Polymorphism and divergence of multilocus DNA markers in sibling species Chironomus riparius Meigen and Chironomus piger Strenzke (Diptera, Chironomidae)

Intra- and interspecific variation and divergence of multilocus markers for genomic DNA of the sibling species from the thimmi group, Chironomus riparius and C. piger, were studied by PCR with arbitrary primers (RAPD). A high level of RAPD polymorphism was determined in both laboratory and natural populations of these species. The genetic distances were estimated between the C. riparius populations and between the sibling species C. riparius and C. piger. The genetic distance between C. riparius and C. piger was 4 to 5 times higher than that between the C. riparius populations. A comparison of the variation and divergence for the RAPD markers with those for other genomic markers--enzyme-coding genes and chromosomes (linked gene groups)--showed that different components of the genome differed in their contribution to the genome divergence.     

Development and localization of microsatellite markers for the sibling species Chironomus riparius and Chironomus piger (Diptera: Chironomidae)

Five variable microsatellite loci are reported for the nonbiting midge species Chironomus riparius and Chironomus piger. All loci show considerable intraspecific variation and species‐specific alleles, which allow to discriminate among the two closely related species and their interspecific hybrids, and to estimate genetic diversity within and between populations. Additionally, the loci were localized on C. riparius polytene chromosomes to verify their single copy status and investigate possible chromosomal linkage. The described markers are used in different studies with regard to population and ecological genetics and evolutionary ecotoxicology of Chironomus.     

Variation and Divergence of Multilocus Genome Markers in the Species of the Genus <Emphasis Type="Italic">Chironomus</Emphasis> (Diptera,Chironomidae)

Variation and divergence patterns of the multilocus genome markers in twelve Chironomus species belonging to the plumosus and piger sibling-species groups were examined by use of polymerase chain reaction with arbitrary primers (RAPD method). The chironomid species showed high levels of the RAPD markers polymorphism. The genetic distances (GD) were determined between the species within the group of closely related species, as well as between the species from different groups. The estimates obtained characterized the divergence levels between the sibling species (GD = 0.739) and morphologically distinct species (GD = 0.935). Comparison of the variation and divergence levels of the RAPD markers with those for the other genome markers, namely, the enzyme-coding genes and chromosomes (gene linkage groups) have demonstrated different divergence rates for different genome components during speciation of chironimids.     

Variation and divergence of multilocus genome markers in the species of the genus Chironomus (Diptera, Chironomidae)

Variation and divergence patterns of the multilocus genome markers in twelve Chironomus species belonging to the plumosus and piger sibling-species groups were examined by use of polymerase chain reaction with random primers (RAPD method). The chironomid species showed high levels of the RAPD markers polymorphism. The genetic distances (GD) were determined between the species within the group of closely related species, as well as between the species from different groups. The estimates obtained characterized the divergence levels between the sibling species (GD = 0.739) and morphologically distinct species (GD = 0.935). Comparison of the variation and divergence levels of the RAPD markers with those for the other genome markers, namely, the enzyme-coding genes and chromosomes (gene linkage groups) have demonstrated different divergence rates for different genome components during speciation of Chironimids.     

Divergence of genomic DNA in species of the subgenus Camptochironomus (Diptera, Chironomidae) differing in their cytogenetic similarity levels

The variation and divergence of genomic DNA in four species of the subgenus Camptochironomus (C. tentans, C. dilutus, C. pallidivittatus, and C. setivalva) differing in the level of their cytological similarity were analyzed using the RAPD (Randomly Amplified Polymorphic DNA) method. A high level of variation in the RAPD markers was found in the species studied. Genetic distances (GD) were assessed between natural C. tentans populations, between different species of the camptochironomus sibling species group (C. tentans, C. dilutus, and C. pallidivittatus), and between these species and C. setivalva which is outside this sibling species group. The GD values obtained characterize the levels of genomic differentiation among natural populations (GD = 0.248), among sibling species (GD = 0.635), and between incipient species (GD = 0.784) of the subgenus Camptochironomus. The degree of genomic DNA divergence between sibling and incipient species in the subgenus Camptochironomus was found to be lower than that in the genus Chironomus. The rate of genomic DNA divergence appears to be lower than the rate of chromosomal divergence in species of the subgenus Camptochironomus.     

Complex relations between freshwater macro- and meiobenthos: interactions between Chironomus riparius and Chydorus piger

• 1 The potential importance of interactions between macro- and meiobenthos in the littoral zone of takes was studied in a series of laboratory experiments with a chironomid and a chydorid.
• 2 In a situation of intraspecific density dependence, Chydorus piger Sars inhibited the growth of Chironomus riparius Meigen, while both second- and fourth-instar C, riparius stimulated the parthenogenetic reproduction of C. piger.
• 3 The meiofaunal chydorids may thus have a structuring effect on macrofaunal chironomid populations by interfering with the early-instar larvae.
• 4 With macro- and meiofaunal species both operating at the primary consumer-level of the food web, this kind of complex interaction will affect the magnitude of secondary production available to higher trophic levels.

     

Chironomus riparius (Diptera) genome sequencing reveals the impact of minisatellite transposable elements on population divergence

Active transposable elements (TEs) may result in divergent genomic insertion and abundance patterns among conspecific populations. Upon secondary contact, such divergent genetic backgrounds can theoretically give rise to classical Dobzhansky–Muller incompatibilities (DMI), thus contributing to the evolution of endogenous genetic barriers and eventually causing population divergence. We investigated differential TE abundance among conspecific populations of the nonbiting midge Chironomus riparius and evaluated their potential role in causing endogenous genetic incompatibilities between these populations. We focussed on a Chironomus‐specific TE, the minisatellite‐like Cla‐element, whose activity is associated with speciation in the genus. Using a newly generated and annotated draft genome for a genomic study with five natural C. riparius populations, we found highly population‐specific TE insertion patterns with many private insertions. A significant correlation of the pairwise F_ST estimated from genomewide single‐nucleotide polymorphisms (SNPs) and the F_ST estimated from TEs is consistent with drift as the major force driving TE population differentiation. However, the significantly higher Cla‐element F_ST level due to a high proportion of differentially fixed Cla‐element insertions also indicates selection against segregating (i.e. heterozygous) insertions. With reciprocal crossing experiments and fluorescent in situ hybridization of Cla‐elements to polytene chromosomes, we documented phenotypic effects on female fertility and chromosomal mispairings. We propose that the inferred negative selection on heterozygous Cla‐element insertions may cause endogenous genetic barriers and therefore acts as DMI among C. riparius populations. The intrinsic genomic turnover exerted by TEs may thus have a direct impact on population divergence that is operationally different from drift and local adaptation.     

Differentiation of chromosome banding sequence pools and genomic dna in Holarctic natural populations of <Emphasis Type="Italic">Chironomus entis</Emphasis> Shobanov (Diptera,Chironomidae)

Polymorphism and differentiation of the chromosome banding sequence pools and genomic DNA were studied in three natural populations of Chironomus entis from Europe and North America. These populations showed a moderate level of chromosomal polymorphism and high RAPD polymorphism of genomic DNA. The Palearctic and Nearctic populations of this species did not differ significantly in the levels of chromosome and genomic DNA polymorphism. Estimation of the cytogenetic (GD_cg) and genetic (GD_DNA) distances between these C. entis populations showed that their chromosome banding sequence pools and cytogenetic structures are differentiated to a greater extent than genomic DNA. The values of cytogenetic and genetic distances between the Palearctic and Nearctic populations of C. entis are higher than the values of the corresponding distances between the Nearctic populations, but they do not reach the level of divergence between species.     

Differential staining of polytene chromosome bands in Chironomus by Giemsa banding methods

Two Giemsa banding methods (C banding and RB banding) are described which selectively stain the centromere bands of polytene salivary gland chromosomes in a number of Chironomus species. — By the C banding method the polytene chromosome appearance is changed grossly. Chromosome bands, as far as they are identifiable, are stained pale with the exception of the centromere bands and in some cases telomeres, which then are intensely stained reddish blue. — By the RB method the centromere bands are stained bright blue, whereas the remainder of the polytene bands stain red to red-violet. — Contrary to all other species examined, in Chironomus th. thummi numerous interstitial polytene chromosome bands, in addition to the centromere regions, are positively C banded and blue stained by RB banding. In the hybrid of Ch. th. thummi x Ch. th. piger only those interstitial thummi bands which are known to have a greater DNA content than their homologous piger bands are C banding positive and blue stained by the RB method whereas the homologous piger bands are C banding negative and red stained by RB banding. Ch. thummi and piger bands with an equal amount of DNA both show no C banding and stain red by RB banding. — It seems that the Giemsa banding methods used are capable of demonstrating, in addition to centromeric heterochromatin, heterochromatin in those interstitial polytene chromosome bands whose DNA content has been increased during chromosome evolution.     

Somatic breakpoints,distribution of repetitive DNA and non-LTR retrotransposon insertion sites in the chromosomes of <Emphasis Type="Italic">Chironomus piger</Emphasis> Strenzke (Diptera,Chironomidae)

Structural aberrations, their frequency and distribution as well as distribution of the tandem repetitive minisatellite DNA clusters of Alu and Hinf elements and two retroelements, the LINE NLRCth1 and the SINE CTRT1, were analyzed in the genome of the chironomid C. piger Strenzke larvae from a Bulgarian population. A consistent somatic variability in the structure of the polytene chromosomes was detected, showing that the C. piger genome is more actively rearranging than supposed before. Breakpoints were concentrated in proximal parts of chromosomes significantly more often than in distal parts. By FISH analysis we could detect only one locus containing Alu elements and 38 Hinf cluster loci which appear to be dispersed equally all over the chromosomes. The retrotransposons NLRCth1 and CTRT1 are present only in a few loci, but highly variant among different individuals. The mean number of NLRCth1 sites per individual was 18.4 ± 2.09 and of CTRT1 was 54.8 ± 8.42. A third of breakpoint locations were close to or coincide with a locus occupied by a retroelement (either NLRCth1 or CTRT1). Nineteen percent of breakpoints coincided with Hinf repetitive DNA elements. Some breakpoints were identical in the two sibling species C. piger and C. riparius Meigen (syn.: C. thummi thummi) and are considered as conserved hot spots of chromosome breakage.     

Phylogenetic study of bisexual Artemia using random amplified polymorphic DNA

Study of polymorphisms in the eukaryotic genome is an important way to discover the evolutionary relationships between species. Artemia (Crustacea, Anostraca) offers a very interesting model for evolutionary studies. In fact the genus, distributed all over the world in hundreds of known biotopes, comprises both bisexual sibling species and parthenogenetic populations easily available from the Artemia Reference Center of Ghent. In spite of great interest in it and its extensive use in aquaculture, little is known about relationships between the different species and intraspecific populations. Recently it has been demonstrated that polymorphisms in genomic fingerprints generated by arbitrarily primed polymerase chain reaction (PCR) can distinguish between strains in many organisms. We have used this technique to estimate the phylogenetic relationships existing between 14 populations living in the American continent, in the Mediterranean area, and in China. The principal coordinate analysis (PCO) obtained from 86 random amplified polymorphic DNA (RAPD) markers indicates that the populations analyzed can be divided into homogeneous clusters representing the four known bisexual species—the American A. franciscana and A. persimilis, the Mediterranean A. salina, and the A. species from China.     

The genomic footprint of climate adaptation in Chironomus riparius

The gradual heterogeneity of climatic factors poses varying selection pressures across geographic distances that leave signatures of clinal variation in the genome. Separating signatures of clinal adaptation from signatures of other evolutionary forces, such as demographic processes, genetic drift and adaptation, to nonclinal conditions of the immediate local environment is a major challenge. Here, we examine climate adaptation in five natural populations of the harlequin fly Chironomus riparius sampled along a climatic gradient across Europe. Our study integrates experimental data, individual genome resequencing, Pool‐Seq data and population genetic modelling. Common‐garden experiments revealed significantly different population growth rates at test temperatures corresponding to the population origin along the climate gradient, suggesting thermal adaptation on the phenotypic level. Based on a population genomic analysis, we derived empirical estimates of historical demography and migration. We used an F_ST outlier approach to infer positive selection across the climate gradient, in combination with an environmental association analysis. In total, we identified 162 candidate genes as genomic basis of climate adaptation. Enriched functions among these candidate genes involved the apoptotic process and molecular response to heat, as well as functions identified in studies of climate adaptation in other insects. Our results show that local climate conditions impose strong selection pressures and lead to genomic adaptation despite strong gene flow. Moreover, these results imply that selection to different climatic conditions seems to converge on a functional level, at least between different insect species.     

Genetic analysis of Pinus strobus and Pinus monticola populations from Canada using ISSR and RAPD markers: development of genome-specific SCAR markers

Pinus is the largest genus of conifers, containing over 100 species and is also the most widespread genus in the Northern Hemisphere. Pinus monticola and P. strobus are two closely related and economically important species in Canada. Morphological and allometric characteristics have been used to assess genetic variation within these two species but these markers are not reliable due to ecological variations. The purpose of the present study was to determine the level of genetic diversity within and among Canadian populations from the two species using molecular markers and to identify and characterize genome-specific inter-simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers. The level of genetic variation among populations was much lower for P. monticola than P. strobus. For both species, the among population variation values were smaller than within population variation. The populations from P. monticola were more closely genetically related than populations from P. strobus based on ISSR and RAPD analyses. Six ISSR and four RAPD markers specific to either P. monticola or P. strobus were cloned and sequenced. Primer pairs flanking these specific sequences were designed and genome specific SCAR markers for P. monticola and P. strobus were developed and characterized.     

Experimental evidence for niche segregation in a sister species pair of non-biting midges

The principle of limiting similarity states that closely related species need to partition resources of the habitat in order to coexist in the same general area. We tested this hypothesis experimentally with a sister species pair of non-biting midges (Chironomus riparius and C. piger) by assessing their relative larval fitness under several concentrations of nitrite and temperature regimes, as suggested by the observed habitat segregation in a previous field study. Both chironomid species often occur in eutrophic habitats like agricultural areas or industrial point source effluents. Based on field observations, we hypothesised C. piger to tolerate higher nitrite concentrations, higher temperatures and larger temperature ranges than C. riparius. As predicted, C. piger coped better with higher nitrite concentrations. Against the expectations, C. riparius had a tendentially higher fitness at both higher constant temperatures and larger daily temperature ranges. However, the interaction of both stressors favoured C. piger in warm high-nitrite habitats thus concurring to the field observations. The complex interaction of candidate environmental factors with antagonistic effects found here emphasises thus the necessity to experimentally assess field observations of niche segregation.     

Genetic variation within and among North Atlantic and Baltic populations of the benthic alga Phycodrys rubens (Rhodophyta)

Genetic variation was examined within and among North Atlantic, North Sea and Baltic populations of the benthic red alga Phycodrys rubens using allozymes and random amplified polymorphic DNA (RAPD) markers. On western and eastern North Atlantic coasts distinct allozyme types were found, with the exception of western Newfoundland where East and West Atlantic types co-occur. Along the European coasts, two genetic groups were distinguished by fixed allelic differences: an outer oceanic group and a North Sea/Baltic group. The two genetic types co-occur in the Skagerrak and Kattegat region. Reproductive isolation between the two types is suggested by the lack of hybrids in the overlap zones, and they may therefore represent sibling species. Unexpectedly, an analysis of RAPD variation was unable to recover the two cryptic species identified using allozymes. Within-population RAPD variation was similar to or greater than between-population variation. The lack of structure in the RAPD data cannot be attributed solely to technical artefacts of the method but appears to reflect real biological variability. Within-population genomic polymorphisms caused by frequent mutational events are discussed, as are high amounts of genetic drift and possible disruptive selection brought about by stressed habitats. Finally, Baltic and extra-Baltic salinity ecotypes are known to exist in P. rubens. However, no correlation between ecotypic variation and allozyme groups was detected.     

The population genomic signature of environmental association and gene flow in an ecologically divergent tree species Metrosideros polymorpha (Myrtaceae)

Genomewide markers enable us to study genetic differentiation within a species and the factors underlying it at a much higher resolution than before, which advances our understanding of adaptation in organisms. We investigated genomic divergence in Metrosideros polymorpha, a woody species that occupies a wide range of ecological habitats across the Hawaiian Islands and shows remarkable phenotypic variation. Using 1659 single nucleotide polymorphism (SNP) markers annotated with the genome assembly, we examined the population genetic structure and demographic history of nine populations across five elevations and two ages of substrates on Mauna Loa, the island of Hawaii. The nine populations were differentiated into two genetic clusters distributed on the lower and higher elevations and were largely admixed on the middle elevation. Demographic modelling revealed that the two genetic clusters have been maintained in the face of gene flow, and the effective population size of the high‐altitude cluster was much smaller. A F_ST‐based outlier search among the 1659 SNPs revealed that 34 SNPs (2.05%) were likely to be under divergent selection and the allele frequencies of 21 of them were associated with environmental changes along elevations, such as temperature and precipitation. This study shows a genomic mosaic of M. polymorpha, in which contrasting divergence patterns were found. While most genomic polymorphisms were shared among populations, a small fraction of the genome was significantly differentiated between populations in diverse environments and could be responsible for the dramatic adaptation to a wide range of environments.     

Polymorphism and differentiation of multilocus DNA markers in natural populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Using multilocus (RAPD) markers, variation and divergence of genomic DNA was examined in two Drosophila melanogaster populations from Russia and three populations from Ukraine. The populations were found to exhibit high polymorphism at RAPD markers. Estimation of genetic distances between the populations showed low differentiation of geographically distant populations of D. melanogaster. Significant gene flow between the D. melanogaster populations was found, which depended on the geographical distance between them.     

RAPD Analysis of the Genome in Species of the Genus Hordeum

Random amplification of polymorphic DNA (RAPD) was used to analyze six species, three populations, and seven regional cultivars of barley. A unique pattern of amplified DNA products was obtained for each species of the genus Hordeum.High polymorphism of barley species was revealed. Specific fragments were found in most RAPD patterns; the fragments can be used as molecular markers of corresponding species and subspecies. Several other DNA fragments were shown to serve as molecular markers of the H genome. Specific RAPD patterns were obtained for each population and each cultivar of H. vulgaresensu lato. In total, variation between the populations and between the cultivars was substantially lower than between species. Cluster analysis (UPGMA) was used to estimate genetic distances between theHordeumspecies, between the H. spontaneumpopulations, and between regional H. vulgarecultivars and a dendrogram was constructed.     

Geographic differentiation of genomic DNA of <Emphasis Type="Italic">Chironomus plumosus</Emphasis> (Diptera,Chironomidae) in natural holarctic populations

Using RAPD markers, polymorphism and differentiation of genomic DNA was examined in seven natural populations of Chironomus plumosus from Europe, Siberia, and North America. All these populations showed high polymorphism of genomic DNA. The Palearctic and Nearctic populations of this species were not statistically significantly different in the genomic DNA polymorphism level. The genetic distance (GD), which characterizes the extent of intraspecific differentiation of population genetic structure, was determined among the natural populations of C. plumosus. The genetic distance was on average 0.245. It was demonstrated that genetic structures of the Palearctic and Nearctic populations of C. plumosus was differentiated to a higher extent than in Palearctic. However, the genetic distances between the populations from different zoogeographical zones (0.313) did not exceed the level characteristic of the among-population differences, which do not disturb the species genetic integrity.     

RAPD variation within and among natural populations of outcrossing buffalograss [Buchloë dactyloides (Nutt.) Engelm.]

RAPD markers provide a powerful tool for the investigation of genetic variation in natural and domesticated populations. Recent studies of strain/cultivar identification have shown extensive RAPD divergence among, but little variation within, inbred species or cultivars. In contrast, little is known about the pattern and extent of RAPD variation in heterogeneous, outcrossing species. We describe the population genetic variation of RAPD markers in natural, diploid sources of dioecious buffalograss [Buchloë dactyloides (Nutt.) Engelm.]. Buffalograss is native to the semi-arid regions of the Great Plains of North America, where it is important for rangeland forage, soil conservation, and as turfgrass. Most sources of buffalograss germplasm are polyploid; diploid populations are previously known only from semi-arid Central Mexico. This is the first report of diploids from humid Gulf Coastal Texas. These two diploid sources represent divergent adaptive ecotypes. Seven 10-mer primers produced 98 polymorphic banding sites. Based on the presence/ absence of bands, a genetic distance matrix was calculated. The new Analysis of Molecular Variance (AMOVA) technique was used to apportion the variation among individuals within populations, among populations within adaptive regions, and among regions. There was considerable variation within each of the four populations, and every individual was genetically distinct. Even so, genetic divergence was found among local populations. Within-population variation was larger and among-population variation smaller in Mexico than in Texas. The largest observed genetic differences were those between the two regional ecotypes. These patterns of genetic variation were very different from those reported for inbred species and provide important baseline data for cultivar identification and continuing studies of the evolution of polyploid races in this species.