High Y-chromosomal diversity and low relatedness between paternal lineages on a communal scale in the Western European Low Countries during the surname establishment.

There is limited knowledge on the biological relatedness between citizens and on the demographical dynamics within villages, towns and cities in pre-17th century Western Europe. By combining Y-chromosomal genotypes, in-depth genealogies and surname data in a strict genetic genealogical approach, it is possible to provide insights into the genetic diversity and the relatedness between indigenous paternal lineages within a particular community at the time of the surname adoption. To obtain these insights, six Flemish communities were selected in this study based on the differences in geography and historical development. After rigorous selection of appropriate DNA donors, low relatedness between Y chromosomes of different surnames was found within each community, although there is co-occurrence of these surnames in each community since the start of the surname adoption between the 14th and 15th century. Next, the high communal diversity in Y-chromosomal lineages was comparable with the regional diversity across Flanders at that time. Moreover, clinal distributions of particular Y-chromosomal lineages between the communities were observed according to the clinal distributions earlier observed across the Flemish regions and Western Europe. No significant indication for genetic differences between communities with distinct historical development was found in the analysis. These genetic results provide relevant information for studies in historical sciences, archaeology, forensic genetics and genealogy.

Increasing phylogenetic resolution still informative for Y chromosomal studies on West-European populations.

Many Y-chromosomal lineages which are defined in the latest phylogenetic tree of the human Y chromosome by the Y Chromosome Consortium (YCC) in 2008 are distributed in (Western) Europe due to the fact that a large number of phylogeographic studies focus on this area. Therefore, the question arises whether newly discovered polymorphisms on the Y chromosome will still be interesting to study Western Europeans on a population genetic level. To address this question, the West-European region of Flanders (Belgium) was selected as study area since more than 1000 Y chromosomes from this area have previously been genotyped at the highest resolution of the 2008 YCC-tree and coupled to in-depth genealogical data. Based on these data the temporal changes of the population genetic pattern over the last centuries within Flanders were studied and the effects of several past gene flow events were identified. In the present study a set of recently reported novel Y-SNPs were genotyped to further characterize all those Flemish Y chromosomes that belong to haplogroups G, R-M269 and T. Based on this extended Y-SNP set the discrimination power increased drastically as previous large (sub-)haplogroups are now subdivided in several non-marginal groups. Next, the previously observed population structure within Flanders appeared to be the result of different gradients of independent sub-haplogroups. Moreover, for the first time within Flanders a significant East-West gradient was observed in the frequency of two R-M269 lineages, and this gradient is still present when considering the current residence of the DNA donors. Our results thus suggest that an update of the Y-chromosomal tree based on new polymorphisms is still useful to increase the discrimination power based on Y-SNPs and to study population genetic patterns in more detail, even in an already well-studied region such as Western Europe.

Low historical rates of cuckoldry in a Western European human population traced by Y-chromosome and genealogical data.

Recent evidence suggests that seeking out extra-pair paternity (EPP) can be a viable alternative reproductive strategy for both males and females in many pair-bonded species, including humans. Accurate data on EPP rates in humans, however, are scant and mostly restricted to extant populations. Here, we provide the first large-scale, unbiased genetic study of historical EPP rates in a Western European human population based on combining Y-chromosomal data to infer genetic patrilineages with genealogical and surname data, which reflect known historical presumed paternity. Using two independent methods, we estimate that over the last few centuries, EPP rates in Flanders (Belgium) were only around 1­2% per generation. This figure is substantially lower than the 8­30% per generation reported in some behavioural studies on historical EPP rates, but comparable with the rates reported by other genetic studies of contemporary Western European populations. These results suggest that human EPP rates have not changed substantially during the last 400 years in Flanders and imply that legal genealogies rarely differ from the biological ones. This result has significant implications for a diverse set of fields, including human population genetics, historical demography, forensic science and human sociobiology.

In the name of the migrant father--analysis of surname origins identifies genetic admixture events undetectable from genealogical records.

Patrilineal heritable surnames are widely used to select autochthonous participants for studies on small-scale population genetic patterns owing to the unique link between the surname and a genetic marker, the Y-chromosome (Y-chr). Today, the question arises as to whether the surname origin will be informative on top of in-depth genealogical pedigrees. Admixture events that happened in the period after giving heritable surnames but before the start of genealogical records may be informative about the additional value of the surname origin. In this context, an interesting historical event is the demic migration from French-speaking regions in Northern France to the depopulated and Dutch-speaking region Flanders at the end of the sixteenth century. Y-chr subhaplogroups of individuals with a French/Roman surname that could be associated with this migration event were compared with those of a group with autochthonous Flemish surnames. Although these groups could not be differentiated based on in-depth genealogical data, they were significantly genetically different from each other. Moreover, the observed genetic divergence was related to the differences in the distributions of main Y-subhaplogroups between contemporary populations from Northern France and Flanders. Therefore, these results indicate that the surname origin can be an important feature on top of in-depth genealogical results to select autochthonous participants for a regional population genetic study based on Y-chromosomes.

Pitfalls in the analysis of mitochondrial DNA from ancient specimens and the consequences for forensic DNA analysis: the historical case of the putative heart of Louis XVII.

Amplification of mtDNA D-loop fragments with a length of 200 bp or more from ancient and even from fairly recent biological samples, can lead to erroneous results. This was clearly illustrated in our investigation of the putative heart of Louis XVII. By selecting different sets of primers which amplified shorter fragments of mtDNA (length 109 bp-201 bp), authentic polymorphisms could be visualised which remained undetected with the more classical primers for fragment sizes > 210 bp. Here we have extended those findings to other biological materials. A competitive PCR assay for quantitation of the amount of mtDNA for different fragment lengths, using a 10 bp deletion construct, was applied to ancient material and on a set of hairs of various ages of sampling (1966 up to the present). The results showed that DNA degradation started a few years after sampling. In the DNA extracts of the older hair shafts (1983-1995), the proportion of the number of short fragments to the number of long fragments is on average 4 in contrast to the most recent hair shafts. The numbers of amplifiable mtDNA copies for the hairs from 1975 and older were too small to show a clear difference. Use of long PCR fragments in such cases can yield misleading results. Use of short PCR fragments for the analysis of mtDNA from shed hair, in combination with a competitive PCR assay to determine the state of degradation, should improve the reliability of forensic mtDNA analysis considerably.

Regulation of the plasma membrane potential in Pneumocystis carinii.

Many protists use a H(+) gradient across the plasma membrane, the proton motive force, to drive nutrient uptake. This force is generated in part by the plasma membrane potential (DeltaPsi). We investigated the regulation of the DeltaPsi in Pneumocystis carinii using the potentiometric fluorescent dye bisoxonol. The steady state DeltaPsi in a buffer containing Na(+) and K(+) (standard buffer) was found to be -78+/-8 mV. In the absence of Na(+) and K(+) (NMG buffer) or Cl(-) (gluconate buffer), DeltaPsi was not significantly changed suggesting that cation and anion conductances do not play a significant role in the regulation of DeltaPsi in P. carinii. The DeltaPsi was also not affected by inhibitors of the Na(+)/K(+)-ATPase, ouabain (1 mM), and the K(+)/H(+)-ATPase, omeprazole (1 mM). In contrast, inhibitors of the plasma membrane H(+)-ATPase, dicyclohexylcarbodiimide (100 microM), N-ethylmaleimide (100 microM) and diethylstilbestrol (25 microM), significantly depolarized the DeltaPsi to -43+/-7, -56+/-5 and -40+/-12 mV, respectively. The data support that the plasma membrane H(+)-ATPase plays a significant role in the regulation of DeltaPsi in P. carinii.

A pyruvate-proton symport and an H+-ATPase regulate the intracellular pH of Trypanosoma brucei at different stages of its life cycle.

Regulation of intracellular pH (pH(i)) and H(+) efflux were investigated in Trypanosoma brucei bloodstream and procyclic trypomastigotes using the fluorescent dyes 2', 7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester and free BCECF respectively. pH(i) in bloodstream and procyclic trypomastigotes was 7.47+/-0.06 and 7. 53+/-0.07 respectively. Differences in the mechanisms for the regulation of pH(i) were noted between bloodstream and procyclic forms. Procyclic trypomastigotes maintained their pH(i) at neutral over a wide range of external pH values from 6 to 8, and in the absence of K(+) or Na(+). The H(+)-ATPase inhibitors N, N'-dicyclohexylcarbodi-imide (DCCD), diethylstilboestrol and N-ethylmaleimide substantially decreased the steady-state pH(i) and inhibited its recovery from acidification. The rate of H(+) efflux in these forms was determined to be 62+/-6.5 nmol/min per mg of protein, and was substantially decreased by H(+)-ATPase inhibitors. The data support the presence of an H(+)-ATPase as the major regulator of pH(i) in procyclic trypomastigotes. In contrast, bloodstream trypomastigotes were unable to maintain a neutral pH under acidic conditions, and their steady-state pH(i) and recovery from acidification were unaffected by H(+)-ATPase inhibitors, except for DCCD (100 microM). Their steady-state pH(i) was markedly decreased in glucose-free buffer or by >/=10 mM pyruvate, whereas procyclic trypomastigotes were unaffected by similar treatments. The rate of H(+) efflux in bloodstream trypomastigotes was 534+/-38 nmol/min per mg of protein, and was decreased in the absence of glucose and by the addition of pyruvate or DCCD. Pyruvate efflux in these forms was calculated to be 499+/-34 nmol/min per mg of protein, and was significantly inhibited by DCCD, 4, 4'-di-isothiocyanatodihydrostilbene-2,2'-disulphonic acid and alpha-cyanohydroxycinnamic acid. The pyruvate analogues beta-hydroxypyruvate, 3-bromopyruvate, 3-oxoglutarate, oxaloacetate, 3-oxoisovalerate and 3-oxoisohexanoate significantly decreased pH(i), as well as proton and pyruvate efflux, whereas lactate had only a small effect, and no effect was observed with citrate or fumarate. The inhibition by pyruvate analogues of pyruvate efflux, proton efflux and acidification of pH(i) supports the hypothesis that pyruvate efflux is accompanied by proton efflux and that this is the major pH(i) control mechanism in bloodstream forms. Inhibition by H(+)-ATPase inhibitors of residual H(+) efflux in the absence of glucose or in the presence of high extracellular pyruvate indicates a minor role for H(+)-ATPase(s) in control of pH(i) in bloodstream forms.

Functional expression of a vacuolar-type H+-ATPase in the plasma membrane and intracellular vacuoles of Trypanosoma cruzi.

Acid-loaded Trypanosoma cruzi amastigotes and trypomastigotes regained normal cytoplasmic pH (pHi), as measured in cells loaded with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), by a process that was sensitive to bafilomycin A1 at concentrations comparable to those that inhibited vacuolar (V) H+-ATPases from different sources. Steady-state pHi was also decreased by similar concentrations of bafilomycin A1 in a concentration-dependent manner. The efflux of H+ equivalents from amastigotes and trypomastigotes was measured by following changes in the fluorescence of extracellular BCECF. Basal H+ extrusion in the presence of glucose was 15.4+/-2.8 (S.D.) nmol of H+/min per 10(8) amastigotes and 6. 37+/-0.8 nmol of H+/min per 10(8) trypomastigotes. Bafilomycin A1 treatment significantly decreased the efflux of H+ equivalents by amastigotes (8.9+/-2.2 nmol of H+/min per 10(8) cells), but not by trypomastigotes (5.1+/-1.7 nmol of H+/min per 10(8) cells). The localization of the V-H+-ATPase of T. cruzi was investigated by immunocytochemistry. Confocal and electron microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the V-H+-ATPase of different stages of T. cruzi is also located in the plasma membrane. However, no labelling was detected in the plasma membrane lining the flagellar pocket of the different developmental stages. Surface localization of the V-H+-ATPase was confirmed by experiments involving the biotinylation of cell surface proteins and immunoprecipitation with antibodies against the V-H+-ATPase. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of amastigotes and with an important role for intracellular acidic compartments in the maintenance of pHi in different stages of T. cruzi.

The role of a H(+)-ATPase in the regulation of cytoplasmic pH in Trypanosoma cruzi epimastigotes.

Cytoplasmic pH (pHi) regulation was studied in Trypanosoma cruzi epimastigotes using fluorescent probes. Steady-state pHi was maintained even in the absence of extracellular Na+ or K+, but was significantly decreased in the absence of Cl-. Acid-loaded epimastigotes regained normal pHi by a process that was ATP-dependent and sensitive to N-ethylmaleimide, dicyclohexyl-carbodi-imide and diethylstiboestrol, suggesting involvement of a H(+)-pumping ATPase. Recovery from an acid load was independent of extracellular Na+ or K+ and insensitive to omeprazole, vanadate and low concentrations of bafilomycin A1. Using the fluorescent probe bisoxonol to measure the membrane potential of intact cells, acid loading of epimastigotes was shown to result in a dicyclohexylcarbodi-imide-sensitive hyperpolarization, which suggests electrogenic pumping of protons across the plasma membrane. Addition of glucose, but not of 6-deoxyglucose, produced a transient cellular acidification of possible metabolic origin, and increased the rate of recovery from an acid load. Taken together, these results are consistent with an important role of a H(+)-ATPase in the regulation of pHi homoeostasis in T. cruzi.

An H(+)-ATPase regulates cytoplasmic pH in Pneumocystis carinii trophozoites.

Pneumocystis carinii is an opportunistic fungus which causes interstitial pneumonia in patients with acquired immunodeficiency syndrome (AIDS). Cytoplasmic pH (pHi) regulation in short-term-cultured P. carinii trophozoites was studied using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein. With an extracellular pH of 7.4, the mean baseline pHi of P. carinii trophozoites was 7.40 +/- 0.10 (n = 8). This steady-state pHi was not significantly affected in the absence of extracellular Na+ or K+. Moreover, steady-state pHi was maintained in the nominal absence of HCO3- and was not affected by the Cl-/HCO(3-)-exchanger inhibitor 4, 4'-di-isothiocyanato-dihydrostilbene-2, 2'-disulphonic acid (100 microM), or the Na+/H(+)-exchanger inhibitor N-ethyl-N-isopropylamiloride (100 microM). In contrast, the general inhibitors of ATPases, N-ethylmaleimide (1 mM), and dicyclohexylcarbodi-imide (100 microM), and the inhibitor of yeast H(+)-ATPase, diethylstilbestrol (12.5-100 microM), decreased pHi, while the K+/H(+)-ATPase inhibitor omeprazole (50-400 microM), and the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (1-5 microM) only produced a dose-dependent acidification of the cells when used at high concentrations. In addition, steady-state pHi depended on the availability of cellular ATP, since it was decreased by the ATP synthase inhibitors oligomycin (1 microgram/ml) and sodium azide (1 mM), and by the uncoupler of oxidative phosphorylation carbonyl cyanide p-trifluorophenylhydrazone (1 microM), agents that were able to deplete significantly the intracellular ATP levels. Taken together, these results are consistent with an important role of an H(+)-ATPase similar to those found in other fungi in the regulation of pHi homoeostasis in P. carinii trophozoites.