Origin of a sudden mass occurrence of the stolbur phytoplasma vector Hyalesthes obsoletus (Cixiidae) in Austria

The grapevine disease ‘bois noir’ is widespread in European viticulture, but in many regions there is a lack of correspondence between disease spread and abundance of the main insect vector, the planthopper Hyalesthes obsoletus. This was the situation in Austria until 2012, when a mass occurrence of the vector was observed on Urtica dioica, a new host plant for the vector and reservoir plant for the pathogen, stolbur phytoplasma, in this area. Here we analyse the origin of the Austrian vector populations using genetic markers. The origin was unambiguously assigned to two regional populations, and two causes for the population expansion: immigration of East Central European populations and local demographic expansion. The observed population increase was thus independent of phylogenetic ancestry, but linked to the host plant and the exchange of a specific stolbur phytoplasma strain between the two vector populations. These circumstances are identical to but independent of the emergence of bois noir west of the European Alps, where an exchange between other vector populations associated with U. dioica of another stolbur phytoplasma genotype has led to disease outbreaks. Combined, the independent outbreaks in Austria and Europe west of the Alps are suggestive of an active role for stolbur phytoplasma in the vector–plant interaction and thus the host distribution of the vector.     

Sympatric diversification vs. immigration: deciphering host‐plant specialization in a polyphagous insect,the stolbur phytoplasma vector Hyalesthes obsoletus (Cixiidae)

The epidemiology of vector transmitted plant diseases is highly influenced by dispersal and the host‐plant range of the vector. Widening the vector's host range may increase transmission potential, whereas specialization may induce specific disease cycles. The process leading to a vector's host shift and its epidemiological outcome is therefore embedded in the frameworks of sympatric evolution vs. immigration of preadapted populations. In this study, we analyse whether a host shift of the stolbur phytoplasma vector, Hyalesthes obsoletus from field bindweed to stinging nettle in its northern distribution range evolved sympatrically or by immigration. The exploitation of stinging nettle has led to outbreaks of the grapevine disease bois noir caused by a stinging nettle‐specific phytoplasma strain. Microsatellite data from populations from northern and ancestral ranges provide strong evidence for sympatric host‐race evolution in the northern range: Host‐plant associated populations were significantly differentiated among syntopic sites (0.054 < F_HT < 0.098) and constant over 5 years. While gene flow was asymmetric from the old into the predicted new host race, which had significantly reduced genetic diversity, the genetic identity between syntopic host‐race populations in the northern range was higher than between these populations and syntopic populations in ancestral ranges, where there was no evidence for genetic host races. Although immigration was detected in the northern field bindweed population, it cannot explain host‐race diversification but suggests the introduction of a stinging nettle‐specific phytoplasma strain by plant‐unspecific vectors. The evolution of host races in the northern range has led to specific vector‐based bois noir disease cycles.     

Infection of Bois‐Noir tuf‐type‐I stolbur phytoplasma in Hyalesthes obsoletus (Hemiptera: Cixiidae) larvae and influence on larval size

Recent dramatic spread of the grapevine yellows disease Bois Noir (BN) in Germany is above all explained by highly increased abundances of the vector Hyalesthes obsoletus (Hemiptera: Cixiidae) associated to the plant Urtica dioica, the reservoir of the BN pathogen stolbur tuf‐type‐I. The vector acquires BN‐phytoplasma as larvae whilst feeding on the roots of infected U. dioica. To understand the dynamics of the Urtica‐cycle, we tested at what instar larvae become infected and whether infection affects larvae size (i.e. growth) at two sites in the Mosel Valley, Germany. Larvae were tested from infected plants and collected at instar‐stages 3, 4 and 5. Larvae at stage 3 were already infected but infection rates increased significantly between stage 3 and 5, mean infection rates: 0.12–0.62. There was no effect of infection on larval size at any instar stage.     

Survival relative to new and ancestral host plants,phytoplasma infection,and genetic constitution in host races of a polyphagous insect disease vector

Dissemination of vectorborne diseases depends strongly on the vector's host range and the pathogen's reservoir range. Because vectors interact with pathogens, the direction and strength of a vector's host shift is vital for understanding epidemiology and is embedded in the framework of ecological specialization. This study investigates survival in host‐race evolution of a polyphagous insect disease vector, Hyalesthes obsoletus, whether survival is related to the direction of the host shift (from field bindweed to stinging nettle), the interaction with plant‐specific strains of obligate vectored pathogens/symbionts (stolbur phytoplasma), and whether survival is related to genetic differentiation between the host races. We used a twice repeated, identical nested experimental design to study survival of the vector on alternative hosts and relative to infection status. Survival was tested with Kaplan–Meier analyses, while genetic differentiation between vector populations was quantified with microsatellite allele frequencies. We found significant direct effects of host plant (reduced survival on wrong hosts) and sex (males survive longer than females) in both host races and relative effects of host (nettle animals more affected than bindweed animals) and sex (males more affected than females). Survival of bindweed animals was significantly higher on symptomatic than nonsymptomatic field bindweed, but in the second experiment only. Infection potentially had a positive effect on survival in nettle animals but due to low infection rates the results remain suggestive. Genetic differentiation was not related to survival. Greater negative plant‐transfer effect but no negative effect of stolbur in the derived host race suggests preadaptation to the new pathogen/symbiont strain before strong diversifying selection during the specialization process. Physiological maladaptation or failure to accept the ancestral plant will have similar consequences, namely positive assortative mating within host races and a reduction in the likelihood of oviposition on the alternative plant and thus the acquisition of alternative stolbur strains.     

Invasion biology and host specificity of the grapevine yellows disease vector Hyalesthes obsoletus in Europe

Within the past 10 years, the yellows disease ‘bois noir’ (BN) has become one of the commercially most important diseases of grapevine [Vitis vinifera L. (Vitaceae)] in Europe. Infection pressure is caused by phytoplasmas of the stolbur 16SrXII‐A group that are transmitted by a planthopper vector, Hyalesthes obsoletus Signoret (Homoptera: Auchenorrhyncha). Infestation happens as an accidental side‐effect of the feeding behaviour of the vector, as vector and pathogen proliferation is dependent on other plants. In Germany, the increase of BN is correlated with the use of a new host plant by the vector, increase in abundance of the vector on the new host plant, and dissemination of host plant‐specific pathogen strains. In this article, we investigate geographic and host‐associated range expansion of the vector. We test whether host‐plant utilization in Germany, hence the increase in BN, is related to genetic host races of the vector and, if so, whether these have evolved locally or have immigrated from southern populations that traditionally use the new host plant. The genetic population analysis demonstrates a recent expansion and circum‐alpine invasion of H. obsoletus into German and northern French wine‐growing regions, which coincides with the emergence of BN. No H. obsoletus mitochondrial DNA haplotype host‐plant affiliation was found, implying that the ability to use alternative host plants is genetically intrinsic to H. obsoletus. However, subtle yet significant random amplified polymorphic DNA (RAPD) genetic differentiation was found among host plant populations. When combined, these results suggest that a geographic range expansion of H. obsoletus only partly explains the increase of BN, and that interactions with host plants also occur. Further possible beneficial factors to H. obsoletus, such as temperature increase and phytoplasma interactions, are discussed.     

Inferring the patterns and causes of geographic variation in Ephippiger ephippiger (Orthoptera, Tettigoniidae) using geographical information systems (GIS)

Ephippiger ephippiger is a tcltigoniid cricket that is fairly widespread in Europe. In southern France, between the Alps and the Pyrenees, it is geographically highly variable lor a number of different traits including morphology, allozymcs, behaviour and genetic markers. This variation has resulted in considerable confusion over the taxonomic and evolutionary status of forms of the organism. Through the use of geographical information systems (GIS) it has been possible to integrate both trait and environmental data sets from a variety of sources. The GIS is used to interpolate trait and environmental surfaces that are then examined for coincidence. Four general spatial patterns are identified. These can be explained through a combination of secondary contact, environmental adaptation and drift. Covariance between trait and environmental surfaces point to patterns which are primary in origin, whereas other dines probably reflect secondary contact following divergence in rcfugiae. Multivariate statistics support the validity of a major subdivision identified by GIS. This study is an example of the considerable potential for GIS as an investigative tool in evolutionary studies.     

Population genetic structure of the endangered crayfish Austropotamobius pallipes in France based on microsatellite variation: biogeographical inferences and conservation implications

1. One important goal in conservation biology is to characterise evolutionary lineages within endangered species before management decisions are taken. Here, we assess population differentiation in the freshwater crayfish Austropotamobius pallipes, an endangered species endemic to western Europe and provide valuable information for the conservation of French populations. 2. Analysis of five microsatellite loci in 44 populations revealed very different within population levels of genetic diversity (0.000 < H₀ < 0.564). Two groups, corresponding to northern and southern French populations, showed a high degree of genetic differentiation in both allele frequencies and allele sizes. Comparison of these results with previous studies of A. pallipes strongly suggests that the divergence between northern and southern populations could have occurred during the last glaciation period of the Pleistocene from one Atlantic and one Mediterranean refuge. 3. Evidence for genetic admixture between these two lineages was revealed by correspondence analyses in southern populations, probably as the result of artificial translocations. 4. French populations appeared significantly differentiated among the different river drainages and were highly structured within rivers. The impact of population size, population bottlenecks and founder events on the population genetic differentiation are discussed. 5. Based on these results, we propose the designation of two evolutionarily significant units for A. pallipes in France. Our data also support the maintenance of separate demographic management strategies for crayfish inhabiting different river systems. However, genetic analyses will have to be combined with demographic and ecological data for sustainable conservation programmes.     

Geminivirus transmission and biological characterisation of Bemisia tabaci (Gennadius) biotypes from different geographic regions

Eighteen populations of Bemisia tabaci, collected from different geographic locations (North & Central America, the Caribbean, Africa, the Middle East, Asia and Europe), were studied to identify and compare biological and genetic characteristics that can be used to differentiate biotypes. The morphology of the fourth instar/pupal stage and compound eye structures of adults were investigated using scanning electron microscopy and found to be typical of the species among all biotypes and populations studied. Setae and spines of B. tabaci larval scales from the same colony were highly variable depending on the host plant species or leaf surface characteristics. The location and the morphology of caudal setae, characteristic of all B. tabaci studied to date, were present in all colonies. However, differences in adult body lengths and in the ability to induce phy to toxic disorders in certain plant species were found between biotypes or populations. The recently identified “B” biotype, characterised by a diagnostic esterase banding pattern and by its ability to induce phytotoxic responses in squash, honeysuckle and nightshade was readily distinguished from non-“B” biotype populations. None of the non-“B” biotypes studied, were found to induce phytotoxic responses. Nine populations examined showed typical “B” biotype characteristics, regardless of country of origin. All tested populations, determined as “B” or “B”-like biotypes successfully mated with other “B” biotype colonies from different geographic areas. Non-“B” biotype colonies did not interbreed with other biotypes. The B. tabaci populations were tested for their ability to transmit 15 whitefly-transmitted geminiviruses (WTGs) from different geographic areas with a wide range of symptom types. All WTGs were transmitted by the “B” biotype colonies and by most non-“B” biotype colonies, with the exception of three viruses found in ornamental plants which were non-transmissible by any colony. Some non-“B” biotypes would not transmit certain geminiviruses and some geminiviruses were more efficiently transmitted than were others.     

The influence of the arid Andean high plateau on the phylogeography and population genetics of guanaco (Lama guanicoe) in South America

A comprehensive study of the phylogeography and population genetics of the largest wild artiodactyl in the arid and cold‐temperate South American environments, the guanaco (Lama guanicoe) was conducted. Patterns of molecular genetic structure were described using 514 bp of mtDNA sequence and 14 biparentally inherited microsatellite markers from 314 samples. These individuals originated from 17 localities throughout the current distribution across Peru, Bolivia, Argentina and Chile. This confirmed well‐defined genetic differentiation and subspecies designation of populations geographically separated to the northwest (L. g. cacsilensis) and southeast (L. g. guanicoe) of the central Andes plateau. However, these populations are not completely isolated, as shown by admixture prevalent throughout a limited contact zone, and a strong signal of expansion from north to south in the beginning of the Holocene. Microsatellite analyses differentiated three northwestern and 4–5 southeastern populations, suggesting patterns of genetic contact among these populations. Possible genetic refuges were identified, as were source‐sink patterns of gene flow at historical and recent time scales. Conservation and management of guanaco should be implemented with an understanding of these local population dynamics while also considering the preservation of broader adaptive variation and evolutionary processes.     

Population structure in relation to host-plant ecology and Wolbachia infestation in the comma butterfly

Experimental work on Polygonia c‐album, a temperate polyphagous butterfly species, has shown that Swedish, Belgian, Norwegian and Estonian females are generalists with respect to host‐plant preference, whereas females from UK and Spain are specialized on Urticaceae. Female preference is known to have a strong genetic component. We test whether the specialist and generalist populations form respective genetic clusters using data from mitochondrial sequences and 10 microsatellite loci. Results do not support this hypothesis, suggesting that the specialist and generalist traits have evolved more than once independently. Mitochondrial DNA variation suggests a rapid expansion scenario, with a single widespread haplotype occurring in high frequency, whereas microsatellite data indicate strong differentiation of the Moroccan population. Based on a comparison of polymorphism in the mitochondrial data and sequences from a nuclear gene, we show that the diversity in the former is significantly less than that expected under neutral evolution. Furthermore, we found that almost all butterfly samples were infected with a single strain of Wolbachia, a maternally inherited bacterium. We reason that indirect selection on the mitochondrial genome mediated by a recent sweep of Wolbachia infection has depleted variability in the mitochondrial sequences. We also surmise that P. c‐album could have expanded out of a single glacial refugium and colonized Morocco recently.     

Genetic diversity in water frog hybrids (Pelophylax esculentus) varies with population structure and geographic location

Pelophylax esculentus is a hybridogenetic frog originating from matings between P. ridibundus (RR) and P. lessonae (LL). Typically, diploid hybrids (LR) live in sympatry with one of their parental species, upon which they depend for successful reproduction. In parts of their range, however, pure hybrid populations can be found. These hybrid populations have achieved reproductive independence from their parental species by using triploid hybrids (LLR, LRR) rather than LL and RR as their sexual hosts. These different breeding systems also entail differences in reproduction (clonal versus sexual) and hence offer the opportunity to study how genetic diversity is affected by reproductive mode, population structure and geographic location. We investigated 33 populations in the Scania region (South Sweden) and 18 additional populations from Northern and Central Europe. Within both genomes (L, R), genetic variability increases with the potential for recombination and declines from the main species distribution area southeast of the Baltic Sea to the fringe populations northwest of the Baltic Sea. Within the main study area in Scania, genetic diversity is low and decreases from a core area to the periphery. Genetic differentiation between Scania populations is small but significant and best explained by ‘isolation by distance’. Despite the low genetic variability within the discrete genomes, all‐hybrid P. esculentus populations in southern Sweden are apparently not suffering from direct negative fitness effects. This is probably because of its somatic hybrid status, which increases diversity through the combination of genomes from two species.     

The relationship between diet and the size of the midgut caeca in grasshoppers (Insecta: Orthoptera: Acridoidea)

A survey of the size and form of the midgut caeca in relation to diet has been carried out on 173 species from 21 families and subfamilies of Acridoidea (grasshoppers). Although differences exist in the size of the anterior caecal arms relative to body length, these differences are not related to the type of food eaten. Assuming that the anterior arms have a key role in digestive and absorptive processes, this suggests that different foods make similar demands on these processes. The posterior caecal arms are smaller in graminivorous species than in species eating other types of plants as a whole or part of their diet. This is true across all the taxa, including those families and subfamilies that are predominantly forb-feeding. It is suggested that the posterior caecal arms have a special role in the detoxification of plant secondary compounds and that the requirement for this is reduced in graminivorous species because of the lower levels of toxic secondary compounds in grasses. A specialized pocket region is present in the posterior caecal arms of some forb-feeding species. Its occurrence across the taxa is spasmodic. It may be concerned with the removal of phenolic compounds.     

Numts help to reconstruct the demographic history of the ocellated lizard (Lacerta lepida) in a secondary contact zone

In northwestern Iberia, two largely allopatric Lacerta lepida mitochondrial lineages occur, L5 occurring to the south of Douro River and L3 to the north, with a zone of putative secondary contact in the region of the Douro River valley. Cytochrome b sequence chromatograms with polymorphisms at nucleotide sites diagnostic for the two lineages were detected in individuals in the region of the Douro River and further north within the range of L3. We show that these polymorphisms are caused by the presence of four different numts (I-IV) co-occurring with the L3 genome, together with low levels of heteroplasmy. Two of the numts (I and II) are similar to the mitochondrial genome of L5 but are quite divergent from the mitochondrial genome of L3 where they occur. We show that these numts are derived from the mitochondrial genome of L5 and were incorporated in L3 through hybridization at the time of secondary contact between the lineages. The additional incidence of these numts to the north of the putative contact zone is consistent with an earlier postglacial northward range expansion of L5, preceding that of L3. We show that genetic exchange between the lineages responsible for the origin of these numts in L3 after secondary contact occurred prior to, or coincident with, the northward expansion of L3. This study shows that, in the context of phylogeographic analysis, numts can provide evidence for past demographic events and can be useful tools for the reconstruction of complex evolutionary histories.     

Mitochondrial and nuclear DNA phylogeography of European grayling (Thymallus thymallus): evidence for secondary contact zones in central Europe

Mitochondrial and microsatellite DNA markers were applied to infer the phylogeography, intraspecific diversity and dynamics of the distributional history of European grayling (Thymallus thymallus) with focus on its central and northern European distribution range. Phylogenetic and nested clade analyses revealed at least four major mtDNA lineages, which evolved in geographical isolation during the Pleistocene. These lineages should be recognized as the basic evolutionary significant units (ESUs) for grayling in central and northern Europe. In addition, and in contrast to previous work on grayling, the results of Bayesian analysis of individual admixture coefficients, two-dimensional scaling analysis and spatial analysis of molecular variance provided evidence for a high level of admixture among major lineages in contact zones between drainages (e.g. the low mountain range of Germany), most likely resulting from glacial perturbations and ancient river connections between drainages during the Pleistocene glaciations. Even within river systems, a high level of differentiation among populations was revealed as indicated by the microsatellite data. Grayling sampled from 29 sites displayed high levels of differentiation (overall F(ST) = 0.367), a high number of private alleles and high bootstrap support for the genetic distance-based population clusters across 12 loci. We specifically discuss our results in context of phylogeograpic studies on other European freshwater fish species with habitat preferences similar to those of grayling. Our study shows that both large-scale phylogeographical and detailed genetic analyses on a fine scale are mandatory for developing appropriate conservation guidelines of endangered species.     

Divergent host plant adaptation drives the evolution of sexual isolation in the grasshopper Hesperotettix viridis (Orthoptera: Acrididae) in the absence of reinforcement

Early stages of lineage divergence in insect herbivores are often related to shifts in host plant use and divergence in mating capabilities, which may lead to sexual isolation of populations of herbivorous insects. We examined host preferences, degree of differentiation in mate choice, and divergence in cuticular morphology using near‐infrared spectroscopy in the grasshopper Hesperotettix viridis aiming to understand lineage divergence. In Kansas (USA), H. viridis is an oligophagous species feeding on Gutierrezia and Solidago host species. To identify incipient mechanisms of lineage divergence and isolation, we compared host choice, mate choice, and phenotypic divergence among natural grasshopper populations in zones of contact with populations encountering only one of the host species. A significant host‐based preference from the two host groups was detected in host‐paired feeding preference studies. No‐choice mate selection experiments revealed a preference for individuals collected from the same host species independent of geographic location, and little mating was observed between individuals collected from different host species. Female mate choice tests between males from the two host species resulted in 100% fidelity with respect to host use. Significant differentiation in colour and cuticular composition of individuals from different host plants was observed, which correlated positively with host choice and mate choice. No evidence for reinforcement in the zone of contact was detected, suggesting that divergent selection for host plant use promotes sexual isolation in this species. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 866–878.     

The history of Seasonally Dry Tropical Forests in eastern South America: inferences from the genetic structure of the tree Astronium urundeuva (Anacardiaceae)

Today, the Seasonally Dry Tropical Forests (SDTF) of eastern South America occur as large, well-defined nuclei (e.g. Caatinga in the northeast) and as smaller enclaves within other vegetations (e.g. Cerrado and Chaco). In order to infer the way the present SDTF distribution was attained, the genetic structure of Astronium urundeuva, a tree confined to SDTF, was assessed using two chloroplast spacers and nine microsatellite loci. Five haplotypes were identified, whose distribution was spatially structured. The distribution of the two most common and divergent haplotypes suggested former vicariance and progressive divergence due to isolation. More recent range expansions of these two lineages subsequently occurred, leading to a secondary contact at the southern limit of the Caatinga SDTF nucleus. The multilocus-Bayesian approach using microsatellites consistently identified three groups of populations (Northeast, Central and Southwest). Isolation by distance was found in Northeast and Southwest groups whereas admixture was detected in the Central group, located at the transition between Caatinga and Cerrado domains. All together, the results support the existence of range expansions and secondary contact in the Central group. This study provides arguments that favour the existence of a previously more continuous formation of SDTF in eastern South America.     

Geographic distribution and food habits of Leopardus tigrinus and L. geoffroyi (Carnivora,Felidae) at their geographic contact zone in southern Brazil

The objective of this study is to define the geographic distribution of Leopardus tigrinus and L. geoffroyi in one of the few regions of South America where they co-occur, the state of Rio Grande do Sul (RS), in southernmost Brazil. We compiled 133 records for both species and constructed a distribution map, which shows sharp geographic segregation between them. Leopardus tigrinus was found to be associated more with forested ecoregions in the northern part of the state, while L. geoffroyi records were mainly associated with open habitats of the Pampas biome in southern RS. We present data on the diet of these two species that indicate trophic niche separation between them in this region of geographic contact. Our results thus suggest that these species exhibit ecological partitioning with respect to habitat and prey, and that these factors may influence the observed pattern of limited spatial overlap in this region.     

Interplay of host specificity and biogeography in the population structure of a cosmopolitan endoparasite: microsatellite study of Ligula intestinalis (Cestoda)

Organisms with wide geographical or phenotypic diversity often constitute assemblages of genetically distinct species or lineages. Within parasites, an emergence of host-specific lineages is assumed to create such cryptic variability; however, empirical evaluation of these processes is scarce. Here, we analyse populations of a parasite with a complex life cycle, wide host spectrum and global distribution, with the aim to reveal factors underlying the evolution of host- or geography-dependent lineages. Using 15 microsatellite loci, deep genetic distances were observed between populations from distant geographical areas. On the local scale, host-mediated genetic structure was found among sympatric samples. Two lineages differing in the spectrum of infected hosts co-occurred in the Euro-Mediterranean area, and two distinct lineages were recovered from Lake Tana in Ethiopia. Although sampled across several host taxa and multiple localities, a lack of marked genetic structure was seen in the populations belonging to one of the European lineages. Only weak genetic differentiation between sympatric samples from two host species was found. Complexity of the parasite life-cycle contributed to such a stratified pattern. Differences in the immune response between fish hosts were suggested as the factor diversifying the populations locally; conversely, high mobility of the parasite due to migration with its bird (definitive) host were assessed to homogenize populations across the area of distribution. However, despite the high mobility, large bodies of salt water prevent the parasite from long-distance migrations, as was demonstrated in an example of the Mediterranean Sea which represented an effective barrier to gene flow.     

Population genetic structure of the lemon shark (Negaprion brevirostris) in the western Atlantic: DNA microsatellite variation

DNA microsatellite markers were used to characterize the population genetic structure of the lemon shark, Negaprion brevirostris, in the western Atlantic. This study demonstrates for the first time the usefulness of microsatellites to study population genetic structure and mating systems in the Chondricthyes. Lemon sharks (mostly juveniles) were sampled non-destructively from four locations, Gullivan Bay and Marquesas Key in Florida, Bimini, Bahamas, and Atol das Rocas, Brazil. At least 545 individuals were genotyped at each of four dinucleotide loci. The number of alleles per locus ranged from 19 to 43, and expected heterozygosities ranged from 0.69 to 0.90. Relatively little genetic structure was found in the western Atlantic, with small but significant values for estimators of F(ST) and R(ST) among populations, theta (0.016) and rho (0.026), respectively. No sharp discontinuities were found between the Caribbean sites and Brazil, and most alleles were found at all four sites, indicating that gene flow occurs throughout the western Atlantic with no evidence for distinct stocks.