Harumi Torii

The Influence of Pleistocene Refugia on the Evolutionary History of the Japanese Hare, Lepus brachyurus

We performed a phylogeographic analysis of the Japanese hare, Lepus brachyurus, using the mitochondrial cytochrome b gene (1140 bp). In total, 119 haplotypes were recovered from 197 samples isolated from 82 localities on three main islands of the Japanese archipelago: Honshu, Sikoku, Kyushu, Sado Island and the Oki Islands. Results showed two distinct clades at a genetic distance of 3.5%, equivalent to an estimated 1.2 million years. The two clades, encompassing seven subclades, showed an apparent geographic affinity to Kyushu, Shikoku and the nearby area of Honshu (southern group) by one clade, whereas the other clade covered the remaining area of Honshu (northern group). The landscape shape interpolation analysis exhibited a higher genetic diversity in the southern parts of central Honshu (northern group) and Shikoku and Kyushu regions (southern group), suggesting the existence of multiple geographical origins of population expansion in each clade. The Bayesian skyline plot analysis showed that lineage diversifications occurred about 0.35, 0.20 and 0.05 million years ago (Mya), which coincide closely with the glacial—interglacial cycles during the Pleistocene. Therefore, we suggest that the Japanese hare population once inhabited northern and southern refugia, and subsequently developed several populations through local demographic fluctuations. The present day demarcation in the northern and southern geographic groups is considered to be a temporal remnant of Pleistocene population dynamics and the geographic boundary between them could move or fade away in time.

A NEW RHABDITOID NEMATODE SPECIES IN ASIAN SCIURIDS, DISTINCT FROM STRONGYLOIDES ROBUSTUS IN NORTH AMERICAN SCIURIDS

Strongyloides callosciureus n. sp. (Nematoda: Rhabditoidea), from Asian sciurids, is described based on morphology, morphometry, and the small and large subunit (SSU/LSU) ribosomal RNA gene (rDNA) sequences. This new species was collected from Pallass squirrels (Callosciurus erythraeus) in the central part of mainland Japan (Honshu), which were originally introduced from Taiwan some decades ago, and plantain squirrels (Callosciurus notatus) imported from Malaysia as personal pets. For comparison, Strongyloides robustus Chandler, 1942 was collected from American red squirrels (Tamiasciurus hudsonicus) and southern flying squirrels (Glaucomys volans) imported from the United States as personal pets. The parasitic females found in North American and Asian sciurids shared some key morphological features such as the ovary running spirally around the gut, and the shapes of the stoma in the apical view and the tail. However, morphometric features of parasitic females in North American and Asian sciurids differed significantly from each other; the former was larger than the latter, and the relative position of the vulva to the whole body length from the mouth was different. The SSU/LSU rDNA sequences supported the division of sciurid Strongyloides isolates by geographical distribution of the host and morphological features, leading us to propose the erection of new species.

Genetic variation of Gongylonema pulchrum from wild animals and cattle in Japan based on ribosomal RNA and mitochondrial cytochrome c oxidase subunit I genes.

The gullet worm (Gongylonema pulchrum) has been recorded from a variety of mammals worldwide, including monkeys and humans. Due to its wide host range, it has been suggested that the worm may be transmitted locally to any mammalian host by chance. To investigate this notion, the ribosomal RNA gene (rDNA), mainly regions of the internal transcribed spacers (ITS) 1 and 2, and a cytochrome c oxidase subunit I (COI) region of mitochondrial DNA of G. pulchrum were characterized using parasites from the following hosts located in Japan: cattle, sika deer, wild boars, Japanese macaques, a feral Reevess muntjac and captive squirrel monkeys. The rDNA nucleotide sequences of G. pulchrum were generally well conserved regardless of their host origin. However, a few insertions/deletions of nucleotides along with a few base substitutions in the ITS1 and ITS2 regions were observed in G. pulchrum from sika deer, wild boars and Japanese macaques, and those differed from G. pulchrum in cattle, the feral Reevess muntjac and captive squirrel monkeys. The COI sequences of G. pulchrum were further divided into multiple haplotypes and two groups of haplotypes, i.e. those from a majority of sika deer, wild boars and Japanese macaques and those from cattle and zoo animals, were clearly differentiated. Our findings indicate that domestic and sylvatic transmission cycles of the gullet worm are currently present, at least in Japan.

A preliminary study on origin of Callosciurus squirrels introduced into Japan

ABSTRACT To examine the origin of Callosciurus erythraeus introduced to Japan, we compared mitochondrial DNA control region sequences of eight haplotypes from Japan with those of 42 haplotypes from Taiwan. There were two distinct phylogroups in Japan. Six haplotypes from Japan were included in the Taiwan population, suggesting that they could have been introduced from Taiwan. Two haplotypes, however, were distantly related to the cluster consisting of all Taiwanese haplotypes and six Japanese haplotypes. The uncorrected genetic distances (5.2–5.7%) between these two haplotypes and the out-group (Callosciurus finlaysonii from Laos) were less than those (8.3–9.8%) between these two haplotypes and the cluster consisting of the other six Japanese haplotypes and all Taiwanese haplotypes. Both C. erythraeus and C. finlaysonii have variable external characteristics, such as pelage color, and include many variable subspecific forms, so that it is difficult to evidently identify both species on the basis of their external characteristics. Callosciurus finlaysonii may have been also introduced in Japan with both species now coexisting in Japan.

The stomal morphology of parasitic females of Strongyloides spp. by scanning electron microscopy

For the discrimination of the species of the genus Strongyloides, the shape of the stoma in the apical view of parasitic females is the most important feature. Microscopical interpretations divided the shape of the stoma into four groups (simple, angular, complex, and simple but with esophageal teeth); however, the stomas classified in the latter two groups, in particular, remained unclear. In the present study, parasitic females of eight representative Strongyloides spp. with the angular stoma (S. procyonis and S. martis), complex stoma (S. robustus, S. callosciureus, S. planiceps, S. papillosus and S. myopotami), and stoma with esophageal teeth (S. ransomi) were studied by scanning electron microscopy. All species examined in this study having complex stoma or with esophageal teeth possessed at least four linguiform or verruciform projections submarginally in the stomal cavity. Namely, the number, size, shape, and the site of attachment of intra-stomal projections, along with the depth of oral cavity, determine a range of complex images of the stoma such as X-shaped or ornate stomas under light microscopy. Similarly, with the standard examination, which usually uses a lateral view of the head, erroneous interpretations of the number of lobes on the circumoral elevation of parasitic females might occur due to such intra-stomal projections in some Strongyloides spp.

Redescription of Enterobius (Enterobius) macaci Yen, 1973 (Nematoda: Oxyuridae: Enterobiinae) Based on Material Collected from Wild Japanese Macaque, Macaca fuscata (Primates: Cercopithecidae)

abstract:  Enterobius (Enterobius) macaci Yen, 1973 (Nematoda: Oxyuridae: Enterobiinae) was collected from a Japanese macaque, Macaca fuscata, in Nara and Yamaguchi Prefectures, Honshu Island, Japan, for the first time. A redescription is presented along with DNA sequence data. This pinworm is a typical member of the subgenus Enterobius and is characteristic in the spicule morphology, being readily distinguished from other congeners. Phylogenetic analyses based on 18S ribosomal RNA gene (rDNA) and mitochondrial DNA (mtDNA) Cox1 gene assign its position in the pinworm lineage adapted to the Old World primates, showing divergence before the splitting of the chimpanzee and human pinworms.

Mitochondria DNA Sequences of Finlayson's Squirrel Found in Hamamatsu, Shizuoka Prefecture, Japan

Alien Callosciurus squirrels cause severe damage to trees in forests, gardens, and commercial plantations (Jouanin 1986; Setoguchi 1990; Torii 1993; Aprile and Chicco 1999; Gurnell and Wauters 1999). Pallas’s squirrel (Callosciurus erythraeus) originally occurs in eastern India, Bhutan, southeast China, Taiwan, Indochina, and Malaya (Corbet and Hill 1992; Wilson and Reeder 2005). This squirrel was introduced into other countries as an exotic pet. At present, C. erythraeus populations are found in Argentina (e.g., Aprile and Chicco 1999), France (Jouanin 1986; Gurnell and Wauters 1999), and Japan (Ishii 2005; Tamura 2002, 2009; Ikeda et al. 2011). Under law number 78 of the Invasive Alien Species Act (Ministry of the Environment, Government of Japan, 2004: http://www.env.go.jp/nature/intro/index. html), import of C. erythraeus to Japan is strictly prohibited. This species is treated as ‘invasive alien species’ in Japan. Finlayson’s squirrel (C. finlaysonii), which originally occurs in the Indochina Peninsula (Corbet and Hill 1992; Wilson and Reeder 2005), was also introduced to Italy (Bertolino et al. 1999; Currado et al. 1999). Similar to C. erythraeus, C. finlaysonii is thought to cause damage to environments (Bertolino et al. 2004). It is difficult to distinguish this species from C. erythraeus based on external characteristics such as pelage patterns (e.g., Lekagul and McNeely 1988). Based on mitochondrial (mt) DNA control region sequences, Oshida et al. (2007) reported that C. finlaysonii was probably introduced to Hamamatsu, Shizuoka Prefecture, Japan (Fig. 1). They captured eight Callosciurus individuals in Hamamatsu. Of them, seven individuals had haplotypes closely related to that of C. finlaysonii from Laos (two C. finlaysonii haplotypes were detected), and one individual had a C. erythraeus haplotype. Therefore, we expect these two Callosciurus species exist sympatrically in Hamamatsu. Callosciurus finlaysonii, however, is not treated as an invasive alien species in Japan (law number 78 of the Invasive Alien Species Act, Ministry of the Environment, Government of Japan, 2004). Currently this species is treated as unidentified alien species. If the population of C. finlaysonii increases, this species should be added to the list of invasive alien species. To estimate whether C. finlaysonii is increasing, we investigated ratios of C. finlaysonii haplotype and C. erythraeus haplotype in the Hamamatsu population by using mtDNA control region sequences. We discuss the present situation of introduced C. finlaysonii in Hamamatsu. In addition, we compare control region sequences of specimens from Hamamatsu, Laos and Thailand, to identify the origin of introduced C. finlaysonii. The exact origin would be difficult to determine because we do not have sequence data from all C. finlaysonii populations occurring in the Indochina Peninsula. We, however, obtained successfully C. finlaysonii sequences from Thailand and Laos. Identifying whether the Hamamatsu population is more closely related to specimens from Thailand or Laos may help to prevent subsequent introductions of this squirrel into Japan with international legal and political action.

Local Variation in Diet Composition of the Japanese Serow During Winter

Abstract We investigated local geographical variation in the plant composition and quality (crude protein) of the diet of the Japanese serow (Capricornis crispus) by analyzing rumen contents. We collected the rumen contents from 111 Japanese serows culled for pest control between January and March 1997 in Shizuoka Prefecture, central Japan. The composition of the diet, characterized by a high proportion of browse (65.2%), including green plant leaves (55.3%), confirmed that the Japanese serow is a typical browser, at least in winter. Although the composition of the diet varied geographically even within Shizuoka Prefecture (i.e. the northern populations included less green broad leaves, and more grasses and Sasa spp.), altitude did not affect the composition. Latitudinal variation in plant composition of the diet was explained by a shift in plant availability within vegetation zones from evergreen broad-leaf to deciduous broad-leaf forests. Lack of variation in the diet plant composition with changes in altitude may be explained by probably greater altitudinal than latitudinal variation in the territories of the Japanese serow, and by greater individual variation in food habits. Diet quality remained at a constant high level regardless of altitudinal and latitudinal variation in diet plant composition. This suggests that the Japanese serow maintains a high-quality diet by selective feeding, which is possible only when the serow is at low population density and in possession of a territory.

Induced response to herbivory in stinging hair traits of Japanese nettle (Urtica thunbergiana) seedlings in two subpopulations with different browsing pressures by sika deer

Evolutionary theories predict that natural selection favors inducible defense when the risk of predation is unpredictable. In this context, the magnitude of the induced defense in populations experiencing intermittent herbivory is predicted to be larger than that in populations experiencing constant herbivory when there is genetic differentiation between populations. To test this prediction, we conducted a clipping experiment to investigate induced response to shoot damage by the stinging hair traits of Japanese nettle (Urtica thunbergiana) seedlings. For this purpose, we studied two nettle subpopulations, one under constant browsing and another under intermittent browsing by sika deer in Nara Park, central Japan. The clipping experiment demonstrates that both subpopulations exhibited induced defenses in response to the clipping of the shoot apex as the number and length of stinging hairs increased after clipping. The subpopulation experiencing intermittent browsing exhibited smaller trait values and larger induced defenses, indicated by the number of stinging hairs on the upper leaf surface and the length of stinging hairs on both leaf surfaces compared with the subpopulation experiencing constant browsing. These results are consistent with the prediction and suggest that genetic differentiation of the induced defense between subpopulations is caused by adaptation to the herbivory regime. We discuss other plausible factors affecting the magnitude of the induced defense of the nettle subpopulations.

Induced response to herbivory in stinging hair traits of Japanese nettle (Urtica thunbergiana ) seedlings in two subpopulations with different browsing pressures by sika deer: INDUCED RESPONSE OF JAPANESE NETTLE

Evolutionary theories predict that natural selection favors inducible defense when the risk of predation is unpredictable. In this context, the magnitude of the induced defense in populations experiencing intermittent herbivory is predicted to be larger than that in populations experiencing constant herbivory when there is genetic differentiation between populations. To test this prediction, we conducted a clipping experiment to investigate induced response to shoot damage by the stinging hair traits of Japanese nettle (Urtica thunbergiana) seedlings. For this purpose, we studied two nettle subpopulations, one under constant browsing and another under intermittent browsing by sika deer in Nara Park, central Japan. The clipping experiment demonstrates that both subpopulations exhibited induced defenses in response to the clipping of the shoot apex as the number and length of stinging hairs increased after clipping. The subpopulation experiencing intermittent browsing exhibited smaller trait values and larger induced defenses, indicated by the number of stinging hairs on the upper leaf surface and the length of stinging hairs on both leaf surfaces compared with the subpopulation experiencing constant browsing. These results are consistent with the prediction and suggest that genetic differentiation of the induced defense between subpopulations is caused by adaptation to the herbivory regime. We discuss other plausible factors affecting the magnitude of the induced defense of the nettle subpopulations.