Outlook

The evolution of extra-tropical tree genera involves dynamic speciation processes. Fagus species are wind-pollinated but animal-dispersed and have a narrow ecological niche. Therefore, beech populations are prone to becoming isolated during phases of area disruption. This can lead to speciation accompanied by lineage sorting. At the same time, species boundaries remain permeable: Fagaceae are known for both ancient and recent gene flow between lineages that diverged tens of millions of years ago (e.g. McVay et al., 2017; López-Heredia et al., 2020). Phases of area expansion likely led to secondary contacts and lineage mixing. Auto- and allopolyploidisation, hybridisation, introgression, and incomplete lineage sorting may leave complex genetic imprints. Single-copy or uniparentally inherited genetic markers can only capture some aspects of this complexity. Biparentally inherited, multi-copy, multi-locus markers, can instead provide direct evidence for past and recent reticulation. In this context, the 5S intergenic spacers are a unique source of information, being the only currently known region that (i ) is highly divergent, (ii ) can be easily amplified due to the conservation of the 5S rRNA genes, and (iii ) can be analysed using High-Throughput Sequencing because of its relatively short length. In this work, the O- and I/X-types found in F. japonica point towards a hybrid (allopolyploid) origin, and the distribution of A- and B-types in the crenata-sylvatica lineage reflect speciation processes predating the establishment of the modern species.
Beeches are ideal study objects because of the low species number and well-documented fossil record. Better understanding the diversity we observed and the putatively ancestral variants will require material from East Asian species not included in the present study: F. engleriana, F. hayatae, F. longipetiolata, and F. lucida , as well as from the yet poorly studied (eastern) North American F. grandifolia (but see Galván-Hernández et al., 2020). Similarly, nothing is known about 5S-IGS gene pools of East Asian and American oaks (and little about their ITS diversity); nevertheless, phylogenomic data of North American and French oaks have anticipated the existence of complex species relationships (Lepoittevin et al., 2015; Hipp et al., 2019). Non-trivial phylogenetic signals that may be the product of reticulate evolution also characterize all other Fagaceae (even complete plastomes cannot resolve the monophyly of oaks) and Fagales in general.
Further applications of 5S-IGS HTS data include taxonomy (co-)informed by genetics (e.g. Grimm & Denk, 2014; Liede-Schumann et al., 2020) and the assessment of global biodiversity in addition to the traditional counting of numbers of species (e.g. Forest et al., 2018). The species-status of F. sylvatica vs. F. orientalis has long been discussed but with focus on the wrong populations: while the western (here: Greek) F. orientalis may be addressed as a subspecies of F. sylvatica, the Iranian populations deserve the recognition as a species and represent a most valuable genetic resource.