Odd-Paired is Involved in Morphological Divergence of Snail-Feeding Beetles.

Body shape and size diversity and their evolutionary rates correlate with species richness at the macroevolutionary scale. However, the molecular genetic mechanisms underlying the morphological diversification across related species are poorly understood. In beetles, which account for one-fourth of the known species, adaptation to different trophic niches through morphological diversification appears to have contributed to species radiation. Here, we explored the key genes for the morphological divergence of the slender to stout body shape related to divergent feeding methods on large to small snails within the genus Carabus. We show that the zinc-finger transcription factor encoded by odd-paired (opa) controls morphological variation in the snail-feeding ground beetle Carabus blaptoides. Specifically, opa was identified as the gene underlying the slender to stout morphological difference between subspecies through genetic mapping and functional analysis via gene knockdown. Further analyses revealed that changes in opa cis-regulatory sequences likely contributed to the differences in body shape and size between C. blaptoides subspecies. Among opa cis-regulatory sequences, single nucleotide polymorphisms on the transcription factor binding sites may be associated with the morphological differences between C. blaptoides subspecies. opa was highly conserved in a wide range of taxa, especially in beetles. Therefore, opa may play an important role in adaptive morphological divergence in beetles.

Recent origin and diversification accompanied by repeated host shifts of thallus-mining flies (Diptera: Agromyzidae) on liverworts and hornworts.

Despite the vast diversity of phytophagous insects that feed on vascular plants (tracheophytes), insects that feed on bryophytes remain understudied. Agromyzidae, one of the most species-rich phytophagous clades in Diptera, consists mainly of leaf-mining species that feed on tracheophytes. However, a recent discovery of thallus-mining species on liverworts and hornworts within the Liriomyza group of Phytomyzinae provides an opportunity to study host shifts between tracheophytes and bryophytes. This study aimed to explore the origin and diversification of thallus-miners and estimate the pattern and timing of host shifts. Phylogenetic analysis of Phytomyzinae has revealed that the thallus-mining agromyzids formed a separate clade, which was sister to a fern pinnule-miner. The diversification of bryophyte-associated agromyzids since the Oligocene involved multiple host shifts across various bryophyte taxa. The diversification of the thallus-mining Phytoliriomyza may have occurred at the same time as the leaf-mining agromyzid flies on herbaceous plants, indicating a dynamic history of interactions between bryophytes and herbivores in angiosperms-dominated ecosystems.

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.

Female-limited responses in remating rate and mating duration in the experimental evolution of a beetle Callosobruchus chinensis.

Mating rate optima often differ between the sexes: males may increase their fitness by multiple mating, but for females multiple mating confers little benefit and can often be costly (especially in taxa without nuptial gifts or mala parental care). Sexually antagonistic evolution is thus expected in traits related to mating rates under sexual selection. This prediction has been tested by multiple studies that applied experimental evolution technique, which is a powerful tool to directly examine the evolutionary consequences of selection. Yet, the results so far only partly support the prediction. Here, we provide another example of experimental evolution of sexual selection, by applying it for the first time to the mating behaviour of a seed beetle Callsorobruchus chinensis. We found a lower remating rate in polygamy-line females than in monogamy-line (i.e. no sexual selection) females after 21 generations of selection. Polygamy-line females also showed a longer duration of first mating than monogamy-line females. We found no effect of male evolutionary lines on the remating rate or first mating duration. Though not consistent with the original prediction, the current and previous studies collectively suggest that the observed female-limited responses may be a norm, which is also consistent with the conceptual advances in the last two decades of the advantages and limitations of experimental evolution technique.

Reproductive isolation via divergent genital morphology due to cascade reinforcement in Ohomopterus ground beetles.

Secondary contact between incipient species and selection against maladaptive hybridization can drive reinforcement between populations in contact and result in reproductive character displacement (RCD). Resultant divergence in mating traits within a species may generate downstream reproductive isolation between populations with displaced and non-displaced traits, referred to as the cascade reinforcement hypothesis. We examined this hypothesis using three allopatric populations of the ground beetle Carabus maiyasanus with a genital lock-and-key system. This species shows RCD in male and female genital morphologies in populations in contact with the sister species C. iwawakianus. In a reciprocal mating experiment using three allopatric populations with differences in male and female genital sizes, insemination failure increased as the difference in genital size increased. Based on the reproductive isolation index, insemination failure was the major postmating-prezygotic isolation barrier, at least in one population pair with comparable total isolation to those of other species pairs. By contrast, there was only incomplete premating isolation among populations. These results suggest that RCD in genital morphologies drives incipient allopatric speciation, supporting the cascade reinforcement hypothesis. These findings provide insight into the roles of interspecific interactions and subsequent trait diversification in speciation processes.

Advances in the Evolution and Ecology of 13- and 17-Year Periodical Cicadas.

Apart from model organisms, 13- and 17-year periodical cicadas (Hemiptera: Cicadidae: Magicicada) are among the most studied insects in evolution and ecology. They are attractive subjects because they predictably emerge in large numbers; have a complex biogeography shaped by both spatial and temporal isolation; and include three largely sympatric, parallel species groups that are, in a sense, evolutionary replicates. Magicicada are also relatively easy to capture and manipulate, and their spectacular, synchronized mass emergences facilitate outreach and citizen science opportunities. Since the last major review, studies of Magicicada have revealed insights into reproductive character displacement and the nature of species boundaries, provided additional examples of allochronic speciation, found evidence for repeated and parallel (but noncontemporaneous) evolution of 13- and 17-year life cycles, quantified the amount and direction of gene flow through time, revealed phylogeographic patterning resulting from paleoclimate change, examined the timing of juvenile development, and created hypotheses for the evolution of life-cycle control and the future effects of climate changeon Magicicada life cycles. New ecological studies have supported and questioned the role of prime numbers in Magicicada ecology and evolution, found bidirectional shifts in population size over generations, quantified the contribution of Magicicada to nutrient flow in forest ecosystems, and examined behavioral and biochemical interactions between Magicicada and their fungal parasites and bacterial endosymbionts.

Evolutionary changes in gene expression profiles associated with the coevolution of male and female genital parts among closely related ground beetle species.

BACKGROUND: The coevolutionary dynamics of corresponding male and female sexual traits, including genitalia, may be driven by complex genetic mechanisms. Carabus (Ohomopterus) ground beetles show correlated evolution in the size of their functionally corresponding male and female genital parts. To reveal the genetic mechanisms involved in the evolution of size, we investigated interspecific differences in gene expression profiles in four closely related species (two species each with long and short genital parts) using transcriptome data from genital tissues in the early and late pupal stages. RESULTS: We detected 1536 and 1306 differentially expressed genes (DEGs) among the species in males and 546 and 1959 DEGs in females in the two pupal stages, respectively. The DEGs were clustered by species-specific expression profiles for each stage and sex to identify candidate gene clusters for genital size based on the expression patterns among the species and gene ontology. We identified one and two gene clusters in females and males, respectively, all from the late pupal stage; one cluster of each sex showed similar expression profiles in species with similar genital size, which implies a common gene expression change associated with similar genital size in each sex. However, the remaining male cluster showed different expression profiles between species with long genital parts, which implies species-specific gene expression changes. These clusters did not show sex-concordant expression profiles for genital size differences. CONCLUSION: Our study demonstrates that sex-independent and partly species-specific gene expression underlies the correlated evolution of male and female genital size. These results may reflect the complex evolutionary history of male and female genitalia.

Role of Sex-Concordant Gene Expression in the Coevolution of Exaggerated Male and Female Genitalia in a Beetle Group.

Some sexual traits, including genitalia, have undergone coevolutionary diversification toward exaggerated states in both sexes among closely related species, but the underlying genetic mechanisms that allow correlated character evolution between the sexes are poorly understood. Here, we studied interspecific differences in gene expression timing profiles involved in the correlated evolution of corresponding male and female genital parts in three species of ground beetle in Carabus (Ohomopterus). The male and female genital parts maintain morphological matching, whereas large interspecific variation in genital part size has occurred in the genital coevolution between the sexes toward exaggeration. We analyzed differences in gene expression involved in the interspecific differences in genital morphology using whole transcriptome data from genital tissues during genital morphogenesis. We found that the gene expression variance attributed to sex was negligible for the majority of differentially expressed genes, thus exhibiting sex-concordant expression, although large variances were attributed to stage and species differences. For each sex, we obtained co-expression gene networks and hub genes from differentially expressed genes between species that might be involved in interspecific differences in genital morphology. These gene networks were common to both sexes, and both sex-discordant and sex-concordant gene expression were likely involved in species-specific genital morphology. In particular, the gene expression related to exaggerated genital size showed no significant intersexual differences, implying that the genital sizes in both sexes are controlled by the same gene network with sex-concordant expression patterns, thereby facilitating the coevolution of exaggerated genitalia between the sexes while maintaining intersexual matching.

Reproductive Character Displacement in Genital Morphology in Ohomopterus Ground Beetles.

AbstractGenital morphology reveals rapid diversification among species, and species-specific divergence in genital morphology may result in reproductive isolation and promote speciation. Natural selection against maladaptive hybridization may cause species-specific genital divergence. In this context, divergence in mating traits is expected to be greater between sympatric populations than between allopatric populations in a pair of species, known as reproductive character displacement (RCD). However, there are few examples of RCD in the genital morphology of closely related species. Additionally, processes leading to RCD have rarely been inferred. In this study, we examined RCD and its underlying mechanisms by focusing on species-specific genital morphologies of closely related Ohomopterus ground beetle species. A morphological analysis showed patterns of RCD in species-specific genital parts in both sexes. Interspecific hybridization was confirmed by a mate choice experiment and by a population genetic analysis indicating extensive interspecific gene flow, suggesting that reinforcement is the most plausible process underlying the observed RCD. We found variation in the degree of displacement in contact zones, which may correspond with the ongoing process of genital evolution and speciation. Our results provide support for the lock-and-key hypothesis of genital evolution in closely related Ohomopterus species.

Global dispersal and diversification in ground beetles of the subfamily Carabinae.

The origin and diversification process of lineages of organisms that are currently widely distributed among continents is an interesting subject for exploring the evolutionary history of global species diversity. Ground beetles of the subfamily Carabinae are flightless except for one lineage, but nevertheless occur on all continents except Antarctica. Here, we used sequence data from ultraconserved elements to reconstruct the phylogeny, divergence time, biogeographical history, ancestral state of hind wings and changes in the speciation rate of Carabinae. Our results show that Carabinae originated in the Americas and diversified into four tribes during the period from the late Jurassic to the late Cretaceous, with two in South America (Celoglossini) and Australasia (Pamborini) and two in Laurasia (Cychrini and Carabini). The ancestral Carabinae were inferred to be winged; three of four tribes (Cychrini, Ceglossini and Pamborini) have completely lost their hind wings and flight capability. The remaining tribe, Carabini, diverged into the subtribes Carabina (wingless) and Calosomina (winged) in the Oligocene. Carabina originated in Europe, spread over Eurasia and diversified into approximately 1000 species, accounting for around 60% of all Carabinae species. Calosomina that were flight-capable dispersed from North America or Eurasia to South America, Australia, and Africa, and then flightless lineages evolved on oceanic islands and continental highlands. The speciation rate increased in the Cychrini and Carabini clades in Eurasia. Within Carabini, the speciation rate was higher for wingless than winged states. Our study showed that the global distribution of Carabinae resulted from ancient dispersal before the breakup of Gondwana and more recent dispersal through flight around the world. These patterns consequently illustrate the causal relationships of geographical history, evolution of flightlessness, and the global distribution and species diversity of Carabinae.

Mapping of quantitative trait loci underlying a magic trait in ongoing ecological speciation.

BACKGROUND: Telmatochromis temporalis is a cichlid fish endemic to Lake Tanganyika. The normal and dwarf morphs of this fish are a clear example of ongoing ecological speciation, and body size plays an important role in this speciation event as a magic trait. However, the genetic basis underlying this trait has not been studied. RESULTS: Based on double-digested restriction-site associated DNA (ddRAD) sequencing of a hybrid cross between the morphs that includes F0 male, F0 female, and 206 F2 individuals, we obtained a linkage map consisting of 708 ddRAD markers in 22 linkage groups, which corresponded to the previously reported Oreochromis niloticus chromosomes, and identified one significant and five suggestive quantitative trait loci (QTL) for body size. From the body-size distribution pattern, the significant and three of the five suggestive QTL are possibly associated with genes responsible for the difference in body size between the morphs. CONCLUSIONS: The QTL analysis presented here suggests that multiple genes, rather than a single gene, control morph-specific body size. The present results provide further insights about the genes underlying the morph specific body size and evolution of the magic trait during ecological speciation.

Population genetic structure underlying the geographic variation in beetle structural colour with multiple transition zones.

We studied the population genetic structure underlying the geographic variation in the structural colour of the geotrupid dung beetle, Phelotrupes auratus, which exhibits metallic body colours of different reflectance wavelengths perceived as red, green and indigo. These forms occur parapatrically in an area of Japan. The colour variation was not related to variation in climatic factors. Using single nucleotide polymorphisms (SNPs) from restriction-site-associated DNA sequences, we discriminated five groups of populations (west/red, south/green, south/indigo, south/red and east/red) by a combination of genetic clusters (west, south and east) and three colour forms. There were three transition zones for the colour forms: two between the red and green forms were hybrid zones with steep genetic clines, which implies the existence of barriers to gene flow between regions with different colours. The remaining transition zone between the green and indigo forms lacked genetic differentiation, despite the evident colour changes, which implies regionally specific selection on the different colours. In a genomewide association study, we identified four SNPs that were associated with the red/green or indigo colour and were not linked with one another, which implies that the coloration was controlled by multiple loci, each affecting the expression of a different colour range. These loci may have controlled the transitions between different combinations of colours. Our study demonstrates that geographic colour variation within a species can be maintained by nonuniform interactions among barriers to gene flow, locally specific selection on different colours, and the effects of different colour loci.

Evolution of host use in fungivorous ciid beetles (Coleoptera: Ciidae): Molecular phylogeny focusing on Japanese taxa.

Consumer-resource interactions between trophic levels are ubiquitous and important factors in shaping the diversity of insects. However, dietary patterns such as host specificity and conservatism have been insufficiently examined in fungivorous insects. Here we reconstructed the evolutionary history of host use in fungivorous ciid beetles (Coleoptera: Ciidae) and tested for host conservatism. Phylogenetic relationships among 49 species from Japan were inferred by using a large sequence data set from ultraconserved elements (UCEs). In addition, sequences of three genes (COI, 28S rRNA, 18S rRNA) were analyzed to reconstruct the phylogeny for 130 OTUs from a broader range of taxa and geographic regions using the UCE tree as a backbone topology. We found that Ciini and Orophiini are not recovered as reciprocally monophyletic groups. As previously suggested, the largest genus Cis Latreille was also not monophyletic. Ancestral-state reconstruction of host use in both datasets showed that Ciidae species were clustered by host-use group across the tree. This pattern was confirmed by the significantly lower transition rate compared with expectations under the random shift hypothesis. The observed conservatism in host use implied these beetles possess unique adaptations to specific fungal taxa, just as herbivorous insects are adapted to specific plant taxa.

Sexual selection increased offspring production via evolution of male and female traits.

Phenotypic evolution driven by sexual selection can impact the fitness of individuals and thus population performance through multiple mechanisms, but it is unresolved how and when sexual selection affects offspring production by females. We examined the effects of sexual selection on offspring production by females using replicated experimental evolutionary lines of Callosobruchus chinensis that were kept under polygamy (with sexual selection) or monogamy (without sexual selection) for 21 generations. We found that polygamous-line pairs produced more offspring than monogamous-line pairs, because polygamous-line beetles evolved to be larger than monogamous-line beetles, and larger females were more fecund. Egg hatchability did not differ between polygamous- and monogamous-line pairs, as a result of the positive and negative effects of sexual selection cancelling out. When mated with an individual from a common tester line, both polygamous-line females and males showed higher hatchability in resultant eggs than monogamous ones. Further, cohabitation with a male reduced egg hatchability, and this effect was more pronounced in polygamous-line than in monogamous-line males. These results demonstrate multiple mechanisms by which sexual selection affects female fitness, with the net effect being positive. Analyses of how development time, body size and male genital morphology were influenced by selection regime suggest that these results arose from both evolution via good-gene processes and sexually antagonistic selection. Our results are also consistent with the hypothesis that the fitness consequences of sexual selection for females are dependent on the evolutionary history of the population.

Mitochondrial Genomics Reveals Shared Phylogeographic Patterns and Demographic History among Three Periodical Cicada Species Groups.

The mass application of whole mitogenome (MG) sequencing has great potential for resolving complex phylogeographic patterns that cannot be resolved by partial mitogenomic sequences or nuclear markers. North American periodical cicadas (Magicicada) are well known for their periodical mass emergence at 17- and 13-year intervals in the north and south, respectively. Magicicada comprises three species groups, each containing one 17-year species and one or two 13-year species. Within each life cycle, single-aged cohorts, called broods, of periodical cicadas emerge in different years, and most broods contain members of all three species groups. There are 12 and three extant broods of 17- and 13-year cicadas, respectively. The phylogeographic relationships among the populations and broods within the species groups have not been clearly resolved. We analyzed 125 whole MG sequences from all broods and seven species within three species groups to ascertain the divergence history of the geographic and allochronic populations and their life cycles. Our mitogenomic phylogeny analysis clearly revealed that each of the three species groups had largely similar phylogeographic subdivisions (east, middle, and west) and demographic histories (rapid population expansion after the last glacial period). The mitogenomic phylogeny also partly resolved the brood diversification process, which could be explained by hypothetical temporary life cycle shifts, and showed that none of the 13- and 17-year species within the species groups was monophyletic, possibly due to gene flow between them. Our findings clearly reveal phylogeographic structures in the three Magicicada species groups, demonstrating the advantage of whole MG sequence data in phylogeographic studies.

Evolutionary fine-tuning of background-matching camouflage among geographical populations in the sandy beach tiger beetle.

Background-matching camouflage is a widespread adaptation in animals; however, few studies have thoroughly examined its evolutionary process and consequences. The tiger beetle Chaetodera laetescripta exhibits pronounced variation in elytral colour pattern among sandy habitats of different colour in the Japanese Archipelago. In this study, we performed digital image analysis with avian vision modelling to demonstrate that elytral luminance, which is attributed to proportions of elytral colour components, is fine-tuned to match local backgrounds. Field predation experiments with model beetles showed that better luminance matching resulted in a lower attack rate and corresponding lower mortality. Using restriction site-associated DNA (RAD) sequence data, we analysed the dispersal and evolution of colour pattern across geographical locations. We found that sand colour matching occurred irrespective of genetic and geographical distances between populations, suggesting that locally adapted colour patterns evolved after the colonization of these habitats. Given that beetle elytral colour patterns presumably have a quantitative genetic basis, our findings demonstrate that fine-tuning of background-matching camouflage to local habitat conditions can be attained through selection by visual predators, as predicted by the earliest proponent of natural selection.

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.

Genetic Structure of Dytiscus sharpi in North and South Hokuriku in Japan Inferred from Mitochondrial and Nuclear Gene Sequence.

Dytiscus sharpi is a critically endangered diving beetle endemic to Japan that is distributed in five distant areas today. Information on the population genetics of this species is crucial for its conservation. We investigated the genetic differentiation and divergence of the D. sharpi in seven populations in North and South Hokuriku on the Sea of Japan side, using mitochondrial cytochrome oxidase subunit I (COI) and nuclear Histone 3 and 28S rRNA gene sequences. Although the nuclear markers showed little geographic genetic differentiation, nine COI haplotypes were identified from 31 individuals, and these haplotypes were divided into two distinct networks A and B. The network A consisted of a single haplotype, and network B consisted of eight haplotypes. The populations in North Hokuriku possessed the haplotypes of both networks, whereas those in South Hokuriku possessed only the haplotypes of network B. The genetic diversity was relatively high in South and North Hokuriku. However, populations in South Hokuriku showed low genetic diversity and the isolation-by-distance pattern was observed among the populations, suggesting a restricted gene flow. Phylogenetic analysis including the data from a previous study revealed that the network A was closely related to a haplotype from Kanto, which is on the Pacific side. These results suggested that in the past this species was distributed widely across the Hokuriku area and the fragmentation of its distribution areas is a recent event. These results will contribute to strategies for the preservation of the current genetic diversity of D. sharpi.

Phylogeography of Endangered Bitterling Acheilognathus melanogaster Endemic to Eastern Japan.

The bitterling Acheilognathus melanogaster is a critically endangered primary freshwater fish endemic to the Pacific side of eastern Japan. To elucidate A. melanogaster genetic structure, we investigated phylogeography in nine populations, using gene sequences of mitochondrial Cytochrome b (Cytb), as well as nuclear Rhodopsin (Rho) and glycosyltransferase (Glyt). We found four Cytb-based geographical clusters unevenly divided between the northern and southern regions, with smaller groups in the south. Of the nuclear genes, Glyt did not show geographical differentiation, whereas Rho formed two clusters: one widely occurring and another restricted to central regions. Genetic diversity was generally higher in southern than in northern populations. Our results suggest that conservation of southern local populations is particularly important in maintaining the genetic diversity of this endangered fish.