Mitochondrial variants of complex I genes associated with leprosy clinical subtypes.

Leprosy is a chronic bacterial infection mainly caused by Mycobacterium leprae that primarily affects skin and peripheral nerves. Due to its ability to absorb carbon from the host cell, the bacillus became dependent on energy production, mainly through oxidative phosphorylation. In fact, variations in genes of Complex I of oxidative phosphorylation encoded by mtDNA have been associated with several diseases in humans, including bacterial infections, which are possible influencers in the host response to leprosy. Here, we investigated the presence of variants in the mtDNA genes encoding Complex I regarding leprosy, as well as the analysis of their pathogenicity in the studied cohort. We found an association of 74 mitochondrial variants with either of the polar forms, Pole T (Borderline Tuberculoid) or Pole L (Borderline Lepromatous and Lepromatous) of leprosy. Notably, six variants were exclusively found in both clinical poles of leprosy, including m.4158A>G and m.4248T>C in MT-ND1, m.13650C>A, m.13674T>C, m.12705C>T and m.13263A>G in MT-ND5, of which there are no previous reports in the global literature. Our observations reveal a substantial number of mutations among different groups of leprosy, highlighting a diverse range of consequences associated with mutations in genes across these groups. Furthermore, we suggest that the six specific variants exclusively identified in the case group could potentially play a crucial role in leprosy susceptibility and its clinical differentiation. These variants are believed to contribute to the instability and dysregulation of oxidative phosphorylation during the infection, further emphasizing their significance.

The mtDNA replication-related genes TFAM and POLG are associated with leprosy in Han Chinese from Southwest China.

BACKGROUND: The pathogen Mycobacterium leprae of leprosy is heavily dependent on the host energy metabolites and nutritional products for survival. Previously we and others have identified associations of several mitochondrion-related genes and mitochondrial DNA (mtDNA) copy number alterations with leprosy and/or its subtype. We hypothesized that genetic variants of mtDNA replication-related genes would affect leprosy. OBJECTIVE: We aimed to identify genetic associations between the mtDNA replication-related genes TFAM, POLG and leprosy. METHODS: Genetic association study was performed in 2898 individuals from two independent sample sets in Yunnan Province, China. We first screened 7 tag SNPs of TFAM and POLG in 527 leprosy cases and 583 controls (Sample I). Expression quantitative trait loci (eQTL) analysis and differential mRNA expression were analyzed to discern potential effect of risk variants. The entire exon region of TFAM and POLG were further analyzed in 798 leprosy cases and 990 controls (Sample II; 4327 East Asians from the ExAC dataset was included as a reference control) by using targeted gene sequencing for fine mapping potentially causal variants. RESULTS: Two tag SNPs of TFAM (rs1049432, P=0.007) and POLG (rs3176238, P=0.006) were associated with multibacillary leprosy (MB) in Sample I and the significance survived correction for multiple comparisons. SNPs rs1937 of TFAM (which was linked with rs1049432) and rs61756401 of POLG were associated with leprosy, whereas no potentially causative coding variants were identified in Sample II. The eQTL analysis showed that rs1049432 was a significant cis eQTL for TFAM in nerve tissue (P=1.20×10-12), and rs3176238 was a significant cis eQTL for POLG in nerve (P=3.90×10-13) and skin tissues (P=2.50×10-11). Consistently, mRNA level of POLG was differentially expressed in leprotic skin lesions. CONCLUSIONS: Genetic variants of TFAM and POLG were associated with leprosy in Han Chinese, presumably by affecting gene expression.

Genetic evidence of African slavery at the beginning of the trans-Atlantic slave trade.

An archaeological excavation in Valle da Gafaria (Lagos, Portugal), revealed two contiguous burial places outside the medieval city walls, dating from the 15(th)-17(th) centuries AD: one was interpreted as a Leprosarium cemetery and the second as an urban discard deposit, where signs of violent, unceremonious burials suggested that these remains may belong to slaves captured in Africa by the Portuguese. We obtained random short autosomal sequence reads from seven individuals: two from the latter site and five from the Leprosarium and used these to call SNP identities and estimate ancestral affinities with modern reference data. The Leprosarium site samples were less preserved but gave some probability of both African and European ancestry. The two discard deposit burials each gave African affinity signals, which were further refined toward modern West African or Bantu genotyped samples. These data from distressed burials illustrate an African contribution to a low status stratum of Lagos society at a time when this port became a hub of the European trade in African slaves which formed a precursor to the transatlantic transfer of millions.

Saccharomyces eubayanus and Saccharomyces uvarum associated with the fermentation of Araucaria araucana seeds in Patagonia.

Mudai is a traditional fermented beverage, made from the seeds of the Araucaria araucana tree by Mapuche communities. The main goal of the present study was to identify and characterize the yeast microbiota responsible of Mudai fermentation as well as from A. araucana seeds and bark from different locations in Northern Patagonia. Only Hanseniaspora uvarum and a commercial bakery strain of Saccharomyces cerevisiae were isolated from Mudai and all Saccharomyces isolates recovered from A. araucana seed and bark samples belonged to the cryotolerant species Saccharomyces eubayanus and Saccharomyces uvarum. These two species were already reported in Nothofagus trees from Patagonia; however, this is the first time that they were isolated from A. araucana, which extends their ecological distribution. The presence of these species in A. araucana seeds and bark samples, led us to postulate a potential role for them as the original yeasts responsible for the elaboration of Mudai before the introduction of commercial S. cerevisiae cultures. The molecular and genetic characterization of the S. uvarum and S. eubayanus isolates and their comparison with European S. uvarum strains and S. eubayanus hybrids (S. bayanus and S. pastorianus), allowed their ecology and evolution us to be examined.

Tracking climate change in a dispersal-limited species: reduced spatial and genetic connectivity in a montane salamander.

Tropical montane taxa are often locally adapted to very specific climatic conditions, contributing to their lower dispersal potential across complex landscapes. Climate and landscape features in montane regions affect population genetic structure in predictable ways, yet few empirical studies quantify the effects of both factors in shaping genetic structure of montane-adapted taxa. Here, we considered temporal and spatial variability in climate to explain contemporary genetic differentiation between populations of the montane salamander, Pseudoeurycea leprosa. Specifically, we used ecological niche modelling (ENM) and measured spatial connectivity and gene flow (using both mtDNA and microsatellite markers) across extant populations of P. leprosa in the Trans-Mexican Volcanic Belt (TVB). Our results indicate significant spatial and genetic isolation among populations, but we cannot distinguish between isolation by distance over time or current landscape barriers as mechanisms shaping population genetic divergences. Combining ecological niche modelling, spatial connectivity analyses, and historical and contemporary genetic signatures from different classes of genetic markers allows for inference of historical evolutionary processes and predictions of the impacts future climate change will have on the genetic diversity of montane taxa with low dispersal rates. Pseudoeurycea leprosa is one montane species among many endemic to this region and thus is a case study for the continued persistence of spatially and genetically isolated populations in the highly biodiverse TVB of central Mexico.

Nuclear and chloroplast DNA phylogeography reveals Pleistocene divergence and subsequent secondary contact of two genetic lineages of the tropical rainforest tree species Shorea leprosula (Dipterocarpaceae) in South-East Asia.

Tropical rainforests in South-East Asia have been affected by climatic fluctuations during past glacial eras. To examine how the accompanying changes in land areas and temperature have affected the genetic properties of rainforest trees in the region, we investigated the phylogeographic patterns of a widespread dipterocarp species, Shorea leprosula. Two types of DNA markers were used: expressed sequence tag-based simple sequence repeats and chloroplast DNA (cpDNA) sequence variations. Both sets of markers revealed clear genetic differentiation between populations in Borneo and those in the Malay Peninsula and Sumatra (Malay/Sumatra). However, in the south-western part of Borneo, genetic admixture of the lineages was observed in the two marker types. Coalescent simulation based on cpDNA sequence variation suggested that the two lineages arose 0.28-0.09 million years before present and that following their divergence migration from Malay/Sumatra to Borneo strongly exceeded migration in the opposite direction. We conclude that the genetic structure of S. leprosula was largely formed during the middle Pleistocene and was subsequently modified by eastward migration across the subaerially exposed Sunda Shelf.

Re-evaluating the taxonomic status of Chiromantis in Thailand using multiple lines of evidence (Amphibia: Anura: Rhacophoridae).

Because of general phenotypic similarities and distribution of species across two continents, the genus Chiromantis has proven somewhat enigmatic. Among Indochinese species, the validity of C. hansenae has been questioned by some who consider it a junior synonym of C. vittatus. We employ three lines of evidence to elucidate the taxonomic status and phylogenetic relationships of four congeneric species of Chiromantis frogs from Thailand. Results of molecular, morphological, and bioacoustic data analyses support at least four evolutionarily distinct and monophyletic clades: C. doriae, C. nongkhorensis, C. vittatus and C. hansenae. Genetic divergence between C. vittatus and C. hansenae is > 10%, significantly greater than C. doriae and C nongkhorensis (4.5%). Our results support the taxonomic validity of C. hansenae and suggest that there may be more diversity within C. hansenae and C. vittatus than is currently recognized.

Mitochondrial DNA copy number, but not haplogroup, confers a genetic susceptibility to leprosy in Han Chinese from Southwest China.

BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, an unculturable pathogen with an exceptionally eroded genome. The high level of inactivation of gene function in M. leprae, including many genes in its metabolic pathways, has led to a dependence on host energy production and nutritional products. We hypothesized that host cellular powerhouse--the mitochondria--may affect host susceptibility to M. leprae and the onset of clinical leprosy, and this may be reflected by mitochondrial DNA (mtDNA) background and mtDNA copy number. METHODS: We analyzed the mtDNA sequence variation of 534 leprosy patients and 850 matched controls from Yunnan Province and classified each subject by haplogroup. mtDNA copy number, taken to be proportional to mtDNA content, was measured in a subset of these subjects (296 patients and 231 controls) and 12 leprosy patients upon diagnosis. RESULTS: Comparison of matrilineal components of the case and control populations revealed no significant difference. However, measurement of mtDNA copy number showed that lepromatous leprosy patients had a significantly higher mtDNA content than controls (P = 0.008). Past medical treatments had no effect on the alteration of mtDNA copy number. CONCLUSIONS: Our results suggested that mtDNA content, but not haplogroup, affects leprosy and this influence is limited to the clinical subtype of lepromatous leprosy.

Efficiency of mitochondrial DNA restriction analysis and RAPD-PCR to characterize yeasts growing on dry-cured Iberian ham at the different geographic areas of ripening.

The efficiency of mitochondrial DNA (mtDNA) restriction analysis and random amplification of polymorphic DNA (RAPD)-PCR to characterize yeasts growing on dry-cured Iberian ham was evaluated. Besides, the distribution of the main species and biotypes of yeasts in the different ripening areas of this product was investigated. MtDNA restriction analysis allowed yeast characterization at species and strain level. RAPD-PCR with the primers (GACA)(4) and (GAC)(5) was inappropriate for characterization at species level. Most of the mtDNA restriction patterns detected in dry-cured Iberian ham were consistent with Debaryomyces hansenii. Several yeasts biotypes were associated to specific geographic areas of dry-cured Iberian ham ripening.

Effect of selected strains of Debaryomyces hansenii on the volatile compound production of dry fermented sausage "salchichón".

Different biotypes of Debaryomyces hansenii, characterized by mitochondrial DNA (mtDNA) restriction analysis, were inoculated in dry fermented sausages to evaluate their influence as single starter culture on volatile compound generation throughout the ripening process. Similar evolution of physicochemical parameters and microbial population was observed in both uninoculated and inoculated sausages. The tested biotypes modified the volatile compound profile of sausages specially in esters, branched alcohols and aldehydes. The biotype of D. hansenii with the E mtDNA restriction pattern is the most suitable to be used as starter culture since it produced volatile compounds involved in flavour development of dry-cured meat products such as 3-methylbutanol, 3-methylbutanal and 2-propanone. Moreover, the use of D. hansenii strains with the B, C2 and E mtDNA restriction patterns, as a mixed starter culture, should be also considered to generate low amount of sulphur compounds in dry-cured meat products.

Population polymorphism of nuclear mitochondrial DNA insertions reveals widespread diploidy associated with loss of heterozygosity in Debaryomyces hansenii.

Debaryomyces hansenii, a yeast that participates in the elaboration of foodstuff, displays important genetic diversity. Our recent phylogenetic classification of this species led to the subdivision of the species into three distinct clades. D. hansenii harbors the highest number of nuclear mitochondrial DNA (NUMT) insertions known so far for hemiascomycetous yeasts. Here we assessed the intraspecific variability of the NUMTs in this species by testing their presence/absence first in 28 strains, with 21 loci previously detected in the completely sequenced strain CBS 767(T), and second in a larger panel of 77 strains, with 8 most informative loci. We were able for the first time to structure populations in D. hansenii, although we observed little NUMT insertion variability within the clades. We determined the chronology of the NUMT insertions, which turned out to correlate with the previously defined taxonomy and provided additional evidence that colonization of nuclear genomes by mitochondrial DNA is a dynamic process in yeast. In combination with flow cytometry experiments, the NUMT analysis revealed the existence of both haploid and diploid strains, the latter being heterozygous and resulting from at least four crosses among strains from the various clades. As in the diploid pathogen Candida albicans, to which D. hansenii is phylogenetically related, we observed a differential loss of heterozygosity in the diploid strains, which can explain some of the large genetic diversity found in D. hansenii over the years.

Molecular exploration of the first-century Tomb of the Shroud in Akeldama, Jerusalem.

The Tomb of the Shroud is a first-century C.E. tomb discovered in Akeldama, Jerusalem, Israel that had been illegally entered and looted. The investigation of this tomb by an interdisciplinary team of researchers began in 2000. More than twenty stone ossuaries for collecting human bones were found, along with textiles from a burial shroud, hair and skeletal remains. The research presented here focuses on genetic analysis of the bioarchaeological remains from the tomb using mitochondrial DNA to examine familial relationships of the individuals within the tomb and molecular screening for the presence of disease. There are three mitochondrial haplotypes shared between a number of the remains analyzed suggesting a possible family tomb. There were two pathogens genetically detected within the collection of osteological samples, these were Mycobacterium tuberculosis and Mycobacterium leprae. The Tomb of the Shroud is one of very few examples of a preserved shrouded human burial and the only example of a plaster sealed loculus with remains genetically confirmed to have belonged to a shrouded male individual that suffered from tuberculosis and leprosy dating to the first-century C.E. This is the earliest case of leprosy with a confirmed date in which M. leprae DNA was detected.

Promiscuous DNA in the nuclear genomes of hemiascomycetous yeasts.

Abstract Transfer of fragments of mtDNA to the nuclear genome is a general phenomenon that gives rise to NUMTs (NUclear sequences of MiTochondrial origin). We present here the first comparative analysis of the NUMT content of entirely sequenced species belonging to a monophyletic group, the hemiascomycetous yeasts (Candida glabrata, Kluyveromyces lactis, Kluyveromyces thermotolerans, Debaryomyces hansenii and Yarrowia lipolytica, along with the updated NUMT content of Saccharomyces cerevisiae). This study revealed a huge diversity in NUMT number and organization across the six species. Debaryomyces hansenii harbors the highest number of NUMTs (145), half of which are distributed in numerous large mosaics of up to eight NUMTs arising from multiple noncontiguous mtDNA fragments inserted at the same chromosomal locus. Most NUMTs, in all species, are found within intergenic regions including seven NUMTs in pseudogenes. However, five NUMTs overlap a gene, suggesting a positive impact of NUMTs on protein evolution. Contrary to the other species, K. lactis and K. thermotolerans harbor only a few diverged NUMTs, suggesting that mitochondrial transfer to the nuclear genome has decreased or ceased in these phylogenetic branches. The dynamics of NUMT acquisition and loss are illustrated here by their species-specific distribution.

Sources of the adventitious microflora of a smear-ripened cheese.

AIMS: To determine the relationships between the major organisms from the cheese-making personnel and environment and the surface of a smear cheese. METHODS AND RESULTS: 360 yeast and 593 bacteria from the cheese surface, the dairy environment and the hands and arms of personnel were collected. Pulsed-field gel electrophoresis, repetitive sequence-based polymerase chain reaction and 16S rDNA sequencing were used for typing and identifying the bacteria, and mitochondrial DNA restriction fragment length polymorphism and Fourier-transform infrared spectroscopy for typing and identifying the yeast. The three most dominant bacteria were Corynebacterium casei, Corynebacterium variabile and Staphylococcus saprophyticus, which were divided into three, five and seven clusters, respectively, by macrorestriction analysis. The same clones from these organisms were isolated on the cheese surface, the dairy environment and the skin of the cheese personnel. Debaryomyces hansenii was the most dominant yeast. CONCLUSIONS: A 'house' microflora exists in the cheese plant. Although the original source of the micro-organisms was not identified, the brines were an important source of S. saprophyticus and D. hansenii and, additionally, the arms and hands of the workers the sources of C. casei and C. variabile. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the major contribution of the house microflora to the ripening of a smear-ripened cheese has been demonstrated.

Survival of surface ripening cultures during storage and monitoring their development on cheese.

AIMS: To study the survival of bacteria isolated from the surface of smear cheese and monitor their development during cheese ripening. METHODS AND RESULTS: The storage of five potential bacterial surface-ripening cheese cultures, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variable, Microbacterium gubbeenense and Staphylococcus saprophyticus, in maximum recovery diluent (MRD), containing 0.85% w/v or 5% w/v NaCl, at 21 or 4 degrees C for 40 days, was investigated. All five strains studied survived well with a maximum decrease of c. 2.5 log(10) CFU ml(-1) after storage for 40 days at 4 degrees C in 0.85% or 5% w/v NaCl. Survival, especially of C. variable, was less at 21 degrees C. The development of defined ripening cultures containing C. casei and Debaryomyces hansenii on two farmhouse cheeses was also evaluated. Using pulsed-field gel electrophoresis (PFGE) for the bacteria and mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) for the yeast, it was shown that the ripening cultures could be re-isolated in high numbers, 10(8) CFU cm(-2) for C. casei and 10(6) CFU cm(-2) for D. hansenii, from the cheese surface after 2.5 weeks of ripening. CONCLUSIONS: Ripening strains of surface ripening cultures can be stored in MRD containing 5% w/v salt at 4 degrees C for at least 40 days. Such cultures are recovered in high numbers from the cheese during ripening. SIGNIFICANCE AND IMPACT OF STUDY: This study has provided a low-cost and efficient way to store bacteria that could be used as ripening cultures for smear cheese. Such cultures can be recovered in high numbers from the cheese surface during ripening.

The mitochondrial genome of the wine yeast Hanseniaspora uvarum: a unique genome organization among yeast/fungal counterparts.

The complete sequence of the apiculate wine yeast Hanseniaspora uvarum mtDNA has been determined and analysed. It is an extremely compact linear molecule containing the shortest functional region ever found in fungi (11 094 bp long), flanked by Type 2 telomeric inverted repeats. The latter contained a 2704-bp-long subterminal region and tandem repeats of 839-bp units. In consequence, a population of mtDNA molecules that differed at the number of their telomeric reiterations was detected. The functional region of the mitochondrial genome coded for 32 genes, which included seven subunits of respiratory complexes and ATP synthase (the genes encoding for NADH oxidoreductase subunits were absent), two rRNAs and 23 tRNA genes which recognized codons for all amino acids. A single intron interrupted the cytochrome oxidase subunit 1 gene. A number of reasons contributed towards its strikingly small size, namely: (1) the remarkable size reduction (by >40%) of the rns and rnl genes; (2) that most tRNA genes and five of the seven protein-coding genes were the shortest among known yeast homologs; and (3) that the noncoding regions were restricted to 5.1% of the genome. In addition, the genome showed multiple changes in the orientation of transcription and the gene order differed drastically from other yeasts. When all protein coding gene sequences were considered as one unit and were compared with the corresponding molecules from all other complete mtDNAs of yeasts, the phylogenetic trees constructed robustly supported its placement basal to the yeast species of the 'Saccharomyces complex', demonstrating the advantage of this approach over single-gene or multigene approaches of unlinked genes.

Inferring the pattern of spontaneous mutation from the pattern of substitution in unitary pseudogenes of Mycobacterium leprae and a comparison of mutation patterns among distantly related organisms.

The pattern of spontaneous mutation can be inferred from the pattern of substitution in pseudogenes, which are known to be under very weak or no selective constraint. We modified an existing method (Gojobori T, et al., J Mol Evol 18:360, 1982) to infer the pattern of mutation in bacteria by using 569 pseudogenes from Mycobacterium leprae. In Gojobori et al.'s method, the pattern is inferred by using comparisons involving a pseudogene, a conspecific functional paralog, and an outgroup functional ortholog. Because pseudogenes in M. leprae are unitary, we replaced the missing paralogs by functional orthologs from M. tuberculosis. Functional orthologs from Streptomyces coelicolor served as outgroups. We compiled a database consisting of 69,378 inferred mutations. Transitional mutations were found to constitute more than 56% of all mutations. The transitional bias was mainly due to C-->T and G-->A, which were also the most frequent mutations on the leading strand and the only ones that were significantly more frequent than the random expectation. The least frequent mutations on the leading strand were A-->T and T-->A, each with a relative frequency of less than 3%. The mutation pattern was found to differ between the leading and the lagging strands. This asymmetry is thought to be the cause for the typical chirochoric structure of bacterial genomes. The physical distance of the pseudogene from the origin of replication (ori) was found to have almost no effect on the pattern of mutation. A surprising similarity was found between the mutation pattern in M. leprae and previously inferred patterns for such distant taxa as human and Drosophila. The mutation pattern on the leading strand of M. leprae was also found to share some common features with the pattern inferred for the heavy strand of the human mitochondrial genome. These findings indicate that taxon-specific factors may only play secondary roles in determining patterns of mutation.

Surface microflora of four smear-ripened cheeses.

The microbial composition of smear-ripened cheeses is not very clear. A total of 194 bacterial isolates and 187 yeast isolates from the surfaces of four Irish farmhouse smear-ripened cheeses were identified at the midpoint of ripening using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR, and 16S rRNA gene sequencing for identifying and typing the bacteria and Fourier transform infrared spectroscopy and mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis for identifying and typing the yeast. The yeast microflora was very uniform, and Debaryomyces hansenii was the dominant species in the four cheeses. Yarrowia lipolytica was also isolated in low numbers from one cheese. The bacteria were highly diverse, and 14 different species, Corynebacterium casei, Corynebacterium variabile, Arthrobacter arilaitensis, Arthrobacter sp., Microbacterium gubbeenense, Agrococcus sp. nov., Brevibacterium linens, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus saprophyticus, Micrococcus luteus, Halomonas venusta, Vibrio sp., and Bacillus sp., were identified on the four cheeses. Each cheese had a more or less unique microflora with four to nine species on its surface. However, two bacteria, C. casei and A. arilaitensis, were found on each cheese. Diversity at the strain level was also observed, based on the different PFGE patterns and mtDNA RFLP profiles of the dominant bacterial and yeast species. None of the ripening cultures deliberately inoculated onto the surface were reisolated from the cheeses. This study confirms the importance of the adventitious, resident microflora in the ripening of smear cheeses.

Mitochondrial DNA and RAPD polymorphisms in the haploid mite Brevipalpus phoenicis (Acari: Tenuipalpidae).

Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae) is recognized as the vector of citrus leprosis virus that is a significant problem in several South American countries. Citrus leprosis has been reported from Florida in the past but no longer occurs on citrus in North America. The disease was recently reported in Central America, suggesting that B. phoenicis constitutes a potential threat to the citrus industries of North America and the Caribbean. Besides B. phoenicis, B. obovatus Donnadieu, and B. californicus (Banks) have been incriminated as vectors of citrus leprosis virus and each species has hundreds of host plants. In this study, Brevipalpus mite specimens were collected from different plants, especially citrus, in the States of Florida (USA) and São Paulo (Brazil), and reared on citrus fruit under standard laboratory conditions. Mites were taken from these colonies for DNA extraction and for morphological species identification. One hundred and two Random Amplified Polymorphic DNA (RAPD) markers were scored along with amplification and sequencing of a mitochondrial cytochrome oxidase subunit I gene fragment (374 bp). Variability among the colonies was detected with consistent congruence between both molecular data sets. The mites from the Florida and Brazilian colonies were morphologically identified as belonging to B. phoenicis, and comprise a monophyletic group. These colonies could be further diagnosed and subdivided geographically by mitochondrial DNA analysis.

Analysis of yeast populations during alcoholic fermentation: a six year follow-up study.

Wine yeasts were isolated from fermenting Garnatxa and Xarel.lo musts fermented in a newly built and operated winery between 1995 and 2000. The species of non-Saccharomyces yeasts and the Saccharomyces cerevisiae strains were identified by ribosomal DNA and mitochondrial DNA RFLP analysis respectively. Non-Saccharomyces yeasts, particularly Hanseniaspora uvarum and Candida stellata, dominated the first stages of fermentation. However Saccharomyces cerevisiae was present at the beginning of the fermentation and was the main yeast in the musts in one vintage (1999). In all the cases, S. cerevisiae took over the process in the middle and final stages of fermentation. The analysis of the S. cerevisiae strains showed that indigenous strains competed with commercial strains inoculated in other fermentation tanks of the cellar. The continuous use of commercial yeasts reduced the diversity and importance of the indigenous S. cerevisiae strains.