between sequence read and species abundance, develop methods for determining confidence limits for detection and taxonomic classification, increase method standardisation, and improve of gaps in reference databases. Future research expanding the method to intraspecific identification, analysis of DNA in ancient pollen samples, and increased use of museum and herbarium specimens could open further avenues for research. Ongoing developments in sequencing technologies can accelerate progress towards these goals. Global ecological change is happening rapidly, and we anticipate that high-throughput methods such as pollen DNA metabarcoding are critical for assessing these changes and providing timely management recommendations to preserve biodiversity and the evolutionary and ecological processes that support it.