Origin and diversification of pheretimoid megascolecid earthworms in the Japanese Archipelago as revealed by mitogenomic phylogenetics.

Megascolecid earthworms of the pheretimoid group are dominant detritivores of soil ecosystems in the Japanese Archipelago and East Asia. However, their diversity and phylogenetic relationships are poorly understood. We assembled whole mitogenome sequences for 197 megascolecid earthworms collected throughout Japan to study the phylogenetic relationships, phylogeography, divergence times, and diversification of important morphological characteristics among pheretimoid earthworms. Using 197 mitogenome sequences and 24 published mitogenome sequences from the East Asian mainland (221 sequences in total), we constructed a maximum likelihood tree and found that the pheretimoid earthworms currently assigned to Amynthas, Metaphire, Duplodicodrilus, and Manus are involved in the most senior genus Amynthas; thus, Amynthas can be treated as the sole genus encompassing all of the above genera. Within the Amynthas group, we identified three major lineages that led to four groups of endemic species in Japan. These lineages originated from different lineages on the East Asian mainland and Taiwan Island, indicating multiple colonization events from the East Asian mainland by different ancestral lineages, possibly after the Miocene. We also assembled nuclear ribosomal DNA sequences encompassing the 18S to 28S rRNA genes. The nuclear gene tree showed major groups consistent with the mitogenome tree except for different (and not well-resolved) relationships among major clades. Our molecular data covered 115-158 native and 7 non-native Amynthas group species in Japan in terms of DNA-based species delimitation. Our findings provide a basis for understanding the evolutionary relationships among diversified megascolecid earthworms in the Amynthas group in Japan and adjacent regions.

Effects of body size divergence on male mating tactics in the ground beetle Carabus japonicus.

Animal body size is involved in reproduction in various ways. Carabus japonicus exhibits considerable variation in adult body size across geographical locations depending on the larval environment. To investigate the effects of body size divergence on male mating traits, spermatophore deposition and weight, copulation duration, and post-copulatory mounting were observed using male-female pairs from C. japonicus populations with different body sizes. Then, variables with high predictive power on the mating traits were identified from individual characteristics. When the male was slightly smaller than his mate, spermatophore deposition likely succeeded, suggesting that mechanical size-assortative insemination determined male body size. Although male reproductive organ size was positively correlated with male body size, spermatophore weight was not significantly affected by male body size, whereas copulation duration decreased with increasing male body size. Enlarged males, with a high capacity for spermatophore production, could increase paternity by decreasing copulation duration and increasing mating frequency. Such shifts in mating tactics would alter selection pressures of intra- and intersexual interactions (e.g., sperm competition and sexual conflict). Genital dimensions also affected mating traits other than copulatory duration. Thus, ecological heterogeneity has the potential to lead to divergences in sexual traits, such as genital morphology, through body size divergence.

Taxonomic and nomenclatural changes in three species of Pterostichus Bonelli (Coleoptera: Carabidae) from the Far East.

The ground beetle genus Pterostichus Bonelli has diversified in regions including the Far East, but taxonomic issues remain even at the species level. This study presents taxonomic and nomenclatural changes in three species of Pterostichus from the Far East: P. (Petrophilus) eximius Morawitz, P. (Lenapterus) wellschmiedi Kirschenhofer, and P. (L.) subrugosus Straneo. The analyses are based on comparative studies of the endophallus of male genitalia, which is taxonomically useful in Carabidae but has not been examined in these species. The first species, P. eximius, has been treated as a monotypic species that is widely distributed in Eastern Siberia, the Russian Far East, and adjacent regions, but our results revealed that populations from Rishiri-tô and the Sakhalin islands are distinct from the nominotypical population from Transbaikalia. The name for the Sakhalin population, which was synonymized with P. eximius, is reinstated as subspecies P. eximius sachalinensis stat. nov., and the Rishiri-tô population is described as Pterostichus eximius rishiridakensis ssp. nov. Conspecificity (syn. nov.) was confirmed for P. wellschmiedi, which was described from southern Sakhalin, and P. marginatus Matsumura, which was described earlier from southern Sakhalin. No conspicuous differences in the endophallus structure were found between P. subrugosus, which was described from Hokkaidô, and P. marginatus, although differences are recognized in the elytral sculptures and in the metallic luster of the dorsal surface. The taxon was thus downgraded to subspecies P. marginatus subrugosus stat. nov.

Predator size divergence depends on community context.

Body size is a multi-functional trait related to various fitness components, but the relative importance of different selection pressures is seldom resolved. In Carabus japonicus beetles, of which the larvae exclusively prey on earthworms, adult body size is related to the presence/absence of a larger congener and habitat temperature. In sympatry, C. japonicus consistently exhibits smaller body size which is effective for avoiding interspecific mating, but in allopatry, it shows size variation unrelated to temperature. Here, we show that this predator-size variation is attributed to prey-size variation, associated with high phylogenetic diversity in earthworm communities. In allopatry, the predator size was larger where larger prey occurred. Larger adult size may have been selected because larger females produce larger larvae, which can subdue larger prey. Thus, in the absence of a larger congener, variation in prey body size had a pronounced effect on geographic body size divergence in C. japonicus.

Genomic regions and genes related to inter-population differences in body size in the ground beetle Carabus japonicus.

Body size is a key trait in diversification among animal species, and revealing the gene regions responsible for body size diversification among populations or related species is important in evolutionary biology. We explored the genomic regions associated with body size differences in Carabus japonicus ground beetle populations by quantitative trait locus (QTL) mapping of F2 hybrids from differently sized parents from two populations using restriction site-associated DNA sequencing and de novo assembly of the beetle whole genome. The assembled genome had a total length of 191 Mb with a scaffold N50 of 0.73 Mb; 14,929 protein-coding genes were predicted. Three QTLs on different linkage groups had major effects on the overall size, which is composed chiefly of elytral length. In addition, we found QTLs on autosomal and X chromosomal linkage groups that affected head length and width, thoracic width, and elytral width. We determined the gene loci potentially related to control of body size in scaffolds of the genome sequence, which contained the QTL regions. The genetic basis of body size variation based on a small number of major loci would promote differentiation in body size in response to selection pressures related to variations in environmental conditions and inter-specific interactions.

Factors Related to Altitudinal Body Size Variation in the Earthworm-Eating Ground Beetle Carabus japonicus.

To understand geographic body size variation in an insect species, various factors including habitat temperature, correlation between life history traits, and food availability must be examined. Carabus (Ohomopterus) japonicus is univoltine, feeds exclusively on earthworms during its larval stage, and shows a clinal body size variation along the habitat temperature gradient in northern Kyushu, Japan. Carabus japonicus occurs at both high and low altitudes; at high altitudes it coexists with a larger species, C. (O.) dehaanii. At low altitudes, C. japonicus shows larger body sizes. We sought to determine whether this increase in body size is only an adaptation to high habitat temperatures in the absence of C. dehaanii, and examined the life history of C. japonicus and seasonal trends in prey earthworms at six sites between altitudes of 30 and 980 m. While high-altitude populations used the entire warm season for reproduction and larval development, low-altitude populations showed shorter periods of reproductive activity and larval incidence coincided with seasonal trends in earthworm abundance. Thus, C. japonicus attained larger body sizes at lower altitudes without a notable extension of their juvenile period. At lower altitudes, earthworms grew faster and reached sizes, which may be too large for predation by small carabid larvae. Large females had higher fecundity, and laid larger eggs, from which larger first instar larvae hatch. The large body size of C. japonicus in warm habitats may thus be an adaptation not only for high fecundity, but also for producing large first instar larvae to more efficiently prey on large earthworms.

Mating behavior and the function of the male genital spine in the ground beetle Carabus clathratus.

The morphologies of male genitalia often appear harmful or aggressive, as if they may inflict physical damage upon females during copulation. Such male genitalia are often thought to function in intra- and intersexual interactions during mating. In the carabid genus Carabus, division Spinulati, males possess a spine (spinula) on the intromittent organ, of which function is unknown. To reveal the function of the spinula, we studied the mating behavior and genital coupling of a Spinulati species, Carabus (Limnocarabus) clathratus. The males positioned the spinula along the inner wall of the vaginal opening throughout copulation. This placement created a small dent and subsequently a melanized patch (wound) on the vaginal wall, but the spinula rarely penetrated the vaginal wall. The spinula did not reach the innermost part of the vagina where the spermatophore is deposited. These results suggest that the spinula is not used for inflicting damage on female genitalia or manipulating spermatophores of rival males. During spermatophore formation, the male partially withdrew the aedeagus, and only the aedeagal tip and endophallus remained within the female. By placing the spinula against the vaginal wall, the male could hold the endophallus within the vaginal chamber in the unstable copulatory posture. Thus, our observations suggest that the spinula primarily functions as an "anchor" to maintain the coupling of the male and female genitalia and thereby ensure insemination.

Resource partitioning or reproductive isolation: the ecological role of body size differences among closely related species in sympatry.

1. Body size differences among coexisting related species are common, but the actual effect of these differences in mitigating interspecific interactions, such as resource competition and reproductive interference, is poorly understood. 2. Local assemblages of the ground beetle genus Carabus (subgenus Ohomopterus) typically consist of two or more species of varying sizes. Through foraging and mating experiments using four Ohomopterus species in parapatry and sympatry, we examined whether interspecific body size differences are effective in partitioning food resources or reducing reproductive interference. 3. Because larval Ohomopterus feed exclusively on earthworms, body size differences may be related to partitioning earthworms of different sizes. However, larvae did not exhibit differences in selectivity or attack success on earthworms of different sizes based on larval body size, indicating little possibility of partitioning food by body size. 4. In contrast, interspecific mating behaviours, such as mate recognition, mounting, and copulation, were hindered when body size differences were large; copulation was frequently accomplished between parapatric species with smaller body size differences. 5. These results suggest that body size differences between species effectively reduce reproductive interference, rather than resource competition. Although body size differences in coexisting closely related species have been considered to function in resource partitioning, they may function primarily in reproductive isolation and thereby facilitate coexistence of species.