Phylogeography of the poison frog Mantella viridis (Amphibia: Mantellidae) reveals chromatic and genetic differentiation across ecotones in northern Madagascar

Mantella viridis is a threatened poison frog species endemic to the ecologically very heterogeneous northern region of Madagascar. The existence of several colour morphs within M. viridis and its very low genetic differentiation to the allopatrically distributed Mantella ebenaui raise questions about the processes driving the differentiation between these poison frog populations and about their taxonomic status. Using a DNA fragment of 476 nucleotides of the mitochondrial cytochrome b gene from 240 individuals of this species complex, we investigated the genetic variability of all known colour morphs of M. viridis, sampling this species throughout its known range, as well as several populations of M. ebenaui. Our genetic results confirm that M. viridis and M. ebenaui are closely related but reveal that no haplotype sharing occurs between these two taxa. Further, our molecular analyses provided evidence for barriers to gene flow among some of the colour morphs. Estimates of overlap of bioclimatic envelopes as assessed by ecological niche modelling also suggest a distinct bioclimatic niche of some of the lineages studied.     

Stress protein response in two sibling species of Marenzelleria (Polychaeta: Spionidae): is there an influence of acclimation salinity?

The induction and synthesis of stress proteins in the polychaete sibling species Marenzelleria viridis and M. neglecta was investigated at two different acclimation salinities (10 and 25 ppt). By in vitro labeling of dissected metameres with ³⁵S-methionine/cysteine and electrophoretic separation, four size classes of heat shock proteins (Hsps) were detected corresponding to 86, 78, 75 and 27 kDa. All Hsps, with the exception of Hsp86, represent a complex of multiple isoforms. The sibling species differed in three aspects of their heat shock response: (1) the induction temperature for Hsp75 synthesis was 25 °C and 30 °C in M. viridis and M. neglecta, respectively; (2) the relative level of synthesis of Hsp75 was higher in M. viridis; (3) the heat shock response was inactivated at a higher temperature in M. neglecta compared to M. viridis. The results showed that acclimation salinity had no explicit effect on Hsp synthesis in either species and that M. viridis was thermally more sensitive than its sibling species. We proposed that temperature, alone or in combination with other abiotic factors, plays a far greater role in the biogeographic distribution in Marenzelleria spp. than has been estimated so far.     

Physiological Aspects of Adaptation of the Marine Microalga Tetraselmis (Platymonas) viridis to Various Medium Salinity

We studied the capability of the marine microalga Tetraselmis (Platymonas) viridis to adapt to low and high medium salinity. The normal NaCl concentration for growth of this alga is 0.5 M. It was shown that T. viridis cells could actively grow and maintain osmoregulation and cytoplasmic ion homeostasis in the wide range of external salt concentrations, from 0.01 to 1.2 M NaCl. Using the plasma membrane vesicles isolated from T. viridis cells grown at various NaCl concentrations (0.01, 0.05, 0.5, 0.9, and 1.2 M), we studied the formation of the phosphorylated intermediate of Na⁺-ATPase, the enzyme responsible for Na⁺ export from the cells with a mol wt of ca. 100 kD. Na⁺-ATPase was shown to function in the plasma membrane even in the cells growing at an extremely low NaCl concentration (0.01 M). When alga was grown in high-salt media, the synthesis of several proteins with molecular weights close to 100 kD was induced. The data obtained argue for the hypothesis, which was put forward earlier, that a novel Na⁺-ATPase isoform is induced by T. viridis growing at high NaCl concentrations.     

Strong effects of amoebae grazing on the biomass and genetic structure of a Microcystis bloom (Cyanobacteria)

Despite its importance for bloom toxicity, the factors determining the population structure of cyanobacterial blooms are poorly understood. Here, we report the results of a two‐year field survey of the population dynamics of Microcystis blooms in a small hypertrophic urban pond. Microscopic enumeration of Microcystis and its predators and parasites was combined with pigment and microcystin analysis and denaturing gradient gel electrophoresis of the ITS rDNA region to assess population dynamics and structure. Two main Microcystis morpho‐ and ITS types were revealed, corresponding to M. aeruginosa and M. viridis. In both years, high population densities of naked amoebae grazing on Microcystis coincided with rapid decreases in Microcystis biomass. In one year, there was a shift from heavily infested M. aeruginosa to the less‐infested M. viridis, allowing the bloom to rapidly recover. The preference of amoebae for M. aeruginosa was confirmed by grazing experiments, in which several amoeba strains were capable of grazing down a strain of M. aeruginosa, but not of M. viridis. Zooplankton and chytrid parasites appeared to be of minor importance for these strong and fast reductions in Microcystis biomass. These findings demonstrate a strong impact of small protozoan grazers on the biomass and genetic structure of Microcystis blooms.     

Microalgal biotechnology: Carotenoid and glycerol production by the green algae <Emphasis Type="Italic">Dunaliella</Emphasis> isolated from the Gave-Khooni salt marsh,Iran

In this study, carotenoid and glycerol production in two unicellular green algae (Dunaliella salina and D. viridis) isolated from the Gave-Khooni salt marsh grown in media containing five different salt concentrations (0.17, 1, 2, 3, and 4 M NaCl) were evaluated under sterile conditions. Algae growth decreased as the medium salinity increased. Optimum growth of D. salina and D. viridis were obtained at 2 and 1 M NaCl, respectively. As salinity increased, glycerol and carotenoid production were increased in D. salina, whereas lower values for these products were produced in D. viridis under the same conditions. Furthermore, the cell color of D. salina changed from green to orange-red following accumulation of carotenoid, but the color of D. viridis was not changed. Thereby, it seems that the Iranian D. salina may be suitable for carotenoid production (betacarotene) on a large scale. In addition, since carotenoid compounds enhance the efficiency of photosynthesis and glycerol synthesis, it appears that the pathway for glycerol production and mechanisms of salt tolerance in D. viridis are unique from those of D. salina.     

Detection of Microcystis in Lake Sediment using Molecular Genetic Techniques

Summary Microcystis, which are toxic microcystin-producing cyanobacteria, normally bloom in summer and drop in numbers during the winter season in Senba Lake, Japan. Recently, this lake has been treated by ultrasonic radiation and jet circulation which were integrated with flushing with river water. This treatment was most likely sufficient for the destruction of cyanobacterial gas vacuoles. In order to confirm whether Microcystis viridis was still present, a molecular genetic monitoring technique on the basis of DNA direct extraction from the sediment was applied. Three primer sets were used for polymerase chain reaction (PCR) based on rRNA intergenic spacer analysis (RISA), the DNA dependent RNA polymerase (rpoC1) and a Microcystis sp.-specific rpoC1 fragment. The results from each primer were demonstrated on the basis of single strand conformation polymorphisms (SSCP). Using the RISA primer showed different results from the rpoC1 and Microcystis sp.-specific rpoC1 fragment; meanwhile, the rpoC1 Microcystis sp.-specific fragment was more specific than the RISA primer. Therefore, the Microcystis sp.-specific rpoC1 fragment was further analysed by denaturing gradient gel electrophoresis (DGGE). The DNA pattern representing M. viridis could not be detected in any of the sediment samples. However, the results were confirmed with another technique, terminal restriction fragment length polymorphisms (T-RFLP). Although T-RFLP patterns of 16S rDNA in sediment at 91 bp and 477 bp lengths were matched with the T-RFLP of M. viridis (HhaI and MspI endonuclease digestion, respectively), the T-RFLP pattern of 75 bp length was not matched with M. viridis (both of HhaI and MspI endonuclease digestion) which were the major T-RFLP pattern of M. viridis. Therefore, the results most likely indicated that M. viridis seems to have disappeared because of the addition of the ultrasonic radiation and jet circulation to the flushing treatment.     

Effects of Environmental Factors on Byssal Thread Formation in Some Members of the Family Mytilidae from the Sea of Japan

The ability to attach repeatedly to a substrate (glass, boulders, sand) in three common mussel species of the upper sublittoral zone of the Sea of Japan, Grayan's mussel Crenomytilus grayanus, the Korean mussel Mytilus coruscus, and northern horse mussel Modiolus modiolus, was studied under experimental conditions. It was found that during 120 h of the experiment C. grayanus and M. modiolus produced more byssal threads than M. coruscus. A decrease in the water temperature from 20 to 0°C slowed the rate of production of byssal threads down to full passivity in some experimental mollusks. This was more typical of M. coruscus and less typical of C. grayanus. Renewed threads differed in their length, thick, size of the adhesive plate, and strength. M. coruscus formed the shortest, thickest, and strongest threads with rather a large adhesive disk. The observed differences are discussed from the position of morphophysiological adaptations of species for colonization of different natural substrata under contrasting conditions of the upper sublittoral zone.     

Distribution and life cycle of the North American Spionid polychaeteMarenzelleria viridis (Verrill, 1873) in the Ems estuary

In 1983 the first specimens of the North American spionid polychaeteMarenzelleria viridis were found along the European mainland shore in the Ems estuary. Since then, this polychaete has spread over several estuaries around the North Sea and the Baltic. In the inner part of the Ems estuary juveniles were predominantly present in muddy sediments high in the intertidal zone; in more sandy sediments at higher salinities juveniles and adults co-occured. Detailed information was obtained at a muddy and at a sandy station. Gametes were present in the coelomic fluid from November through March. In May new recruits were found in the sediment samples, reaching densities of over 10⁵ M⁻² at the muddy station, andc. 2000 m⁻² at the sandy station. During summer, densities decreased at the muddy station, coinciding with a density increase at the sandy station, suggesting migration of juveniles from a nursery to the adult habitat. Cage experiments showed that the decrease of juveniles at the muddy station could be attributed to migration and not to mortality due to predation. At the sandy habitat palps and anterior parts ofM. viridis made up 4–11% of the stomach content of juvenile plaice (Pleuronectes platessa). In juvenile flounder (Platichthys flesus) only in Aprilc. 10% of the stomach content consisted ofM. viridis. During 1983–1990 increasing densities ofM. viridis at the sandy habitat coincided with a reduced abundance ofNereis diversicolor, however, this inverse relationship was not found to be statistically significant. Density fluctuations ofM. viridis andCorophium volutator showed a significant positive relationship, the cause of which is not yet understood.     

Effects of iron on growth and reflectance spectrum of the bloom‐forming cyanobacterium Microcystis viridis

Iron is an important factor in algal blooms because it is involved in cyanobacterial pigment biosynthesis and therefore has the ability to influence the pigment status of algal cells. This role in pigment biosynthesis offers the opportunity for rapid monitoring of iron availability to cyanobacteria through spectral reflectance characterization. In the present study, the freshwater cyanobacterium Microcystis viridis was cultured with different levels of iron. Cell density, cellular content of iron and photosynthetic pigments, and spectral reflectivity of M. viridis were determined daily during the course of the culture experiment. The results showed that at the lowest iron concentration (0.01 μM) the growth of M. viridis was seriously limited, and the maximal cell density was only approximately 6.4% of the density observed with an iron concentration of 18 μM. Iron availability dramatically affected chlorophyll a, carotenoid and phycocyanin content, with the greatest impact on chlorophyll a. The iron‐induced changes in content and ratios of pigments were detectable through spectral reflectance. Eleven spectral indices previously developed for the estimation of concentrations and/or ratios of pigments and a newly proposed chlorophyll a/phycocyanin index were found to be suitable for generating sensitive regression models between cellular iron content and spectral parameters. The comprehensive application of key sensitive spectral indices and regression equations should help to support monitoring and diagnosis of iron availability to cyanobacteria via remote sensing.     

Continuous underwater light measurement near Helgoland (North Sea) and its significance for characteristic light limits in the sublittoral region

Underwater irradiance was measured at intervals of 20 min for one year at 2 water depths (2.5 and 3.5 m below M.L.W.S.) and in 3 spectral regions in the sublittoral region of the rocky island of Helgoland. Data are presented for spectral and total irradiance at water depths ranging from 2 to 15 m (below M.L.W.S.). 90% of the total annual light reaching sublittoral habitats is received during the period from April to September, when Jerlov water type 7 (occasionally water type 5) dominates. During the other half of the year, the water is very turbid, and transparency is so low that long dark periods occur even at moderate water depths. The total annual light received at the lower kelp limit (Laminaria hyperborea), at 8 m water depth, is 15 MJ m^–2 year^–1 or 70 E m^–2 year^–1, which corresponds to 0.7% of surface irradiance (visible). At the lower algal limit (15 m water depth) these values are 1 MJ m^–2 year^–1 or 6 E m^–2 year^–1, corresponding to 0.05% of surface irradiance. These data are similar to measurements at the same limits in several different geographical areas, and may determine the depth at which these limits occur.     

Phylogenetic analysis of ITS2 sequences suggests the taxonomic re‐structuring of Dunaliella viridis (Chlorophyceae,Dunaliellales)

We analyzed the ITS2 primary and secondary structure (including Compensatory Base Changes (CBCs)) of 17 new Dunaliella strains (11 D. viridis, two D. tertiolecta, and four Dunaliella sp.), and compared these with other Dunaliella sequences available from the ITS2 database to circumscribe their taxonomic position. The ITS2 primary and secondary structure analysis positioned the majority of D. viridis strains in four main clades, showing that D. viridis is polyphyletic. The detection of at least one CBC among these clades strongly suggests that they could correspond to different biological species. Unexpectedly, while D. viridis var. euchlora (CCAP19/21) was positioned within the subgenus Dunaliella, D. viridis var. palmelloides (CCAP11/34) was positioned clearly outside this subgenus, suggesting that this taxon may not be properly placed in Dunaliella. Furthermore, the detection of at least three compensatory base changes (CBCs) between D. viridis var. palmelloides (CCAP11/34) and the other strains analyzed, confirm that this strain is a different species. For these reasons we propose re‐naming D. viridis var. palmelloides (CCAP11/34) to incertae sedis, and D. viridis var. euchlora (CCAP19/21) to Dunaliella sp. Therefore, the ITS2 primary and secondary structure data suggest a taxonomic re‐structuring of D. viridis.     

Predicting the distribution of a rare species of moss: The case of Buxbaumia viridis (Bryopsida,Buxbaumiaceae)

Buxbaumia viridis is a rare policy species restricted to decaying woods in forests. Although Member States of EU are required to monitor its conservation status, specific models able to predict species distribution are still lacking. However, the availability of such models would strongly improve the efficiency in collection additional data and consequently lead to a better knowledge of its ecology. Aims of this work were (i) to provide a model for species distribution assessing the importance of different environmental variables thought to be important in setting the occurrence of Buxbaumia viridis and (ii) to test the effect of imperfect detection in defining the environmental space where the species occur. With this work, records of B. viridis increased twofold in the Alpine region of Italy, passing from 13 records to 26. We showed that on the Alps, occurrence of Buxbaumia viridis was best predicted by northness, rainfall, canopy closure and necromass. Necromass was the single most important variable. A volume of 48–61 m³/ha of necromass was identified as the threshold value determining the high probability of species occurrence. The imperfect detection probability of the species (p = 0.25), biased towards zero the importance of the environmental variables.     

PHYLOGENETIC ANALYSIS OF THE GENUS EUGLENA (EUGLENOPHYCEAE) WITH PARTICULAR REFERENCE TO THE TYPE SPECIES EUGLENA VIRIDIS1

Euglena viridis (subgenus Euglena) serves as the type species for the genus Euglena. In this study, molecular phylogenetic analyses using a small subunit (SSU) and a combined SSU–partial large subunit rDNA data set for members of the genus Euglena showed that strains identified as E. viridis on the basis of morphology are distributed between two separate nonsister clades. Although all the E. viridis strains examined were morphologically indistinguishable and possessed spherical mucocysts and stellate chloroplasts with one paramylon center, there was a high degree of sequence divergence between the E. viridis strains in different clades, making this a cryptic species. Like E. viridis, all taxa from the subgenus Euglena are characterized by having one or more stellate chloroplasts with paramylon grains clustered around the center of the chloroplast. These additional taxa were divided into four clades in all the molecular analyses. Strains of Euglena stellata formed two nonsister clades whose members had a single aggregate chloroplast with paramylon center and spindle‐shaped mucocysts. A geniculata clade included species with one or two stellate chloroplasts with paramylon centers and spherical mucocysts, and the cantabrica clade had members with one stellate chloroplast with paramylon center and spherical mucocysts often arranged in spiral rows. Interspersed among these were three additional clades bearing taxa from the subgenus Calliglena that contains members with discoid plastids and pyrenoids that may or may not be capped with paramylon. These taxa formed a laciniata clade, mutabilis clade, and gracilis clade. This study demonstrates that E. viridis and E. stellata are cryptic species that can only be distinguished at the molecular level. Because E. viridis is the designated type species for the genus Euglena, we designated an epitype for E. viridis.     

Intraguild predation and interference competition on the endangered dragonfly <Emphasis Type="Italic">Aeshna viridis</Emphasis>

We examined the effects of intraguild predation (IGP) and interference competition on an endangered dragonfly, Aeshna viridis Eversm. (Odonata: Anisoptera). A. viridis is rare in Europe due to the decrease in suitable habitats harboring the macrophyte Stratiotes aloides L. Stratiotes plants are the principal oviposition substrate for A. viridis females and protect the larvae of A. viridis from fish predation. In our study lakes A. viridis larvae are sympatric with larvae of Aeshna grandis and Aeshna juncea. The susceptibility of A. viridis larvae to IGP by similar-sized larvae of A. grandis and A. juncea was tested in a laboratory predation experiment. Microhabitat use of A. viridis and A. grandis was studied in the laboratory to determine the possible effects of interference competition on the spatial distribution of A. viridis larvae. Our results show that at least in laboratory conditions, A. viridis is susceptible to IGP and interference competition. In competition, A. grandis larvae dominated the middle and outer portion of S. aloides rosettes whereas A. viridis stayed in the inner parts. When A. grandis larvae were absent, A. viridis colonized the middle and outer parts of the rosettes. We conclude that asymmetric predation between odonate larvae of equal size can be intense, and that both IGP and interference competition affect A. viridis. Although natural habitat complexity diminishes their impact, these interactions may nevertheless influence the distribution of A. viridis in S. aloides waters and restrict its microhabitat use in S. aloides rosettes.     

Phylogeography and cryptic variation within the Lacerta viridis complex (Lacertidae, Reptilia)

It is well known that the current genetic pattern of many European species has been highly influenced by climatic changes during the Pleistocene. While there are many well known vertebrate examples, knowledge about squamate reptiles is sparse. To obtain more data, a range‐wide sampling of Lacerta viridis was conducted and phylogenetic relations within the L. viridis complex were analysed using an mtDNA fragment encompassing part of cytochrome b, the adjacent tRNA genes and the noncoding control region. Most genetic divergence was found in the south of the distribution range. The Carpathian Basin and the regions north of the Carpathians and Alps are inhabited by the same mitochondrial lineage, corresponding to Lacerta viridis viridis. Three distinct lineages occurred in the south‐eastern Balkans — corresponding to L. v. viridis, L. v. meridionalis, L. v. guentherpetersi— as well as a fourth lineage for which no subspecies name is available. This distribution pattern suggests a rapid range expansion of L. v. viridis after the Holocene warming, leading to a colonization of the northern part of the species range. An unexpected finding was that a highly distinct genetic lineage occurs along the western Balkan coast. Phylogenetic analyses (Bayesian, maximum likelihood, maximum parsimony) suggested that this west Balkan lineage could represent the sister taxon of Lacerta bilineata. Due to the morphological similarity of taxa within the L. viridis complex this cryptic taxon was previously assigned to L. v. viridis. The distribution pattern of several parapatric, in part highly, distinct genetic lineages suggested the existence of several refuges in close proximity on the southern Balkans. Within L. bilineata sensu stricto a generally similar pattern emerged, with a high genetic diversity on the Apennine peninsula, arguing for two distinct refuges there, and a low genetic diversity in the northern part of the range. Close to the south‐eastern Alps, three distinct lineages (L. b. bilineata, L. v. viridis, west Balkan taxon) occurred within close proximity. We suggest that the west Balkan lineage represents an early offshoot of L. bilineata that was isolated during a previous Pleistocene glacial from the more western L. bilineata populations, which survived in refuges on the Apennine peninsula.     

Impact of the nemertean Lineus viridis on its polychaete prey on an intertidal sandflat

Interactions of the predator Lineus viridis (O. F. Muller 1774) with infaunal polychaetes were investigated in the German Wadden Sea. L. viridis is the dominant heteronemertean in this intertidal area with a mean abundance of 14 individuals per square meter. Predator enclosure experiments in the field revealed that L. viridis preyed on predatory polychaetes. The preferred prey species was Nereis diversicolor; others were Anaitides maculata. Nephtys hombergii and Harmothoe sarsi. These results were supported by a series of feeding experiments in the laboratory. The rate of consumption was estimated by laboratory and field experiments. The feeding rate of Lineus viridis was estimated by laboratory enclosure experiments to 5.6 g ashfree dry weight (AFDW) per square meter annually.     

The salt relations of Dunaliella

Dunaliella tertiolecta (marine) and D. viridis (halophilic) were each trained by serial transfer to growth at salt concentrations previously regarded as the other's domain. D. viridis then had a salt optimum at 1.0–1.5 M sodium chloride whereas that for D. tertiolecta was less than 0–2 M. Nevertheless D. tertiolecta grew faster than the halophil at all salt concentrations up to 3.5 M, the highest at which they were compared.Both species accumulate glycerol, which is necessary for growth at elevated salinities and which responds in its content to water activity (a _w ) rather than specifically to salt concentration. Variation in glycerol content is a metabolic process which occurs in the dark from accumulated starch as well as photosynthetically. Regulation of glycerol content by a _w does not require protein synthesis. The NADP-specific glycerol dehydrogenase of each of the algae is likely to be directly involved in the regulation of glycerol content. Kinetic studies, together with those described in an earlier publication, show that the enzyme has regulatory properties, and that both glycerol and dihydroxyacetone act as effectors as well as reactants. A mechanism of the reaction is tentatively proposed.     

Effects of diet on growth and development of three species of cyclopoid copepods

Megacyclops viridis (Jurine, 1820), Macrocyclops albidus (Jurine, 1820) and Acanthocyclops vernalis (Fischer, 1853) were raised in the laboratory at six temperatures (5, 8, 10, 12, 15 and 20 °C) and fed five different diets. The diets consisted of: a large ciliate Paramecium caudatum, a small ciliate Colpidium campylum, a mixture of P. caudatum and C. campylum, the cladoceran Daphnia hyalina, and nauplii of brine shrimp Artemia salina. Data was collected on development time, growth and adult size achieved. The results indicated that in all three species development and growth are faster on the diet of crustacea and Paramecium than on Colpidium. Female M. viridis and M. albidus do not develop beyond the stage V copepodid at low temperatures on the diet of Colpidium.     

Influence of temperature and nitrogen concentration on photosynthesis ofDunaliella viridis Teodoresco

The photosynthetic behaviour ofDunaliella viridis has been studied under a combination of three variables: irradiance (0–900 mol m^–2 s^–1), temperature (15, 23, 31, 38, 42 °C) and nitrogen concentration (0.05, 0.5, 1.5, 5, 10 mM NO ₃ ^- ) at a salinity of 2 M NaCl.The highest rates of photosynthesis have been found at 31 °C and a nitrate concentration of 10 mM. There exists a synergistic effect between temperature and nitrogen availability on the photosynthesis ofD. viridis; under nitrogen deficiency oxygen evolution is low, even null at high temperature. The interaction between these two variables of control occurs in a multiplicative way. There is also a general increase in photosynthetic pigments following the increase in nitrogen concentration in the culture medium. The normalization of net photosynthesis data in relation to chlorophylla shows that nitrogen concentration makes an indirect control of the photosynthetic rate ofD. viridis through the variation of pigment concentration.