Development of microsatellite markers for the geographically parthenogenetic stick insect Phraortes elongatus (Insecta: Phasmatodea).

Many plant and animal species exhibit geographic parthenogenesis, wherein unisexual (= parthenogenetic) lineages are more common in their marginal habitats such as high latitude or altitudes than their closely related bisexual counterparts. The Japanese stick insect, Phraortes elongatus (Thunberg) (Insecta: Phasmatodea), is known as a geographically parthenogenetic species due to the existence of both bisexual and unisexual populations. Here, we developed microsatellite markers to infer the genetic variation among populations of P. elongatus. Totally, 13 primer pairs were developed for the species, and they were tested on 47 samples collected from both a bisexual population and a unisexual population. All 13 loci were polymorphic in the bisexual population, whereas no loci were polymorphic in the unisexual population. The loss of variation in the unisexual population implies automixis with terminal fusion or gamete duplication as the mode of parthenogenesis. The markers developed in this study will be helpful for further comprehensive analysis of the genetic diversity and gene flow between bisexual and parthenogenetic lineages of P. elongatus.

Intracellular localization of histone deacetylase HDA6 in plants.

Histone modification is an important epigenetic mechanism in eukaryotes. Histone acetyltransferase and deacetylase regulate histone acetylation levels antagonistically, leading to dynamic control of chromatin structure. One of the histone deacetylases, HDA6, is involved in gene silencing in the heterochromatin regions, chromocenter formation, and metabolic adaptation under drought stress. Although HDA6 plays an important role in chromatin control and response to drought stress, its intracellular localization has not been observed in detail. In this paper, we generated transformants expressing HDA6-GFP in the model plant, Arabidopsis thaliana, and the crops, rice, and cassava. We observed the localization of the fusion protein and showed that HDA6-GFP was expressed in the whole root and localized at the nucleus in Arabidopsis, rice, and cassava. Remarkably, HDA6-GFP clearly formed speckles that were actively colocalized with chromocenters in Arabidopsis root meristem. In contrast, such speckles were unlikely to be formed in rice or cassava. Because AtHDA6 directly binds to the acetate synthesis genes, which function in drought tolerance, we performed live imaging analyses to examine the cellular dynamics of pH in roots and the subnuclear dynamics of AtHDA6 responding to acetic acid treatment. The number of HDA6 speckles increased during drought stress, suggesting a role in contributing to drought stress tolerance.

Endangered island endemic plants have vulnerable genomes.

Loss of genetic diversity is known to decrease the fitness of species and is a critical factor that increases extinction risk. However, there is little evidence for higher vulnerability and extinction risk in endangered species based on genomic differences between endangered and non-endangered species. This is true even in the case of functional loci, which are more likely to relate to the fitness of species than neutral loci. Here, we compared the genome-wide genetic diversity, proportion of duplicated genes (PD), and accumulation of deleterious variations of endangered island endemic (EIE) plants from four genera with those of their non-endangered (NE) widespread congeners. We focused on exhaustive sequences of expressed genes obtained by RNA sequencing. Most EIE species exhibited significantly lower genetic diversity and PD than NE species. Additionally, all endangered species accumulated deleterious variations. Our findings provide new insights into the genomic traits of EIE species.

Development of microsatellite markers for an endangered fern in the Ryukyus, Plagiogyria koidzumii (Plagiogyriaceae).

We developed 10 microsatellite markers for Plagiogyria koidzumii, a critically endangered fern species found on Iriomotejima Island in the Ryukyu Archipelago, Japan and in Taiwan. These markers showed polymorphism among 65 wild individuals from Iriomotejima Island; the number of alleles per locus was 2-14, and mean observed and expected heterozygosity in the largest population were 0.276 and 0.277, respectively. A genetic structure analysis using these markers indicated clear genetic differentiation even within the narrow geographic range (ca. 10 × 8 km) on Iriomotejima Island. These microsatellite markers should be valuable for measuring genetic diversity and comparing genetic structure within and between populations.

Taxonomic reassessment of two subspecies of Chinese skink in Taiwan based on morphological and molecular investigations (Squamata, Scincidae).

The Chinese skink, Plestiodon chinensis (Gray, 1838), is widely distributed across continental China, Taiwan, the Korean Peninsula, and offshore islets, and consists of several subspecies. Here morphological and molecular methods have been used to reassess the taxonomic status and distributions of P. c. formosensis (Van Denburgh, 1912) and P. c. leucostictus (Hikida, 1988), which are endemic to Taiwan and Green Island (an islet off the east coast of Taiwan), respectively. It can be confirmed that the eastern Taiwanese populations of P. c. formosensis exhibit similar juvenile color patterning and genetic composition to the islet subspecies P. c. leucostictus, and are distinct from consubspecific populations in western Taiwan. Therefore, the eastern Taiwanese populations are assigned to P. c. leucostictus, and this subspecies is recognized as a distinct species, Plestiodon leucostictus (Hikida, 1988), based on their unique juvenile coloration and highly divergent DNA sequences. Our results also revealed that P. c. formosensis in western Taiwan is close to nominotypical subspecies from the continent, suggesting the necessity of a comprehensive taxonomic analysis in the future.

Genetic interaction of DISC1 and Neurexin in the development of fruit fly glutamatergic synapses.

Originally identified at the breakpoint of a (1;11)(q42.1; q14.3) chromosomal translocation in a Scottish family with a wide range of mental disorders, the DISC1 gene has been a focus of intensive investigations as an entry point to study the molecular mechanisms of diverse mental dysfunctions. Perturbations of the DISC1 functions lead to behavioral changes in animal models, which are relevant to psychiatric conditions in patients. In this work, we have expressed the human DISC1 gene in the fruit fly (Drosophila melanogaster) and performed a genetic screening for the mutations of psychiatric risk genes that cause modifications of DISC1 synaptic phenotypes at the neuromuscular junction. We found that DISC1 interacts with dnrx1, the Drosophila homolog of the human Neurexin (NRXN1) gene, in the development of glutamatergic synapses. While overexpression of DISC1 suppressed the total bouton area on the target muscles and stimulated active zone density in wild-type background, a partial reduction of the dnrx1 activity negated the DISC1-mediated synaptic alterations. Likewise, overexpression of DISC1 stimulated the expression of a glutamate receptor component, DGLURIIA, in wild-type background but not in the dnrx1 heterozygous background. In addition, DISC1 caused mislocalization of Discs large, the Drosophila PSD-95 homolog, in the dnrx1 heterozygous background. Analyses with a series of domain deletions have revealed the importance of axonal localization of the DISC1 protein for efficient suppression of DNRX1 in synaptic boutons. These results thus suggest an intriguing converging mechanism controlled by the interaction of DISC1 and Neurexin in the developing glutamatergic synapses.

A new species of Plestiodon (Squamata: Scincidae) from the Senkaku Group, Ryukyu Archipelago, Japan.

A new scincid lizard, Plestiodon takarai sp. nov., is described from the Senkaku Group, Ryukyu Archipelago, Japan. The Plestiodon lizards in this island group have previously been identified as P. elegans because they all exhibit a patch of enlarged and irregularly arranged scales on the posterior surface of their femurs. However, detailed molecular analyses revealed that the Senkaku population, although closely related to P. elegans and other species in the P. latiscutatus species group, is substantially diverged from all other recognized species. Furthermore, although the Senkaku population largely exhibits the characteristic morphological features of this species group, it can be differentiated from all recognized species by the scutellation and hatchling tail coloration. The biogeographical and conservation implications of these findings are briefly discussed.

Live imaging of H3K9 acetylation in plant cells.

Proper regulation of histone acetylation is important in development and cellular responses to environmental stimuli. However, the dynamics of histone acetylation at the single-cell level remains poorly understood. Here we established a transgenic plant cell line to track histone H3 lysine 9 acetylation (H3K9ac) with a modification-specific intracellular antibody (mintbody). The H3K9ac-specific mintbody fused to the enhanced green fluorescent protein (H3K9ac-mintbody-GFP) was introduced into tobacco BY-2 cells. We successfully demonstrated that H3K9ac-mintbody-GFP interacted with H3K9ac in vivo. The ratio of nuclear/cytoplasmic H3K9ac-mintbody-GFP detected in quantitative analysis reflected the endogenous H3K9ac levels. Under chemically induced hyperacetylation conditions with histone deacetylase inhibitors including trichostatin A, Ky-2 and Ky-14, significant enhancement of H3K9ac was detected by H3K9ac-mintbody-GFP dependent on the strength of inhibitors. Conversely, treatment with a histone acetyltransferase inhibitor, C646 caused a reduction in the nuclear to cytoplasmic ratio of H3K9ac-mintbody-GFP. Using this system, we assessed the environmental responses of H3K9ac and found that cold and salt stresses enhanced H3K9ac in tobacco BY-2 cells. In addition, a combination of H3K9ac-mintbody-GFP with 5-ethynyl-2'-deoxyuridine labelling confirmed that H3K9ac level is constant during interphase.

The role of ecological factors in determining phylogeographic and population genetic structure of two sympatric island skinks (Plestiodon kishinouyei and P. stimpsonii).

We conducted comparative phylogeographic and population genetic analyses of Plestiodon kishinouyei and P. stimpsonii, two sympatric skinks endemic to islands in the southern Ryukyus, to explore different factors that have influenced population structure. Previous phylogenetic studies using partial mitochondrial DNA indicate similar divergence times from their respective closest relatives, suggesting that differences in population structure are driven by intrinsic attributes of either species rather than the common set of extrinsic factors that both presumably have been exposed to throughout their history. In this study, analysis of mtDNA sequences and microsatellite polymorphism demonstrate contrasting patterns of phylogeography and population structure: P. kishinouyei exhibits a lower genetic variability and lower genetic differentiation among islands than P. stimpsonii, consistent with recent population expansion. However, historical demographic analyses indicate that the relatively high genetic uniformity in P. kishinouyei is not attributable to recent expansion. We detected significant isolation-by-distance patterns among P. kishinouyei populations on the land bridge islands, but not among P. stimpsonii populations occurring on those same islands. Our results suggest that P. kishinouyei populations have maintained gene flows across islands until recently, probably via ephemeral Quaternary land bridges. The lower genetic variability in P. kishinouyei may also indicate smaller effective population sizes on average than that of P. stimpsonii. We interpret these differences as a consequence of ecological divergence between the two species, primarily in trophic level and habitat preference.

An investigation for population maintenance mechanism in a miniature garden: genetic connectivity or independence of small islet populations of the Ryukyu five-lined skink.

The Ryukyu five-lined skink (Plestiodon marginatus) is an island lizard that is even found in tiny islets with less than half a hectare of habitat area. We hypothesized that the island populations are maintained under frequent gene flow among the islands or independent of each other. To test our hypotheses, we investigated genetic structure of 21 populations from 11 land-bridge islands that were connected during the latest glacial age, and 4 isolated islands. Analyses using mitochondrial cytochrome b gene sequence (n = 67) and 10 microsatellite loci (n = 235) revealed moderate to high levels of genetic differentiation, existence of many private alleles/haplotypes in most islands, little contemporary migration, a positive correlation between genetic variability and island area, and a negative correlation between relatedness and island area. These evidences suggest a strong effect of independent genetic drift as opposed to gene flow, favoring the isolation hypothesis even in tiny islet populations. Isolation-by-distance effect was demonstrated and it became more prominent when the 4 isolated islands were excluded, suggesting that the pattern is a remnant of the land-bridge age. In a few island populations, however, the possibility of occasional overwater dispersals was partially supported and therefore could not be ruled out.

A new species of Plestiodon (Squamata: Scincidae) from Kuchinoshima Island in the Tokara Group of the Northern Ryukyus, Japan.

A scincid lizard of the genus Plestiodon from Kuchinoshima Island in the Tokara Group of the Northern Ryukyus, Japan, has proved to be genetically and morphologically differentiated from any previously recognized species in the genus. We thus describe this island population as a new species, Plestiodon kuchinoshimensis. The new species shows characteristics of the P. latiscutatus species group, but differs from other species of this group by the combination of the following character states: postnasal absent; hatchling with five longitudinal light lines on dorsum; lateral light line on each side passing over ear opening and the sixth to eighth scale rows at midbody; dorsolateral light line beginning from behind supraoculars; patch of enlarged irregular scales on posterior femur absent; scale rows around midbody 27-32; and brownish background on the dorsal surface of the juvenile.

Divergence and long-distance overseas dispersals of island populations of the Ryukyu five-lined skink, Plestiodon marginatus (Scincidae: Squamata), in the Ryukyu Archipelago, Japan, as revealed by mitochondrial DNA phylogeography.

We assessed the historical biogeography of the Ryukyu five-lined skink, Plestiodon marginatus, and related species (P. stimpsonii and P. elegans). Our specific aims were to reveal the origin, tim- ing, and route of the colonization to three volcanic islands in the northern Tokara Group of the northern Ryukyus: Kuchinoshima, Nakanoshima, and Suwanosejima. We conducted phylogenetic analyses and divergence time estimation using a partial sequence of the mitochondrial cytochrome b gene for P. marginatus collected from across its whole range (the northern and central Ryukyus), and for P. stimpsonii (from the Yaeyama Group of the southern Ryukyus) and P. elegans (from Taiwan). Our results suggest three major clades (A, B, and C). Clades A and B consist of P. marginatus, excluding the Kuchinoshima population, and Clade C consisted of the Kuchinoshima population, P. stimpsonii, and P. elegans. These clades are estimated to have diverged during the Late Miocene to the Late Pliocene. Among the three examined northern Tokara populations, the Kuchinoshima population was shown to be a sister group of P. stimpsonii. The two other populations from Nakanoshima and Suwanosejima Islands were closely related to P. marginatus from the northern part of the Okinawa Group and that from Kodakarajima Island in the southern Tokara Group, respectively. These populations are estimated to have diverged from their respective related spe cies in various ages of the Early to Late Pleistocene, suggesting that they colonized the islands by independent overseas dispersals of approximately 50-850 km via the Kuroshio Current. Taxonomic implications for P. marginatus are also discussed.

Modelling the global distribution of fungal species: new insights into microbial cosmopolitanism.

Microbes are usually believed to have cosmopolitan distributions. However, for estimating the global distributions of microorganisms, discriminating among cryptic species and eliminating undersampling biases are important challenges. We used a novel approach to address these problems and infer the global distribution of a given fungal ecological guild. We collected mushroom-forming fungi from Yakushima, Japan. We sequenced the internal transcribed spacer 2 (ITS2) from these samples and queried their sequences against GenBank. After identifying similar sequences, we tracked down the geographical origins of samples that yielded those sequences. We used Bayesian zero-inflated models to allow for species whose DNA sequences have not yet been deposited in GenBank. Results indicated that the geographical distribution of ectomycorrhizal (ECM) fungi was strongly constrained by host specificity, resulting in the occurrence of these fungi intensively in the neighbouring regions. On the other hand, saprotrophic (SAP) fungi were less constrained by climatic conditions, resulting in a much broader distribution range. We inferred that differences in constraints during colonization between ECM and SAP fungi were responsible for the different geographical distribution ranges. We hypothesize that the degree of host/habitat specificity and the degree of isolation of potentially suitable habitats determine microbial biogeographic patterns.

Functional analysis of the role of eyes absent and sine oculis in the developing eye of the cricket Gryllus bimaculatus.

In the cricket Gryllus bimaculatus, a hemimetabolous insect, the compound eyes begin to form in the embryo and increase 5-6 fold in size during the postembryonic development of the nymphal stage. Retinal stem cells in the anteroventral proliferation zone (AVPZ) of the nymphal eye proliferate to increase retinal progenitors, which then differentiate to form new ommatidia in the anterior region of the eye. However, mechanisms underlying this type of eye formation have not been well elucidated yet. Here, we found that the homologues of the retinal determination transcription factor genes of eyes absent (eya) and sine oculis (so) are expressed during the cricket embryonic eye formation. eya is also expressed intensely in the AVPZ of the nymphal eye. To explore their functions, we performed knockdown by RNA interference (RNAi). Knockdown of Gb'eya resulted in loss of the embryonic eye. In the nymphal eye, RNAi against Gb'eya or Gb'so impaired retinal morphology by apparently transforming cornea structures into head cuticle. These results imply that Gb'eya and Gb'so are essential for the differentiation of the retinal progenitor cells and maintaining retinal structures during eye development.

Ancestral functions of Delta/Notch signaling in the formation of body and leg segments in the cricket Gryllus bimaculatus.

Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, we analyzed the function of the Delta (Gb'Delta) and Notch (Gb'Notch) genes in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. We found that Gb'Delta and Gb'Notch were expressed in developing legs, and that RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. Our results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, we found that Gb'Delta was expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries.

Lowfat, a mammalian Lix1 homologue, regulates leg size and growth under the Dachsous/Fat signaling pathway during tissue regeneration.

In the cricket Gryllus bimaculatus, missing distal parts of amputated legs are regenerated from blastemas based on positional information. The Dachsous/Fat (Ds/Ft) signaling pathway regulates blastema cell proliferation and positional information along the longitudinal axis during leg regeneration. Herein, we show that the Gryllus homologue of Lowfat (Gb'Lft), which modulates Ds/Ft signaling in Drosophila, is involved in leg regeneration. Gb'lft is expressed in regenerating legs, and RNAi against Gb'lft (Gb'lft(RNAi)) suppressed blastema cell hyperproliferation caused by Gb'ft(RNAi) or Gb'ds(RNAi) but enhanced that caused by Gb'kibra(RNAi) or Gb'warts(RNAi). In Gb'lft(RNAi) nymphs, missing parts of amputated legs were regenerated, but the length of the regenerated legs was shortened depending on the position of the amputation. Both normal and reversed intercalary regeneration occurred in Gb'lft(RNAi) nymphs, suggesting that Gb'Lft is involved in blastema cell proliferation and longitudinal leg regeneration under the Ds/Ft signaling pathway, but it is not required for intercalary regeneration.