Wolbachia effects in natural populations of Chorthippus parallelus from the Pyrenean hybrid zone.

We evaluate for the first time the effect of Wolbachia infection, involving two different supergroups, on the structure and dynamics of the hybrid zone between two subspecies of Chorthippus parallelus (Orthoptera) in the Pyrenees. Wolbachia infection showed no effects on female fecundity or a slight increment in females infected by F supergroup, although in the last case it has to be well established. Cytoplasmic incompatibility (CI) is confirmed in crosses carried out in the field between individuals from a natural hybrid population. This CI, registered as the relative reduction in embryo production (sh ), was of sh = 0.355 and sh = 0.286 in unidirectional crosses involving B and F supergroups, respectively. CI also occurred in bidirectional crosses (sh = 0.147) but with a weaker intensity. The transmission rates of the two Wolbachia strains (B and F) were estimated by the optimization of a theoretical model to reach the infection frequencies observed in certain population. To fit this scenario, both supergroups should present transmission rates close to 1. Further, we have simulated the infection dynamics, and hence, the capacity of Wolbachia to structure the population of the host insects and to affect to reproduction and genetic introgression in the hybrid zone. This represents a first example of the influence of Wolbachia in an insect natural hybrid zone.

Cryptic or mystic? Glacial tree refugia in northern Europe.

Here, we examine the evidence for tree refugia in northern Europe during the Late Pleniglacial (LPG) interval of maximum tree-range contraction. Our review highlights the often equivocal nature of genetic data and a tendency to overestimate potential tree distributions due to warm climate-model bias, and also reveals a convergence of macrofossil and pollen evidence. What emerges is the absence of temperate trees north of 45°N and a west-east (W-E) asymmetry in boreal tree distribution, with a treeless Western Europe north of 46°N, while restricted boreal populations persisted in Eastern Europe up to 49°N, and higher latitudes east of the Fennoscandian ice-sheet. These results have implications for current thinking on European genetic diversity patterns, species migration capacity, and conservation strategies.

Genetic structure of European sheep breeds.

Large-scale evaluations of genetic diversity in domestic livestock populations are necessary so that region-specific conservation measures can be implemented. We performed the first such survey in European sheep by analysing 820 individuals from 29 geographically and phenotypically diverse breeds and a closely related wild species at 23 microsatellite loci. In contrast to most other domestic species, we found evidence of widespread heterozygote deficit within breeds, even after removing loci with potentially high frequency of null alleles. This is most likely due to subdivision among flocks (Wahlund effect) and use of a small number of rams for breeding. Levels of heterozygosity were slightly higher in southern than in northern breeds, consistent with declining diversity with distance from the Near Eastern centre of domestication. Our results highlight the importance of isolation in terms of both geography and management in augmenting genetic differentiation through genetic drift, with isolated northern European breeds showing the greatest divergence and hence being obvious targets for conservation. Finally, using a Bayesian cluster analysis, we uncovered evidence of admixture between breeds, which has important implications for breed management.

Evolution of spider silks: conservation and diversification of the C-terminus.

Analysis of DNA sequences coding for the C-terminus of spider silk proteins from a range of spiders suggests that many silk C-termini share a common origin, and that their physical properties have been highly conserved over several hundred million years. These physical properties are compatible with roles in protein synthesis, silk function and in recruiting accessory proteins. Phylogenetic relationships among different silk genes suggest that any recombination has been insufficient to homogenize the different types of silk gene, which appear to have evolved independently of one another. The types of nucleotide substitutions that have occurred suggest that selection may have operated differently in the various silk lineages. Amino acid sequences of flagelliform silk C-termini differ substantially from the other types of spider silk studied, but they are expected to have very similar physical properties and may perform a similar function.

Disjunct distributions during glacial and interglacial periods in mountain butterflies: Erebia epiphron as an example.

Over several decades, the distribution patterns and evolution of alpine disjunct species has become an increasingly discussed subject. Large scale genetic analysis has allowed the resolution of the past range changes and intraspecific evolution of many species, in Europe especially of Mediterranean origin. However, the phylogeographic structures of species with arctic-alpine disjunct distribution patterns are relatively poorly studied. The existing phylogeographic analysis (mostly of alpine plant species) supports disjunct distributions during glacial as well as post-glacial periods for a number of species. However, several questions still remain unresolved and we therefore analysed the Mountain Ringlet Erebia epiphron as a model for such alpine disjunct species. We found strong differentiation into five different lineages supporting five differentiation centres: (i) the eastern Pyrenees, (ii) the mountain ranges between the central Pyrenees and south-western Alps, (iii, iv) two areas along the southern Alps margin and (v) the northern Alps margin. We propose that these patterns evolved due to the humidity requirements of this species, which did not allow survival in the dry glacial steppes, but along the margins of the wetter glaciated high mountain ranges.

Understanding the phylogeographic patterns of European hedgehogs, Erinaceus concolor and E. europaeus using the MHC.

The genome of the European hedgehog, Erinaceus concolor and E. europaeus, shows a strong signal of cycles of restriction to glacial refugia and postglacial expansion. Patterns of expansion, however, differ for mitochondrial DNA (mtDNA) and preliminary analysis of nuclear markers. In this study, we determine phylogeographic patterns in the hedgehog using two loci of the major histocompatibility complex (MHC), isolated for the first time in hedgehogs. These genes show long persistence times and high polymorphism in many species because of the actions of balancing selection. Among 84 individuals screened for variation, only two DQA alleles were identified in each species, but 10 DQB alleles were found in E. concolor and six in E. europaeus. A strong effect of demography on patterns of DQB variability is observed, with only weak evidence of balancing selection. While data from mtDNA clearly subdivide both species into monophyletic subgroups, the MHC data delineate only E. concolor into distinct subgroups, supporting the preliminary findings of other nuclear markers. Together with differences in variability, this suggests that the refugia history and/or expansion patterns of E. concolor and E. europaeus differ.

Genetic consequences of climatic oscillations in the Quaternary.

An appreciation of the scale and frequency of climatic oscillations in the past few million years is modifying our views on how evolution proceeds. Such major events caused extinction and repeated changes in the ranges of those taxa that survived. Their spatial effects depend on latitude and topography, with extensive extinction and recolonization in higher latitudes and altitudinal shifts and complex refugia nearer the tropics. The associated population dynamics varied with life history and geography, and the present genetic constitution of the populations and species carry attenuated signals of these past dynamics. Phylogeographic studies with DNA have burgeoned recently and studies are reviewed from the arctic, temperate and tropical regions, seeking commonalities of cause in the resulting genetic patterns. Arctic species show distinct shallow genetic clades with common geographical boundaries. Thus Beringia is distinct phylogeographically, but its role as a refugial source is complex. Arctic taxa do not show the common genetic pattern of southern richness and northern purity in north-temperate species. Temperate refugial regions in Europe and North America show relatively deep DNA divergence for many taxa, indicating their presence over several Ice Ages, and suggesting a mode of speciation by repeated allopatry. DNA evidence indicates temperate species in Europe had different patterns of postglacial colonization across the same area and different ones in previous oscillations, whereas the northwest region of North America was colonized from the north, east and south. Tropical montane regions contain deeply diverged lineages, often in a relatively small geographical area, suggesting their survival there from the Pliocene. Our poor understanding of refugial biodiversity would benefit from further combined fossil and genetic studies.

The genetic pattern of population threat and loss: a case study of butterflies.

Recent decreases in biodiversity in Europe are commonly thought to be due to land use and climate change. However, the genetic diversity of populations is also seen as one essential factor for their fitness. Genetic diversity in species across the continent of Europe has been recognized as being in part a consequence of ice age isolation in southern refugia and postglacial colonization northwards, and these phylogeographical patterns may themselves affect the adaptability of populations. Recent work on butterfly species with different refugia, colonization paths and genetic structures allows this idea to be examined. The 'chalk-hill blue' pattern is one of decreasing genetic diversity from south to north, whereas the 'woodland ringlet' pattern shows greater genetic diversity in eastern than in western lineages. Comparison of population demographic trends in species with these biogeographical patterns reveals higher rates of decrease with lower genetic diversity. This indicates reduced adaptability due to genetic impoverishment as a result of glacial and postglacial range changes. Analysis of phylogeographical pattern may be a useful guide to interpreting demographic trends and in conservation planning.

No evidence for parallel sympatric speciation in cichlid species of the genus Pseudotropheus from north-western Lake Malawi.

To test the hypothesis of parallel speciation by sexual selection, we examined length variation at six microsatellite loci of samples from four sites of four to six putative species belonging to two subgenera of rocky shore mbuna cichlids from Lake Malawi. Almost all fixation indices were significantly different from zero, suggesting that there is presently little or no gene flow among allopatric populations or sympatric species. Analysis of variance indicated that genetic distances among allopatric populations of putative conspecifics were significantly lower than among sympatric populations of heterospecifics. The topology of trees based on distance matrices was also largely consistent with the hypothesis that the putative species are monophyletic and have thus not evolved in parallel in their present locations. If parallel speciation does occur in Malawi cichlids, it may be on a larger spatial scale than investigated in our study.

Buffered tree population changes in a quaternary refugium: evolutionary implications.

A high-resolution pollen record from western Greece shows that the amplitude of millennial-scale oscillations in tree abundance during the last glacial period was subdued, with temperate tree populations surviving throughout the interval. This provides evidence for the existence of an area of relative ecological stability, reflecting the influence of continued moisture availability and varied topography. Long-term buffering of populations from climatic extremes, together with genetic isolation at such refugial sites, may have allowed lineage divergence to proceed through the Quaternary. Such ecologically stable areas may be critical not only for the long-term survival of species, but also for the emergence of new ones.

Phylogenetic relationships among the Canary Island Steganacaridae (Acari, Oribatida) inferred from mitochondrial DNA sequence data.

The mite genus Steganacarus is represented in the Canary Islands by three endemic species, one recently discovered species, and several morphotypes of uncertain taxonomic position. We used a fragment of the mitochondrial cytochrome oxidase I gene to reconstruct the phylogenetic relationships among representatives of the different taxa from the three central islands of the archipelago, Tenerife, La Gomera and Gran Canaria. Sequence data were analysed by both maximum parsimony and maximum likelihood methods. The inferred phylogenetic relationships do not correlate well with current morphological taxonomy but reveal four deeply divergent and geographically coherent lineages, one each on Gran Canaria and La Gomera and two on Tenerife. No pattern of molecular differentiation was observed among different morphotypes. Possible explanations for this incongruence are suggested in relation to the ecology and biogeography of the group. A recently discovered Steganacarus species from La Gomera, morphologically quite distinct from the other Canarian Steganacarus, is clearly identified as a taxon distantly related to all the other Canarian samples.

Testing for recombination in a short nuclear DNA sequence of the European meadow grasshopper, Chorthippus parallelus.

Single-copy nuclear DNA sequences have high potential as a source of genetic markers for population analyses. However, the difficulties that arise when haplotypes that are the product of recombinational rearrangements are present require additional consideration. Two statistical methods for identifying potential recombinants by detecting anomalies in the distribution of variable sites along sequences were used to screen sequences from a single-copy nuclear DNA fragment, cpnl-1, of the European meadow grasshopper (Chorthippus parallelus). Five of the 71 haplotypes in the cpnl-1 data set showed nonrandom distribution of polymorphic sites using both methods. The second method pinpointed an additional four haplotypes. Estimates of the rate of recombination in the entire data set were obtained using standard methods. It is concluded that cpnl-1 haplotypes have been involved in recombination or gene conversion events at a rate more than twice the mutation rate. This confirms that recombination and gene conversion are significant factors in the generation of haplotype variation in nuclear gene sequences. The cpnl-1 haplotypes identified by the tests were present only in populations that have had recent contact; the Balkan and Turkish refugial populations and their post-glacial colonies to the north. This is discussed in relation to the phylogenetic inferences drawn from the same data in a previous report.

DNA footprints of European hedgehogs, Erinaceus europaeus and E. concolor: Pleistocene refugia, postglacial expansion and colonization routes.

European hedgehogs, Erinaceus europaeus and E. concolor, are among the many European plant and animal taxa that have been subjected to cyclical restriction to glacial refugia and interglacial expansion. An analysis of 95 mitotypes, comprising partial cytochrome b and control region sequences, shows deep divergence between the two hedgehog species. Three europaeus and two concolor clades are clearly identified and are consistent with previously identified refugia for Europe: the Iberian peninsula, Italy, and the Balkans. The degree of mitochondrial divergence among these clades suggests pre-Pleistocene separation of the refugial populations. In contrast, analysis of two nuclear introns clearly separates the two concolor clades, as in the mitochondrial data, but cannot discriminate the three europaeus clades. This discrepancy between nuclear and mitochondrial data is attributed to historical differences in the refugial population size of europaeus and concolor. The geographical distribution of mitotypes is analysed using nested clade analysis. This method, by including unobserved ('missing') mitotypes, can identify mitotype groupings that remain undetected in conventional analyses. However, the application of nested clade analysis to the study of refugial populations may be hampered by such factors as the loss of haplotypes from the refugial areas by repeated contractions of the population and the recent time scale of colonization relative to mutation rate.

Mitochondrial pseudogenes: evolution's misplaced witnesses.

Nuclear copies of mitochondrial DNA (mtDNA) have contaminated PCR-based mitochondrial studies of over 64 different animal species. Since the last review of these nuclear mitochondrial pseudogenes (Numts) in animals, Numts have been found in 53 of the species studied. The recent evidence suggests that Numts are not equally abundant in all species, for example they are more common in plants than in animals, and also more numerous in humans than in Drosophila. Methods for avoiding Numts have now been tested, and several recent studies demonstrate the potential utility of Numt DNA sequences in evolutionary studies. As relics of ancient mtDNA, these pseudogenes can be used to infer ancestral states or root mitochondrial phylogenies. Where they are numerous and selectively unconstrained, Numts are ideal for the study of spontaneous mutation in nuclear genomes.

Mitochondrial DNA, ecology and morphology: interpreting the phylogeography of the Nesotes (Coleoptera: Tenebrionidae) of Gran Canaria (Canary Islands).

The genus Nesotes (Coleoptera: Tenebrionidae) is represented in the Canary Islands by 19 endemic species, the majority of which are single island endemics. Nesotes conformis and N. fusculus are described on four and three islands, respectively, but each forms a paraphyletic assemblage between Gran Canaria and the other islands. The other described species for Gran Canaria are N. quadratus, N. lindbergi and N. piliger. Thirty-six individuals representing the five species on Gran Canaria have been sequenced for 675 bp of the mitochondrial DNA (mtDNA) cytochrome oxidase II gene. Neighbour-joining analysis of maximum likelihood distances resulted in five distinct mtDNA lineages for N. quadratus, two of which also include mitotypes of N. conformis. Each of the other three species is found on only one mtDNA lineage. We propose from the molecular data that differentiation in a widespread N. quadratus-type ancestor was followed by morphological adaptation to coastal, pine and laurel forest habitats.

Speciation, hybrid zones and phylogeography - or seeing genes in space and time.

The origins and development of the study of speciation, hybrid zones and phylogeography are outlined using evolutionary iconography. This traces the ideas in this field from Lamarck and Darwin through to the present as represented in diagrams and figures. A 'tree of trees' summarizes this growth and current vitality. The new facility to use various DNA sequences from nuclear, mitochondrial and chloroplast genomes to determine genetic variation throughout a species range is examined particularly. There is great genomic subdivision across species distributions, which can be interpreted in the light of the recent demonstrations of severe palaeoclimatic oscillations. Refugia and postglacial colonization routes are proposed for several organisms across Europe. The role of geography in speciation through the Pleistocene is considered. These emerging principles and analyses are applied to data available on a variety of organisms in other regions of the world, such as the Arctic, North America and the Tropics, and including the progress of Homo sapiens through the last ice age. Some suggestions are made for future research directions.

Genomic gigantism: DNA loss is slow in mountain grasshoppers.

Several studies have shown DNA loss to be inversely correlated with genome size in animals. These studies include a comparison between Drosophila and the cricket, Laupala, but there has been no assessment of DNA loss in insects with very large genomes. Podisma pedestris, the brown mountain grasshopper, has a genome over 100 times as large as that of Drosophila and 10 times as large as that of Laupala. We used 58 paralogous nuclear pseudogenes of mitochondrial origin to study the characteristics of insertion, deletion, and point substitution in P. pedestris and Italopodisma. In animals, these pseudogenes are "dead on arrival"; they are abundant in many different eukaryotes, and their mitochondrial origin simplifies the identification of point substitutions accumulated in nuclear pseudogene lineages. There appears to be a mononucleotide repeat within the 643-bp pseudogene sequence studied that acts as a strong hot spot for insertions or deletions (indels). Because the data for other insect species did not contain such an unusual region, hot spots were excluded from species comparisons. The rate of DNA loss relative to point substitution appears to be considerably and significantly lower in the grasshoppers studied than in Drosophila or Laupala. This suggests that the inverse correlation between genome size and the rate of DNA loss can be extended to comparisons between insects with large or gigantic genomes (i.e., Laupala and Podisma). The low rate of DNA loss implies that in grasshoppers, the accumulation of point mutations is a more potent force for obscuring ancient pseudogenes than their loss through indel accumulation, whereas the reverse is true for Drosophila. The main factor contributing to the difference in the rates of DNA loss estimated for grasshoppers, crickets, and Drosophila appears to be deletion size. Large deletions are relatively rare in Podisma and Italopodisma.

Reconciling gene trees with organism history: the mtDNA phylogeography of three Nesotes species (Coleoptera: Tenebrionidae) on the western Canary Islands.

The processes of island colonization and speciation are investigated through mtDNA studies on Canary Island beetles. The genus Nesotes (Coleoptera: Tenebrionidae) is represented by 19 endemic species on the Canary Islands, the majority of which are single island endemics. Nesotes conformis is the most widespread, occurring on Gran Canaria, Tenerife, La Palma and El Hierro. Nesotes conformis forms a paraphyletic assemblage, with a split between Gran Canaria and the other three islands. Nesotes conformis of the western Canary Islands cluster with Nesotes altivagans and Nesotes elliptipennis from Tenerife. Fifty-two individuals from this western islands species complex have been sequenced for 675 base pairs of the mtDNA cytochrome oxidase II gene, representing Tenerife, La Palma and El Hierro. A neighbour joining analysis of maximum likelihood distances resulted in three distinct mtDNA lineages for N. conformis, two of which also include mitotypes of N. altivagans and N. elliptipennis. Through application of parametric bootstrap tests, we are able to reject hypotheses of monophyly for both N. conformis and N. altivagans. Nesotes altivagans and N. elliptipennis are poorly separated morphologically and mtDNA sequence data adds support to this being one species with a highly variable morphology. We propose that N. altivagans/N. elliptipennis is recently derived from two ancestral mtDNA lineages within N. conformis from the Teno region of Tenerife. We further propose colonization of the younger islands of La Palma and El Hierro by N. conformis from a mitochondrial lineage within the Teno massif (colonization; diversification; mitochondrial DNA; Canary Islands; Coleoptera).

Temporal and spatial genetic variation in spawning grounds of European hake (Merluccius merluccius) in the Bay of Biscay.

Polymorphism at five microsatellite loci were screened to determine the genetic variability and the temporal stability of population structure in natural populations of European hake (Merluccius merluccius, L.) within the Bay of Biscay. In addition, the control region (900 bp) and two protein coding genes (ATPase, subunits 6 and 8, 842 bp and a partial sequence of the ND1, 800 bp) of the mitochondrial DNA (mtDNA) were sequenced from geographically distant populations from the extremes of the species range. One hundred individuals from either side of a supposed stock boundary within the bay were collected in autumn 1997. This sampling strategy was repeated during hake spawning seasons in late spring of 1998 and 1999. Low levels of population subdivision were found between putative populations within years. Similarly, low levels of differentiation were found between autumn 1997 northern samples and spring 1998 southern samples which were collected 7 months later on spawning grounds. These results are discussed in relation to ecological, behavioural and oceanographic information. Sampling effects, which may influence these results, are also discussed. Theta (theta) estimates were significantly different from zero in every other pairwise comparison between geographical areas (north and south of the Bay of Biscay) and between years within the same area (P<0.05). Hierarchical analysis of molecular variance (AMOVA) does not confirm the temporal persistence of population structure. These results are discussed in relation to variance in reproductive success, and temporal spawning patterns, which may exist within the bay. mtDNA variability was very low between geographically distant samples from Norway and the Mediterranean Sea with only 10 variable sites found in a total of 2542 bp of mtDNA, these differences being exclusively in the D-loop.