Jelena Mlinarec

Evolution of the tetraploid Anemone multifida (2n = 32) and hexaploid A. baldensis (2n = 48) (Ranunculaceae) was accompanied by rDNA loci loss and intergenomic translocation: evidence for their common genome origin.

BACKGROUND AND AIMS In the genus Anemone two small groups of taxa occur with the highest ploidy levels 2n = 6x = 48, belonging to the closely related clades: the montane/alpine Baldensis clade and the more temperate Multifida clade. To understand the formation of polyploids within these groups, the evolution of allohexaploid A. baldensis (AABBDD, 2n = 6x = 48) from Europe and allotetraploid Anemone multifida (BBDD, 2n = 4x = 32) from America was analysed. METHODS Internal transcribed spacer and non-transcribed spacer sequences were used as molecular markers for phylogenetic analyses. Cytogenetic studies, including genomic in situ hybridization with genomic DNA of potential parental species as probe, fluorescence in situ hybridization with 5S and 18S rDNA as probes and 18S rDNA restriction analyses, were used to identify the parental origin of chromosomes and to study genomic changes following polyploidization. KEY RESULTS This study shows that A. multifida (BBDD, 2n= 4x = 32) and A. baldensis (AABBDD, 2n = 6x = 48) are allopolyploids originating from the crosses of diploid members of the Multifida (donor of the A and B subgenomes) and Baldensis groups (donor of the D subgenome). The A and B subgenomes are closely related to the genomes of A. sylvestris, A. virginiana and A. cylindrica, indicating that these species or their progeny might be the ancestral donors of the B subgenome of A. multifida and A and B subgenomes of A. baldensis. Both polyploids have undergone genomic changes such as interchromosomal translocation affecting B and D subgenomes and changes at rDNA sites. Anemone multifida has lost the 35S rDNA loci characteristic of the maternal donor (B subgenome) and maintained only the rDNA loci of the paternal donor (D subgenome). CONCLUSIONS It is proposed that A. multifida and A. baldensis probably had a common ancestor and their evolution was facilitated by vegetation changes during the Quaternary, resulting in their present disjunctive distribution.

Cytogenetic and phylogenetic studies of diploid and polyploid members of Tribe Anemoninae (Ranunculaceae)

The ancestry, phylogenetic differentiation and systematic classification of the worldwide-distributed genus Anemone have been debated for many years. In this paper 11 Anemone, three Pulsatilla species and Hepatica nobilis were subjected to detailed karyotype analysis with the aim of obtaining new cytogenetic data that will contribute to karyotype evolutionary studies of the tribe Anemoninae. The results are interpreted in a phylogenetic context, established from the intergenic nontranscribed spacer (NTS) of 5S rDNA and internal transcribed spacer (ITS) of 35S rDNA. One to three 35S and one to three 5S rDNA loci are present in diploid and polyploid taxa. The 35S rDNA loci are located terminally on the short arm of acrocentric chromosomes, while for 5S rDNA there is no preferential chromosomal position as it exhibits terminal, subterminal, interstitial or pericentromeric positions, and is located either on acrocentric or metacentric chromosomes. The karyotype of hexaploid A. baldensis (2n = 6x = 48) is presented for the first time, and A. sylvestris is proposed as one of its putative parental species. Chromosome fusion/translocation is proposed as the key mechanism involved in reduction of the basic chromosome number from 8 in the Anemone subgenus to 7 in the Anemonidium subgenus. The cytogenetic data obtained are mainly supported by ITS and NTS phylogeny. Diversification of the genus Anemone was accompanied by a large reduction of heterochromatin, from the Mediterranean anemones that have large amounts of heterochromatin to the New World anemones without any detectable heterochromatic blocks.

Comparative Karyotype Investigations in the European Crayfish Astacus astacus and A. leptodactylus (Decapoda, Astacidae)

This study reports on the chromosome number and karyological characteristics of the endangered species of European crayfish, Astacus astacus and A. leptodactylus (Decapoda, Astacidae), both native to Croatian freshwater habitats. The karyotype of A. astacus and A. leptodactylus consists of 2n = 176 and 2n = 180 chromosomes, respectively. The haploid chromosome complement of A. astacus consists of 52 metacentric, 35 metacentric-submetacentric, and 1 acrocentric chromosomes. Fluorochrome staining with 4,6-diamino-2-phenylindole (DAPI) has revealed that the karyotypes of A. astacus and A. leptodactylus are characterized by large heterochromatic blocks located at centromeric and intercalary positions on the chromosomes. Interstitial heterochromatic blocks were more frequent in A. astacus than in A. leptodactylus. In both species pairing of chromosomes in meiosis was regular with the majority of bivalents in a ring- and a dumbbell-form. Fluorescence in situ hybridization (FISH) has revealed that two 45S rDNA loci were present in the investigated species. In A. astacus one of the two 45S rDNA-bearing chromosome pairs was highly heteromorphic, exhibiting a three-fold size difference between 45S rDNA sites on homologous chromosomes. Such a size difference was significantly less pronounced in A. leptodactylus. The karyotype differences between A. astacus and A. leptodactylus suggest changes in chromosome number as well as position of repetitive DNAs have played a role in the karyotype evolution of the species of Astacus.

Comparative karyotype investigations in the white-clawed crayfish Austropotamobius pallipes (Lereboullet, 1858) species complex and stone crayfish A. torrentium (Schrank, 1803) (Decapoda: Astacidae)

We report for the first time the chromosome number and karyological characteristics of the endangered European freshwater crayfish genus Austropotamobius (Decapoda, Astacidae). The karyotypes of the white-clawed crayfish ( Austropotamobius pallipes species complex) and of the stone crayfish ( A. torrentium ) consisted of 2 n = 176 chromosomes arranged into 76 metacentric, 11 metacentric-submetacentric, and one heteromorphic acrocentric/metacentric chromosome pair. Fluorochrome staining with 4,6-diamino-2-phenylindole (DAPI) revealed distinctive centromeric/pericentromeric as well as interstitial AT-rich blocks in A. pallipes and A. torrentium . Centromeric/pericentromeric AT-rich blocks were observed on all chromosomes, whereas interstitial AT-rich blocks were observed on some chromosomes, mostly on the larger ones. Fluorescence in situ hybridization (FISH) with the 45S rDNA probe on mitotic metaphases of A. pallipes and A. torrentium have revealed four major Texas-red signals, corresponding to four sites of 45S rDNA. One of the two 45S rDNA-bearing chromosome pairs was highly heteromorphic, considering the length of chromosomes as well as the size and intensity of 45S rDNA FISH signal. We speculate that the heteromorphic chromosome pair might represents X and Y sex chromosomes, suggesting the presence of XX-XY sex determination system in the A. pallipes species complex and A. torrentium .

Multilocus PCR assay reveals high diversity of vegetative compatibility types in populations of Cryphonectria parasitica in Croatia

The ascomycete fungus Cryphonectria parasitica, causal agent of chestnut blight, is probably one of the best known invasive fungal pathogens in forests of Europe and North America. Mycovirus that reduces virulence of C. parasitica can be used as a biocontrol agent of the chestnut blight. However, anastomosis-mediated virus transmission is limited by a vegetative (in)compatibility (vc) system involving at least six known diallelic vic genetic loci. This study looked at vegetative compatibility (vc) diversity in two populations of C. parasitica in Croatia. For that purpose, a PCR assay was validated and implemented using already known/published and newly designed primers for amplification of six known vic loci. The vc genotypes determined by PCR for 158 C. parasitica isolates investigated in this study were in complete agreement with the vc genotypes determined by pairwise co-culturing of the same isolates, revealing the specificity and accuracy of the PCR-based molecular vic genotyping assay. Twenty-six unique vc genotypes were found among 158 isolates, and 19 vc types per population, which makes Croatian C. parasitica populations among the most diverse in Europe regarding the number of vc types and genetic diversity. Low values of multilocus linkage disequilibrium suggest sexual reproduction as a major contributor to high C. parasitica genetic diversity in studied populations.

Molecular evolution and invasion pattern of Cryphonectria hypovirus 1 in Europe: Mutation rate, and selection pressure differ between genome domains

Understanding virus evolution is a fundamental goal of virology, evolutionary biology, and disease epidemiology. We provide a detailed analysis of evolution and origin of Cryphonectria hypovirus 1 (CHV1) populations in Europe, based on the complete genome sequence of all European subtypes. Phylogenetic analyses divided European strains into two closely related clades. Strains of the subtype I belong to the first, while strains of the subtypes F1, D and E belong to the second clade suggesting that the subtypes F1, D and E are more closely related than previously thought. Strains of the subtype F2 appeared to be recombinant; subtypes F1/D/E contributed a larger fraction of sequence while subtype I contributed a smaller fraction. The p29 was the most variable domain, while the replication-associated large ORF B protein was the most conserved domain within the CHV1. Low sequence similarity, predominant negative selection and frequent recombination characterise the evolution of CHV1.

Changes in Cryphonectria parasitica populations affects natural biological control of chestnut blight

Invasive species, especially plant pathogens, have a potential to completely eradicate native plant species and remodel landscapes. Tripartite interactions among sweet chestnut (Castanea sativa), chestnut blight-causing invasive fungus Cryphonectria parasitica, and hyperparasitic virus Cryphonectria hypovirus 1 (CHV1) were studied in two populations. The number of different vegetative compatibility (vc) types of C. parasitica more than doubled over the 10 years, while the hypovirulence incidence dropped in one population and slightly increased in the other one. Over the course of our 3-year monitoring experiment, the prevalence of hypovirulent isolates obtained from monitored cankers increased slowly (i.e., more hypovirulent isolates were being obtained from the same cankers over time). Within studied cankers, considerable changes in vc type and CHV1 presence were observed, indicating a highly dynamic system in which virulent and hypovirulent mycelia, sometimes of discordant vc types, often appeared together. The increase in hypovirulence prevalence did not have any observable curative effect on the cankers and, occasionally, reactivation of healed cankers by new, virulent C. parasitica isolates was observed. Both short- and long-term observations and revalidation of the infected plant populations are necessary to accurately estimate disease progress and formulate an adequate disease management strategy.

Cryphonectria hypovirus 1-Induced Epigenetic Changes in Infected Phytopathogenic Fungus Cryphonectria parasitica

Biotic stress caused by virus infections induces epigenetic changes in infected plants and animals, but this is the first report on methylation pattern changes in a fungus after mycovirus infection. As a model pathosystem for mycovirus-host interactions, we used Cryphonectria hypovirus 1 (CHV1) and its host fungus Cryphonectria parasitica, in which deregulation of methylation cycle enzymes upon virus infection was observed previously. Six CHV1 strains of different subtypes were transferred into three different C. parasitica isolates in order to assess the effect of different CHV1 strains and/or subtypes on global cytosine methylation level in infected fungus, using methylation-sensitive amplification polymorphism (MSAP). Infection with CHV1 affected the methylation pattern of the C. parasitica genome; it increased the number and diversity of methylated, hemi-methylated, and total MSAP markers found in infected fungal isolates compared to virus-free controls. The increase in methylation levels correlated well with the CHV1-induced reduction of fungal growth in vitro, indicating that C. parasitica genome methylation upon CHV1 infection, rather than being the defensive mechanism of the fungus, is more likely to be the virulence determinant of the virus. Furthermore, the severity of CHV1 effect on methylation levels of infected C. parasitica isolates depended mostly on individual CHV1 strains and on the combination of host and virus genomes, rather than on the virus subtype. These novel findings broaden our knowledge about CHV1 strains which could potentially be used in human-aided biocontrol of chestnut blight, a disease caused by C. parasitica in chestnut forest ecosystems and orchards.

Negative regulators of Wnt signaling pathway SFRP1 and SFRP3 expression in preterm and term pathologic placentas

Abstract Objective: Since Wnt signaling pathway plays a pivotal role in the placental development, we explored the expression of its negative regulators, SFRP1 and SFRP3 proteins in placentas from pathological pregnancies and compared their levels with those in healthy placentas. Methods: Placentas (n = 79) were stained for SFRP1, and SFRP3 proteins by immunohistochemistry and their expression levels were quantified by stereological variable of volume density (Vv, mm°). Results: Significantly higher expressions of SFRP1 and SFRP3 were found in all investigated groups of term and preterm pathologic placentas as well as in preterm control placentas in comparison with normal-term placentas. Conclusions: Our findings indicate the active involvement of negative Wnt regulators SFRP1/SFRP3 in placental development and important role in pathology of pregnancy.