R.antirrhiniCOII-AlignmentNexus.txt

Nexus file of COII sequence

Sampled locality data and summarized per population genetic diversity of <i>Hyalesthes obsoletus</i> sorted by corresponding host-plant and country of origin.

<p>Sampled locality data and summarized per population genetic diversity of <i>Hyalesthes obsoletus</i> sorted by corresponding host-plant and country of origin.</p

Comparison of microsatellite-based mean genetic diversity (<i>A</i>_<i>R</i>—allelic richness and <i>H</i>_<i>E</i>—expected heterozygosity) among four host-plant associated <i>Hyalesthes obsoletus</i> metapopulations.

<p>Comparison of microsatellite-based mean genetic diversity (<i>A</i>_<i>R</i>—allelic richness and <i>H</i>_<i>E</i>—expected heterozygosity) among four host-plant associated <i>Hyalesthes obsoletus</i> metapopulations.</p

R.antirrhiniEF1-AlignmentNexus.txt

Nexus file of Elongation Factor sequence

Bar plots of the Bayesian clustering analysis performed using Structure software on microsatellite data.

<p>(A) 702 <i>Hyalesthes obsoletus</i> individuals from the 50 populations genotyped in this study and a single 20-member population associated with <i>Vitex agnus-castus</i> from Israel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196969#pone.0196969.ref007" target="_blank">7</a>], suggesting a ΔK = 3 as the most likely number of genetic clusters; (B) 132 <i>H</i>. <i>obsoletus</i> individuals from the 8 populations associated with <i>Vitex agnus-castus</i> in Montenegro and Greece genotyped in this study and the aforementioned Israeli population [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196969#pone.0196969.ref007" target="_blank">7</a>], suggesting a ΔK = 2 as the most likely number of clusters; and (C) the ten syntopic localities of the <i>H</i>. <i>obsoletus</i> populations associated with two host-plants. Each column on the plots represents a single individual and the vertical black lines divide individuals by population. Colors represent proportional membership in each genetic cluster (green = <i>Convolvulus arvensis</i> and <i>Urtica dioica</i>, blue = <i>Vitex agnus-castus</i>, and red = <i>Crepis foetida</i>). Population membership assignments to the suggested clusters are designated above bar plots (A) and (B).</p

AccessionsList-R.antirrhini.xlsx

the list of samples analyses in the paper with relevant accession code

Widespread plant specialization in the polyphagous planthopper <i>Hyalesthes obsoletus</i> (Cixiidae), a major vector of stolbur phytoplasma: Evidence of cryptic speciation

<div><p>The stolbur phytoplasma vector <i>Hyalesthes obsoletus</i> is generally considered as a polyphagous species associated with numerous wild and cultivated plants. However, recent research in southeastern Europe, the distribution centre of <i>H</i>. <i>obsoletus</i> and the area of most stolbur-inflicted crop diseases, points toward specific host-plant associations of the vector, indicating specific vector-based transmission routes. Here, we study the specificity of populations associated with four host-plants using mitochondrial and nuclear genetic markers, and we evaluate the evolution of host-shifts in <i>H</i>. <i>obsoletus</i>. Host-plant use was confirmed for <i>Convolvulus arvensis</i>, <i>Urtica dioica</i>, <i>Vitex agnus-castus</i> and <i>Crepis foetida</i>. Mitochondrial genetic analysis showed sympatric occurrence of three phylogenetic lineages that were ecologically delineated by host-plant preference, but were morphologically inseparable. Nuclear data supported the existence of three genetic groups (Evanno’s ΔK(3) = 803.72) with average genetic membership probabilities > 90%. While populations associated with <i>C</i>. <i>arvensis</i> and <i>U</i>. <i>dioica</i> form a homogenous group, populations affiliated with <i>V</i>. <i>agnus-castus</i> and <i>C</i>. <i>foetida</i> constitute two independent plant-associated lineages. The geographical signal permeating the surveyed populations indicated complex diversification processes associated with host-plant selection and likely derived from post-glacial refugia in the eastern Mediterranean. This study provides evidence for cryptic species diversification within <i>H</i>. <i>obsoletus sensu lato</i>: i) consistent mitochondrial differentiation (1.1–1.5%) among host-associated populations in syntopy and in geographically distant areas, ii) nuclear genetic variance supporting mitochondrial data, and iii) average mitochondrial genetic distances among host-associated meta-populations are comparable to the most closely related, morphologically distinguishable species, i.e., <i>Hyalesthes thracicus</i> (2.1–3.3%).</p></div

Maximum-likelihood phenogram based on allele frequencies calculated with four microsatellite loci for the 51 populations of <i>H</i>. <i>obsoletus</i> (722 individuals).

<p>Individuals were associated with <i>Convolvulus arvensis</i> and <i>Urtica dioica</i> (green branches), <i>Vitex agnus-castus</i> (blue branches) and <i>Crepis foetida</i> (red branches) from Southeastern Europe and Turkey analyzed in this study and for the Israeli population associated with <i>V</i>. <i>agnus-castus</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196969#pone.0196969.ref007" target="_blank">7</a>]. <i>Hyalesthes luteipes</i> was used as an outgroup to root the tree. The two most divergent populations of the <i>Crepis foetida</i>-associated genotype group from east Turkey (Cf-TR) and the <i>Vitex agnus-castus</i>-associated genotype group from Israel (V-IL) are designated.</p

Sampling localities of <i>Hyalesthes obsoletus</i> populations and associated host-plants.

<p>Numbers refer to localities listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196969#pone.0196969.t001" target="_blank">Table 1</a>. The number’s color refers to the <i>H</i>. <i>obsoletus</i> population host-plant association as given on the map. Syntopic localities are designated with the same number in two host-plant corresponding colors. The sampling localities of two previously reported <i>H</i>. <i>obsoletus</i> populations collected on crop plants in Romania and Russia (Radovanu and Mayak) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196969#pone.0196969.ref007" target="_blank">7</a>] are designated with black outlined circles. Reprinted from d-maps <a target="_blank">http://d-maps.com/carte.php?num_car=2068&lang=en</a> and <a target="_blank">http://d-maps.com/carte.php?num_car=2232&lang=en</a> under a CC BY license, with permission from Daniel Dalet, original copyright 2007–2018.</p

Schematic representation of <i>COI</i>-<i>tRNA(Leu)</i>-<i>COII</i> and <i>16S</i>-<i>tRNA(Leu)</i>-<i>ND1</i> mtDNA gene regions showing the binding site positions of the primers used for amplification of freshly collected <i>Hyalesthes obsoletus</i> specimens and dry museum <i>H</i>. <i>thracicus</i> specimen.

<p>The amplicon length (bp) for each primer pair combination is given below the scheme (length excluding primers is given in parentheses). Primers marked with the symbol "§" were designed in this study and used for the amplification of short DNA fragments of the <i>H</i>. <i>thracicus</i> paratype specimen. Scheme not drawn to scale. Primer sequences are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196969#pone.0196969.s004" target="_blank">S2 Table</a>.</p