Genetic diversity of wine Saccharomyces cerevisiae strains in an experimental winery from Galicia (NW Spain)

Genetic diversity of wine Saccharomyces cerevisiae strains involved in spontaneous fermentations was studied by analysis of mitochondrial DNA restriction patterns. Yeasts were isolated at different stages of fermentations with must from three different white grapevine varieties, Albariño, Godello and Treixadura, which are autochthonous from Galicia. Nineteen different patterns out of a total of 446 strains analysed were identified, but only a few of them appeared at high frequency and therefore were able to lead the fermentation process. Some strains were common to all fermentations; however, most of them were a minority being only found at low frequency for one or two specific grape varieties. The dominant strain was different for each variety except in one case, suggesting that some strains are better adapted to certain must conditions.     

New insights on Cricetodontini (Rodentia,Mammalia) from the Duero Basin,Spain

Abstract

The tribe Cricetodontini is a common cricetid group found in several European basins from the Miocene. Here we present a study of the fossils of this group found in the Duero Basin. We updated the biostratigraphical assignation of some of the localities in which several species of Cricetodontini have been found. Cricetodontini remains from eight localities from the central sector of the Duero Basin have been described, measured and assigned to a species. The presence in these localities of Hispanomys aguirrei, H. lavocati, H. nombrevillae and H. aragonensis has allowed correlating them to the biostratigraphic scale built for the Miocene in Calatayud-Daroca Basin, identifying biozones G3, H and I (MN7/8 – MN10, Late Aragonian – Early Vallesian, Middle – Late Miocene). Furthermore, this study constitutes the first citation of this species in this basin, except H. aguirrei, previously described in the Duero Basin. After this work, the biostratigraphical assignation of the studied sites is now well known. We evidenced the resemblance of Duero and Calatayud-Daroca basins.     

Molecular characterization of the human red cell Rho(D) antigen

Human red cells of Rh blood groups -D-/-D- ('super-D'), -/- (Rhnull) and normal Rho(D)+ cells were radioactively surface-labeled using the lactoperoxidase 125I method. Polyacrylamide gel electrophoresis in the presence of SDS followed by fluorography showed a strong enrichment of a polypeptide with an apparent mol. wt. of 28,0000-33,000 in the 125I-labeled -D-/-D- membranes. This polypeptide was specifically immune precipitated with anti-Rho(D) antiserum. Treatment of intact cells with trypsin or Pronase did not digest the protein. The Rho polypeptide migrated identically on polyacrylamide gel electrophoresis under reducing and non-reducing conditions. It was not phosphorylated after in vitro incubation of red cells with 32P. When whole labeled membranes were solubilized in neutral detergent and applied to lectin-Sepharose columns the Rho(D) polypeptide adsorbed to Ricinus communis lectin but not to wheat germ lectin or Lens culinaris lectin. The purified molecule did not adsorb to R. communis lectin-Sepharose. Treatment of the Rho(D) antigen with endo-N-acetyl glucosaminidase H, endo-beta-galactosidase or mild alkali did not lower its apparent mol. wt.     

Molecular polymorphism distribution in phenotypically distinct populations of wine yeast strains.

Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to analyze natural yeast populations from fermenting musts in El Penedès, Spain. Both analyses revealed a considerable degree of polymorphism, indicating heterogeneous natural populations. By specifically designed genetic selection protocols, strains showing potentially interesting phenotypes, such as high tolerance to ethanol and temperature or the ability to grow and to ferment in wine-water-sugar mixtures, were isolated from these natural populations. Genetic analysis showed a strong correlation between the selected phenotypes and mitochondrial DNA polymorphisms. Karyotype analysis revealed several genetically similar yeast lineages in the natural yeast microflora, which we interpret as genetically isolated subpopulations of yeast strains with distinct genetic traits, which may correspond to specific microenvironments. Thus, molecular polymorphism analysis may be useful not only to study the geographical distribution of natural yeast strains but also to identify strains with specific phenotypic properties.     

Onchocercosis in red deer (Cervus elaphus) from Spain.

Onchocercosis, caused by Onchocerca flexuosa, was observed in red deer (Cervus elaphus) from Spain for the first time. Adult specimens of P. flexuosa were found in nodules in subcutaneous tissues in 42 of 125 (33%) red deer between October 1994 and September 1995; intensity of infection +/- SD was 3.93 +/- 5.26 nodules per infected host. A clear seasonal pattern in the distribution of nodules was observed, with higher values of prevalence and intensity in fall and winter in contrast to spring and summer. Significant differences were found among age groups in prevalence, but not in the mean intensity. No differences in infection were apparent between male and female adult red deer.     

Molecular identification and characterization of wine yeasts isolated from Tenerife (Canary Island, Spain)

AIMS: The present study was aimed at the identification, differentiation and characterization of indigenous yeasts isolated from Tenerife vineyards (viticulture region that has never been characterized before). Microbiota were studied from 14 samples taken during fermentations carried out in the 2002 vintage, from 11 wineries belonging to five wine regions on Tenerife Island. METHODS AND RESULTS: Yeasts' strains were identified and characterized through restriction analysis of the 5.8S-internal transcribed spacer region and the mitochondrial DNA. At the beginning of alcoholic fermentation, 26 yeast species were found, where 14 species were present in significant frequencies in only one sample. Likewise, the Saccharomyces cerevisiae strains isolated are very specific, as they were only present in one wine region. CONCLUSIONS: There were isolated specific yeasts from each region on Tenerife Island. The founded yeasts may be responsible for distinctive and interesting properties of the studied wines. SIGNIFICANCE AND IMPACT OF THE STUDY: This study forms part of an extensive taxonomic survey within the ecological framework of vineyards in Tenerife. This investigation is an essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in the grape growing regions of this island. The results obtained demonstrate the value of using molecular genetic methods in taxonomic and ecological surveys. The results also shed some light on the ecology and oenological potential of S. cerevisiae strains isolated from this unique environment.     

Goussia polylepidis n. sp. (Apicomplexa: Sporozoea: Coccidia) from Chondrostoma polylepis (Pisces: Cyprinidae) of the Duero Basin (NW Spain)1

In the examination of 90 Chondrostoma polylepis caught in the Esla River, a coccidium of the genus Goussia was found in the swimbladder, peritoneum, kidney, and ureter. It is described as Goussia polylepidis n. sp. and its taxonomic affinities are discussed. Data on its prevalence and seasonality are also given.     

Goussia carpelli (Protozoa, Apicomplexa) in cyprinid fish of the Duero basin (NW Spain). Aspects of host-parasite relationships

Goussia carpelli was found parasitizing the intestine and gall bladder of Cyprinus carpio, Carassius carassius and Barbus barbus bocagei, and less frequently Leuciscus cephalus cabeda and Chondrostoma polylepis, from the Esla River. The coccidian is described and data on its prevalence, intensity and pathogenicity are given. In histological sections, the oocysts were detected in the lamina propria or submucosa. We did not observe a clear seasonal pattern nor an influence of the host sex on the infections. On the contrary, there was a tendency to a decrease in the prevalence with the age of the host.     

Reproductive and growth strategies of the red roach, Rutilus arcasii (Steindachner, 1866), in two contrasting tributaries of the River Duero, Spain

The life-histories of red roach were assessed in the R. Moros and the R. Ucero, two contrasting tributaries of the R. Duero. Growth occurred from April to October and Ucero red roach were larger than those in the Moros from their second year onwards. Condition increased with gonad development and abruptly decreased after spawning in both populations. The condition of Ucero fish recovered during the summer, but that of the Moros fish remained low. The same year classes (1980 and 1982) dominated both populations. River Moros red roach spawned in a 4-week period between the end of April and early June, while the spawning season of Ucero red roach lasted until August. The length to fecundity relationship was similar for both populations, and fecundity and ovum size increased with fish length. The life-history traits of these red roach populations are discussed in relation to current life-history theories.     

Molecular differentiation and diversity among the California red oaks (Fagaceae; Quercus section Lobatae)

A recent epidemic of Phytopthora (Sudden Oak Death) in coastal woodlands of California is causing severe mortality in some oak species belonging to the red oak (Lobatae) group. To predict the risks of spread of this disease, an understanding of the relationships among California's red oak species and of their population genetic structure is needed. We focus here on relationships among the four species of red oak. Whereas morphological distinction of Quercus wislizeni and Quercus parvula can pose problems, Quercus kelloggii and Quercus agrifolia in pure forms are easily distinguishable from one another and from Q. wislizeni and Q. parvula in the field. However, hybrids among all species combinations are known to occur in nature and these can confound data from ecological studies. Our results revealed greatest differentiation of the deciduous Q. kelloggii, with only weak AFLP fragment differentiation of the three remaining evergreen species. The molecular data suggest a closer affinity of Q. agrifolia with Q. wislizeni and Q. parvula contrary to earlier suggestions that its origins are likely to have been with northern deciduous oaks probably through a common ancestor with Q. kelloggii. Interior and coastal populations of Q. wislizeni separated in dendrograms based on phenetic and genetic distances suggesting probable isolation in different glacial refugia. The position of Q. parvula remains ambiguous, having a closer affinity with interior populations of Q. wislizeni and with Q. agrifolia, than with coastal populations of Q. wislizeni. Mean population differentiation in Q. wislizeni was 0.18, which is somewhat higher than the average for other oak species, suggesting that range fragmentation has occurred in the past, resulting in a metapopulation structure. Our results provide evidence that introgression among these species may be causing reticulation, further confounding species separation. Whereas Phytopthora has been reported on Q. agrifolia, Q. parvula and Q. kelloggii, it has not yet been detected in natural populations of Q. wislizeni. The species relationships that our molecular data show suggest that this is more likely a result of escape due to ecological tolerances than to genetic differences.     

Molecular diversity analysis of eggplant (Solanum melongena) genetic resources

Eggplant (Solanum melongena), a vegetable that is cultivated worldwide, is of considerable importance to agriculture in China. We analyzed the diversity of this plant using inter-simple sequence repeat (ISSR) and RAPD procedures to subdivide 143 Chinese-cultivated eggplants based on coefficient of parentage, genetic diversity index (GDI) and canonical discriminant analysis. ISSR markers were more effective than RAPD markers for detecting genetic diversity, which ranged from 0.10-0.51, slightly lower than what is known from other crops. Our ISSR/RAPD data provide molecular evidence that coincides with morphological-based classification into three varieties and further subdivision into eight groups, except for two groups. Intensive use of elite parents and extensive crossing within groups have resulted in increased coefficient of parentage and proportional contribution but decreased GDI during the past decades. The mean coefficient of parentage and proportional contribution increased from 0.05 to 0.10% and from 3.22 to 6.46% during 1980-1991 and 1992-2003, respectively. The GDI of landraces was 0.21, higher than the 0.09 and 0.08 calculated for the hybrid cultivars released during the two periods. The recent introduction of alien genotypes into eggplant breeding programs may broaden the genetic base.     

Molecular diversity of phospholipase D in angiosperms

Background  

The phospholipase D (PLD) family has been identified in plants by recent molecular studies, fostered by the emerging importance of plant PLDs in stress physiology and signal transduction. However, the presence of multiple isoforms limits the power of conventional biochemical and pharmacological approaches, and calls for a wider application of genetic methodology.     

Molecular typing of wine yeast strains Saccharomyces bayanus var. uvarum using microsatellite markers

The Saccharomyces bayanus var. uvarum yeasts are associated with spontaneous fermentation of must. Some strains were shown to be enological yeasts of interest in different winemaking processes. The molecular typing of S. bayanus var. uvarum at the strain level has become significant for wine microbiologists. Four microsatellite loci were defined from the exploration of genomic DNA sequence of S. bayanus var. uvarum. The 40 strains studied were homozygote for the locus considered. The discriminating capacity of the microsatellite method was found to be equal to that of karyotypes analysis. Links between 37 indigenous strains with the same geographic origin could be established through the analysis of microsatellite patterns. The analysis of microsatellite polymorphism is a reliable method for wine S. bayanus var. uvarum strains and their hybrids with Saccharomyces cerevisiae identification in taxonomic, ecological studies and winemaking applications.     

Decomposition rate of Quercus rotundifolia leaves in an evergreen oak forest of the Duero Basin (Province of Zamora,Spain)

Experimental litter decomposition of Quercus rotundifolia leaves in an evergreen oak forest of the semiarid area of the Duero Basin has been carried out. The loss of weight of the decomposing oak leaves was 62% after 24 months. In general, it is physical factors (mainly precipitation) which limit the decomposition of the evergreen oak leaves. Different patterns of bioelement losses were observed, increasing the relative content of N and P, and decreasing the amounts of Mg, K and Na in the decomposing leaves.     

Muridae from the late Miocene site of Venta del Moro (Eastern Spain)

Murids are usually the dominant faunas of the late Miocene and early Pliocene micromammal assemblages. The present work deals with the murid faunas of the well-known late Miocene locality of Venta del Moro, comprising over 2700 molars ascribed to the taxa Apodemus gorafensis, Occitanomys alcalai, Paraethomys meini and Stephanomys dubari. This list differs from those of previous studies by the presence of A. gorafensis instead of A. gudrunae. The presence of A. gudrunae in younger localities than Venta del Moro implies that both species, considered ancestor and descendant, coexisted for some time. In addition, the analysis of such an extensive collection has allowed us to check the variability of the studied species in great detail, such as the development of the longitudinal connections of O. alcalai, to a greater extent than previously described. Finally, we discuss the biometrical changes of the lineages of Paraethomys in Southern Europe from MN13 to MN15, and hypothesize about the mechanisms behind these variations in size.     

Sedimentary multiproxy response to hydroclimatic variability in Lagunillo del Tejo (Spain)

Lagunillo del Tejo is a small groundwater-fed sinkhole lake in the karst region of the Iberian Range (central-eastern Spain), which undergoes significant lake level fluctuation in response to rainfall variability. The aim of this study is to understand the record of water level fluctuations in Lagunillo del Tejo over the last two-and-a-half centuries. This information could be used in future studies to interpret longer sedimentary sequences. We analysed photosynthetic pigments, diatoms and cladoceran remains in sediment sequences recovered from the deepest part of the lake. The paleoecological proxies traced two different communities which have switched their prevalence during the past: (1) a planktonic community of algae, including diatoms, chlorophytes, cryptophytes and cyanobacteria, and phototrophic bacteria associated with higher lake level and water column seasonal stratification; (2) a littoral community with the higher levels of macrophyte pigments and associated epiphytic diatoms and chydorids, all of which indicate lower lake level. The levels of coherence between different proxies, each having an independent mechanistic link to lake-level variability, enhance the reliability of palaeolimnological inferences. The high-resolution stratigraphical data from the upper part of the core was compared with lake-level inferences from instrumental rainfall series (1859–2005) to establish the correspondence between Lagunillo del Tejo sediment sequences and climate record. Guest editors: K. Buczkó, J. Korponai, J. Padisák & S. W. Starratt Palaeolimnological Proxies as Tools of Environmental Reconstruction in Fresh Water     

Xylazine hydrochloride-ketamine hydrochloride immobilization of free-living red foxes (Vulpes vulpes) in Spain.

A combination of xylazine hydrochloride-ketamine hydrochloride was used to immobilize 83 wild red foxes (Vulpes vulpes) (15 pups and 68 adults) at Do?ana National Park (Spain). Mean ketamine hydrochloride doses were 17.1 mg/kg (SE = 1.53) and 12.3 mg/kg (SE = 0.4) for pups and adults, respectively, and mean xylazine hydrochloride doses for the same groups were 6.2 mg/kg (SE = 0.63) and 4.7 mg/kg (SE = 0.14), respectively. Mean induction times and first reaction times were 1.6 minutes and 22.5 minutes for pups and 3.8 minutes and 39.4 minutes for adults, respectively. Recommended doses for wild adult foxes of unknown weight are 75 mg of ketamine hydrochloride and 20 mg of xylazine hydrochloride.     

Genome survey sequencing of the wine spoilage yeast Dekkera (Brettanomyces) bruxellensis

The hemiascomycete yeast Dekkera bruxellensis, also known as Brettanomyces bruxellensis, is a major cause of wine spoilage worldwide. Wines infected with D. bruxellensis develop distinctive, unpleasant aromas due to volatile phenols produced by this species, which is highly ethanol tolerant and facultatively anaerobic. Despite its importance, however, D. bruxellensis has been poorly genetically characterized until now. We performed genome survey sequencing of a wine strain of D. bruxellensis to obtain 0.4x coverage of the genome. We identified approximately 3,000 genes, whose products averaged 49% amino acid identity to their Saccharomyces cerevisiae orthologs, with similar intron contents. Maximum likelihood phylogenetic analyses suggest that the relationship between D. bruxellensis, S. cerevisiae, and Candida albicans is close to a trichotomy. The estimated rate of chromosomal rearrangement in D. bruxellensis is slower than that calculated for C. albicans, while its rate of amino acid evolution is somewhat higher. The proteome of D. bruxellensis is enriched for transporters and genes involved in nitrogen and lipid metabolism, among other functions, which may reflect adaptations to its low-nutrient, high-ethanol niche. We also identified an adenyl deaminase gene that has high similarity to a gene in bacteria of the Burkholderia cepacia species complex and appears to be the result of horizontal gene transfer. These data provide a resource for further analyses of the population genetics and evolution of D. bruxellensis and of the genetic bases of its physiological capabilities.     

Additional analysis of chemical diversity of the red algal genus Laurencia (Rhodomelaceae) from Japan

The halogenated secondary metabolite constitution of four species of the red algal genus Laurencia (Rhodomelaceae) from southern Japan is reported. Laurencia composita Yamada from Tanegashima Island (Kagoshima Prefecture) bears five sesquiterpenoids (2,10‐dibromo‐3‐chloro‐α‐chamigrene and 2,10‐dibromo‐3‐chloro‐9‐hydroxy‐α‐chamigrene, in addition to pre‐pacifenol epoxide, johnstonol and pacifenol, which are known in other populations of this species). Laurencia intricata Lamouroux from Chinzei (Saga Prefecture) and Oomura Bay (Nagasaki Prefecture) bear a C₁₅ aceto‐genin, okamurallene. Laurencia majuscula (Harvey) Lucas from Tanegashima Island produces three sesquiterpenoids, (Z)‐10,15‐dibromo‐9‐hydroxy‐chamigra‐1, 3(15),7(14)‐triene, 10‐bromo‐7‐hydroxylaurene and 10,11‐dibromo‐7‐hydroxylaurene, corresponding to those of one of its chemical races. Laurencia venusta Yamada from Tanegashima Island produces two sesquiterpenoids, cupalaurenol and cyclolaurenol, which were known only from a sea hare, Aplysia dactylomela Rang. This strongly suggests that Aplysia consumes L. venusta and concentrates these halogenated compounds.