Symptomatology, (Co)occurrence and Differential Diagnostic PCR Identification of 'Ca. Phytoplasma solani' and 'Ca. Phytoplasma convolvuli' in Field Bindweed.

Field bindweed (Convolvulus arvensis) is one of the major natural plant hosts and reservoirs of 'Candidatus Phytoplasma solani' ('Ca. P. solani'), the causal agent of plant diseases in diverse agricultural crops, including Bois noir (BN) disease of grapevine. Phylogenetically, the most closely related phytoplasma to 'Ca. P. solani', the 'Ca. P. convolvuli', induces disease in field bindweed that is known by its symptoms as bindweed yellows (BY). The occurrence, coinfection and symptoms association of the two phytoplasmas in shared host plants were the subject of this study. Specific primers for the amplification of the elongation factor Tu gene (tuf) were developed for the identification of 'Ca. P. convolvuli' (by conventional nested PCR), as well as primers for simultaneous detection of 'Ca. P. solani' and 'Ca. P. convolvuli' by duplex SYBR Green real-time PCR. Among symptomatic bindweed plants, 25 and 41% were infected with a single phytoplasma species, 'Ca. P. solani' and 'Ca. P. convolvuli', respectively, while 34% were infected with both phytoplasmas. None of the non-symptomatic control plants carried phytoplasma, while non-symptomatic plants from our previous epidemiological studies in BN-affected vineyards were confirmed to be infected solely with 'Ca. P. solani'. Stamp gene typing revealed Rqg50 and Rqg31 'Ca. P. solani' genotypes in plants coinfected with 'Ca. P. convolvuli', while three diverse genotypes (Rqg50, GGY and Rpm35) were identified in a single locality with symptomatic bindweeds infected solely with 'Ca. P. solani'. Variations in symptoms and their association with each of the phytoplasmas are described and documented. The symptom of bushy appearance could be single out as specific for 'Ca. P. convolvuli' infection, while occurrence of 'Ca. P. solani' could not be unequivocally associated with specific alterations in infected bindweeds. The results are discussed in the context of the epidemiological and ecological complexity of 'Ca. P. solani'-induced diseases and the relationship between the two phytoplasma relatives in shared host plant.

Phylogeny of the Subtribe Monoctonina (Hymenoptera, Braconidae, Aphidiinae).

Members of the Monoctonina subtribe have long been neglected in applied studies of the subfamily Aphidiinae, due to their low economic importance, as they do not parasitize pests of cultivated plants. Consequently, data about this group are scarce, including its taxonomy and phylogeny. In the present study, we explore inter- and intraspecific genetic variation of Monoctonina species, including genera Monoctonus Haliday 1833, Monoctonia Starý 1962, Falciconus Mackauer 1959 and Harkeria Cameron 1900. We employ two molecular markers, the barcode region of the mitochondrial cytochrome c oxidase subunit I (COI) and the D2 region of the 28S nuclear gene (28S rDNA), to analyze genetic structuring and phylogeny of all available Monoctonina species, and combine them with morphological data for an integrative approach. We report one new species, and three potentially new species which can be formally described when further specimens are available. Analysis of phylogenetic relationships within the subtribe shows a basal position for the genera Falciconus and Monoctonia, and the close relatedness of Harkeria and Monoctonus.

Phylogenetic relationships and subgeneric classification of European Ephedrus species (Hymenoptera, Braconidae, Aphidiinae).

In this study two molecular markers were used to establish taxonomic status and phylogenetic relationships of Ephedrus subgenera and species distributed in Europe. Fifteen of the nineteen currently known species have been analysed, representing three subgenera: Breviephedrus Gärdenfors, 1986, Lysephedrus Starý, 1958 and Ephedrus Haliday, 1833. The results of analysis of COI and EF1α molecular markers and morphological studies did not support this classification. Three clades separated by the highest genetic distances reported for the subfamily Aphidiinae on intrageneric level. Ephedrus brevis is separated from persicae and plagiator species groups with genetic distances of 19.6 % and 16.3 % respectively, while the distance between persicae and plagiator groups was 20.7 %. These results lead to the conclusion that the traditional subgeneric classification of Ephedrus needs revision. Species from persicae species group are raised to subgenus level as Fovephedrus Chen, 1986 and Lysephedrus syn. nov. is assigned as a junior synonym of subgenus Ephedrus. Key for identification of Ephedrus subgenera is provided. Ephedrus hyadaphidis Kocic & Tomanovic sp. nov. is described and several species are confirmed as valid species for the first time. Furthermore, two species are synonymised: E. dysaphidis syn. nov. as a junior synonym of E. cerasicola and E. blattnyi syn. nov. as a junior synonym of E. plagiator.

Integrative taxonomy of root aphid parasitoids from the genus Paralipsis (Hymenoptera, Braconidae, Aphidiinae) with description of new species.

Species from the genus Paralipsis are obligatory endoparasitoids of root aphids in the Palaearctic. It is known that these species are broadly distributed, parasitizing various aphid hosts and showing great biological and ecological diversity. On the other hand, this group of endoparasitoids is understudied and was thought to be represented by a single species in Europe, viz., Paralipsisenervis (Nees). However, recent description of two new species indicated the possibility of cryptic speciation and recognition of additional Paralipsis species in Europe. In this research, Paralipsis specimens collected during the last 60 years from eight European countries, as well as one sample from Morocco, were subjected to molecular and morphological characterization. Newly designed genus-specific degenerative primers successfully targeted short overlapping fragments of COI of the mitochondrial DNA. Molecular analyses showed clear separation of four independent lineages, two of which are the known species P.enervis and P.tibiator, while two new species are described here, viz., P.brachycaudi Tomanovic & Starý, sp. n. and P.rugosa Tomanovic & Starý, sp. n. No clear specialization of the taxa to a strict root aphid host has been determined. The recognized mitochondrial lineages were distinct one from another, but with a substantial within-lineage divergence rate, clearly indicating the complexity of this group of parasitoids, on which further research is required in order to clarify the factors triggering their genetic differentiation. We reviewed literature data and new records of Paralipsisenervis aphid host associations and distributions. A key for the identification of all known Paralipsis species is provided and illustrated.

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

The stolbur phytoplasma vector Hyalesthes obsoletus is generally considered as a polyphagous species associated with numerous wild and cultivated plants. However, recent research in southeastern Europe, the distribution centre of H. obsoletus 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 H. obsoletus. Host-plant use was confirmed for Convolvulus arvensis, Urtica dioica, Vitex agnus-castus and Crepis foetida. 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 C. arvensis and U. dioica form a homogenous group, populations affiliated with V. agnus-castus and C. foetida 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 H. obsoletus sensu lato: 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., Hyalesthes thracicus (2.1-3.3%).

Re-visiting the Aphidius urticae s. str. group: re-description of Aphidius rubi Starý and A. silvaticus Starý (Hymenoptera: Braconidae: Aphidiinae).

Here we tested Aphidius urticae s. str. host-associated lineages from Microlophium carnosum (Buckton), Amphorophora rubi (Kaltenbach), Macrosiphum funestum (Macchiati) and Aulacorthum vaccinii Hille Ris Lambers with the barcoding region of the mitochondrial cytochrome oxidase subunit I gene used to analyse population differences and elucidate phylogenetic relationships between the separated taxa. This molecular marker has been shown to be the most informative molecular marker in resolving species complexes in aphidiine parasitoids. Analyses of the mitochondrial sequences revealed the existence of three clearly separated mitochondrial lineages of A. urticae s. str. group associated with: i) Macrosiphum funestum and Aulacorthum vaccinii aphid hosts, ii) Microlophium carnosum and iii) Amphorophora rubi. This corresponds to the initial descriptions of A. rubi, A. silvaticus and A. urticae and their aphid host associations prior to synonymization of A. rubi and A. silvaticus with A. urticae. On the other hand, significant evolutionary distances ranging from 2.3 to 9.2% between the three mitochondrial lineages were not accompanied by clear morphological differences. Therefore, re-descriptions of A. rubi and A. silvaticus are presented, together with their morphological differentiation in a key, as well as their phylogenetic relationships and genetical differentiation.

Genetic and morphological variation in sexual and asexual parasitoids of the genus Lysiphlebus - an apparent link between wing shape and reproductive mode.

BACKGROUND: Morphological divergence often increases with phylogenetic distance, thus making morphology taxonomically informative. However, transitions to asexual reproduction may complicate this relationship because asexual lineages capture and freeze parts of the phenotypic variation of the sexual populations from which they derive. Parasitoid wasps belonging to the genus Lysiphlebus Foerster (Hymenoptera: Braconidae: Aphidiinae) are composed of over 20 species that exploit over a hundred species of aphid hosts, including many important agricultural pests. Within Lysiphlebus, two genetically and morphologically well-defined species groups are recognised: the "fabarum" and the "testaceipes" groups. Yet within each group, sexual as well as asexual lineages occur, and in L. fabarum different morphs of unknown origin and status have been recognised. In this study, we selected a broad sample of specimens from the genus Lysiphlebus to explore the relationship between genetic divergence, reproductive mode and morphological variation in wing size and shape (quantified by geometric morphometrics). RESULTS: The analyses of mitochondrial and nuclear gene sequences revealed a clear separation between the "testaceipes" and "fabarum" groups of Lysiphlebus, as well as three well-defined phylogenetic lineages within the "fabarum" species group and two lineages within the "testaceipes" group. Divergence in wing shape was concordant with the deep split between the "testaceipes" and "fabarum" species groups, but within groups no clear association between genetic divergence and wing shape variation was observed. On the other hand, we found significant and consistent differences in the shape of the wing between sexual and asexual lineages, even when they were closely related. CONCLUSIONS: Mapping wing shape data onto an independently derived molecular phylogeny of Lysiphlebus revealed an association between genetic and morphological divergence only for the deepest phylogenetic split. In more recently diverged taxa, much of the variation in wing shape was explained by differences between sexual and asexual lineages, suggesting a mechanistic link between wing shape and reproductive mode in these parasitoid wasps.

Lysiphlebus orientalis (Hymenoptera, Braconidae), a new invasive aphid parasitoid in Europe - evidence from molecular markers.

We report the occurrence of Lysiphlebus orientalis in Serbia, an aphid parasitoid from the Far East that is new to Europe and has the potential to become invasive. Our finding based on morphological characters is confirmed by analyses of mitochondrial cytochrome oxidase subunit I sequences. An increase in number and an expansion of the host range were observed during field studies over the past two years, and it is determined that the current host range encompasses nine aphid hosts on 12 different host plants, forming 13 tri-trophic associations. A host range determined for European populations of L. orientalis appears wider compared with that in its Far Eastern native habitats where Aphis glycines Mats. is the sole known host. Moreover, it overlaps considerably with the host ranges of European parasitoids that play an important role in the natural control of pest aphids.

Host-associated genetic differentiation in a seed parasitic weevil Rhinusa antirrhini (Coleptera: Curculionidae) revealed by mitochondrial and nuclear sequence data.

Plant feeding insects and the plants they feed upon represent an ecological association that is thought to be a key factor for the diversification of many plant feeding insects, through differential adaptation to different plant selective pressures. While a number of studies have investigated diversification of plant feeding insects above the species level, relatively less attention has been given to patterns of diversification within species, particularly those that also require plants for oviposition and subsequent larval development. In the case of plant feeding insects that also require plant tissues for the completion of their reproductive cycle through larval development, the divergent selective pressure not only acts on adults, but on the full life history of the insect. Here we focus attention on Rhinusa antirrhini (Curculionidae), a species of weevil broadly distributed across Europe that both feeds on, and oviposits and develops within, species of the plant genus Linaria (Plantaginaceae). Using a combination of mtDNA (COII) and nuclear DNA (EF1-alpha) sequencing and copulation experiments we assess evidence for host associated genetic differentiation within R. antirrhini. We find substantial genetic variation within this species that is best explained by ecological specialisation on different host plant taxa. This genetic differentiation is most pronounced in the mtDNA marker, with patterns of genetic variation at the nuclear marker suggesting incomplete lineage sorting and/or gene flow between different host plant forms of R. antirrhini, whose origin is estimated to date to the mid-Pliocene (3.77 Mya; 2.91-4.80 Mya).