Niche differences may explain the geographic distribution of cytotypes in Erysimum mediohispanicum.

Polyploidisation has played an important role in plant diversification, and variation in ploidy level may be found not only between species of the same genus, but also within a single species. Although establishing the adaptive significance of polyploidy to explain the geographic distribution of cytotypes is challenging, the occurrence of different cytotypes in different ecological niches may suggest an adaptive role of genome duplication. We studied the adaptive significance of the geographic distribution of cytotypes across the entire distribution range of the endemic Erysimum mediohispanicum (Brassicaceae). For that, we have used climate variables, population elevation and soil properties to model ecological niches for the different cytotypes. In addition, we analysed the effect that ploidy level has on the floral phenotype. We found a clear geographic pattern in the distribution of cytotypes, with diploid individuals occurring in the southernmost part of the distribution range, while tetraploids were found in the northern area. A contact (mosaic) zone between both cytotypes was identified, but diploids and tetraploids occur in sympatry in only one population (although in a highly unbalanced proportion). Gene flow between different cytotypes seems to be negligible, as evident from an almost complete absence of triploids and other minority cytotypes. Niches occupied by both cytotypes showed subtle, but significant differences, even in the contact zone. Precipitation was higher in regions occupied by tetraploid individuals, which present wider corolla tubes and thinner but taller stalks than diploids. Our findings highlight the potential role of polyploidy in the ecological adaptation of E. mediohispanicum to both abiotic factors and biotic interactions.

Drivers of genetic differentiation in a generalist insect-pollinated herb across spatial scales.

The isolation-by-distance model (IBD) predicts that genetic differentiation among populations increases with geographic distance. Yet, empirical studies show that a variety of ecological, topographic and historical factors may override the effect of geographic distance on genetic variation. This may particularly apply to species with narrow but highly heterogeneous distribution ranges, such as those occurring along elevational gradients. Using nine SSR markers, we study the genetic differentiation of the montane pollination-generalist herb, Erysimum mediohispanicum. Because the effects of any given factor may depend on the geographic scale considered, we investigate the contribution of different environmental and historical factors at three different spatial scales. We evaluate five competing models that put forward the role of geographic distance, local environmental factors [biotic interactions (IBEb) and climatic variables (IBEa)], landscape resistance (IBR) and phylogeographic patterns (IBP), respectively. We find significant IBD regardless of the spatial scale and the genetic distance estimator considered. However, IBEa and IBP also play a prominent role in shaping genetic differentiation patterns at the larger spatial scales, and IBR is significant at the fine spatial scale. Overall, our results highlight the importance of combining different estimators, statistical approaches and spatial scales to disentangle the relative importance of the various ecological factors contributing to the shaping of genetic divergence patterns in natural populations.

Non-random expression of ribosomal DNA units in a grasshopper showing high intragenomic variation for the ITS2 region.

We analyse intragenomic variation of the ITS2 internal transcribed spacer of ribosomal DNA (rDNA) in the grasshopper Eyprepocnemis plorans, by means of tagged PCR 454 amplicon sequencing performed on both genomic DNA (gDNA) and RNA-derived complementary DNA (cDNA), using part of the ITS2 flanking coding regions (5.8S and 28S rDNA) as an internal control for sequencing errors. Six different ITS2 haplotypes (i.e. variants for at least one nucleotide in the complete ITS2 sequence) were found in a single population, one of them (Hap4) being specific to a supernumerary (B) chromosome. The analysis of both gDNA and cDNA from the same individuals provided an estimate of the expression efficiency of the different haplotypes. We found random expression (i.e. about similar recovery in gDNA and cDNA) for three haplotypes (Hap1, Hap2 and Hap5), but significant underexpression for three others (Hap3, Hap4 and Hap6). Hap4 was the most extremely underexpressed and, remarkably, it showed the lowest sequence conservation for the flanking 5.8-28S coding regions in the gDNA reads but the highest conservation (100%) in the cDNA ones, suggesting the preferential expression of mutation-free rDNA units carrying this ITS2 haplotype. These results indicate that the ITS2 region of rDNA is far from complete homogenization in this species, and that the different rDNA units are not expressed at random, with some of them being severely downregulated.

Association between inbreeding depression and floral traits in a generalist-pollinated plant.

Individual variation in the magnitude of inbreeding depression (ID) in plants and its association with phenotypic traits may have important consequences for mating system evolution. This association has been investigated only scarcely, and always considering traits functionally related to autogamy. Here, we explore the association between individual variation in ID and plant traits associated with pollinator attractiveness (related to plant size, corolla size and corolla shape) in two populations of Erysimum mediohispanicum (Brassicaceae). ID was calculated along the entire life cycle of the plants. In addition, we also explored the relationship between phenotypic traits and the individual levels of heterozygosity. We found significant associations between ID and corolla diameter and stalk height, being taller plants with larger corollas those undergoing a lower intensity of ID. Furthermore, we found a negative relationship between corolla diameter and heterozygosity, suggesting that plants with large flowers have purged their genetic load. Finally, we found a significant effect of corolla diameter on the intrapopulation genetic structure. All these findings suggest that plants with large flowers have secularly suffered frequent inbreeding in the study populations. Because corolla diameter is a trait frequently selected by pollinators in E. mediohispanicum, we believe that the observed relationship between this trait and ID could be mediated by pollinators, probably throughout an increasing in biparental inbreeding, geitonogamy or autogamy.

Disparate molecular evolution of two types of repetitive DNAs in the genome of the grasshopper Eyprepocnemis plorans.

Wide arrays of repetitive DNA sequences form an important part of eukaryotic genomes. These repeats appear to evolve as coherent families, where repeats within a family are more similar to each other than to other orthologous representatives in related species. The continuous homogenization of repeats, through selective and non-selective processes, is termed concerted evolution. Ascertaining the level of variation between repeats is crucial to determining which evolutionary model best explains the homogenization observed for these sequences. Here, for the grasshopper Eyprepocnemis plorans, we present the analysis of intragenomic diversity for two repetitive DNA sequences (a satellite DNA (satDNA) and the 45S rDNA) resulting from the independent microdissection of several chromosomes. Our results show different homogenization patterns for these two kinds of paralogous DNA sequences, with a high between-chromosome structure for rDNA but no structure at all for the satDNA. This difference is puzzling, considering the adjacent localization of the two repetitive DNAs on paracentromeric regions in most chromosomes. The disparate homogenization patterns detected for these two repetitive DNA sequences suggest that several processes participate in the concerted evolution in E. plorans, and that these mechanisms might not work as genome-wide processes but rather as sequence-specific ones.

Evolution of pollination niches and floral divergence in the generalist plant Erysimum mediohispanicum.

BACKGROUND AND AIMS: How generalist plants diverge in response to pollinator selection without becoming specialized is still unknown. This study explores this question, focusing on the evolution of the pollination system in the pollination generalist Erysimum mediohispanicum (Brassicaceae). METHODS: Pollinator assemblages were surveyed from 2001 to 2010 in 48 geo-referenced populations covering the entire geographic distribution of E. mediohispanicum. Bipartite modularity, a complex network tool, was used to find the pollination niche of each population. Evolution of the pollination niches and the correlated evolution of floral traits and pollination niches were explored using within-species comparative analyses. KEY RESULTS: Despite being generalists, the E. mediohispanicum populations studied can be classified into five pollination niches. The boundaries between niches were not sharp, the niches differing among them in the relative frequencies of the floral visitor functional groups. The absence of spatial autocorrelation and phylogenetic signal indicates that the niches were distributed in a phylogeographic mosaic. The ancestral E. mediohispanicum populations presumably belonged to the niche defined by a high number of beetle and ant visits. A correlated evolution was found between pollination niches and some floral traits, suggesting the existence of generalist pollination ecotypes. CONCLUSIONS: It is conjectured that the geographic variation in pollination niches has contributed to the observed floral divergence in E. mediohispanicum. The process mediating this floral divergence presumably has been adaptive wandering, but the adaptation to the local pollinator faunas has been not universal. The outcome is a landscape where a few populations locally adapted to their pollination environment (generalist pollination ecotypes) coexist with many populations where this local adaptation has failed and where the plant phenotype is not primarily shaped by pollinators.

Level of heat shock proteins decreases in individuals carrying B-chromosomes in the grasshopper Eyprepocnemis plorans.

We analyzed the effect of B-chromosome presence on expression level of heat shock protein 70 (Hsp70) in cerebral ganglion and gonad in both males and females of the grasshopper Eyprepocnemis plorans. Two natural Spanish populations, Salobreña (Granada) and Torrox (Málaga) were assayed, the former harbouring a neutralized (non-driving) B-chromosome (B(2)) and the latter a parasitic (driving) B-chromosome (B(24)). The analysis was performed by Western blotting, immunostaining and densitometric measuring expression level of the Hsp70 family in adult individuals. The results showed that Hsp70 levels of testis were significantly higher in Salobreña than Torrox, and were significantly lower in testes of B-carrying males from both populations. A similar effect was observed in the ovary of females from Torrox. No effect was, however, observed in cerebral ganglia in any sex or population. B-chromosome effects in Torrox showed a dose-dependent pattern. The results point to an interesting interaction between B-chromosome and stress protein expression in reproductive tissue.

The genetic basis of interspecies host preference differences in the model parasitoid Nasonia.

The genetic basis of host preference has been investigated in only a few species. It is relevant to important questions in evolutionary biology, including sympatric speciation, generalist versus specialist adaptation, and parasite-host co-evolution. Here we show that a major locus strongly influences host preference in Nasonia. Nasonia are parasitic wasps that utilize fly pupae; Nasonia vitripennis is a generalist that parasitizes a diverse set of hosts, whereas Nasonia giraulti specializes in Protocalliphora (bird blowflies). In laboratory choice experiments using Protocalliphora and Sarcophaga (flesh flies), N. vitripennis shows a preference for Sarcophaga, whereas N. giraulti shows a preference for Protocalliphora. Through a series of interspecies crosses, we have introgressed a major locus affecting host preference from N. giraulti into N. vitripennis. The N. giraulti allele is dominant and greatly increases preference for Protocalliphora pupae in the introgression line relative to the recessive N. vitripennis allele. Through the utilization of a Nasonia genotyping microarray, we have identified the introgressed region as 16 Mb of chromosome 4, although a more complete analysis is necessary to determine the exact genetic architecture of host preference in the genus. To our knowledge, this is the first introgression of the host preference of one parasitoid species into another, as well as one of the few cases of introgression of a behavioral gene between species.

Abnormal spermatid formation in the presence of the parasitic B(24) chromosome in the grasshopper Eyprepocnemis plorans.

Morphology and size of spermatids were analysed in the grasshopper Eyprepocnemis plorans by means of light and electron microscopy. At light microscopy, normal and abnormal (macro- and micro-) spermatids differed in size and number of centriolar adjuncts (CAs): 1 CA in normal spermatids and 2 or more CAs, depending on ploidy level, in macrospermatids. Males carrying the additional B(24) chromosome showed significantly more macro- and microspermatids than 0B males. The frequency of macro- and microspermatids showed an odd-even pattern in respect to the number of B chromosomes, with a higher frequency of abnormal spermatids associated with odd B numbers. Transmission electron microscopy showed that macrospermatids carried more than one axoneme, depending on ploidy level: 2 for diploid, 3 for triploid, and 4 for tetraploid spermatids. In 0B males, the most frequent abnormal spermatids were diploid, whereas in 1B males they were the tetraploid spermatids and, to a lesser extent, triploid ones. This suggests that most macrospermatids derived from cytokinesis failure and nucleus restitution. The implications of aberrant spermatids on B chromosome transmission and male fertility are discussed.

Microdissection and chromosome painting of X and B chromosomes in the grasshopper Eyprepocnemis plorans.

The relative location of 2 repetitive DNAs, i.e. ribosomal (rDNA) and a tandemly repeated satellite DNA (satDNA), with respect to the centromere, suggested that B chromosomes in the grasshopper Eyprepocnemis plorans derived intraspecifically from the X chromosome. To test this hypothesis, we microdissected X and B chromosomes and amplified the obtained DNA by 2 different procedures, the conventional DOP-PCR method and the single-cell whole-genome amplification GenomePlex method. We then generated DNA probes to perform chromosome painting. Our results have confirmed that X and B chromosomes share many DNA sequences between them and with most of the autosomes, especially at locations where the satDNA and rDNA reside, in consistency with previous information. This supports the hypothesis of an intraspecific origin of B chromosomes in E. plorans. Nevertheless, the present results did not help to clarify whether Bs were derived from the X chromosome or else from 1 or more autosomes.

Local adaptation and maladaptation to pollinators in a generalist geographic mosaic.

The Geographic Mosaic Theory of Coevolution predicts the occurrence of mosaics of interaction-mediated local adaptations and maladaptations. Empirical support to this prediction has come mostly from specialist interactions. In contrast, local adaptation is considered highly unlikely in generalist interactions. In this study, we experimentally test local adaptation in a generalist plant-pollinator geographic mosaic, by means of a transplant experiment in which plants coming from two evolutionary hotspots and two coldspots were offered to pollinators at the same four localities. Plants produced in the hotspots attracted more pollinators in all populations, whereas coldspot plants attracted fewer pollinators in all populations. Differences in adaptation were not related to genetic similarity between populations, suggesting that it was mainly due to spatial variation in previous selective regimes. Our experiment provides the first strong support for a spatially structured pattern of adaptation and maladaptation generated by a generalist free-living mutualism.

Rapid suppression of drive for a parasitic B chromosome.

The persistence of parasitic B chromosomes in natural populations depends on both B ability to drive and host response to counteracting it. In the grasshopper Eyprepocnemis plorans, the B24 chromosome is the most widespread B chromosome variant in the Torrox area (Málaga, Spain). Its evolutionary success, replacing its ancestral neutralized B variant, B2, was based on meiotic drive in females, as we showed in a sample caught in 1992. In females collected six years later, mean B24 transmission ratio (k(B)) was 0.523, implying a very rapid decrease from the 0.696 observed in 1992. This shows that B24 neutralization is running very fast and suggests that it might most likely be based on a single gene of major effect.

The odd-even effect in mitotically unstable B chromosomes in grasshoppers.

The odd-even effect, by which B chromosomes are more detrimental in odd numbers, has been reported in plants and animals. In grasshoppers, there are only a few reports of this effect and all were referred to as traits related to the formation of aberrant meiotic products (AMPs). Here we review the existing information about B chromosome effects on AMPs, chiasma frequency and the number of active nucleolus organizer regions (NORs) per cell. Polysomy for A chromosomes and B chromosomes are two kinds of chromosome polymorphism frequently found in grasshoppers. In some aspects, e.g. meiotic behaviour and mitotic instability leading to individual mosaicism (in the case of mitotically unstable Bs), polysomic As show similar characteristics to B chromosomes. In fact, polysomy is regarded as one of the main mechanisms for B chromosome origin. Here we review some features of meiotic behaviour in known cases of polysomy and mitotically unstable Bs in grasshoppers, in looking for possible causes for the odd-even effect. In all these traits, the odd-even effect was apparent, although its appearance was not universal in any case, with variation among species or populations within the same species. The equational division and lagging of the extra chromosomes, when univalents, could favour the appearance of abnormal meiotic products, and the formation of bivalents, when there are two or more extra chromosomes, inhibits this process. Therefore, the odd-even effect might be a consequence of the concomitant operation of both aspects of extra chromosome meiotic behaviour. The possibility that the odd-even effect might result from an increase in cell stress generated by odd numbers is suggested.

Spatio-temporal dynamics of a neutralized B chromosome in the grasshopper Eyprepocnemis plorans.

Spatial and temporal patterns of frequency variation for a neutralized B chromosome in the grasshopper Eyprepocnemis plorans were analyzed along six transects in the east of Spain to explore possible factors affecting the population dynamics of this polymorphism. Three parameters were employed to quantify B frequency: prevalence, load and mean frequency. Of them, load seemed to be the less sensitive parameter, probably due to its small range of variation. Prevalence, however, shows ample variation, but the mean frequency of B chromosomes per individual is the best parameter to characterize B frequency. Only river transects revealed significant differences among populations, and the use of two geographic explicit approaches (Mantel test and distograms) revealed significant isolation by distance (IBD), especially at the Segura River mouth, presumably due to low gene flow and drift. No temporal trend was found in the Segura River transects, which is consistent with the slow changes in B frequency expected during the random walk for neutralized B chromosomes. But these transects showed a clear spatial pattern, with B1 showing lower frequency in the upper course of this river. The present results provide the first empirical evidence of IBD in the evolution of a neutralized B chromosome, and support the notion that B dynamics at this evolutionary stage is best explained by a metapopulation approach.

Multiregional origin of B chromosomes in the grasshopper Eyprepocnemis plorans.

Analysis of chromosome localization of three molecular markers, 18S-5.8S-28S rDNA, 5S rDNA and a 180 bp satDNA, showed that B chromosomes in the grasshopper Eyprepocnemis plorans originated independently in Eastern (Caucasus) and Western (Spain and Morocco) populations. Eastern B chromosomes are most likely derived from the smallest autosome, which is the only A chromosome carrying the three markers, in coincidence with Caucasian B chromosomes. Western B chromosomes, however, lack 5S rDNA and are most likely derived from the X chromosome, which is the only A chromosome carrying the two remaining markers, always in the same order with respect to the centromere, as the B chromosome.

Population variation in the A chromosome distribution of satellite DNA and ribosomal DNA in the grasshopper Eyprepocnemis plorans.

The double FISH analysis of two repetitive DNAs (a satellite DNA and ribosomal DNA) in 12 natural populations of the grasshopper Eyprepocnemis plorans collected at the south (Granada and Málaga provinces) and south-east (Albacete and Murcia provinces) of the Iberian Peninsula has shown their wide-spread presence throughout the whole genome as well as extensive variation among populations. Both DNAs are found in most A chromosomes. Regularly, both DNAs occurred in the S11 and X chromosomes, rDNA in the S10 and satDNA in the L2 and M3. No correlation was found between the number of satDNA and rDNA clusters in the A genomes of the 12 populations analysed, and both figures were independent of the presence of B chromosomes. The genomic distribution of both DNAs showed no association with the geographical localization of the populations analysed. Finally, we provide evidence that the supernumerary chromosome segment proximally located on the S11 chromosome is, in most cases, the result of satDNA amplification but, in some cases, it might also derive from amplification of both satDNA and rDNA.

Comparative FISH analysis in five species of Eyprepocnemidine grasshoppers.

The chromosomal localization of ribosomal DNA, and a 180 bp satellite DNA isolated from Spanish Eyprepocnemis plorans specimens, has been analysed in five Eyprepocnemidinae species collected in Russia and Central Asia. Caucasian E. plorans individuals carried each of the two DNAs, but the rDNA was limited to only two chromosomes (S(9) and S(11)) in sharp contrast to Spanish specimens that show 4-8 rDNA clusters and to Moroccan specimens which carry rDNA in almost all chromosomes. The four remaining species, however, lacked the 180 bp tandem repeat, and showed rDNA clusters in one (S(9) in Thisoicetrinus pterostichus), two (S(9) and S(10) in Eyprepocnemis unicolor; M(8) and S(11) in Heteracris adspersa), or three (S(9), S(10), and S(11) in Shirakiacris shirakii) chromosome pairs. The implications of these findings for the evolution of these two chromosome markers in this group of species are discussed.

The B chromosomes of the grasshopper Eyprepocnemis plorans and the intragenomic conflict.

The grasshopper Eyprepocnemis plorans harbours an extremely widespread polymorphism for supernumerary (B) chromosomes, which is found in almost all circum-Mediterranean and Caucasian populations hitherto analysed. B chromosomes in this species have been shown to evolve through several stages of parasitic and near-neutral nature, presumably because of an arms race between the standard (A) and B chromosomes. This intragenomic conflict can either be solved with the extinction of the neutralised B chromosome or, more interestingly, with the replacement of the neutralised B by a mutant version being parasitic again and thus prolonging B chromosome life. This species thus provides a complete view of the long-term life-cycle of parasitic B chromosomes.

Genetic load caused by variation in the amount of rDNA in a wasp.

Extensive variation in the size of the short (heterochromatic) arm of chromosome 14 was found in the wasp Trypoxylon (Trypargilum) albitarse. Ten different variants were differentiated by size and C-banding pattern. Fluorescent in-situ hybridization (FISH) revealed that ribosomal DNA in this species is clustered in the darkly C-banded parts of the heterochromatic short arm of chromosome 14. On this basis, we got an indirect estimate of the amount of rDNA from the area of these dark C-bands. The significant absence in males of the three chromosome variants with lower amounts of rDNA indicates that these three variants are lethal in this sex, and suggests the existence of a threshold marking the minimum amount of rDNA which is tolerable in haploidy. This implies about 4% genetic load in the population caused by variation in rDNA amount.

Host recombination is dependent on the degree of parasitism.

Parasites and hosts are involved in a continuous coevolutionary process leading to genetic changes in both counterparts. To understand this process, it is necessary to track host responses, one of which could be an increase in sex and recombination, such as is proposed by the Red Queen hypothesis. In this theoretical framework, the inducible recombination hypothesis states that B-chromosomes (genome parasites that prosper in natural populations of many living beings) elicit an increase in host chiasma frequency that is favoured by natural selection because it increases the proportion of recombinant progeny, some of which could be resistant to both B-chromosome effects and B-accumulation in the germline. We have found a clear parallelism between host recombination and the evolutionary status of the B-chromosome polymorphism, which provides explicit evidence for inducible recombination and strong support for the Red Queen hypothesis.