From Western Asia to the Mediterranean Basin: Diversification of the Widespread Euphorbia nicaeensis Alliance (Euphorbiaceae)

Abstract

The Mediterranean Basin is an important biodiversity hotspot and one of the richest areas in the world in terms of plant diversity. Its flora parallels in several aspects that of the Eurasian steppes and the adjacent Irano-Turanian floristic region. The Euphorbia nicaeensis alliance spans this immense area from the western Mediterranean to Central Asia. Using an array of complementary methods, ranging from phylogenomic and phylogenetic data through relative genome size (RGS) estimation to morphometry, we explored relationships and biogeographic connections among taxa of this group. We identified the main evolutionary lineages, which mostly correspond to described taxa. However, despite the use of highly resolving Restriction Site Associated DNA (RAD) sequencing data, relationships among the main lineages remain ambiguous. This is likely due to hybridisation, lineage sorting triggered by rapid range expansion, and polyploidisation. The phylogenomic data identified cryptic diversity in the Mediterranean, which is also correlated with RGS and, partly, also, morphological divergence, rendering the description of a new species necessary. Biogeographic analyses suggest that Western Asia is the source area for the colonisation of the Mediterranean by this plant group and highlight the important contribution of the Irano-Turanian region to the high diversity in the Mediterranean Basin. The diversification of the E. nicaeensis alliance in the Mediterranean was triggered by vicariance in isolated Pleistocene refugia, morphological adaptation to divergent ecological conditions, and, to a lesser extent, by polyploidisation.

Keywords: Eurasian steppes; Irano-Turanian region; Mediterranean Basin; RAD sequencing; morphometry; phylogeny; polyploidy; taxonomy.

FIGURE 1

Distribution of Euphorbia nicaeensis alliance populations sampled and used in this study. Black symbols indicate populations used in genetic (RADseq and/or ITS) and, mostly, also, in relative genome size (RGS) and morphometric analyses. Grey symbols indicate additional populations used in RGS and/or morphometric analyses; white symbols indicate populations used only in morphometric analyses. The corresponding population numbers are presented in Supplementary Figure 1, and details are given in Supplementary Table 1. Euphorbia adriatica and E. japygica have been previously included in E. nicaeensis but are, based on our results, independent species.

FIGURE 2

(A) Geographic areas and geographic provenance of the investigated populations. (B) The time-calibrated species tree inferred with SNAPP and used in the biogeographic analysis with BioGeoBEARS. Numbers above branches are posterior probabilities > 0.80, and the horizontal bars correspond to 95% highest posterior densities (HPD) for the age estimates. Pie charts at each node show the marginal probabilities of alternative ancestral ranges obtained from the BioGeoBEARS analysis under the DEC + J model. In addition, smaller pie charts resulting from the DEC model are presented in cases where the reconstruction between the models differed. Colours in pie charts correspond to the geographic areas in A. The trees, including outgroup taxa, are presented in Supplementary Figure 3. Population numbers correspond to Supplementary Table 1.

FIGURE 3

(A) Phylogenetic relationships within the Euphorbia nicaeensis alliance and between this alliance and its closest relatives within E. sect. Pithyusa, as inferred by maximum likelihood analysis of RADseq loci, with indicated bootstrap values above 50%. (B) Geographic provenance of the investigated populations with colour coding as in (A). Two outgroup species, E. seguieriana (from Austria; C) and E. triflora (from Slovenia; D) from the E. seguieriana and the E. barrelieri groups, and the ingroup E. petrophila (from Turkey; E) are shown in situ. Photos: B. Frajman (C,D), C. Gilly (E). Population numbers correspond to Supplementary Table 1. The tree, including outgroup taxa, is presented in Supplementary Figure 2.

FIGURE 4

Phylogenetic relationships within the Euphorbia nicaeensis alliance based on RADseq data. (A) The time-calibrated species tree inferred with SNAPP. Numbers above branches are posterior probabilities, and the horizontal bars correspond to 95% highest posterior densities (HPD) of the age estimates. (B) Alternative topologies visualised with DensiTree and represented by different colours. (C) Division of all populations into two groups (blue and red) with Bayesian clustering using fastSTRUCTURE. (D) Euphorbia nicaeensis in its natural habitat northwest of Carcassonne in France (Photo: B. Frajman). Population numbering corresponds to Supplementary Table 1.

FIGURE 5

Phylogenetic relationships within the Euphorbia nicaeensis alliance and between this alliance and its closest relatives within E. sect. Pithyusa as inferred by Internal Transcribed Spacer (ITS) sequences. (A) Bayesian consensus phylogram with posterior probabilities > 0.50 above, and parsimony bootstrap values > 50% below branches; country codes follow the accession names. (B) NeighborNet and (C) geographic position of the ITS ribotype groups revealed by the NeighborNet and indicated by different colours. Population numbers in (A,B) are presented in Supplementary Figure 1 and in Supplementary Table 1. Population numbers of populations that are based on our revised taxonomic treatment belong to E. adriatica and are in (B) in bold italics (45, 47, 59, 63), and the one corresponding to E. japygica (58) is in bold. (D) Euphorbia adriatica from Italy and (E) E. macroclada from Turkey in their natural habitats. Photos: B. Frajman (D), C. Gilly (E).

FIGURE 6

Relative genome size (RGS) variation in the Euphorbia nicaeensis alliance. Outliers putatively belonging to the same ploidy level as the majority of the samples are presented as dots, whereas putatively ploidy-divergent outliers are presented as lines, including their population numbers, which correspond to Supplementary Figure 1 and Supplementary Table 1.

FIGURE 7

Morphological differentiation amongst Euphorbia glareosa s.l. (blue), E. hercegovina (red), E. macroclada (green), and E. nicaeensis s.l. (yellow). Principal component analysis (PCA; A) and discriminant analyses (DA) based on (B) 21 metric and nine ratio vegetative and cyathium characters, (C) four metric and two ratio fruit characters, and (D) six metric and four ratio seed characters.

FIGURE 8

Morphological differentiation amongst Euphorbia adriatica (yellow), E. hercegovina (red), and E. nicaeensis (green). Principal component analysis (PCA; A) and discriminant analyses (DA) based on (B) 21 metric and nine ratio vegetative and cyathium characters, (C) five metric and two ratio fruit characters, and (D) six metric and four ratio seed characters.

FIGURE 9

Relations amongst taxa within the E. nicaeensis alliance as a result of the outcomes of this study, combined with previous treatments within E. glareosa s.l., which were not addressed in this study.