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.     

Fitness differences among diploids, tetraploids, and their triploid progeny in Chamerion angustifolium: mechanisms of inviability and implications for polyploid evolution

Abstract. Theoretical models indicate that the evolution of tetraploids in diploid populations will depend on both the relative fitness of the tetraploid and that of the diploid-tetraploid hybrids. Hybrids are believed to have lower fitness due to imbalances in either the ploidy (endosperm imbalance) or the ratio of maternal to paternal genomes in their endosperm (genomic imprinting). In this study we created diploids, tetraploids, and hybrid triploids of Chamerion angustifolium from crosses between field-collected diploid and tetraploid plants and evaluated them at six life stages in a greenhouse comparison. Diploid offspring (from 2 x × 2 x crosses) had significantly higher seed production and lower biomass than tetraploid offspring (from 4 x × 4 x crosses). Relative to the diploid, the cumulative fitness of tetraploids was 0.67. In general, triploids (from 2 x × 4 x , 4 x × 2 x crosses) had significantly lower seed production, lower pollen viability, and higher biomass than diploid individuals. Triploid offspring derived from diploid maternal parents had lower germination rates, but higher pollen production than those with tetraploid mothers. Relative to diploids, the cumulative fitness of 2 x × 4 x triploids and 4 x × 2 x triploids was 0.12 and 0.06, respectively, providing some support for effect of differing maternal:paternal ratios and endosperm development as a mechanism of hybrid inviability. Collectively, the data show that tetraploids exhibit an inherent fitness disadvantage, although the partial viability and fertility of triploids may help to reduce the barrier to tetraploid establishment in sympatric populations.     

Call degradation in diploid and tetraploid green toads

According to the 'environmental selection' hypothesis, the physical characteristics of environments influence the evolution of long-range acoustic signals by favouring those properties that decrease sound attenuation and distortion with distance. Different environments could favour different acoustic properties and therefore contribute to the intra- and interspecific variation of calls. In the present paper, we investigate whether this hypothesis could explain the differences between the advertisement calls of three taxa of central-Asian green toads: lowland diploids, highland tetraploids and lowland tetraploids. The pattern of propagation of 12 natural calls (four for each taxon) was analysed in nine localities of Kyrgyzstan and Kazakstan. We broadcast the calls and recorded them along a trasect at distances of 2, 4, 8, 16, and 32 m from the speaker, to estimate sound attenuation and propagation. Attenuation was quantified from the oscillogram (by directly calculating the SPL of calls) and from the power spectrum (by measuring the relative amplitude of the fundamental frequency) , whereas degradation was estimated by cross-correlating spectrograms of the same call at different distances. Results show that: (1) the pattern of sound propagation significantly differs among localities in relation to the different vegetation and environmental noise; (2) in most localities, call attenuation and degradation differ significantly among the three taxa ; (3) such differences are not consistent to those expected under the hypothesis of environmental selection: independent of altitude, lowland tetraploid calls fare worse than both diploid and highland tetraploid calls, whereas diploid and highland tetraploid calls show different patterns of propagation in a few localities only.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78 , 11–26.     

Possible pathways of the gene flow inTaraxacum sect.Ruderalia

Reproductive behaviour and the pathways of gene flow among ploidy levels were studied experimentally inTaraxacum sect.Ruderalia. Diploid, triploid and tetraploid individuals were sampled from mixed diploid — polyploid natural populations. 136 experimental hybridizations between the plants of different ploidy levels were performed. Seeds resulting from these crosses, those obtained from isolated anthodia as well as from open pollinated anthodia (both from cultivated and wild plants) were subjected to the flow-cytometric seed screening (FCSS) to determine ploidy levels in the progeny and to infer breeding behaviour of maternal plants. Three possible pathways of the gene flow were studied: (A) fertilization of sexuals by pollen of apomicts, (B) B_III hybrid formation, (C) facultative apomixis. Diploid maternal plants when experimentally crossed with triploid pollen donors produced diploids and polyploid progeny, while when pollinated with a mixture of the pollen of diploids and triploids or insect pollinated, no polyploids were discovered. It seems that in the mixture with the pollen of diploids, the pollen of triploids is ineffective. Tetraploids produce hybrids much easier with diploid mothers and their role in wild populations requires further study. Triploid mothers, even those with subregular pollen did not show traces of facultative apomixis. B_III hybrids were present in the progeny of both triploids and tetraploids, in tetraploids in quite high percentages (up to 50% of the progeny in some crosses).     

Inter-embryo competition in the progeny of autotriploid Aloineae (Liliaceae)

In reciprocal crosses between diploid and triploid Aloineae the progeny are largely diploid or diploid plus one or two chromosomes, but in reciprocal crosses between triploids and tetraploids they are tetraploid or nearly so. Thus the triploids contribute circa haploid gametes to the progeny when crossed with diploids but circa diploid gametes when crossed with tetraploids. These results are compared with those of a number of earlier workers. It is concluded that the bias in the frequency of progeny types towards diploidy or tetraploidy, depending on the ploidy level of the plant which is crossed with the triploid, is caused by inter-embryo competition. Those embryos with an endosperm/embryo factor of 1.5, the value found in normal diploid/diploid crosses having triploid endosperms, are selected in preference to those with factors higher or lower than 1.5.Inter-gamete competition also occurs among the euploid and aneuploid gametes produced by the triploids. This is more pronounced on the male side, because the degree of survival of aneuploid pollen from the triploids into the next generation is much lower than that of aneuploid egg nuclei.Non-reduction in the triploids gives rise to occasional pentaploid progeny in crosses with tetraploids, but it is more probable that in diploid/triploid crosses tetraploid progeny are the products of non-reduction in the diploid.     

The role of triploid hybrids in the evolutionary dynamics of mixed-ploidy populations

Theory suggests that the evolution of autotetraploids within diploid populations will be opposed by a minority-cytotype mating disadvantage. The role of triploids in promoting autotetraploid establishment is rarely considered, yet triploids are often found in natural populations and are formed in experimental crosses. Here, I evaluate the effects of triploids on autotetraploid evolution using computer simulations and by synthesizing research on the evolutionary dynamics of mixed-ploidy populations in Chamerion angustifolium (Onagraceae). Simulations show that the fate of a tetraploid in a diploid population varies qualitatively depending on the relative fitness of triploids, the ploidy of their gametes and the fitness of diploids relative to tetraploids. In general, even partially fit triploids can increase the likelihood of diploid–tetraploid coexistence and, in some cases, facilitate tetraploid fixation. Within the diploid–tetraploid contact zone of C. angustifolium , mixed populations are common (43%), and often (39%) contain triploids. Greenhouse and field studies indicate that triploid fitness is low (9% of diploids) but variable. Furthermore, euploid gametes produced by triploids can be x , 2 x or 3 x and contribute the majority (62%) of new polyploids formed in each generation (2.3 × 10⁻³). Although triploid bridge, alone, may not account for the evolution of autotetraploidy in C. angustifolium , it probably contributes to the prevalence of mixed-ploidy populations in this species. Therefore, in contrast to hybrids in homoploid species, triploids may actually facilitate rather than diminish the fixation of tetraploids by enhancing the rate of formation.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 537–546.     

Directional and Fluctuating Asymmetry in Sexual and Asexual Otiorhynchus alpicola Populations

This study concerns the contribution of directional asymmetry (DA) and fluctuating asymmetry (FA) as a characterization of variation in six sexual (diploid) and two asexual (triploid and tetraploid) populations of the weevil Otiorhynchus alpicola. It is shown that DA in sexual populations is about 1 % of the mean length of each of the seven bilateral traits and the average contribution of DA to trait variation is even lower in asexual populations (about 0.85 in triploids and 0.65 in tetraploids forms). The average contribution of FA to the total phenotypic variance is about 23 %, 12 % and 19 % in diploid, triploid and tetraploid populations, respectively. Since FA is generally regarded as a measure of developmental stability, our data indicate that triploid forms of O. alpicola are developmentally more stable than diploid and tetraploid forms. The relationship between the level of ploidy and FA is discussed.     

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.     

二倍体鲫鲤F2产生不同倍性卵子的证据

在检测到鲫鲤F2产生3种不同大小(直径分别为0.13 cm,0.17cm和0.2 cm)类型的卵子基础上,进行了F2(♀)×红鲫(♂)及F2(♀)×四倍体鲫鲤(♂)的交配实验.通过染色体计数和流式细胞仪分析,在F2(♀)×红鲫(♂)后代中获得了四倍体、三倍体、二倍体鱼;在F2(♀)×四倍体鲫鲤(♂)后代中获得了四倍体和三倍体鱼.这两个交配组合后代中出现的不同倍性的鱼类为证明鲫鲤F2能产生三倍体、二倍体和单倍体卵子提供了进一步证据.F2(♀)×红鲫(♂)中雄性四倍体鱼的存在说明在四倍体后代中存在基因型为XXXY的个体.对上述两个交配组合后代的四倍体鱼和三倍体鱼的性腺结构观察表明四倍体鱼是可育的,而三倍体鱼是不育的.作者认为鲫鲤F2能够产生二倍体和三倍体卵子与核内复制机制和生殖细胞的融合有关.     

The evolution of release and advertisement calls in green toads (Bufo viridis complex)

In the present paper we tested the hypothesis that differences in the acoustic communication system of diploid and tetraploid green toads ( Bufo viridis complex) might be due to selection for reproductive character displacement. We recorded two acoustic signals of the toad repertoire − the advertisement call (a long range mate-attracting signal) and the release call (a short-range signal mediating male–male interactions) − from six sympatric Central Asian populations (three diploid and three tetraploid populations) as well as from three allopatric diploid populations from Italy, and compared their patterns of variation with the pattern of among-population genetic distances. Although release and advertisement calls share the same morpho-physiological constraints, they show significantly different patterns of variation. Release calls vary congruently with the pattern of genetic distances, suggesting that mutation and genetic drift have been the major forces responsible for their change both in time and space. By contrast, the pattern of advertisement-call variation is not consistent with the phylogeny of the group, because the advertisement calls of Asian diploid and tetraploid populations differ from each other more than their genetic distances would predict. These results strongly support the hypothesis that selection acted on the advertisement calls of either or both Asian taxa , possibly, to favour reproductive isolation.  © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 77, 379–391.     

Triploid bridge and role of parthenogenesis in the evolution of autopolyploidy

Autopolyploidization is considered to play an important role in plant evolution. In polyploidization, the polyploid evolves from the original diploid cytotype, in which the triploid state is considered to mediate the process (triploid bridge). Nevertheless, the fitness of triploid individuals seems to be too low to facilitate the polyploidization process (triploid block). The evolutionary condition of autopolyploidy was analyzed using a mathematical model focusing on the role of parthenogenesis in triploid and tetraploid individuals. In addition, offspring were assumed to arise by sexual reproduction by conjugations between haploid, diploid, and triploid gametes produced by diploid, tetraploid, and triploid individuals. According to the analysis, even if triploid block suppresses the fitness of sexually produced triploids, the polyploidization process can proceed when parthenogenesis occurs frequently. If only triploids frequently reproduce parthenogenetically, the evolutionary consequences tend to depend on the fitness of the tetraploid individuals. On the basis of a predetermined parameter set, if tetraploid fitness is relatively low, all three ploidies can coexist. Otherwise, tetraploidization occurs. In this case, triploid parthenogenesis promotes not only triploidization but also tetraploidization. However, if both triploids and tetraploids frequently reproduce parthenogenetically, the ploidy levels with the highest fitness are likely to dominate in the population through direct competition among cytotypes.     

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.     

Distribution and reproductive capacity of natural triploid individuals and occurrence of unreduced eggs as a cause of polyploidization in the loach,Misgurnus anguillicaudatus

Loaches (Misgurnus anguillicaudatus) were collected from 35 localities in Japan and assayed by flow cytometry to determine ploidy status. No tetraploids were found, with samples from 33 localities having no or few (1.2–3.2%) triploids. Samples collected from Ichinomiya Town, Aichi Prefecture, showed a relatively high rate of triploidy (7.7%). Samples collected from a fish farm in Hirokami Village, Niigata Prefecture, also showed high proportions of triploids (2.0–15.8%), these triploid males being sterile, but the females producing both large-sized triploid and small-sized haploid eggs. Such eggs developed bisexually rather than gynogenetically, giving rise to viable tetraploid and diploid offspring after normal fertilization. Of eight diploid females obtained from the same locality, one produced a high incidence of viable diploid gynogens (55%) after gynogenetic induction by fertilization with UV-irradiated spermatozoa. These observations indicated the presence of diploid fish which produced both diploid and haploid eggs. Thus, triploid and diploid individuals were also produced after fertilization with haploid spermatozoa. These results suggested that the occurrence of such unreduced eggs may be a cause of natural polyploidization in this species.     

Cytotype distribution at a diploid-tetraploid contact zone in Chamerion (Epilobium) angustifolium (Onagraceae)

In North America, the geographic distributions of diploid and tetraploid Chamerion (formerly Epilobium) angustifolium overlap in a narrow zone along the southern border of the boreal forest and along the Rocky Mountains. We examined the frequency and distribution of diploid and tetraploid cytotypes in a narrow (5 km) zone of sympatry across an elevational gradient and in putatively uniform diploid and tetraploid reference populations on the Beartooth Pass, in the Rocky Mountains of southern Montana-northern Wyoming. All five reference populations sampled were dominated by a single cytotype, but only one was completely uniform. In the zone of sympatry, 27 transects were sampled every 2 m for a total of 238 plants. Reproductive status (vegetative, flower buds, open flowers) was recorded, and the ploidy of each plant was determined by flow cytometry. Diploid and tetraploid plants predominated (36 and 55%, respectively) but were heterogeneously distributed among the transects. Six of the 27 transects were fixed for a single cytotype (four transects, diploid; two transects, tetraploid), and in seven others either diploids or tetraploids predominated (frequency >75%). Triploids represented 9% of the total sample and occurred most frequently in transects containing both diploids and tetraploids (G = 3.4, df = 2, P = 0.07). Diploids were more often reproductive (in bud, flower, or fruit) than either triploids or tetraploids (G = 12.0, df = 2, P < 0.001) and were the only cytotype to have produced open flowers. These results suggest that the zone of sympatry is best characterized as a mosaic rather than a cline, with diploid and tetraploids in close proximity and that the distribution of polyploidy is regulated by ecological sorting in a heterogeneous physical environment.     

Cytotype distribution inEmpetrum (Ericaceae) at various spatial scales in the Czech Republic

Ploidy levels inEmpetrum (crowberry) from the Czech Republic and from one adjacent locality in Poland were estimated by flow cytometry to examine cytotype distribution patterns at large (within the country), medium (within mountain ranges) and small (within particular localities) spatial scales. Diploid, triploid, and tetraploid individuals were found. Triploids are reported from Central Europe for the first time; they occurred in the Krkono?e Mts. Exclusively diploid plants were observed in three mountain ranges (the Kru?né hory Mts., Labské pískovce Mts., Adr?pa?sko-Teplické skály Mts.), exclusively tetraploids were observed in the Jeseníky Mts., and both cytotypes were observed in the ?umava Mts., Jizerské hory Mts. and Krkono?e Mts. Except for the latter mountain range, diploids and tetraploids were always found in different habitats. Spatial isolation is supposed to be the main barrier preventing cytotype mating. A mosaic-like sympatric occurrence of different cytotypes was demonstrated in the Krkono?e Mts., where peat bogs and rocky places were not spatially separated. Eight of 11 localities studied there were inhabited by diploids and tetraploids (five localities), diploids and triploids (one locality) or all three ploidy levels (two localities). Diploid and triploid plants occasionally intermingled at 0.3 × 0.3 m. Flower sex in crowberries was strongly associated with ploidy level: diploids usually had unisexual flowers, the tetraploids bore exclusively bisexual flowers. However, a few diploid plants with hermaphrodite flowers occurred in one population in the Krkono?e Mts.     

Evidence of Different Ploidy Eggs Produced by Diploid F2 Hybrids of Carassius auratus (♀) × Cyprinus carpio (♂)

Based on the presence of three types of eggs with different diameters 0.13, 0.17 and 0.2 cm, we made two crosses: F₂ (♀) × diploid red crucian carp (♂), and F₂ (♀) × F₁₀ tetraploid (♂). The ploidy levels of the progeny of the two crosses were examined by chromosome counting and DNA content measurement by flow cytometer. In the offspring of the former cross, tetraploids, trip-loids, and diploid were obtained. In the progeny of the latter cross, tetraploids and triploids were observed. The production of the different ploidy level fish in the progeny of the two crosses provided a further evidence that F₂ might generate triploid, diploid and haploid eggs. The presence of the male tetraploid found in F₂ (♀) × diploid red crucian carp (♂) suggested that the genotype of XXXY probably existed in the tetraploid progeny. The gonadal structures of the tetraploids and triploids indicated that both female and male tetraploids were fertile and the triploids were sterile. We concluded that the formations of different ploidy level eggs from F₂ were contributed by endoreduplication and fusion of germ cells.     

Correlation between distyly and ploidy level in Damnacanthus (Rubiaceae)

Somatic chromosomes were observed in 661 individuals of 14 taxa, nine species and five varieties, of Damnacanthus (Rubiaceae). Chromosome numbers are reported for the first time for 13 taxa. Diploid (2n = 22) and tetraploid (2n = 44) counts were obtained. Distyly is reported for the first time for four species, D. angustifolius, D. henryi, D. labordei, and D. officinarum. A strong correlation exists between chromosome number and occurrence of distyly. Regardless of taxa in Damnacanthus, distylous populations are diploid, and monomorphic populations are tetraploid. Flowers of the monomorphic populations observed have a long style and short stamens with few exceptions. Polyploidization may have caused the breakdown of distylous to monomorphic flowers. In D. indicus, leaves from the tetraploid populations tend to be larger than those from the diploid populations. Populations of tetraploid D. indicus were distributed in more northern areas than those of the diploid. Three types of sympatric distribution were found for the varieties of D. indicus in Japan: diploid and tetraploid, two diploids, and two tetraploids. Based on the present chromosome number study, the taxonomy of the varieties of D. indicus should be revised.     

The role of tetraploids in the sexual-asexual cycle in dandelions (Taraxacum)

Apomictic plants often produce pollen that can function in crosses with related sexuals. Moreover, facultative apomicts can produce some sexual offspring. In dandelions, Taraxacum, a sexual-asexual cycle between diploid sexuals and triploid apomicts, has been described, based on experimental crosses and population genetic studies. Little is known about the actual hybridization processes in nature. We therefore studied the sexual-asexual cycle in a mixed dandelion population in the Netherlands. In this population, the frequencies of sexual diploids and triploids were 0.31 and 0.68, respectively. In addition, less than 1% tetraploids were detected. Diploids were strict sexuals, triploids were obligate apomicts, but tetraploids were most often only partly apomictic, lacking certain elements of apomixis. Tetraploid seed fertility in the field was significantly lower than that of apomictic triploids. Field-pollinated sexual diploids produced on average less than 2% polyploid offspring, implying that the effect of hybridization in the 2x-3x cycle in Taraxacum will be low. Until now, 2x-3x crosses were assumed to be the main pathway of new formation of triploid apomicts in the sexual-asexual cycle in Taraxacum. However, tetraploid pollen donors produced 28 times more triploid offspring in experimental crosses with diploid sexuals than triploid pollen donors. Rare tetraploids may therefore act as an important bridge in the formation of new triploid apomicts.     

Production of second generation triploid and tetraploid rainbow trout by mating tetraploid males and diploid females — Potential of tetraploid fish

Summary First generation tetraploids were produced by hydrostatic pressure treatment before the first cleavage and raised until the adult stage. Their survival and growth were severely depressed when compared to the diploid control: after two years, no ovulated females were found although males produced sperm at 1 and 2 years of age and were mated individually with diploid females. The progenies were consistently normal with high survival rates. They were found to be almost all triploids by karyology, which failed to detect a significant rate of aneuploidies. However, the fertilizing ability of tetraploid males was always low (0 to 97% of the control; average 40%). Several arguments presented here support the hypothesis that diploid spermatozoas, which are wider than haploid ones, would be frequently blocked during their penetration through the micropyle canal. Second generation tetraploids were produced after such matings by heat shocks, causing the retention of the second polar body. Their survival and growth were much more satisfactory than in the first generation, although still lower than in diploid and triploid controls issuing from diploid parents. Performances of second generation triploids were comparable to those of diploids, and slightly better than those of conventional triploids issuing from diploid parents. 94.5% of the second generation tetraploids were male.     

Spontaneous polyploids in marginal populations of Alopecurus bulbosus Gouan (Poaceae)

Natural populations of A bulbosus from three sites in southern England have been investigated and two were found to contain triploid and tetraploid plants in addition to normal diploids. Meiosis in the triploids was characterized by a high frequency of trivalent formation but, in the tetraploids, multivalents were formed at low frequencies and bivalents were the most common configurations. It is suggested that these are true autopolyploids that have probably arisen through somatic doubling since tetraploid cell lines were found in some anther loculi. The meiotic behaviour of the tetraploid plants is explained on the basis of bivalent-promoting mechanisms being present in the diploid progenitors.