Dinámica espacial y temporal de los cromosomas b del saltamontes eyprepocnemis plorans

Resumen

We performed a molecular phylogenetic analysis after sequencing three mitochondrial genome regions, i.e. two regions in the cytochrome oxidase I gene (COI) and a region of the NADH dehydrogenase (ND5), in 15 populations of E. plorans plorans from the Iberian Peninsula, Morocco, Greece and Armenia, and one population of E. plorans meridionalis from South Africa. In addition, we included the relative species E. unicolor, Shirakiacris shirakii, Heteracris adspersa and Heteracris pterosticha, and Chorthippus chinensis and Locusta migratoria as outgroups. Our objective was to obtain phylogeographic information in E. plorans, as well as uncovering possible colonization routes for this species from Africa (since the Eyprepocnemis genus is of African origin). In addition, the inclusion of other Eyprepocnemidinae species can provide some phylogenetic information on the origin of this genus and species. Our results showed that Eastern (Greece and Armenia) and Western (Spain and Morocco) populations of E. plorans plorans diverged about a million to 270,000 years ago. In addition, haplotype networks for ND5 and COI showed that the two subspecies could be derived from a common ancestor, probably living in central Africa, which evolved into E. plorans meridionalis towards the south and into E. plorans plorans towards the north, but in two separate (Eastern and Western) lineages. It is also remarkable that the number of mutational steps between the hypothetical African common ancestor and each Eastern or Western lineage is about the same as that between the Eastern and Western lineages themselves, suggesting that these populations have had little gene flow since they separated from the common ancestor. This fact is consistent with the differences previously observed for DNA composition between Eastern and Western B chromosomes. We also estimated a maximum age of about three million years for the B chromosome, which is present only in the E, plorans plorans subspecies, and four million years for the 180 bp tandem repeat DNA, which is exclusive of the E. plorans species. We also analysed ten E. plorans plorans populations from the Iberian Peninsula, for 97 Inter-Simple Sequence Repeats (ISSR) markers. Five populations were collected in coastal localities from the Granada and Malaga provinces, whereas the remaining five populations were collected at the head of the Segura river basin. We first analysed the repeatability and inheritance of these markers to evaluate their validity as population genetics markers. We found a low proportion of markers showing allelic dropout, and this phenomenon explained most segregation distortions observed in controlled crosses. However, when the population genetics analyses were repeated with the 87 ISSR markers less affected by dropout, the results did not differ from those obtained with the 97 markers, thus suggesting that allelic dropout does not produce a significant bias to these results. The populations analysed showed 100% of polymorphic ISSR loci when all ten populations were considered, and 97% for coastal and 96% for Segura populations. The FST value calculated for the ten populations (0.108) indicated significant subdivision among populations, as was also indicated by an AMOVA showing that 17.34% of molecular variation was found among populations. When two regions (coastal and Segura) were defined, the AMOVA showed higher variation between regions (15.27%) than among populations within regions (7.58%). The analysis of population structure, by the software STRUCTURE, showed that the ten populations belong to two genetically differentiated groups, one including the five populations from the coast and the other including the five populations from the Segura river basin. Analyses performed, at lower scale, separately in each region, revealed higher genetic differentiation among the coastal populations than among the Segura populations. The Nm value estimated from the average FST was NM= 2 for the ten populations, suggesting that gene flow is slightly more intense than genetic drift. At lower scale, however, Nm values were higher (4 in the coastal and 6 in the Segura regions) suggesting higher rates of gene flow within each region, and very low between regions. This was consistent with the significant isolation by distance (IBD) found among the ten populations. When IBD was tested at each region, we found significant IBD in the coastal populations but not in those of the Segura river basin. In fact, Nm values between pairs of populations were higher in the the Segura region that in the Granada and Malaga coast. The low FST values found among Segura populations could suggest that these populations have recently been colonized from a common ancestor population. The possibility that this could have occurred in association with Pleistocene glaciations is discussed. To investigate the biological role of B chromosomes in E. plorans, we performed an experiment of artificial invasion of a B chromosome in a natural population (El Gallego, Albacete) where B chromosomes had not previously been found. In 1994, fifty males from the Salobreña (Granada) population were released in El Gallego after performing a triple mark-recapture experiment to estimate population size (N= 1439). The male sample carried an average of 0.8 B chromosomes per individual (as deduced from previous analyses in the Salobreña population), implying a mean number of Bs equal to 0.03 Bs per individual at experiment start. The most frequent B variant in Salobreña is B2, which was the one presumptly introduced in El Gallego. We analysed B frequence at six years between 1996 and 2008, and found that B frequency apparently increased in the 1996 and 1999 samples but decreased from 2000 onwards. The analysis of three controlled crosses involving B-carrying females caught at El Gallego, showed B transmission rates significantly lower than the Mendelian one. In addition, these three females laid egg-pods containing less eggs than those laid by 0B females from the same population. All these facts explain why B frequency has decreased in the last years. The possibility that this could be due to the introduction of A chromosome genes being able to suppress B drive in the donor population, is discussed. To ascertain whether the males released had effectively crossed to resident females, we analysed 97 ISSR markers in El Gallego, Salobreña and four other populations from the Segura river basin. This indicated the presence of two individuals captured at El Gallego in 2006 showing an ISSR profile from Salobreña. The B chromosomes found in El Gallego were larger than the B2 variant from Salobreña and B1 from other populations (e.g. Calasparra) being close to El Gallego, and showed intermediate characteristics in respect to the relative amount of rDNA and satDNA. This suggests the possibility that interaction with the new host genome has provoked changes in the B implying unequal amplification of these two types of repetitive DNAs. The possibility that these B chromosome changes were promoted by activation of mobile elements is discussed. In an attempt to uncover the causes why a newly parasitic B chromosome variant (i.e. B24) is able to replace the old neutralized variant (i.e. B2), we analysed the B chromosome distribution of these two variants in several populations located towards the east (Nerja, Tetuan and Maro) and the west (Algarrobo) of the Torrox population, which is the place were it is thought that this variant originated. In Algarrobo, we found an average transmission ratio for B24 (0.6) significantly higher than the Mendelian one, but lower than the 0.696 observed in a sample from Torrox collected in 1992 by Zurita et al. (1998). The additional fact that B24 has not reached in Algarrobo frequencies as high as those reported in Torrox (1.5 Bs per individual, on average) suggest that B24 invasion in Algarrobo has been less virulent than in Torrox. This might be due to the joint introduction in Algarrobo of B24 and the suppressor genes against this variant that evolved very fast in Torrox (Perfectti et al. 2004). Our analysis of populations towards the east of Torrox suggest temporal changes in B frequency which are mostly estocastic, although a tendency to increase of B24 frequency was observed in two populations collected at Nerja. Remarkably, The frequency of B24 was higher in Nerja (the closest location to Torrox towards the east) than Tetuan and Maro, suggesting that B24 is currently spreading towards the east. In looking for DNA sequences being located on B chromosomes, we analysed the possible association of the 97 ISSR markers with B chromosome presence. A total of 12 ISSR markers showed positive association with B presence. Since this association was partial, it is most likely that they are also present in the A chromosomes, as it is logical for B chromosomes arisen intraspecifically, which is the most accepted hypothesis in E. plorans. This opens the possibility to analyse microsatellite DNA sequences from B chromosomes, as well as DNA sequences close to them. This would provide additional markers to investigate the origin and late differentiation of B chromosomes in this species.