Frequencies and cytogenetic mechanisms of gynogenetic reproduction and sterility in artificially produced Cobitis F1 hybrids from parental species with different degree of phylogenetic relatedness
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1
Laboratory of Fish Genetics, Institute of Animal Physiology and Genetics (ASCR), Czechia
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2
Charles University, Czechia
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3
Institute of Zoology, Academy of Sciences of Moldova (ASM), Moldova
Most asexually reproducing lineages among teleost fish are the result of an interspecific hybridization between sexual species. In certain F1 hybrid specimen the females produce gametes (eggs) with unreduced chromosomal set; these eggs are genetically identical to somatic cells. These eggs are capable of development after initialisation, while males are usually sterile. In unisexual fishes, premeiotic genome duplication is one of the most widespread modifications of gametogenesis which provides homospecific pairing of identical chromosomal copies. Various asexual hybrids were reported to employ quite different reproductive strategies ranging from hybridogenesis and kleptogenesis, where only part of the genome is clonally transmitted, to parthenogenesis and gynogenesis, which ensures almost complete clonality. Gynogenesis and hybridogenesis are the most related modes of clonal and hemiclonal reproduction in fish. In gynogenesis, the sperm is only required for triggering the development of the zygote. Consequently, all offspring are clonal copies of their mother. However, sometimes the sperm nucleus is entering the zygote and the male DNA is incorporated, increasing the ploidy of the offspring. . Until now many gynogenetic fishes were reported to be of hybrid origin, but the exact mechanisms that triggers asexual reproduction are poorly studied.
Spined loaches of the genus Cobitis offer excellent models to study hybridization, polyploidization, asexuality and sterility since they include several independent cases of polyploidisation, including 7 hybrid zones with at least 4 independently evolved hybrid sexual-asexual complexes. These sexual-asexual complexes dominate most Cobitis populations in Central and Eastern Europe and it is likely that the majority of Cobitis in Europe are of hybrid origin. This evolutionary success challenged the theories predicting the inability of asexuals to form long-term persisting lineages. The age of known clonal lineages is substantially variable ranging from quite recent clones, ~10 000 years, to considerably old lineages, ~300 000 years, with a genetic distance between parental species varying between 7 to 20 mya.
In this study, we aim to answer three questions: 1) Does the genetic distance between parental species trigger the appearance of asexuality in offspring or not? 2) What is the percentage of clonality and sterility within F1 hybrids originated from parental species with different genetic distance? 3) Do asexual F1 hybrids from crosses between different parental species have the same or different mechanism(s) of gametogenesis? To study these questions, we performed bidirectional crosses between closely as well as distantly related species: C. taenia, C. elongatoides, C. taurica and C. tanaitica from the Cobitis s. str. lineage and C. ohridana and C. biliniata from the Adriatic lineage –. Some of these crosses simulated hybridisation events that are known to have happened in nature (from here onwards referred to as ‘specific hybrids’: C. taenia x C. elongatoides, C. elongatoides x C. tanaitica/taurica, C. ohridana x C. sp.), while others crossed species that are not known to hybridize because of geographical isolation (‘non-specific hybrids’: bidirectional crosses between C. ohridana, C. bilineata and C. elongatoides). As a control we propagated wild caught diploid and triploid unisexual hybrids by mating them with males of the co-occurring parental species. Altogether we obtained about 50 specific and non-specific F1 hybrid families.
We analysed the gonadal development of F1 hybrids during ontogenesis and their gametogenesis by using confocal microscopy; gonads of adult specimens were analysed for comparison. In all investigated gonads of hybrid offspring we proofed the presence of germ cell line by staining the gonad with antibodies against vasa protein. The occurrence of meiosis in specific and non-specific hybrids was confirmed by immunofluorescence detection of main proteins of the synaptonemal complexes of chromosomes during the pachytene stage (SYCP1 and SYCP3). To evaluate the rate of clonality vs sterility we performed cytological investigation of different meiotic stages. In contrast to naturally occurring unisexual hybrids (control), not all F1 hybrid individuals overcame the pachytene stage of meiosis and were unable to produce gametes. The main cause of meiotic abortion is due to chromosomal mispairing, by that failing to form bivalents and showing a low rate of recombination. Notably, depending on the genetic divergence between parental species, a variable number of paired chromosomes were observed during pachytene stage in specific and unspecific F1 hybrids. According to morphological and cytological analysis, in majority of hybrid fish which reached the size of an adult individual, gonads are sterile in both sexes.
Only a few F1 hybrid females from each cross were able to overcome pachytene stage and develop oocytes during diplotene. After analysis of diplotenic oocytes from such females we observed a higher number of bivalents than in sexual species. This fact indicates occurrence of a premeiotic genome duplication event allowing overcoming chromosomal mispairing. To confirm the presence of genomic duplication, we applied chromosome specific markers using 3D FISH approach. Following that, we identified a pool of germ cells and early stage oocytes with duplicated genomes, which basically rescued the gametogenesis allowing chromosomes to pair between their identical homologous.
To sum up, regardless the genetic distance and direction of crosses, F1 hybrids show normal gonadal development during early stage of gametogesis up to first meiotic prophase, more precisely pachytene stage. Chromosomal mispairing during pachytene stage leads to sterility in the majority of females and all males. However, comparable to naturally occurring unisexual fish, few F1 hybrid females from each cross were rescued from sterility by specific alteration of canonical gametogenesis via premeiotic genome duplication.
Keywords:
Cobitis,
Hybrid complex,
Meiosis,
clonality,
genome duplication,
Sterility
Conference:
XVI European Congress of Ichthyology, Lausanne, Switzerland, 2 Sep - 6 Sep, 2019.
Presentation Type:
Oral
Topic:
EVOLUTION AND ECOLOGY OF FISH WITH ASEXUAL REPRODUCTION, HYBRID COMPLEXES AND POLYPLOIDY, WITH SPECIAL FOCUS ON LOACHES (COBITOIDEI)
Citation:
Marta
A,
Dedukh
D and
Janko
K
(2019).
Frequencies and cytogenetic mechanisms of gynogenetic reproduction and sterility in artificially produced Cobitis F1 hybrids from parental species with different degree of phylogenetic relatedness.
Front. Mar. Sci.
Conference Abstract:
XVI European Congress of Ichthyology.
doi: 10.3389/conf.fmars.2019.07.00153
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Received:
26 Jun 2019;
Published Online:
14 Aug 2019.
*
Correspondence:
MD. Anatolie Marta, Laboratory of Fish Genetics, Institute of Animal Physiology and Genetics (ASCR), Liběchov, 277 21, Czechia, anatolmarta@gmail.com