Kudoa sp. (Myxozoa, Multivalvulida): first report in five commercial fish species from the Canary Islands-FAO 34 (Macaronesia-Spain).

Kudoid myxozoans have been reported causing serious chronic problems in marine fisheries, by reducing the market value of infected fish through pathological damage to the host musculature. We report here the overall prevalence of a Kudoa species in 84/277 (30.3%) fishes from 20 different species of high commercial value captured between October 2011 and December 2013 from the United Nations Food and Agriculture Organization (FAO) 34 commercial fishing area, near the coast of the Canary Islands (Spain). Macroscopic examination showed myxozoan-like cysts in skeletal muscle from 5 of the 20 fish species examined, with the following prevalences: Pagellus acarne (86.7%), Pagellus erythrinus (46.5%), Serranus cabrilla (27.8%), Spondyliosoma cantharus (19.4%), and Sarpa salpa (28.6%). Infection intensity was determined based on spore counts following muscle tissue digestion. Morphometric studies to characterize the species and DNA sequence analysis results suggest that these infections are attributable to a Kudoa species closely related to Kudoa trachuri. This paper reports the first study on a multivalvulidan species to be identified from the Canary Islands. Furthermore, this is the first report of Kudoa parasites in all of the hosts mentioned above, with the exception of P. acarne.

Lethal Influenza in Two Related Adults with Inherited GATA2 Deficiency.

The pathogenesis of life-threatening influenza A virus (IAV) disease remains elusive, as infection is benign in most individuals. We studied two relatives who died from influenza. We Sanger sequenced GATA2 and evaluated the mutation by gene transfer, measured serum cytokine levels, and analyzed circulating T- and B-cells. Both patients (father and son, P1 and P2) died in 2011 of H1N1pdm IAV infection at the ages of 54 and 31 years, respectively. They had not suffered from severe or moderately severe infections in the last 17 (P1) and 15 years (P2). A daughter of P1 had died at 20 years from infectious complications. Low B-cell, NK- cell, and monocyte numbers and myelodysplastic syndrome led to sequence GATA2. Patients were heterozygous for a novel, hypomorphic, R396L mutation leading to haplo-insufficiency. B- and T-cell rearrangement in peripheral blood from P1 during the influenza episode showed expansion of one major clone. No T-cell receptor excision circles were detected in P1 and P3 since they were 35 and 18 years, respectively. Both patients presented an exuberant, interferon (IFN)-γ-mediated hypercytokinemia during H1N1pdm infection. No data about patients with viremia was available. Two previously reported adult GATA2-deficient patients died from severe H1N1 IAV infection; GATA2 deficiency may predispose to life-threatening influenza in adulthood. However, a role of other genetic variants involved in immune responses cannot be ruled out. Patients with GATA2 deficiency can reach young adulthood without severe infections, including influenza, despite long-lasting complete B-cell and natural killer (NK) cell deficiency, as well as profoundly diminished T-cell thymic output.

Simvastatin down-regulates differential genetic profiles produced by organochlorine mixtures in primary breast cell (HMEC).

Women all over the world are exposed to an unavoidable contamination by organochlorine pesticides and other chemical pollutants. Many of them are considered as xenoestrogens and have been associated with the development and progression of breast cancer. We have demonstrated that the most prevalent pesticide mixtures found in healthy women and in women diagnosed with breast cancer modulates the gene expression in human epithelial mammary cells. Statins are well-known cholesterol-depleting agents acting as inhibitors of cholesterol synthesis. Since the early 1990s, it has been known that statins could be successfully used in cancer therapy, including breast cancer, but the exact mechanism behind anti-tumor activity of the statins remains unclear. In the present study we evaluated the effect of simvastatin in the gene expression pattern induced by realistic organochlorine mixtures found in breast cancer patients. The gene expression of 94 genes related with the cell signaling pathways were assessed. Our results indicate that simvastatin exerts a global down regulating effect on successfully determined genes (78.7%), thus attenuating the effects induced by organochlorine mixtures on the gene profile of human mammary epithelial cells. This effect was more evident on genes whose function is the ATP-binding process (that also were particularly up-regulated by pesticide mixtures). We also found that MERTK (a proto-oncogene which is overexpressed in several malignancies) and PDGFRB (a member of the platelet-derived growth factor family whose expression is high in breast-cancer cells that have become resistant to endocrine therapy) were among the genes with a higher differential regulation by simvastatin. Since resistance to treatment with tyrosine kinase inhibitors is closely related to MERKT, our findings would enhance the possible utility of statins in breast cancer treatment, i.e. improving therapeutic results combining statins with tyrosine Kinase inhibitors.

Differential gene expression pattern in human mammary epithelial cells induced by realistic organochlorine mixtures described in healthy women and in women diagnosed with breast cancer.

Organochlorine pesticides (OCs) have been associated with breast cancer development and progression, but the mechanisms underlying this phenomenon are not well known. In this work, we evaluated the effects exerted on normal human mammary epithelial cells (HMEC) by the OC mixtures most frequently detected in healthy women (H-mixture) and in women diagnosed with breast cancer (BC-mixture), as identified in a previous case-control study developed in Spain. Cytotoxicity and gene expression profile of human kinases (n=68) and non-kinases (n=26) were tested at concentrations similar to those described in the serum of those cases and controls. Although both mixtures caused a down-regulation of genes involved in the ATP binding process, our results clearly indicate that both mixtures may exert a very different effect on the gene expression profile of HMEC. Thus, while BC-mixture up-regulated the expression of oncogenes associated to breast cancer (GFRA1 and BHLHB8), the H-mixture down-regulated the expression of tumor suppressor genes (EPHA4 and EPHB2). Our results indicate that the composition of the OC mixture could play a role in the initiation processes of breast cancer. In addition, the present results suggest that subtle changes in the composition and levels of pollutants involved in environmentally relevant mixtures might induce very different biological effects, which explain, at least partially, why some mixtures seem to be more carcinogenic than others. Nonetheless, our findings confirm that environmentally relevant pollutants may modulate the expression of genes closely related to carcinogenic processes in the breast, reinforcing the role exerted by environment in the regulation of genes involved in breast carcinogenesis.

In vitro evaluation of oestrogenic/androgenic activity of the serum organochlorine pesticide mixtures previously described in a breast cancer case-control study.

Some organochlorine pesticides (OCs) have been individually linked to breast cancer (BC) because they exert oestrogenic effects on mammary cells. However, humans are environmentally exposed to more or less complex mixtures of these organochlorines, and the biological effects of these mixtures must be elucidated. In this work we evaluated the in vitro effects exerted on human BC cells by the OC mixtures that were most frequently detected in two groups of women who participated in a BC case-control study developed in Spain: healthy women and women diagnosed with BC. The cytotoxicity, oestrogenicity, and androgenicity of the most prevalent OC mixtures found in healthy women (H-mixture) and in BC patients (BC-mixture) were tested at concentrations that resembled those found in the serum of the evaluated women. Our results showed that both OC mixtures presented a similar oestrogenic activity and effect on cell viability, but BC-mixture showed an additional anti-androgenic effect. These results indicate that although the proliferative effect exerted by these mixtures on human breast cells seems to depend mainly on their oestrogenic action, the BC-mixture might additionally induce cell proliferation due to its anti-androgenic activity, therefore increasing the carcinogenic potential of this mixture. The findings of this study demonstrate that subtle variations in the composition of a mixture may induce relevant changes in its biological action.

Mitochondrial DNA haplogroup phylogeny of the dog: Proposal for a cladistic nomenclature.

Canis lupus familiaris mitochondrial DNA analysis has increased in recent years, not only for the purpose of deciphering dog domestication but also for forensic genetic studies or breed characterization. The resultant accumulation of data has increased the need for a normalized and phylogenetic-based nomenclature like those provided for human maternal lineages. Although a standardized classification has been proposed, haplotype names within clades have been assigned gradually without considering the evolutionary history of dog mtDNA. Moreover, this classification is based only on the D-loop region, proven to be insufficient for phylogenetic purposes due to its high number of recurrent mutations and the lack of relevant information present in the coding region. In this study, we design 1) a refined mtDNA cladistic nomenclature from a phylogenetic tree based on complete sequences, classifying dog maternal lineages into haplogroups defined by specific diagnostic mutations, and 2) a coding region SNP analysis that allows a more accurate classification into haplogroups when combined with D-loop sequencing, thus improving the phylogenetic information obtained in dog mitochondrial DNA studies.

Isolation and prominent aboriginal maternal legacy in the present-day population of La Gomera (Canary Islands).

The present-day population structure of La Gomera is outstanding in its high aboriginal heritage, the greatest in the Canary Islands. This was earlier confirmed by both mitochondrial DNA and autosomal analyses, although genetic drift due to the fifteenth century European colonization could not be excluded as the main factor responsible. The present mtDNA study of aboriginal remains and extant samples from the six municipal districts of the island indeed demonstrates that the pre-Hispanic colonization of La Gomera by North African people involved a strong founder event, shown by the high frequency of the indigenous Canarian U6b1a lineage in the aboriginal samples (65%). This value is even greater than that observed in the extant population (44%), which in turn is the highest of all the seven Canary Islands. In contrast to previous results obtained for the aboriginal populations of Tenerife and La Palma, haplogroups related to secondary waves of migration were not detected in La Gomera aborigines, indicating that isolation also had an important role in shaping the current population. The rugged relief of La Gomera divided into several distinct valleys probably promoted subsequent aboriginal intra-insular differentiation that has continued after the European colonization, as seen in the present-day population structure observed on the island.

The history of the North African mitochondrial DNA haplogroup U6 gene flow into the African, Eurasian and American continents.

BACKGROUND: Complete mitochondrial DNA (mtDNA) genome analyses have greatly improved the phylogeny and phylogeography of human mtDNA. Human mitochondrial DNA haplogroup U6 has been considered as a molecular signal of a Paleolithic return to North Africa of modern humans from southwestern Asia. RESULTS: Using 230 complete sequences we have refined the U6 phylogeny, and improved the phylogeographic information by the analysis of 761 partial sequences. This approach provides chronological limits for its arrival to Africa, followed by its spreads there according to climatic fluctuations, and its secondary prehistoric and historic migrations out of Africa colonizing Europe, the Canary Islands and the American Continent. CONCLUSIONS: The U6 expansions and contractions inside Africa faithfully reflect the climatic fluctuations that occurred in this Continent affecting also the Canary Islands. Mediterranean contacts drove these lineages to Europe, at least since the Neolithic. In turn, the European colonization brought different U6 lineages throughout the American Continent leaving the specific sign of the colonizers origin.

Multiple ethnic origins of mitochondrial DNA lineages for the population of Mauritius.

This article reports on the first genetic assessment of the contemporary Mauritian population. Small island nodes such as Mauritius played a critical role in historic globalization processes and revealing high-resolution details of labour sourcing is crucial in order to better understand early-modern diaspora events. Mauritius is a particularly interesting case given detailed historic accounts attesting to European (Dutch, French and British), African and Asian points of origin. Ninety-seven samples were analysed for mitochondrial DNA to begin unravelling the complex dynamics of the island's modern population. In corroboration with general demographic information, the majority of maternal lineages were derived from South Asia (58.76%), with Malagasy (16.60%), East/Southeast Asian (11.34%) and Sub-Saharan African (10.21%) also making significant contributions. This study pinpoints specific regional origins for the South Asian genetic contribution, showing a greater influence on the contemporary population from northern and southeast India. Moreover, the analysis of lineages related to the slave trade demonstrated that Madagascar and East Asia were the main centres of origin, with less influence from West Africa.

Introducing the Algerian mitochondrial DNA and Y-chromosome profiles into the North African landscape.

North Africa is considered a distinct geographic and ethnic entity within Africa. Although modern humans originated in this Continent, studies of mitochondrial DNA (mtDNA) and Y-chromosome genealogical markers provide evidence that the North African gene pool has been shaped by the back-migration of several Eurasian lineages in Paleolithic and Neolithic times. More recent influences from sub-Saharan Africa and Mediterranean Europe are also evident. The presence of East-West and North-South haplogroup frequency gradients strongly reinforces the genetic complexity of this region. However, this genetic scenario is beset with a notable gap, which is the lack of consistent information for Algeria, the largest country in the Maghreb. To fill this gap, we analyzed a sample of 240 unrelated subjects from a northwest Algeria cosmopolitan population using mtDNA sequences and Y-chromosome biallelic polymorphisms, focusing on the fine dissection of haplogroups E and R, which are the most prevalent in North Africa and Europe respectively. The Eurasian component in Algeria reached 80% for mtDNA and 90% for Y-chromosome. However, within them, the North African genetic component for mtDNA (U6 and M1; 20%) is significantly smaller than the paternal (E-M81 and E-V65; 70%). The unexpected presence of the European-derived Y-chromosome lineages R-M412, R-S116, R-U152 and R-M529 in Algeria and the rest of the Maghreb could be the counterparts of the mtDNA H1, H3 and V subgroups, pointing to direct maritime contacts between the European and North African sides of the western Mediterranean. Female influx of sub-Saharan Africans into Algeria (20%) is also significantly greater than the male (10%). In spite of these sexual asymmetries, the Algerian uniparental profiles faithfully correlate between each other and with the geography.

Polymorphisms of glutathione S-transferase µ and θ, MDR1 and VEGF genes as risk factors of bladder cancer: a case-control study.

OBJECTIVE: The present study was aimed at examining the local distribution of GSTM1, GSTT1, MDR1, and VEGF gene polymorphisms as possible risk factors contributing to the development of bladder cancer among the population from Canary Islands, Spain. MATERIALS AND METHODS: The genotypes were determined by PCR-based methods in a hospital-based case-control study consisting of 119 cases and 110 controls. The socio-demographic and clinicopathologic data were collected, including the smoking habits of the population covered in the study. RESULTS: The observed allelic frequencies were (%): GSTM1-GSTT1, (positive) 54 and (null) 46 in cases, and 65 and 35, respectively, in controls (P = 0.144); MDR1 C3435T, (C) 57 and (T) 43 in cases, and 54 and 46, respectively, in controls (P = 0.633); VEGF A2578C, (A) 40 and (C) 60 in cases, and 51 and 49, respectively, in controls (P = 0.221). Among Canary Islands subjects, GSTT1-null genotype appeared as a significant risk factor for bladder cancer (odds ratio (OR) 2.0; 95% confidence interval (CI), 1.0-3.7; P = 0.041), in multivariate analysis adjusted by age and smoking habits. No statistical changes in genotype distribution of GSTM1, MDR1 C3435T, and VEGF A2578C gene polymorphisms were observed between cases and controls. The distribution of the initial clinical stage, clinical grade, or recurrence status was not significantly different among the polymorphic variants in the case group (P = NS). CONCLUSIONS: Subjects with the GSTT1-null genotype might be at an increased risk of bladder cancer in Canary Islands, Spain. However, extensive studies are required for accurate confirmation of these results.

Partial recessive IFN-γR1 deficiency: genetic, immunological and clinical features of 14 patients from 11 kindreds.

We report a series of 14 patients from 11 kindreds with recessive partial (RP)-interferon (IFN)-γR1 deficiency. The I87T mutation was found in nine homozygous patients from Chile, Portugal and Poland, and the V63G mutation was found in five homozygous patients from the Canary Islands. Founder effects accounted for the recurrence of both mutations. The most recent common ancestors of the patients with the I87T and V63G mutations probably lived 1600 (875-2950) and 500 (200-1275) years ago, respectively. The two alleles confer phenotypes that are similar but differ in terms of IFN-γR1 levels and residual response to IFN-γ. The patients suffered from bacillus Calmette-Guérin-osis (n= 6), environmental mycobacteriosis (n= 6) or tuberculosis (n= 1). One patient did not suffer from mycobacterial infections but had disseminated salmonellosis, which was also present in two other patients. Age at onset of the first environmental mycobacterial disease differed widely between patients, with a mean value of 11.25 ± 9.13 years. Thirteen patients survived until the age of 14.82 ± 11.2 years, and one patient died at the age of 7 years, 9 days after the diagnosis of long-term Mycobacterium avium infection and the initiation of antimycobacterial treatment. Up to 10 patients are currently free of infection with no prophylaxis. The clinical heterogeneity of the 14 patients was not clearly related to either IFNGR1 genotype or the resulting cellular phenotype. RP-IFN-γR1 deficiency is, thus, more common than initially thought and should be considered in both children and adults with mild or severe mycobacterial diseases.

Temporal evolution of the ABO allele frequencies in the Canary Islands: the impact of the European colonization.

The indigenous Canary Islands population suffered a strong cultural and genetic impact when they were colonized by Europeans in the fifteenth century. The molecular analysis of the ABO blood group gene on aboriginal and seventeenth to eighteenth century remains confirms the demographic history of the islands depicted by previous archaeological, anthropological, and genetic studies. ABO allele frequencies clearly related Canarian aborigines with North African Berber populations, its most probable source of origin, and is far related to Iberian and to the current population of the archipelago. The historical sample shows a congruent intermediate position testifying already a strong European influence that would go in augment since then to present times, affecting all the islands with the important exception of La Gomera.

Phylogeography and genetic structure of the Canarian common chaffinch (Fringilla coelebs) inferred with mtDNA and microsatellite loci.

The widespread common chaffinch (Fringilla coelebs) inhabits five of the seven Canary Islands. Sequences of the mitochondrial cytochrome b gene (1002bp) revealed new insights into the systematics and phylogeography of this taxon. Additionally, a set of microsatellite loci were analyzed to examine the structure of these populations. Our results suggest that a new species of the genus Fringilla is present in the Canary Islands, which comprises at least three subspecies, but with a different distribution to that which has been morphologically accepted. The specimens from Gran Canaria are genetically distinct from those of La Gomera and Tenerife (F. c. canariensis), which suggests the existence of an undescribed taxon. Furthermore, nuclear microsatellite data suggest an ongoing incipient speciation process in this population. This study provides both important conservationist implications and a basis for re-evaluating the taxonomic status of the Canarian Fringilla coelebs populations.

Differential effects exerted on human mammary epithelial cells by environmentally relevant organochlorine pesticides either individually or in combination.

Organochlorine pesticides (OCs) have been associated with breast cancer development and progression. However, the deleterious mechanisms exerted by these contaminants are yet unclear and need to be further elucidated. In the present study, we investigated the effects of a number of OCs (previously detected in human serum from a Spanish population), individually or in combination, on normal human mammary epithelial cells (HMEC) at concentrations close to those found in human beings. The results obtained after a 96-h exposure indicated that OCs exert a clear cytotoxic effect on these cells at higher concentrations than those found in human beings. DDT-derivative organochlorines (DDT and its metabolites, DDE and DDD) are individually more cytotoxic than non-DDT-derivative organochlorines (aldrin and dieldrin). On the contrary, combinations of non-DDT organochlorines were clearly more cytotoxic than combinations of DDT-derivative organochlorines at concentrations close to those described in human serum. Additionally, transcriptional regulation arrays showed that the exposure of HMEC to an environmentally relevant mixture of OCs (p,p'-DDD plus p,p'-DDE plus o,p'-DDE plus aldrin plus dieldrin) sharply upregulated the expression of a number of protein kinases genes, such as ACVRL1, ALK-1, KIT, ERBB3, and ALK-1 at concentrations close to those detected in human populations. Taken together, these findings show a detrimental effect of OCs on human breast cells and indicate a possible association between exposure to organochlorine pesticide combinations and the induction of transformation processes in human breast cells.

The maternal aborigine colonization of La Palma (Canary Islands).

Teeth from 38 aboriginal remains of La Palma (Canary Islands) were analyzed for external and endogenous mitochondrial DNA control region sequences and for diagnostic coding positions. Informative sequences were obtained from 30 individuals (78.9%). The majority of lineages (93%) were from West Eurasian origin, being the rest (7%) from sub-Saharan African ascription. The bulk of the aboriginal haplotypes had exact matches in North Africa (70%). However, the indigenous Canarian sub-type U6b1, also detected in La Palma, has not yet been found in North Africa, the cradle of the U6 expansion. The most abundant H1 clade in La Palma, defined by transition 16260, is also very rare in North Africa. This means that the exact region from which the ancestors of the Canarian aborigines came has not yet been sampled or that they have been replaced by later human migrations. The high gene diversity found in La Palma (95.2+/-2.3), which is one of the farthest islands from the African continent, is of the same level than the previously found in the central island of Tenerife (92.4+/-2.8). This is against the supposition that the islands were colonized from the continent by island hopping and posterior isolation. On the other hand, the great similarity found between the aboriginal populations of La Palma and Tenerife is against the idea of an island-by-island independent maritime colonization without secondary contacts. Our data better fit to an island model with frequent migrations between islands.

Mitochondrial lineage M1 traces an early human backflow to Africa.

BACKGROUND: The out of Africa hypothesis has gained generalized consensus. However, many specific questions remain unsettled. To know whether the two M and N macrohaplogroups that colonized Eurasia were already present in Africa before the exit is puzzling. It has been proposed that the east African clade M1 supports a single origin of haplogroup M in Africa. To test the validity of that hypothesis, the phylogeographic analysis of 13 complete mitochondrial DNA (mtDNA) sequences and 261 partial sequences belonging to haplogroup M1 was carried out. RESULTS: The coalescence age of the African haplogroup M1 is younger than those for other M Asiatic clades. In contradiction to the hypothesis of an eastern Africa origin for modern human expansions out of Africa, the most ancestral M1 lineages have been found in Northwest Africa and in the Near East, instead of in East Africa. The M1 geographic distribution and the relative ages of its different subclades clearly correlate with those of haplogroup U6, for which an Eurasian ancestor has been demonstrated. CONCLUSION: This study provides evidence that M1, or its ancestor, had an Asiatic origin. The earliest M1 expansion into Africa occurred in northwestern instead of eastern areas; this early spread reached the Iberian Peninsula even affecting the Basques. The majority of the M1a lineages found outside and inside Africa had a more recent eastern Africa origin. Both western and eastern M1 lineages participated in the Neolithic colonization of the Sahara. The striking parallelism between subclade ages and geographic distribution of M1 and its North African U6 counterpart strongly reinforces this scenario. Finally, a relevant fraction of M1a lineages present today in the European Continent and nearby islands possibly had a Jewish instead of the commonly proposed Arab/Berber maternal ascendance.

Results of the 2003-2004 GEP-ISFG collaborative study on mitochondrial DNA: focus on the mtDNA profile of a mixed semen-saliva stain.

We report here a review of the seventh mitochondrial DNA (mtDNA) exercise undertaken by the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) corresponding to the period 2003-2004. Five reference bloodstains from five donors (M1-M5), a mixed stain of saliva and semen (M6), and a hair sample (M7) were submitted to each participating laboratory for nuclear DNA (nDNA; autosomal STR and Y-STR) and mtDNA analysis. Laboratories were asked to investigate the contributors of samples M6 and M7 among the reference donors (M1-M5). A total of 34 laboratories reported total or partial mtDNA sequence data from both, the reference bloodstains (M1-M5) and the hair sample (M7) concluding a match between mtDNA profiles of M5 and M7. Autosomal STR and Y-STR profiling was the preferred strategy to investigate the contributors of the semen/saliva mixture (M6). Nuclear DNA profiles were consistent with a mixture of saliva from the donor (female) of M4 and semen from donor M5, being the semen (XY) profile the dominant component of the mixture. Strikingly, and in contradiction to the nuclear DNA analysis, mtDNA sequencing results yield a more simple result: only the saliva contribution (M4) was detected, either after preferential lysis or after complete DNA digestion. Some labs provided with several explanations for this finding and carried out additional experiments to explain this apparent contradictory result. The results pointed to the existence of different relative amounts of nuclear and mtDNAs in saliva and semen. We conclude that this circumstance could strongly influence the interpretation of the mtDNA evidence in unbalanced mixtures and in consequence lead to false exclusions. During the GEP-ISFG annual conference a validation study was planned to progress in the interpretation of mtDNA from different mixtures.

Mitochondrial DNA error prophylaxis: assessing the causes of errors in the GEP'02-03 proficiency testing trial.

We report the results of the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) Collaborative Exercise 2002-2003 on mitochondrial DNA (mtDNA) analysis. Six different samples were submitted to the participating laboratories: four blood stains (M1-M2-M3-M4), one mixture blood sample (M5), and two hair shaft fragments (M6). Most of the labs reported consensus results for the blood stains, slightly improving the results of previous collaborative exercises. Although hair shaft analysis is still carried out by a small number of laboratories, this analysis yielded a high rate of success. On the contrary, the analysis of the mixture blood stain (M5) yielded a lower rate of success; in spite of this, the whole results on M5 typing demonstrated the suitability of mtDNA analysis in mixture samples. We have found that edition errors are among the most common mistakes reported by the different labs. In addition, we have detected contamination events as well as other minor problems, i.e. lack of standarization in nomenclature for punctual and length heteroplasmies, and indels. In the present edition of the GEP-ISFG exercise we have paid special attention to the visual phylogenetic inspection for detecting common sequencing errors.