Tetsuo I. Kohyama

Spatial and temporal variation at major histocompatibility complex class IIB genes in the endangered Blakiston's fish owl

IntroductionQuantifying intraspecific genetic variation in functionally important genes, such as those of the major histocompatibility complex (MHC), is important in the establishment of conservation plans for endangered species. The MHC genes play a crucial role in the vertebrate immune system and generally show high levels of diversity, which is likely due to pathogen-driven balancing selection. The endangered Blakiston’s fish owl (Bubo blakistoni) has suffered marked population declines on Hokkaido Island, Japan, during the past several decades due to human-induced habitat loss and fragmentation. We investigated the spatial and temporal patterns of genetic diversity in MHC class IIβ genes in Blakiston’s fish owl, using massively parallel pyrosequencing.ResultsWe found that the Blakiston’s fish owl genome contains at least eight MHC class IIβ loci, indicating recent gene duplications. An analysis of sequence polymorphism provided evidence that balancing selection acted in the past. The level of MHC variation, however, was low in the current fish owl populations in Hokkaido: only 19 alleles were identified from 174 individuals. We detected considerable spatial differences in MHC diversity among the geographically isolated populations. We also detected a decline of MHC diversity in some local populations during the past decades.ConclusionsOur study demonstrated that the current spatial patterns of MHC variation in Blakiston’s fish owl populations have been shaped by loss of variation due to the decline and fragmentation of populations, and that the short-term effects of genetic drift have counteracted the long-term effects of balancing selection.

Demographic Properties Shape Tree Size Distribution in a Malaysian Rain Forest

Different mechanisms have been proposed to explain how vertical and horizontal heterogeneity in light conditions enhances tree species coexistence in forest ecosystems. The foliage partitioning theory proposes that differentiation in vertical foliage distribution, caused by an interspecific variation in mortality-to-growth ratio, promotes stable coexistence. In contrast, successional niche theory posits that horizontal light heterogeneity, caused by gap dynamics, enhances species coexistence through an interspecific trade-off between growth rate and survival. To distinguish between these theories of species coexistence, we analyzed tree inventory data for 370 species from the 50-ha plot in Pasoh Forest Reserve, Malaysia. We used community-wide Bayesian models to quantify size-dependent growth rate and mortality of every species. We compared the observed size distributions and the projected distributions from size-dependent demographic rates. We found that the observed size distributions were not simply correlated with the rate of population increase but were related to demographic properties such as size growth rate and mortality. Species with low relative abundance of juveniles in size distribution showed high growth rate and low mortality at small tree sizes and low per-capita recruitment rate. Overall, our findings were in accordance with those predicted by foliage partitioning theory.

Geographic variation of host use in the leaf beetle Agelasa nigriceps suggests host range expansion

Host range expansion is an important event in the evolution of host use in phytophagous insects. Herein, we report geographic variation of host use in the chrysomelid leaf beetle, Agelasa nigriceps Motschulsky (Coleoptera: Chrysomelidae), and suggest that this beetle is expanding its host range. This beetle has been recently recorded on Pterostyrax hispidus Sieb. et Zucc. (Styracaceae) in addition to its common host plant Actinidia arguta (Sieb. et Zucc.) Planch. ex Miq. (Actinidiaceae). The A. arguta‐associated populations were widely found in Japan, whereas the P. hispidus‐associated populations were found only in central and southwestern Japan. In the present study, we examined adult feeding behavior and larval performance of 12 A. nigriceps populations collected from eight localities, four localities where beetles occurred only on A. arguta (allopatric localities) and four localities where A. arguta‐ and P. hispidus‐associated populations occurred sympatrically (sympatric localities). Beetles of all populations, irrespective of their host plants and localities, showed high acceptance of and high larval performance on A. arguta leaves. In contrast, we found considerable variation in the beetle response to P. hispidus leaves. The A. arguta‐associated populations of allopatric localities scarcely accepted P. hispidus leaves, whereas those of sympatric localities, particularly those of P. hispidus‐associated populations, accepted and grew on P. hispidus leaves, although the degree of acceptance and larval performance varied among localities. These results strongly suggest that A. arguta is the ancestral host for A. nigriceps, and host range expansion to the P. hispidus has occurred in this beetle.

Isolation and characterization of major histocompatibility complex class II B genes in cranes.

In this study, we isolated and characterized the major histocompatibility complex (MHC) class II B genes in cranes. Genomic sequences spanning exons 1 to 4 were amplified and determined in 13 crane species and three other species closely related to cranes. In all, 55 unique sequences were identified, and at least two polymorphic MHC class II B loci were found in most species. An analysis of sequence polymorphisms showed the signature of positive selection and recombination. A phylogenetic reconstruction based on exon 2 sequences indicated that trans-species polymorphism has persisted for at least 10 million years, whereas phylogenetic analyses of the sequences flanking exon 2 revealed a pattern of concerted evolution. These results suggest that both balancing selection and recombination play important roles in the crane MHC evolution.

Intra-specific variations in reaction norms of predator-induced polyphenism in the water flea Daphnia pulex

Phenotypic plasticity is considered an important factor leading to phenotypic evolution through adaptations to changing environments. Interactions between environmental inputs and expressed phenotypes can be evaluated as “reaction norms”. Comparisons of these reaction norms among geographically distinct populations may provide evidence for genetic diversity and, moreover, important clues into understanding how the interactions affect evolutionary processes. The water flea Daphnia pulex exhibits predator-induced polyphenism in response to a kairomone released by predatory phantom midge larvae (Chaoborus spp.). The polyphenism, a defensive structure formed on the head that enables juvenile larvae to avoid predation, is referred to as neckteeth. Differences in shape and/or plasticity of defensive structures have also been reported among related Daphnia species, suggesting that the species-specific variation in defensive morphology is derived from intra-specific differences in reaction norms. By determining reaction norms based on the incidence and number of neckteeth, we clarified differences in patterns of neckteeth formation in response to varying concentrations of Chaoborus kairomone. We then compared these differences among populations (strains) collected from six locations in Japan. Our findings showed that the reaction norms of both traits differed among strains. Furthermore, a molecular phylogeny using mitochondrial ND5 and COI sequences suggested that differences in the reaction norms of Japanese strains corresponded to their genetically different lineages, which likely originated from North American populations that were probably introduced to Japan independently.

Deep phylogeographical structure and parallel host range evolution in the leaf beetle Agelasa nigriceps

To understand the mechanisms behind the diversification of herbivorous insects through insect–plant interactions, it is important to know how the insects change their diet breadth in response to environmental changes. In this study, we investigated the phylogeographical pattern of the leaf beetle Agelasa nigriceps to infer the evolutionary history of its host range. While this beetle commonly uses Actinidia arguta (Actinidiaceae) as a host plant, it has been recorded recently on Pterostyrax hispidus (Styracaceae), which is now increasing in abundance at some localities in Japan due to the indirect effects of high population size of a mammalian herbivore. Considerable variation among populations in the ability of Ag. nigriceps to use P. hispidus suggests that P. hispidus is a newly acquired host plant for this beetle. Phylogenetic analyses using mitochondrial DNA sequences and amplified fragment length polymorphism (AFLP) revealed a high degree of phylogeographical structure in Ag. nigriceps throughout Japan, which is consistent with the hypothesis that several glacial refugia existed in the Japanese archipelago. In contrast, no genetic structure associated with the host plants was detected. Both the mitochondrial DNA and AFLP analyses showed that populations that can use P. hispidus are polyphyletic. These results and geographical variation in host use suggest that the host range expansion to a novel host, P. hispidus, is a very recent and possibly ongoing phenomenon and has occurred independently in several regions. Our study illustrates that the host range of herbivorous insects can evolve repeatedly in response to similar environmental changes.

Genetic variation of major histocompatibility complex genes in the endangered red-crowned crane

Populations that have drastically decreased in the past often have low genetic variation, which may increase the risk of extinction. The genes of major histocompatibility complex (MHC) play an important role in the adaptive immune response of jawed vertebrates. Maintenance of adaptive genetic diversity such as that of MHC genes is important for wildlife conservation. Here, we determined genotypes of exon 3 of MHC class IA genes (MHCIA) and exon 2 of MHC class IIB genes (MHCIIB) to evaluate genetic variation of the endangered red-crowned crane population on Hokkaido Island, Japan, which experienced severe population decline in the past. We identified 16 and 6 alleles of MHCIA and MHCIIB, respectively, from 152 individuals. We found evidence of a positive selection at the antigen-binding sites in MHCIA exon 3 and MHCIIB exon 2. The phylogenetic analyses indicated evidence of trans-species polymorphism among the crane MHC genes. The genetic variability in both classes of MHC genes at the population level was low. No geographic structure was found based on the genetic diversity of microsatellite and MHC genes. Our study provides useful data for the optimal management of the red-crowned crane population in Hokkaido and can contribute to future studies on MHC genes of the continental populations of the red-crowned crane and other crane species.

Toxicity of venom of Asobara and Leptopilina species to Drosophila species

The Drosophila parasitoid Asobara japonica Belokobylskij (Hymenoptera: Braconidae) has highly toxic venom that kills host larvae if its injection is not followed by an injection of lateral oviduct components along with egg‐laying. In the present study, the venoms of seven other Drosophila parasitoids (Asobara rossica, Asobara rufescens, Asobara pleuralis, Leptopilina heterotoma, Leptopilina japonica, Leptopilina ryukyuensis, and Leptopilina victoriae) are tested against three kinds of Drosophila species (i.e. Drosophila species that are suitable as host for focal parasitoids, those that are resistant to the parasitoids, and a cosmopolitan species, Drosophila simulans). Venoms of the three Asobara species are not toxic to any of Drosophila species, whereas those of the four Leptopilina species are toxic to some Drosophila species. The toxicity of venom varies among Leptopilina species, and the susceptibility to venom also varies among host Drosophila species. Furthermore, toxicity and paralytic effects of venom are not correlated. Because the toxicity of venom is not adaptive for parasitoids, it may be an inevitable side effect of some components that play an essential role in parasitism.

Genetic divergence with ongoing gene flow is maintained by the use of different hosts in phytophagous ladybird beetles genus Henosepilachna

Adaptation to different environments can promote population divergence via natural selection even in the presence of gene flow – a phenomenon that typically occurs during ecological speciation. To elucidate how natural selection promotes and maintains population divergence during speciation, we investigated the population genetic structure, degree of gene flow and heterogeneous genomic divergence in three closely related Japanese phytophagous ladybird beetles: Henosepilachna pustulosa, H. niponica and H. yasutomii. These species act as a generalist, a wild thistle (Cirsium spp.) specialist and a blue cohosh (Caulophyllum robustum) specialist, respectively, and their ranges differ accordingly. The two specialist species widely co‐occur but are reproductively isolated solely due to their high specialization to a particular host plant. Genomewide amplified fragment‐length polymorphism (AFLP) markers and mitochondrial cytochrome c oxidase subunit I (COI) gene sequences demonstrated obvious genomewide divergence associated with both geographic distance and ecological divergence. However, a hybridization assessment for both AFLP loci and the mitochondrial sequences revealed a certain degree of unidirectional gene flow between the two sympatric specialist species. Principal coordinates analysis (PCoA) based on all of the variable AFLP loci demonstrated that there are genetic similarities between populations from adjacent localities irrespective of the species (i.e. host range). However, a further comparative genome scan identified a few fractions of loci representing approximately 1% of all loci as different host‐associated outliers. These results suggest that these three species had a complex origin, which could be obscured by current gene flow, and that ecological divergence can be maintained with only a small fraction of the genome is related to different host use even when there is a certain degree of gene flow between sympatric species pairs.

Heterospecific sperm reduction in interspecific crosses between two closely related phytophagous ladybird beetles, Henosepilachna vigintioctomaculata and H.pustulosa (Coleoptera: Coccinellidae)

In interspecific crosses, a mismatch in internal physiological conditions between two species can reduce sperm viability in the interval from insemination to fertilization, leading to gametic isolation. Two closely related Japanese phytophagous ladybird beetles, Henosepilachna vigintioctomaculata and H. pustulosa, show several isolating barriers, including reduction in the number of heterospecific sperm in the female reproductive tract and low egg‐hatching rates in interspecific matings. However, the mechanisms of these two potential isolating barriers and the association between them are unknown. Here we investigated temporal changes in the number of sperm stored in the female reproductive tract and egg‐hatching rates in inter‐ and intraspecific crosses between these species. Although the number of sperm decreased after both inter‐ and intraspecific crosses, the reduction was more drastic in inter‐ than in intraspecific crosses for females of both species. Most of the sperm reduction occurred early on, during sperm transfer from the bursa copulatrix to the paired ampullae of the common oviduct (the sperm storage organs). These two species also demonstrated stably low egg‐hatching rates in interspecific crosses. Since the degree and timing of the sperm reduction did not correlate with egg‐hatching rates, the reduction in heterospecific sperm in interspecific crosses may not directly cause the low hatching rates. These two isolating barriers could be different expressions of the physiological mismatch and/or genetic incompatibility between gametes of these species.