Fig 1.
Sampling localities of Hyalesthes obsoletus populations and associated host-plants.
Numbers refer to localities listed in Table 1. The number’s color refers to the H. obsoletus 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 H. obsoletus populations collected on crop plants in Romania and Russia (Radovanu and Mayak) [7] are designated with black outlined circles. Reprinted from d-maps http://d-maps.com/carte.php?num_car=2068&lang=en and http://d-maps.com/carte.php?num_car=2232&lang=en under a CC BY license, with permission from Daniel Dalet, original copyright 2007–2018.
Table 1.
Sampled locality data and summarized per population genetic diversity of Hyalesthes obsoletus sorted by corresponding host-plant and country of origin.
Fig 2.
Schematic representation of COI-tRNA(Leu)-COII and 16S-tRNA(Leu)-ND1 mtDNA gene regions showing the binding site positions of the primers used for amplification of freshly collected Hyalesthes obsoletus specimens and dry museum H. thracicus specimen.
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 H. thracicus paratype specimen. Scheme not drawn to scale. Primer sequences are given in S2 Table.
Fig 3.
Mitochondrial diversity indices calculated 316 Hyalesthes obsoletus genotyped specimens of the three host-associated metapopulations.
Convolvulus arvensis and Urtica dioica (177 specimens), Vitex agnus-castus (56 specimens) and Crepis foetida (83 specimens). Indices are designated in colors: black = number of alleles (θk), red = expected homozygosity (θH), blue = segregating sites (θS) and green = pairwise differences (θπ).
Table 2.
Comparison of microsatellite-based mean genetic diversity (AR—allelic richness and HE—expected heterozygosity) among four host-plant associated Hyalesthes obsoletus metapopulations.
Fig 4.
Bayesian phylogenetic tree inferred from 1180 bp of the COI-tRNA(Leu)-COII and 16S-tRNA(Leu)-ND1 concatenated mitochondrial gene regions sampled from the Hyalesthes obsoletus host-associated populations in this and previous studies.
The Bayesian posterior probabilities are noted above branches, and the maximum parsimony and neighbor-joining bootstrap support values appear below the branches in that order. Bootstrap values below 70% and posterior probabilities below 0.95 are omitted. The nonrecovered nodes are marked with an em dash "–". The main nodes are designated with colored circles corresponding to host-plant associated haplogroups as follows: green = Convolvulus arvensis and Urtica dioica, blue = Vitex agnus-castus, and red = Crepis foetida. Average within-group genetic distances for each of the three host-plant clusters are noted left of the corresponding colored circle, while between-group distances are given on the right. The range of within- and between-group distances is given in parentheses. Distances are calculated with correction by applying the HKY+I nucleotide substitution model (Hasegawa-Kishino-Yano, pinvar = 0.881) among three genotype groups for all 51 haplotypes according to the three major host-associated clusters; therefore, the Israeli GE and HE haplotypes are considered as Crepis foetida-cluster members, while the QB, ψC and PC collected on crop plant are members of the Convolvulus-Urtica group. Distances between each H. obsoletus host-associated haplogroup and H. thracicus used as the outgroup are presented in the figure’s bottom right corner. Haplotypes detected in previous studies [8, 11, 15, 37] are marked with an asterisk (*).
Fig 5.
The phylogenetic haplotype network obtained using median-joining and statistical parsimony algorithms on concatenated COI-tRNA(Leu)-COII and 16S-tRNA(Leu)-ND1 mitochondrial gene regions of the 51 Hyalesthes obsoletus haplotypes identified in this and previous studies.
Haplotype colors correspond to the host-plant associations. Haplotypes detected in previous studies [8, 11, 15, 37] are marked with an asterisk (*). The most common haplotypes within each haplogroup are noted with enlarged circles. Dashed lines represent alternative variants of network formations obtained using both algorithms. Black dot vertices represent missing or unsampled haplotypes. Distribution maps are given above each host-associated H. obsoletus haplogroup. Each detected haplogroup’s country is designated on maps in the color corresponding to the associated host-plant. Because Convolvulus arvensis and Urtica dioica share a number of H. obsoletus haplotypes, BB-AB and EC haplogroup distribution is designated in two shades. Haplotypes detected in the two previously reported H. obsoletus populations collected on crop plants in Romania and Russia (Radovanu and Mayak) [7] are not colored.
Fig 6.
Plots representing parameters of mtDNA genetic differentiation between Hyalesthes obsoletus host-associated populations based on (A) pairwise FST values and (B) Nei’s average number of pairwise differences.
Associated host-plants: Convolvulus arvensis (Ca), Urtica dioica (Ud), Vitex agnus-castus (Vac), and Crepis foetida (Cf). Populations are listed in the same order as in Table 1. The three populations marked with arrows show genetic differentiation compared to all others in the same host-associated group (Ca-Ud). The blue elements below the diagonal of FST values range from 0 to 1, with 0 (including < 0) indicating no divergence between the populations and 1 indicating that two populations are completely separated. The green elements above the diagonal denote Nei’s average number of pairwise differences among populations, the orange diagonal elements denote Nei’s average number of pairwise differences within populations, and blue elements below the diagonal denote the net number of nucleotide differences among populations (Nei’s distance).
Fig 7.
Bar plots of the Bayesian clustering analysis performed using Structure software on microsatellite data.
(A) 702 Hyalesthes obsoletus individuals from the 50 populations genotyped in this study and a single 20-member population associated with Vitex agnus-castus from Israel [7], suggesting a ΔK = 3 as the most likely number of genetic clusters; (B) 132 H. obsoletus individuals from the 8 populations associated with Vitex agnus-castus in Montenegro and Greece genotyped in this study and the aforementioned Israeli population [7], suggesting a ΔK = 2 as the most likely number of clusters; and (C) the ten syntopic localities of the H. obsoletus 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 = Convolvulus arvensis and Urtica dioica, blue = Vitex agnus-castus, and red = Crepis foetida). Population membership assignments to the suggested clusters are designated above bar plots (A) and (B).
Fig 8.
Maximum-likelihood phenogram based on allele frequencies calculated with four microsatellite loci for the 51 populations of H. obsoletus (722 individuals).
Individuals were associated with Convolvulus arvensis and Urtica dioica (green branches), Vitex agnus-castus (blue branches) and Crepis foetida (red branches) from Southeastern Europe and Turkey analyzed in this study and for the Israeli population associated with V. agnus-castus [7]. Hyalesthes luteipes was used as an outgroup to root the tree. The two most divergent populations of the Crepis foetida-associated genotype group from east Turkey (Cf-TR) and the Vitex agnus-castus-associated genotype group from Israel (V-IL) are designated.