\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[ngerman,polish,greek,english]{babel}



\usepackage{float}






% Edit this header.tex file to include frontmatter definitions and global macros

% Add here any LaTeX packages you would like to load in all document blocks
% \usepackage{xspace}

% Add here any LaTeX macros you would like to load in all document blocks
% \def\example{This is an example macro.}

% -----

\iflatexml
% Add here any LaTeXML-specific commands

% -----

\else
% Add here any export style-specific LaTeX commands. These will only be loaded upon document export.
% \paperfield{Subject domain of my document}
% \keywords{keyword1, keyword2}
% \corraddress{Author One PhD, Department, Institution, City, State or Province, Postal Code, Country}
% \fundinginfo{Funder One, Funder One Department, Grant/Award Number: 123456.}
\fi


\begin{document}

\title{Genomic,~ spatial and morphometric data for discrimination of four
species in the~ Mediterranean Tamus clade of yams (\emph{Dioscorea},
Dioscoreaceae)~~~~~~~~~~~~}



\author[1]{Miguel Campos}%
\author[1]{Emma Kelley}%
\author[1]{Barbara GRAVENDEEL}%
\author[1]{Frédéric Médail}%
\author[1]{J. M. Maarten Christenhusz}%
\author[1]{Michael F. Fay}%
\author[1]{Pilar Catalán}%
\author[1]{Ilia J. Leitch}%
\author[1]{Félix Forest}%
\author[1]{Paul Wilkin}%
\author[1]{Juan Viruel}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
\textbf{Background and aims.} Among the numerous pantropical species of
the yam genus, \emph{Dioscorea}, only a small group occurs in the
Mediterranean basin, including two narrow Pyrenean endemics (Borderea
clade), and two Mediterranean-wide species (\emph{D. communis} and
\emph{D. orientalis}, Tamus clade). However, several currently
unrecognized species and infraspecific taxa have been described in the
Tamus clade due to significant morphological variation associated with
\emph{D. communis}. Our overarching aim was to investigate taxon
delimitation in the Tamus clade using an integrative approach combining
phylogenomic, spatial and morphological data.

\textbf{Methods.} We analysed 76 herbarium samples using Hyb-Seq genomic
capture to sequence 260 low-copy nuclear genes and plastomes, together
with morphometric and environmental modelling approaches.

\textbf{Key results.} Phylogenomic reconstructions confirmed that the
two previously accepted species of the Tamus clade, \emph{D. communis}
and \emph{D. orientalis}, are monophyletic and form sister clades. Three
subclades showing distinctive geographic patters were identified within
\emph{D. communis}. These subclades were also identifiable from
morphometric and climatic data, and introgression patterns were inferred
between subclades in the eastern part of the distribution of \emph{D.
communis.}

\textbf{Conclusions.} We propose a taxonomy that maintains \emph{D.
orientalis}, endemic to the eastern Mediterranean region, and splits
\emph{D. communis} \emph{sensu lato} into three species: \emph{D.
edulis}, endemic to Macaronesia (Canary Islands and Madeira); \emph{D.
cretica}, endemic to the eastern Mediterranean region; and \emph{D.
communis} \emph{sensu stricto}, widespread across western and central
Europe. Introgression inferred between \emph{D. communis s.s.} and
\emph{D. cretica} is likely to be explained by their relatively recent
speciation at the end of the Miocene, disjunct isolation in eastern and
western Mediterranean glacial refugia and a subsequent westward
recolonization of \emph{D. communis s.s}. Our study shows that the use
of integrated genomic, spatial and morphological approaches allows a
more robust definition of species boundaries and the identification of
species that previous systematic studies failed to uncover.%
\end{abstract}%



\sloppy




\par\null

\textbf{Introduction}

The yam genus, \emph{Dioscorea} L. (Dioscoreaceae) is a diverse group
currently containing 631 accepted species (POWO, 2022) possessing
underground storage organs and, in most, a climbing habit. Species with
starchy tubers constitute a food staple for millions of people,
resulting in seven to ten species being cultivated on a large scale
(Asiedu and Sartie, 2010), including two (\emph{D. alata} L. and\emph{D.
cayenensis} Lam.) which together are the most widely cultivated crops
(Price \emph{et al} ., 2016). More than 40 wild species are harvested as
food sources (Martin and Degras, 1978). In addition, some yams have been
used in traditional medicine and as a source of steroidal precursors (De
Luca \emph{et al.,} 2012; Hua \emph{et al.,} 2017; Price\emph{et al} .,
2016). While most wild yam species are found in tropical regions
(Caddick \emph{et al.,} 2002), a few species are distributed in
temperate regions and exhibit unique morphological traits
(Viruel\emph{et al.,} 2010). For example, only six species occur in the
Mediterranean-Macaronesian region: two species of the Stenophora clade
(\emph{D. balcanica} Ko\selectlanguage{polish}š\selectlanguage{english}anin native to Montenegro and Albania, and
\emph{D. caucasica} Lipsky, found in Georgia and Caucasian Russia), the
Borderea clade, which contains two well-defined and narrow endemic
species from the Pyrenean mountains (\emph{D. chouardii} Gaussen and
\emph{D. pyrenaica} Bubani \& Bord\selectlanguage{ngerman}ère ex Gren.), and the Tamus clade,
which is defined by having berries rather than winged capsules and is
more widely distributed across the Mediterranean Basin, Macaronesia and
Atlantic Europe (Viruel \emph{et al.,} 2016).

The Tamus clade currently comprises two species (Wilkin \emph{et
al.,}2005): \emph{D. communis} (L.) Caddick \& Wilkin, distributed
throughout the Mediterranean Basin and the Macaronesian Islands (Canary
Islands and Madeira), and with infraspecific variation in ploidy (Viruel
\emph{et al.,} 2019); and \emph{D. orientalis} (J. Thiébaut) Caddick \&
Wilkin, restricted to Lebanon and Israel. However, like in many
\emph{Dioscorea}clades (Viruel \emph{et al.,} 2010), the Tamus clade has
had multiple previous taxonomic circumscriptions. The Tamus clade was
considered as a separate genus, \emph{Tamus} L., distinct from
\emph{Dioscorea} , until 2002 (Caddick \emph{et al} ., 2002). Linnaeus
(1753) recognized two species: \emph{T. communis} L. with cordate leaves
and a Mediterranean distribution, and \emph{T. cretica} L\emph{,} with
trilobed leaves and typified with material from the Greek island of
Crete. In the 19\textsuperscript{th} and early 20\textsuperscript{th}
centuries, four Macaronesian endemic species were described (\emph{T.
edulis}Lowe\emph{, T. parviflora} Kunth\emph{, T. norsa} Lowe and
\emph{T. canariensis} Willd. ex Kunth), while \emph{T. cirrhosa}
Hausskn. ex Bornm., \emph{T. cordifolia} Stokes and \emph{T. racemosa}
Gouan were treated as distinct Mediterranean species. In the late
20\textsuperscript{th} Century, \emph{T. cretica} was placed as a
subspecies in \emph{T. communis} (\emph{T. communis}
subsp.\emph{cretica} (L.) Nyman), and \emph{T. communis} f.
\emph{subtriloba}(Guss.) O. Bolòs \& Vigo was described as a variety
with trilobed leaves found in the Balearic Islands and northeastern
Spain (Catalonia). All these names were subsequently united under the
currently accepted\emph{D. communis} (Caddick \emph{et al.,} 2002). The
second species currently recognized in the Tamus clade, \emph{D.
orientalis} , was originally described as \emph{T. orientalis}
J.Thièbaut named after its eastern Mediterranean distribution.

From the above, it is clear that species concepts have undergone many
changes since Linnaeus described two \emph{Tamus} species using
morphology, especially reproductive traits (De Queiroz, 2007). As for
many other species in other plant genera and families, integrative
taxonomic and systematic approaches combining genetic data,
morphometrics and climatic envelope data have successfully helped to
delimit species in challenging groups of plants (e.g., Frajman \emph{et
al.,} 2019). The emergence of high-throughput sequencing (HTS)
techniques and the production of thousands of molecular markers have
massively increased our ability to resolve relationships between and
within species, and subsequently redefine species boundaries (e.g.,
Fay\emph{et al.,} 2019; Escudero \emph{et al.,} 2020). Among these HTS
methods, Hyb-Seq has become widely adopted across plant phylogenomic
studies due to its ability to generate data from degraded herbarium
materials (e.g., Brewer \emph{et al.,} 2019; Viruel \emph{et al.,} 2019)
and to resolve relationships at different taxonomic scales (e.g.,
Villaverde \emph{et al.,} 2018). Hyb-Seq techniques rely on genome skim
data and target capture probes either designed specifically for some
genera or families (e.g., Soto Gomez \emph{et al.,} 2019) or more widely
across larger groups, including all angiosperms (e.g., Johnson \emph{et
al.,} 2019). In this study, we use a multidisciplinary approach
combining genomic, morphometrics, and environmental niche modelling data
generated from herbarium specimens to identify taxon boundaries in the
challenging Tamus clade of \emph{Dioscorea} , and to explore their
phylogeographic patterns across the Mediterranean.

\textbf{Material and methods}

\textbf{Plant material}

Seventy-six herbarium specimens identified as \emph{D. communis} or
\emph{D. orientalis} were used to obtain genomic, spatial, and
morphometric data. They were selected as being representative of the
macromorphological diversity and geographical distribution ranges of the
two species as currently circumscribed (Supplementary Data Table S1).
The genomic sampling also included material that were used as outgroup
taxa, comprising the two species from the Borderea clade (\emph{D.
chouardii} and \emph{D. pyrenaica} ), which is sister to the Tamus clade
(Viruel \emph{et al} ., 2016), and two members of the more distantly
related African clade (\emph{D. elephantipes} (L'Hér.) Engl. and
\emph{D. sylvatica} Eckl.).

\textbf{Phylogenomics}

Total genomic DNA was extracted from herbarium specimens using a
modified CTAB protocol (Doyle and Doyle, 1987). Nuclear target
enrichment was used to capture 260 low- to single-copy nuclear (LSCN)
genes using RNA baits design for \emph{Dioscorea} (Soto Gomez \emph{et
al.,} 2019). Genomic libraries were prepared using NEBNext® Ultra\selectlanguage{english} II
DNA Library Prep Kit for Illumina(r) (New England Biolabs, Ipswich,
Massachusetts, USA) with AMPure XP magnetic beads and NEBNext(r) Multiplex
Oligos for Illumina(r) (Dual Index Primer Sets I and II) as tags for
simultaneous sequencing. Subsequently, the enriched libraries were
multiplexed and sequenced on a HiSeq X platform (Illumina, Inc.) lane.

We filtered raw paired-end reads by removing adapter sequences and
low-quality reads using Trimmomatic v.0.36 (Bolger \emph{et al.,} 2014).
We used HybPiper v.1.3.1 (Johnson \emph{et al.,} 2016) to recover 260
nuclear genes and associated introns (Soto Gomez \emph{et al.,} 2019)
using sequence data from the transcriptome of \emph{D. communis} (SRA
SAMN11290810) as a reference file following Viruel \emph{et al.} (2019).
We used nQuire (Weiss \emph{et al} ., 2018) to calculate the number of
read counts for each allele per single nucleotide polymorphism (SNP),
and estimated the median value of allelic ratios per sample to classify
each individual as diploid (\textless{}2) or polyploid (\textgreater{}2)
as described and optimized for \emph{Dioscorea} in Viruel \emph{et
al.}(2019). The percentage of polymorphic sites was calculated as the
percentage of SNP positions compared to the total number of base pairs
retrieved for each sample. Plastome data were recovered using HybPiper
and the plastome of \emph{D. elephantipes} as reference (GenBank
NC\_009601).

Sequences were aligned with MAFFT v.7 (Katoh, \emph{et al.,} 2002) using
the \emph{--auto} parameter, and debugged with trimAl v.1.4.1
(Capella-Gutierrez \emph{et al.,} 2009) using the
\emph{-automated1}command. Phylogenomic trees were reconstructed using
the concatenated nuclear DNA (nDNA) and plastid DNA (pDNA) datasets
independently, and for each nuclear gene independently, using RAxML-NG
(Katoh \emph{et al.,} 2019) and IQ-TREE (Nguyen \emph{et al.,} 2015),
with a GTR+GAMMA substitution model and 1,000 bootstrap replicates. We
used ASTRAL\selectlanguage{english}-III (Zhang \emph{et al.,} 2018) to construct a species tree
based on the independent nuclear gene trees, and SVDquartets to evaluate
10,000,000 random quartets -or all possible quartets if lower- and
10,000 bootstrap replicates, as implemented in PAUP* 4.0a146 (Swofford,
2002). Haplotype networks were reconstructed with plastid data using the
TCS method as implemented in Popart v1.7 (Clement \emph{et al.,} 2002;
Leigh and Bryant, 2015).

We used Structure (Pritchard \emph{et al.,} 2000) to further investigate
the genetic clusters within and between taxa based on filtered SNP data
from the concatenated nDNA dataset. We tested one to six genetic groups
(\emph{K} =1--6) allowing admixture at individual level, and correlated
allele frequencies, by running five replicates with a 100,000 burn-in
and a chain length of 1,000,000 simulations each. Structure Harvester
(Earl and vonHoldt, 2012) was used to obtain likelihood values for the
multiple values of \emph{K} and to apply the delta \emph{K} criterion to
select the optimal \emph{K} . We plotted Structure results for
each\emph{K} value using StructuRly (Criscuolo and Angelini, 2020).

Divergence times were estimated using a Bayesian relaxed-clock approach
implemented in BEAST 1.10.4 (Drummond and Rambaut, 2007) and a penalized
likelihood approach as implemented in treePL (Smith and O'Meara, 2012)
using the concatenated nDNA dataset containing one representative per
taxon. In both analyses, the crown node of the African/Mediterranean
clade was used as calibration by applying a minimum age of 24 MY and a
maximum age of 40 MY, based on the age estimates from Viruel \emph{et
al.} (2016; 95\% HPD of 24.3469--39.2223 MY). We applied the GTR+I+G
substitution model, Yule tree prior, and an uncorrelated lognormal
molecular clock and ran the analysis for 1 billion generations, sampling
every 100,000 generations. The treePL analysis was conducted in two
consecutive runs: i) applying the ``prime'' option to select the most
optimal parameter values, and ii) a ``thorough'' analysis by
setting\emph{opt} = 1, \emph{optad} = 2 and \emph{optcvad} = 5.

\textbf{Spatial analysis}

Occurrence records were obtained from 287 observations from the Global
Biodiversity Information Facility (211 occurrences validated by
morphology, http://www.gbif.org/) and data from herbarium specimens (76
occurrences). For modelling purposes, the dataset was reduced to keep
only georeferenced data.

We used environmental niche modelling (ENM) approaches to reconstruct
the potential distribution of the four main clades uncovered in the
phylogenomic analyses (see Results) under current and past climatic
conditions using the maximum entropy algorithm implemented in the R
package `Maxent' (Phillips, 2021). Nineteen bioclimatic variables were
extracted from the Bioclim dataset, provided by WorldClim 1.4 in a
GIS-based raster format (2.5 min resolution). The correlations between
environmental variables were determined with a Pearson's correlation
matrix and subsequent realization of a dendrogram cluster for its
visualization (Supplementary Data Figure S1). We selected a different
set of uncorrelated variables for each geographical region with a high
percentage of contribution (PC): bio4 (temperature seasonality), bio8
(mean temperature of wettest quarter), bio9 (mean temperature of driest
quarter), bio15 (precipitation seasonality) and bio16 (precipitation of
wettest quarter) for the circum-Mediterranean region; bio3
(isothermality), bio6 (min temperature of coldest month), bio8 (mean
temperature of wettest quarter), bio14 (precipitation of driest month),
bio15 (precipitation seasonality) and bio16 (precipitation of wettest
quarter) for the Eastern Mediterranean region; bio3 (isothermality),
bio4 (temperature seasonality), bio16 (precipitation of wettest quarter)
and bio18 (precipitation of warmest quarter) for the Macaronesian
region. ENM analyses were carried out under current climatic conditions,
and projected to climatic conditions of the Mid Holocene (MH, about
6,000 years ago), the Last Glacial Maximum (LGM, about 22,000 years ago;
Braconnot \emph{et al.,} 2007) and the Last Interglacial (LIG,
\textasciitilde{}120,000 - 140,000 years BP; Otto-bliesner \emph{et
al.,} 2006) using the palaeoclimatic `Community Climate System Model'
(CCSM; Gent \emph{et al.,} 2011). Layers were cropped to represent the
distribution range of each phylogenetic group (i.e., DC1, DC2 and DC3
for \emph{D. communis} , and \emph{D. orientalis} ; see Results) to
maximize the reliability of the results and discard false occurrences
using the package `raster' (version 3.5-15; R v.4.0.5). We used the
Schoener's D and Hellinger's I indices as implemented in ENMtools
v.1.0.4 to evaluate niche overlap (Warren \emph{et al.,} 2008, 2010).
Equivalence and similarity tests with 1,000 replicates were carried out
to assess if the overlap between ENMs is higher than expected under
randomized ENMs.

A multivariate ordination analysis (principal component analysis, PCA)
was carried out using uncorrelated bioclimatic variables obtained from
WorldClim using the packages `ade4', `factoextra', `magrittr', `dismo'
and `HH' in R v.4.0.5. The correlation analysis was performed with a
Pearson correlation matrix and the subsequent visualization and
selection of variables using a dendrogram cluster. The selection of
uncorrelated variables with the highest contribution to the PCA were 10:
bio1 (annual mean temperature), bio2 (mean diurnal range), bio3
(isothermality), bio7 (temperature annual range), bio8 (mean temperature
of wettest quarter), bio9 (mean temperature of driest quarter), bio10
(mean temperature of warmest quarter), bio12 (annual precipitation),
bio15 (precipitation seasonality) and bio19 (precipitation of coldest
quarter).

\textbf{Morphometrics}

We studied vegetative and reproductive traits of 76 herbarium specimens
(60 males and 16 females), previously used to delimit taxa boundaries in
other \emph{Dioscorea} species (e.g., Viruel \emph{et al.,} 2010), using
a 150 mm calliper, a stereomicroscope and ImageJ 1.52a
(Schneider\emph{et al.,} 2012). Traits were measured and treated
independently for male and female individuals (Supplementary Data Table
S2). Pollen grains were sputter coated with platinum and examined using
a Hitachi S4700 cold field emission scanning electron microscope (SEM)
at 2kV (Hitachi High-Technologies Corporation, Tokyo, Japan).

A value of \textgreater{}0.7 Pearson correlation was used as a threshold
to exclude correlated variables from the analysis. The normality
Kolmogorov-Smirnov test with Lilliefors correction and the
homoscedasticity Levene's test were applied to the variables following a
normal distribution. Subsequently, comparisons among taxonomic units
identified in phylogenomic analyses (see Results) were conducted using
Kruskal-Wallis and Bonferroni post-hoc tests. Statistical analyses were
performed using `nortest', `Hmisc', `corrplot', `PerformanceAnalytics'
and `car' packages in in R v.4.0.5 (R Core Team, 2022).

\textbf{Results}

\textbf{Data recovery and phylogenomic results}

Target capture data recovery for 76 samples of the Tamus clade
of\emph{Dioscorea} and the four outgroup samples included in this study
are summarized in Table 1. Sequence data are available at SRA
repository, PRJNA525269. An average of 2,240,287 quality filtered
paired-end reads were retrieved per sample, ranging between 43,612 and
16,196,619 reads. While the samples from herbarium specimens dated from
1788 to recently collected material, the differences in number of
retrieved reads were not related to the age of the specimens. On
average, the proportion of reads on target (enrichment efficiency) was
0.33 (0.09--0.60), and although sequences were assigned on average to
258 genes per sample, assemblies at 50\% of the expected size of each
gene were retrieved on average for 215 genes per sample.

Our target capture approach allowed us to recover an average of 326,149
bp (45,171--394,977) of nuclear data per sample, which corresponds to a
recovery rate of 76.9\% (10.6--93.1\%), while the off-target reads
contained plastid data that permitted the assembly of 131,543 bp on
average (30,666--151,239) of the plastome per sample. No differences
were observed in recovery rates between the clades reconstructed in our
analysis (see below); the overall sequencing and target capture data
obtained for the four outgroup samples were in the range of the
remaining samples.

Both nuclear- and plastid-based phylogenomic reconstructions support the
monophyly of the Tamus clade in \emph{Dioscorea} (Figure 1), with two
highly supported clades that corresponded to the two currently
recognized species (\emph{D. communis} and \emph{D. orientalis} ). Three
highly supported subclades were reconstructed in the \emph{D.
communiss.l.} clade in the nuclear tree (Figure 1). A first split
separated the samples of \emph{D. communis} from Macaronesia (clade
DC1). The remaining samples of \emph{D. communis} fell into two sister
subclades corresponding to samples of \emph{D. communis} from the
eastern Mediterranean (clade DC2) and Mediterranean and Europe (clade
DC3), respectively. The latter clade DC3 was subsequently further
subdivided into (eastern Mediterranean, (central Mediterranean, western
Europe)) subclades. Overall bootstrap support was \textgreater{}90\% for
most of the nodes (Figure 1).

\par\null\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}
\caption{{Phylogenomic trees reconstructed using the concatenated nuclear (left)
and plastid data (right) obtained using Hyb-Seq for 76 samples of the
Tamus clade of \emph{Dioscorea}. Samples included are representative of
its distribution range across the Mediterranean region. Filled circles
represent branches with support values over 90\%, while lower values are
shown on branches. Colours represent the main clades found in the
nuclear tree, see Results (\emph{D. orientalis} violet, DC1 blue, DC2
green, DC3 red), and roman numbers identifying the subclades in \emph{D.
communis} in the plastid tree (I, II, III; subclades within each clade
were indicated with lowercase letters a and b). Dashed lines connect the
same samples in the nuclear and plastid trees.
{\label{846065}}%
}}
\end{center}
\end{figure}

The plastid tree was generally congruent with the nuclear tree. However,
the plastid topology differed in the resolution of the most recent
Mediterranean subclades. In addition, one DC2 sample (R32) was placed in
a different subclade (see Figure 1). The remaining Mediterranean\emph{D.
communis} samples (DC3) were intermingled in three plastid subclades (I,
II and III in Figure 1). In the nuclear tree, the subclade DC3 from the
eastern Mediterranean contained samples with plastid haplotypes from all
plastid subclades I, II and III, and the subclades of DC3 from central
Mediterranean and western Europe contained samples with those from
plastid subclades I and II, and II and III, respectively (Figure 1).

To better understand the source of topological incongruence between the
nuclear and plastid phylogenetic trees of the Mediterranean samples
of\emph{D. communis} , we used STRUCTURE to investigate whether
introgression between samples from different subclades may have
occurred. The highest delta\emph{K} value (15821.9) was obtained
for\emph{K} = 3 genetic groups (Supplementary Data Figure S2). All
samples in clade DC2 of \emph{D. communis} showed genetic profiles
corresponding to one genetic group (Cluster 1), and five samples also
showed a percentage of membership \textless{}20\% to Cluster 2 (i.e.,
samples R16, R17, S69 and S70) or Cluster 3 (sample R32, 26\%). The
samples of\emph{D. communis} in clade DC3 showed genetic profiles
corresponding predominantly to Cluster 3 and multiple patterns of
admixture with Clusters 1 and 2 (Figure 2). The genetic profile of one
DC3 sample from the eastern Mediterranean subclade showed a percentage
of membership \textgreater{}20\% to Cluster 1, and nine DC3 samples had
membership percentages of \textgreater{}20\% to Cluster 2 (five from the
eastern Mediterranean, one from the central Mediterranean and three from
the western Europe subclades). Other alternative \emph{K} groups did not
increase the number of clusters in DC2 and showed higher admixture in
the DC3 subclades (Supplementary Data Figure S3). In all cases, the
sample R32 was always resolved with ca. 20\% admixture with the main
genetic cluster of the DC3.

\par\null\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}
\caption{{Admixture proportions for~\emph{K} = 3 genetic groups obtained from
genetic structure analysis of nuclear data of Mediterranean
~\emph{Dioscorea communis} samples through ten replicates in STRUCTURE
(see text for details). Each sample is shown by a vertical bar
partitioned according to its membership to one of the~\emph{K} clusters,
represented in red, green and blue. The samples follow the same order as
in the nuclear phylogenetic tree (Figure 1) and organized by clades (DC2
and DC3). The plastid clades (I, II and III, Figure 1) were labelled
next to the sample codes. An asterisk (*) was used to represent the only
sample of~\emph{D. communis} (R32) that was resolved as part of clade
DC1 based on nuclear data, and in a different clade with plastid data
(see Figure 1).
{\label{632367}}%
}}
\end{center}
\end{figure}

Based on our phylogenomic and genetic structure analyses, we performed
divergence time analysis using treePL and BEAST (Supplementary Data
Figure S4) using one representative sample of each defined clade and
genetic group: \emph{D. orientalis} , DC1, DC2 and DC3. Both approaches
reconstructed an early Oligocene origin for the crown node of the Tamus
and Borderea clades (BEAST 28.5 MY and treePL 28.2 MY), and a late
Miocene split for the two species of the Borderea clade (BEAST 8.1 MY
and treePL 10.6 MY). The split of \emph{D. orientalis} from \emph{D.
communis} was inferred to have occurred during the early Miocene; BEAST
18.2 MY and treePL 20.6 MY), and the Macaronesian clade (DC1) likely
diverged from the Mediterranean lineage of \emph{D. communis} during the
mid-Miocene; BEAST 13.5 MY and treePL 16.0 MY). The most recent split
between the clades DC2 and DC3 was estimated to have taken place during
the late Miocene (BEAST 5.6 MY and treePL 6.6 MY).

The mean and median values of allelic ratios were \textgreater{}2 in all
cases for \emph{D. orientalis} and clade DC1, and only ten samples
showed allelic ratio values \textless{}2: eight samples from clade DC2
and two samples from the eastern Mediterranean subclade of DC3 (Table
1). The lowest incidence of estimated polyploidy based on allelic ratio
estimates was found in DC2, with 50\% of the samples classified as
diploids (Table 1).

\textbf{Current and past overlaps in species distribution ranges between
the Tamus lineages}

ENM analysis was performed for each of the four lineages in the Tamus
clade (Figure 3) to explore whether differences exist in (former)
distribution between the four clades identified within the Tamus clade
of \emph{Dioscorea} . Predicted distribution models were estimated using
available occurrence data points (DC1: 27, DC2: 26, DC3: 218, \emph{D.
orientalis} : 17) and selected bioclimatic variables (DC1: bio16, bio9,
bio15, bio3 and bio8; DC2: bio16, bio14, bio6, bio15, bio3, bio8; DC3:
bio4, bio8, bio16, bio15 and bio9; \emph{D. orientalis} : bio16, bio14,
bio6, bio15, bio3 and bio8; in order of importance); and showed high AUC
values (DC1: 0.9989 \selectlanguage{ngerman}± 0.0003, DC2: 0.980 ± 0.005, DC3: 0.937 ±
0.002,\emph{D. orientalis} : 0.9994 ± 0.0001) and a 10\% threshold was
applied (DC1: 0.24, DC2: 0.29, DC3: 0.20, \emph{D. orientalis} : 0.50
probability).

\par\null\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}
\caption{{Environmental niche models (ENM) constructed using a current climate
envelop and occurrence data for the samples of the Tamus clade of
\emph{Dioscorea} analysed in this study. ENMs were estimated for each of
the four lineages identified in the Tamus clade with background maps
adjusted to reflect their estimated potential distributions: DC3 clade
of \emph{D. communis}; DC2 clade of \emph{D. communis}; the Macaronesian
DC1 lineage of \emph{D. communis}; and \emph{D. orientalis}. A 10\%
threshold cropping was applied for each distribution range model (see
Results). The legend represents the prediction of probability of
occurrence.
{\label{946229}}%
}}
\end{center}
\end{figure}

The group formed by DC3 samples had its highest distribution probability
in several Mediterranean areas, central and southern Europe (including
England and Belgium to Crimea), north-western Africa and western Asia
(Turkey, Syria, Caucasus, Caspian shores). The most optimal distribution
for DC2 was in the eastern Mediterranean, specifically in the eastern
Aegean islands and the Mediterranean coastal zone of Turkey, Lebanon and
Israel. The distribution model of the latter group overlapped with the
distribution range inferred for \emph{D. orientalis} , which also
presented its optimum in the eastern Mediterranean area, along the
coasts of Lebanon, Syria, Palestine and Israel. The highest
probabilities of potential distribution ranges for the DC1 group are
restricted to the humid areas of the western Canary Isles (Figure 3),
with Madeira and western Morocco showing a lower probability of
occurrence. Overlap was observed between DC2, DC3 and \emph{D.
orientalis} in the eastern Mediterranean region, and between DC1 and DC3
in the Canary Islands.

The highest niche breadth obtained corresponded to the clade with the
largest modelled distribution projection (i.e., 0.875 for DC3 clade),
followed by DC2 (0.731), \emph{D. orientalis} (0.516) and DC1 (0.499).
We calculated Schoener's \emph{D} and Hellinger's \emph{I} indexes as
metrics of niche overlap between pairs of distribution models. The
highest overlaps between current niches were found between the DC3 clade
with DC1 (\emph{D} = 0.52, \emph{I} = 0.64) and DC2 (\emph{D} =
0.48,\emph{I} = 0.67), whereas \emph{D. orientalis} showed lower overlap
values with DC2 (\emph{D} = 0.32, \emph{I} = 0.60) and DC3 (\emph{D} =
0.36, \emph{I} = 0.48). Although niche overlap between DC2 and \emph{D.
orientalis} clades showed the lowest values, a PCA using the raw
bioclimatic data obtained from the studied samples did not differentiate
between them (Figure 4); a better separation was, however, found between
DC3 and DC1. These results are supported by observed differences between
the four clades for each of the bioclimatic variables (Supplementary
Data Figure S5).

\par\null\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}
\caption{{Principal Component Analysis (PCA) conducted with 19 bioclimatic
variables for samples of the Tamus clade of \emph{Dioscorea} across the
Mediterranean region separated into the four main lineages (see
Results): Macaronesian DC1 clade of \emph{D. communis} in light blue;
\emph{D. communis} clade DC2 in green; \emph{D. communis} DC3 clade in
red; and \emph{D. orientalis} in purple.
{\label{124112}}%
}}
\end{center}
\end{figure}

Hindcast species distribution models projected to the past
(Supplementary Data Figure S6) support a long-term presence of \emph{D.
orientalis} and DC2 clades in the eastern Mediterranean region, and a
prevalence of the DC3 clade in the western Mediterranean since the Last
Interglacial (LIG, \textasciitilde{}120,000 - 140,000 years BP). The
overlap between current and Mid Holocene (MH, about 6000 years ago)
distribution models was high for DC2 (0.907), \emph{D.
orientalis}(0.798) and DC3 (0.859), slightly lower between MH and the
Last Glacial Maximum (LGM, about 22,000 years ago) for DC2 (0.835),
\emph{D. orientalis} (0.564) and DC3 (0.719), and moderately to
drastically lower between LGM and LIG for DC2 (0.216), \emph{D.
orientalis} (0.419) and DC3 (0.558).

\textbf{Identification of morphological traits defining taxa based on
lineage divergences in the Tamus clade}

We explored whether differences may exist between the four clades
identified in the Tamus clade of \emph{Dioscorea} in macro- and
micromorphological characteristics (Figure 5). Only three traits (male
flower pedicel, female inflorescence length and leaf coverage) were
found to have a normal distribution (Supplementary Data Table S3). An
assumption of homoscedasticity was corroborated using a Levene test for
these variables. In the vegetative traits analysed (Supplementary Data
Table S2), we found significant differences between clades in leaf area,
including leaf length, leaf width, leaf perimeter and petiole, while
leaf coverage and the main nerve length and leaf length ratio did not
show significant differences between groups (Supplementary Data Table
S3). Regarding reproductive traits (Supplementary Data Table S2), we
found significant differences in male individuals for the total length
of the inflorescence, the length of pedicels and the number of
fascicles, but the number of flowers did not show significant
differences (Supplementary Data Table S3B). The following morphological
traits showed Pearson correlations \textgreater{}0.7 (Supplementary Data
Figure S7): leaf length, leaf width, leaf area, leaf perimeter, petiole,
leaf coverage, main nerve length, leaf length ratio, inflorescence
length and number of flowers. We therefore selected leaf area, leaf
coverage and male inflorescence length as potential diagnostic
variables. Post-hoc analyses were performed (Supplementary Data Table
S3), and we used these statistical differences to describe the
morphological variability of each genetic group in Table 2.

\par\null\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}
\caption{{Phylogenetic tree reconstructed using the concatenated nuclear data
obtained using Hyb-Seq for 76 samples of the Tamus clade of
\emph{Dioscorea}. Morphological, ploidy and geographic distribution
trait variation is represented with circles for each sample. Leaf:
filled circle, trilobed; empty circle, cordate; ploidy level: filled
circle, allelic ratio \textgreater{}2 (estimated as polyploid); empty
circle, allelic ratio \textless{}2 (estimated as diploid; see Results);
geographic distribution: filled circle, W and C Mediterranean; empty
circle, E Mediterranean; inflorescence: filled circle, sessile solitary;
empty circle, pedunculated fascicule.
{\label{772446}}%
}}
\end{center}
\end{figure}

Morphological differences were found between the four clades and served
as diagnostic characters in an identification key to distinguish the
four distinct species (see Identification key below); these
morphological features matched the genetic and niche data of these taxa.
Our multidisciplinary approach combining genetic, spatial and
morphometric data has delimited four taxa at species level, three
species in the previously recognized \emph{Dioscorea communis sensu
lato} , namely \emph{D. communis sensu stricto} , \emph{D. edulis}
and\emph{D. cretica} , plus \emph{D. orientalis} .

\textbf{Identification key for \emph{Dioscorea communis}, \emph{D.
cretica}, \emph{D. edulis} and \emph{D. orientalis}}

1a. Flowers sessile, solitary; perigonium white or violet in male
inflorescences; leaf pedicel up to 1.6 cm \ldots{}. \emph{D. orientalis}

1b. Flowers pedicellate in fascicles (2--4 flowers each); perigonium
whitish-greenish or violet; leaf pedicel usually longer than leaf
\ldots{}. 2

2a. Leaves cordate-sagittate; perigonium violet; Macaronesia \ldots{}.

\emph{D. edulis}

2b. Leaves cordate-trilobed; perigonium whitish-greenish; not in
Macaronesia\ldots{}. 3

3a. Leaves cordate (rarely trilobed in Balearic Islands); flowers
turbinate infundibuliform \ldots{}. \emph{D. communis}

3b. Leaves trilobed; flowers urceolate \ldots{}. \emph{D. cretica}

\textbf{Taxonomic treatment and descriptions}

\selectlanguage{ngerman}\textbf{1. \emph{Dioscorea orientalis} (Thiébaut) Caddick \& Wilkin}

Basionym = \emph{Tamus orientalis} Thiébaut., Bull. Soc. Bot. France 81:
119. 1934.

-- Holotype: Lebanon. Between Batroun and Saïda, January
1933,\emph{Thiébaut s.n.} (P00301666).

Perennial herb, glabrous. Stems simple or little branched. Leaves ovate,
acuminate, cordate at the base of up to 56 × 48 mm, petiole with
glandular base. Flowers sessile, in axillary spikes, hanging 1--23
(male), 1--4 (female) flowers. Whitish purple (i.e., including a range
of shades of purple) perigonium; six lobes, recurved ovals. Six stamens,
three stigmas and six naked filaments (female). Bracteoles 1--2 widely
ovate, with final peak, adpressed perigonium, with a hull in the outer
surface. Fleshy fruit, oblong, 7--11 × 6--10 mm, red at maturity. Pollen
grains (Supplementary Data Figure S8) with two apertures with longer
axis up to 40 \selectlanguage{greek}µ\selectlanguage{english}m and shorter axis up to 30 \selectlanguage{greek}µ\selectlanguage{english}m. Perforated with 1.9
perforations per \selectlanguage{greek}µ\selectlanguage{english}m and perforation size up to 0.85 \selectlanguage{greek}µ\selectlanguage{english}m, with spines.
Eastern Mediterranean region. 2\emph{n} = unknown.

\textbf{2. \emph{Dioscorea edulis} (Lowe) Campos, Wilkin \&
Viruel,\emph{comb. nov}. (= Clade DC1)}

=Basionym: \emph{Tamus edulis} Lowe, Trans. Cambridge Philos. Soc. 4: 1.
1833.

--Type: Portugal, Madeira, Pta Moniz, 7 May 1828, Herb. Lowe 504 (K;
K000099334!, K001081657!).

Heterotypic synonyms:

= \emph{Tamus canariensis} Willd. ex Kunth, Enum. Pl. 5: 455. 1850, nom.
illeg. pro syn. Type: Spain, Canary Islands, \emph{Herbarium Willdenow
no 18374} (B-W).

= \emph{Tamus parviflora} Kunth, Enum. Pl. 5: 454. 1850. Holotype:
Spain, Canary Islands, Teneriffa, ex Museo Paris, 1821 (B 10 0160963!),
male plant.

Perennial herb, glabrous. Leaves varying from cordiform to sagittate,
petiole 1.60--3.88 cm, leaves up to 125 \selectlanguage{ngerman}× 110 mm, slightly wavy, coarse,
chartaceous and secondary veins visible, but not prominent. Male
inflorescence compound is arranged in fascicles of three or four
flowers, compound inflorescence up to 12 cm in length and bearing up to
65 flowers in total. Female flowers composed of six violaceous tepals
with erect filaments. Fruit a globose berry, 6--12 × 1--8 mm,
reddish-orange. Pollen grains (Supplementary Data Figure S8) with two
apertures with longer axis up to 36 \selectlanguage{greek}µ\selectlanguage{english}m and shorter axis up to 18 \selectlanguage{greek}µ\selectlanguage{english}m.
Perforated with 3.9 perforations per \selectlanguage{greek}µ\selectlanguage{english}m and perforation size up to 0.37
\selectlanguage{greek}µ\selectlanguage{english}m, without spines. Macaronesia (Madeira, Gran Canaria, Tenerife,
Gomera, Hierro, La Palma). 2\emph{n} = 36, 48.

\textbf{3. \emph{Dioscorea cretica} (L.) Campos, Wilkin \&
Viruel,\emph{comb. nov}. (= Clade DC2)}

Basionym: \emph{Tamus cretica} L., Sp. Pl., 1028. 1753.

= \emph{Tamus communis} L. subsp. \emph{cretica} (L.) Kit Tan, Notes
Roy. Bot. Gard. Edinburgh 41(1): 47. 1983.

-- Neotype (designated by Kit Tan in Davis, 1984, as isotype, corrected
by Jarvis, 2007): ''Habitat in Creta'', Herb. Tournefort no. 283
(P-00665856!).

Perennial herb, glabrous. Leaves deeply trilobate, or apex or basal
lobes elongated from a cordate leaf, up to 70 \selectlanguage{ngerman}× 70 mm, with 3-9
prominent main nerves and secondary reticulated nerves not visible. Male
inflorescence branched, to 25 cm long with up to 50 flowers in total,
short female inflorescence up to 7 cm with 1--6 urceolate,
greenish-yellow flowers. Fruit a globose berry, 7--11 × 6--10 mm,
reddish-orange. Pollen grains (Supplementary Data Figure S8) with two
apertures with longer axis up to 36 \selectlanguage{greek}µ\selectlanguage{english}m and shorter axis up to 29 \selectlanguage{greek}µ\selectlanguage{english}m.
Perforated with 0.5 perforations per \selectlanguage{greek}µ\selectlanguage{english}m and perforation size up to 1.2
\selectlanguage{greek}µ\selectlanguage{english}m, without spines. Eastern Mediterranean. 2\emph{n} = unknown.

\textbf{4. \emph{Dioscorea communis} (L.) Caddick \& Wilkin (= Clade
DC3)}

Basionym: \emph{Tamus communis} L., Sp. Pl.: 1028. 1753.

= \emph{Tamus communis} (L.) f. \emph{subtriloba} (Guss.) O.Bol\selectlanguage{ngerman}òs \&
Vigo -- Fl. Països Catalans 4: 171 (2001).

- Lectotype (designated by Ferrer-Gallego \& Boisset, 2016): ''habitat
in Europa australi'', \emph{anon} ., Herb. Linn. No. 1181.2 (LINN!).

Heterotypic synonym: \emph{Tamus communis} L. var.
\emph{subtriloba}Guss., \emph{Fl. Siculae Syn.} 2(2): 880 (1884).
\emph{Tamus communis}L. f. \emph{subtriloba} (Guss.) O.Bolòs \& Vigo,
\emph{Fl. Països Catalans} 4: 171 (2001). Lectotype (designated by
Ferrer-Gallego \& Boisset, 2016): Italy, Sicily, Palermo, Vergine Maria,
April--May,\emph{anon.} (NAP).

Perennial herb, glabrous. Stem, striated, branched. Leaves cordate,
rarely trilobed, up to 180 × 150 mm, with 3--9 prominent main nerves and
secondary reticulated nerves not visible. Male inflorescence branched,
up to 35 cm with up to 73 flowers in total, short female inflorescence
up to 7 cm with 1--14 flowers, turbinate-infundibuliform,
greenish-yellow. Fruit a somewhat tapered, globose berry, 7 to 12 mm,
reddish-orange. Pollen grains (Supplementary Data Figure S8) with two
apertures with longer axis up to 36 \selectlanguage{greek}µ\selectlanguage{english}m and shorter axis up to 30 \selectlanguage{greek}µ\selectlanguage{english}m.
Perforated with 0.5 perforations per \selectlanguage{greek}µ\selectlanguage{english}m and perforation size up to 1.2
\selectlanguage{greek}µ\selectlanguage{english}m, without spines. Western and central Mediterranean, western Europe.
2\emph{n} = 96.

\textbf{Discussion}

\textbf{Species discovery based on integrative approaches}

The biological species concept (Mayr, 1942) defines a species as a group
of populations reproductively isolated from others. This concept is
difficult to apply to species delimitation in flowering plants due to
the high incidence of hybridization and introgression (Mitchell \emph{et
al} ., 2019). Although plant taxonomy has relied on morphological traits
to differentiate and discover new taxa for centuries using a typological
species concept (Haider, 2018), biological processes such as
hybridization can obscure the morphological attributes used to
differentiate species. Combined morphological and molecular approaches
have been used to identify cryptic species in angiosperms (Maguilla and
Escudero, 2016). Alternative species concepts have been proposed in
plants to accommodate a broad spectrum of approaches, such as cytology,
phytochemistry, anatomy, embryology or phylogenetics (De Queiroz, 2007;
Aldhebiani, 2018). These are method-based concepts, such as the
evolutionary species concept using phylogenetic inference, or the
ecological species concept based on niche differentiation.

Here, we use an integrative approach combining morphological,
phylogenetic, and ecological niche data to decipher species delimitation
in the Tamus clade of \emph{Dioscorea} and uncovered the existence of
introgression in some individuals (Figure 2) that could at least partly
explain the overlap in some of the morphological characteristics between
taxa. The discovery of cryptic species in this group shapes our current
understanding of it, specifically for what has been to date accepted
as\emph{D. communis} (Caddick \emph{et al.,} 2002). Based on our
results, we propose the maintenance of \emph{D. orientalis} as a species
and divide \emph{D. communis} \emph{sensu lato} into three distinct
species:\emph{D. communis sensu stricto} , \emph{D. edulis} and \emph{D.
cretica}(see Results; Figure 6).

\par\null\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-6/Figure-6}
\caption{{Geographic distribution of the 76 samples of the Tamus clade of
\emph{Dioscorea} coded according to their respective lineage in the
phylogenetic tree based on concatenated nuclear data (Figure 1),
\emph{D. cretica} (DC2) in green, and subclade; and pink, orange, and
red for western European, central Mediterranean and eastern
Mediterranean clades in \emph{D. communis s.s.} (DC3), respectively. For
\emph{D. orientalis}, in purple, codes in the map indicate their
phylogenetic position named ``Leb'' (Lebanon) and ``S23''. For \emph{D.
edulis} (DC1), in light blue, codes in the map indicate their
phylogenetic position named ``Mad'' (Madeira), ``Ten'' (Tenerife) and
``GC'' (Gran Canaria). Roman numbers in the mapped dot samples represent
their respective plastid tree lineage (Figure 1). Asterisks indicate
cultivated samples in Botanic Gardens of unknown geographic origin.
{\label{214160}}%
}}
\end{center}
\end{figure}

The emergence of HTS methodologies has allowed the detection of cryptic
species (Carstens and Satler, 2013), the resolution of complex
phylogenetic trees (Bogar\selectlanguage{ngerman}ín \emph{et al.,} 2018; Frajman \emph{et
al.,}2019; Hassemer \emph{et al.,} 2019; Yang \emph{et al.,} 2019) and
the reconstruction of evolutionary patterns in extinct species
(Moreno-Aguilar \emph{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 \emph{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 \emph{et al.,} 2019, Viruel \emph{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 \emph{et al.,} 2019), to reveal the evolutionary patterns
and relationships between taxa belonging to the Tamus clade
of\emph{Dioscorea} (Figure 1). By sequencing 76 samples of
the\emph{Tamus} clade, the phylogenomic and genetic clustering
approaches revealed extensive infraspecific variability in \emph{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 \emph{Tamus edulis} and \emph{T. cretica}
, which were recently placed within the large morphological variability
and wide distribution of \emph{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 \emph{D. communis
s.l.} into\emph{D. edulis} , \emph{D. cretica} and \emph{D. communis} ,
and to maintain \emph{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 \emph{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 \emph{et al.,} 2009; Zhan \emph{et al.,} 2016). Polyploidy
is a common phenomenon, which has been frequently reported in
several\emph{Dioscorea} species (Viruel \emph{et al.,} 2008), although
defining ploidy of the Tamus and Borderea clades has been challenging.
The two\emph{Dioscorea} species belonging to the Borderea clade,
\emph{D. chouardii} and \emph{D. pyrenaica} , have chromosome counts of
2\emph{n}= 24. Based on the discovery of allotetraploidy using
microsatellite markers (Segarra-Moragues \emph{et al} ., 2003), it was
proposed that the chromosome base number for the Borderea clade was
\emph{x} = 6 (see also Viruel \emph{et al} ., 2008). Extrapolating this
find to the sister Tamus clade, the known chromosome counts reported for
\emph{D. communiss.s.} of 2\emph{n} = 36 and 48 (Al-Shehbaz and
Schubert, 1989; Viruel \emph{et al} ., 2019) would therefore represent
hexaploid and octoploid forms, respectively. Similarly, the Macaronesian
\emph{D. edulis} , with 2\emph{n} = 96, would be 16-ploid assuming a
base chromosome number of \emph{x} = 6. Using flow cytometry to estimate
ploidy in \emph{D. communis} \emph{s.s.} , multiple ploidies were
observed (1C-values ranging from 0.41 to 1.36 pg; Viruel \emph{et al} .,
2019). The chromosome number and genome size of \emph{D. orientalis}
and\emph{D. 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 \emph{et al} ., 2019). Median and mean
values of allelic ratios based on the number of reads supporting each
SNP were recently proposed to classify \emph{Dioscorea} samples as
diploid forms when the allelic ratio is \textless{}2, and polyploids
when \textgreater{}2 (Viruel \emph{et al} ., 2019). For example, all
samples of \emph{D. edulis} had mean and median allelic ratios
\textgreater{}2 (Table 1), confirming the polyploid nature of this
species based on chromosome data. In all cases, \emph{D.
orientalis}samples studied here showed allelic ratios \textgreater{}2
and would therefore be estimated to be a polyploid species (Table 1).
For \emph{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 \textless{}2
(samples S67 and R12, Table 1). Samples estimated to be diploid based on
allelic ratios were also observed in \emph{D. cretica} , with half of
the samples (eight) having average and median allelic ratios
\textless{}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.

\textbf{Evolutionary patterns of the Tamus clade of \emph{Dioscorea} in
the Mediterranean}

Overall evolutionary patterns of \emph{Dioscorea} lineages have been
thoroughly studied using plastid markers (e.g., Wilkin \emph{et
al.,}2005; Hsu \emph{et al.,} 2013; Maurin \emph{et al.,} 2016;
Viruel\emph{et al.,} 2016) and low copy nuclear genes (e.g. Viruel
\emph{et al.,} 2018, Soto Gomez \emph{et al.,} 2019). Previous studies
determined that yams diverged and expanded since the Late Cretaceous,
probably from Laurasia, and that a split of the African-Mediterranean
lineage, which includes the Tamus clade, likely occurred in the
Oligocene, following a westward migration ca. 33 MY (Viruel \emph{et al}
., 2016). Fossil records indicate that \emph{Dioscorea} ancestors
persisted in Europe during the Oligocene (Andreànzky, 1959). Based on
data from four plastid markers, the split between the two Mediterranean
clades, Borderea and Tamus, was estimated to have occurred during the
late Oligocene (ca. 25.7 MY) (Viruel \emph{et al} ., 2016), a similar
divergence time to the one we obtained with our analyses based on 260
nuclear genes, which indicate that this divergence took place in the
early Oligocene (28.2--28.4 MY); Supplementary Data Figure S4). Two
narrowly endemic species of the Borderea clade survived in refugia in
the Pyrenean mountains, \emph{D. chouardii} , a critically endangered
species with only one known locality (Martínez \& Otano, 2011), and
\emph{D. pyrenaica} , with a slightly wider distribution in the central
Pyrenees and Pre-Pyrenees (Segarra \& Catalán, 2005; Catalan \emph{et
al} ., 2006; Segarra-Moragues \& Catalan, 2008; García \emph{et al.,}
2012). A previous study (Viruel \emph{et al.,} 2016) estimated a split
between these two species during the early Pliocene (ca. 4.3 MY),
whereas our results indicated that this divergence likely occurred
during the late Miocene (8.1--10.6 MY).

The differences observed in divergence times for the Tamus clade in
comparison with previous studies are a consequence of the newly
recognized species (\emph{D. cretica} ). In Viruel \emph{et al} .
(2016), the crown node of the Tamus clade was estimated to be 15.3 MY
(\emph{D. edulis} (\emph{D. communis} , \emph{D. orientalis} )), and the
subsequent split between \emph{D. orientalis} and \emph{D. communis} at
10.4 MY. However, the samples of \emph{D. orientalis} included in Viruel
\emph{et al} . (2016) have now been reidentified as \emph{D. cretica} ,
and thus the older age estimates herein for the crown node of the Tamus
clade (20.6--18.2 MY) demonstrate an early split of \emph{D. orientalis}
in the eastern Mediterranean, followed by a split of \emph{D. edulis}
ca. 16.0-13.5 MY, and the divergence of \emph{D. communis} \emph{s.s.}
and\emph{D. cretica} ca. 6.6--5.6 MY. Given the findings presented here,
with a sampling representative of the whole distribution range of the
Tamus clade across the circum-Mediterranean region, we conclude that the
divergence times estimated here are more robust and taxonomically more
representative, which allowed us to reassess the species delimitation in
this group.

The Mediterranean region is considered one of the major biodiversity
hotspots of the world (Médail and Quézel, 1997). Fossil records and
evolutionary studies have confirmed that the ancestors of several plant
lineages were part of a tropical flora that occupied the Mediterranean
region during the Miocene and early Pliocene (Suc \emph{et al.,} 2018).
The drastic subsequent climatic changes that came during the Pliocene
(3.5--2.4 MY), with a significant drop in temperature and a marked
seasonality in thermal and rainfall regimes, impacted the
diversification patterns of plant lineages and resulted in narrow
endemics in the margins of the distribution range of their sister
species (e.g., \emph{Ceratonia oreothauma} Hillc., G.P.Lewis \& Verdc.;
Viruel \emph{et al.,} 2020). The diversification of species in the Tamus
clade likely occurred during the Miocene when subtropical climatic
conditions were present across the Mediterranean (Suc \emph{et al} .,
2018). The most recent common ancestor of all Tamus clade taxa likely
diversified during the early Miocene (20.6--18.2 MY), when the lineages
that gave rise to the current \emph{D. orientalis} and the clade
comprising the three lineages of \emph{D. communis s.l.} likely split.
This was followed by a subsequent split of the Macaronesian \emph{D.
edulis} that would have taken place in the mid-Miocene (16.0--13.5 MY),
after the formation of some of the Canary Islands, which has been
estimated to start around 23 MY (Sanmartín \emph{et al.,} 2008;
Florencio \emph{et al} ., 2021). The most recent split between \emph{D.
communis} \emph{s.s.} and \emph{D. cretica} is estimated to have
occurred during the Messinian (Miocene, 6.6--5.6 MY). During this
period, the significant and rapid lowering of the sea level of the
Mediterranean also resulted in new terrestrial biogeographical
connections allowed by the formation of land-bridges.

Several phylogeographic studies have attempted to explain the
biodiversity patterns and processes that shaped the Mediterranean region
and its development into one of the world's biodiversity hotspot (e.g.,
Nieto Feliner, 2014; Thompson, 2021). Two main areas of high plant
endemism were identified in the western (Iberian Peninsula and Morocco)
and eastern Mediterranean (including Turkey and Greece) (Médail and
Quézel, 1997). In both these areas, Quaternary glaciations likely played
a major role shaping the distribution of species and left a footprint in
the genetic structure of many Mediterranean species, particularly in
refugia (Médail and Diadema, 2009). Western and eastern genetic groups
have been identified in phylogeographic patterns of several
Mediterranean plants, leading to disjunct distributions in some cases,
such as in \emph{Microcnemum} Ung.-Sternb (Amaranthaceae)
and\emph{Mandragora} L. (Solanaceae) (Kadereit and Yaprak, 2008;
Volis\emph{et al.,} 2018), or by differentiating morphotypes that later
hybridized in intermediate zones (e.g., \emph{Quercus ilex} L.,
Lumaret\emph{et al.,} 2002). The strong geographic influence in the
genetic structuring of \emph{D. communis} across the Mediterranean may
have also been slightly influenced by bird dispersal. Bird dispersals
have contributed to shaping the postglacial recolonization of the
Mediterranean, such as seen in \emph{Frangula} \emph{alnus} Mill.
(Hampe\emph{et al} ., 2003). The birds that consume berries produced by
species of the Tamus clade, mainly blackbirds (\emph{Turdus merula} L.),
robins (\emph{Erithacus rubecula} L.) and blackcaps (\emph{Sylvia
communis}Latham) (Chiscano, 1983; Herrera, 1984), are predominantly
sedentary birds or have modern migratory routes that do not strictly
coincide with the past and current distribution patterns estimated in
this study (Adriaensen, 1988; Burfield and Van Bommel, 2004). It would
thus be useful to analyse the patterns of genetic structure at the
population level of \emph{D. communis s.s.} in more detail and in
connection with the possible magnitude of ornithochory, which has never
been studied in detail to our knowledge.

Changes in ploidy, morphological differences and introgression between
the central Mediterranean and western European populations have been
shown to have occurred between the \emph{D. communis s.s.} and \emph{D.
cretica} lineages (Figures 2, 5). The central-eastern Mediterranean area
constitutes the contact region between these two species and is
congruent with the introgression patterns found in our study (Figure 2).
Five out of 16 samples studied of \emph{D. cretica} exhibited
\textless{}20\% of admixture index with \emph{D. communis s.s.} , and
all individuals of \emph{D. communis} belonging to the eastern clade
of\emph{D. communis s.s.} showed \textless{}20\% of admixture index
with\emph{D. cretica} (Figure 2). However, only four individuals from
the central Mediterranean and western European subclades of \emph{D.
communis} were detected as introgressing with a sister species, and one
sample of \emph{D. cretica} was placed in a clade of \emph{D. communis
s.s.} in the phylogenetic tree based on plastid data (R32, Figure 1).
These results are congruent with their potential distribution in
disjunct refugia followed by secondary contact through recolonization,
and by maintaining some capacity of interspecific gene flow between
closely related species (see Viruel \emph{et al.,} 2021). The
topological incongruencies found between the nuclear and plastid
phylogenetic trees, indicative of plastid capture events (Figure 1), are
congruent with these hypothesized introgression patterns: plastid clades
I and II are found in the central and eastern Mediterranean lineages
without a clear geographic separation (Figure 6), whereas clade III is
uniquely found in the western part of the Mediterranean where lower
introgression events have been inferred.

\textbf{Conclusions}

The identification of new plant species usually requires a broad
understanding of taxon boundaries applying multidisciplinary
methodologies. Our study exemplifies the complexity of identifying new
species by integrating different types of data: target capture
sequencing data from herbarium specimens to reveal phylogenomic patterns
and introgression between clades, differences in allelic ratios to
estimate ploidy, spatial analysis estimating current and past
distribution ranges and niche overlaps, and macro- and micromorphometric
comparisons. By integrating all these results, we have newly
corroborated the existence of four species in the Mediterranean Tamus
clade of \emph{Dioscorea} , maintaining \emph{D. orientalis} as a
distinct species, and demonstrating that \emph{D. edulis} and \emph{D.
cretica} are species discrete from the synonymy of the morphologically
variable \emph{D. communis} .

\textbf{Acknowledgements}

We are thankful to the herbaria of Kew (K) and Seville (SEV) for
providing access to their collections for this study and to the
colleagues who collected samples in the field. This work was supported
by a Marie Sk\selectlanguage{polish}ł\selectlanguage{english}odowska-Curie Individual Fellowship (704464 -- YAMNOMICS
-- MSCA-IF-EF-ST) and the RBG Kew-funded pilot study to Juan Viruel; and
Erasmus+ Programme for Miguel Campos. Emma Kelley received funding from
the Muriel Kohn Pokross '34 Travel/Internship Fund, Smith College,
Massachusetts. Pilar Catal\selectlanguage{ngerman}án was supported by a European Social Fund and
Spanish Aragón Government Bioflora grant.

\textbf{Literature cited}

Adriaensen F. 1988. An analysis of recoveries of robins (\emph{Erithacus
rubecula} ) ringed or recovered in Belgium: winter
distributions.\emph{Le Gerfaut} 781: 25--42.

Aldhebiani AY. 2018. Species concept and speciation. \emph{Saudi Journal
of Biological Sciences} 25: 437--440. doi: 10.1016/j.sjbs.2017.04.013

Andreàndzky G. 1959. Contributions à la connaissance de la flore de
l'Oligocène inférieur de la Hongrie et un essai sur la reconstruction de
la flore contemporaine. \emph{Acta Botanica Academiae Scientarum
Hungaricae} 5: 1--37.

Asiedu R, Sartie A. 2010. Crops that feed the world 1. Yams. \emph{Food
Security} 2: 305--315.

Bogarín D, Pérez-Escobar OA, Groenenberg D, \emph{et al.,} 2018.
Anchored hybrid enrichment generated nuclear, plastid and mitochondrial
markers resolve the \emph{Lepanthes horrida} (Orchidaceae:
Pleurothallidinae) species complex. \emph{Molecular Phylogenetics and
Evolution} 129: 27--47. doi: 10.1016/j.ympev.2018.07.014

Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for
Illumina sequence data. \emph{Bioinformatics} 30: 2114--2120. doi:
10.1093/bioinformatics/btu170

Braconnot P, Otto-Bliesner B, Harrison S, \emph{et al.} 2007. Results of
PMIP2 coupled simulations of the Mid-Holocene and last glacial maximum -
Part 1: experiments and large-scale features. \emph{Climate of the
Past}3: 261--277. doi: 10.5194/cp-3-261-2007

Brewer GE, Clarkson JJ, Maurin O, \emph{et al.} 2019. Factors Affecting
Targeted Sequencing of 353 Nuclear Genes From Herbarium Specimens
Spanning the Diversity of Angiosperms. \emph{Frontiers in Plant
Science}10: 1102. doi: 10.3389/fpls.2019.01102

Burfield I, van Bommel F. 2004. Birds in Europe: population estimates,
trends and conservation status: 12 (BirdLife Conservation Series).
Cambridge, GB: BirdLife International.

Caddick LR, Wilkin P, Rudall PJ, Hedderson TAJ, Chase MW. 2002b. Yams
reclassified: a recircumscription of Dioscoreaceae and
Dioscoreales.\emph{Taxon} 51: 103--114. doi: doi.org/10.2307/1554967

Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. 2009. trimAl: a tool
for automated alignment trimming in large-scale phylogenetic
analyses.\emph{Bioinformatics} 25: 1972--1973. doi:
10.1093/bioinformatics/btp348

Carstens BC, Satler JD. 2013. The carnivorous plant described
as\emph{Sarracenia alata} contains two cryptic species. \emph{Biological
Journal of the Linnean Society} 109: 737--746. 10.1111/bij.12093

Catalán P, Segarra-Moragues JG, Palop-Esteban M, Moreno C,
Gonzalez-Candelas F. 2006. A Bayesian approach for discriminating among
alternative inheritance hypotheses in plant polyploids: the
allotetraploid origin of genus \emph{Borderea}
(Dioscoreaceae).\emph{Genetics} 172: 1939-1953.

Chiscano JLP. 1983. La ornitocoria en la vegetación de
Extremadura.\emph{Studia Botanica} 2: 155--168.

Clement M, Snell Q, Walker P, Posada D, Crandall K. 2002. TCS:
estimating gene genealogies. \emph{16th International Parallel and
Distributed Processing Symposium} (\emph{IPDPS 2002} ), 15--19 April
2002, Fort Lauderdale, FL, USA, CD-ROM/Abstracts Proceedings.

Criscuolo NG, Angelini C. 2020. StructuRly: a novel shiny app to produce
comprehensive, detailed and interactive plots for population genetic
analysis. \emph{PLoS One} 15: e0229330. doi:
10.1371/journal.pone.0229330

Davis PH. (ed.) 1984. \emph{Flora of Turkey and the East Aegean Islands}
, vol. 8. Edinburgh: Edinburgh University Press, pp. 552--554.

De Luca V, Salim V, Atsumi SM, Yu F. 2012. Mining the biodiversity of
plants: a revolution in the making. \emph{Science} 336: 1658--1661. doi:
10.1126/science.1217410

De Queiroz K. 2007. Species concepts and species
delimitation.\emph{Systematic Biology} 56: 879--886. doi:
10.1080/10635150701701083

Doyle J, Doyle JL. 1987. Genomic plant DNA preparation from fresh
tissue-CTAB method. \emph{Phytochemical Bulletin} 19: 11--15.

Drummond AJ, Rambaut A. 2007. BEAST: Bayesian evolutionary analysis by
sampling trees. \emph{BMC Evolutionary Biology} 7: 1--8. doi:
10.1186/1471-2148-7-214

Earl DA, vonHoldt BM. 2011. STRUCTURE HARVESTER: a website and program
for visualizing STRUCTURE output and implementing the Evanno
method.\emph{Conservation Genetic Resources} 4: 359--361. doi:
10.1007/s12686-011-9548-7

Escudero M, Nieto Feliner G, Pokorny L, Spalink D, Viruel J. 2020.
Phylogenomic approaches to deal with particularly challenging plant
lineages. \emph{Frontiers in Plant Science} 11: 591762. doi:
10.3389/fpls.2020.591762

Fay MF, Gargiulo R, Viruel J. 2019. The present and future for
population genetics, species boundaries, biogeography and
conservation.\emph{Botanical Journal of the Linnean Society} 191:
299--304. doi: 10.1093/botlinnean/boz076

Ferrer-Gallego PP, Boisset F. 2016. Typification of \emph{Dioscorea
communis} and its synonym \emph{Tamus communis} var.
\emph{subtriloba}(Dioscoreaceae). \emph{Phytotaxa} 260(3): 258--266.

Florencio M, Patiño J, Nogué S, \emph{et al} . 2021. Macaronesia as a
Fruitful Arena for Ecology, Evolution, and Conservation
Biology.\emph{Frontiers in Ecology and Evolution} 9: 718169. doi:
10.3389/fevo.2021.718169

Frajman B, Záveská E, Gamisch A, Moser T, The STEPPE Consortium,
Schönswetter P. 2019. Integrating phylogenomics, phylogenetics,
morphometrics, relative genome size and ecological niche modelling
disentangles the diversification of Eurasian \emph{Euphorbiaseguieriana}
s.l. (Euphorbiaceae). \emph{Molecular Phylogenetics and Evolution} 134:
238--252. doi: 10.1016/j.ympev.2018.10.046

García MB, Espadaler X, Olesen JM. 2012. Extreme reproduction and
survival of a true cliffhanger: the endangered plant \emph{Borderea
chouardii} (Dioscoreaceae). \emph{PLoS One} 7: e44657. doi:
10.1371/journal.pone.0044657

Gent PR, Danabasoglu G, Donner LJ, \emph{et al.} 2011. The community
climate system model version 4. \emph{Journal of Climate} 24:
4973--4991. doi: 10.1175/2011JCLI4083.1

Goñi Martínez D, Guzmán Otano D. 2011. \emph{Borderea chouardii}
.\emph{The IUCN Red List of Threatened Specie} s 2011:
e.T162110A5540643.

Haider N. 2018. A Brief Review on Plant Taxonomy and its
Components.\emph{Journal of Plant Science Research} 34: 275--290. doi:
10.32381/JPSR.2018.34.02.17

Hampe A, Arroyo J, Jordano P, Petit RJ. 2003. Rangewide phylogeography
of a bird-dispersed Eurasian shrub: contrasting Mediterranean and
temperate glacial refugia. \emph{Molecular Ecology} 12: 3415--3426. doi:
10.1046/j.1365-294X.2003.02006.x

Hassemer G, Bruun-Lund S, Shipunov AB, \emph{et al} . 2019. The
application of high-throughput sequencing for taxonomy: the case
of\emph{Plantago} subg. \emph{Plantago} (Plantaginaceae).
\emph{Molecular Phylogenetics and Evolution} 138: 156--173. doi:
10.1016/j.ympev.2019.05.013

Hasumi H, Emori S. 2004. \emph{K-1 coupled model (MIROC) description
(K-1 Technical Report 1)} . Tokyo: Center for Climate System Research,
University of Tokyo.

Herrera CM. 1984. A study of avian frugivores, bird-dispersed plants,
and their interaction in Mediterranean scrublands. \emph{Ecological
Monographs} 54: 2--23. doi: 10.2307/1942454

Hsu KM, Tsai JL, Chen MY, Ku HM, Liu SC. 2013. Molecular phylogeny
of\emph{Dioscorea} (Dioscoreaceae) in East and Southeast
Asia.\emph{Blumea: Journal of Plant Taxonomy and Plant Geography} 58:
21--27. doi: 10.3767/000651913X669022

Hua W, Kong W, Cao X, \emph{et al.} 2017. Transcriptome analysis
of\emph{Dioscorea zingiberensis} identifies genes involved in diosgenin
biosynthesis. \emph{Genes \& Genomics} 39: 509--520. doi:
10.1007/s13258-017-0516-9

Jarvis CE. 2007. \emph{Order out of chaos} . \emph{Linnaean plant names
and their types} . London: Linnean Society of London with the Natural
History Museum, 1016 pp.

Johnson MG, Gardner EM, Liu Y, \emph{et al.} 2016. HybPiper: Extracting
Coding Sequence and Introns for Phylogenetics from High-Throughput
Sequencing Reads Using Target Enrichment. \emph{Applications in Plant
Sciences} 4: 1600016. doi: 10.3732/apps.1600016

Johnson MG, Pokorny L, Dodsworth S, \emph{et al.} 2019. A Universal
Probe Set for Targeted Sequencing of 353 Nuclear Genes from Any
Flowering Plant Designed Using k-Medoids Clustering. \emph{Systematic
Biology} 68: 594--606. doi: 10.1093/sysbio/syy086

Kadereit G, Yaprak AE. 2008. \emph{Microcnemum
coralloides}(Chenopodiaceae-Salicornioideae): An example of
intraspecific East-West disjunctions in the Mediterranean region. Anales
del Jardín Botánico de Madrid 65: 415--426. doi:
10.3989/ajbm.2008.v65.i2.303

Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel method for
rapid multiple sequence alignment based on fast Fourier transform.
Nucleic Acids Research 30: 3059--3066. doi: 10.1093/nar/gkf436

Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. 2019. RAxML-NG: a
fast, scalable and user-friendly tool for maximum likelihood
phylogenetic inference. \emph{Bioinformatics} 35: 4453--4455. doi:
10.1093/bioinformatics/btz305

Leigh JW, Bryant D. 2015. POPART: full-feature software for haplotype
network construction. \emph{Methods in Ecology and Evolution} 6:
1110--1116. doi: 10.1111/2041-210X.12410

Linnaeus C. 1753. \emph{Species plantarum vol. 1} . Stockholm: L.
Salvius.

Lumaret R, Mir C, Michaud H, Raynal V. 2002. Phylogeographical variation
of chloroplast DNA in holm oak (\emph{Quercus ilex} L.). \emph{Molecular
Ecology} 11: 2327--2336. doi: 10.1046/j.1365-294X.2002.01611.x

Maguilla E, Escudero M. 2016. Cryptic Species Due to Hybridization: A
Combined Approach to Describe a New Species (\emph{Carex} :
Cyperaceae).\emph{PLoS One} 11: e0166949. doi:
10.1371/journal.pone.0166949

Martin FW, Degras L. 1978. \emph{Tropical yams and their potential. Part
6. Minor cultivated} Dioscorea \emph{species.} Washington, DC.: USDA,
Science and Education Administration.

Martínez GD, Otano GD. 2011. \emph{Borderea chouardii} . The IUCN Red
List of Threatened Species 2011: e.T162110A5540643.

Maurin O, Muasya, AM, Catalán P, \emph{et al.} 2016. Diversification
into novel habitats in the Africa clade of
\emph{Dioscorea}(Dioscoreaceae): erect habit and elephant's foot tubers.
\emph{BMC Evolutionary Biology} 16: 1--17. doi:
10.1186/s12862-016-0812-z

Mayr E. 1942. \emph{Systematic and the origin of species} . New York:
Columbia University Press.

Médail F, Quézel P. 1997. Hot-Spots Analysis for Conservation of Plant
Biodiversity in the Mediterranean. \emph{Annals of the Missouri
Botanical Garden} 84: 112--127. doi: 10.2307/2399957

Médail F, Diadema K. 2009. Glacial refugia influence plant diversity
patterns in the Mediterranean Basin. \emph{Journal of Biogeography} 36:
1333--1345.

Mitchell N, Campbell LG, Ahern JR, \emph{et al} . 2019. Correlates of
hybridization in plants. \emph{Evolution Letters} 3: 570--585. doi:
https://doi.org/10.1002/evl3.146

Moreno-Aguilar MF, Arnelas I, Sánchez-Rodríguez A, Viruel J, Catalán P.
2020. Museomics Unveil the Phylogeny and Biogeography of the Neglected
Juan Fernandez Archipelago \emph{Megalachne} and
\emph{Podophorus}Endemic Grasses and Their Connection With Relict
Pampean-Ventanian fescues. \emph{Frontiers in Plant Science} 11: 819.
doi: 10.3389/fpls.2020.00819

Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. 2015. IQ-TREE: A fast
and effective stochastic algorithm for estimating maximum likelihood
phylogenies. \emph{Molecular Biology and Evolution} 32: 268--274. doi:
10.1093/molbev/msu300

Nieto Feliner G. 2014. Patterns and processes in plant phylogeography in
the Mediterranean Basin, a review. \emph{Perspectives in Plant Ecology,
Evolution and Systematics} 16: 265--278. doi:
10.1016/j.ppees.2014.07.002

Otto-Bliesner ABL, Marshall SJ, Overpeck JT, \emph{et al.} 2006.
Simulating Arctic Climate Warmth and Icefield Retreat in the Last
Interglaciation. \emph{Science} 311: 1751--1753. doi:
10.1126/science.1120808

POWO. 2022. \emph{Plants of the world online} . Richmond, UK: Royal
Botanic Gardens, Kew. http://www.plantsoftheworldonline.org/

Price EJ, Wilkin P, Sarasan V, Frase PD. 2016. Metabolite profiling
of\emph{Dioscorea} (yam) species reveals underutilised biodiversity and
renewable sources for high-value compounds. \emph{Scientific Reports} 6:
29136. doi: 10.1038/srep29136

Pritchard JK, Stephens M, Donnelly P. 2000. Inference of Population
Structure Using Multilocus Genotype Data. \emph{Genetics} 155: 945--959.
doi: 10.1093/genetics/155.2.945

R Core Team. 2022. \emph{R: a language and environment for statistical
computing} . Vienna: R Foundation for Statistical Computing.
https://www.R-project.org/

Sanmartín I, Van der Mark P, Ronquist F. 2008. Inferring dispersal: a
Bayesian approach to phylogeny-based island biogeography, with special
reference to the Canary Islands. \emph{Journal of Biogeography} 35:
428--449. doi: 10.1111/j.1365-2699.2008.01885.x

Schneider C, Rasband W, Eliceiri K. 2012. ImageJ. \emph{Fundamentals of
Digital Imaging in Medicine} 9: 185--188. doi:
10.1007/978-1-84882-087-6\_9

Segarra JG, Catalán P. 2005. Borderea Miègeville in Aedo C. and A.
Herrero (eds.). \emph{Flora Iberica} 21: 11--14. Real Jardín Botánico de
Madrid, CSIC.

Segarra-Moragues JG, Catalán P. 2008. Glacial survival, phylogeography,
and a comparison of microsatellite evolution models for explaining
population structure in two species of dwarf yams (\emph{Borderea} ,
Dioscoreaceae) endemic to the central Pyrenees. \emph{Plant Ecology and
Diversity} 1: 229--243.

\selectlanguage{polish}Š\selectlanguage{english}marda P. 2008. DNA ploidy level variability of some fescues
(\emph{Festuca} subg. \emph{Festuca} , Poaceae) from Central and
Southern Europe measured in fresh plants and herbarium specimens.
\emph{Biologia}63: 349--367. doi: 10.2478/s11756-008-0052-9

Smith SA, O'Meara BC. 2012. TreePL: Divergence time estimation using
penalized likelihood for large phylogenies. \emph{Bioinformatics} 28:
2689--2690. doi: 10.1093/bioinformatics/bts492

Soltis DE, Albert VA, Leebens-Mack J, \emph{et al.} 2009. Polyploidy and
angiosperm diversification. \emph{American Journal of Botany} 96:
336--348. doi: 10.3732/ajb.0800079

Soto Gomez M, Pokorny L, Kantar, MB, \emph{et al.} 2019. A customized
nuclear target enrichment approach for developing a phylogenomic
baseline for \emph{Dioscorea} yams (Dioscoreaceae). \emph{Applications
in Plant Sciences} 7: 1--13. doi: 10.1002/aps3.11254

Suc J-P, Popescu S-M, Fauquette S, \emph{et al} . 2018. Reconstruction
of Mediterranean flora, vegetation and climate for the last 23 million
years based on an extensive pollen dataset. \emph{Ecologia
Mediterranea}44: 53--85. doi: 10.3406/ecmed.2018.2044

Swofford DL. 2002. \emph{PAUP*. Phylogenetic analysis using parsimony
(*and other methods).} Version 4.0b10. Sunderland; Sinauer Associates.

Thompson JD. 2021. \emph{Plant evolution in the Mediterranean: insights
for conservation (2nd edn).} Oxford: Oxford University Press. doi:
10.1093/oso/9780198835141.001.0001

Urtubey E, Baeza CM, L\selectlanguage{ngerman}ópez-Sepúlveda P, \emph{et al.} 2018. Systematics
of \emph{Hypochaeris} section \emph{Phanoderis} (Asteraceae,
Cichorieae). \emph{Systematic Botany Monographs} 106: 1--204.

Villaverde T, Pokorny L, Olsson S, \emph{et al.} 2018. Bridging the
micro\selectlanguage{english}- and macroevolutionary levels in phylogenomics: Hyb\selectlanguage{english}-Seq solves
relationships from populations to species and above. \emph{New
Phytologist} 220: 36--650. doi: 10.1111/nph.15312

Viruel J, Segarra\selectlanguage{english}-Moragues JG, Perez\selectlanguage{english}-Collazos E, \emph{et al} . 2008.
The diploid nature of the Chilean \emph{Epipetrum} and a new base number
in the Dioscoreaceae, \emph{New Zealand Journal of Botany} 46: 327--339.

Viruel J, Segarra-Moragues JG, Perez-Collazos E, \emph{et al.} 2010.
Systematic revision of the \emph{Epipetrum} group of
\emph{Dioscorea}(Dioscoreaceae) endemic to Chile. \emph{Systematic
Botany} 35: 40--63. doi: 10.1600/036364410790862579

Viruel J, Segarra-Moragues JG, Raz L, \emph{et al.} 2016. Late
Cretaceous-Early Eocene origin of yams (\emph{Dioscorea} ,
Dioscoreaceae) in the Laurasian Palaearctic and their subsequent
Oligocene-Miocene diversification. \emph{Journal of Biogeography} 43:
750--762. doi: 10.1111/jbi.12678

Viruel, J., Conejero, M., Hidalgo, O., \emph{et al.} 2019. A target
capture-based method to estimate ploidy from herbarium
specimens.\emph{Frontiers in Plant Science} 10: 937. doi:
10.3389/fpls.2019.00937

Viruel J, Forest F, Paun O, \emph{et al.} 2018. A nuclear Xdh
phylogenetic analysis of yams (\emph{Dioscorea} : Dioscoreaceae)
congruent with plastid trees reveals a new Neotropical
lineage.\emph{Botanical Journal of the Linnean Society} 187: 232--246.
https://doi.org/10.1093/botlinnean/boy013

Viruel J, Le Galliot N, Pironon S, \emph{et al.} 2020. A strong
east--west Mediterranean divergence supports a new phylogeographic
history of the carob tree (\emph{Ceratonia siliqua} , Leguminosae) and
multiple domestications from native populations. \emph{Journal of
Biogeography} 47: 460--471. doi: 10.1111/jbi.13726

Viruel J, Kantar MB, Gargiulo R, \emph{et al.} 2021. Crop wild
phylorelatives (CWPs): phylogenetic distance, cytogenetic compatibility
and breeding system data enable estimation of crop wild relative gene
pool classification. \emph{Botanical Journal of the Linnean Society}195:
1--33. doi: 10.1093/botlinnean/boaa064

Volis S, Fogel K, Tu T, Sun H, Zaretsky M. 2018. Evolutionary history
and biogeography of \emph{Mandragora} L. (Solanaceae). \emph{Molecular
Phylogenetics and Evolution} 129: 85--95. doi:
10.1016/j.ympev.2018.08.015

Waltari E, Hijmans RJ, Peterson AT, \emph{et al} . 2007. Locating
Pleistocene refugia: comparing phylogeographic and ecological niche
model predictions. \emph{PLoS One} 2: e563. doi:
10.1371/journal.pone.0000563

Warren DL, Glor RE, Turelli M. 2008. Environmental niche equivalency
versus conservatism: quantitative approaches to niche
evolution.\emph{Evolution} 62: 2868--2883. doi:
10.1111/j.1558-5646.2008.00482.x

Warren DL, Glor RE, Turelli M. 2010. ENMTools: a toolbox for comparative
studies of environmental niche models. \emph{Ecography} 33: 607--611.
doi: 10.1111/j.1600-0587.2009.06142.x

Weiss CL, Pais M, Cano LM, Kamoun S, Burbano HA. 2018. nQuire: a
statistical framework for ploidy estimation using next generation
sequencing. \emph{BMC Bioinformatics} 19: 122. doi:
10.1186/s12859-018-2128-z

Wendel JF, Lisch D, Hu G, Mason AS. 2018. The long and short of doubling
down: polyploidy, epigenetics, and the temporal dynamics of genome
fractionation. \emph{Current Opinion in Genetics \& Development} 49:
1--7. doi: 10.1016/j.gde.2018.01.004

Wilkin P, Schols P, Chase MW, \emph{et al.} 2005. A plastid gene
phylogeny of the yam genus, \emph{Dioscorea} : Roots, Fruits and
Madagascar. \emph{Systematic Botany} 30: 736--749. doi:
10.1600/036364405775097879

Wood TE, Takebayashi N, Barker MS, \emph{et al} . 2009. The frequency of
polyploid speciation in vascular plants. \emph{Proceedings of the
National Academy of Sciences of the U.S.A.} 106: 13875--13879. doi:
10.1073/pnas.0811575106

Yang L, Kong H, Huang JP, Kang M. 2019. Different species or genetically
divergent populations? Integrative species delimitation of
the\emph{Primulina hochiensis} complex from isolated karst
habitats.\emph{Molecular Phylogenetics and Evolution} 132: 219--231.
10.1016/j.ympev.2018.12.011

Zhan SH, Drori M, Goldberg EE, \emph{et al} . 2016. Phylogenetic
evidence for cladogenetic polyploidization in land plants.
\emph{American Journal of Botany} 103: 1252--1258. doi:
10.3732/ajb.1600108

Zhang C, Rabiee M, Sayyari E, Mirarab S. 2018. ASTRAL-III: Polynomial
time species tree reconstruction from partially resolved gene
trees.\emph{BMC Bioinformatics} 19: 15--30. doi:
10.1186/s12859-018-2129-y

\textbf{Figure 1.} Phylogenomic trees reconstructed using the
concatenated nuclear (left) and plastid data (right) obtained using
Hyb-Seq for 76 samples of the Tamus clade of \emph{Dioscorea} . Samples
included are representative of its distribution range across the
Mediterranean region. Filled circles represent branches with support
values over 90\%, while lower values are shown on branches. Colours
represent the main clades found in the nuclear tree, see Results
(\emph{D. orientalis} violet, DC1 blue, DC2 green, DC3 red), and roman
numbers identifying the subclades in \emph{D. communis} in the plastid
tree (I, II, III; subclades within each clade were indicated with
lowercase letters a and b). Dashed lines connect the same samples in the
nuclear and plastid trees.

\textbf{Figure 2.} Admixture proportions for \emph{K} = 3 genetic groups
obtained from genetic structure analysis of nuclear data of
Mediterranean \emph{Dioscorea communis} samples through ten replicates
in STRUCTURE (see text for details). Each sample is shown by a vertical
bar partitioned according to its membership to one of the
\emph{K}clusters, represented in red, green and blue. The samples follow
the same order as in the nuclear phylogenetic tree (Figure 1) and
organized by clades (DC2 and DC3). The plastid clades (I, II and III,
Figure 1) were labelled next to the sample codes. An asterisk (*) was
used to represent the only sample of \emph{D. communis} (R32) that was
resolved as part of clade DC1 based on nuclear data, and in a different
clade with plastid data (see Figure 1).

\textbf{Figure 3.} Environmental niche models (ENM) constructed using a
current climate envelop and occurrence data for the samples of the Tamus
clade of \emph{Dioscorea} analysed in this study. ENMs were estimated
for each of the four lineages identified in the Tamus clade with
background maps adjusted to reflect their estimated potential
distributions: DC3 clade of \emph{D. communis} ; DC2 clade of \emph{D.
communis} ; the Macaronesian DC1 lineage of \emph{D. communis} ;
and\emph{D. orientalis} . A 10\% threshold cropping was applied for each
distribution range model (see Results). The legend represents the
prediction of probability of occurrence.

\textbf{Figure 4.} Principal Component Analysis (PCA) conducted with 19
bioclimatic variables for samples of the Tamus clade of
\emph{Dioscorea}across the Mediterranean region separated into the four
main lineages (see Results): Macaronesian DC1 clade of \emph{D.
communis} in light blue; \emph{D. communis} clade DC2 in green; \emph{D.
communis} DC3 clade in red; and \emph{D. orientalis} in purple.

\textbf{Figure 5.} Phylogenetic tree reconstructed using the
concatenated nuclear data obtained using Hyb-Seq for 76 samples of the
Tamus clade of \emph{Dioscorea} . Morphological, ploidy and geographic
distribution trait variation is represented with circles for each
sample. Leaf: filled circle, trilobed; empty circle, cordate; ploidy
level: filled circle, allelic ratio \textgreater{}2 (estimated as
polyploid); empty circle, allelic ratio \textless{}2 (estimated as
diploid; see Results); geographic distribution: filled circle, W and C
Mediterranean; empty circle, E Mediterranean; inflorescence: filled
circle, sessile solitary; empty circle, pedunculated fascicule.

\textbf{Figure 6.} Geographic distribution of the 76 samples of the
Tamus clade of \emph{Dioscorea} coded according to their respective
lineage in the phylogenetic tree based on concatenated nuclear data
(Figure 1), \emph{D. cretica} (DC2) in green, and subclade; and pink,
orange, and red for western European, central Mediterranean and eastern
Mediterranean clades in \emph{D. communis s.s.} (DC3), respectively.
For\emph{D. orientalis} , in purple, codes in the map indicate their
phylogenetic position named ``Leb'' (Lebanon) and ``S23''. For \emph{D.
edulis} (DC1), in light blue, codes in the map indicate their
phylogenetic position named ``Mad'' (Madeira), ``Ten'' (Tenerife) and
``GC'' (Gran Canaria). Roman numbers in the mapped dot samples represent
their respective plastid tree lineage (Figure 1). Asterisks indicate
cultivated samples in Botanic Gardens of unknown geographic origin.

\textbf{Supplementary Data}

\textbf{Table S1.} \emph{Dioscorea} samples used with the name of the
species, collector's name and collection number, date of collection and
location of the individuals. *DNA extracted from fresh material.

\textbf{Table S2.} Traits used for morphometric analyses of the Tamus
clade of \emph{Dioscorea} samples divided into macromorphological
(vegetative and reproductive) and micromorphological (pollen) variables,
with units and description of the trait measured.

\textbf{Table S3.} Table showing the results of Lilliefors test, Levene
test and the subsequent ANOVA or Kruskall-Wallis test for the selected
variables analysed in the Tamus clade of \emph{Dioscorea} samples;
asterisk indicates the level of signification (p \textless{} 0.05 = one
asterisk; p \textless{} 0.01 = two asterisks). Bonferroni post-Hoc test
showing the difference between groups, the level of significance is
shown with asterisks (p \textless{} 0.05 = one asterisk; p \textless{}
0.01 = two asterisks). For a full description of the traits and
abbreviations used, see Supplementary Data Table S2.

\textbf{Figure S1.} Dendrograms constructed from correlation values
(Pearson's correlation matrices) between bioclimatic variables for the
samples belonging to the \emph{D. communis} and \emph{D.
orientalis}clades. Three cluster dendrograms are shown corresponding to
samples identified as belonging to the different phylogenetic clades
identified (see Results). Clade DC1: \emph{D. communis} samples from
Macaronesia; DC2: samples of \emph{D. communis} from the Eastern
Mediterranean, DC3: samples of D. communis from western and central
Europe.

\textbf{Figure S2.} Results obtained in Structure Haverster based on
genetic structure analysis of \emph{D. communis} nuclear data though 10
replicates of Structure for each \emph{K} (see Materials and Methods).
The best delta \emph{K} value is highlighted in bold and represented in
a graphic below.

\textbf{Figure S3.} Genetic structure analysis of nuclear SNP data of
the studied Mediterranean \emph{D. communis} samples with STRUCTURE
imposing ancestral admixture and correlated allelic frequencies models
for \emph{K} =2 to \emph{K} =6 hypothetical genetic groups.

\textbf{Figure S4.} Divergence time estimations and chronograms of the
Tamus clade of \emph{Dioscorea} and outgroup based on nuclear sequence
data and compiled with treePL (a) and BEAST (b) showing mean node ages
in MY. BEAST maximum credibility tree (b) includes bars showing the 95\%
highest posterior density intervals for nodes.

\textbf{Figure S5.} Boxplots representing the differences between the
four main clades: DC1, DC2, DC3 (\emph{D. communis} ) and \emph{D.
orientalis} in the Tamus clade of \emph{Dioscorea} for each of the 19
analysed WorldClim current climate bioclimatic variables.

\textbf{Figure S6.} Past projections of the environment niche model
(ENMs) constructed for the four Tamus clades of \emph{Dioscorea} with a
10\% lower probability threshold cropping applied for each plot. Mid
Holocene (MH), Last Glacial Maximum (LGM) and Last Interglacial (LIG)
climate envelopes for the DC3 clade of \emph{D. communis} ; DC2 clade
of\emph{D. communis} ; for \emph{D. orientalis} clade, and for the
Macaronesian DC1 clade.

\textbf{Figure S7.} Pairwise Pearson correlation matrix calculated
between the morphological variables measured to distinguish between
Tamus clade taxa.

\textbf{Figure S8.} SEM images of the pollen grains of \emph{D.
communis} DC3 clade (A, B), \emph{D. cretica} (C, D), \emph{D.
edulis}(E, F) and \emph{D. orientalis} (G, H).

\textbf{Table 1.} Target capture efficiency for the 76 herbarium samples
of the Tamus clade of \emph{Dioscorea} and four outgroup taxa studied.
*DNA extracted from fresh material. `Paired-end reads' is the number of
reads obtained after quality trimming with Trimmomatic. `Length \%' is
the recovery length percentage calculated based on the nucleotides
retrieved per sample relative to the full length of the reference target
gene set. `Enrich. eff.' is the proportion of reads on target; `bp nDNA'
corresponds to the total number of bp retrieved for nuclear genes; `bp
pDNA' is the total number of bp retrieved for the plastome; `P(\%)' is
the percentage of polymorphic sites (P) calculated as the coefficient
between the SNPs and the total bp recovered for each sample; AR, allelic
ratio; `\%\textgreater{}2' the percentage of SNPs with an allelic ratio
\textless{}2.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllllll@{}}
\toprule
Species & Clade & Sample & Year collected & Paired-end reads & Enrich.
eff. & Assembled target genes & Genes at 50\% & Length \% & bp nDNA & bp
pDNA & SNPs & P(\%) & Mean AR & Median AR &
\%\textless{}2\tabularnewline
\emph{D. orientalis} & \emph{D. orientalis} & S23 & 1909 & 2980360 &
0.526 & 260 & 252 & 34.5 & 146415 & 146223 & 1652 & 1.13 & 2.79 & 2.96 &
18.71\tabularnewline
& & R34 & 1958 & 43612 & 0.516 & 208 & 34 & 34.1 & 144729 & 76269 & 1474
& 1.02 & 2.28 & 2.00 & 45.9\tabularnewline
& & S85 & 2008 & 1020080 & 0.448 & 260 & 134 & 50.3 & 213231 & 66768 &
835 & 0.39 & 2.71 & 2.80 & 31.9\tabularnewline
& & S84 & 1957 & 2068305 & 0.528 & 260 & 244 & 82.1 & 348456 & 149412 &
779 & 0.22 & 2.42 & 2.05 & 47.0\tabularnewline
& & S86 & 1957 & 795799 & 0.513 & 260 & 245 & 77.9 & 330582 & 110763 &
697 & 0.21 & 2.57 & 2.40 & 34.7\tabularnewline
\emph{D. communis} & Clade DC1 (Macaronesia) & S92 & 1960 & 299677 &
0.311 & 255 & 0 & 10.6 & 45171 & 34242 & 427 & 0.95 & 2.73 & 2.67 &
22.0\tabularnewline
& & S91 & 1960 & 1637342 & 0.455 & 260 & 239 & 79.2 & 335889 & 141522 &
388 & 0.12 & 2.13 & 2.01 & 41.8\tabularnewline
& & S40 & 2008 & 10339082 & 0.525 & 260 & 256 & 93.1 & 394977 & 145812 &
991 & 0.25 & 2.16 & 2.05 & 41.9\tabularnewline
& & W33 & 2003 & 1370314 & 0.429 & 260 & 253 & 91.1 & 386241 & 145767 &
1025 & 0.27 & 2.83 & 3.05 & 19.5\tabularnewline
& & S37 & 2008 & 10212202 & 0.502 & 260 & 257 & 92.6 & 392901 & 145089 &
1299 & 0.33 & 2.07 & 2.04 & 44.8\tabularnewline
& & S41 & 2008 & 16196619 & 0.492 & 260 & 256 & 92.6 & 392970 & 141618 &
1459 & 0.37 & 2.04 & 2.02 & 46.8\tabularnewline
& & R171 & 1788 & 5772951 & 0.381 & 260 & 240 & 80.4 & 341028 & 133494 &
1129 & 0.33 & 2.80 & 2.75 & 14.3\tabularnewline
& & S95 & 1975 & 663825 & 0.454 & 260 & 142 & 51.1 & 216750 & 143307 &
705 & 0.33 & 2.49 & 2.27 & 34.9\tabularnewline
& & S93 & 1867 & 2521444 & 0.460 & 260 & 230 & 72.5 & 308190 & 50046 &
1327 & 0.43 & 2.87 & 2.81 & 17.8\tabularnewline
& Clade DC2 (Trilobed leaves, Eastern Mediterranean) & R32 & 1956 &
1238692 & 0.426 & 260 & 252 & 88.7 & 376359 & 144702 & 2304 & 0.61 &
2.62 & 2.74 & 22.6\tabularnewline
& & S69 & 1905 & 782006 & 0.114 & 257 & 169 & 56.9 & 241176 & 119904 &
833 & 0.35 & 2.63 & 2.50 & 27.6\tabularnewline
& & R15 & 1979 & 2481398 & 0.248 & 260 & 254 & 90.9 & 385665 & 141975 &
1466 & 0.38 & 1.46 & 1.22 & 85.3\tabularnewline
& & R22 & 1955 & 1206365 & 0.297 & 260 & 255 & 91.1 & 386331 & 141084 &
0606 & 0.16 & 1.63 & 1.36 & 78.5\tabularnewline
& & R95 & 1951 & 1483426 & 0.382 & 260 & 251 & 89.4 & 379077 & 143058 &
803 & 0.21 & 2.45 & 2.18 & 47.4\tabularnewline
& & R14 & 1967 & 3918262 & 0.334 & 260 & 253 & 91.3 & 387393 & 141879 &
2230 & 0.58 & 1.86 & 1.62 & 67.9\tabularnewline
& & S68 & 2007 & 782006 & 0.114 & 257 & 169 & 90.8 & 385149 & 77787 &
1404 & 0.36 & 1.64 & 1.29 & 81.5\tabularnewline
& & W79 & 2010 & 1637458 & 0.485 & 260 & 254 & 91.5 & 387951 & 140592 &
1065 & 0.27 & 1.48 & 1.21 & 77.56\tabularnewline
& & R18 & 1942 & 578726 & 0.604 & 260 & 246 & 84.3 & 357756 & 59802 &
1186 & 0.33 & 3.03 & 3.28 & 15.1\tabularnewline
& & R16 & 1943 & 164691 & 0.330 & 256 & 172 & 76.6 & 324924 & 70617 &
3367 & 1.04 & 2.56 & 2.50 & 30.7\tabularnewline
& & R17 & 1943 & 281364 & 0.595 & 260 & 231 & 58.3 & 247470 & 136824 &
3805 & 1.54 & 2.95 & 3.00 & 8.86\tabularnewline
& & R20 & 2004 & 1925906 & 0.265 & 260 & 253 & 90.5 & 383769 & 141294 &
871 & 0.23 & 1.63 & 1.25 & 74.3\tabularnewline
& & S78 & 1955 & 2705587 & 0.233 & 260 & 249 & 85.4 & 362205 & 144948 &
525 & 0.14 & 1.94 & 1.34 & 61.7\tabularnewline
& & R96 & 1970 & 1514421 & 0.410 & 260 & 246 & 86.1 & 365292 & 143595 &
772 & 0.21 & 2.83 & 3.18 & 30.3\tabularnewline
& & S71 & 1963 & 3234283 & 0.220 & 260 & 251 & 87.1 & 369312 & 145470 &
808 & 0.22 & 1.94 & 1.53 & 65.6\tabularnewline
& & S70 & 1971 & 120618 & 0.245 & 243 & 34 & 29.4 & 124917 & 144003 &
259 & 0.21 & 2.59 & 2.50 & 39.0\tabularnewline
& Clade DC3 (Eastern Mediterranean) & S67 & 1962 & 1879003 & 0.587 & 260
& 247 & 83.9 & 355812 & 145620 & 1064 & 0.30 & 1.47 & 1.27 &
83.7\tabularnewline
& & R12 & 1957 & 2716763 & 0.355 & 260 & 251 & 89.6 & 380058 & 145545 &
1411 & 0.37 & 1.65 & 1.29 & 81.7\tabularnewline
& & R21 & 1937 & 1624504 & 0.226 & 260 & 250 & 86.9 & 368490 & 146073 &
472 & 0.13 & 2.25 & 2.29 & 40.5\tabularnewline
& & S72 & 2004 & 198920 & 0.266 & 256 & 161 & 55.0 & 233424 & 143145 &
878 & 0.38 & 2.67 & 2.67 & 32.9\tabularnewline
& & S21 & 2004 & 991796 & 0.301 & 260 & 181 & 61.4 & 260340 & 147450 &
641 & 0.25 & 2.64 & 2.83 & 32.8\tabularnewline
& & S36 & 1957 & 298300 & 0.537 & 259 & 160 & 54.6 & 231513 & 93522 &
512 & 0.22 & 2.44 & 2.40 & 30.7\tabularnewline
& & R11 & 1936 & 1955793 & 0.256 & 260 & 251 & 87.5 & 370965 & 146694 &
4029 & 1.09 & 2.30 & 2.27 & 41.1\tabularnewline
& & R10 & 1931 & 1859291 & 0.187 & 260 & 251 & 86.8 & 368178 & 145881 &
1079 & 0.29 & 2.90 & 2.94 & 13.7\tabularnewline
& & S22 & 1969 & 1300587 & 0.233 & 259 & 55 & 89.3 & 378783 & 112770 &
859 & 0.23 & 2.58 & 2.66 & 23.6\tabularnewline
& & R26 & 1858 & 759663 & 0.095 & 258 & 173 & 60.4 & 256383 & 147990 &
2769 & 1.08 & 2.81 & 2.80 & 19.5\tabularnewline
& & S33 & 1932 & 1895199 & 0.103 & 260 & 218 & 71.9 & 304818 & 150111 &
972 & 0.32 & 2.62 & 2.58 & 23.9\tabularnewline
& & S63 & 1999 & 1742636 & 0.226 & 260 & 252 & 89.5 & 379584 & 143289 &
1452 & 0.38 & 2.59 & 2.74 & 24.4\tabularnewline
& & S18 & 1900 & 1014934 & 0.181 & 256 & 2 & 11.2 & 47622 & 31500 & 332
& 0.70 & 2.33 & 2.00 & 38.0\tabularnewline
& & S34 & 1959 & 1441193 & 0.191 & 260 & 250 & 83.8 & 355620 & 144501 &
775 & 0.22 & 2.84 & 2.90 & 12.1\tabularnewline
& Clade DC3 (Central Mediterranean) & R23 & 1978 & 1319618 & 0.238 & 260
& 253 & 87.4 & 370599 & 143055 & 814 & 0.22 & 2.76 & 2.89 &
23.6\tabularnewline
& & R06 & 1965 & 2526479 & 0.280 & 260 & 251 & 88.0 & 373401 & 148890 &
3306 & 0.89 & 2.35 & 2.42 & 35.8\tabularnewline
& & S61 & 1990 & 1760430 & 0.090 & 259 & 233 & 73.6 & 312126 & 151014 &
812 & 0.26 & 2.32 & 2.19 & 36.1\tabularnewline
& & R08 & 1931 & 1169102 & 0.192 & 260 & 242 & 79.4 & 337011 & 146019 &
2148 & 0.64 & 3.03 & 3.17 & 9.73\tabularnewline
& & R24 & 1930 & 895451 & 0.121 & 259 & 214 & 71.1 & 301512 & 144822 &
1340 & 0.44 & 2.77 & 2.75 & 20.7\tabularnewline
& & S79 & 2016 & 3929348 & 0.283 & 260 & 256 & 92.5 & 392268 & 151239 &
3010 & 0.77 & 2.48 & 2.67 & 27.3\tabularnewline
& & R30 & 2016 & 1677314 & 0.256 & 260 & 253 & 91.3 & 387120 & 144900 &
2464 & 0.64 & 2.59 & 2.73 & 24.8\tabularnewline
& & R28 & 2016 & 1999986 & 0.342 & 260 & 253 & 91.3 & 387408 & 142863 &
3335 & 0.86 & 2.51 & 2.67 & 26.7\tabularnewline
& & S28 & 1967 & 2114903 & 0.288 & 260 & 250 & 85.6 & 363141 & 148317 &
1107 & 0.30 & 2.66 & 2.74 & 25.6\tabularnewline
& & R07 & 1847 & 1912677 & 0.136 & 260 & 244 & 79.9 & 339057 & 104967 &
1365 & 0.40 & 2.81 & 3.00 & 21.5\tabularnewline
& & R100 & 1906 & 1536342 & 0.114 & 260 & 202 & 66.0 & 279993 & 146673 &
903 & 0.32 & 2.51 & 2.43 & 30.6\tabularnewline
& & S35 & 1929 & 1846641 & 0.125 & 260 & 226 & 73.7 & 312624 & 148557 &
902 & 0.29 & 2.57 & 2.67 & 33.0\tabularnewline
& & S20 & 1929 & 776828 & 0.172 & 260 & 9 & 21.1 & 89439 & 144906 & 520
& 0.58 & 2.37 & 2.00 & 36.9\tabularnewline
& Clade DC3 (Western European) & P06* & 2018 & 793772 & 0.576 & 260 &
253 & 91.6 & 388401 & 142761 & 3371 & 0.87 & 2.51 & 2.72 &
25.8\tabularnewline
& & R84* & 2018 & 1525356 & 0.226 & 260 & 240 & 86.2 & 365586 & 148377 &
5476 & 1.50 & 2.89 & 2.90 & 12.9\tabularnewline
& & R76 & 2004 & 1653906 & 0.448 & 260 & 239 & 86.3 & 365946 & 144525 &
2086 & 0.57 & 3.15 & 3.38 & 13.8\tabularnewline
& & R27 & 2017 & 2060161 & 0.340 & 260 & 253 & 91.7 & 388935 & 143238 &
2810 & 0.72 & 2.46 & 2.66 & 33.0\tabularnewline
& & S42 & 2017 & 1432342 & 0.336 & 260 & 254 & 92.3 & 391608 & 143334 &
2576 & 0.66 & 2.48 & 2.57 & 28.7\tabularnewline
& & R04 & 1956 & 2921528 & 0.304 & 260 & 253 & 89.1 & 377823 & 149163 &
2165 & 0.57 & 2.60 & 2.73 & 23.2\tabularnewline
& & R05 & 1948 & 2418558 & 0.322 & 260 & 251 & 87.4 & 370722 & 144150 &
3443 & 0.93 & 2.22 & 2.19 & 44.2\tabularnewline
& & S64 & 2015 & 4389479 & 0.307 & 260 & 255 & 92.3 & 391437 & 142944 &
2165 & 0.55 & 2.34 & 2.54 & 37.7\tabularnewline
& & R129 & 1932 & 1893572 & 0.262 & 260 & 243 & 78.7 & 333720 & 146958 &
1221 & 0.37 & 2.43 & 2.13 & 42.6\tabularnewline
& & S54 & 1976 & 1744816 & 0.164 & 260 & 214 & 68.7 & 291213 & 112173 &
662 & 0.23 & 2.50 & 2.40 & 33.8\tabularnewline
& & S55 & 1980 & 124177 & 0.576 & 254 & 26 & 28.6 & 121518 & 30666 & 294
& 0.24 & 2.37 & 2.00 & 42.2\tabularnewline
& & S53 & 1971 & 1811439 & 0.130 & 260 & 221 & 72.3 & 306672 & 145788 &
714 & 0.23 & 2.38 & 2.30 & 33.5\tabularnewline
& & R25 & 1934 & 1189706 & 0.151 & 260 & 251 & 83.5 & 354219 & 141843 &
758 & 0.21 & 2.49 & 2.46 & 31.5\tabularnewline
& & S76 & 1972 & 1882846 & 0.589 & 260 & 247 & 85.2 & 361521 & 145740 &
1424 & 0.39 & 2.72 & 2.82 & 16.2\tabularnewline
& & S62 & 2003 & 2401716 & 0.200 & 260 & 249 & 88.4 & 375072 & 150357 &
1452 & 0.39 & 2.24 & 2.18 & 43.6\tabularnewline
& & R29 & 2016 & 2093906 & 0.341 & 260 & 252 & 91.5 & 388347 & 142563 &
2415 & 0.62 & 2.57 & 2.75 & 24.14\tabularnewline
& & R01 & 2016 & 5236808 & 0.415 & 260 & 255 & 92.0 & 390150 & 142731 &
3043 & 0.78 & 2.41 & 2.68 & 32.3\tabularnewline
& & R31 & 2016 & 8920132 & 0.566 & 260 & 255 & 92.1 & 390780 & 143364 &
2972 & 0.76 & 2.41 & 2.72 & 31.1\tabularnewline
& & S25 & 2016 & 3546012 & 0.358 & 260 & 252 & 90.6 & 384462 & 147363 &
1004 & 0.26 & 2.43 & 2.55 & 42.5\tabularnewline
& & S80 & 2016 & 1341416 & 0.231 & 260 & 252 & 88.6 & 375798 & 142617 &
764 & 0.20 & 2.46 & 2.50 & 34.0\tabularnewline
\emph{D. elephantipes} & Outgroup & W32 & 2001 & 1020073 & 0.467 & 260 &
254 & 95.2 & 403968 & 152598 & & & & &\tabularnewline
\emph{D. sylvatica} & Outgroup & S49* & 2018 & 2658330 & 0.541 & 260 &
255 & 94.4 & 400443 & 150822 & & & & &\tabularnewline
\emph{D. chouardii} & Borderea & D66 & 2003 & 5285968 & 0.355 & 260 &
257 & 92.5 & 392553 & 146505 & & & & &\tabularnewline
\emph{D. pyrenaica} & Borderea & W34 & 1956 & 1648853 & 0.298 & 260 &
237 & 76.8 & 325572 & 146466 & & & & &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2}. Morphological traits and variability in the four
morphological groups identified in the Tamus clade of \emph{Dioscorea},
one corresponding to \emph{D. orientalis}, and three to the currently
recognized \emph{D. communis} \emph{s.l}.: Macaronesian clade DC1 (=
\emph{D. edulis}), trilobed leaf Eastern Mediterranean clade (DC2) (=
\emph{D. cretica}) and the DC3 cordate leaf clade of \emph{D. communis}
(= \emph{D. communis} \emph{s.s}.) (see Figure 1).\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
& & \emph{D. communis} \textbf{DC3} & \emph{D. communis} \textbf{DC2} &
\emph{D. communis} \textbf{DC1} & D. orientalis\tabularnewline
\textbf{Leaf} & \textbf{Petiole (cm)} & 0.24--10.1 & 1.7--8.8 &
1.6--3.88 & 0.6--1.6\tabularnewline
& \textbf{Shape} & Cordate & Trilobed & Cordate to sagittate &
Cordate\tabularnewline
& \selectlanguage{ngerman}\textbf{Size (mm) length × width} & Up to 180 \selectlanguage{ngerman}\textbf{×} 150 & Up to
70 \selectlanguage{ngerman}\textbf{×} 70 & Up to 125 \selectlanguage{ngerman}\textbf{×} 110 & Up to 56 \selectlanguage{ngerman}\textbf{×}
48\tabularnewline
& \textbf{Margin} & Thickened, not undulate & Thickened, not undulate &
Thickened, slightly undulate & Thickened and undulate\tabularnewline
& \textbf{Consistency} & Chartaceous & Chartaceous & Thinly chartaceous
& Thinly coriaceous\tabularnewline
& \textbf{Secondary veins} & Reticulated, usually not evident &
Reticulated, usually not evident & Dark, visible but not prominent &
Concolorous and prominent\tabularnewline
\textbf{Male inflorescence} & \textbf{Disposition} & In a fascicle, up
to four flowers & In a fascicle, up to four flowers & In a fascicle, up
to four flowers & Solitary\tabularnewline
& \textbf{Branching} & Compound & Compound & Compound &
Simple\tabularnewline
& \textbf{Size (cm)} & Up to 35 & Up to 25 & Up to 12 &
6.7\tabularnewline
& \textbf{Number of flowers} & 3--4 per fascicle, up to 73 per
inflorescence & 1--4 per fascicle, up to 50 per inflorescence & 3--4 per
fascicle, 65 per inflorescence & 1--23\tabularnewline
& \textbf{Pedicel (mm)} & 0.5--5.4 & 1.04--5.17 & 1.4--5.4 & Sessile or
subsessile\tabularnewline
\textbf{Female inflorescence} & \textbf{Number of flowers} & 1--14 per
inflorescence & 1--6 per inflorescence & 1--8 per inflorescence &
1--4\tabularnewline
& \textbf{Pedicel (mm)} & 4.7--11.3 & 2.5--9.3 & 3.8--7.3 & Sessile or
subsessile\tabularnewline
\textbf{Fruit} & \textbf{Size (mm)} & 7--11, 6--10 & 7--11, 6--10 &
6--12, 1--8 & 5.5--11, 1--4\tabularnewline
& \textbf{Shape} & Globose to ellipsoid & Globose to ellipsoid & Globose
to ellipsoid & Ellipsoid\tabularnewline
\textbf{Seed} & \textbf{Number} & 1--6 & 1--6 & 1--6 &
1--6\tabularnewline
& \textbf{Color} & Dark brown & Dark brown & Dark brown & Dark
brown\tabularnewline
\textbf{Pollen} & \textbf{Apertures} & 2 & 2 & 2 & 2\tabularnewline
& \selectlanguage{greek}\textbf{Longest axis (µm)}\selectlanguage{english} & 31--36 & 31--36 & 28--36 &
35--40\tabularnewline
& \selectlanguage{greek}\textbf{Shorter axis (µm)}\selectlanguage{english} & 27--30 & 26--29 & 14--18 &
27--30\tabularnewline
& \selectlanguage{greek}\textbf{Perforation size (µm)}\selectlanguage{english} & 0.6--1.2 & 0.6--1.2 & 0.18--0.37 &
0.31--0.85\tabularnewline
& \selectlanguage{greek}\textbf{Perforations/µm\textsuperscript{2}}\selectlanguage{english} & 0.5 & 0.5 & 3.9 &
1.9\tabularnewline
& \textbf{Spines} & No & No & No & Yes\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{english}
\FloatBarrier
\end{document}

