In the rolling woodlands of Serbia, a small bat has quietly rewritten a cytogenetic record. Researchers analysing Leisler’s bats (Nyctalus leisleri) captured at two Serbian localities have found individuals carrying up to five B chromosomes — supernumerary chromosomes that sit outside the standard genome set — the highest number ever recorded in this species and among the highest documented in any bat. The finding, published in Comparative Cytogenetics, offers a rare window into one of genetics’ most enigmatic phenomena and raises fresh questions about why these extra chromosomes flourish in some mammals but are vanishingly rare in others.

B chromosomes, often abbreviated to Bs, are additional, dispensable components of the karyotype. Unlike the standard A chromosomes, they are not required for survival, do not follow Mendelian rules of inheritance, and typically do not recombine with the regular chromosome set, instead pursuing what researchers describe as their own evolutionary destiny. They have now been detected in roughly three percent of all analysed species across animals, plants, and fungi, and in 85 mammalian species. Yet their distribution is strikingly uneven: some lineages carry them routinely, while others — including most bats — almost never do. Usually Bs originate from A chromosomes of the same species, though hybridization between closely related species can also generate them, and recent studies suggest many are transcriptionally active and may even shape the traits of their carriers.

The new study focused on four adult male Leisler’s bats captured with mist-nets over water bodies at Bebića Luka in Western Serbia and Zlot in Eastern Serbia during 2020. Tissue samples from the plagiopatagium, the membrane stretching between body and wing, were taken with sterile biopsy punches and transported to the laboratory within 24 hours, where primary fibroblast cell cultures were established. Once sufficient cells had grown, researchers arrested cell division with colchicine, treated the cultures with ethidium bromide to lengthen the chromosomes, and prepared chromosome spreads for staining. Every animal was safely released immediately after sampling under permits from Serbia’s Ministry of Environmental Protection.

The cytogenetic results were unambiguous. The standard complement of Nyctalus leisleri counts 44 chromosomes — 42 autosomes plus a pair of sex chromosomes — and all four males exceeded this baseline. Three bats from Bebića Luka carried karyotypes ranging from 44 plus one to two Bs, and 44 plus three to five Bs, while the single male from Zlot carried between two and four. All the extra chromosomes were heterochromatic microchromosomes, tiny elements composed largely of constitutive heterochromatin concentrated at their centromeres. Crucially, the number of Bs varied not only between individuals but also between cells within the same individual, a hallmark of B chromosome biology that reflects their unstable, non-Mendelian behaviour during cell division.

Detailed banding analyses added texture to the picture. G-banding, which reveals the characteristic light and dark stripes of condensed and less condensed chromatin, confirmed the morphology of the standard set: three pairs of large metacentrics, one small submetacentric pair, and 17 pairs of acrocentrics, with a medium-sized metacentric X and a small acrocentric Y. C-banding showed heterochromatin in the centromeric regions of all autosomes, sex chromosomes, and Bs alike. Silver staining identified active nucleolus organizer regions — the sites of ribosomal RNA genes — on two chromosome pairs, numbered 8 and 11 in the karyotype. Notably, earlier work on specimens from Greece and Turkey had placed these regions on pairs 8 and 15, a discrepancy the authors attribute either to genuine spatial diversity among populations or to differences in chromosome condensation during preparation, a known pitfall when karyotypes contain many similarly sized acrocentrics.

With this study, Leisler’s bat becomes the holder of the highest B chromosome count recorded in the species, surpassing the maximum of three Bs documented by Volleth in 1992 in males from Turkey, Germany, and Greece. Only four bat species are known to carry Bs at all: three vespertilionids — Pipistrellus tenuis, Myotis macrodactylus, and Nyctalus leisleri — and the megadermatid Megaderma spasma, whose Bs, though among the smallest chromosomes in its karyotype, are not microchromosomes and do not appear fully heterochromatic. In the least pipistrelle, Bs take metacentric form, while the big-footed Myotis carries a single micro B that can be acrocentric or metacentric. Intriguingly, every analysed Leisler’s bat karyotype reported to date — all of them males — has contained Bs, and the only female karyotype on record, from Poland, probably carried two micro Bs that the original authors, analysing a single animal, did not report as such.

The rarity of Bs in bats compared with rodents is the study’s central puzzle. Rodents, the largest mammalian order, boast 61 species with B chromosomes; bats, the second-largest order with more than 1440 species, have just four. The authors propose that genome size may hold part of the answer. Bats average a remarkably small genome of about 2.35 picograms of DNA, ranging from 1.3 to 3.2 pg — a range comparable to birds, whose genomes span a narrow 2 to 4 pg and whose repeated sequence content, at 15 to 20 percent, is the lowest among vertebrates. Both birds and bats are the only living vertebrates capable of active flight, and it has long been hypothesized that the extreme metabolic demands of flight impose a constraint on genome size. Smaller genomes mean smaller cells, and smaller cells have a higher surface-area-to-volume ratio, improving gas exchange to satisfy metabolic needs. A streamlined genome, the reasoning goes, may simply have no room to tolerate the genomic baggage of Bs — a pattern echoed in flowering plants, where B chromosome presence correlates positively with total genome size and small-genomed species rarely feature them.

Life history may reinforce the genomic argument. Bats are among the longest-lived mammals for their size, with slow reproductive rates and small litters, typically one or two young per year. Rodents, by contrast, live fast and die young, producing large litters that give B chromosomes far more opportunities to establish, spread, and persist within populations. A dispensable chromosome that confers no immediate benefit needs many generations and many reproductive events to gain a foothold; a bat’s leisurely life cycle offers few such chances. The authors also note that the apparent rarity of Bs in bats may partly reflect how few bat karyotype studies have been conducted compared with the extensive cytogenetic literature on rodents.

The Serbian setting adds ecological intrigue of its own. Leisler’s bat is a medium-sized woodland species distributed across Europe up to 57 degrees north, considered rare almost everywhere except Ireland, where it thrives. It roosts in tree holes, forages over canopy, forest trails, and water, hibernates in tree cavities, and undertakes long seasonal migrations — movements exceeding 1500 kilometres have been recorded in females breeding in Germany and wintering on the Iberian Peninsula. In Serbia, the species has been recorded at seven localities, almost always as single males captured at foraging grounds between July and September, with no known roosts and no evidence of reproduction. This male-dominated pattern likely reflects the species’ migration dynamics, in which males occur sporadically in northern regions but dominate southern populations, and it explains why the only published female karyotype comes from Poland.

For the researchers, the combination of a record-breaking B chromosome count, the absence of female karyotype data, and scarce ecological information makes Nyctalus leisleri a compelling model for future B chromosome research. The modern view of Bs frames them as an extra genomic compartment with huge potential and still unknown biological significance — elements once dismissed as inert heterochromatic leftovers now suspected of contributing actively to the phenotypes of their carriers. Whether the five Bs of a Serbian Leisler’s bat carry genes, influence traits, or simply ride the waves of non-Mendelian inheritance remains to be discovered, but this small woodland bat has made clear that even the most streamlined genomes can still surprise us.

Subject of Research: Cytogenetics

Article Title: The new highest number of B chromosomes ( Bs ) in Leisler’s bat Nyctalus leisleri (Kuhl, 1817)

Article References: Rajičić, M., Budinski, I., Miljević, M., Bajić, B., Paunović, M., Vujošević, M., & Blagojević, J. (2022). The new highest number of B chromosomes (Bs) in Leisler’s bat Nyctalus leisleri (Kuhl, 1817). Comparative Cytogenetics, 16(3), 173-184. https://doi.org/10.3897/compcytogen.v16i3.89911

Image Credits: AI Generated

DOI: 10.3897/CompCytogen.v16i3.89911

Keywords: B chromosomes, Leisler's bat, Nyctalus leisleri, cytogenetics, karyotype, Chiroptera, heterochromatin, supernumerary chromosomes, genome size, Serbia, highest, number