Meiotic chromosome configurations in oocytes of <Emphasis Type="Italic">Cobitis taenia</Emphasis> and its polyploid hybrids

Diploid and triploid hybrid females of Cobitis as a rule produce unreduced eggs which mainly develop gynogenetically, but some of the eggs incorporate sperm genome and develop into triploids and tetraploids, respectively. Here, we observed for the first time the meiotic chromosomes in the germinal vesicles (GVs) of mature oocytes of three diploid C. taenia (2n = 48) and 20 allopolyploid females of Cobitis (18 triploid 3n = 74 and 2 tetraploid 4n = 99). The majority of GVs in diploid, triploid and tetraploid females contained 24, 74 and 96 or 99 bivalents, respectively. These results directly indicated premeiotic endomitosis as a mechanism underlying the formation of unreduced eggs in allopolyploid females of Cobitis.     

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.     

Reproductive capacity of triploid loaches obtained from Hokkaido Island, Japan

Reproductive capacity was investigated in naturally occurring triploid individuals of the loach Misgurnus anguillicaudatus collected from Memanbetsu Town, Abashiri County, Hokkaido Island, Japan. These triploids have been considered to appear by accidental incorporation of the haploid sperm genome from normal diploid into unreduced diploid eggs from the clonal lineage that usually reproduces unisexually. By fertilization with sperm from the normal male, one triploid female gave many inviable aneuploid (2.1–2.7n) and very few tetraploid progeny, whereas the other produced both diploid and triploid progeny. The results suggest that at least four different types of eggs can be formed in triploid females in this locality. In contrast, no progeny hatched when eggs of the normal female were fertilized with sperm or sperm-like cells obtained from triploid males. These gametes exhibited inactive or no motility after adding ambient water. They had larger head sizes than those of normal haploid sperm and had a short or no tail. Although their ploidy was triploid or hexaploid, a small number of haploid cells were detected in the semen by flow cytometry. Thus, triploid males were generally sterile, but they have a little potential for producing very few haploid sperm.     

Chromosomal inheritance of parental rDNAs distribution pattern detected by FISH in diploid F1 hybrid progeny of Cobitis (Teleostei,Cobitidae) species has non-Mendelian character

This study was conducted to describe the major and the minor rDNA chromosome distribution in the spined loach Cobitis taenia (2n = 48) and the Danubian loach Cobitis elongatoides (2n = 50), and their laboratory-produced diploid reciprocal F₁ hybrid progeny. It was tested by fluorescence in situ hybridisation (FISH) whether the number of 28s and 5s rDNA sites in the karyotypes of diploid hybrids corresponds to the expectations resulting from Mendelian ratio and if nucleolar organiser regions (NOR)were inherited from both parents or nucleolar dominance can be observed in the induced F₁ hybrid progeny. Ten (females) or twelve (males) 28s rDNA loci were located in nine uniarm chromosomes of C. taenia. Two of such loci terminally bounded on one acrocentric chromosome were unique and indicated as specific for this species. Large 5s rDNA clusters were located on two acrocentric chromosomes. In C. elongatoides of both sexes, six NOR sites in terminal regions on six meta-submetacentric chromosomes and two 5s rDNA sites on large submetacentrics were detected. The F₁ hybrid progeny (2n = 49) was characterised by the intermediate karyotype with the sites of ribosome synthesis on chromosomes inherited from both parents without showing nucleolar dominance. 5s rDNA sites were detected on large submetacentric and two acrocentric chromosomes. The observed number of both 28s and 5s rDNAs signals in F₁ diploid Cobitis hybrids was disproportionally inherited from the two parental species, showing inconsistency with the Mendelian ratios. The presented rDNA patterns indicate some marker chromosomes that allow the species of the parental male and female to be recognised in hybrid progeny. The 5s rDNA was found to be a particularly effective diagnostic marker of C. elongatoides to partially discern genomic composition of diploid Cobitis hybrids and presumably allopolyploids resulting from their backcrossing with one of the parental species. Thus, the current study provides insight into the extent of rDNA heredity in Cobitis chromosomes and their cytotaxonomic character.     

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.     

The Genetic Structure of the Diploid–Polyploid Complex of the Spined Loach Cobitis taenia (Cypriniformes: Cobitidae) from the Middle Dnieper Basin

Biochemical genetic typing and cytometry showed that polyploid females account for 87% of the spined loachCobitis taeniapopulation from the middle Dnieper basin. The polyploidy series included triploids, tetraploids, and, possibly, a few pentaploids. A characteristic feature of the genetic structure of polyploids was that their genetic variation was due to the clonal variation in the haploid portion of the genome originating from Cobitis sp. and to polymorphism of the diploid portion originating fromC. taenia. The results are discussed with regard to comparative evolution of alloploid complexes in fish and terrestrial vertebrates.     

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

The gynogenetic reproduction of diploid and triploid hybrid spined loaches (<Emphasis Type="Italic">Cobitis</Emphasis>: Teleostei), and their ability to establish successful clonal lineages—on the evolution of polyploidy in asexual vertebrates

Polyploidisation is assumed to have played a significant role in the evolution of hybrid asexual lineages. The virtual absence of natural asexual systems in which more than a single ploidy level successfully establishes successful independent clonal lineages is generally explained by the strong effects of polyploidisation on fitness. Experimental crosses were made between diploid and triploid asexual Cobitis elongatoides × C. taenia hybrids (female) and both parental spined loach species (male). Genotyping of the progeny using allozymes and multilocus DNA fingerprinting, along with flow cytometric measurement of ploidy level, demonstrated the occurrence of gynogenetic reproduction in both female biotypes. The incorporation of the sperm genome occurred in some progeny, giving rise to a higher ploidy level, but the rate of polyploidisation differed significantly between the diploid and triploid females. These outcomes are consistent with the existence of developmental constraints on tetraploidy, which determine the rarity of tetraploids in natural populations. No cases of ploidy level reduction were observed. Since diploid and triploid hybrid populations occur where the lack of potential progenitor excludes the possibility of de novo origin, it is probable that both diploid and triploid females can establish successful clonal lineages. Spined loaches represent a unique example, among asexual vertebrates, where more than one ploidy level can establish persistent clonal lineages, which are reproductively independent of one another.     

Contents of Trace Elements as Indicators of Ecological Divergence between Sympatric Spined Loaches (Teleostei,Cobitidae) from the Upper Dnieper

The contents of eight trace elements (Ti, Mn, Ni, Cu, Zn, Sr, Ba, and Pb) in muscles of syntopic sexual and clonal spined loaches (the golden loach Sabanejewia baltica, diploid males and females of Cobitis taenia, and congeneric triploid clonal females) from the upper Dnieper River and in the syntopic spined loaches (males and females of C. melanoleuca and males of C. taenia) from the upper Volga River basin were determined using the X-ray fluorescence spectroscopy technique. The contents of Cu in the clonal triploid Cobitis females and diploid C. taenia females from the Dnieper are different. The intersexual differences in the contents of Pb and Zn between C. taenia males and females, as well as the interspecific differences in the content of Ti between the spined loaches from the Dnieper and Volga basins were revealed. The concentrations of Cu and Pb correlated with the individual fish size. The potential for the use of the revealed differences in the trace element contents as an indicator of the divergence of ecological niches in the syntopic spined loaches is discussed.     

Genetic relationships among the Japanese and Korean striated spined loach complex (Cobitidae: Cobitis) and their phylogenetic positions

The striata complex, a group of spined loaches included in the genus Cobitis and characterized by a striped coloration pattern on the lateral midline, is distributed in rivers in northeastern Asia to western Japan. The complex comprises 2 continental species (Cobitis tetralineata and Cobitis lutheri) and 3 Japanese races of species rank (large race, middle race, and small race), the small race further comprising 6 local forms of subspecific rank (Tokai form, Biwa form, Yodo form, Sanyo form, San-in form, and Kyushu form). Previous karyological studies have revealed that the large race is an allotetraploid, the others being diploid. In this study, mitochondrial (mt) DNA analyses were conducted for 30 diploid populations of the Cobitis striata complex from Japan and Korea to examine: (1) their phylogenetic relationships and the position of the complex among the major lineages of Cobitis; and (2) the genetic relationships among the Japanese and Korean populations. The results, based on cytochrome b sequences (724 base pairs) analyzed with those of the main lineage of European and Japanese Cobitis, indicated that the striata complex should be considered as a monophyletic group, which evolved in northeastern Asia. Initially considered as a subspecies of Cobitis taenia, widely distributed from Europe to Asia, the striata complex does not have a sister-relationship with the former. Although the Korean species C. tetralineata was formerly believed to be closely related to the middle race in Japan, and a second continental species, C. lutheri, closely related to the Kyushu or San-in forms of the small race in Japan, the trees resulting from the present study revealed that the two Korean species were clustered with each other and separated from all Japanese races.     

Triploid females and diploid males: underreported phenomena in <Emphasis Type="Italic">Polistes</Emphasis> wasps?

Summary In hymenopteran species, males are usually haploid and females diploid. However, in species that have complementary sex determination (CSD), diploid males arise when a female produces offspring that are homozygous at the sex-determining locus. Although diploid males are often sterile, in some species they have been shown to produce diploid sperm, thus producing triploid daughters if they mate successfully. Diploid males have been observed in very few species of social wasps, and we know of no published reports of triploid females. In this paper, we review the existing literature on diploid males and triploid females in the Hymenoptera, and report the observation of triploid females in three species of Polistes paper wasps. Although polyploid offspring may be produced parthenogenetically, the more likely scenario is that Polistes wasps have CSD and produce diploid males via homozygosity at the sex-determining locus. Therefore, female triploidy indicates that diploid males do exist in Polistes species where they are presumed to be absent, and are likely to be even more frequent among species that have experienced a genetic bottleneck. We conclude by cautioning against the assumption of a selective advantage to the production of early males, and by discussing the implications of male diploidy and female triploidy for measurement of sex ratio investment and assumptions of reproductive skew theory.Received 5 December 2003; revised 20 March 2004; accepted 19 April 2004.     

Viable diploid progeny induced from sperm of Chinese tetraploid pond loach by cold‐shock androgenesis

Natural tetraploid loach (Misgurnus anguillicaudatus) living in the Changjiang River basin and adjacent area in China has been considered a true genetic tetraploidy (4n = 100) with four sets of homologous chromosomes. Here, we reported its further supporting evidence provided by the cold‐shock androgenesis. Viable androgenetic progeny appeared when eggs were cold‐shocked at 3°C for 60 min, starting 5 min after fertilization with sperm of natural tetraploid males, although the survival rate was significantly lower than control group. The majority (87%) of androgenetic embryos were diploid (2n = 50) and all‐paternal inheritance was verified in larval stage by microsatellite genotypes. Microscopic observations confirmed the elimination of both egg nucleus and second polar body from a fertilized egg, followed by the cleavage exclusively with sperm nucleus. Thus, the appearance of normal diploid androgenetic progeny from sperm of natural tetraploid loach revealed the presence of four chromosome sets in tetraploid males.     

A Complementary Method for Production of Tetraploid Crassostrea gigas Using Crosses Between Diploids and Tetraploids with Cytochalasin B Treatments

We present a new method to produce tetraploid Crassostrea gigas by cytochalasin B inhibition of polar body 2 expulsion in diploid females crossed with tetraploid males. This offers a means of direct introgression of genetic characters from selected diploid to tetraploid lines, avoiding a triploid step. Offspring larval ploidy shifted over time and depended on size, with tetraploids more frequent among the smaller larvae and triploids among the large. Viable tetraploids were found at 4 and 6 months, indicating the technique was successful. The possibility that gynogenesis occurred was tested by microsatellite analysis to confirm the presence of paternally inherited alleles. These were present in all animals of the 2n × 4n + CB (female first) cross. However, a 4n × 2n + CB cross produced triploids, including some gynogens. Our method illustrates for the first time that diploid C. gigas eggs, if selected for large size, can give viable tetraploid offspring.     

Some factors influencing the microdistribution of a population of spined loach,Cobitis taenia (L.)

Monthly observations (October 1972–October 1974) of a natural population of spined loach, Cobitis taenia (L.), in the River Great Ouse at Newport Pagnell indicated a patchy microdistribution, which varied seasonally. The distribution was clearly linked to the areas of fine substrate, which altered seasonally in position.Flow rates measured at monthly intervals in areas where Cobitis taenia (L.) were present were found to be approximately half those of areas in which Cobitis taenia L. were not found (0.148 m/sec and 0.293 m/sec). Substrate samples from areas where Cobitis taenia L. were present had a fine organic component, in contrast to the remainder of the river bed, which consisted of hard-packed gravel.In the laboratory, choice chamber experiments demonstrated a positive selection for fine organic deposits.     

Haematological characterization of loach Misgurnus anguillicaudatus: comparison among diploid, triploid and tetraploid specimens

The purpose of this study was to determine whether diploid, triploid and tetraploid loach (Misgurnus anguillicaudatus) differed in terms of their main haematological and physiological characteristics. Diploid and tetraploid fish were produced by crossing of natural diploids (2n x 2n) and natural tetraploids (4n x 4n), respectively. Triploid fish were produced by hybridization between diploid males and tetraploid females. The blood cells were significantly larger in polyploids, and the volumetric ratios of erythrocytes and leucocytes (thrombocyte and neutrophil) in tetraploids, triploids and diploids were consistent with the ploidy level ratio of 4:3:2. No significant differences were observed in haematocrit among polyploids. The erythrocyte count decreased with increased ploidy level, while total haemoglobin, mean cell volume, mean cellular haemoglobin content, and mean cell haemoglobin concentration all increased with increase in ploidy level. Erythrocyte osmotic brittleness declined in polyploids so that polyploid erythrocytes were more resistant to osmotic stress than diploid ones. Overall, loach with higher ploidy levels showed evidence of some advantages in haematological characteristics.     

Genetic diversity, origin, and geographic distribution tendencies of biotypes of polyploid spined loaches (Cypriniformes, Cobitidae, Cobitis) in Ukraine

Sixteen of 28 possible biotypes of C. elongatoides-taenia s.lato (C. taenia, C. tanaitica, C. species-1) have been found in Ukrainian waters. Presumably, the biotypes of C. aff. melanoleuca-tanaitica has also been identified. The share of polyploids among Cobitis on average was 65%, where males made up less than 1% with the same ratio among tri- and tetra-polyploids. The absence of amphydiploidy and presence of individuals with recombinant genotype and abnormal electrophoretic specters, whose frequency sometimes is considerable, are attracting increasing interest. There are two centers of polyploid biotypes creation: southern (the Lower Danube) and northern (the Upper Danube, Oder, and Rhine). The C. taenia and C. species-1 type, which is typical for the northwestern basins of Ukraine, participates in the formation of polyploids in the Rhine alongside with C. elongatoides and C. tanaitica. It is determined that biotypes (C. 2(3) elongatoidestanaitica, C. elongatoides-2(3) tanaitica) in comparison with biotypes whose genome includes the chromosome set of C. taenia are limited in distribution towards the East. This is due to the hybridization of females from these biotypes with males of C. taenia afflicted by genetic instability and reduced viability of posterity.     

Diploid clone produces unreduced diploid gametes but tetraploid clone generates reduced diploid gametes in the Misgurnus loach

Most individuals of the loach Misgurnus anguillicaudatus reproduce bisexually, but cryptic clonal lineages reproduce by natural gynogenesis of unreduced diploid eggs that are genetically identical to maternal somatic cells. Triploid progeny often occur by the accidental incorporation of a sperm nucleus into diploid eggs. Sex reversal from a genetic female to a physiological male is easily induced in this species by androgen treatment and through environmental influences. Here, we produced clonal tetraploid individuals by two methods: 1) fertilization of diploid eggs from a clonal diploid female with diploid sperm of a hormonally sex-reversed clonal diploid male and 2) artificial inhibition of the release of the second polar body in eggs of clonal diploid females just after initiation of gynogenetic development. There is no genetic difference between the clonal diploid and tetraploid individuals except for the number of chromosome sets or genomes. Clonal tetraploid males never produced unreduced tetraploid sperm, only diploid sperm that were genetically identical to those of a clonal diploid. Likewise, clonal tetraploid females did not form unreduced tetraploid eggs, just diploid eggs. However, the eggs' genotypes were identical to those of the original clone, and almost all the eggs initiated natural gynogenesis. Thus, gametogenesis of the clonal tetraploid loach is controlled by the presence of two chromosome sets to pair, thereby preserving the normal meiotic process, i.e., the formation of bivalents and subsequently two successive divisions.