Genetic structure of the populations of <Emphasis Type="Italic">Dactylorhiza ochroleuca</Emphasis> and <Emphasis Type="Italic">D. incarnata</Emphasis> (Orchidaceae) in the area of their joint growth in Russia and Belarus

We carried out an allozyme analysis to investigate polymorphism and genetic structure of the populations of D. incarnata and D. ochroleuca in regions of their joint growth in Russia and Belarus. We found that D. ochroleuca individuals in the populations of the Urals and Siberia, which are distant fragments from the main range of the species, do not differ significantly from individuals within the main part of the area (Belarus) on the basis of the allelic composition of eight gene loci. We revealed that D. ochroleuca and D. incarnata are differentiated by different alleles of the GDH locus. Thus, we established a genetic marker suitable to distinguish these closely related taxa. In addition to the GDH locus, D. ochroleuca and D. incarnata in the places of their joint growth, differ in the allelic structure of the PGI and NADHD loci. D. incarnata from the Urals and Siberia were polymorphic for both loci, and individuals from Belarus were polymorphic for one locus (PGI). In contrast, all D. ochroleuca individuals growing in sympatric populations with polymorphic D. incarnata were homozygous for the same alleles. Thus, comparison of the genetic structure of D. ochroleuca and D. incarnata points to the existence of a genetic isolation and a functioning isolation mechanism even under conditions of their joint growth. We found that the GDH locus in D. incarnata is polymorphic only in populations which grow together with D. ochroleuca, with exception a few examples. Thus, we conclude that variability of the GDH locus in D. incarnata is associated with hybridization with D. ochroleuca.     

Sympatric reinforcement of reproductive barriers between <Emphasis Type="Italic">Neotinea tridentata</Emphasis> and <Emphasis Type="Italic">N. ustulata</Emphasis> (Orchidaceae)

Reinforcement is the process by which selection favors traits that decrease mating between two incipient species in response to costly mating or the production of maladapted hybrids, causing the evolution of greater reproductive isolation between emerging species. I have studied a pair of orchids, Neotinea tridentata and N. ustulata, to examine the level of postmating pre- and post-zygotic isolating mechanisms that maintain these species, and the degree to which the boundary may still be permeable to gene flow. In this study, I performed pollen tube growth rate experiments and I investigated pre- and post-zygotic barriers by performing hand pollination experiments in order to evaluate fruit set, embryonate seed set and seed germination rates by intra- and interspecific crosses. Fruit set, the percentage of embryonate seeds and germinability of interspecific crosses were reduced compared to intraspecific pollinations, showing significant differences between sympatric and allopatric populations. While in allopatric populations the post-pollination isolation index ranged between 0.40 and 0.11, in sympatric populations orchid pairs showed total isolation due to post-pollination prezygotic barriers, guaranteed at the level of pollen–stigma interactions. Indeed, in sympatric populations, pollen tubes reached the ovary after 24 h in only 8 out of 45 plants; in the remaining cases, the pollen tubes did not enter the ovary, and thus no fruit set occurred. This pair of orchids is characterized by postmating pre-zygotic reproductive isolation in sympatric populations that prevents the formation of hybrids. This mechanism of speciation, starting in allopatry and triggering the reinforcement mechanisms of reproductive isolation in secondary sympatry, is the most likely explanation for the pattern of evolutionary transitions found in this pair of orchids.     

Detection of hybridization between two loach species (<Emphasis Type="Italic">Paramisgurnus dabryanus</Emphasis> and <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>) in wild populations

Artificial interspecific hybrids between large scale loach P. dabryanus and tetraploid pond loach M. anguillicaudatus (Cobitidae, Cypriniformes) are viable. To detect the occurrence of possible natural hybridization, genetic analyses by using microsatellite markers were performed for natural populations of large scale loach and pond loach, the reciprocal laboratory hybrids, and “supposed hybrids” with ambiguous morphology. The fertility of the artificial hybrids was also tested. At one diagnostic microsatellite (Mac50), one out of 20 “supposed hybrids” was identified to be F₁ hybrid between the two loach species because it had the same genotype as that of the laboratory hybrids. The triploid hybrids between the two species were confirmed to be female-sterile. The results show that rare hybridization has occurred between diploid large scale loach and tetraploid pond loach in nature although it may have little effect in genetic introgression. This study is helpful for fish conservation and encourages further investigation on natural hybridization and introgression of loaches.     

Molecular evidence for a natural diploid hybrid between <Emphasis Type="Italic">Miscanthus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis> (Poaceae) and <Emphasis Type="Italic">M. sacchariflorus</Emphasis>

The hybrid origin of Miscanthus purpurascens has previously been proposed, primarily because of its intermediate morphology. In this study, phylogenies based on the DNA sequences from the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS), on the DNA sequences of the trnL intron and trnL-F intergenic spacer of chloroplast DNA, and on amplified fragment length polymorphism (AFLP) fingerprinting confirm that M. purpurascens originated through homoploid hybridization between M. sinensis and M. sacchariflorus. Two different types of ITS sequences were identified from almost all plants of M. purpurascens. One type was found to be closely related to M. sinensis and the other to M. sacchariflorus. Miscanthus purpurascens was found to possess many M. sinensis- and M. sacchariflorus-specific AFLP bands but no band specific to itself. Clustering with the Unweighted Pair Group Method with Arithmetic Mean and principal coordinate analysis based on the AFLP data also demonstrated that M. purpurascens is an approximate intermediate of the two species. In addition, M. purpurascens has the plastid genome of M. sinensis or M. sacchariflorus, suggesting that either species could be its maternal parent. All specimens of M. purpurascens and its coexisting parental species are identified as diploids (2n = 2x = 38). Possible mechanisms of natural hybridization, hybrid status, chloroplast DNA recombination, and evolutionary implications of this hybridization are also discussed.     

Genetic structure of <Emphasis Type="Italic">Varroa destructor</Emphasis> populations infesting <Emphasis Type="Italic">Apis mellifera</Emphasis> colonies in Argentina

Although mitochondrial DNA mapping of Varroa destructor revealed the presence of several haplotypes, only two of them (Korean and Japanese haplotypes) were capable to infest Apis mellifera populations. Even though the Korean haplotype is the only one that has been reported in Argentina, these conclusions were based on mites sampled in apiaries from a specific geographical place (Buenos Aires province). To study mites from several sites of Argentina could reveal the presence of the Japanese genotype, especially considering sites near to Brazil, where Japanese haplotype was already detected. The aim of this work was to study the genetic structure of V. destructor populations from apiaries located in various provinces of Argentina, in order to determine the presence of different haplotypes. The study was carried out between January 2006 and December 2009. Phoretic adult Varroa mites were collected from honey bee workers sampled from colonies of A. mellifera located in Entre Ríos, Buenos Aires, Corrientes, Río Negro, Santa Cruz and Neuquén provinces. Twenty female mites from each sampling site were used to carry out the genetic analysis. For DNA extraction a nondestructive method was used. DNA sequences were compared to Korean haplotype (AF106899) and Japanese haplotype (AF106897). All DNA sequences obtained from mite populations sampled in Argentina, share 98% of similitude with Korean Haplotype (AF106899). Taking into account these results, we are able to conclude that Korean haplotype is cosmopolite in Argentina.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

Carotenoid fluorescence in <Emphasis Type="Italic">Dunaliella salina</Emphasis>

Dunaliella salina is a halotolerant green alga that is well known for its carotenoid producing capacity. The produced carotenoids are mainly stored in lipid globules. For various research purposes, such as production and extraction kinetics, we would like to determine and/or localise the carotenoid globules in vivo. In this study, we show that the carotenoid-rich globules emit clear green fluorescence, which can be used in, for example, fluorescence microscopy (e.g. CLSM) to obtain pictures of the cells and their carotenoid content.     

Genetic homeostasis and developmental stability in natural populations of bisexual (<Emphasis Type="Italic">Carassius auratus</Emphasis>) and unisexual (<Emphasis Type="Italic">C. gibelio</Emphasis>) goldfishes

One amphymictic diploid Carassius auratus, three apomictic triploids C. gibbelio, and a hybrid triploid Carassius auratus forms of goldfish were compared with respect to a set of characteristics of developmental stability: morphological variation, fluctuating asymmetry, and phenodeviations. It was found that C. gibbelio forms are characterized by lower levels both of morphological variation and fluctuating symmetry and more rare morphological abnormalties. A hybrid form Carassius auratus-gibelio was characterized by the intermediate values of the above-listed features. It is emphasized that phenotypic stabilization of apomictic forms is caused by two factors: its clone structure and actual developmental canalization. It is specified that promoted genetic homeostasis of the C. gibelio form does not give obvious adaptive advantages, as in the basic river basins of East and Central Europe. they are everywhere ousted by an amphimictic C. auratus.     

The polytene dot chromosome of <Emphasis Type="Italic">Drosophila</Emphasis>: <Emphasis Type="Italic">D. melanogaster</Emphasis> and <Emphasis Type="Italic">D. subobscura</Emphasis>

The chromosome arms are assumed to be homologous within the genus Drosophila. Homology at the level of the polytene chromosome banding pattern between non-sibling species is, however, almost impossible to establish as different processes such as inversion, transposition and unequal crossing over, have disturbed it. Even though the band sequences cannot be followed, we may ask whether there is a correlation in the total number of bands between species. The polytene dot chromosome is an excellent starting point for such an approach. Here we present the detailed cytology of polytene chromosome 4 of D. melanogasterand the polytene dot chromosome of D. subobscura using electron microscopy. The results show that the number of bands is about the same, around 30, in both species. We predict that by using thin sections and electron microscopy for the longer polytene chromosome arms, both species will turn out to have approximately equal band numbers.     

<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.     

Temporal alterations of <Emphasis Type="Italic">Nannochloropsis</Emphasis><Emphasis Type="Italic">salina</Emphasis> (Eustigmatophyceae) grown under aqueous diesel fuel stress

The influence of an aqueous extract of diesel fuel was tested on growth of the marine eustigmatophyte Nannochloropsis (Monallantus) salina Hibberd. An increase in the concentration of the pollutant led to a decrease in growth rate as measured by optical density, with maximum effect observed (33% of control) at 100% aqueous pollutant. Spectrophotometric examination of cell viability (using Evan’s blue dye) showed a significant negative effect of the diesel extract (p ≤ 0.05, r = −0.92). Infrared spectra showed a slight change in the absorbance of contaminated compared with controlled cells. Proteome analysis (sodium dodecyl sulfate polyacrylamide gel electrophoresis – “SDS-PAGE”) indicated that cell protein profiles depended on the pollutant concentration. Some of the resultant bands were characteristic to the pollutant concentration applied, indicating a distinct effect of the pollutant on the proteome structure. Iodine and toluidine blue dyes were applied using light microscopy to detect starch and mucilage, respectively. This indicated the presence of starch during all treatments, while the mucilage has been reduced. Transmission electron microscopy showed alterations to cell walls and membranes with different degrees of plasmolysis leading to a gradual increase in cell volume. However, the nucleus, the nucleolus and the pyrenoid remained unaffected. Similar results were obtained when the alga was cultured for 25 days in the 100% aqueous diesel extract indicating that long-term culture does not affect the degree of pollutant stress. Further, these cells recovered their normal appearance and characteristics within two days of being transferred to culture medium free of extract, indicating that N. salina shows a high tolerance to aqueous diesel fuel pollution.     

Palatability and fatality of the dinoflagellate <Emphasis Type="Italic">Prorocentrum lima</Emphasis> to <Emphasis Type="Italic">Artemia salina</Emphasis>

Prorocentrum lima is a toxic alga that produces both intra-cellular and extra-cellular toxins, including okadaic acid (OA) and dinophysistoxins (DTXs). Nauplii of the brine shrimp Artemia salina were exposed to both the cell and cell-free culture medium of P. lima in order to test the hypotheses that the extra-cellular medium is toxic to brine shrimp and that the P. lima cell is palatable but fatal to it. Artemia cysts incubated in the cell-free medium hatched, but mortalities were recorded for nauplii that hatched in, and metanuaplii exposed to, test solutions (autoclaved filtered seawater + cell-free medium) that contained at least 50% of the cell-free medium. Animals exposed to cells of P. lima readily fed on the cells. Some, especially among the Day 1 nauplii, ingested only one cell before dying, while others ingested more than one cell, up to six cells in the case of Day 3 nauplii, before dying. Day 3 nauplii were readily and heavily impacted by the P. lima cells. Survival analysis was used to evaluate survivorship of Day 1 to Day 3 nauplii exposed to cells of P. lima. Estimates were made of tD50s for the different age groups. Comparisons of the tD50s showed that the tD50s for Day 1 and Day 2 nauplii did not vary significantly, but they each varied significantly from the tD50 for the Day 3 nauplii. The possible ecological implications of the findings are discussed.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

Genetic characterization of asymmetric reciprocal hybridization between the flatfishes <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> and <Emphasis Type="Italic">Paralichthys dentatus</Emphasis>

Interspecific reciprocal crosses between the two flatfishes Paralichthys olivaceus and P. dentatus yielded hybrids with different viabilities. Specifically, the hybrids of P. olivaceus female and P. dentatus male (HI) were found to be viable, while the reciprocal hybrids from P. dentatus female and P. olivaceus male (HII) were completely inviable. All the HII individuals showed morphological deformities and died before first feeding. The chromosome analysis showed that HI individuals had the same chromosome number as parents. However, two chromosomes were missing in HII offspring indicating that the latter were aneuploids. Genomic inheritance from the parents to F₁ progeny was also examined by amplified fragment length polymorphism (AFLP) analyses, and the results showed differences between reciprocal hybrids. Almost all AFLP bands (97.71%) observed in parents were passed on to HI individuals. In contrast, only 86.64% of the AFLP bands from parents were scored in HII individuals. Frequency of lost parental bands was thus significantly higher in HII than that in HI and intraspecific crosses, which was probably associated with chromosomal elimination. In addition, higher segregation distortions were found in hybrids than in controls, although these differences were not significant. The present study indicates that chromosomal elimination and loss of AFLP loci occurred in inviable HII individuals, while such genomic changes were not found in viable HI individuals. Possible implications of such difference on genomic changes for asymmetric viability in reciprocal hybrids are discussed.     

<Emphasis Type="Italic">Anacamptis</Emphasis> versus <Emphasis Type="Italic">Orchis</Emphasis> (Orchidaceae): seed micromorphology and its taxonomic significance

Seed micromorphology of 24 taxa of the genera Anacamptis and Orchis was examined under light and scanning electron microscopy. Seed qualitative characters appear very useful at the supraspecific level in the subtribe Orchidinae. Based on our observations, the sculpturing of the periclinal walls of the medial testa cells, the seed shape and several features of the anticlinal walls of the apical cells showed variability between the studied taxa, with special relevance of the first character. According to this factor, we found one type of seed for Anacamptis and two for Orchis. The taxonomic value of seed coat characters is compared with the last taxonomic proposals for the genera Anacamptis and Orchis.     

Comparative <Emphasis Type="Italic">in vitro</Emphasis> germination ecology of <Emphasis Type="Italic">Calopogon tuberosus</Emphasis> var. <Emphasis Type="Italic">tuberosus</Emphasis> (Orchidaceae) across its geographic range

Seed responses to temperature are often essential to the study of germination ecology, but the ecological role of temperature in orchid seed germination remains uncertain. The response of orchid seeds to cold stratification have been studied, but the exact physiological role remains unclear. No studies exist that compare the effects of either cold stratification or temperature on germination among distant populations of the same species. In two separate experiments, the role of temperature (25, 22/11, 27/15, 29/19, 33/24°C) and chilling at 10°C on in vitro seed germination were investigated using distant populations of Calopogon tuberosus var. tuberosus. Cooler temperatures promoted germination of Michigan seeds; warmer temperatures promoted germination of South Carolina and north central Florida seeds. South Florida seed germination was highest under both warm and cool temperatures. More advanced seedling development generally occurred at higher temperatures with the exception of south Florida seedlings, in which the warmest temperature suppressed development. Fluctuating diurnal temperatures were more beneficial for germination compared to constant temperatures. Cold stratification had a positive effect on germination among all populations, but South Carolina seeds required the longest chilling treatments to obtain maximum germination. Results from the cold stratification experiment indicate that a physiological dormancy is present, but the degree of dormancy varies across the species range. The variable responses among populations may indicate ecotypic differentiation.