Decay of homologous chromosome pairs and discovery of males in the thelytokous fungus-growing ant Mycocepurus smithii | Scientific Reports -…

Karyotypes in asexually reproducing M. smithii

Our comparative cytogenetic study of asexually and sexually reproducing M. smithii colonies reveals that in an asexual population from Minas Gerais karyotypes were constant within M. smithii colonies but variable between colonies. We identified intraspecific variation between sympatric M. smithii colonies with three distinct karyomorphs where the number of diploid chromosomes equaled 9, 10, and 11. In addition, the karyomorphs differed not only in their numbers but also in the morphology of the chromosomes. Although the chromosome numbers were constant within the same colonies, the described variation was observed between colonies that were located in the same population.

Upon examining the first sample, we first believed that the karyomorph 2n=11 was a haploid karyotype because it presented an uneven chromosome number and almost all the chromosomes were unpaired. Closer study revealed that this karyotype belonged to female individuals, which are expected to be diploid in arrhenotokous Hymenoptera. However, studying the prepupae and larvae of queens confirmed that the observed individuals were in fact females due to the absence of testes.

In contrast to the asexual individuals, the karyotypes of M. smithii females from sexually reproducing colonies were constant. The worker and gyne larvae from the sexually reproducing M. smithii population in Belm, Par had identical diploid chromosome numbers (2n=14), whereas male karyotypes were characterized by half the chromosome number (n=7), which is expected for haploid males produced via arrhenotokous parthenogenesis. Female karyotypes also exhibited the proper homologous chromosome pairings, which is consistent with female karyotypes reported for most sexually reproducing ant species40,41. In addition, variation in chromosome number was not observed between individuals of different sexually reproducing M. smithii colonies, which contrasts with our observation in the asexual population.

The karyotype differences observed between asexual and sexual M. smithii populations, as well as within the asexual M. smithii population, were characterized by numerical and morphological differences probably resulting from centric fission and other chromosome rearrangements. In the absence of meiosis, centric fission can increase the chromosome number, whereas further minor and independently occurring rearrangements on both the rearranged chromosome and the larger ancestral chromosome removes traces of homology between them, leading to the decay of the diploid chromosome structure. The observed karyotypes in asexual M. smithii populations were consistent with this expectation because the karyomorph 2n=9 was characterized by three pairs of homologous chromosomes, whereas the karyomorph 2n=10 presented only two homologous chromosome pairings, and the karyotype 2n=11 showed only a single set of properly paired homologous chromosomes. The loss of homologous chromosome pairings is indicative of a decay in the diploid structure of the individual karyotypes, and the most extreme form of decay was observed in karyotype 2n=11. In contrast, a decay of homologous chromosome pairs could not be observed in the sexually reproducing population of M. smithii, and in that population the chromosomes were perfectly paired with a haploid chromosome number of n=7 and a diploid set of 2n=14.

Our results indicate decaying karyotypes in an asexual M. smithii population, which is consistent with the hypothesis that natural selection is relaxed in the absence of meiosis5,42. Under those circumstances, chromosomes accumulate structural rearrangements and can become heterozygous in the absence of forced pairings during meiosis5,7, which has also been discussed as a potential mechanism for sexual chromosome diferentiation43,44. Ultimately, the accumulation of heterozygosity on individual chromosomes should lead to the loss of homologous chromosome pairs. Our results are consistent with this prediction because in asexual M. smithii population, we observe the partial decay of homologous chromosome structure, whereas chromosome structure is preserved in a sexually reproducing M. smithii population in the face of meiosis and genetic recombination.

Mycocepurus smithii constitutes of a mosaic of asexual and sexual populations and the loss of sexual reproduction likely evolved repeatedly and independently from the ancestral sexual population21. Because asexual populations likely evolved convergently, we hypothesize that additional chromosomal variations will be recognized in other asexual populations. In contrast, we would expect that the karyotypes in sexual M. smithii populations are uniform. Among the different asexual colonies from Minas Gerais bearing karyomorphs of 2n=10 and 2n=11, variation in chromosomal morphology was not observed, suggesting a single origin of these karyomorphs in our study population (Table 1).

Cytogenetic data is available for at most three Mycocepurus species including M. goeldii (2n=8)45, Mycocepurus sp. (2n=8)46, and the asexual (2n=9, 10, 11) and sexual (2n=14, n=7) populations of M. smithii studied here. Considering that cytogenetic data is available for two sexually reproducing species with a karyotype of 2n=8, we initially expected that the asexual karyotype would be 2n=8. The asexual karyomorph of 2n=9 represents the smallest amount of decay in diploid chromosome structure. The observed diploid chromosome number of 2n=14 in the sexual M. smithii population is the highest number of chromosomes observed in Mycocepurus ants so far.

Chromosome rearrangements were previously reported for ants such as in sexually reproducing bulldog ants in the Myrmecia pilosula species complex25,47. Interestingly, variation in chromosome number was reported from individuals belonging to the same colony even when only few individuals were sampled. Imai and colleagues25,47 suggested that the observed variation in chromosome number was a consequence of variable chromosome numbers in the parental generation, which is different from the variation observed in the asexual M. smithii where variation is likely caused by the decay of chromosome structure due to the lack of meiosis and where variation occurred between but not within colonies.

Outside the Hymenoptera, cyclic parthenogenesis is observed in aphids where sexually and asexually reproducing generations alternate with each other. In contrast, many members of the aphid Tribe Tramini reproduce exclusively asexually48, and a decay of karyotype diploidy with a high degree of chromosome diversification can also be observed in these species.

Among ants, thelytokous parthenogenesis has been documented for at least 20 species in four subfamilies including the Dorylinae, Formicinae, Myrmicinae, and Ponerinae10,12,13,14,15. Cytogenetic information is available for six thelytokous ant species including Ooceraea biroi (Forel, 1907), Paratrechina longicornis (Latreille, 1802), Platythyrea punctata (Smith, 1858), Pristomyrmex punctatus (Smith, 1860), Vollenhovia emeryi Wheeler, 1906, and Wasmannia auropunctata (Roger, 1863)40,49,50,51,52,53,54,55. In contrast to the results obtained here for M. smithii, all of the abovementioned thelytokous ant species maintain their chromosome numbers and their homologous chromosome pairings40. In addition, all of these species are characterized by regular or occasional male production via arrhenotoky, except for P. longicornis where males are clones of their fathers56, suggesting that meiosis is still functional in those species10. In contrast, the production of males was exclusively observed in sexually reproducing M. smithii populations, and males do not seem to be produced in asexually reproducing M. smithii colonies suggesting that meiosis may be dysfunctional in these asexual populations.

Heterochromatin plays an important architectural role in chromosome structure and it is enriched in tandem repetitive sequences. The heterochromatin distribution on chromosomes is related to specific functions. For example, centromeric heterochromatin is important for the accurate segregation of chromosomes57. In addition, other regions with accumulations of highly repetitive DNA can be observed throughout the genome extending beyond the centromere. Heterochromatin can influence the frequency of structural rearrangements in neighboring regions58, and consequently, it is essential for chromosome stability47. According to the Minimum Interaction Theory (MIT) proposed by Imai and colleagues47, chromosome fissions are the principal rearrangements in the karyotype evolution of ants because they reduce chromosome sizes and thus reduce deleterious chromosome interactions in the interphase nucleus. The posterior heterochromatin growth after fission plays an important role in allowing telomeric maintenance and chromosome stability, yielding chromosomes with heterochromatic arms.

The repetitive DNA sequences that constitute the heterochromatin in fungus-growing ant chromosomes, including M. goeldii, show richness of GC-base pairs, which is an uncommon trait in other ant species. It was suggested that this GC-rich heterochromatin originated in the common ancestor of the fungus-growing ants, but this hypothesis requires further study41,45,59. Notwithstanding, the results obtained in this study demonstrate that both asexual and sexual populations of M. smithii also possess a GC-rich heterochromatin composition similar to M. goeldii, which adds to the number of attine species with this characteristic trait and strengthens the hypothesis of a common origin of GC-rich heterochromatin in fungus-growing ants.

The heterochromatin distribution pattern on chromosomes aides to recognize possible chromosomal pairs in asexual M. smithii individuals. In addition, it strongly suggests the occurrence of centric fission in M. smithii according to Imais Minimum Interaction Theory (MIT)47 because we observed heterochromatin on short arms of rearranged chromosomes in sexual (2n=14) and in asexual populations (2n=9, 10, 11). In addition, the karyomorph 2n=9, i.e., the karyotype with less chromosomal decay, presented heterochromatin on centromeric or pericentromeric regions of all the chromosomes and this pattern is similar to the one observed in M. goeldii (2n=8)45. Thus, the information about heterochromatin distribution on the chromosomes provides important insights into the pathways of karyotype evolution in ants27,47.

Ribosomal genes are important chromosomal markers used in different organisms, including ants, because these genes follow specific patterns of chromosomal organization. A single chromosome pair bearing all the rDNA genes is the most common and plesiomorphic pattern observed in diploid karyotypes of ants60. In M. smithii, 18S rDNA genes were located on a single chromosome pair in asexual individuals with the karyomorphs 2n=9 (chromosome pair 1) and 2n=11 (chromosomes1 and 1b), as well as in the sexual individuals with the karyomorph 2n=14 (chromosome pair 3). In M. smithii males the rDNA was located on a single chromosome (chromosome 3). These data corroborate our findings that asexual individuals are indeed diploid. In asexual individuals with the karyomorph 2n=11, the homologous chromosomes showed the same morphology (submetacentric), but we also observed a size difference between them suggesting the occurrence of centric fission in the rDNA-bearing chromosome pair.

Both asexual and sexual populations had a secondary constriction in at least one of the homologous chromosomes that were colocalized with the 18S rDNA clusters and GC-rich heterochromatic bands. According to Teixeira et al.60, the presence of single rDNA sites on the chromosomes is influenced by their position on the chromosome because intrachromosomal regions are less prone to rearrangements compared to terminal regions. The analysis of rDNA clusters in further asexual populations should be promising to better understand the selective pressures on rDNA clusters in the genome.

A prior population genetic study of M. smithii suggested that individuals in asexual populations were genetically identical across multiple generations and that males and sexual recombination were absent from asexual populations21. The strict clonality of asexual M. smithii populations suggested either apomixis or automixis with central fusion, i.e., a form of asexual reproduction with low recombination rates, as the cytological mechanism underlying thelytokous parthenogenesis in M. smithii21. Our cytogenetic results are consistent with this general interpretation and lend further support to the apomixis hypothesis because the karyotypes of asexual M. smithii individuals show that heterozygous chromosome configurations likely resulted from chromosome rearrangements in the absence of meiosis. In contrast, the presence of aneuploid individuals would strengthen the hypothesis that asexual M. smithii reproduce via automixis with central fusion, however, such individuals were not detected. Improbable and unpaired chromosomal rearrangements are not expected to be maintained in the absence of meiosis in eukaryotic genomes and its loss would allow for the decay of diploid structures, as observed in asexual M. smithii individuals.

Mycocepurus smithii reproduces via thelytokous parthenogenesis and only few populations in the center of the species distribution range in the Brazilian Amazonas region are known to reproduce sexually21. Previously, the existence of M. smithii males was inferred from the haploid sperm extracted from the spermathecae of M. smithii queens21. The male individuals described here finally provide evidence that recombinant M. smithii workers are the result of syngamy resulting from M. smithii males inseminating queens of the same species. The putative males of M. smithii from Rio Claro, So Paulo36 were previously identified as males of M. obsoletus20, and the morphological comparison between males of M. smithii and M. obsoletus could further identify morphological characters that are diagnostic of each species.

The comparative study of spermatozoa is useful for understanding the reproductive biology of ants in more detail and to obtain additional morphological characteristics that could be used for comparative studies of closely related species. The sperm length in ants varies from 53m in Pseudomyrmex termitarius (Smith, 1855)61 to 230m in Apterostigma fungus-growing ants62. Information related to spermatozoa in ants is still scarce62,63. Nevertheless, comparing the structure of these cells can be insightful when characterizing a species and solving taxonomic problems of cryptic species existing in sympatry, such as Neopoera inversa (Smith, 1858) and Neoponera villosa (Fabricius, 1804) that showed species specific differences in the length of the sperm nucleus64.

The spermatozoa of attine ants are highly variable in length with 67.06m in Atta sexdens (Linnaeus, 1758), being the smallest known sperm cell in the fungus-growing ants, to 230.49m in an unidentified Apterostigma species, being the longest known sperm cell in fungus-growing ants62. With a total length of 69m, the spermatozoa of M. smithii are among the smallest observed in the fungus-growing ants, similar in length to spermatozoa of Atta sexdens. In general, in the lower attine ants spermatozoa length decreases with the increase of colony size for the queens that copulate with a single male, which suggests that the production and storage of sperm affect the evolution of sperm length62. The genus Apterostigma is a member of the paleoattine fungus-growing ants which also includes Mycocepurus and Myrmicocrypta65,66, and the size difference of sperm length between different species of Apterostigma ants is remarkable, ranging from 138.06 to 230.49 m62.

The nucleus length of M. smithii spermatozoa measures 12.2 (06.4) m. The nucleus length in other ant species ranges from 9 to 50m in Dolichoderus and Nesomyrmex, respectively63. The available data for fungus-growing ant sperm refers exclusively to the total length, preventing further comparisons.

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Decay of homologous chromosome pairs and discovery of males in the thelytokous fungus-growing ant Mycocepurus smithii | Scientific Reports -...

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