In cold-blooded species such as fish, reptiles and amphibians, their sex chromosomes are not static entities, but over the course of their evolving into new, closely related species, can be swapped out for non-sex chromosomes, and vice versa. For Tago’s Brown Frog, this happens a lot, but until now, scientists had no idea why or how.

A new study of the small frog has solved the mystery, however, and shone a light on this strange phenomenon right across the vertebrate world. The paper describing these findings was published in February in the journal Genes.

Sex chromosomes in animals determine whether the organism is male or female. In mammals, a sex-chromosome pair is normally composed of some combination of X and Y chromosomes, and in birds, of Z and W chromosomes. All the other, non-sex-determining chromosomes are called autosomes.

There are, however, some vertebrate species, especially cold-blooded vertebrates such as fish, reptiles and amphibians, where the sex chromosomes are not static entities, but can evolve into being autosomes. Meanwhile, some autosomes can evolve into being the sex chromosomes.

Biologists call this evolutionary switching back and forth sex-chromosome turnover. And in frogs in particular, sex-chromosome turnover tends to happen very often.

“But until now, we did not know why frog sex-chromosome turnover is so frequent,” said Ikuo Miura, lead author of the study and a herpetologist with the Hiroshima University Amphibian Research Center, “or how it happens.”

One idea has been that sex-chromosome turnover is associated in some way with speciation—evolution into a new species. It is well known that Tago’s Brown Frog, or Rana tagoi—a type of frog often found amongst brown fallen leaves and that is endemic to Japan—and its sister species, including Rana sakuraii, have recently speciated. Indeed, the speciation process is still ongoing. This made the Tago frogs a great model for investigating the mysterious relationship between sex-chromosome turnover and speciation.

To investigate what was going on, the scientists used a “chromosome banding” technique that allowed them to visualize the structure of chromosomes under a microscope, helping to identify specific chromosomes and their characteristics, such as size and shape. By doing this, the researchers could distinguish between the different chromosomes and observe any differences in chromosome structure between the sexes.

In addition, a molecular analysis looked at sex-linked single nucleotide polymorphisms, or SNPs—a difference in a single one of the building blocks of DNA, such as the nucleotide adenine being replaced by the nucleotide guanine—to confirm the identity of the sex chromosomes. By analyzing all the genetic variations associated with these SNPs, the researchers could determine which chromosomes were linked to sex determination in the different populations of the frogs.

Through these combined techniques, the researchers were able to map out the sex chromosomes in the populations studied, leading to the identification of the distinct sex chromosomes present amongst the closely related Tago's Frog species.

In so doing, they found three distinct sex chromosomes, numbered 3, 7, and 13, in the various geographic populations of the Rana tagoi and Rana sakuraii running from northern to southern Japan. In particular, the scientists found that chromosome 7 has been involved in multiple transitions between being a Y chromosome and an autosome.

So, how does this swap occur? The answer lies in hybridization—the process where two different species interbreed. The researchers found that hybridization between two or three different populations belonging to the two species, Rana tagoi and Rana sakuraii, has led to the introduction of new genetic material, allowing for this reshuffling of chromosomes. This can then be followed by the introduction of a restricted chromosome from a different population or species.

Moreover, this swapping is not just a quirky side effect of mating; it plays a crucial role in the evolution of these frogs, enabling them to adapt to their environments in new geographies with new conditions and potentially giving rise to new species.

For the researchers, the next step is to figure out the precise molecular mechanisms of sex chromosome turnover. They believe that they can uncover these mechanisms by reading the whole genomes of the frogs and then compare them before and after the sex-chromosome turnover.

The research team also includes Chiao Kuwana and Chiyo Nanba from Hiroshima University’s Amphibian Research Center and Associate Professor Norio Shimizu from the Hiroshima University Museum.

Other institutes that took part in the study were the Institute for Applied Ecology, Centre for Conservation Ecology and Genomics, Faculty of Science and Technology, University of Canberra in Canberra, Australia; the Natural History Museum and Institute in Chiba, Japan; the Gifu World Freshwater Aquarium in Gifu, Japan; the Saitama Museum of Rivers in Saitama, Japan; the Department of Preschool Education, Nagasaki Women’s Junior College in Nagasaki, Japan; the Preservation and Research Center in Yokohama, Japan; and the National Institute of Advanced Industrial Science and Technology (AIST) in Tsukuba, Japan.

Journal: Genes
Title: Multiple Transitions between Y Chromosome and Autosome in Tago’s Brown Frog Species Complex
Author: Ikuo Miura, Foyez Shams, Jun’ichi Ohki, Masataka Tagami, Hiroyuki Fujita, Chiao Kuwana, Chiyo Nanba, Takanori Matsuo, Mitsuaki Ogata, Shuuji Mawaribuchi, Norio Shimizu, and Tariq Ezaz
DOI: 10.3390/genes15030300

Journal Citation Indicator (JCI) Quartile: Q2