Philippe Audiot

Isolation by distance and sharp discontinuities in gene frequencies: implications for the phylogeography of an alpine insect species, Carabus solieri

Analysis of genetic isolation by distance (IBD) is of prime importance for the study of processes responsible for spatial population genetic structure and is thus frequently used in case studies. However, the identification of a significant IBD pattern does not necessarily imply the absence of sharp discontinuities in gene frequencies. Therefore, identifying barriers to gene flow and/or secondary contact between differentiated entities remains a major challenge in population biology. Geographical genetic structure of 41 populations (1080 individuals) of an alpine insect species, Carabus solieri, was studied using 10 microsatellite loci. All populations were significantly differentiated and spatially structured according to IBD over the entire range. However, clustering analyses clearly identified three main clusters of populations, which correspond to geographical entities. Whereas IBD also occurs within each cluster, population structure was different according to which group of populations was considered. The southernmost cluster corresponds to the most fragmented part of the range. Consistently, it was characterized by relatively high levels of differentiation associated with low genetic diversity, and the slope of the regression of genetic differentiation against geographical distances was threefold those of the two other clusters. Comparisons of within‐cluster and between‐cluster IBD patterns revealed barriers to gene flow. A comparison of the two approaches, IBD and clustering analyses, provided us with valuable information with which to infer the phylogeography of the species, and in particular to propose postglacial colonization routes from two potential refugia located in Italy and in southeastern France. Our study highlights strongly the possible confounding contribution of barriers to gene flow to IBD pattern and emphasizes the utility of the model‐based clustering analysis to identify such barriers.

Genetic structure and gene flow in French populations of two Ostrinia taxa: host races or sibling species?

Most models of ecological speciation concern phytophagous insects in which speciation is thought to be driven by host shifts and subsequent adaptations of populations. Despite the ever‐increasing number of studies, the current evolutionary status of most models remains incompletely resolved, as estimates of gene flow between taxa remain extremely rare. We studied the population genetics of two taxa of the Ostrinia genus — one feeding mainly on maize and the other on mugwort and hop — occurring in sympatry throughout France. The actual level of divergence of these taxa was unknown because the genetic structure of populations had been investigated over a limited geographical area and the magnitude of gene flow between populations had not been estimated. We used 11 microsatellite markers to investigate the genetic structure of populations throughout France and the extent of gene flow between the two Ostrinia taxa at several sites at which they are sympatric. We observed clear genetic differentiation between most populations collected on the typical respective hosts of each taxon. However, populations displaying intermediate allelic frequencies were found on hop plants in southern France. Individual assignments revealed that this result could be accounted for by the presence of both taxa on the same host. Gene flow, estimated by determining the proportion of hybrids detected, was low: probably < 1% per generation, regardless of site. This indicates that the two Ostrinia taxa have reached a high level of genetic divergence and should be considered sibling species rather than host races.

Molecular differentiation at nuclear loci in French host races of the European corn borer (Ostrinia nubilalis)

French populations of the European corn borer consist of two sympatric and genetically differentiated host races. As such, they are well suited to study processes that could be involved in sympatric speciation, but the initial conditions of host-race divergence need to be elucidated. Gene genealogies can provide insight into the processes involved in speciation. We used DNA sequences of four nuclear genes to (1) document the genetic structure of the two French host races previously delineated with allozyme markers, (2) find genes directly or indirectly involved in reproductive isolation between host races, and (3) estimate the time since divergence of the two taxa and see whether this estimate is compatible with this divergence being the result of a host shift onto maize after its introduction into Europe ∼500 years ago. Gene genealogies revealed extensive shared polymorphism, but confirmed the previously observed genetic differentiation between the two host races. Significant departures from the predictions of neutral molecular evolution models were detected at three loci but were apparently unrelated to reproductive isolation between host races. Estimates of time since divergence between French host races varied from ∼75,000 to ∼150,000 years, suggesting that the two taxa diverged recently but probably long before the introduction of maize into Europe.

Genetic structure of European and Mediterranean maize borer populations on several wild and cultivated host plants

Target pests may become resistant to Bacillus thuringiensis (Bt) toxins produced by trangenic maize (Zea mays L.). Untreated refuge areas are set aside to conserve high frequencies of susceptibility alleles: a delay in resistance evolution is expected if susceptible individuals from refuges mate randomly with resistant individuals from Bt fields. In principle, refuges can be toxin‐free maize or any other plant, provided it hosts sufficiently large pest populations mating randomly with populations from Bt‐maize fields. Our aim was to examine the suitability of several cultivated or weedy plants [pepper (Capsicum frutescens L.), sorghum (Sorghum spec.), sunflower (Helianthus annuus L.), cocklebur (Xanthium spec.), cantaloupe (Cucumis melo L.), and hop (Humulus lupulus L.)] as refuges for Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) and Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae), two major maize pests in southern Europe. Larvae of both species were collected on these plants. Their genetic population structure was examined at several allozyme loci. We found little or no evidence for an influence of geographic distance, but detected a significant host‐plant effect on the genetic differentiation for both species. Ostrinia nubilalis populations from sunflower, pepper, cocklebur, and sorghum appear to belong to the same genetic entity as populations collected on maize, but to differ from populations on hop. Accordingly, females from pepper and cocklebur produced exclusively the ‘Z’ type sexual pheromone, which, in France, characterizes populations developing on maize. Qualitatively, these plants (except hop) could thus serve as refuges for O. nubilalis; however, they may be of little use quantitatively as they were found much less infested than maize. Sesamia nonagrioides populations on maize and sorghum reached comparable densities, but a slight genetic differentiation was detected between both. The degree of assortative mating between populations feeding on both hosts must therefore be assessed before sorghum can be considered as a suitable refuge for this species.

Assortative Mating between European Corn Borer Pheromone Races: Beyond Assortative Meeting

Background Sex pheromone communication systems may be a major force driving moth speciation by causing behavioral reproductive isolation via assortative meeting of conspecific individuals. The ‘E’ and ‘Z’ pheromone races of the European corn borer (ECB) are a textbook example in this respect. ‘Z’ females produce and ‘Z’ males preferentially respond to a ‘Z’ pheromone blend, while the ‘E’ race communicates via an ‘E’ blend. Both races do not freely hybridize in nature and their populations are genetically differentiated. A straightforward explanation would be that their reproductive isolation is a mere consequence of “assortative meeting” resulting from their different pheromones specifically attracting males towards same-race females at long range. However, previous laboratory experiments and those performed here show that even when moths are paired in a small box – i.e., when the meeting between sexual partners is forced – inter-race couples still have a lower mating success than intra-race ones. Hence, either the difference in attractivity of E vs. Z pheromones for males of either race still holds at short distance or the reproductive isolation between E and Z moths may not only be favoured by assortative meeting, but must also result from an additional mechanism ensuring significant assortative mating at close range. Here, we test whether this close-range mechanism is linked to the E/Z female sex pheromone communication system. Methodology/Principal Findings Using crosses and backcrosses of E and Z strains, we found no difference in mating success between full-sisters emitting different sex pheromones. Conversely, the mating success of females with identical pheromone types but different coefficients of relatedness to the two parental strains was significantly different, and was higher when their genetic background was closer to that of their male partners pheromone race. Conclusions/Significance We conclude that the close-range mechanism ensuring assortative mating between the E and Z ECB pheromone races is unrelated to the difference in female sex pheromone. Although the nature of this mechanism remains elusive, our results show that it is expressed in females, acts at close range, segregates independently of the autosome carrying Pher and of both sex chromosomes, and is widely distributed since it occurs both in France and in the USA.

‘Becoming a species by becoming a pest’ or how two maize pests of the genus Ostrinia possibly evolved through parallel ecological speciation events

New agricultural pest species attacking introduced crops may evolve from pre‐existing local herbivores by ecological speciation, thereby becoming a species by becoming a pest. We compare the evolutionary pathways by which two maize pests (the Asian and the European corn borers, ACB and ECB) in the genus Ostrinia (Lepidoptera, Crambidae) probably diverged from an ancestral species close to the current Adzuki bean borer (ABB). We typed larval Ostrinia populations collected on maize and dicotyledons across China and eastern Siberia, at microsatellite and mitochondrial loci. We found only two clusters: one on maize (as expected) and a single one on dicotyledons despite differences in male mid‐tibia morphology, suggesting that all individuals from dicotyledons belonged to the ABB. We found evidence for migrants and hybrids on both host plant types. Hybrids suggest that field reproductive isolation is incomplete between ACB and ABB. Interestingly, a few individuals with an ‘ABB‐like’ microsatellite profile collected on dicotyledons had ‘ACB’ mtDNA rather than ‘ABB‐like’ mtDNA, whereas the reverse was never found on maize. This suggests asymmetrical gene flow directed from the ACB towards the ABB. Hybrids and backcrosses in all directions were obtained in no‐choice tests. In laboratory conditions, they survived as well as parental strain individuals. In Xinjiang, we found ACB and ECB in sympatry, but no hybrids. Altogether, our results suggest that reproductive isolation between ACB and ABB is incomplete and mostly prezygotic. This points to ecological speciation as a possible evolutionary scenario, as previously found for ECB and ABB in Europe.

Introgression in natural populations of bioindicators: a case study of Carabus splendens and Carabus punctatoauratus.

The evolutionary importance of hybridization in wild plants and animals has become increasingly widely recognized in the last decade. In practical terms, hybridization provides an exceptionally tough set of problems for conservation biologists. We illustrate this in a case study of two Carabidae species widely used to evaluate the impact of human activities on biodiversity. These two species live in a complex mosaic of sympatry/allopatry and are known to hybridize in controlled conditions. Hybridization has not been quantified in natural populations to date due to the lack of a simple set of phenotypic traits for identifying hybrids. We thus screened for hybrids in natural populations, by multilocus genotyping at nine microsatellite loci. A high level of genetic differentiation between these two taxa was observed, as shown by allelic frequency distributions. Two Bayesian assignment procedures without obligatory pure taxon references were used to infer different classes of hybrids (F1, F2 and backcrosses) and mixture proportions between the two species. A low level of hybridization (F1 genotypes) was observed in natural populations, contrasting with results obtained in controlled conditions. A high level of introgression was, however, detected at three of 12 sites, as revealed by the detection of backcrossed genotypes. This interspecific gene flow was detected in a limited zone of the common geographical range of the two species and was not related to the pattern of sympatry/allopatry. We then considered the origin and repercussions of this introgression, based on intraspecific genetic diversity and geographical structure.

Genetic survey of two endangered grasshopper subspecies, Prionotropis hystrix rhodanica and Prionotropis hystrix azami (Orthoptera, Pamphagidae): within- and between-population dynamics at the regional scale

The fragmentation and destruction of natural habitats is one of the major causes of extinction of wild flora and fauna due to human activities. The area around the Mediterranean Basin is particularly affected: this region is one of the world’s richest in terms of biodiversity and is undergoing substantial modification due to extensive changes in land use. We investigated the demographic state (through a genetic survey) of an endangered grasshopper, the geographical distribution of which is restricted to arid open land or glades in south-eastern France. We compared two subspecies: one is severely threatened by the fragmentation of its specific and restricted habitat (Prionotropis hystrix rhodanica in the ‘Crau’ steppe) whereas the other, P.␣h.␣azami, occurs more widely in glades or clearings in the evergreen Mediterranean forest. Genetic diversity at eight microsatellite loci was highly structured, indicating substantial isolation of populations. Within-population diversity was high at all but one site. Genetic drift was the major force involved in this genetic structure, with very little gene flow at the regional scale of observation, consistent with both the limited dispersal of this flightless species and the patchy configuration of its habitat. No significant differences in the extent of genetic diversity or population dynamics were observed between the two subspecies. The observed genetic pattern calls into question the validity of the subspecies status, and shows no strong impact of recent anthropogenic modification of the Crau steppe. The evolution of the Mediterranean xeric and open habitats under both natural and anthropogenic processes is discussed.

Parallel evolution of behaviour during independent host‐shifts following maize introduction into Asia and Europe

Maize was introduced into opposite sides of Eurasia 500 years ago, in Western Europe and in Asia. This caused two host‐shifts in the phytophagous genus Ostrinia; O. nubilalis (the European corn borer; ECB) and O. furnacalis (the Asian corn borer; ACB) are now major pests of maize worldwide. They originated independently from Dicot‐feeding ancestors, similar to O. scapulalis (the Adzuki bean borer; ABB). Unlike other host‐plants, maize is yearly harvested, and harvesting practices impose severe mortality on larvae found above the cut‐off line. Positive geotaxis in the ECB has been proposed as a behavioural adaptation to harvesting practices, allowing larvae to move below the cut‐off line and thus escape harvest mortality. Here, we test whether the same behavioural adaptation evolved independently in Europe and in Asia. We sampled eight genetically differentiated ECB, ACB and ABB populations in France and China and monitored geotaxis through the entire larval development in artificial stacks mimicking maize stems. We find that all ECB and ACB populations show a similar tendency to move down during the latest larval stages, a behaviour not observed in any European or Asian ABB population. The behaviour is robustly expressed regardless of larval density, development mode or environmental conditions. Our results indicate that maize introduction triggered parallel behavioural adaptations in Europe and Asia, harvest selection presumably being the main driver.

Transcriptomic response to host and non-host plants during oviposition in two closely related moth species

We present here a comprehensive analysis of the transcriptomic response to plant environments in ovipositing females of two sibling species of phytophagous moths affiliated to different host ranges: the European corn borer (ECB) and the adzuki bean borer (ABB). We first assembled and annotated a de novo reference transcriptome based on a high throughput RNA sequencing of females placed in different plant environments, then we measured differences in gene expression between ECB and ABB, and also within each moth species between environments. We further related the differentially expressed (DE) genes to the host preference in ECB and ABB and highlighted the functional categories involved. More specifically, we conducted an analysis on chemosensory genes previously characterized in ECB, ABB and other related Ostrinia species, as these genes are considered as good candidates for the host recognition before oviposition. Overall, we recorded more DE genes in ECB than in ABB samples, what could highlight the higher strength of the host specialization in ECB compared to ABB as observed at the behavioral level. We also noticed that the genes involved in the preference for their respective host were different between ECB and ABB. At the functional level, the response to plant environment in ECB and ABB during oviposition involved many processes, including the chemosensory repertoire as expected, but also metabolism of carbohydrates, lipids, proteins, and amino acids, detoxification mechanisms and immunity. All together, our results allowed identifying genes and functions candidates for specialization and also for the species divergence between ECB and ABB. By ad-hoc categorization, we discriminated some genes responding to the environment with similar or divergent pattern in ECB and ABB. Among them, we highlighted new lines of research like carbohydrates metabolism or virus and retrovirus dynamics.