Disturbances in the ploidy level in the gynogenetic sterlet <Emphasis Type="Italic">Acipenser ruthenus</Emphasis>

Artificial mitotic gynogenesis, a chromosome set manipulation, is applied to provide the homozygous progeny with only maternal inheritance. Here, gynogenetic development was induced in the sterlet Acipenser ruthenus L. (Acipenseridae) by activation of the eggs originating from albino females with the UV-irradiated spermatozoa from wild-coloured males, followed by the heat shock applied to suppress the first mitotic division in the haploid zygotes. All experimentally obtained gynogenetic offspring possessed recessive albino coloration. Moreover, the genetic verification, based on three microsatellite DNA markers, confirmed the only maternal inheritance in the albino progeny. Cytogenetic screening enabled identification of the aneuploids, haploids, diploids, triploids, tetraploids and mosaic individuals among the gynogenetic larvae that hatched from the eggs subjected to the heat shock. Furthermore, 40% of the larvae from the haploid variants of the research that were not exposed to the temperature shock showed the diploid chromosome number. A variation of the ploidy level observed in the gynogenetic sterlets may be the consequence of the spontaneous polyploidisation that occurred in the haploid zygotes. Moreover, observation during embryogenesis showed varied stages of eggs development and the asynchronous cell cleavages that may have resulted in the chromosomal disturbances observed in the gynogenetic sterlets here.     

Genetic and selective factors in reduction of the hallux in Pongo pygmaeus

Extreme reduction of the hallux is unique to the orang-utan (Pongo pygmaeus) among Primates. Hallucal diminution has advanced so far that 60% of orang-utans lack both distal phalanges and nails. Absence of these structures occurs significantly more often in females than in males. Hypotheses on possible genetic control of the condition have been tested and indicate that either single gene inheritance or polygenic inheritance with variable expressivity is involved. Reduction of pollex and of hallux in Pongo have advanced with selection for a specialized four-digit grasp in hands and feet. Diminution has progressed farther in the great toe than in the thumb due to selection for fine manipulation in the latter digit.     

Genetic Evidence for Gonochoristic Reproduction in Gynogenetic Silver Crucian Carp (Carassius auratus gibelio Bloch) as Revealed by RAPD Assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp. Received: 5 January 2000 / Accepted: 3 August 2000     

Female-biased operational sex ratio of sexual host fish in a gynogenetic complex of Carassius auratus

To study the coexistence of sexual and gynogenetic forms, we examined the population structure of a gynogenetic complex of the Japanese crucian carp, Carassius auratus Temminck et Schlegel, during the April–June reproductive season by collecting 1225 mature fish that migrated from Lake Suwa to a tributary river for spawning. There were more sexual fish (about 80%) than gynogenetic fish in this complex, and the operational sex ratio in the sexual form was female biased (males were about 20%). Mean standard length and body weight of sexual females were larger than those of sexual males. Sex ratio was male biased in smaller fish (standard length, <8.5 cm) but female biased in larger fish (standard length, ≥8.5 cm). We determined age by scale ring marks; the average age of sexual females was higher than that of males, but there was no significant difference in the average age between sexual and gynogenetic females. Sex ratio in the sexual form was more female biased for old than for young fish, and the mean size of sexual females was larger than that of males of the same age. The clear female-biased sex ratio and age difference between sexual females and males can be explained either by (1) higher mortality of males or by (2) female-biased sex allocation. The latter process reduces the disadvantage of sex and contributes to the coexistence of sexual and gynogenetic forms. Received: November 24, 2000 / Accepted: March 6, 2001     

Mating Preferences of the Gynogenetic Amazon Molly Differ Between Populations Sympatric with Different Host Species

Gynogenetic species rely on sperm from heterospecifics for reproduction but do not receive genetic benefits from mating because none of the paternal genome is incorporated into offspring. The gynogenetic Amazon molly (Poecilia formosa) is a species of hybrid origins that are sympatric with one of the two parent species that provide sperm for reproduction, P. latipinna or P. mexicana. Amazons should not prefer to mate with one species over the other because sperm from both species will trigger embryogenesis, but mating preferences may be present in Amazons through other mechanisms. Amazons may prefer the more familiar species (males found in sympatry), or Amazons may prefer males with the greatest lateral projection area (LPA), a preference that is present in the parent species and may be retained within the Amazon hybrid genome. We tested association preferences of two populations of Amazons sympatric with either P. mexicana or P. latipinna. We first performed live trials to test whether Amazons preferred one host species over the other and found that neither population of Amazons showed a preference. We then tested whether Amazons preferred sympatric male (familiar) host or the male with the greatest lateral projection area (LPA) using four animated male models that varied in host species and manipulation of LPA. We found Amazons from a population sympatric with P. latipinna showed no variation in their association preference across the different models. In contrast, Amazons from a population sympatric with P. mexicana (naturally small LPA) spent more time associating with the male models that had smaller LPA, which is more familiar to this population of Amazons. We suggest that Amazons may have population differences in mating preferences, where Amazons sympatric with P. latipinna may not show mating preference for host species, but Amazons sympatric with P. mexicana may show preferences for more familiar‐shaped males.     

Features of inheritance of several morphological characters in the hybrids between Siberian sturgeon <Emphasis Type="Italic">Acipenser baerii</Emphasis> and beluga <Emphasis Type="Italic">A. huso</Emphasis> (Acipenseridae) and in gynogenetic individuals of Siberian sturgeon

Comparative analysis of several morphological characters is conducted in experimentally obtained hybrids between Siberian sturgeon Acipenser baerii and beluga A. huso, in parental species, and in gynogenetic progeny of Siberian sturgeon. The gynogenetic individuals are similar to Siberian sturgeon by all characters used for differentiation between Siberian sturgeon and beluga that supports gynogenetic origin of these individuals. Based on the data obtained on the hybrid specimens, the features of inheritance of the studied characters in sturgeons and their variation in postlarval ontogeny are discussed.     

Evidence for the Formation of the Male Gynogenetic Fish

The females and unexpected males of gynogenetic red crucian carps (GRCC) with the 1:1 sex ratio were found in the progeny of the distant crossing of red crucian carp (RCC; ♀, 2n?=?100) × blunt snout bream (BSB; ♂, 2n?=?48). The females and males of GRCC were fertile, and they mated each other to generate the red crucian carps (GRCC₁) and another variational gray crucian carps (GGCC). The GRCC and their offspring were proved to be diploids (2n?=?100) with one to three microchromosomes by examining the chromosomal metaphases. The evidences for the male’s genetic effect in GRCC were provided by means of fluorescence in situ hybridization, Sox-HMG DNA markers, and microsatellite DNA markers. The genotypic variances of GRCC resulted in their phenotypic variances which were quite different from their maternal parent. It was concluded that the formation of the male gynogenetic fish in GRCC resulted from the genetic leakage of the paternal fish in the form of the microchromosomes including the paternal male-determining gene. After being activated by the sperm of BSB, which was inactivated and finally degraded but left the microchromosomes, the egg of RCC, in which the 50 chromosomes were spontaneously doubled to 100 chromosomes, developed into the diploid male gynogenetic fish. The formation of the bisexual GRCC and their progeny indicated that the distant hybridization could generate the bisexual diploid gynogenetic fish with genetic variation derived from the paternal fish, which is of great significance in both fish genetic breeding and evolutionary biology.     

Choice of microsatellite markers for identifying homozygosity of mitotic gynogenetic diploids in Japanese flounder Paralichthys olivaceus

A set of 72 microsatellite markers distributed evenly among 24 linkage groups were selected from the published genetic linkage maps of Japanese flounder Paralichthys olivaceus. In two normal diploid full‐sib families, the test for Mendelian inheritance showed that genotypic segregation deviations were not significant at all analysed loci. To estimate microsatellite‐centromere map distances, four meiotic gynogenetic diploid lines were produced by the activation of eggs using UV irradiated sperm of red seabream Pagrus major and cold‐shock treatment to block the extrusion of the second polar body. Under the assumption of complete interference, 21 markers were located in the centromeric region, 39 in the telomeric region and the rest in the intermediate region of linkage groups. A total of 192 mitotic gynogenetic diploids from one spawn were identified by these markers. Genotype analysis showed that the number of homozygous individuals decreased as microsatellite‐centromere map distance increased on each linkage group.     

Inheritance of microsatellite DNA in bisexual gynogenesis complex of Fangzheng silver crucian carp,Carassius auratus gibelio (Bloch)

Five gynogenetic progeny groups of silver crucian carp Carassius auratus gibelio were produced and sex ratios (males:total progeny) of each of the progeny groups were analysed. About 110 males and 366 females were genotyped at 15 microsatellite loci for comparison with their parents to (1) verify the gynogenesis status of Fangzheng C. auratus gibelio, (2) detect the incorporation of paternal genetic material into the offspring and (3) study the possible association of genetic exchange at microsatellite loci with the existence of sex. The sex ratios in progenies of five groups were highly variable, but all had significant female bias. The sex ratio ranged from 0 to 0·37. Significant differences in the sex ratio within and between groups were also found. Microsatellite genotyping at 15 loci showed that 100 and 97% of the progeny shared the same genotype with the mother in four groups and in one group, respectively, confirming that gynogenesis is the general mechanism of reproduction in C. auratus gibelio. However, 0·63% of all offspring did show incorporation of paternal genetic material. No single loci tested were associated with the occurrence of male progeny, indicating unknown genetic mechanisms for sex determination in C. auratus gibelio.     

Inter-populational difference in microsatellite–centromere map distances in the loach, <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>

Microsatellite–centromere recombination rates were estimated at 21 loci in relation to centromere of chromosomes in gynogenetic diploid lines induced from loaches Misgurnus anguillicaudatus of two different populations in Japan. All the microsatellite loci gave allelic segregation according to the Mendelian manner of inheritance in normal diploid families. Since loaches from the Kita population in the southern area of Hokkaido Island and those from the Memanbetsu population in the northern area, Hokkaido, Japan, were reported by previous genetic studies to be genetically diversified, map distances were compared between loaches from the two different populations. Three (Mado7, Mac3 and Mac49) of five loci, which could be compared inter-populationally, gave significantly different recombination rates, i.e., map distances. The results support the presence of genetic difference between the two populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular characterization of different preproGnRHs in Astyanax altiparanae (Characiformes): Effects of GnRH on female reproduction

Gonadotropin‐releasing hormone (GnRH) is a key molecule in the initiation of the hypothalamic–pituitary–gonadal axis. Thus, knowledge about GnRH may contribute to the effectiveness of species reproduction. Using a Neotropical tetra Astyanax altiparanae as a fish model species, the GnRH forms were characterized at the molecular level and the role of injected GnRHs in vivo was evaluated. The full‐length complementary DNA (cDNA) sequences of preproGnRH2 (612 bp) and preproGnRH3 (407 bp) of A. altiparanae were obtained, and the GnRH1 form was not detected. The cDNA sequences of preproGnRH2 and preproGnRH3 were found to be conserved, but a change in the amino acid at position 8 of the GnRH3 decapeptide of A. altiparanae was observed. All the injected GnRHs stimulated lhβ messenger RNA (mRNA) expression but not fshβ mRNA expression, and only GnRH2 was able to increase maturation‐inducing steroid (MIS) levels and possibly stimulate oocyte release. Furthermore, only GnRH2 was able to start the entire reproductive hormonal cascade and induce spawning.     

Biosystematics of the Muellerianella complex (Homopters,Delphacidae), hybridization studies

Crosses were made between three closely related species of the planthopper genus Muellerianella, M. brevipennis, M. extrusa and M. fairmairei. Most hybrids are produced in crosses between M. extrusa and M. fairmairei. Most of the hybrid females in these crosses and some of the hybrid males are fertile and can be successfully backcrossed with the parental species. Crosses of M. brevipennis with either M. fairmairei or M. extrusa yield less progeny, of which all males are sterile. Backcrosses of the hybrid females with the parental species are partly successful. The possible evolutionary consequences of mating readiness and interfertility between the three species under field conditions are discussed. Efforts to resynthesize triploid gynogenetic forms by backcrossing hybrid females of M. fairmairei and M. brevipennis with males of M. fairmairei, as was reported by Drosopoulos (Evolution 32: 916–920, 1978), failed. To produce offspring, the existing gynogenetic forms have to mate with males of one of the three bisexual species.     

Induction of meiotic gynogenesis in Atlantic cod,Gadus morhua (L.)

Gynogenesis is one of several chromosome‐manipulating techniques used in fish. In gynogenesis the male does not contribute to the genetic material of the offspring, and the sperm cells act only as stimulators in order for the egg to start development. This technique has several applications, both in aquaculture and in biological research: gynogenetic fish may be used as a step in the production of all‐female populations, the production of isogenetic‐ and inbred lines, revealing of the sex determination mechanism, construction of genetic maps, and testing of environmental vs genetic control of different traits. The aim of this study was to develop a simple protocol for production of gynogenetic cod (Gadus morhua L.) for further use in aquaculture research. Various milt dilutions and UV‐irradiation doses were tested, in order to inactivate the sperm without destroying its ability to induce egg development. This was followed by pressure treatment of the eggs shortly after ‘fertilization’ to suppress the completion of meiosis II, and thereby restoring diploidy. A dose of 9000 erg mm^?2, followed by a 5‐min pressure treatment (58.6 MPa) 180 min‐degrees after fertilization gave 100% gynogenetic larvae. Histologically, sexual differentiated fish were all females, possibly confirming female homogamety in Atlantic cod. No particular signs of reduced growth, survival or enhanced deformity rates were observed after the fish had reached the juvenile phase. Mortality was, however, high during the egg and larval stages. This protocol has made capable the production of gynogenetic cod juveniles in significant amounts using relatively simple means; the next step will be to elaborate on the technique in order to produce mitotic gynogenetic (double haploid) individuals, which are 100% homozygous.     

Gynogenesis Assessment Using Microsatellite Genetic Markers in Turbot (<Emphasis Type="Italic">Scophthalmus maximus</Emphasis>)

Gynogenesis was assessed by different methods in 2 families of gynogenetic offspring in turbot (Scophthalmus maximus). Karyotype analysis in embryos and larvae demonstrated high accuracy in estimation of ploidy level, but performance was uneven given the low quality and number of plates obtained. The use of silver staining to estimate the number of nucleoli per nucleus resulted in a straightforward and easy method to evaluate the ploidy of the samples studied. However, the existence of a nucleolus organizer region polymorphism in turbot determined a small error in ploidy estimation, important when checking ploidy in specific individuals. The use of a set of 11 highly variable microsatellite loci proved to be a powerful method to confirm the exclusive maternal inheritance to gynogenetic offspring in turbot, with probabilities of detection of putative paternal genetic contribution above 99.99%.     

Evidence of doubly uniparental inheritance of the mitochondrial DNA in Polititapes rhomboides (Bivalvia,Veneridae): Evolutionary and population genetic analysis of F and M mitotypes

Doubly uniparental inheritance (DUI) is a particular mitochondrial DNA inheritance mode reported in a number of bivalves. DUI species show two types of mtDNA, one transmitted from females to daughters and sons (F mitotype) and another one from males to sons (M mitotype). In Veneridae, the existence of DUI has been investigated in several species but it was found in only two of them. In this study, we obtained partial sequences of rrnL, cytb and cox1 genes of males and females of Polititapes rhomboides from NW Spain and we demonstrated the existence of heteroplasmy in males, as expected under DUI. F and M mitotypes showed a taxon-specific phylogenetic pattern and similar evolutionary rates. We focused on cox1 for population genetic analysis, examining separately F and M mitotypes, but also F mitotypes from females (F_♀) and males (F_♂). In all cases, cox1 bears signs of strong purifying selection, with no apparent evidence of relaxed selection in the M genome, while the divergence between F and M genomes is in agreement with the neutral model of evolution. The cox1 polymorphism, higher at the M than at the F genome, also shows clear footprints of genetic hitchhiking with favourable mutations at other mtDNA loci, except for F_♂. In terms of population structure, results suggest that the pattern depends on the examined mitotype (F, F_♀, F_♂ or M).     

New microsatellite loci confirm hybrid origin,parthenogenetic inheritance,and mitotic gene conversion in the gynogenetic Amazon molly (Poecilia formosa)

We describe the first microsatellite loci for the gynogenetic Amazon molly, Poecilia formosa, an all‐female species arisen through hybridization of the bisexual species Poecilia mexicana and Poecilia latipinna. The loci showed one to six alleles and an expected heterozygosity between zero and 0.75. As expected with parthenogenetic inheritance, most loci were either constantly homozygous (five loci) or constantly heterozygous (eight loci). For six loci, both heterozygotes and homozygotes occurred. This and the fact that some loci only showed alleles of one of the ancestral species could indicate genome homogenization through mitotic gene conversion. Our new loci conformed to the hybrid origin of Amazon molly and are also applicable to both ancestral bisexual species.     

Isotopic variation in five species of stream fishes under the influence of different land uses

The aim of this study was to test if changes in land use alter the isotopic signature of fish species, promoting changes in the trophic position and food resource partitioning between these consumers. Three different systems were investigated: pasture streams (n = 3), streams in sugar cane plantations (n = 3) and reference streams (n = 3). Fish species Aspidoras fuscoguttatus, Astyanax altiparanae, Characidium zebra, Hisonotus piracanjuba and Knodus moenkhausii were selected, and their nitrogen and carbon isotopic compositions were estimated to assess changes in the trophic level and partitioning of food items consumed. The composition of δ¹³C (‰) only differed among the land use categories for A. altiparanae, H. piracanjuba and K. moenkhausii. Resource partitioning was different for all species, with changes in the sources or proportions they consumed in each land use category, but only A. altiparanae introduced new food sources in large quantity in altered land uses. It is important to note, however, that the results from the resource partitioning analysis are limited due to large overlapping of isotopic signatures between the analysed food resources. All fish species exhibited variation in δ¹⁵N (‰), with the highest values found in streams under sugar cane or pasture influence. Despite the variation in nitrogen isotopic values, only C. zebra and H. piracanjuba displayed changes in trophic level. Therefore, it is believed that the increase in the δ¹⁵N (‰) value of the individuals collected in streams under the influence of sugar cane or pasture was due to the greater influence of livestock dung and chemical and organic fertilizers. The results also highlight the importance of studying consumer species along with all forms of resources available at each location separately, because the signatures of these resources also vary within different land uses.     

Inheritance of Microsatellite DNA Markers in the Pacific Abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>

Microsatellite markers have been developed for a variety of abalones, and locus-specific homozygote excesses at population level have been recorded for microsatellite loci. To ascertain whether null alleles exist at microsatellite loci in the Pacific abalone, we studied the mode of inheritance of 7 microsatellite loci in 4 families with a reciprocal cross of 2 females × 2 males. All loci segregated codominantly, but only 3 loci (Hdh1321, Hdh78, and Hdd108C) conformed to Mendelian segregation and can be used for parental analysis and population genetic studies. When null alleles were considered, 2 loci (Hdh1761 and Hdh1457) confirmed Mendelian expectations in all families, while the remaining 2 loci (Hdd114B and Hdd229) showed deviation from Mendelian segregation in at least one family even though null alleles were considered. These results indicated the need to test the inheritance pattern for microsatellite markers in abalones before using them for population genetic or parentage analysis.     

Amplified fragment length polymorphism analysis to assess crossover interference and homozygosity in gynogenetic diploid Pacific abalone (Haliotis discus hannai)

Recombination analysis in gynogenetic diploids is a powerful tool for assessing the degree of inbreeding, investigating crossover events and understanding chiasma interference during meiosis. To estimate the marker–centromere recombination rate, the inheritance pattern of 654 amplified fragment length polymorphism (AFLP) markers was examined in the 72‐h veliger larvae of two meiogynogenetic diploid families in the Pacific abalone (Haliotis discus hannai). The second‐division segregation frequency (y) of the AFLP loci ranged from 0.00 to 0.96, with 23.9% of loci showing y‐values higher than 0.67, evidencing the existence of interference. The average recombination frequency across the 654 AFLP loci was 0.45, allowing estimation of the fixation index of 0.55, indicating that meiotic gynogenesis could provide an effective means of rapid inbreeding in the Pacific abalone. The AFLP loci have a small proportion (4.4%) of y‐values greater than 0.90, suggesting that a relatively low or intermediate degree of chiasma interference occurred in the abalone chromosomes. The information obtained in this study will enhance our understanding of the abalone genome and will be useful for genetic studies in the species.     

Biological traits of rare males in the population of Carassius gibelio (Actinopterygii: Cyprinidae) from Lake Pamvotis (north‐west Greece)

The gynogenetic population of Prussian carp Carassius gibelio in Lake Pamvotis (north‐west Greece) included a small percentage of males (2–3%). Cytogenetic analysis revealed heterogeneity of the population at the ploidy level: all females were triploid (modal chromosome number 156–162 chromosomes) while the males were either triploid (modal number 156–158 chromosomes) or tetraploid (modal number 200–214 chromosomes). Morphometric analysis revealed significant differences in body shape among these three groups. The males were more slender than females, while the triploid males could be discriminated from the tetraploid by the morphology of their caudal peduncle. The males attained a significantly lower asymptotic standard length (L_S) (25·04 cm) than females (29·75 cm) and had lower exponent b of the L_S and mass relationship. Under laboratory conditions, intraspecific crossings of C. gibelio males with females could give viable all‐female offspring; in comparison with goldfish Carassius auratus males, the fertility of the C. gibelio males was generally reduced but remained highly variable among individuals.