Evolution and phylogenetic information content of the ITS-1 region in the tiger beetle Cicindela dorsalis

Sequence divergence in the internal transcribed spacer region 1 (ITS-1) of the ribosomal DNA locus was assessed in subspecies of the coastal North American tiger beetle, Cicindela dorsalis. The spacer region was amplified using the polymerase chain reaction and cloned for sequencing. Of a total of 50 clones obtained from 12 specimens, 42 clones were different in at least one nucleotide position. In a parsimony analysis of these sequences, the main phylogenetic distinction was found to separate sequences from the Gulf of Mexico and the Atlantic Ocean. Within these two assemblages phylogenetic resolution was low, and the variation within individuals was almost as high as the variation within the entire lineage. The pattern of sequence variation suggests the existence of two forms of the ITS-1 that are maintained on different chromosomes. Polymorphisms of limited geographical distribution could be detected, and 41 additional clones were partly sequenced, to assess the geographic distribution of these polymorphisms in more detail. In a population aggregation analysis, the geographic pattern of ITS-1 distribution was basically congruent with that obtained in earlier studies from mitochondrial DNA in the same C. dorsalis populations.      

Phylogeographic patterns of two tiger beetle species at both sides of the strait of Gibraltar (Coleoptera: Cicindelini)

The Strait of Gibraltar is one of the major barriers to gene flow between land masses of Europe and Africa at the western end of the Mediterranean. Since the opening of the Strait at the end of the Miocene 5.33 million years ago (Mya) it has exerted a strong influence on the dispersal and colonization of whole biotas, particularly with regard to the retreat of organisms during glaciation peaks and the northward colonization events during the warm periods of Pleistocene. The aim of this study is to elucidate the influence of the Strait of Gibraltar in the gene flow among populations of two tiger beetle species collected in Morocco, Cicindela campestris (L. 1758) and Lophyra flexuosa (Fabricius 1787), with regard to both new and published data from populations of southern Iberia. The phylogeographic analysis showed that Moroccan haplotypes of L. flexuosa belonged to a single coastal mitochondrial clade and that populations at both sides of` the Strait of Gibraltar were genetically well connected. The haplotype network of C. campestris showed that Moroccan populations made up a robust cluster clearly differentiated from those of Iberian and other European populations. These differences are thought to reflect the distinct evolutionary history (dispersal capacity, ecological strategies) of both species, as L. flexuosa shows an almost continuous distribution on the coasts located at both sides of the West Mediterranean, whereas C. campestris shows a patchy distribution and prefers montane habitats in the Western Palaearctic.     

rDNA site number polymorphism and NOR inactivation in natural populations ofAllium schoenoprasum

Nucleolar-organiser activity has been studied by silver staining and byin situ hybridization with an rDNA probe in two populations ofAllium schoenoprasum. One population is monomorphic with NORs and rDNA sites terminal on the short arm of pair 8 in all individuals. The other populations is monomorphic for pair 8 NORs but is also polymorphic for NORs on the long arm of pair 7. All plants in this population carry ribosomal cistrons on both chromosomes of pair 7 but 0, 1 or 2 of these sites can be active in rRNA synthesis. Cis-acting nucleolar-suppression affects the pair 7 locus. We suggest that there has been progressive reduction in the number of NORs during the evolution ofA. schoenoprasum.     

The evolution of genome size and rDNA in diploid species of Chenopodium s.l. (Amaranthaceae)

The evolution of genome size and ribosomal DNA (rDNA) locus organization was analysed in 23 diploid species of Chenopodium s.l., all of which share the same base chromosome number of x = 9. Phylogenetic relationships among these species were inferred from plastid and nuclear ribosomal internal transcribed spacer (nrITS) DNA sequences. The molecular phylogenetic analyses assigned all analysed species of Chenopodium s.l. to six evolutionary lineages, corresponding to the recent new generic taxonomic treatment of Chenopodium s.l. The distribution of rDNA loci for four species is presented here for the first time using fluorescence in situ hybridization (FISH) with 5S and 35S rDNA probes. Most of the 23 analysed diploid Chenopodium spp. possessed a single subterminally located 35S rDNA locus, except for three species which possessed two 35S rDNA loci. One or two 5S rDNA loci were typically localized subterminally on chromosomes, rarely interstitially. Analyses of rDNA locus numbers in a phylogenetic context resulted in the reconstruction of one locus each of 35S rDNA and 5S rDNA, both in subterminal positions, as the ancestral state. Genome sizes determined using flow cytometry were relatively small (2C value < 2.8 pg), ranging from 0.734 pg in C. schraderianum to 2.721 pg in C. californicum (nearly four‐fold difference), and were often conserved within major phylogenetic lineages, suggesting an adaptive value. The reconstructed ancestral genome size was small for all evolutionary lineages, and changes have probably coincided with the divergence of major lineages. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 218–235.     

Gene mapping of 28S and 5S rDNA sites in the spined loach Cobitis taenia (Pisces, Cobitidae) from a diploid population and a diploid–tetraploid population

We compare the chromosomal 28S and 5S rDNA patterns of the spined loach C. taenia (2n = 48) from an exclusively diploid population and from a diploid–polyploid population using 28S and 5S rDNA probe preparation and labelling, and fluorescence in situ hybridization (FISH). The 5S rDNA was located in two to three chromosome pairs, and separated from the 28S loci for the males and one female (F1) from the diploid population. Loaches from a diploid–polyploid population, and one female (F2) from the diploid population were characterized by at least one chromosome pair with 5S and 28S overlapping signals. The fishes differed mainly in their number of 28S rDNA loci, located on 3–6 chromosomes. All individuals from both populations were characterized by one acrocentric chromosome bearing a 28S rDNA signal on the telomeres of its long arm. The number of major ribosomal DNA in the karyotype of C. taenia by FISH was always higher than the number of Ag-NORs. Our data confirm the extensive polymorphism of NORs in both populations, as already has been observed in closely related Cobitis species, and less polymorphic 5S rDNA pattern. However, this preliminary result highlights the need for a wider scale study.     

Isolation and characterization of polymorphic microsatellites in Iris ensata (Iridaceae)

• Premise of the study: Microsatellite primers were developed in Iris ensata (Iridaceae) to provide polymorphic markers for further studies into population genetics. • Methods and Results: Thirteen polymorphic microsatellite loci were isolated from I. ensata. These loci were successfully amplified in two natural populations of I. ensata from eastern China (Longwangshan, Zhejiang Province) and northeastern China (Jinchuan, Jilin Province). There was no significant linkage disequilibrium found for any pair of loci. These loci contained between two and 12 alleles per locus across all 48 individuals of I. ensata. The number of alleles per locus varied from two to 10 at the population level and the observed and expected heterozygosities ranged from 0.167 to 0.958 and from 0.284 to 0.853, respectively. • Conclusions: These loci showed high levels of polymorphism and could be used to study the population genetic structure, genetic relationships, and phylogeography of I. ensata.     

Development and genetic mapping of 127 new microsatellite markers in barley

To enhance the marker density of existing genetic maps of barley (Hordeum vulgare L.), a new set of microsatellite markers containing dinucleotide motifs was developed from genomic clones. Out of 254 primer pairs tested, a total of 167 primer pairs were classifed as functional in a panel of six barley cultivars and three H. spontaneum accessions, and of those, 127 primer pairs resulting in 133 loci were either mapped or located onto chromosomes. The polymorphism information content (PIC) ranged from 0.05 to 0.94 with an average of 0.60. The number of alleles per locus varied from 1 to 9. On average, 3.9 alleles per primer pair were observed. The RFLP frameworks of two previously published linkage maps were used to locate a total of 115 new microsatellite loci on at least one mapping population. The chromosomal assignment of 48 mapped loci was corroborated on a set of wheat-barley chromosome addition lines; 18 additional loci which were not polymorphic in the mapping populations were assigned to chromosomes by this method. The microsatellites were located on all seven linkage groups with four significant clusters in the centromeric regions of 2H, 3H, 6H and 7H. These newly developed microsatellites improve the density of existing barley microsatellite maps and can be used in genetic studies and breeding research.Communicated by G. Wenzel     

Chromosome evolution in ground beetles: localization of the rDNA loci in the tribe Harpalini (Coleoptera,Carabidae)

The pattern of localization of the ribosomal genes was studied by means of fluorescence in situ hybridization in 39 species of the tribe Harpalini. Most of them show one pair of autosomes carrying the ribosomal genes in a distal position of a single chromosome arm. This pattern is hypothesized to be ancestral for the whole tribe. Both, chromosome number and the number and localization of rDNA loci, show little variation and are therefore of little phylogenetic value. Only in the subtribe Ditomina is there enough variation to characterize phyletic relationships. The stability of rDNA loci is even higher than the constancy of chromosome number, as most species of Ditomina (genera Dixus, Eocarterus, Carterus, Odontocarus and Ditomus) have the usual pair of autosomes with rDNA loci, in spite of remarkable differences in the diploid number. Only Dixus sphaerocephalus and Dixus clypeatus have two autosomal pairs with a fluorescent signal. These results do not support the hypothesis that the high chromosome numbers found within Ditomina are the result of polyploid change from the ancestral 2n = 37 karyotype of the tribe Harpalini. Chromosomal translocations or the presence of mobile genetic elements are plausible sources of the few cases of intraspecific polymorphism in the rDNA loci found in species of Harpalus.     

Variability in rDNA loci in the genus Oryza detected through fluorescence in situ hybridization

The 17s-5.8s-25s ribosomal RNA gene (rDNA) loci in Oryza spp. were identified by the fluorescence in-situ hybridization (FISH) method. The rDNA loci were located on one-to-three chromosomes (two-to-six sites) within the eight diploid Oryza spp. One of the rDNA loci gave the weakest hybridization signal. This locus is reported for the first time in the genus Oryza. The chromosomes containing the rDNA loci were determined to be numbers 9, 10 and 11 in descending order of the copy number of rDNA. The application of image analysis methods, after slide preparation treatments (post-treatments), and the use of a thermal cycler, greatly improved the reproducibility of the results. The evolutionary significance of the variability of rDNA loci among the Oryza spp. is discussed.     

Variations of 18S rDNA Loci Among Six Populations of Paeonia obovata Maxim. （Paeoniaceae） Revealed by Fluorescence In Situ Hybridization

The localization of 18S ribosomal RNA genes （rDNA） by fluorescence in situ hybridization （FISH） had been performed for some species of Paeonla. However, the pattern of 18S rDNA loci among populations Is Indistinct. In the present study, we localized 18S rDNA loci on meiotic or mitotic chromosomes of six populations of Paeonla obovata Maxim. （Paeonlaceae）. Different numbers of rDNA loci were found with different diploid （2n=10） populations, namely eight （Lushl and Mt. JIuhua populations）, 10 （Mt. Talbal population）, and seven （Mt. Guandl population）, whereas tetraplold （2n=20） populations were all found with 16 loci. Aii rDNA loci were mapped near teiomeres of mitotic chromosomes and there was no chromosome with two loci. The present results show that molecular cytological polymorphlsm exists among P. obovata diploid populations, Indicating that structural variations occurred frequently during the evolutionary history of this species, accompanied with differentiation among populations.     

Cytogenetic analysis of Australian tiger beetles (Coleoptera: Cicindelidae): chromosome number, sex-determining system and localization of rDNA genes

The chromosomes of four species of Australian tiger beetle are documented. The male meioformulae of three species of Cicindela (tribe Cicindelini) were determined as follow: Cicindela cardinalba Sumlin 1987, n = 10 + X₁ X₂ X₃Y; C. sp. ( saetigera group) and Cicindela gillesensis Hudson 1994, n = 11 + X₁ X₂ X₃Y. The male meioformula of Megacephala whelani Sumlin 1992 (tribe Megacephalini) is n = 12 + XY. Fluorescence in situ hybridization (FISH) was used to localize the 18S-28S ribosomal gene clusters on male mitotic and meiotic chromosomes and nuclei using a polymerase chain reaction-amplified ribosomal probe. Fluorescence in mitosis and meiotic first prophase stages in the three Cicindela species showed that rDNA genes are in two of the four chromosomes that form the sex vesicle. In M. whelani hybridization in mitosis and first metaphase stages indicate that rDNA genes are in three, medium- to small-sized, autosomal pairs. Silver staining of male meiotic nuclei reveals the presence of active nucleolar organizing regions (NORs) in the sex vesicle of the three species of Cicindela . The cytogenetic and evolutionary implications of these findings are discussed.     

The chromosome banding pattern in two cytotypes (2n = 36 and 38) of blind mole rats from Turkey (Mammalia: Spalaxidae)

Two cytotypes (2n = 36 and 38) of blind mole rats, Nannospalax xanthodon (Nordmann, 1840), from the Ayd?n and Manisa provinces in Turkey were investigated. Conventional chromosome staining, Ag-NOR staining and C-banding analysis were carried out. From the cytogenetic point of view, the particular phylogenetic position of these populations is supported by their low diploid numbers only, and the Cbanding pattern and the NORs distribution seem generally similar to populations with higher chromosome numbers. Several autosomal pairs with centromeric dark Cbands were observed in the 2n=36 cytotype. One autosomal pair possessed an interstitial dark C-band on the short arm; another pair possessed an interstitial dark Cband on the long arm. Whole C-heterochromatic short arms were observed in three subtelocentric autosomal pairs in the 2n=38 cytotype. Most of the other autosomal pairs possessed centromeric dark C-bands. Distinct dark C-bands were observed also in the presumed X chromosomes of both the cytotypes. The Ag-NOR regions were found on three autosomal pairs of both the cytotypes. These sites were located in telomeric areas of the short arms of two subtelocentric and one submetacentric pair.     

Effect of location,organization, and repeat-copy number in satellite-DNA evolution

Here, we analyze the evolutionary dynamics of a satellite-DNA family in an attempt to understand the effect of factors such as location, organization, and repeat-copy number in the molecular drive process leading to the concerted-evolution pattern found in this type of repetitive sequences. The presence of RAE180 satellite-DNA in the dioecious species of the plant genus Rumex is a noteworthy feature at this respect, as RAE180 satellite repeats have accumulated differentially, showing a distinct distribution pattern in different species. The evolution of dioecious Rumex gave rise to two phylogenetic clades: one clade composed of species with an ancestral XX/XY sex chromosome system and a second, derived clade of species with a multiple sex–chromosome system XX/XY₁Y₂. While in the XX/XY dioecious species, the RAE180 satellite-DNA is located only in a small autosomal locus, the RAE180 repeats are present also in a small autosomal locus and additionally have been massively amplified in the Y chromosomes of XX/XY₁Y₂ species. Here, we have found that the RAE180 repeats of the autosomal locus of XX/XY species are characterized by intra-specific sequence homogeneity and inter-specific divergence and that the comparison of individual nucleotide positions between related species shows a general pattern of concerted evolution. On the contrary, both in the autosomal and the Y-linked loci of XX/XY₁Y₂ species, ancestral variability has remained with reduced rates of sequence homogenization and of evolution. Thus, this study demonstrates that molecular mechanisms of non-reciprocal exchange are key factors in the molecular drive process; the satellite DNAs in the non-recombining Y chromosomes show low rates of concerted evolution and intra-specific variability increase with no inter-specific divergence. By contrast, freely recombining loci undergo concerted evolution with genetic differentiation between species as occurred in the autosomal locus of XX/XY species. However, evolutionary periods of rapid sequence change might alternate with evolutionary periods of stasis with variability remaining by the reduced action of molecular mechanisms of non-reciprocal exchange as occurred in XX/XY₁Y₂ species, which could depend on repeat-copy number and the processes involved in their amplification.     

Variability of Minisatellite Loci and mtDNA in Individuals with and without B Chromosomes from Populations of the Grasshopper <Emphasis Type="Italic">Dichroplus elongatus</Emphasis>

Dichroplus elongatus is an extensively distributed South American grasshopper considered a pest of major crops. Argentinean populations show a widespread B-chromosome polymorphism which could be maintained as the result trade-offs among opposite selective effects and interactions with their mitotic instability. The main objective of this study was to evaluate the relationships between B chromosomes and mtDNA sequences coupled with minisatellites loci, and verify the genotype/karyotype covariation in 12 populations located at both sides of Paraná River (Eastern and Western Regions). B carrier individuals showed significantly higher genetic diversity (H_E and X) respect to standard individuals. AMOVAs based on nuclear loci and mtDNA sequence datasets showed statistically significant levels of differentiation among karyotypes in the Eastern Region. Cluster analysis through Bayesian procedure considering nuclear loci splits B carriers and standard individuals into different genetic clusters in some Eastern populations. The Bayesian phylogenetic analysis showed two divergent mtDNA clades. Haplogroup 1 is composed exclusively of standard individuals, however all B chromosome carriers are included in haplogroup 2. There is an association between some haplotypes and B chromosomes and a strong effect of phylogenetic signal on B chromosome population structure. Genetic differentiation between karyotypes at Eastern Region revealed by AMOVA, Bayesian approaches and clustering analysis based on uniparental and biparental inherited markers may be due to the inherent nature of the B chromosome, to karyotype biased dispersal or to difference tolerance of B chromosomes on different genetic background. The combination of molecular and chromosome analysis performed in this study indicated that B chromosomes in D. elongatus is an important factor in explaining the genetic population structure at minisatellite and mitochondrial DNA levels.     

Population structure, mitochondrial polyphyly and the repeated loss of human biting ability in anopheline mosquitoes from the southwest Pacific

Australia and New Guinea contain high levels of endemism and biodiversity, yet there have been few evaluations of population‐level genetic diversity in fauna occurring throughout the Australo‐Papuan region. Using extensive geographical sampling, we examined and compared the phylogenetic relationships, phylogeography and population structure of Anopheles farauti, An. hinesorum and An. irenicus throughout their ranges in the southwest Pacific using mitochondrial (mtDNA COI) and nuclear (ribosomal protein S9 and ribosomal DNA ITS2) loci. Phylogenetic analyses suggest that the ability to utilize humans as hosts has been lost repeatedly, coincident with independent colonizations of the Solomon Islands. As some of the species under investigation transmit malaria in the region, this is a medically important finding. Maximum likelihood and Bayesian phylogenetic analyses of nuclear loci also showed that the three species are monophyletic. However, putative introgression of An. hinesorum mtDNA onto a nuclear background of An. farauti was evident in populations from Queensland, Torres Strait and southern New Guinea. Haplotype networks and pairwise F_ST values show that there is significant genetic structure within New Guinea and Australia in both An. farauti and An. hinesorum, consistent with a long‐term history of low gene flow among populations.     

Glacial refuges and cryptic speciation in a Southern Palearctic tiger beetle (Coleoptera: Cicindelidae)

Pleistocene glaciations had a determining role for shaping the current distribution and diversity of organisms, especially in the Palearctic region. In this work, we carry out a phylogeographic analysis of Iberian and two Eastern European populations of the tiger beetle Calomera littoralis (Fabricius, 1787) in order to infer the processes that may have affected their evolutionary history. According to our results, the genetic diversity of central Iberian C. littoralis populations is very low. The haplotype networks also suggest that these populations experienced a genetic bottleneck in the past, possibly related to the last glacial maxima, similar to that observed in other cicindelid taxa. These results highlight the remarkable dispersal capacity of this species, being able to move freely from one locality to another, despite the relatively long distances of sub-optimal habitat that separates them. The genetic data of central Iberian populations contrast with those of the Eastern European populations, with higher genetic diversity and no hints of any past bottleneck. This can be explained by the different characteristics of both (Iberian and Pontic) glacial refuges. The high degree of genetic differentiation between the three C. littoralis clades, and the inclusion of C. lunulata between them, suggests that the three analysed populations could be considered as different cryptic species. In that case, C. littoralis may correspond to a species complex that is still undergoing a process of speciation, similar to that observed in Cicindela campestris.     

Evolution of the Larval Cuticle Proteins Coded by the Secondary Sex Chromosome Pair: X2 and Neo-Y of Drosophila miranda: II. Comparison at the Amino Acid Sequence Level

The larval cuticle proteins (LCPs) are encoded by a multigene family, Lcp1–4, located at the right arm of the metacentric autosome 2 (2R) in Drosophila melanogaster. Due to a chromosome fusion the Lcp locus of Drosophila miranda is situated on a pair of secondary sex chromosomes, the X2 and neo-Y chromosomes. Comparing the deduced amino acid sequences of the autosomal D. melanogaster loci with the sex-chromosomal loci of D. miranda, we were able to trace the evolution of the Lcp loci with respect to their different chromosomal inheritance. The length of the signal peptide is conserved in all four LCPs, while the size of the mature LCPs varies. Conserved protein motifs became obvious from the alignment, indicating regions of structural and functional importance. Analyzing intra- and interspecific sequence similarities of the Lcp gene families allowed us to reconstruct the phylogeny of the gene cluster. Alignment with cuticle amino acid sequences originating from divergent insect species reveals motifs already present in the primordial insect LCPs. These motifs indicate different levels of constraint acting during the evolution of the LCPs. Received: 27 December 1995 / Accepted: 30 April 1996     

Mapping the nucleolus organizer region,seed protein loci and isozyme loci on chromosome 1R in rye

Summary The nucleolus organizer region located on the short arm of chromosome 1R of rye consists of a large cluster of genes that code for ribosomal RNA (designated the Nor-R1 locus). The genes in the cluster are separated by spacer regions which can vary in length in different rye lines. Differences in the spacer regions were scored in two families of F₂ progeny. Segregation also occurred, in one or both of the families, at two seed protein loci and at two isozyme loci also located on chromosome 1R. The seed protein loci were identified as the Sec 1 locus controlling -secalins located on the short arm of chromosome 1R and the Sec 3 locus controlling high-molecular-weight secalins located on the long arm of 1R. The two isozyme loci were the Gpi-R1 locus controlling glucose-phosphate isomerase isozymes and the Pgd 2 locus controlling phosphogluconate dehydrogenase isozymes. The data indicated linkage between all five loci and map distances were calculated. The results indicate a gene order: Pgd 2 ... Sec 3 ... [centromere] ... Nor-R1 ... Gpi-R1 ... Sec 1. Evidence was obtained that rye possesses a minor 5S RNA locus (chromosome location unknown) in addition to the major 5S RNA locus previously shown to be located on the short arm of chromosome 1R.     

Isolation and characterization of microsatellite loci in the beaver (Castor canadensis)

We have isolated and characterized 10 microsatellite loci in the beaver (Castor canadensis). Sixty individuals from southern and central Illinois were screened at each locus. All loci exhibited moderate levels of polymorphism, ranging from five to 13 alleles per locus with average heterozygosity ranging from 0.317 to 0.867. Locus Cca5 deviated significantly from HWE (P < 0.001). The locus pair Cca4/Cca5 was shown to be in linkage disequilibrium in southern Illinois, but not in the central Illinois population. The remaining eight loci will be useful in investigations of mating and kinship patterns in beaver populations in Illinois.     

Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and (TTAGGG)n by one-color and double-color FISH in the toadfish Halobatrachus didactylus (Teleostei: Batrachoididae)

The karyotype of Halobatrachus didactylus presents 46 chromosomes, composed of eight metacentric, 18 submetacentric, four subtelocentric, and 16 acrocentric chromosomes. The results of FISH showed that the major ribosomal genes were located in the terminal position of the short arm of a large submetacentric chromosome. They also showed a high variation in the hybridization signals. The products of amplification of 5S rDNA produced bands of about 420 pb. The PCR labeled products showed hybridization signals in the subcentromeric position of the long arm of a submetacentric chromosome of medium size. Double-color FISH indicated that the two ribosomal families are not co-located since they hybridizated in different chromosomal pairs. Telomeres of all the chromosomes hybridized with the (TTAGGG) _n probe. The GATA probe displayed a strong signal in the long arm of a submetacentric chromosome of medium size, in the subcentromeric position. The double-color FISH showed that the microsatellite GATA and the 5S rDNA gene are located in different chromosomal pairs. The majority presence of GATA probes in one pair of chromosomes is unusual and considering its distribution through different taxa it could be due to evolutionary mechanisms of heterochromatine accumulation, leading to the formation of differentiated sex chromosomes.