The emergence of HTS methodologies has allowed the detection of cryptic
species (Carstens and Satler, 2013), the resolution of complex
phylogenetic trees (BogarÃn et al., 2018; Frajman et al.,2019; Hassemer et al., 2019; Yang et al., 2019) and the
reconstruction of evolutionary patterns in extinct species
(Moreno-Aguilar et al., 2020). Thus, HTS methods provide new
sources of useful data to clarify phylogenetic enigmas that classical
molecular methods could not decipher (e.g., Urtubey et al.,2018). Among HTS methods, target capture is currently being used in a
broad number of plant systematics and evolutionary studies due to its
versatility to successfully sequence hundreds of loci from highly
degraded DNA samples (Brewer et al., 2019, Viruel et al.,2019). Herbarium samples constitute a valuable and vast source of
information for morphological and niche modelling approaches, and
recently proved to be equally important for phylogenetic studies based
on DNA sequence data obtained using HTS methods. In our study, we used
herbarium material, with the oldest specimen sequenced collected in
1788, and a custom bait capture kit targeting 260 low copy nuclear genes
(Soto Gomez et al., 2019), to reveal the evolutionary patterns
and relationships between taxa belonging to the Tamus clade ofDioscorea (Figure 1). By sequencing 76 samples of theTamus clade, the phylogenomic and genetic clustering approaches
revealed extensive infraspecific variability in D. communis sensu
lato , clearly dividing it into three genetic groups, each showing a
distinct geographic distribution across the Mediterranean and western
Europe (Figure 6). Two of these genetic groups are congruent with the
previously recognized Tamus edulis and T. cretica , which
were recently placed within the large morphological variability and wide
distribution of D. communis s.l . HTS methodologies applied to
herbarium material allowed us to recognize the species rank for these
genetic groups and to support the split of D. communis s.l. intoD. edulis , D. cretica and D. communis , and to
maintain D. orientalis as a species.
Whole-genome duplication events (i.e., polyploidy) have been commonly
reported across flowering plants and have been correlated with
diversification of gene functions and new genetic architecture, which
could be linked with adaptative traits (Wendel et al., 2018).
Increased speciation events have been observed in some angiosperm
lineages reported to have a high incidence of whole-genome duplication
events (Wood et al., 2009; Zhan et al., 2016). Polyploidy
is a common phenomenon, which has been frequently reported in severalDioscorea species (Viruel et al., 2008), although defining
ploidy of the Tamus and Borderea clades has been challenging. The twoDioscorea species belonging to the Borderea clade, D.
chouardii and D. pyrenaica , have chromosome counts of 2n= 24. Based on the discovery of allotetraploidy using microsatellite
markers (Segarra-Moragues et al ., 2003), it was proposed that the
chromosome base number for the Borderea clade was x = 6 (see also
Viruel et al ., 2008). Extrapolating this find to the sister Tamus
clade, the known chromosome counts reported for D. communiss.s. of 2n = 36 and 48 (Al-Shehbaz and Schubert, 1989;
Viruel et al ., 2019) would therefore represent hexaploid and
octoploid forms, respectively. Similarly, the Macaronesian D.
edulis , with 2n = 96, would be 16-ploid assuming a base
chromosome number of x = 6. Using flow cytometry to estimate
ploidy in D. communis s.s. , multiple ploidies were
observed (1C-values ranging from 0.41 to 1.36 pg; Viruel et al .,
2019). The chromosome number and genome size of D. orientalis andD. cretica remain unknown, but allelic ratios estimated for each
SNP per sample using HTS data can be used as a proxy to distinguish
between diploid and polyploid forms when multiple ploidies are expected
in a group of plants (Viruel et al ., 2019). Median and mean
values of allelic ratios based on the number of reads supporting each
SNP were recently proposed to classify Dioscorea samples as
diploid forms when the allelic ratio is <2, and polyploids
when >2 (Viruel et al ., 2019). For example, all
samples of D. edulis had mean and median allelic ratios
>2 (Table 1), confirming the polyploid nature of this
species based on chromosome data. In all cases, D. orientalissamples studied here showed allelic ratios >2 and would
therefore be estimated to be a polyploid species (Table 1). For D.
communis s.s ., all samples were estimated to be polyploids except for
two samples of the clade DC3 from the eastern Mediterranean with mean
and median allelic ratio values <2 (samples S67 and R12, Table
1). Samples estimated to be diploid based on allelic ratios were also
observed in D. cretica , with half of the samples (eight) having
average and median allelic ratios <2 (Table 1). The incidence
of diploid forms, as estimated using allelic ratio values, in the
eastern Mediterranean will require further investigation applying
cytological and flow cytometry methodologies.