The Okinawa spiny rat, Tokudaia muenninki, is the only species with a Y chromosome in the genus Tokudaia. Its phylogenic relationship with two XO/XO species, Tokudaia osimensis and Tokudaia tokunoshimensis, lacking a Y chromosome and the mammalian sex-determining gene SRY, is unknown. Furthermore, there has been little cytogenetic analysis of the sex chromosomes in T. muenninki. Therefore, we constructed molecular phylogenetic trees with nucleotide sequences of cyt b, RAG1, and IRBP. All trees strongly supported that T. muenninki was the first to diverge from the Tokudaia ancestor, indicating that loss of the Y chromosome and SRY occurred in the common ancestor of the two XO/XO species after T. muenninki diverged. We found that the X and Y chromosomes of T. muenninki consisted of large euchromatic and heterochromatic regions by conducting G- and C-banding analyses. PCR, Southern blotting, and FISH revealed that T. muenninki males had multiple SRY copies on the long arm of the Y chromosome. At least three of 24 SRY sequences contained a complete open reading frame (ORF). A species-specific substitution from alanine to serine was found in all copies at the DNA-binding surface within the HMG-box, suggesting that it occurred in an original SRY.

The genus Tokudaia comprises three species, two of which have lost their Y chromosome and have an XO/XO sex chromosome constitution. Although Tokudaia muenninki (Okinawa spiny rat) retains the Y chromosome, both sex chromosomes are unusually large. We conducted a molecular cytogenetic analysis to characterize the sex chromosomes of T. muenninki. Using crossspecies fluorescence in situ hybridization (Zoo-FISH), we found that both short arms of the T. muenninki sex chromosomes were painted by probes from mouse chromosomes 11 and 16. Comparative genomic hybridization analysis was unable to detect sex-specific regions in the sex chromosomes because both sex probes highlighted the large heterochromatic blocks on the Y chromosome as well as five autosomal pairs. We then performed comparative FISH mapping using 29 mouse complementary DNA (cDNA) clones of the 22 X-linked genes and the seven genes linked to mouse chromosome 11 (whose homologue had fused to the sex chromosomes), and FISH mapping using two T. muenninki cDNA clones of the Y-linked genes. This analysis revealed that the ancestral gene order on the long arm of the X chromosome and the centromeric region of the short arm of the Y chromosome were conserved. Whereas six of the mouse chromosome 11 genes were also mapped to Xp and Yp, in addition, one gene, CBX2, was also mapped to Xp, Yp, and chromosome 14 in T. muenninki. CBX2 is the candidate gene for the novel sex determination system in the two other species of Tokudaia, which lack a Y chromosome and SRY gene. Overall, these results indicated that the Y chromosome of T. muenninki avoided a loss event, which occurred in an ancestral lineage of T. osimensis and T. tokunoshimensis, through fusion with an autosome. Despite retaining the Y chromosome, sex determination Chromosome Res (2012) 20:111-125

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Abstract. The Okinawa spiny rat, Tokudaia muenninki, is a critically endangered species endemic to the northern part of Okinawa Island and may be extinct in the wild as there have been no recent sightings of the animal in its natural habitat. We initiated the present search to determine whether the spiny rat still exists in the northern part of Okinawa Island. Sensor cameras and traps were distributed across areas in which past studies had identified the location of occurrence of spiny rats. From a total of 1,276 camera-nights and 2,096 trap-nights from 2007 to 2009, we captured 24 spiny rats; however, we were only successful in identifying spiny rats in the northernmost of the areas sampled, with no indications of the spiny rat in the more southerly areas. The area in which the spiny rats were still present was estimated to be only 1-3 km 2 and is comprised of forest dominated by Castanopsis sieboldii, Lithocarpus edulis, Distylium racemosum and Schima wallichii. The trees range in age from about 30 to more than 100 years old, and have an average height of 12 m (range 7 m-16 m). Our rediscovery of the spiny rat in 2008 comes after an interval of 30 years since the previous trapping study in 1978 and seven years since indirect survey evidence from analysis of feral cat feces 2001. Measures for conservation of the location of the spiny rats are urgently required.

Okinawajima Island; thus, our results provide a warning about potential negative impact on such animals when E. t. friesi expands its range to the northern part of the island.