Introduction
Oaks (Quercus L., Fagaceae) are anemophilous trees and shrubs of
the Northern Hemisphere and are of high ecological, scientific and
economic importance (Menitsky 2005). They occur in a wide range of
habitats and climates ranging from semi-arid Mediterranean areas and
subtropical rainforests to boreal (cold temperate) continental regions
(Kremer and Hipp 2019). Oak taxonomy is still in flow; only recently the
genus has been divided into two monophyletic subgenera and eight
sections accommodating the 300–600 reported oak species (Denk et al.
2017). Discrepancies in species counts are due to the considerable
morphological variation and phenotypic plasticity exhibited by oaks, the
result of the complex interplay of strong ecological adaptation,
convergence of morphological traits, hybridization, introgression, and
reticulate evolution (Burger 1975; Van Valen 1976; Cavender-Bares et al.
2015; Simeone et al. 2016; McVay et al. 2017a, b; Hipp et al. 2019a).
Consequently, oaks are a big challenge for modern taxonomists (Denk et
al. 2017) and provide an ideal model for the development of a holistic
taxonomy that accounts for complex ecological, biogeographic and
evolutionary processes (Kremer et al. 2012), thereby improving our
understanding of Earth’s biodiversity. At the same time, key questions
about oak diversification and evolutionary history, especially in the
Old World, are just about to be answered (Denk et al. 2017; Hipp et al.
2019; Yan et al. 2019; Jiang et al. 2019).
After three decades of research (since Whittemore and Schaal 1991), it
has been widely demonstrated that oak plastid genomes are decoupled from
species identity (Simeone et al. 2016, 2018; Pham et al. 2017; Vitelli
et al. 2017) and that nuclear genes and regions with sufficient levels
of variation for solid species delineation and phylogenetic inferences
are still unavailable (Oh and Manos 2008; Hubert et al. 2014). Based on
extensive phylogenomic data, Hipp (2018a) concluded that oaks can be
defined as phylogenomic mosaics, meaning that every individual genome
actually represents an assembly of different histories reflecting as
well selection (ecological adaptation) and origin, or multiple origins
(divergence, reticulation, and lineage sorting). Species-level
phylogenetic reconstructions are affected by such genomic blending.
Cohesive intra-specific gene flow is counterbalanced by local gene flow
among individuals and populations in different parts of a species’
range, introgression, sorting of ancestral traits and divergence (Eaton
et al. 2015; McVay et al. 2017b; Hipp et al. 2018b, 2019b; Crowl et al.
2020; Leroy et al. 2020).
However, detection and distinction of horizontal gene transfer,
introgression and random sorting of ancestral genetic variation can be
difficult. The labor and costs associated with the necessary
comprehensive datasets (both at the inter- and intraspecific level) will
likely prevent a wide application of phylogenomic tools beyond species
trees and selected case studies (McVay et al. 2017; Hipp et al. 2019;
Jiang et al. 2019). Among nuclear DNA target regions of potentially high
taxonomic resolution, the spacer regions of the ribosomal DNA (35S and
5S nrDNA) are a quite popular choice for taxonomic studies. These
regions have been used for phylogenetic inferences across a broad range
of evolutionary lineages, have a high copy repeat number, and universal
or specific PCR primers are available allowing efficient amplification
in a wide range of taxa (Volkov et al. 2001, 2003; Alvarez and Wendel
2003). In particular, the internal transcribed spacers (ITS1, ITS2) of
the 18S-5.8S-25S cistron (35S rDNA) still are the most widely used
nuclear markers for phylogeny and systematics, including molecular
taxonomy such as DNA barcoding of complex plant groups (Hollingsworth et
al. 2011). However, in many groups the ITS1 and ITS2 can be nearly
invariable. In western Eurasian oaks, for instance, the nuclear
ribosomal 5S DNA intergenic spacer resolved species relationships to a
much higher degree than ITS data (Denk and Grimm 2010).