Playing hide-and-seek with the tiny dragonfly: DNA barcoding discriminates multiple lineages of <Emphasis Type="Italic">Nannophya pygmaea</Emphasis> in Asia

We examined the utility of DNA barcode data for assessing genetic diversity of the tiny dragonfly Nannophya pygmaea Rambur in Asia. Data analyses inferred from the barcode region of cytochrome oxidase subunit I (COI) were performed with Malaysian N. pygmaea, along with the existing COI haplotypes distributed in Asia. We applied four species delimitation analyses [automatic barcode gap discovery (ABGD), generalised mixed yule coalescent (GMYC), poisson tree processes maximum likelihood (PTP_ML) and poisson tree processes simple heuristic solutions (PTP_sh)] to investigate potential lineages in this geographically widespread species. Based on our dataset, we provisionally recognize four distinct lineages or operational taxonomic units of N. pygmaea, which were represented by the taxa from Japan/Korea, China/Laos/Taiwan, Malaysia and Vietnam, respectively. Phylogenetic analyses showed two well-supported assemblages of N. pygmaea: one restricted to the taxa from Malaysia and Vietnam; and the other covering all populations further north (i.e., China, Japan, Korea, Laos and Taiwan). An extraordinarily high degree of genetic distance (up to >12 %) was detected between these two assemblages—suggesting they represent two separate species.     

Short and flat grass preferred by adults of the endangered dragonfly <Emphasis Type="Italic">Sympetrum pedemontanum elatum</Emphasis> (Odonata: Libellulidae)

Sympetrum pedemontanum (Müller in Allioni) (Odonata: Libellulidae) is widely distributed across the Eurasian continent and its neighboring islands. However, the populations of its subspecies S. pedemontanum elatum (Selys) in Japan have been rapidly decreasing with the loss of habitats in rural and suburban areas since the 1970s. For the conservation of this subspecies, which is now listed as endangered in many prefectures, it is important to understand the habitat preferences of the adults. Previous studies indicate that adult males of this species tend to fly on the flat surface of rice paddy fields. Thus, we hypothesized that they preferred short and flat grass. Field experiments in the Sakasegawa River, Hyogo Prefecture, Japan, showed that adult S. p. elatum significantly preferred the trimmed grass of Phragmites japonicus to untreated shaggy grass, regardless of sex. Our results indicate the importance of grass management for the conservation of this species, not only in and around paddy fields but also in fluvial habitats, which are abundant in Japan.     

Development of expressed sequence tag-derived microsatellite markers for <Emphasis Type="Italic">Saccharina</Emphasis> (<Emphasis Type="Italic">Laminaria</Emphasis>) <Emphasis Type="Italic">japonica</Emphasis>

Expressed sequence tag-derived microsatellite markers (EST-SSR) were generated and characterized in Laminaria japonica using data mining from updated public EST databases and polymorphism testing. Fifty-eight of 578 ESTs (10.0%) containing various repeat motifs were used to design polymerase chain reaction (PCR) amplification primers. A total of 12 pairs of primer were generated and used in the PCR amplification. Alleles per locus ranged from two to ten (average of 5.7). The observed heterozygosities and expected heterozygosities were from 0.045 to 0.543 and from 0.056 to 0.814, respectively. All loci were in Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. These robust, informative, and potentially transferable polymorphic markers appear suitable for population, genetic, parentage, and mapping studies of L. japonica.     

Isolation and characterization of microsatellite markers for the endangered <Emphasis Type="Italic">Taxus yunnanensis</Emphasis>

Eleven polymorphic microsatellite loci were characterized for the endangered conifer Taxus yunnanensis. Eight loci were isolated through SSR-anchored PCR, one locus was developed by cross-species amplification tests, while the last two loci were obtained from cross-species microsatellite sequences available in GenBank. Variability of these markers was tested in 48 individuals collected from its main distribution range in Southwest China. The number of alleles per locus ranged from 2 to 9, and the observed and expected heterozygosity varied from 0.000 to 0.625 and from 0.062 to 0.853, respectively. Ten of these eleven loci were significantly deviated from Hardy–Weinberg expectation, except TS07 which showed a distinct heterozygote excess. The availability of these new polymorphic microsatellite markers will provide an ideal marker system for detailed population genetics studies in T. yunnanensis and potentially also for closely related species.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Development of ten microsatellite markers for <Emphasis Type="Italic">Quercus mongolica</Emphasis> var. <Emphasis Type="Italic">crispula</Emphasis> by database mining

Simple sequence repeats (SSRs) in the NCBI dbEST database were surveyed to identify potential SSR markers for Quercus mongolica. In total, 2,691 gene sequences, mainly from expressed sequence tags (ESTs) for Q. robur and Q. petraea had been registered. Twenty-two PCR primers were designed for SSRs in these sequences and screened for polymorphisms in 16 Q. mongolica trees. Ten loci were easily genotyped and showed polymorphism, with numbers of alleles and expected heterozygosity ranging from 3 to 15 and 0.28 to 0.94, respectively. These EST-SSR markers should be useful for studying the genetic diversity of Quercus species.     

Isolation and characterization of microsatellite DNA markers for the flagfish <Emphasis Type="Italic">Jordanella </Emphasis><Emphasis Type="Italic">floridae</Emphasis>

The flagfish (Jordanella floridae) is commonly used in studies of wetlands ecology. Here we describe the isolation of ten microsatellite loci, six of which were polymorphic. The observed number of alleles ranged from 4 to 24 and observed heterozygosities ranged from 0.59 to 0.81 for the polymorphic loci. The isolation of these markers will enable estimations of genetic diversity in natural populations.     

Multiple paternity in <Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis> confirmed by microsatellite analysis

The aim of this study was to determine if individual ticks among the progeny of a single female Rhipicephalus (Boophilus) microplus tick removed from cattle under natural conditions are the result of mating with one or several males. To this end, simulations were run using an existing dataset of genotypes from 8 microsatellite loci to predict the number of samples required and the best locus. Subsequently, 14–22 progeny from each of 15 engorged female ticks removed from three cows, and the engorged females themselves, were genotyped for the BmM1 locus and the minimum number of potential male parents was determined for each progeny group. Of the 15 progeny groups, 10 must have been sired by more than one male, as indicated by the presence of five unique alleles among the progeny or three unique alleles that could not have been contributed by the female. This finding demonstrates multiple paternity in R. microplus.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Dinucleotide microsatellite markers in the genus <Emphasis Type="Italic">Caulerpa</Emphasis>

Caulerpa spp. are clonal green marine algae which often act as invasive species when growing outside their native biogeographical borders. Over the two past decades, Caulerpa taxifolia has spread along the Mediterranean coast, presently occurring at 70 sites and covering nearly 3,000 ha of subtidal area. New genetic markers (microsatellites) have been developed to assess clonal structure and genetic diversity of recently established populations of the invasive species C. taxifolia and Caulerpa racemosa in comparison with populations of the native Caulerpa prolifera in the Mediterranean. Our results show that nine polymorphic markers have been developed for C. prolifera, seven for C. taxifolia, and three for C. racemosa. Genetic diversity in Caulerpa was assessed in two geographical scales: one at a population scale where 40 thalli units were collected from C. prolifera in Cala d’Or, Mallorca, Spain, and another at a species scale, where 30 sample units were analyzed for C. prolifera, 24 for C. taxifolia, and 24 for C. racemosa from different sites in the Mediterranean, Atlantic, and Pacific Ocean. Number of alleles, expected heterozygosity, and marker amplification success are provided in each case.     

Interxylary cork of <Emphasis Type="Italic">Saussurea discolor</Emphasis> and <Emphasis Type="Italic">S. pygmaea</Emphasis> (Asteraceae)

The root anatomy of the subalpine to alpine plant species Saussurea discolor (Willd.) DC., and Saussurea pygmaea (Jacq.) Spreng., (Asteraceae) has been investigated by means of light and fluorescence microscopy on specimens of Austrian provenance. Both species develop a so called interxylary cork which mediates the splitting of the root into various strands. This phenomenon takes place in the secondary xylem and involves the development of a periderm separating the originally solid xylem cylinder. Interxylary cork is currently known from approximately 40 species of the Dicotyledones. This is the first report of this specific anatomical structure from the two studied species.     

Development and characterization of microsatellite markers for <Emphasis Type="Italic">Prunus verecunda</Emphasis> and <Emphasis Type="Italic">Prunus grayana</Emphasis> (Rosaceae)

Simple sequence repeat (SSR) markers were developed for Prunus verecunda and Prunus grayana to help understand the seed dispersal pattern of each species. We isolated and characterized nine microsatellite loci (four from P. verecunda and five from P. grayana). In P. verecunda, the number of alleles detected and the expected heterozygosity of five loci ranged from 11 to 24 and 0.59 to 0.92, respectively. In P. grayana, the number of alleles detected and the expected heterozygosity of five loci ranged from 4 to 14 and 0.62 to 0.86, respectively. These results show that the markers described here will be useful in studying the seed dispersal pattern of Prunus species.     

The ectomycorrhizae of <Emphasis Type="Italic">Lactarius rimosellus</Emphasis> and <Emphasis Type="Italic">Lactarius acatlanensis</Emphasis> with the endangered <Emphasis Type="Italic">Fagus grandifolia</Emphasis> var. <Emphasis Type="Italic">mexicana</Emphasis>

Gut bacterium Pantoea sp. is one of the predominant bacterial species in the larval gut of the diamondback moth, Plutella xylostella. The phenotypic characters of Pantoea sp. were investigated with BIOLOG phenotype MicroArray (PM) in this study. Totally 950 different metabolic phenotypes were tested using the PM plates 1–10. Results exhibited that Pantoea sp. was able to metabolize 37.37 % of the tested carbon sources, 91.32 % of nitrogen sources, 100 % of sulfur sources, and 98.31 % of phosphorus sources. Most informative utilization patterns for carbon sources of Pantoea sp. were organic acids and carbohydrates, and for nitrogen were various amino acids. The bacterium had 94 different biosynthetic pathways. It had a wide range of adaptabilities, and could still metabolize in osmolytes with up to 9 % sodium chloride, 6 % potassium chloride, 5 % sodium sulfate, 20 % ethylene glycol, 4 % sodium formate, 4 % urea, 5 % sodium lactate, 200 mmol/L sodium phosphate (pH 7.0), 100 mmol/L ammonium sulfate (pH 8.0), 100 mmol/L sodium nitrate, and 100 mmol/L sodium nitrite, respectively. It also exhibited active metabolism under pH values between 4.5 and 10. Pantoea sp. showed active decarboxylase activities while poor deaminase activities in the presence of various amino acids. The phenotypic characterization of Pantoea sp. increased our knowledge of the bacterium, in particular its interactions with insect hosts and the adaptability in gut environments, and showed us some possible approaches to controlling diamondback moth through decreasing Pantoea sp. density.     

Thermal factors affecting egg development in the wandering glider dragonfly, <Emphasis Type="Italic">Pantala flavescens</Emphasis> (Odonata: Libellulidae)

The wandering glider dragonfly, Pantala flavescens (Fabricius), arrives in Japan from tropical regions every spring. The offspring colonize areas throughout Japan, with rapid increases in populations in the autumn, but all individuals die in the winter, suggesting low tolerance to low temperatures. However, few quantitative data on egg development and water temperature have been reported for this species. Females at the reproductive stage were collected from fields throughout the flying season and their eggs released using an artificial oviposition technique. Almost all of the eggs were fertilized. Egg size was stable throughout the seasons. Most eggs hatched within a period of 5 days at high water temperatures (35 and 30 °C), which were recorded in the shallow ponds and rice paddy fields from summer to early autumn. However, the egg-stage duration increased with declining water temperature. All eggs in water at 15 °C had failed to hatch by 90 days. The calculated critical temperature of water was determined to be approximately 14.3 °C; the total effective temperature for the egg stage was about 80 degree-days. Thus, low water temperatures in winter may prevent P. flavescens overwintering in Japan.     

<Emphasis Type="Italic">Funastrum rupicola</Emphasis> (<Emphasis Type="Italic">Apocynaceae:</Emphasis><Emphasis Type="Italic">Asclepiadoideae</Emphasis>), a new species from Bolivia

Summary   Funastrum rupicola Goyder, a new species of Apocynaceae: Asclepiadoideae from Bolivia, is described and illustrated. The conservation status of this species is assessed.     

Incidence of <Emphasis Type="Italic">Wolbachia</Emphasis> and <Emphasis Type="Italic">Cardinium</Emphasis> endosymbionts in the <Emphasis Type="Italic">Osmia</Emphasis> community in Korea

Sex ratio distorting endosymbionts induce reproductive anomalies in their arthropod hosts. They have recently been paid much attention as firstly texts of evolution of host-symbiont relationships and secondly potential biological control agents to control arthropod pests. Among such organisms, Wolbachia and Cardinium bacteria are well characterized. This study aims at probing such bacteria in the Osmia community to evaluate their potential utilization to control arthropod pests. Among 17 PCR tested species, Osmia cornifrons and a parasitic fly are infected with Wolbachia and a mite species is infected with Cardinium. Phylogenetic tree analyses suggest that horizontal transfer of the bacteria occurred between phylogenetically distant hosts.     

Microhabitat use by larvae of the endangered dragonfly <Emphasis Type="Italic">Sympetrum pedemontanum</Emphasis><Emphasis Type="Italic">elatum</Emphasis> (Selys) in Japan

Sympetrum pedemontanum (Müller in Allioni) (Odonata: Libellulidae), which is distributed widely in the Eurasian continent and its neighboring islands, is listed as a Least Concern species in the International Union for Conservation of Nature Red List (2015). In Japan, however, the population of its subspecies S. pedemontanum elatum (Selys) has been rapidly decreasing since the 1970s. In order to conserve this subspecies, it is important to understand the seasonal microhabitat use by its larvae. However, this has been a difficult task because larvae of S. pedemontanum elatum often coexist with those of a common congener, S. eroticum (Selys), and cannot be morphologically distinguished from the latter. Thus, in this study, we first established a molecular technique based on the polymerase chain reaction to accurately identify each species. In the subsequent field survey in 2015 with its application in the Sakasegawa River, Hyogo Prefecture, we found that S. pedemontanum elatum larvae hatch in stagnant water and subsequently advance into weakly flowing water. Our results indicated a change in the microhabitats during the larval developmental process, reflecting the need for a continuous spectrum of stagnant, transitional, and flowing water. Such aquatic environments with a spectrum of water conditions are disappearing in Satoyama, a rural farming area in Japan. This has endangered species such as S. pedemontanum elatum and Oryzias latipes (Beloniformes: Adrianichthyidae) by depriving them of their favorable habitats. For their conservation, it is necessary to develop methods to recover the traditional aquatic environments in Satoyama.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.