Abstract: An adaptive role of corolla shape has been often asserted without an empirical demonstration of how natural selection acts on this trait. In generalist plants, in which flowers are visited by diverse pollinator fauna that commonly vary spatially, detecting pollinator-mediated selection on corolla shape is even more difficult. In this study, we explore the mechanisms promoting selection on corolla shape in the generalist crucifer Erysimum mediohispanicum Polatschek (Brassicaceae). We found that the main pollinators of E. mediohispanicum (large bees, small bees and bee flies) discriminate between different corolla shapes when offered artificial flowers without reward. Importantly, different pollinators prefer different shapes: bees prefer flowers with narrow petals, whereas bee flies prefer flowers with rounded overlapping petals. We also found that flowers with narrow petals (those preferred by bees) produce both more pollen and nectar than those with rounded petals. Finally, different plant populations were visited by different faunas. As a result, we found spatial variation in the selection acting on corolla shape. Selection favoured flowers with narrow petals in the populations where large or small bees are the most abundant pollinator groups. Our study suggests that pollinators, by preferring flowers with high reward, exert strong selection on the E. mediohispanicum corolla shape. The geographical variation in the pollinator-mediated selection on E. mediohispanicum corolla shape suggests that phenotypic evolution and diversification can occur in this complex floral trait even without specialization.

Abstract: BACKGROUND AND AIMS: Floral rewards may be associated with certain morphological floral traits and thus act as underlying factors promoting selection on these traits. This study investigates whether some traits that are under pollinator-mediated selection (flower number, stalk height, corolla diameter, corolla tube length and corolla tube width) in the Mediterranean herb E. mediohispanicum (Brassicaceae) are associated with rewards (pollen and nectar). METHODS: During 2005 the phenotypic traits and the visitation rate of the main pollinator functional groups were quantified in 720 plants belonging to eight populations in south-east Spain, and during 2006 the same phenotypic traits and the reward production were quantified in 400 additional plants from the same populations. KEY RESULTS: A significant correlation was found between nectar production rate and corolla tube length, and between pollen production and corolla diameter. Visitation rates of large bees and butterflies were significantly higher in plants exhibiting larger flowers with longer corolla tubes. CONCLUSIONS: The association between reward production and floral traits may be a factor underlying the pattern of visitation rate displayed by some pollinators.

Abstract: One outstanding and unsolved challenge in ecology and conservation biology is to understand how pollinator diversity affects plant performance. Here, we provide evidence of the functional role of pollination diversity in a plant species, Erysimum mediohispanicum (Brassicaceae). Pollinator abundance, richness and diversity as well as plant reproduction and recruitment were determined in eight plant populations. We found that E. mediohispanicum was generalized both at the regional and local (population) scale, since its flowers were visited by more than 100 species of insects with very different morphology, size and behaviour. However, populations differed in the degree of generalization. Generalization correlated with pollinator abundance and plant population size, but not with habitat, ungulate damage intensity, altitude or spatial location. More importantly, the degree of generalization had significant consequences for plant reproduction and recruitment. Plants from populations with intermediate generalization produced more seeds than plants from populations with low or high degrees of generalization. These differences were not the result of differences in number of flowers produced per plant. In addition, seedling emergence in a common garden was highest in plants from populations with intermediate degree of generalization. This outcome suggests the existence of an optimal level of generalizations even for generalized plant species.

Abstract: The number of nucleoli and nucleolar area were measured in meiotic cells from males of the grasshopper Eyprepocnemis plorans collected in three natural populations. Number of nucleoli per cell showed no significant correlation among cells in different meiotic stages, but there was strong positive correlation for nucleolar area between leptotene and interkinesis cells in individuals from distant populations (Salobreña in Spain, and Smir in Morocco). No correlation was, however, observed for both parameters between the meiotic stages analysed in individuals from the population of Torrox (Spain). The number of nucleoli at leptotene was about double the number at interkinesis, as expected from the double ploidy level at leptotene and the corresponding double number of rDNA clusters. Leptotene nucleolar area, however, was about fourfold that in interkinesis, presumably due to higher requirements for ribosome biogenesis in meiosis I than meiosis II. In Torrox, diplotene cells showed a lower number of nucleoli but larger nucleolar area than in leptotene cells, suggesting an increase in nucleolus size during prophase I. Significant differences were found among populations for nucleolar area but not for number of nucleoli, the smallest nucleolar area being observed in Torrox, which is the population harbouring the most parasitic B chromosome variant. No clear effects on nucleolar area or number of nucleoli were associated with the B-chromosome number. However, B-chromosome effects on the nucleolar area were apparent in the Torrox population when data were analysed with respect to a B-chromosome odd-even pattern in leptotene and interkinesis cells. However, in diplotene cells no odd-even pattern was observed for both nucleolar parameters, suggesting that the increase in nucleolar size from leptotene to diplotene dilutes the leptotene odd-even pattern. The rDNA distally located in the B chromosome was associated with a nucleolus in 6.5% out of the 247 diplotene cells analysed. The implications of these findings are discussed in the context of B chromosomes as stress-causing genome parasites and the nucleolus as a sensor of stress.

Abstract: Paleontological and phylogenetic studies have shown that floral zygomorphy (bilateral symmetry) has evolved independently in several plant groups from actinomorphic (radially symmetric) ancestors as a consequence of strong selection exerted by specialized pollinators. Most studies focused on unraveling the developmental genetics of flower symmetry, but little is known about the adaptive significance of intraspecific flower shape variation under natural conditions. We provide the first evidence for natural selection favoring zygomorphy in a wild population of Erysimum mediohispanicum (Brassicaceae), a plant showing extensive continuous variation in flower shape, ranging from actinomorphic to zygomorphic flowers. By using geometric morphometric tools to describe flower shape, we demonstrate that plants bearing zygomorphic flowers received more pollinator visits and had the highest fitness, measured not only by the number of seeds produced per plant but also by the number of seeds surviving to the juvenile stage. This study provides strong evidence for the existence of significant fitness differences associated with floral shape variation in E. mediohispanicum, thus illuminating a pathway for the evolution of zygomorphy in natural populations.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: A previous study showed that in the haplodiploid solitary wasp Trypoxylon albitarse, most individuals carry one B chromosome per haploid genome, the same dosage as the standard (A) chromosomes, indicating a possible regularization of B-chromosome meiotic behaviour and its integration into the A genome. In a new sampling, we have analysed 15 populations (including 9 out of the 10 previously analysed) to test the evolution of this integration process. The new results provide a direct report of the invasion process in the Porto Firme population, where B frequency has dramatically increased in only four generations. In the populations from the Viçosa region, however, B frequency has remained stable, although the principal B type, the metacentric one, has increased in frequency at the expense of the acrocentric one in several populations. The implications of these new results on the hypothesis of the integration of these B chromosomes, as regular members of the A genome, are discussed.

Abstract: The transmission of the B1 chromosome through females has been analysed in three Moroccan populations (Smir, SO.DE.A. and Mechra) of the grasshopper Eyprepocnemis plorans. We analysed transmission ratio (kB) variation at two levels: intra-individual (to test female age effects) and inter-individual (to test for A chromosome effects). In 81.8% of females, kB did not differ among successive egg-pods, suggesting no effect of female age. The remaining females (18.2%), showed significant differences in kB values among egg-pods, but without clear temporal patterns. In Smir, kB ranged between B elimination (0.244) and B accumulation (0.689) but there was no net accumulation (mean +/- s.e. = 0.463 +/- 0.045). In SO.DE.A., all females analysed transmitted B1 at a Mendelian rate, with a mean kBequal to 0.512 +/- 0.020. In Mechra, kB ranged from 0.341 to 0.972, with mean kB (0.575 +/- 0.029) showing a net B accumulation. All these observations suggest that the B1 chromosome could be at a drive-suppression stage in Smir and Mechra, but that it has already been neutralised in SO.DE.A.

Abstract: 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.

Abstract: 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.

Abstract: B chromosomes are genome symbionts, the presence of which in many eukaryote species is explained, in most cases, by their violation of Mendelian rules, usually based on meiotic or mitotic instability, leading to their accumulation in the germ line (drive). However, B chromosome integration into the genome as a regular member of the chromosome set should imply the loss of drive. A possible way of bypassing this difficulty is to regularize meiosis when the B chromosome is frequent in the population, in order to yield gametes with one B chromosome. In diploid organisms, this task needs to be achieved in the two sexes, but in haplodiploids the problem simplifies to only the diploid sex. We have found, to the authors' knowledge, the first evidence of a B chromosome that is regularizing its meiotic behaviour and limiting its number to one B chromosome per haploid genome, the same dosage as the standard (A) chromosomes, in the solitary wasp Trypoxylon albitarse. It suggests a possible mechanism for B chromosome integration as a regular member of the chromosome complement.

Abstract: A centric fragment was generated during the introgression of a chromosome region from Nasonia giraulti into N. vitripennis. This neo B chromosome carries the N. giraulti or 123+ gene for wild-type eye color. Using this phenotypic effect, the transmission of this chromosome was analyzed. The supernumerary chromosome showed less than Mendelian segregation rate in meiosis and some mitotic instability manifested as mosaic phenotype for eye color. However, transmission rate and mitotic stability increased over successive generations. The transmission rate through male gametogenesis was nearly 100%. These results support the interspecific hybridization model for B chromosome origin and reveal that problems in chromosome stability can persist for several generations after "foreign chromosomes" are introduced into a different species. We suggest that hybrid zones should be investigated as possible sites for neo-B chromosome generation.

Abstract: Fluorescence in situ hybridization revealed the presence of ribosomal RNA genes in paracentromeric regions of all A chromosomes and in the distal half of B chromosomes in embryonic cells from Moroccan specimens of the grasshopper Eyprepocnemis plorans. The expression of these genes was monitored by the presence of nucleoli attached to each chromosome bivalent in diplotene cells from males collected from two different Moroccan populations and was compared to previous data of Spanish populations. Whereas only the nucleolus organizer regions (NORs) on S9-S11 and X chromosomes were active in the Spanish specimens, Moroccan individuals showed NOR activity in all chromosomes. The rRNA genes on the B chromosome were inactive in both populations. The S9 and S10 NORs were less active in Moroccan specimens than in Spanish specimen, which might be partly explained by the negative inter-dependence for expression of the S10 NOR with respect to those on L2 and X chromosomes. On the other hand, the X NOR was more active in Moroccan specimens than in Spanish specimens, and this might be partly due to the positive effect that the presence of B chromosomes has on the expression of this NOR. The implications of these observations on current models of NOR activity regulation are discussed.

Abstract: Several components of fitness were analysed in relation to the presence of a supernumerary chromosome segment (SCS) in two natural populations of the grasshopper Eyprepocnemis plorans, including clutch size, egg fertility, egg and embryo productivity and survivability from embryo to adult, and SCS transmission through males. The results have shown the absence of a significant relationship between SCS presence and these fitness components, with the single exception of egg fertility which decreases significantly in SCS females with mating shortage. This fertility decrease is thus expected to be relevant for the population dynamics of the SCS only in low-density populations, those in which it is difficult for females to find a male to copulate with before each egg-batch is ready to be laid. The analysis of the SCS transmission through males showed no significant differences between expected and observed SCS frequencies. The SCS polymorphism seems to be at a status close to neutrality in respect to fitness, but its slight disadvantage in transmission through females carrying B chromosomes predicts that the polymorphism should tend to disappear, unless SCS recurrent amplification, or another undiscovered force, counteracts this tendency.

Abstract: Six populations of the fish Prochilodus lineatus were analysed for B chromosome frequency. A study of spermatogenesis revealed the absence of B accumulation during the stages analysed. In one of the populations, from the Mogi-Guaçu river where samples have been analysed over a ten-year period, B chromosome frequency doubled between 1979-80 and 1987 89, whereas no additional changes were noticed in samples collected in 1991-92. The analysis of B chromosome mitotic instability, manifested by intraindividual variation in B chromosome number, indicated a very significant decrease during this time period. This suggests that, in the 1980s, this population was in the final stage of B chromosome invasion, and that there was a possible causal relationship between B mitotic instability and the accumulation mechanism that caused its frequency increase. Mitotic stabilization might thus be a way by which a mitotically unstable B chromosome may become neutralized.

Abstract: Polymorphism for B chromosomes has been detected in all nine populations of the grasshopper Eyprepocnemis plorans ssp. plorans sampled in Morocco. The most frequent B chromosome in all populations showed a C-banding pattern and size similar to those of the B1 variant found in the Iberian Peninsula. In addition, other B chromosome variants (B1iso1, B1iso2, B1d1, B1di1, B3 and B1dd1) were discovered in these populations, although at a very low frequency. No significant differences in B chromosome frequency were found either in the nine populations or, for some of them, in up to three consecutive years. These results are discussed in the light of current hypotheses on the evolution of this B chromosome polymorphism in the Iberian Peninsula.