Previous reports revealed that variation in the chromosomal number within a single species might take place in Hymenoptera, as observed in bees of the genus Melipona and in social wasps of the tribe Epiponini (Francini et al. 2011; Menezes et al. 2013; Menezes et al. 2014; Barbosa et al. 2021). In some cases, distinct chromosomal numbers refer to the presence of cryptic forms or a species complex, particularly common in groups of a wide geographic range, as reported in wasps of the genera Metapolybia (Menezes et al. 2013) and Polybia (Marchioro 2019).
For instance, karyotypic variation has been already indicated in Polistes apachus, but the author suggested further detailed analysis to confirm the chromosomal number of this species (Machida 1934). Similarly, the present results obtained in P. canadensis (n = 28) also differ significantly from the previous report by Kerr in 1952 (n = 16), since we identified 12 additional chromosome pairs in this species. Most likely, this divergence is derived from technical artifacts since the former report was based on squashed cells instead of air-drying technique, as presently performed.
In fact, squash techniques might be harmful to chromosomes either by the rupture of sister chromatids or poor spreading of chromosomes, eventually leading to difficulties in counting chromosomes and defining their morphology (Koçak and Okutaner 2017). On the other hand, as presently performed, air-drying techniques provide reliable cytogenetic results, allowing a refined analysis of chromosomal microstructure. Corroborating the reliability of the present data, the 2n value herein reported for P. canadensis is close to the average value in other species of the subgenus Aphanilopterus (Pompolo and Takahashi 1986, 1990).
Even though the karyoevolutionary trends in species of Polistes remain largely unclear by the lack of additional information, the presence of acrocentric pairs, also reported in other species of Polistes (Pompolo and Takahashi 1990) should derive from fissions of metacentric chromosomes, giving rise to unstable one-armed chromosomes (Imai 1991). To counteract such karyotype instability, most acrocentric chromosomes would have undergone heterochromatinization on breakage points, thus determining the appearance of pseudoacrocentric or acrocentric chromosomes (Hoshiba and Imai 1993). Therefore, the presence of conspicuous heterochromatin blocks might mitigate putative telomere instability after centric fissions in species with derived karyotypes characterized by higher 2n values (Imai et al. 1988; Pompolo and Takahashi 1990).
Unfortunately, data about the distribution and composition of heterochromatin are rarely reported in Polistes, jeopardizing comparative results among congeneric species. Nonetheless, the C-banding pattern described in this study (heterochromatin at pericentromeric regions) is similar to that reported in other hymenopterans such as Meliponini bees and other social wasps (Vespidae) (Travenzolli 2018; Marchioro 2019).
In addition, the staining with base-specific fluorochromes revealed CMA3+ signals at euchromatic regions of several chromosomal arms, indicating these regions are GC-rich (Fig. 1c). A similar pattern was found in other species of the genera Mischocyttarrus and Polybia (Menezes et al. 2014; Cunha et al. 2017), suggesting this feature is shared among these taxa and Polistes. According to Menezes et al. (2013), these regions play a key role in some groups of social wasps, once they are supposed to be involved in chromosomal rearrangements. Moreover, GC-rich sites are usually interspersed with 45S rDNA regions, and previous reports associated multiple ribosomal cistrons with the occurrence of centric fissions in Hymenopterans (Menezes et al. 2021). These data reinforce that fissions are actually involved in the speciation process of P. canadensis and further analyses focusing on chromosomal mapping of rDNA genes are highly recommended in this species.
In conclusion, the utilization of additional cytogenetic techniques like C-banding and fluorochrome staining proved to be useful as chromosomal markers to identify, for the first time, the distribution and composition of heterochromatin in the analyzed species. Besides being useful to infer the karyoevolutionary history of Polistes wasps, we reinforce the utilization of cytogenetic data as an additional tool to the systematics of insects by providing a fast characterization of species-specific patterns of genome organization.