A molecular approach to detect hybridisation between crucian carp (Carassius carassius) and non-indigenous carp species (Carassius spp. and Cyprinus carpio)

1. Releases of non‐native fish into the wild is an increasing problem posing considerable ecological and genetic threats through direct competition and hybridisation. 2. We employed six microsatellite markers to identify first generation hybrids and backcrosses between native crucian carp (Carassius carassius) and introduced goldfish (C. auratus) and common carp (Cyprinus carpio) in the U.K. We also investigated the genetic characteristics of the taxonomically controversial gibel carp (Carassius spp.) from sites across Europe. 3. Natural hybridisation between goldfish and crucian carp occurs frequently, although hybrids between all other species pairs were observed. Only 62% of British crucian carp populations (n = 21) consisted exclusively of pure crucian carp. In some populations hybrids were so frequent, that no pure crucian carp were caught, indicating a high competitive ability of hybrids. 4. Most hybrids belonged to the F1 generation but backcrossing was evident at a low frequency in goldfish × crucian carp hybrids and goldfish × common carp hybrids. Furthermore, some local populations had high frequencies of backcrosses, raising the opportunity for introgression. 5. Gibel carp from Germany and Italy belonged to two triploid clonal lineages that were genetically closely related to goldfish, whereas all individuals identified from British populations proved to be crucian carp × goldfish hybrids. 6. Our study suggests that the release of closely related exotic cyprinids not only poses a threat to the genetic integrity and associated local adaptations of native species, but may also contribute to shifts in community structure through competitive interactions.     

Diagnostic features and biometry of head bones for identifying Carassius species in faecal and archaeological remains

The identification of fish species from head bone remains is employed in various sciences, including archaeology, paleontology and field ecology, with the estimation of fish size from biometric relationships being useful in the assessment of predation pressure exerted by increasing numbers of piscivorous species (e.g. Eurasian otter Lutra lutra and great cormorant Phalacrocorax carbo). This is particularly relevant for crucian carp, Carassius carassius, which is in decline in Europe due to changes in land use and to increasing numbers of non‐native Carassius species (i.e. goldfish C. auratus, gibel carp C. gibelio), which hybridize with C. carassius. However, diagnostic keys and biometric relationships are lacking for C. carassius and its most common hybrids, crosses with C. auratus. The present paper addresses this gap in knowledge, providing diagnostic keys and biometric relationships for the head bones of all Carassius species found in Europe as well as for C. carassius × C. auratus hybrids. All bone size to body length relationships were statistically significant. Similarly, all bone size to body weight relationships were significant for C. carassius, C. auratus, and C. gibelio, but none were significant for C. carassius × C. auratus hybrids. Diagnostic structures were found to distinguish easily between the Carassius species and hybrids, which will assist in determining the identity and sizes of prey found in faecal and archaeological remains.     

Fertility of triploid hybrids of Prussian carp (<Emphasis Type="Italic">Carassius gibelio</Emphasis>) with common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Fertility of backcross triploid hybrids containing one genome of Prussian carp and two genomes of common carp is investigated. The females of hybrids of Prussian carp and common carp (Prussian × common carp) are prolific and produce diploid gametes. Since males of such hybrids are sterile, their reproduction is realized by means of induced gynogenesis. Triploid progeny is obtained by backcrossing female Prussian × common carp with carp males. Among triploids obtained from hybrids F₁ and among hybrids of the first gynogenetic generation, there were no prolific specimens. However, in reproduction of diploid hybrids by means of gynogenesis during six generations, the female fertility in the backcross progeny is restored. From backcross triploid females (daughters of Prussian × common carp of the sixth gynogenetic generation), a viable triploid gynogenetic progeny and a tetraploid backcross (by carp) progeny are obtained. The obtained data may be considered as the experimental proof of the hypothesis of reticular speciation.     

Hypoxia tolerance of hybrids of carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> and golden carp <Emphasis Type="Italic">Carassius auratus</Emphasis>

Hypoxia tolerance of diploid gynogenetic and triploid backcross hybrids between golden carp Carassius auratus and carp Cyprinus carpio was investigated in comparison with carp. The more that heredity of golden carp was in their genotype, the better the hybrids survived under hypoxia conditions.     

Genetic structure of silver Prussian carp <Emphasis Type="Italic">Carassius</Emphasis> (Superspecies <Emphasis Type="Italic">auratus</Emphasis>) (Linnaeus, 1758) colonies from the Middle Dnieper Basin

Population genetic structure of silver Prussian carp from the Middle Dnieper basin was established by means of biochemical gene marking and cytometric analysis. In addition to diploid goldfish Carassius auratus, which was the basic species of bisexual colonies, a number of biotypes of triploid silver Prussian carp C. gibelio were identified. These biotypes either formed isolated unisexual populations, or were an admixture to the bisexual colonies. In addition, in bisexual colonies, triploid females of non-clonal origin were identified. Despite the fact that all forms of C. gibelio reproduced by means of gynogenesis, some of them (C. gibelio-1) formed isolated unisexual populations, while the others (C. gibelio-2, -3, and -4) usually coexisted with C. auratus.     

Detection of hybridization between two loach species (<Emphasis Type="Italic">Paramisgurnus dabryanus</Emphasis> and <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>) in wild populations

Artificial interspecific hybrids between large scale loach P. dabryanus and tetraploid pond loach M. anguillicaudatus (Cobitidae, Cypriniformes) are viable. To detect the occurrence of possible natural hybridization, genetic analyses by using microsatellite markers were performed for natural populations of large scale loach and pond loach, the reciprocal laboratory hybrids, and “supposed hybrids” with ambiguous morphology. The fertility of the artificial hybrids was also tested. At one diagnostic microsatellite (Mac50), one out of 20 “supposed hybrids” was identified to be F₁ hybrid between the two loach species because it had the same genotype as that of the laboratory hybrids. The triploid hybrids between the two species were confirmed to be female-sterile. The results show that rare hybridization has occurred between diploid large scale loach and tetraploid pond loach in nature although it may have little effect in genetic introgression. This study is helpful for fish conservation and encourages further investigation on natural hybridization and introgression of loaches.     

Spontaneous polyploidy,gynogenesis and androgenesis in second generation (F2) koi Cyprinus carpio × goldfish Carassius auratus hybrids

The objective of this study was to characterize the genetics of second generation (F₂) koi Cyprinus carpio × goldfish Carassius auratus hybrids. Spermatozoa produced by a novel, fertile F₁ male were found to be diploid by flow‐cytometric analysis. Backcross (F₁ female × C. carpio male and C. carpio female × F₁ male) juveniles were triploid, confirming that female and male F₁ hybrids both produced diploid gametes. The vast majority of surviving F₂ juveniles was diploid and small proportions were aneuploid (2·1n–2·3n and 3·1n–3·9n), triploid (3n) and tetraploid (4n). Microsatellite genotyping showed that F₂ diploids repeated either the complete maternal or the complete paternal genotype. Fish with the maternal genotype were female and fish with the paternal genotype were male. This demonstrates that F₂ diploids were the result of spontaneous gynogenesis and spontaneous androgenesis. Analysis of microsatellite inheritance and the sex ratio in F₂ crosses showed that spontaneous gynogenesis and androgenesis did not always occur in equal proportions. One cross was found to have an approximate equal number of androgenetic and gynogenetic offspring while in several other crosses spontaneous androgenesis was found to occur more frequently than spontaneous gynogenesis.     

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.     

Hybridization of crucian carp, Carassius carassius (Linnaeus, 1758), in Ukrainian reservoirs and genetic structure of hybrids

Hybridization of crucian carps Carassius carassius in polyspecific crucian populations of reservoirs of Ukraine and genetic structure of the hybrids were investigated using biochemical gene marking and cytometric procedure. The fact of wide hybridization between C. auratus and C. carassius was proved to be true by large number of hybrids which can form populations consisting only from hybrid individuals. Hybrids C. auratus x C. carassius were diploid, tryploid and in exceptional cases tetraploid; females and males which most likely breed by hybridogenesis. Besides, some clonal hybrids C. carassius x C. gibelio-1 appearing as tetraploid females, and one triploid female C. carassius x Tinca tinca were revealed. It is supported that hybridization of alien C. auratus with endemic C. carassius became one of mechanisms of replacement and depressions of populations of the last.     

Genetic Analysis of Putative Triploid Miscanthus Hybrids and Tetraploid M. sacchariflorus Collected from Sympatric Populations of Kushima, Japan

Miscanthus ?×giganteus, a triploid hybrid between tetraploid M. sacchariflorus and diploid M. sinensis, has considerable potential as a bioenergy crop. Currently only one genotype is widely cultivated, increasing its vulnerability to diseases during production. Finding new hybrids is important to broaden genetic resources of M. ×giganteus. Three putative triploid hybrids were discovered in a sympatric population of tetraploid M. sacchariflorus and diploid M. sinensis in Kushima, Japan. The hybrid nature of the triploids was determined by morphological analysis and sequencing the ribosomal DNA internal transcribed spacer (ITS) region. The triploids had awns on their florets, which is a common characteristic of diploid M. sinensis, and sheath hairs, which is typical of tetraploid M. sacchariflorus. All triploids showed heterozygosity in their ribosomal DNA ITS sequences. Based on these results, it is confirmed that the triploids are hybrids and novel genotypes of M. ×giganteus. Natural crossing between tetraploid M. sacchariflorus × diploid M. sinensis may also lead to the production of tetraploid hybrids. ITS analysis of tetraploid plants showed that one maternal parent of the triploid hybrids, K-Ogi-1, had heterozygous ITS, which was different than the other analyzed tetraploid, M. sacchariflorus. Thus, K-Ogi-1 was likely of hybrid origin. These tetraploid hybrids can also be utilized as parents in M. ×giganteus breeding. Since all hybrids identified in this study had tetraploid M. sacchariflorus as maternal parents, collecting and analyzing seeds from tetraploid M. sacchariflorus in sympatric areas could be an effective strategy to identify natural Miscanthus hybrids that can be used as bioenergy crops.     

Hybridization success is largely limited to homoploid <Emphasis Type="Italic">Prunus</Emphasis> hybrids: a multidisciplinary approach

Prunus fruticosa is a rare shrub occurring in Eurasian thermophilous forest-steppe alliances. The species frequently hybridizes with cultivated Prunus species in Europe (allochthonous tetraploid P. cerasus and partly indigenous diploid P. avium). Propidium iodide flow cytometry, distance-based morphometrics, elliptic Fourier analysis and embryology were employed to evaluate the extent of hybridization in six Slovak populations. Flow cytometric analyses revealed three ploidy levels: diploid (P. avium), triploid (P. × mohacsyana) and tetraploid (P. fruticosa, P. × eminens and P. cerasus). In addition, P. fruticosa and P. cerasus, at the tetraploid level, were found to differ in absolute genome size. An embryological evaluation suggested the existence of a triploid block in P. × mohacsyana and significant potential for hybridization among tetraploid taxa (indicated also by a continuous distribution of genome size data and further mirrored by morphometrics). Although hybrids significantly differ in ploidy level and embryological characteristics, they are almost indistinguishable using morphological characters. Hybridization with P. cerasus thus turns out to be a significant threat to wild populations of P. fruticosa compared to the relatively weak influence of P. avium.     

In vitro embryo rescue culture of F<Subscript>1</Subscript> progenies from crosses between diploid and tetraploid grape varieties

The purpose of this study was to understand factors affecting in vitro embryo rescue culture from hybrids between diploid and tetraploid varieties of grape in creation new triploid germplasm resources. The effects of different media, removal ages of immature seeds and reciprocal crosses of parents on the germination and seedling survival of immature seeds from crosses between diploid and tetraploid grape varieties by in vitro embryo rescue culture were investigated. The results indicated that the medium consisting of NN-1969 + IAA 1.75 mg l⁻¹ + GA₃ 0.35 mg l⁻¹ + CH 400 mg l⁻¹ + AC 2.0 g l⁻¹ was better than other media. The optimal removal age of immature seeds for the best development of embryos was 35–45 days after pollination (DAP). The percentage of germination (PG) for immature seeds and the percentage of seedling survival (PSS) for immature seeds for diploid varieties used as female parents were 10.72% and 4.35% higher than when tetraploid varieties were used as female parents respectively. A total of 41 hybrid progenies from eight combinations were obtained, made up of 17 diploid, 9 tetraploid, 14 aneuploid, and 1 triploid progeny as determined by root-tip chromosome identification. The triploid progeny was from Fujiminori (2n = 4x = 76) × Jingxiu (2n = 2x = 38). These results implied that it was feasible to extend the hybridization range of grape and to create new germplasm resources by in vitro embryo rescue based on the conventional hybridization. The NN-1969 medium supplemented with GA₃ and IAA was more propitious to the development of immature seeds sampled at about 45 DAP. It was easier to obtain plants using diploid as female parent, but triploid progeny was only obtained using tetraploid as female parent.     

Production of tetraploid plants of non apomictic citrus genotypes

Ploidy manipulation in Citrus is a major issue of current breeding programs aiming to develop triploid seedless mandarins to address consumer demands for seedless fruits. The most effective method to obtain triploid hybrids is to pollinate tetraploid non apomictic cultivars with pollen of diploid varieties. Such non apomictic tetraploid lines are not found in the citrus germplasm and need to be created. In this work we describe a new methodology based on in vitro shoot-tip grafting combined with treatment of the micro-grafted shoot-tip with colchicine and oryzalin to achieve chromosome doubling and a dechimerization procedure assisted by flow cytometry. Stable tetraploid plants of Clemenules, Fina and Marisol clementines and Moncada mandarin have been obtained directly from shoot tip grafting combined with colchicine and oryzalin treatments or after dechimerization of mixoploids plants (2x–4x). These stable tetraploid plants have been used in 4x × 2x hybridizations, to recover over 3,250 triploid hybrids in 3 years.     

The formation of the polyploid hybrids from different subfamily fish crossings and its evolutionary significance

This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp x blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (female symbol) x blunt snout bream (BSB) (male symbol) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.     

Comparative trophic impacts of two globally invasive cyprinid fishes reveal species‐specific invasion consequences for a threatened native fish

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Cytological and Reproductive Studies of Japanese Diplazium (Woodsiaceae; Pteridophyta). II. Polyploidy and Hybridity in the Species Group with Summer-Green Bi- to Tripinnate Leaves

Diplazium with summer-green bi- to tripinnate leaves. Diplazium mesosorum and D. sibiricum var. sibiricum are sexual diploids (2n=82; n=41ll); D. chinense and D. squamigerum are sexual tetraploids (2n=164; n=82ll); and D. sibiricum var. glabrum is a sterile triploid (2n=123; meiosis irregular). Diplazium nipponicum includes both sterile triploid and sexual tetraploid populations. The triploid is larger in relation to several morphological characteristics and occurs in more southern regions than the tetraploid. Allozyme analysis suggests that the triploid is a hybrid of recurrent origin between the tetraploid and an unknown diploid. Diplazium × bittyuense is a sterile tetraploid, and the mitotic chromosome number, meiotic chromosome behavior, and allozyme analysis confirm the working hypothesis that it is a hybrid between D. nipponicum and D. chinense. Apomicts are not found within Diplazium with summer-green bi- to tripinnate leaves, and the taxonomic complexity can be attributed to polyploidy and natural hybridization. Received 3 March 2000/ Accepted in revised form 19 April 2000     

Koi herpesvirus infection in experimentally infected common carp Cyprinus carpio (Linnaeus, 1758) and three potential carrier fish species Carassius carassius (Linnaeus, 1758); Rutilus rutilus (Linnaeus, 1758); and Tinca tinca (Linnaeus, 1758) by quantitative real‐time PCR and in‐situ hybridization

Only single cells in the carrier fish species Carassius carassius (Linnaeus, 1758) for koi herpesvirus (KHV) are infected in contrast to large numbers in the susceptible species common carp Cyprinus carpio (Linnaeus 1758). Several species of the family Cyprinidae have been described as virus carrier species, showing no clinical signs of a KHV disease but able to transmit the virus to other susceptible fish. In this study, 72 common carp Cyprinus carpio (Linnaeus, 1758), 36 tench Tinca tinca (Linnaeus, 1758), 36 crucian carp Carassius carassius (Linnaeus, 1758) and 36 common roach Rutilus rutilus (Linnaeus, 1758) were experimentally infected with KHV (isolate “Israel”) by immersion and kept at 20°C. The fish were euthanized at 12 timepoints over a period of 90 days and virus DNA was quantified in tissues by a real‐time TaqMan PCR. Whereas KHV‐DNA was found in Cyprinus carpio for up to 90 days, the virus DNA was detectable only in single individuals of Rutilus rutilus, Tinca tinca and Carassius carassius for up to 25 days after experimental virus exposure. Tissue samples of Cyprinus carpio and Carassius carassius were screened by in‐situ hybridization. Positive signals were found in various organs of the common carp tested crucian carp. In the latter species a much smaller number of virus‐positive stained cells was detected compared to the infected carp.     

Hybridization and introgression among three Aconitum (Ranunculaceae) species of different ploidy levels in the Tatra Mountains (Western Carpathians)

Hybridization among species of A conitum effects their morphology and ecology. In this study the hybridization between the diploid 2n(2x) = 16 ( A . lasiocarpum and A . variegatum) and tetraploid 2n(4x) = 32 ( A . firmum) species was documented in the Tatra Mountains (Western Carpathians) in a small, local population in a semi‐natural site. The hybrid species were: homoploid A . × pawlowskii ( A . lasiocarpum × A . variegatum), and triploid A . × berdaui ( A . firmum × A . variegatum, 2n(3x) = 24). Chloroplast DNA (cpDNA) alleles formed two distinct haplotypes, one typical for the tetraploid and another for diploid lines, shared between the tetraploid, triploid and diploid groups, indicating introgressive hybridization. The presumed gene flow was from the tetraploid to diploid species via the triploid bridge. The only two specimens of A . × pawlowskii that harbored tetraploid ( A . firmum) type cpDNA possessed bracteoles of A . firmum‐type. The remaining introgressed (cpDNA and Inter Simple Sequence Repeats (ISSR)) specimens ( A . variegatum) were morphologically pure, implying cryptic introgression. ISSR loci shared between the tetraploid A . firmum and diploid A . variegatum support the hypothesis of an ancient allopolyploid origin of A . firmum and the diploid species of A . variegatum‐type as one of its parent.     

Cytological and Reproductive Studies on Japanese Diplazium (Woodsiaceae; Pteridophyta): Apomictic Reproduction in Diplazium with Evergreen Bi- to Tripinnate Leaves

Diplazium , including polymorphic terrestrial species with evergreen bi- to tripinnate leaves. Diplazium hachijoense, D. virescens var. virescens, var. conterminum, var. okinawaense, and two other unnamed varieties, D. kawakamii var. kawakamii, D. dilatatum var. heterolepia, D. taiwanense, D. × kawabatae (=D. dilatatum × taiwanense), D. × takii (=D. hachijoense × virescens var. virescens), and D.× nakamurae (= D. hachijoense × virescens var. conterminum) are apomictic triploids (2n=n=123). Diplazium amamianum and D. esculentum are sexual diploids (2n=82, n=41) and D. subtripinnatum is a sexual tetraploid (2n= 164, n=82). Diplazium dilatatum var. dilatatum includes both sexual diploid and apomictic triploid populations. Cultivated gametophytes of six triploid taxa produced sporophytes apogamously, confirming their apomictic reproduction. All three putative hybrids, D. × kawabatae, D. × takii, and D. × nakamurae, are triploid, apomictic, and fertile taxa, therefore they are not the result of hybridization between known pairs of Japanese Diplazium plants. Received 16 March 1999/ Accepted in revised form 30 September 1999     

OBSERVATIONS OF CHROMOSOMES IN CIRCAEA (ONAGRACEAE)

Observations of meiotic and/or mitotic chromosomes for 16 specific, infraspecific, and hybrid taxa from 56 populations are presented; chromosome numbers for 11 taxa are reported for the first time. All samples reported have 2n = 22, with the exception that 3 of 12 collections of C. × intermedia have at least some individuals that are triploid. North American diploid collections of this hybrid are heterozygous for one reciprocal translocation; only 1 of 5 European diploid collections also exhibited a translocation. Meiosis in four naturally occurring Asian hybrids is also analyzed: C. × ovata (C. cordata × C. mollis), C. × decipiens (C. erubescens × C. lutetiana) and C. x× dubia (C. cordata × C. eurbescens) possess a single translocation; C. × mentiens (C. alpina × C. eurbescens) is structurally homozygous. With the exception of the translocation, chromosome pairing in these hybrids is normal. Early diakinesis chromosomes possess densely staining centric regions and diffusely staining arms that are subequal in length.