Sperm mortality, insemination and fertilization in the damselfly Ischnura senegalensis: comparisons between wild and inbred populations

Inbreeding can have deleterious effects on individual or population fitness. To avoid fitness reduction, individuals may adopt behavioral or physiological mechanisms to reduce their investment in the production of offspring with genetically similar mates. We examined whether insemination by inbred males introduced more dead sperm than insemination by wild males by counting sperm in female Ischnura senegalensis (Rambur) sperm storage organs. If inbred males inseminated fewer or lower-quality sperm, females would avoid inferior sperm. Our results revealed three features of damselfly inbreeding: insemination failed in a larger proportion of inbred pairs than in wild pairs, inbred pairs showed significantly reduced fertility, and the numbers of live and dead sperm in an inbred female’s sperm storage organs did not differ from those in wild females. These results suggested that neither sperm quantity nor sperm quality was responsible for low fertility to a significant extent, but some kind of female quality, such as sperm usage or storing ability, was. Although inbred pairs had lower fertility, there were no significant differences between inbred and wild pairs in the total numbers of live or dead sperm. It thus seemed that female choice at the insemination stage was responsible for low fertility rather than sperm quantity or quality measured by live-to-dead ratio.     

Isolation and validation of eight microsatellite loci in a damselfly Ischnura senegalensis (Odonata: Coenagrionidae) by pyrosequencing a bead-enriched library

Eight microsatellite markers for the population genetics and evolutionary ecology of Ischnura senegalensis (Rambur 1842), which shows body color polymorphism in females, were developed using a streptavidin-bead enrichment library and pyrosequence by a next generation sequencer. The number of alleles per locus and effective number of alleles ranged from 3 to 11 and from 1.24 to 5.51, respectively. Observed and expected heterozygosities were 0.18–0.75 and 0.19–0.77, respectively. No linkage disequilibrium between loci was detected. One locus, IsenAC75, deviated significantly from the Hardy–Weinberg equilibrium, and the locus and additional two loci, IsenAC40 and IsenAC8, were suspected for the presence of null alleles. Altogether, these eight microsatellite loci are considered to be useful for population genetic analyses because of the high polymorphic status and independency.     

Differentiation between subpopulations of a polychromatic damselfly with respect to morph frequencies, but not neutral genetic markers

The damselfly, Nehalennia irene (Hagen), has two distinct female colour morphs. Individuals of one morph have male-like colouration and pattern (androchromes), whereas gynochromes are different from males and androchromes in these respects. In several damselflies, such female-limited polychromatism is attributable to a single genetic locus. We developed six polymorphic genetic markers, which were codominant, to test for genetic differentiation in N. irene, collected from two sites located 8 km from one another in eastern Ontario, Canada. Based on three censuses spanning a 10 year period (1992-2001), morph ratios differed consistently and significantly between these two sites. However, subpopulations at these sites were not genetically differentiated with respect to the putatively neutral markers. Our results suggest that site differences in morph ratios of female N. irene cannot be explained by genetic drift, but are consistent with spatially variable selection operating on different morphs, perhaps mediated by male density. Alternatively, morph type may be a plastic trait and cues for induction may differ between sites.     

Time constraints related to sexual maturation and prolonged copulation in the female‐dimorphic damselfly Ischnura senegalensis

Time constraints are critical for reproductive success. To understand the spatiotemporal dynamics of morph frequency in the female‐dimorphic damselfly Ischnura senegalensis, we compared two different morphs for two important time constraints on female reproductive output, i.e. post‐emergence sexual maturation and prolonged copulation. The females of both morphs achieved sexual maturation 4–5 days after emergence, suggesting that the rate of sexual mutation does not result in morph‐specific fitness. The copulation durations declined with the time of onset of copulation in both morphs. Consequently, all copulations terminated at approximately 12:00 hours. Because females show foraging and oviposition activity only after copulation, the copulation duration does not result in morph‐specific time constraints. These two important time constraints do not account for morph‐specific reproductive success and do not affect the evolutionary equilibrium of morph frequency in I. senegalensis.     

Genome assembly,sex-biased gene expression and dosage compensation in the damselfly Ischnura elegans

The evolution of sex chromosomes, and patterns of sex-biased gene expression and dosage compensation, are poorly known among early winged insects such as odonates. We assembled and annotated the genome of Ischnura elegans (blue-tailed damselfly), which, like other odonates, has a male-hemigametic sex-determining system (X0 males, XX females). By identifying X-linked genes in I. elegans and their orthologs in other insect genomes, we found homologies between the X chromosome in odonates and chromosomes of other orders, including the X chromosome in Coleoptera. Next, we showed balanced expression of X-linked genes between sexes in adult I. elegans, i.e. evidence of dosage compensation. Finally, among the genes in the sex-determining pathway only fruitless was found to be X-linked, while only doublesex showed sex-biased expression. This study reveals partly conserved sex chromosome synteny and independent evolution of dosage compensation among insect orders separated by several hundred million years of evolutionary history.     

Difference in seasonal activity pattern between non-native and native ants in subtropical forest of Okinawa Island,Japan

Biological invasions by non-native tramp ants are reported throughout the world, particularly in island ecosystems. In Okinawa Island, a subtropical island in south-western Japan, many tramp ants including the invasive ant, Anoplolepis gracilipes, already occur. In this study activity patterns of the ground-foraging tramp and native ants were investigated for one year by using food bait traps at the forest edges where both categories of ants coexist. On a seasonal time scale, activity patterns were different between exotic and native ants. Native ants are active and seemingly more dominant from spring to summer whereas tramp ants become more dominant from autumn to winter. These results suggest there might be temporal niche partitioning between tramp and native ants, and native ants might be able to dominate tramp ants in their suitable seasons.     

Patterns of differentiation in a colour polymorphism and in neutral markers reveal rapid genetic changes in natural damselfly populations

The existence and mode of selection operating on heritable adaptive traits can be inferred by comparing population differentiation in neutral genetic variation between populations (often using F(ST) values) with the corresponding estimates for adaptive traits. Such comparisons indicate if selection acts in a diversifying way between populations, in which case differentiation in selected traits is expected to exceed differentiation in neutral markers [F(ST )(selected) > F(ST )(neutral)], or if negative frequency-dependent selection maintains genetic polymorphisms and pulls populations towards a common stable equilibrium [F(ST) (selected) < F(ST) (neutral)]. Here, we compared F(ST) values for putatively neutral data (obtained using amplified fragment length polymorphism) with estimates of differentiation in morph frequencies in the colour-polymorphic damselfly Ischnura elegans. We found that in the first year (2000), population differentiation in morph frequencies was significantly greater than differentiation in neutral loci, while in 2002 (only 2 years and 2 generations later), population differentiation in morph frequencies had decreased to a level significantly lower than differentiation in neutral loci. Genetic drift as an explanation for population differentiation in morph frequencies could thus be rejected in both years. These results indicate that the type and/or strength of selection on morph frequencies in this system can change substantially between years. We suggest that an approach to a common equilibrium morph frequency across all populations, driven by negative frequency-dependent selection, is the cause of these temporal changes. We conclude that inferences about selection obtained by comparing F(ST) values from neutral and adaptive genetic variation are most useful when spatial and temporal data are available from several populations and time points and when such information is combined with other ecological sources of data.     

Diurnal changes and frequency dependence in male mating preference for female morphs in the damselfly Ischnura senegalensis (Rambur) (Odonata: Coenagrionidae)

Ischnura senegalensis females exhibit color dimorphism, consisting of an andromorph and a gynomorph, which might be maintained under a frequency-dependent process of mating harassment by mate-searching males. Males change their mating preference for female morph depending on prior copulation experience. Binary choice experiments between two female morphs were carried out in four local populations in the early morning (07.00–09.00 hours) and the afternoon (12.00–14.00 hours), times which mark the onset and the end of diurnal mating activity, respectively. According to the line census along the water's edge, the proportion of andromorphs in the female population varied from 21 to 67% throughout the survey period for four local populations. Males showed non-biased preference for female morphs in the early morning in each local population, while they chose the common morph in the afternoon. Male mating preference for female morphs was positively correlated to the proportion of female morphs in the population. If the selective mating attacks on the common female morphs inhibit their foraging and/or oviposition behavior, frequency-dependent male mating attacks might provide a selective force for maintaining the female color dimorphism in I. senegalensis .     

An analysis of mutualistic interactions between exotic ants and honeydew producers in the Yanbaru district of Okinawa Island,Japan

Invasive exotic ants often have a mutualistic relationship with other insects excreting honeydew, and this is considered to play a key role in their invasion success. We investigated the multispecies association patterns between ants and hemipteran insects in the Yanbaru forests, Okinawa, Japan, an Asian biodiversity hotspot. We especially focused on roadside environments, which are the frontlines of invasion for exotic ants. We found that only a small number of herbaceous and pioneer plants were predominant on the roadsides. Four honeydew producers, Melanaphis formosana, Dysmicoccus sp. A, Heteropsylla cubana, and Sogata hakonensis, living on these roadside plants accounted for 94.9% of the total honeydew-producer aggregations observed. Only a few exotic ants, such as Technomyrmex brunneus and Anoplolepis gracilipes, were observed with these honeydew-producer aggregations, and densities of these ants and honeydew producers were often positively correlated. An ant exclusion experiment showed that exotic ant occurrence improved the survival of some of the hemipteran colonies. Interestingly, the abundance of native ants was not correlated with the abundance of honeydew producers, and the local density of Pheidole noda was negatively correlated with that of M. formosana. These findings, i.e., only a few ants, all exotic, tended to hemipteran honeydew producers despite the existence of many native ants, and the abundances of those exotic ants and those hemipteran insects had positive correlations, provide some insights into the mechanism of biological invasion and provide information for the management of exotic ants.     

Simulating allele frequencies in a population and the genetic differentiation of populations under mutation pressure

A method is developed for simulating the allele frequencies in an equilibrium or transient population under the effects of neutral mutation and random drift. The method is based on diffusion theory and is fast so that it can be used to study in detail the distribution of heterozygosity or any quantity that can be expressed as a function of allele frequencies. It has been applied to study the distribution of heterozygosity and the distributions of the frequencies of the first three most frequent alleles in a population. It also has been applied to study the distribution of the number of alleles shared by two populations that were derived from a common stock.     

Mixed support for an alignment between phenotypic plasticity and genetic differentiation in damselfly wing shape

The relationship between genetic differentiation and phenotypic plasticity can provide information on whether plasticity generally facilitates or hinders adaptation to environmental change. Here, we studied wing shape variation in a damselfly (Lestes sponsa) across a latitudinal gradient in Europe that differed in time constraints mediated by photoperiod and temperature. We reared damselflies from northern and southern populations in the laboratory using a reciprocal transplant experiment that simulated time-constrained (i.e. northern) and unconstrained (southern) photoperiods and temperatures. After emergence, adult wing shape was analysed using geometric morphometrics. Wings from individuals in the northern and southern populations differed significantly in shape when animals were reared in their respective native environment. Comparing wing shape across environments, we found evidence for phenotypic plasticity in wing shape, and this response differed across populations (i.e. G × E interactions). This interaction was driven by a stronger plastic response by individuals from the northern population and differences in the direction of plastic wing shape changes among populations. The alignment between genetic and plastic responses depended on the specific combination of population and rearing environment. For example, there was an alignment between plasticity and genetic differentiation under time-constrained, but not under non-time-constrained conditions for forewings. We thus find mixed support for the hypothesis that environmental plasticity and genetic population differentiation are aligned. Furthermore, although our laboratory treatments mimicked the natural climatic conditions at northern and southern latitudes, the effects of population differences on wing shape were two to four times stronger than plastic effects. We discuss our results in terms of time constraints and the possibility that natural and sexual selection is acting differently on fore- and hindwings.     

Interspecific crossing between blue‐tailed damselflies Ischnura elegans and I. senegalensis in the laboratory

Ischnura species (Odonata) are among the most common damselflies in the world, which often exhibit female color polymorphisms. One morph, called androchrome, is similar to males in its color pattern, whereas the other morphs, generally referred to as gynochromes, exhibit female‐specific colors. In several Ischnura species, the female polymorphism is heritable, although molecular and genetic mechanisms remain largely unknown. The dominant‐recessive patterns of the female color morphs may differ between species. For example, androchromic females are dominant to gynochromic females in Ischnura elegans, whereas androchromic females are recessive in Ischnura senegalensis. Here we report a case of interspecific hybridization between a gynochrome female of I. elegans and a male of I. senegalensis in the laboratory. We obtained 61 hybrid adult offspring, of which all 31 females were of gynochrome morph. DNA analyses of the hybrids confirmed that nuclear DNA sequences were derived from both parent species, whereas mitochondrial DNA sequences were maternally inherited. In the hybrids, the postocular spots of female heads, the shape of male appendages, and the color of female's cerci resembled those of I. elegans, whereas the size of abdominal blue spots was similar to that of I. senegalensis. The shape of prothorax and basal abdominal markings were intermediate in females. The larval developmental traits and the morphological changes in the final larval instar of the hybrids were similar to those of I. senegalensis. To our knowledge, this is the first report of hybrids between two damselfly species with different dominant‐recessive patterns of female color morphs.     

Habitats of larger foraminifera on the upper reef slope of Sesoko Island,Okinawa, Japan

Larger foraminifera living in the upper 50 m in front of the fringing coral reef northwest off Sesoko Island, Japan show strong habitat differences. This study closely examines the distributions of larger foraminifers and relates these to a number of key environmental factors using rigorous statistical methods. Since all larger foraminifera house symbiotic algae, light attenuation by the water column is the most important limiting factor that must be dealt with wall structures. Water energy is also countered by test structure. The local topography is responsible for different intensities of hydrodynamic forces, which are expressed in various substrates, mostly coral rubble and coarse-grained sand. The genus Peneroplis, very common on the reef flat, clearly prefers hardgrounds of the shallowest slope parts down to 30 m, while Dendritina is restricted to sandy bottoms and avoids the uppermost meters of the slope. It can be found down to 50 m at least. Alveolinella shows a similar depth distribution to Dendritina, but is common on hard bottom. The distribution of Parasorites, which is restricted to sandy substrates, starts at 20 m and extends to 80 m. Sorites, on the other hand, was found only on firm substrates between the reef edge and 50 m. The same depth distribution was recorded for Amphisorus, but this genus is not correlated with specific substrates. Most of the Amphistegina species prefer hardgrounds, while Amphistegina radiata is also common on sand. The calcarinids, capable of withstanding high water energy, are abundant on firm substrates close to the reef edge. Only Baculogypsinoides inhabits deeper parts of the slope on sandy bottom and avoids the shallowest parts. Sections with hard substrates are settled by Heterostegina, even down to 80 m, although this genus was occasionally found on sandy bottoms. Nummulites, in contrast, is restricted to sands between 20 and 70 m. Operculina, starting at 20 m, also prefers sandy substrates, but rare individuals were detected on coral rubble and macroids.     

Reproductive characteristics and gamete development of the soft coral Sclerophytum cf. heterospiculatum in Okinawa Island,Japan

Sexual reproduction data are important to understand how organisms can replenish their populations and proliferate on coral reefs. Despite the importance of such data, the reproductive characteristics of most soft coral species are still unknown. Here, we examined the reproductive strategies of a species from the often-dominant genus Sclerophytum in a coral reef on subtropical Okinawa Island, Japan. DNA barcoding and histological examinations of the tissues were conducted to confirm colony conspecificity and identify reproductive characteristics, respectively, between March 2020 and March 2021. Results indicated that the studied species, identified as Sclerophytum cf. heterospiculatum, exhibits gonochorism with longer oogenesis and shorter spermatogenesis. Female colonies produced immature oocytes throughout the year, with mature oocytes observed from late July to early September, and thus, extended spawning is likely characteristic of this species. In male colonies, spermatogenesis took place over ~5 months, with spermaries present from April through August. Mature spermaries were noted beginning in July and the inferred peak of sperm release was between late August and early September, which suggests that spermatogenesis duration was ~5 months. The largest mean oocyte and spermary sizes (628.45 ± 61.36 and 240.04 ± 49.49 μm, respectively) were both recorded in August. Gamete spawning presumably occurred during the summer season, which suggests seasonality in reproduction as influenced by changes in seawater temperature. However, the proximate cue for exact dates of spawning could be the lunar period because the inferred release of spawning materials seemed to occur between full moon and last-quarter moon phases in both the months of August and September. The results of this study represent the first detailed report of reproductive characteristics of the genus Sclerophytum in Japan.     

Secondary succession and effects of clear-logging on diversity in the subtropical forests on Okinawa Island,southern Japan

In order to clarify the recovery process of the subtropical forest on Okinawa Island, southern Japan, biomass accumulation and the successional trend of species diversity with time were investigated by comparing plots of old-growth and clear logged secondary forests. Self-thinning was an important factor in the development of young secondary forests, and the small variance in tree size within a stand was related to the stand not being fully stratified after clear-cutting. A large variance of size structure in old secondary and old-growth forests implies re-initiation of the understorey. Additionally, the trajectory of stand development indicated that the subtropical forest quickly recovered aboveground biomass, which reached its upper limit at about 50 years after disturbance. However, there was a large distinction in species diversity between the secondary forests and old-growth forests. The diversity of forest floor plants did not recover well after being clear-cut. This indicates that management of the subtropical forest should not only take timber-oriented tree species into account, but also the biodiversity in ground flora. The secondary forests were characterized by Castanopsis sieboldii and Schima wallichii, and the monopolization of C. sieboldii through secondary succession had a negative influence on species diversity. Distylium racemosum dominated at the late development stage and was considered a long-lived competing species that reduced the dominance of C. sieboldii and facilitated the co-occurrence of understorey species. Light-demanding pioneer tree species such as S. wallichii that regenerated after logging should be replaced by competitive effects of climax species, and thus relayed floristic change might increase species diversity along secondary succession.Nomenclature: Hatushima and Amano (1994).     

Heterogeneity in genetic diversity among non-coding loci fails to fit neutral coalescent models of population history

Inferring aspects of the population histories of species using coalescent analyses of non-coding nuclear DNA has grown in popularity. These inferences, such as divergence, gene flow, and changes in population size, assume that genetic data reflect simple population histories and neutral evolutionary processes. However, violating model assumptions can result in a poor fit between empirical data and the models. We sampled 22 nuclear intron sequences from at least 19 different chromosomes (a genomic transect) to test for deviations from selective neutrality in the gadwall (Anas strepera), a Holarctic duck. Nucleotide diversity among these loci varied by nearly two orders of magnitude (from 0.0004 to 0.029), and this heterogeneity could not be explained by differences in substitution rates alone. Using two different coalescent methods to infer models of population history and then simulating neutral genetic diversity under these models, we found that the observed among-locus heterogeneity in nucleotide diversity was significantly higher than expected for these simple models. Defining more complex models of population history demonstrated that a pre-divergence bottleneck was also unlikely to explain this heterogeneity. However, both selection and interspecific hybridization could account for the heterogeneity observed among loci. Regardless of the cause of the deviation, our results illustrate that violating key assumptions of coalescent models can mislead inferences of population history.     

Cellulolytic Microbes in the Yanbaru,a Subtropical Rainforest with an Endemic Biota on Okinawa Island,Japan

Cellulolytic microbes in the soil of the Yanbaru, a subtropical forest with an endemic biota, on Okinawa Island, were isolated and characterized in a search for novel microbial strains with biotechnological potential. Soil samples of the Yanbaru were suspended in sterilized water, inoculated on mineral salt agar overlaid with a filter paper as carbon source, and cultivated aerobically at 30 °C. After 2 weeks of cultivation, emerging colonies were isolated and subjected to phylogenetic and enzyme analyses. The phylogenetic analyses revealed bacterial and fungal isolates belonging to nine and three genera respectively. All isolates possessed cellulase activity, and several strains showed strong activity comparable to Trichoderma cellulase. Many isolates also exhibited xylanase activity.     

Cellulolytic microbes in the Yanbaru, a subtropical rainforest with an endemic biota on Okinawa Island, Japan

Cellulolytic microbes in the soil of the Yanbaru, a subtropical forest with an endemic biota, on Okinawa Island, were isolated and characterized in a search for novel microbial strains with biotechnological potential. Soil samples of the Yanbaru were suspended in sterilized water, inoculated on mineral salt agar overlaid with a filter paper as carbon source, and cultivated aerobically at 30 °C. After 2 weeks of cultivation, emerging colonies were isolated and subjected to phylogenetic and enzyme analyses. The phylogenetic analyses revealed bacterial and fungal isolates belonging to nine and three genera respectively. All isolates possessed cellulase activity, and several strains showed strong activity comparable to Trichoderma cellulase. Many isolates also exhibited xylanase activity.