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The insect mitochondrial control region (=AT-rich-region) is a rarely used genetic marker in phylogeographic studies and population genetic surveys. Reasons for this are that the high AT content and the presence of tandem repeats and indels pose technical and analytical problems. We provide a new pair of primers and the first taxonomically wide-scale description of control region (CR) structure in an insect order after sequencing it in 31 lepidopteran species. We assessed levels of variation occurring in the CR and cytochrome oxidase I (COI) by sequencing and comparison. Intrapopulation analyses in five species of butterflies showed that CR was more variable than COI. Interpopulation variation from three populations of Erebia triaria and E. palarica was slightly lower in the CR than COI with regard to single-nucleotide polymorphisms, but the results were concordant between both markers and highly congruent with regard to population differentiation. Using 15 species of Satyrinae we found that the CR has the same, or stronger, phylogenetic resolution as COI. Our results indicate that the CR can be of importance in addition to COI in population genetic studies. Alignments for the whole CR are direct and unambiguous at the intraspecific level. Indels show phylogenetic signal, but make this marker more complex to use than COI for higher phylogenetic analyses. Nevertheless, alignments at the generic level are straightforward over one part of the CR. The combination CR+COI appears to be a very promising phylogenetic tool to resolve fast-evolving species-level phylogenies.

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Sex attractant pheromones are highly sensitive and selective tools for detecting and monitoring populations of insects, yet there has been only one reported case of pheromones being used to monitor protected species. Here, we report the identification and synthesis of the sex pheromone of a protected European moth species, Graellsia isabellae (Lepidoptera: Saturniidae), as the single component, (4E,6E,11Z)-hexadecatrienal. In preliminary field trials, lures loaded with this compound attracted male moths from populations of this species at a number of widely separated field sites in France, Switzerland, and Spain, clearly demonstrating the utility of pheromones in sampling potentially endangered insect species.

Reproductive character displacement occurs when competition for successful breeding imposes a divergent selection on the interacting species, causing a divergence of reproductive traits. Here, we show that a disputed butterfly taxon is actually a case of male wing colour shift, apparently produced by reproductive character displacement. Using double digest restriction‐site associated DNA sequencing and mitochondrial DNA sequencing we studied four butterfly taxa of the subgenus Cupido (Lepidoptera: Lycaenidae): Cupido minimus and the taxon carswelli, both characterized by brown males and females, plus C. lorquinii and C. osiris, both with blue males and brown females. Unexpectedly, taxa carswelli and C. lorquinii were close to indistinguishable based on our genomic and mitochondrial data, despite displaying strikingly different male coloration. In addition, we report and analysed a brown male within the C. lorquinii range, which demonstrates that the brown morph occurs at very low frequency in C. lorquinii. Such evidence strongly suggests that carswelli is conspecific with C. lorquinii and represents populations with a fixed male brown colour morph. Considering that these brown populations occur in sympatry with or very close to the blue C. osiris, and that the blue C. lorquinii populations never do, we propose that the taxon carswelli could have lost the blue colour due to reproductive character displacement with C. osiris. Since male colour is important for conspecific recognition during courtship, we hypothesize that the observed colour shift may eventually trigger incipient speciation between blue and brown populations. Male colour seems to be an evolutionarily labile character in the Polyommatinae, and the mechanism described here might be at work in the wide diversification of this subfamily of butterflies. [ABSTRACT FROM AUTHOR]

Graellsia isabellae is a protected lepidopteran both in France and Spain; however, there has been considerable debate over its conservation status. Recent literature emphasised the need of monitoring population size in the different mountain ranges where this iconic species occurs.We used mark–capture–recapture and genotypes of nine molecular microsatellite markers to estimate the census (N) and contemporary effective population size (Ne) of two Spanish populations extending over similar size areas (10–15 km2): Puebla (Eastern Spain) and Ordesa (Western Pyrenees). Only adult males were captured and analysed, as sampling was based on the use of the synthesised female sex pheromone.Estimates of N were rather different in the two populations: 3398 males in Puebla (95% CI = 2875–4145) and 1500 in Ordesa (95% CI = 1229–1932), although the area occupied by the populations was larger and more densely forested in Ordesa than in Puebla.Several lines of evidence pointed to a moderate–large contemporary Ne at Puebla (173–178 individuals) and a one‐order of magnitude lower Ne at Ordesa (27–49). Thus, Ne/N ratios were very low (0.026 and 0.01 respectively).We recommend G. isabellae to be classified as of Least Concern under the IUCN criteria; however, the high temporal fragmentation index and the very low values of the Ne/N ratios obtained for this species, as compared with those recorded for most others, are usually taken as indicators of actual threat for their conservation. As a cautionary measure, managers should aim at maintaining gene flow by ensuring connectivity of Pinus sylvestris in these areas. [ABSTRACT FROM AUTHOR]

The phylogeography of montane species often reveals strong genetic differentiation among mountain ranges. Both classic morphological and genetic studies have indicated distinctiveness of Pyrenean populations of the butterfly Erebia euryale. This hypothesis remained inconclusive until data from the westernmost populations of the distribution area (Cantabrian Mountains) were analysed. In the present study, we set out to describe the population structure of Erebia euryale in western Cantabria, where the species occurs in scattered localities. For this goal, we estimate the genetic diversity and differentiation found in 218 individuals from six Cantabrian (North Spain) localities genotyped by 17 allozyme loci. We also sequence 816 bp of the cytochrome oxidase subunit I mitochondrial gene in 49 individuals from Cantabrian localities and 41 specimens from five other European sites. Mitochondrial data support the recognition of four major genetic groups previously suggested for the European populations based on allozyme polymorphisms. Both mitochondrial and nuclear markers reveal genetic distinctiveness of a single Pyrenean-Cantabrian lineage of E. euryale. The lack of geographical structure and the star-like topology displayed by the mitochondrial haplotypes indicate a pattern of demographic expansion in northern Iberia, probably related to Upper Pleistocene climatic oscillations. By contrast, within the Pyrenean-Cantabrian lineage, Cantabrian samples are genetically structured in nuclear datasets. In particular, San Isidro is significantly differentiated from the other five populations, which cluster into two groups. We recognize an evolutionary significant unit for Pyrenean-Cantabrian populations of Erebia euryale. Our results also illustrate that the evolutionary history of a species may be shaped by processes undetectable by using mtDNA alone. [ABSTRACT FROM AUTHOR]

Ten polymorphic microsatellite loci were developed for the butterfly Erebia palarica (Nymphalidae, Satyrinae), endemic to NW Spain. Polymorphism for 20 individuals from a NW Spanish population (Ancares) and 15 more from two other nearby localities (Courel, Trevinca) was assessed. Overall, the number of alleles per locus ranged from six to 24. Ancares showed an average number of alleles per locus of 14.30 (SD = 5.32), observed heterozygosity of 0.753 (SD = 0.14) and unbiased expected heterozygosity of 0.818 (SD = 0.114). Genotypic frequencies conformed to the Hardy-Weinberg equilibrium at Ancares, and there was no evidence of linkage disequilibrium. Multilocus genotypes resulting from this set of markers will be useful for determining genetic diversity and differentiation within and among populations of this local endemic butterfly in NW Spain. Of the six loci amplified in the closely related species, Erebia meolans, five were polymorphic. [ABSTRACT FROM AUTHOR]