Difference in Size at Maturity in Annual and Overwintering Generations in the Tanaidacean Zeuxo sp. in Oshoro Bay, Hokkaido, Japan.

We investigated the life cycle of the tanaidid Zeuxo sp. 1 (Crustacea: Peracarida: Tanaidacea), which lives epiphytically in dwelling tubes on the algae Sargassum spp. and Neorhodomela aculeata in Oshoro Bay, Hokkaido, Japan. We obtained data on its population size distribution, age structure, and reproductive phenology through monthly sampling from April 2011 to June 2012. From these data, we detected an overwintering group (F0) that arises from mancae that are released in autumn, overwinters, reproduces the following spring, and then disappears. The overwintering group produces mancae (F1) that reach maturity and themselves reproduce within six weeks after release; the F1, mancae give rise to an F2 and possibly an F3 generation within a single summer. We refer to individuals that reproduce in the same summer that they were released as the 'annual group'. Reproduction overall was restricted to the period from May to October, when the seasurface temperature exceeded 10°C. During both years, overwintering females first became reproductive in May. Reproduction in the annual group began in June and continued through mid-October. Mancae were observed in samples from June through October. The two groups differed significantly in size at reproduction; compared to the annual group, females in the overwintering group reproduced at larger minimum and average body sizes, and males began to express enlarged chelae, a secondary sexual character, at a larger size. The difference in size at maturity may be related to the differences in water temperature during the main period of growth and maturation.

Introgression and Habitat Segregation in a Pair of Ladybird Beetle Species in the Genus Propylea (Coccinellidae, Coccinellinae) in Northern Japan.

The ladybird beetles Propylea quatuordecimpunctata and P. japonica have largely overlapping distributions in northern Japan, and in the laboratory produce fertile hybrids. In this study, we surveyed the distribution and morphological differentiation of these species and the hybrids in natural populations, with a focus on western Hokkaido, northern Japan. Phenotypic analyses were conducted for 987 individuals collected at 90 localities. In addition, the nuclear internal transcribed spacer-II (ITS2) region (549 bp) and part of the mitochondrial cytochrome c oxidase subunit I (COI) gene (700 bp) were sequenced for 620 individuals from 53 localities. Analyses of both phenotypic and genotypic features discriminated two distinct entities assignable to P. quatuordecimpunctata and P. japonica. However, individuals with intermediate phenotypes and/or genotypes also occurred extensively, indicating natural hybridization. Putative P. quatuordecimpunctata individuals were collected across a wide range of altitudes (30-600 m), whereas those of P. japonica were found mostly lower than 300 m alt. In addition, P. quatuordecimpunctata was dominant in semi-open habitats shaded by canopy foliage, whereas P. japonica was frequent in more open habitats. The perceived altitudinal difference in the distributions may thus in part be a consequence of this different habitat preference, as open habitats are more common at lower altitudes in the study area.

Phylogeny of epilachna, henosepilachna, and some minor genera of phytophagous ladybird beetles (coleoptera: coccinellidae: coccinellinae: epilachnini), with an analysis of ancestral biogeography and host-plant utilization.

Ladybird beetles in the tribe Epilachnini include notorious crop pests and model species studied intensively in various fields of evolutionary biology. From a combined dataset of mitochondrial (ND2) and nuclear (28S) DNA sequences, we reconstructed the phylogeny of 46 species of Epilachnini from Asia, Africa, America, and the Australian region: 16 species in Epilachna, 24 species in Henosepilachna, and one species each in Adira, Afidenta, Afidentula, Afissula, Chnootriba, and Epiverta. In our phylogenetic trees, both Epilachna and Henosepilachna were reciprocally polyphyletic. Asian Epilachna species were monophyletic, except for the inclusion of Afissula sp. Asian and Australian Henosepilachna species likewise formed a monophyletic group, excluding H. boisduvali. African Epilachna and Henosepilachna species did not group with their respective Asian and American congeners, but were paraphyletic to other clades (Epilachna species) or formed a separate monophyletic group (Henosepilachna species) together with Chnootriba similis. The American Epilachna species were monophyletic and formed a clade with American Adira clarkii and Asian Afidentula manderstjernae bielawskii; this clade was the sister group to Asian and Australian Henosepilachna, but was distant from Asian Epilachna. Chnootriba was embedded in the African Henosepilachna clade, and Afissula in the Asian Epilachna clade. Epiverta, which is morphologically unique, was the sister group to Asian Epilachna, although with weak support. From reconstructions of biogeographical distribution and host-plant utilization at ancestral nodes, we inferred an African origin for the common ancestor of the species studied, and found the frequency of host shifts to differ greatly between the two major lineages of Epilachnini examined.

Effects of low temperature on ploidy levels in parthenogenetic and sexual forms of the weevil Scepticus insularis (Coleoptera: Curculionidae).

We examined whether the ploidy level of eggs from sexual and parthenogenetic females of the weevil Scepticus insularis changes when field-collected, egg-bearing females are exposed to low temperature, as suggested by a previous study. We observed no change in ploidy level in eggs laid by sexual females (n = 15) treated by low temperature (1.1-6.7°C). In contrast, eggs laid by parthenogenetic females were not stable in ploidy level, as 11 of 16 females tested laid both diploid and triploid eggs even before the low-temperature treatment. After the low-temperature treatment, the proportion of triploid eggs to total eggs increased in nine individuals and decreased in the rest, and the effect of the treatment on the overall change in frequency was significant. Our results thus show that exposure to cold does not induce a change in egg ploidy in the sexual form of S. insularis, although cold may affect ploidy levels in the eggs of parthenogens. Additionally, eggs laid by laboratory-reared, virgin sexual females (n = 13) did not hatch after the low-temperature treatment, indicating that the treatment did not induce parthenogenetic reproduction in normally sexually reproducing females of S. insularis. We also examined the effect of low temperature on the ploidy level of eggs from parthenogenetic females (n = 4) of another weevil species, Catapionus gracilicornis, and confirmed that the proportion of triploid eggs steeply decreased and that of diploid eggs increased after exposure to cold, being consistent with those of previous studies.

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.

Fecundity of parthenogenetic and sexual forms of the flightless weevil Scepticus insularis (Coleoptera: Curculionidae) with and without effects from mating.

We measured the fecundity (number of eggs laid and number of eggs hatched) of the Parthenogenese and sexual forms of the weevil Scepticus insularis (Roelofs) with and without effects from mating, using one sexual and two parthenogenetic populations (one of the latter sympatric and the other allopatric with sexuals). When mated sexuals and unmated parthenogens were compared, the average number of eggs laid per female was largest for allopatric parthenogens (274 eggs), followed by sympatric parthenogens (223), and smallest for sexuals (169), with a significant difference only between allopatric parthenogens and sexuals (P < 0.05). The average number of eggs hatched per female was largest for allopatric parthenogens (136 hatchlings), followed by sexuals (119), and smallest for sympatric parthenogens (59), with a significant difference only between allopatric and sympatric parthenogens. In addition, mating significantly increased the reproductive output of sympatric parthenogens (59 vs. 188 hatchlings per female) by increasing egg productivity and hatching rate, but not that of allopatric parthenogens (136 vs. 133). The productivity of parthenogens of S. insularis is thus nearly equal to (in unmated sympatric parthenogens) or much higher (in other categories of parthenogens) than that of conspecific sexuals at the start of the larval stage, provided that the sex ratio of sexuals is 0.5 and all else being equal. Our results further showed that fecundity and other important aspects of the demography of parthenogenetic females may vary locally, or depending on whether they are sympatric or allopatric with sexuals.

Geographic distributions of sexual and parthenogenetic forms of the weevil Scepticus insularis (Coleoptera: Curculionidae) in Japan.

Geographic ranges of parthenogenetic animals often differ from those of their sexual conspecifics, providing valuable information about evolutionary histories. We studied the geographic distributions of sexual and parthenogenetic forms of the weevil Scepticus insularis (Roelofs) (Coleoptera, Curculionidae) on Honshu, Shikoku, and Kyushu Islands, Japan. We collected Scepticus insularis at 18 sites in the northern half of Honshu; sequenced 633 bp of the mitochondrial ND2 (NADH dehydrogenase subunit 2) gene for 102 Honshu beetles; and constructed a ML gene tree based on these sequences and sequences previously reported for 104 conspecific beetles from Hokkaido of known reproductive mode. The ND2 haplotypes fell into two distinct clades (I and II) separated by 9.5% nucleotide sequence divergence. The haplotypes from all parthenogenetic females on Hokkaido and those from all-female populations on Honshu invariably belonged to clade I, whereas sexual females on Hokkaido and some of the females in male/female populations on Honshu belonged to clade II. Haplotypes of males were in clade II, except for two Hokkaido males having a clade-I haplotype. Excluding these two enigmatic males, we treated individuals with clade-I haplotypes as parthenogenetic, and those with clade-II haplotypes as sexual. Parthenogenetic beetles were widespread throughout Hokkaido and central and northern Honshu, whereas sexuals were found only in three separate areas on Hokkaido and one on Honshu, often sympatrically with parthenogens. The distribution of S. insularis is congruent with the pattern termed geographic parthenogenesis.

Phylogenetic relationships among bisexual and unisexual lineages of the weevil Scepticus insularis (Coleoptera: Curculionidae) on Hokkaido, Northern Japan.

We examined the geographic distributions and phylogenetic relationships of bisexual and unisexual (parthenogenetic) forms of the weevil Scepticus insularis on Hokkaido Island, northern Japan. Unisexual beetles were widespread throughout Hokkaido, whereas bisexuals were found only in three remote areas. Bisexuals (females and males) and unisexual females occurred sympatrically in two areas. We determined nucleotide sequences for part of the mitochondrial NADH dehydrogenase subunit 2 (ND2; 633 bp) gene for 104 individuals, and for part of the nuclear internal transcribed spacer region 2 (ITS2; 360 bp) for 91 individuals. In an ND2 gene tree, haplotypes of S. insularis fell into two distinct clades (A and B), which were genetically differentiated from one another by 9.1% nucleotide sequence divergence. Haplotypes of females identified as unisexual were all in clade A, whereas those of females identified as bisexual belonged to clade B. Haplotypes of males were in clade B, except for two males having a clade-A haplotype. Circumstantial evidence suggests that these two males were produced by unisexual females. The ND2 tree suggests that the current unisexual form of S. insularis on Hokkaido was of a single origin. In contrast, a gene tree for ITS2 haplotypes show no clear divergence between the two modes of reproduction, with two major haplotypes shared by unisexual females, bisexual females, and males. This incongruence between the nuclear and mitochondrial phylogenies may be attributable to occasional gene flow between the unisexual and bisexual lineages through males occasionally produced by unisexual females, but our results do not exclude the possibility that the two lineages share polymorphic ancestral ITS2 haplotypes.

Population structure of Aegialites beetles (Coleoptera, Salpingidae) on the coasts of Hokkaido, northern Japan.

We conducted a molecular phylogeographic analysis of Aegialites beetles sampled on the coasts of Hokkaido, northern Japan, using a partial sequence (553 bp) of the mitochondrial ND2 gene. The beetles are flightless and are adapted to the supralittoral zone of rocky shores. In all, 249 individuals from 17 populations were included in the molecular analysis. Haplotypes of the Aegialites beetles studied comprised two distinct lineages with different distributional ranges: one (lineage A) on the coasts of Hokkaido facing the Sea of Japan and the Sea of Okhotsk, and the other (lineage B) mostly on the Pacific coast and on the Shiretoko Peninsula in northeastern Hokkaido. These haplotype lineages clearly indicate two groups of populations, one comprising lineage A haplotypes (group A) and the other lineage B haplotypes (group B). Only one population, at Taisei facing the Sea of Japan on the Oshima Peninsula, contained a mixture of haplotypes from the two lineages. Genetic divergence among populations was larger in group B than in group A. Despite the unidimensional nature of the beetles' habitat, no correlation was found between the degree of genetic differentiation and geographic distance in either group of populations. Population genetic parameters suggested that the two lineages originated in the Pliocene, and that the populations in group A underwent a rapid increase in population size, whereas those in group B did not. We present a scenario for the allopatric divergence of the two lineages.

Effects of straits as dispersal barriers for the flightless roving carrion beetle, Silpha perforata (Coleoptera, Silphidae, Silphinae).

Population genetic analyses were conducted for the flightless beetle Silpha perforata in northern Japan to evaluate effects of straits as dispersal barriers. In all, 19 populations on the Hokkaido mainland and one population on each of seven adjacent islands were included in the study. Based on partial sequences (853 bp) of the mitochondrial ND2 gene from 365 individuals, we calculated haplotype diversity (h), nucleotide diversity (pi), Tajima's D, and Fu's F(s) statistics for each population. The genetic distance d(A) and fixation index F(ST) were then estimated between populations, excluding two mainland and two island populations with small sample sizes. The results showed that four island populations (Rebun, Rishiri, Yagishiri, and Teuri) were genetically diverged and isolated from mainland populations, including the closest ones, consistent with the assumption that the straits separating these islands from the mainland have functioned as strong barriers against beetle dispersal. However, some factors other than sea barriers may have resulted in the observed divergence of these island populations, since the straits might be too young to permit such divergence (all are estimated to have been finally established approximately 11,000 to 13,000 years BP). On the other hand, the population on Okushiri Island showed little genetic divergence from the nearest mainland population, suggesting current overseas dispersal across the strait (estimated to have been formed more than 200,000 years BP). The present study thus demonstrated that the effects of straits on dispersal can be diverse, even for species of terrestrial invertebrates with limited dispersal ability.

Reproductive isolation between 2 karyotypes in natural populations of the leaf beetle Chrysolina aurichalcea.

Japanese populations of the leaf beetle, Chrysolina aurichalcea, have 2 main karyotypes, Type 31 and Type 41, which greatly differ in chromosome number and form. We examined their geographical distribution and reproductive isolation in southern Hokkaido, northern Japan. Type 41 was widely distributed in the survey area, whereas Type 31 was restricted to a narrow portion. A transect across each karyotypic habitat revealed that both karyotypes co-occurred in a approximately 5 km wide zone, with few hybrids or hybrid derivatives. Assessment of the fine scale distribution showed that the 2 karyotypes do not occupy separate microhabitats within the sympatric zone. Karyotype combinations of copulating pairs in the sympatric zone showed no evidence of assortative mating according to karyotype. These findings suggest that postmating isolation between the 2 karyotypes is strong, whereas premating isolation is weak. It does not agree with results from previous studies, which reported a substantial degree of both post- and premating isolation between the karyotypes. Possible causes of the discrepancy in premating isolation are discussed.

Contribution of multiple isolating barriers to reproductive isolation between a pair of phytophagous ladybird beetles.

Reproductive isolation between species may often be attained by multiple isolating barriers, but the components are rarely studied in animal taxa. To elucidate the nature of multiple isolating barriers, we quantified the strength of three premating barriers, including ecologically based ones (seasonal, habitat, and sexual), two postmating-prehatching barriers (reduced egg hatchability and conspecific sperm precedence [CSP]), and one posthatching barrier, including four components of F(1) hybrid reduced fitness, between two phytophagous ladybird beetles, Henosepilachna vigintioctomaculata and H. pustulosa. We detected five positive barriers (habitat isolation, sexual isolation, reduced egg hatchability, CSP, and reduced egg hatchability in backcrosses of F(1) hybrids). None of these barriers entirely prevents gene exchange when it acts alone, but jointly they generate nearly complete reproductive isolation even between sympatric populations. Host fidelity contributed most strongly to reproductive isolation by reducing interspecific hybridization through several important types of ecological isolation, including microspatial, habitat, and seasonal isolation. The existence of multiple isolating barriers likely helps keep reproductive isolation stable and robust, by complementing changes in the strength of leaky barriers. This complementarity of multiple isolating barriers yields the concept of robustness of reproductive isolation, which is important when considering the long-term maintenance of species boundaries in coexisting species pairs.

Round-trip catadromous migration in a Japanese amphipod, Sternomoera rhyaca (Gammaridea: Eusiridae).

We conducted a field study of the life cycle of the eusirid gammaridean amphipod Sternomoera rhyaca Kuribayashi, Mawatari, and Ishimaru, 1996 in a stream at Gokibiru, Hokkaido, Japan over the course of two non-consecutive years. This species is biennial; it spends most of its life in freshwater, but undertakes a short catadromous migration to the sea for reproduction. Reproduction occurs from March-June. Mature adults drift downstream to the sea singly and in precopulating pairs. Copulation and oviposition in the marsupium occur in mixed water at the stream mouth. Males die after copulation; ovigerous females return upstream by walking or swimming, where their eggs develop and hatch, after which the females also die. Juveniles remain in the stream, growing until they reach sexual maturity. Laboratory experiments showed that survivorship of all stages was lowest in seawater and highest in freshwater, though juveniles survived equally well in mixed water (50% seawater) and freshwater. Eggs developed to hatching only in freshwater; hatchlings in seawater and mixed water died within one and 21 days, respectively. Thus, S. rhyaca is well adapted to freshwater. Indeed, the only stages that required elevated salinity were copulation and subsequent oviposition, and we speculate that freshwater inhibits the female pre-reproductive molt. Because the life cycle of S. rhyaca has the most ontogenetically and temporally restricted saltwater phase known in any catadromous animal, its origin and maintenance are of evolutionary interest. We discuss two alternative hypotheses for the origin of the migratory life cycle, and discuss its maintenance in terms of fitness costs and benefits.

REPRODUCTIVE ISOLATION BY HOST SPECIFICITY IN A PAIR OF PHYTOPHAGOUS LADYBIRD BEETLES.

Crossing experiments and food-choice tests show that two sympatric species of phytophagous ladybird beetles, Epilachna niponica and E. yasutomii, are reproductively isolated by host-plant specificity. Adult beetles selected their natural hosts when given choices, though some accepted the host of the other species when no choice was offered. In each species, survival of larvae to the second instar was significantly higher on their own host plant. No evidence for sexual isolation, gametic isolation, hybrid inviability, or reduced hybrid fertility was detected. Reproductive isolation by host specificity is an important prerequisite for certain models of sympatric speciation. Although the present example supports the plausibility of such models, an allopatric origin of host-plant specificity cannot be discounted.