Parasexual fusion products in green algae: Enteromorpha and Ulvaria (Ulvales,Chlorophyta)

Enteromorpha linza and Ulvaria oxysperma in North Carolina reproduce exclusively by asexual zoospores. Calcofluor white staining indicated that newly released zoospores lack significant cellulose cell wall material, making them suitable for treatment as protoplasts in a parasexual fusion process using high pH-Ca²⁺, PEG and centrifugation. Presumptive fusion products were identified by their larger size, twin chloroplasts and eyespots, and presence of fluorescence labelled and unlabelled portions. Parasexual fusion and karyogamy were confirmed by elevated levels of nuclear DNA in fusion cell germlings. In addition, aceto-orcein staining of fusion cell products revealed a diploid chromosome complement of 2N = 20 in Enteromorpha linza. Fusion cells were isolated by killing the more numerous adjacent unfused zoospores with 2-3 min exposure to blue light (410–490 nm). Unexposed fusion cells could be readily distinguished and recovered by micropipette at the 10-day stage.Center for Marine Science Research UNC-W Contribution No. 008.     

Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture

A continuous micropropagation was established from protoplasts of thegreen alga Enteromorpha intestinalis. The effects of two differentcrude enzymes and the osmolarity at different concentrations of the enzymesolution on algal protoplast yields were tested. The optimal enzymecomposition for cell wall digestion and protoplast viability was 2%cellulase R 10 Onozuka and 2% Aplysie with 0.5 m mannitol. Largenumbers of Enteromorpha protoplasts were released (10.0 × 10⁶protoplasts from 1 g fresh thalli) and settled on a rangeof substrata. Regeneration of the protoplasts followed the normal patternfor this species. Conditions for pure cultures and efficient systems offloating supports with nets were determined to optimise the product qualityof plantlets of Enteromorpha. A promising storage process has beendeveloped which involves including protoplasts in beads of alginic acid gel.Plants regenerated from protoplasts may also be used as seedstock tofacilitate propagation for macroalgal culture.     

Phylogeographical structure, distribution and genetic variation of the green algae Ulva intestinalis and U. compressa (Chlorophyta) in the Baltic Sea area

The marine algae Ulva intestinalis and U. compressa are morphologically plastic with many overlapping characters and are therefore difficult to distinguish from each other. The present distribution of U. intestinalis and U. compressa is investigated along the salinity gradient in the Baltic Sea area through analyses of internal transcribed spacer (ITS) sequence data. Also, the amount and distribution of intraspecific genetic polymorphism in the ITS region is studied allowing inferences on the phylogeographical pattern and postglacial recolonization of the Baltic Sea area. The data show that of the two species only U. intestinalis occurs in the Baltic Sea. The distribution of U. compressa is more restricted than previously reported, and it was not found in salinities lower than 15 ppt. All of Scandinavia and the Baltic Sea were covered with ice during the last ice age and the organisms in the Baltic Sea must have colonized the area after the ice had started to melt. The genetic diversity of U. intestinalis and U. compressa in the Baltic Sea and the neighbouring area was found to be reduced compared to that in the British Isles. This reduction may be the result of either a historical reduction of diversity or an adaptation of specific clones to the northern environmental conditions.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Model simulations of conditions suitable for the establishment of Enteromorpha sp. (Chlorophyta) macroalgal mats

Floating mats of algae regularly appear in shallow bays in many coastal areas around the world. This paper presents a model that simulates the growth of ephemeral macroalgae in shallow bays, and interprets the growth according to physical and biological prerequisites. Model runs emphasized the role of internal nutrient cycling to provide the enhanced nutrient availability needed to ensure abundant growth of macroalgae. Thus, the presence of macroalgae, as well as long water residence times, influenced the nutrient cycling in the bay to support further growth of macroalgal mats.     

Identification by SDS PAGE of green seaweeds ( Ulva and Enteromorpha) used in the food industry

SDS PAGE was tested as an analytical tool for the identification of fourgreen algae (Ulva rigida, U. rotundata, Enteromorphaintestinalis, E. compressa) used as food ingredients. A referencepattern composed of the bands present all year long was performed foreach species. The pattern for Ulva rotundata consists of 7 bandslocated between 69.9 and 15.5 kDa with the presence of triplicate bandsat 29.5, 26.3 and 22.9 kDa. The pattern for Ulva rigida is consistsof three bands with apparent molecular weights of 68.5, 56.4 and 44.7kDa. The Enteromorpha compressa pattern is characterised by sixbands located between 65.8 and 19.8 kDa. A double band withmolecular weights of 23.1 and 23.9 distinguished this pattern from theothers. Six bands situated between 66.4 and 19.4 kDa with a specifictriplicate band of 25.9, 23.9 and 22.5 kDa constituted the specific patternof Enteromorpha intestinalis. SDS PAGE appears to be suitable forthe identification of green seaweed foods.     

Development of eight microsatellite markers in the parsley frog (Pelodytes punctatus)

We characterized eight microsatellite loci to study spatial and temporal population structure of Pelodytes punctatus, a European anuran that has a peculiar breeding pattern among amphibians. The eight loci proved to be highly polymorphic with the number of alleles per locus ranging from two to 15 within two populations. Cross-amplification indicates that those markers may be also useful for closely related species from the same family.     

Development of microsatellite markers in Byrsonima crassifolia (Malpighiaceae)

? Premise of the study: We developed and characterized microsatellite markers for Byrsonima crassifolia (Malpighiaceae), a widely distributed neotropical fruit tree. ? Methods and Results: Eight polymorphic and two monomorphic microsatellite loci were identified and screened in 60 samples from four geographically disparate populations (Bolivia, Brazil, Mexico, and Panama). Each locus exhibited between two and 11 alleles. Expected heterozygosity ranged from 0 to 0.839. All loci amplify in the congeners B. variabilis and B. basiloba, four amplify in B. bucidaefolia, and seven amplify in B. variabilis, although levels of polymorphism have not been assessed. ? Conclusions: These loci will provide novel tools for comparing genetic diversity present in cultivated and noncultivated populations of B. crassifolia throughout its range, and may prove valuable in related species.     

Ethene (ethylene) production in the marine macroalga Ulva (Enteromorpha) intestinalis L. (Chlorophyta, Ulvophyceae): effect of light-stress and co-production with dimethyl sulphide

Ethene (ethylene; H₂C = CH₂) is one of a range of non-methane hydrocarbons (NMHC) that affect atmospheric chemistry and global climate. Ethene acts as a hormone in higher plants and its role in plant biochemistry, physiology and ecology has been the subject of extensive research. Ethene is also found in seawater, but despite evidence that marine microalgae and seaweeds can produce ethene directly, its production is generally attributed to photochemical breakdown of dissolved organic matter. Here we confirmed ethene production in cultured samples of the macroalga Ulva (Enteromorpha) intestinalis. Ethene levels increased substantially when samples acclimatized to low light conditions were transferred to high light, and ethene addition reduced chlorophyll levels by 30%. A range of potential inhibitors and inducers of ethene biosynthesis were tested. Evidence was found for ethene synthesis via the 1-aminocylopropane-1-acrylic acid (ACC) pathway and ACC oxidase activity was confirmed for cell-free extracts. Addition of acrylate, a potential ethene precursor in algae that contain the compatible solute dimethylsulphoniopropionate, doubled the ethene produced but no acrylate decarboxylase activity was found. Nonetheless the data support active production of ethene and we suggest ethene may play a multifaceted role in algae as it does in higher plants.     

Characterization of microsatellite loci in the marine seaweed Ascophyllum nodosum (Phaeophyceae; Fucales)

Ascophyllum nodosum L. dominates rocky intertidal shores throughout the temperate North Atlantic. Six microsatellite loci were developed for A. nodosum using enriched libraries. The number of alleles ranged from 9 to 24 and heterozygosities from 0.2213 to 0.7785. Ascophyllum is monotypic. There was no cross‐reactivity observed with Fucus serratus, F. vesiculosus or F. evanescens.     

Characterization of polymorphic microsatellite markers for the green turtle (Chelonia mydas)

We describe primers and polymerase chain reaction conditions to amplify 12 microsatellite loci from the green turtle (Chelonia mydas), including one dinucleotide, four trinucleotide and seven tetranucleotide loci. The primers were tested on 78 individuals from a Pacific population nesting in the Hawaiian Islands. The primer pairs developed in this study yielded an average of 8.33 alleles per locus (range of 3-15 alleles), an average observed heterozygosity of 0.668 (range 0.309-0.910), and an average polymorphic information content of 0.647 (range 0.287-0.894).     

Development of microsatellite markers for noninvasive DNA samples of the eastern green lizard Lacerta viridis

We report the isolation and characterization of microsatellite markers for the eastern green lizard (Lacerta viridis) usable for noninvasive samples. Ten polymorphic loci were obtained by screening 3000 recombinant clones and tested on 39 noninvasive DNA samples of individuals from different locations of the Danube area, Germany. Allelic richness ranged from three to nine alleles, the observed heterozygosity from 0.33 to 0.80, and the expected heterozygosity from 0.36 to 0.85. Our 10 loci, along with another 12 loci previously described, deliver effective analytical tools to analyse the genetic variability and to assess the social structure of eastern green lizard populations.     

Development of polymorphic microsatellite markers for the Steere's Liocichla (Liocichla steerii)

For the Steere's Liocichla (Liocichla steerii) we developed seven polymorphic microsatellite markers from a partial genomic library enriched for GATA, tetranucleotide repeats. Among the 27–28 individuals screened, allelic diversity ranged from six to 19 alleles per locus and the observed heterozygosity of each locus ranged from 0.250 to 0.889. All loci conformed to Hardy–Weinberg expectations except for two that appeared to be Z‐Iinked; such Z‐Iinked loci can be used in studies of male dispersal.     

Development of microsatellite markers for the drywood termite Incisitermes minor (Hagen)

Ten polymorphic microsatellite markers were developed in the drywood termite Incisitermes minor (Hagen) by using a genomic DNA extracted from the heads of workers. The microsatellite markers obtained in this study produced between two and seven alleles per locus. The observed and expected heterozygosities among the 10 markers ranged from 0.16 to 0.83 and from 0.43 to 0.84, respectively. These markers were shown to be good molecular tools for identification of the genetic structure and parentage assessment in I. minor.     

Development and characterization of nine new microsatellite markers in Taraxacum (Asteraceae)

This study aims at developing and characterizing new microsatellite primer pairs in Taraxacum officinale auct. to produce polymorphic markers for genetical and evolutionary studies on apomixis in this sexual‐apomictic complex. A total of 24 diploid plants were tested for allelic polymorphism and heterozygosity. Out of nine loci three deviated significantly from the Hardy–Weinberg equilibrium expectations, probably due to the presence of null‐alleles. Successful cross‐species amplification was obtained for all markers in 29 Taraxacum microspecies from five sections.     

Development of polymorphic microsatellite markers for the dung fly (Sepsis cynipsea)

The polyandrous fly Sepsis cynipsea has been used extensively in studies of sexual selection and local adaptation. We isolated and characterized 11 novel microsatellite markers for S. cynipsea from a genomic library and screened 32 flies for polymorphism. All microsatellite markers show high allelic diversity with an average of 9.64 alleles per locus. Two microsatellites were found likely to be X-linked. These novel markers will significantly advance studies of sexual selection and evolutionary genetics of S. cynipsea and related species, especially given the low numbers of markers currently available in this family.     

Development of microsatellite markers for the endangered medicinal plant Launaea arborescens (Asteraceae)

• Premise of the study: Microsatellite loci were developed in Launaea arborescens, an endangered and medicinal Asteraceae species in North Africa, for further investigation of its conservation genetics. • Methods and Results: We isolated and characterized 10 polymorphic and nine monomorphic microsatellite loci from L. arborescens using the protocol of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). For the 10 polymorphic loci, the number of alleles detected per locus varied from two to six, and the observed and expected heterozygosities ranged from 0.000 to 0.833 and 0.059 to 0.713, respectively. • Conclusions: The polymorphic markers provide a useful tool for conservation genetics studies of L. arborescens, including analysis of mating system, estimating gene flow, and identifying discrete genetic units within the species.     

Isolation and characterization of microsatellite loci in the tapeworm Ligula intestinalis (Cestoda: Pseudophyllidea)

Ligula intestinalis is a species with a complex biology. Several strains, distinct in the host specificity and biogeography, are supposed to exist; however, their morphological or molecular identification remains unresolved. We describe the isolation of 16 polymorphic microsatellite markers. The number of alleles ranged from four to 14 in a population of 21 L. intestinalis individuals. Loci showed amplification success across samples of worldwide distribution. A cross‐amplification of 14 markers in Digramma interrupta (Cestoda: Pseudophyllidea) was observed.     

Development of microsatellite markers for parentage analysis in the great tinamou (Tinamus major)

Eighteen microsatellite loci were isolated from great tinamous (Tinamus major), which are large terrestrial birds found in the Neotropics. These are the first primers developed for the Order Tinamiformes. Paternity analyses are possible because the levels of heterozygosity are sufficiently high (0.29-0.90).     

Development of microsatellite markers for the critically endangered Limonium dufourii (Girard) Kuntze (Plumbaginaceae)

Limonium dufourii is an endemic plant from the eastern Mediterranean coast of Spain with a triploid chromosome number and apomictic reproduction. We have isolated and characterized 13 polymorphic microsatellite loci from an enriched library in order to investigate its population genetic structure. Simple sequence repeat (SSR) loci were screened in 120 individuals from the six extant populations of this species. They show an average of 5.76 alleles per locus, ranging from 2 to 18, with seven loci exhibiting heterozygosities larger than 0.60. Three loci present one single allele in each individual, whereas one locus presents three alleles in every individual analysed.