Alicja Boroń

European colonization by the spined loach (Cobitis taenia) from Ponto-Caspian refugia based on mitochondrial DNA variation.

In the last 20 years, new species, asexual reproduction, polyploidy and hybridization have all been reported within the genus Cobitis. An understanding of the current distribution and baseline phylogeographical history of ‘true’ nonhybrid Cobitis species is crucial in order to unravel these discoveries. In the present work, we investigated the phylogeography of the spined loach, Cobitis taenia, using 1126 bp of the mitochondrial cytochrome b gene from 174 individuals collected at 47 sites. In total, 51 haplotypes that differed at 49 positions (4.35%) were detected. We deduce that C. taenia survived European glaciations in at least three refuges in the Ponto‐Caspian area. Two of these refuges each provided a major lineage that recolonized Europe in separate directions: one westward to England and the other spreading north into Russia before moving west. A third (minor) lineage that contributed little to the recolonization of Europe was also revealed — remaining near its Black Sea refuge. However, more recent history was difficult to resolve with colonization from a more western refugium during the last glacial maximum (LGM) a distinct possibility. Nested clade analysis indicates a pattern of restricted gene flow with isolation by distance at the first two levels and overall. Unlike many other European freshwater fish species, the Danube is not part of the current distribution of C. taenia, nor was it used as either a refuge or a source of colonization of Europe. Low genetic diversity within C. taenia suggests that its colonization of Europe is relatively recent. Demographic analyses revealed a history of recent expansion and isolation by distance.

Cryptic clonal lineages and genetic diversity in the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) inferred from nuclear and mitochondrial DNA analyses

In the loach Misgurnus anguillicaudatue, the asexual lineage, which produces unreduced clonal diploid eggs, has been identified. Among 833 specimens collected from 54 localities in Japan and two localities in China, 82 candidates of other lineage(s) of cryptic clones were screened by examining RFLP (restriction fragment length polymorphism)-PCR haplotypes in the control region of mtDNA. This analysis was performed because triploid loaches arise from the accidental incorporation of the sperm nucleus into unreduced diploid eggs of a clone. The categorization of members belonging to three newly identified lineages (clones 2–4) and the previously identified clonal lineage (clone 1) was verified by evaluating the genetic identity between two or more individuals from each clonal lineage based on RAPD (random amplified polymorphic DNA)-PCR and multilocus DNA fingerprints. We detected 75 haplotypes by observing the nucleotide status at variable sites from the control region of mtDNA. Phylogenic trees constructed from such sequences showed two highly diversified clades, A and B, that were beyond the level common for interspecific genetic differentiation. That result suggests that M. anguillicaudatus in Japan is not a single species entity. Two clone-specific mtDNA sequences were included in clade A, and the loaches with such sequences may be the maternal origin of the clones.

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.

Banded karyotype of spined loach Cobitis taenia and triploid Cobitis from Poland.

The present work provides new data on the banding pattern of diploid Cobitis taenia and its triploid hybrid females, which belong to the diploid–polyploid complex in the Vistula River tributary. C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A3 techniques were used to describe the diploid and triploid karyotype. The karyotype of Cobitis taenia of 2n=48 was characterised by one pair of NOR-bearing subtelocentric chromosomes and at least four chromosomes with CMA3-positive sites. The C-positive heterochromatin was present in the centromeres of almost all chromosomes and the pericentromeric regions of several metacentric and submetacentric chromosomes.The triploid females of 3n=74 had two pairs of chromosomes with active NORs. The NORs-sites were located terminally on two biarmed and two uniarmed chromosomes. The CMA3-staining revealed at least six A3-positive sites. The C-banded and A3-stained triploid karyotype was composed of haploid set of Cobitis taenia and diploid set of unidentified species, so heterochromatin pattern confirmed the possibility of their hybrid origin. The characteristics of banded diploid and triploid karyotype, and the hypothetical karyotype of an unknown species of 2n=50 is discussed.

Comparative chromosomal studies on two minnow fish, Phoxinus phoxinus (Linnaeus, 1758) and Eupallasella perenurus (Pallas, 1814); an associated cytogenetic-taxonomic considerations

The present work provides new data on the banding pattern of two cyprinid fish species Phoxinus phoxinus and Eupallasella perenurus from Poland. C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A3 techniques were used to describe the karyotypes. Both of the species karyotypes of 2n = 50 were characterised by one pair of acrocentric chromosomes, the largest in the set, and by two pairs of NOR-bearing chromosomes. In the chromosome set of Ph. phoxinus Ag-stained NORs were located on telomeres of two metacentric and two submetacentric chromosomes, but in most metaphases only one of the two homologous was observed. The karyotype of E. perenurus was characterised by Ag-NOR regions at a telomeric position on the shorter arm of two submetacentric chromosome pairs. In most metaphases only three NOR-bearing chromosomes were observed. In both investigated species the location of the A3 positive signals corresponded with the location of Ag-stained NORs and these sites were associated with heterochromatin shown as C-bands. The results of cytogenetical studies on other related, mainly the North American phoxinins, species are compared and discussed.

Replication banding patterns in the spined loach, Cobitis taenia L. (Pisces, Cobitidae).

The chromosomal complement of Cobitis taenia was analysed by replication banding techniques to determine whether there were specific patterns that could allow distinction of the different chromosomes. The diploid chromosome number of 2n = 48 is diagnostic of this species. In vivo 5-bromodeoxyuridine (5-BrdU) incorporation induced highly reproducible replication bands. Most of the chromosome pairs were distinguishable on the base of their banding patterns. The karyotype, consisting of five pairs of metacentrics, nine pairs of submetacentrics and 10 pairs of subtelocentrics and acrocentrics, was confirmed. C-banding and replication banding patterns were compared, and heterochromatin was both early and later replicating. C-positive heterochromatin in centromeric regions was mainly early replicating, but that located in pericentromeric regions was late replicating. Most of the late-replicating regions found interstitially were C-band negative. The results obtained so far for combined chromosomal staining methods of C. taenia and other Cobitis fish species are discussed.

Ploidy-dependent survival of progeny arising from crosses between natural allotriploid Cobitis females and diploid C. taenia males (Pisces, Cobitidae)

Crosses between 21 triploid hybrid Cobitis females and 19 C. taenia (2n = 48) males led to viable progeny; whereas no embryonic development was observed in crosses with tetraploid males (4n = 98). The ploidy status of 491 progenies randomly selected with flow cytometry (316) or chromosome analysis (175) revealed an average of 55.2 % triploids and 44.8 % tetraploids, but the ratio of 3n versus 4n fish did change during development. In the first 2 days after hatching, approximately 65.1 % of tetraploid larvae were observed. Their number decreased significantly to 30.8 and 6.2 % on average during 2–5 and 10–15 months of life, respectively. The karyotype of tetraploid progeny (4n = 98) included 3n = 74 chromosomes of the parental female and n = 24 of C. taenia male. The number of tetraploid progeny indicated indirectly that about 66 % of eggs from 3n females were fertilized with C. taenia. The rest of the eggs developed clonally via gynogenesis or hemiclonally via hybridogenesis into triploids of the same karyotype structure as parental females. We have documented for the first time that (at least under experimental conditions) tetraploids are commonly formed, but are less viable than triploids, and a ratio similar to what is found under natural conditions is finally attained. The current explanation concerning the ploidy and karyotype structure of the progeny confirms that the eggs of 3n Cobitis females are not only capable of maintaining all chromosomes but are also capable of incorporating the sperm genome, thus creating the potential to produce tetraploids.

Fecundity of the spined loach, Cobitis taenia (Pisces, Cobitidae) and natural allopolyploids of Cobitis from a diploid-polyploid population

Abstract. A comparison was made between the absolute and actual fecundity of C. taenia and allotriploid Cobitis females from a diploid-polyploid population inhabiting the Bug River, of Vistula River basin in Poland. All specimens were measured, weighed and aged. The absolute fecundity was determined by a gravimetric method using 29 ovaries (three of C. taenia, 25 of triploids and one of tetraploid). The actual fecundity was estimated according to the method adopted by Halačka et al. (2000) and used five ovaries of C. taenia and 40 of Cobitis triploids. Absolute fecundity of C. taenia under analysis ranged from 1 819 to 3 302 eggs and equaled 2 487 on average. It was significantly lower than absolute fecundity of an exclusively diploid population in Klawój Lake described previously (Juchno & Boroń 2006a). Absolute fecundity of triploid females ranged from 285 to 3 710, with an average of 1 577 eggs, whereas the fecundity of tetraploid Cobitis female was low, with only 882 eggs. The highest actual fecundity of C. taenia as well as of triploids was observed after they laid their first batch of eggs.

Molecular cytogenetic analysis of the crucian carp, Carassius carassius (Linnaeus, 1758) (Teleostei, Cyprinidae), using chromosome staining and fluorescence in situ hybridisation with rDNA probes

Abstract The crucian carp Carassius carassius (Linnaeus, 1758) is a species with restricted and decreasing distribution in Europe. Six males and six females of the species from the Baltic Sea basin in Poland were examined to show sequentially CMA3/AgNO3 staining pattern, DAPI staining, and, for the first time in literature, molecular cytogenetic analysis using double-colour fluorescence in situ hybridisation (FISH) with 28S and 5S rDNA probes. The karyotype consisted of 20 m, 36 sm and 44 sta chromosomes, NF=156. The AgNO3 stained NORs were most frequently located terminally in the short arms of two sm and two sta elements, and CMA3-positive sites were also observed suggesting abundant GC-rich repetitive DNA in the regions. Other CMA3-positive sites in the short arms of six to ten sm and sta chromosomes were detected. The results based on 28S rDNA FISH confirmed the location of rDNA sites. DAPI-negative staining of NORs suggested the scarcity of AT-rich DNA in the regions. FISH with 5S rDNA probe revealed 8–14 loci (ten and 12 in respectively 49 and 29% of metaphases). They were located in two sm and eight to ten sta chromosomes and six of them were larger than others. Simultaneously, mapping of the two rDNA families on the chromosomes of C. carassius revealed that both 28S and 5S rDNA probes were located in different chromosomes. Molecular cytogenetic data of C. carassius presented here for the first time give an important insight into the structure of chromosomes of this polyploid and declining species and may be useful in its systematics.

Comparison of egg and offspring size of karyologically identified spined loach, Cobitis taenia L., and hybrid triploid Cobitis females (Pisces, Cobitidae)

Abstract The aim of this study was to determine and compare for the first time the size of eggs laid by C. taenia (2n=48) and its naturally occurring triploid (3n=74) hybrid females and the total length of offspring of these females obtained from experimental crosses, in the period from hatch to 22 days post hatch. The eggs laid by triploid females had an average diameter of 1.18mmand were significantly larger than the eggs spawned by C. taenia females, which averaged 0.93 mm in diameter. The size of the triploid female eggs was associated with their level of ploidy. Similarly, the average total length of triploid female progeny from one to ten days post hatch was statistically significantly higher than the average total length of C. taenia offspring in the same period of life. After two weeks, the offspring of diploid and triploid females reached average total lengths of 13.27 mm and 13.60 mm, respectively, which was not a significant difference. The possible causes of triploid Cobitis female domination in diploid-polyploid populations in terms of egg size and other traits associated with polyploid functioning was also investigated.