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The Ryukyu long-furred rat, Diplothrix legata (Muridae) is an endangered large arboreal species endemic to the central Ryukyus, Japan. Previous studies have recorded the food habits of this species through direct observations. However, its observation records are limited. Here, we aimed to clarify the food habits of D. legata using DNA metabarcoding of its stomach contents. The ITS2 intergenic regions in plant nuclear genomes and the COI gene regions in animal mitochondrial genomes were used as DNA markers to identify the prey species of D. legata based on its stomach contents. We successfully identified 63 plant and 36 animal species as the prey species of D. legata. Finally, 84 plant and 46 animal species are listed as food menu in total. Thus, a greater number of its animal prey species were identified in this study than in previous observation-based studies. As this species is omnivorous, the conservation of a wide array of ecological habitats is necessary for maintaining its population. Regardless of the limited sample size and unknown parts consumed, slight differences in food items were observed between different sex- and age-based groups. Relatively more detailed DNA reference databases for the local fauna and flora are required for further analysis.
In the northern part of Okinawajima Island, a large nest box equipped with an automatic camera trap was set up at a height of 5.2 m above the ground from April 2022 to March 2023 to study the reproductive ecology of the Ryukyu long-furred rat Diplothrix legata. This species is endemic to the central Ryukyus and is the largest arboreal murid species in Japan. Videos captured between October and February showed that D. legata was carrying branches and leaves into the nest box and nesting. Calls of newborn rats, sometimes overlapped between different individuals, were recorded from late February to early March, indicating that multiple rats were born in the nest box. These observations suggest that large nest boxes are useful for studying the nesting behavior of D. legata. However, habu snakes Protobothrops flavoviridis were observed entering the nest box and preying on newborns, resulting in no newborns being observed leaving the nests. Although previous studies have recorded the predation of D. legata by habu snakes, this study is the first to provide direct evidence of habu snakes attacking the nesting site and preying on newborns of D. legata.
The Ryukyu Islands have experienced a complex history of island connection and separation since the Late Miocene. Questions persist regarding how organisms have evolved through changes in island configurations over millions of years particularly in the Central Ryukyus, which is characterized by high species endemism. We conducted comparative phylogenetic and phylogeographic analyses among island populations targeting five mammalian species (Pentalagus furnessi, Diplothrix legata, and three Tokudaia species), all of which belong to genera endemic to the Central Ryukyus, employing genome-wide SNP and mitochondrial DNA variation analyses. The SNP and mitochondrial DNA analyses across these genera revealed distinct lineages on each island (Amami-Ohshima, Tokunoshima, and Okinawajima, except for P. furnessi absent from Okinawajima), with the Amami-Ohshima population showing the highest genetic diversity. Divergence times between island populations were estimated to be in the Middle Pleistocene for P. furnessi and D. legata and ranged from the Late Miocene to the Early Pleistocene for Tokudaia. These findings, along with those of previous studies on other terrestrial animals, emphasize the significant impact of pre-Pleistocene island isolation on genetic divergence in species with limited dispersal abilities, while some species exhibited inter-island migration by the Middle Pleistocene.
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