Cytogenetic study of four East Asian dobsonflies finds conserved 24-chromosome karyotypes

A comparative cytogenetic study of four East Asian dobsonfly species found that all had a diploid chromosome number of 2n = 24, with closely corresponding chromosome arrangements within two geographic pairs. The observations were based on larval gonads collected in Japan and Taiwan between June 1994 and February 1995 and analyzed between June 1994 and June 1995, rather than on newly collected specimens.

The four species examined were Protohermes grandis, P. immaculatus, P. disjunctus and P. costalis. The genus contains about 90 recognized species, all occurring in Asia. P. costalis is native to continental China, Taiwan and India, whereas the other three species are known from Japan. P. grandis occurs throughout Japan, P. immaculatus is known from the Ryukyu Islands, and P. disjunctus is known from the Yaeyama Islands. The study also included previously published chromosome observations for comparison.

Researchers collected final-instar larvae from rivers and other collection sites in Japan and Taiwan. Thirteen P. grandis larvae came from the Sugihara River in Hyogo Prefecture on Honshu, collected from June 1994 to June 1995. Five P. immaculatus larvae came from the Kawauchi River on Amami-Oshima Island in Kagoshima Prefecture in November 1994. Six P. disjunctus larvae came from the Nagura River on Ishigaki Island and the Takana River on Iriomote Island in Okinawa Prefecture between August and November 1994, and six P. costalis larvae came from the Tunhou Valley at Wulai, Taiwan, in February 1995.

Each larva was placed individually in a water-filled cup in the field and transported alive to the laboratory, where it was fed aquatic insects until analysis. The gonads of final-instar larvae were used for chromosome preparations. Larval sex was determined from head width. The protocol modified an earlier insect cytogenetic method: dissected testes and ovaries were treated with 0.005% colchicine for 30 minutes, placed on glass slides, fixed and dissociated with solutions containing acetic acid, ethanol, water and lactic acid, and teased with a needle for 10 to 15 seconds. After further fixation, slides were dried in an incubator at 40–50 °C overnight.

Chromosomes were stained with 3% Gimsa solution in Sorensen phosphate buffer at pH 6.8 for 20 minutes. Microscope photographs of selected chromosome spreads were taken with a 100× oil-immersion objective and a scale bar of 5 µm. The study was therefore an animal cytogenetics investigation based on chromosome morphology in four species, rather than a genomic sequence analysis or a direct test of reproductive isolation.

All four species had 11 pairs of autosomes, with XX in females and XY in males. The previously reported P. grandis autosomes included one pair of large submetacentric chromosomes, two pairs of metacentric chromosomes, seven pairs of telocentric chromosomes and one pair of small chromosomes. In P. immaculatus, the autosomes included one large submetacentric pair, two metacentric pairs, two subtelocentric pairs, five telocentric pairs and one pair of small chromosomes. Its X chromosome was about 5 µm long, considerably longer than the X chromosomes in the other three species, and a secondary constriction occurred in the long arm of chromosome 3.

Despite the difference in X-chromosome length, the karyotype of P. immaculatus was similar to that of P. grandis when chromosome number and centromere position were considered. The autosomes of P. disjunctus and P. costalis each comprised one pair of large submetacentric chromosomes, nine pairs of telocentric chromosomes and one pair of small chromosomes; their sex chromosomes consisted of a submetacentric X and a small dot-like Y. These two species consequently had closely matching karyotypes based on chromosome number and centromere position.

Male meiotic preparations showed parachute-type X-and-Y bivalents, termed Xyp, in P. immaculatus, P. disjunctus and P. costalis. The same bivalent type had previously been reported for P. grandis. Xyp bivalents have also been described in two species each of Parachauliodes and Neochauliodes fishflies, in the American fishfly Neohermes fillicornis and in Coleoptera. The observations indicate that this sex-chromosome configuration is shared across the sampled Protohermes species, although the chromosome data alone do not establish the detailed evolutionary history of the mechanism.

Chromosome similarity between P. grandis and P. immaculatus, and between P. costalis and P. disjunctus, was consistent with the treatment of P. immaculatus and P. disjunctus as vicariants of those respective species. The authors connected this pattern with paleogeographic evidence that the Ryukyu Arc was linked from Kyushu to Taiwan several times between 0.4 and 0.02 million years ago and that sea immersion and gaps fragmented the islands after about 0.02 million years ago. Within the limits of this small, geographically focused comparison, the study supports an association between karyotype similarity and the existing interpretation of vicariant relationships, but it does not directly measure how geographic isolation produced the species.

The chromosome count was also not unique to East Asian Protohermes. The North American dobsonfly Corydalus cornutus had previously been reported with 2n = 24, including 11 autosomal pairs, XX in females and an Xyp bivalent in males. Together, the present observations and those earlier reports indicate that chromosome number is highly conserved among the dobsonflies examined across Asia and North America. The principal limitation of the study is its scope: detailed karyotypes were established for four species using small numbers of larvae from selected locations, so the results provide comparative cytological evidence rather than a comprehensive survey of the genus.

Subject of Research: Comparative chromosome morphology in four East Asian Protohermes dobsonfly species

Article Title: Chromosomes of four species of dobsonflies, in the genus Protohermes ( Megaloptera , Corydalidae , Corydalinae ) from East Asia

Article References: Takeuchi, Y., Iizuka, K., Koishi, H., & Hoshiba, H. (2025). Chromosomes of four species of dobsonflies, in the genus Protohermes (Megaloptera, Corydalidae, Corydalinae) from East Asia. Comparative Cytogenetics, 19, 75-82. https://doi.org/10.3897/compcytogen.19.146501

Image Credits: AI Generated

DOI: 10.3897/compcytogen.19.146501

Keywords: dobsonflies, Protohermes, cytogenetics, karyotypes, chromosome number, sex chromosomes, Ryukyu Islands, Megaloptera, Chromosomes, four, species, genus