tRNA Leu Research Articles

Modeling of mitochondrial bioenergetics and autophagy impairment in MELAS-mutant iPSC-derived retinal pigment epithelial cells

BackgroundMitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of age-related macular degeneration (AMD). However, a deeper understanding is required to determine the contribution of mitochondrial dysfunction and impaired mitochondrial autophagy (mitophagy) to RPE damage and AMD pathobiology. In this study, we model the impact of a prototypical systemic mitochondrial defect, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), in RPE health and homeostasis as an in vitro model for impaired mitochondrial bioenergetics.MethodsWe used induced pluripotent stem cells (iPSCs) derived from skin biopsies of MELAS patients (m.3243A > G tRNA leu mutation) with different levels of mtDNA heteroplasmy and differentiated them into RPE cells. Mitochondrial depletion of ARPE-19 cells (p0 cells) was also performed using 50 ng/mL ethidium bromide (EtBr) and 50 mg/ml uridine. Cell fusion of the human platelets with the p0 cells performed using polyethylene glycol (PEG)/suspension essential medium (SMEM) mixture to generate platelet/RPE “cybrids.” Confocal microscopy, FLowSight Imaging cytometry, and Seahorse XF Mito Stress test were used to analyze mitochondrial function. Western Blotting was used to analyze expression of autophagy and mitophagy proteins.ResultsWe found that MELAS iPSC-derived RPE cells exhibited key characteristics of native RPE. We observed heteroplasmy-dependent impairment of mitochondrial bioenergetics and reliance on glycolysis for generating energy in the MELAS iPSC-derived RPE. The degree of heteroplasmy was directly associated with increased activation of signal transducer and activator of transcription 3 (STAT3), reduced adenosine monophosphate-activated protein kinase α (AMPKα) activation, and decreased autophagic activity. In addition, impaired autophagy was associated with aberrant lysosomal function, and failure of mitochondrial recycling. The mitochondria-depleted p0 cells replicated the effects on autophagy impairment and aberrant STAT3/AMPKα signaling and showed reduced mitochondrial respiration, demonstrating phenotypic similarities between p0 and MELAS iPSC-derived RPE cells.ConclusionsOur studies demonstrate that the MELAS iPSC-derived disease models are powerful tools for dissecting the molecular mechanisms by which mitochondrial DNA alterations influence RPE function in aging and macular degeneration, and for testing novel therapeutics in patients harboring the MELAS genotype.

Analysis of mutations in leu tRNA gene in patients of heart diseases

Cardiovascular diseases (CVD) are the leading cause of death all over the world. Beside general risk factors, there are some genetic factors which lead to cardiovascular diseases. Various nuclear DNA mutation and also mitochondrial DNA mutations have been related with cardiovascular diseases. In the present study, a total of 21 samples were collected from different families residing in district Dir. DNA was extracted from buccal epithelial cells using saliva. The mitochondrial tRNA leu (MT TL1) gene was amplified by PCR and 10 samples of different families were sequenced. The sequence was aligned with revised Cambridge Reference Sequence (rCRS) accession # NC-012920.1. It is concluded that cardiovascular diseases in our subjects are not due to mutation in the mitochondrial leucine tRNA gene. However, a large population of subjects with cardiovascular diseases needs to be studied and whole mitochondrial DNA is needed to be sequenced in the subjects with CVD. This will give an idea about the probable DNA marker which can be used to prevent loses due to these diseases at a very early stages.

A wolf spider from South American grasslands: phylogenetic placement and redescription of Paratrochosina amica (Mello-Leitão 1941)

The male of the wolf spider Paratrochosina amica (Mello-Leitão 1941) is redescribed, and the female of this species is described for the first time. Additionally, we evaluate the phylogenetic position of P. amica using the mitochondrial genes cytochrome c oxidase subunit I (cox1), the 12S rRNA (12S) and 16S rRNA (16S), NADH deshydrogenase subunit I (nad1) and the complete tRNA leu (L1). In live specimens we recognized three different morphotypes based on colour patterns. There were no differences in genitalic features of males and females among the three morphotypes. Molecular results through GMYC analyses did not identify independent evolutionary lineages. Genetic and taxonomic results support that the three morphotypes are conspecific. Phylogenetic analysis confirms the position of P. amica in the subfamily Allocosinae.

Mitochondrial Dysfunctions Contribute to Hypertrophic Cardiomyopathy in Patient iPSC-Derived Cardiomyocytes with MT-RNR2 Mutation.

SummaryHypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death in young individuals. A potential role of mtDNA mutations in HCM is known. However, the underlying molecular mechanisms linking mtDNA mutations to HCM remain poorly understood due to lack of cell and animal models. Here, we generated induced pluripotent stem cell-derived cardiomyocytes (HCM-iPSC-CMs) from human patients in a maternally inherited HCM family who carry the m.2336T>C mutation in the mitochondrial 16S rRNA gene (MT-RNR2). The results showed that the m.2336T>C mutation resulted in mitochondrial dysfunctions and ultrastructure defects by decreasing the stability of 16S rRNA, which led to reduced levels of mitochondrial proteins. The ATP/ADP ratio and mitochondrial membrane potential were also reduced, thereby elevating the intracellular Ca2+ concentration, which was associated with numerous HCM-specific electrophysiological abnormalities. Our findings therefore provide an innovative insight into the pathogenesis of maternally inherited HCM.

Population genetic structure of coastal Croatian honeybees (Apis mellifera carnica)

The genetic structure and molecular diversity of Croatian honeybee coastal populations have been investigated with microsatellite and mitochondrial markers. According to sequence data of the mito- chondrial tRNA leu -cox2 intergenic region, all analysed samples belong to the Central Mediterranean and Southeast European evolutionary C-lineage. Four mitochondrial haplotypes have been found in the Croat- ian honeybees, whereas two newly described have been found in Croatia and Greek respectively. Through the Bayesian analysis of microsatellite variation, two groups can be distinguished within the Croatian hon- eybee population, suggesting the existence of two subpopulations of A. m. carnica. The relation of these subpopulations with previously described ecotypes and regional variations is discussed. These results em- phasize the importance of sequencing in the description of new haplotypes and therefore, in the inference of molecular biodiversity within honeybee populations. The description of two subpopulations in coastal Croatian honeybees must be considered in future conservation strategies. honeybees / mitochondrial DNA / microsatellites / population structure / Croatia

Molecular phylogeny of the tribe Sphodrini (Coleoptera: Carabidae) based on mitochondrial and nuclear markers

A phylogenetic analysis of 6.4 kb of nucleotide sequence data from seven genes (mitochondrial cox1-cox2 and tRNA leu, and nuclear Ef-1α C0, Ef-1α C1, 28S, and 18S) was done to reconstruct the phylogenetic relationships of the ground-beetle tribe Sphodrini. Gene regions of variable nucleotide length were aligned using both a secondary structure model, Clustal W, and a combination of the two. Sensitivity analysis was performed in order to explore the effect of alignment methods. The ribosomal and protein-coding genes were largely congruent based on the ILD test and partitioned Bremer support measures. MtDNA analysis provided high resolution and high support for most clades. The tribe Sphodrini and the related tribes Platynini, Pterostichini and Zabrini made up monophyletic clades, but the relationship between them was weakly resolved and sensitive to alignment strategy. Previously suggested relationships between subtribes of Sphodrini were not corroborated, and only the subtribe Atranopsina revealed high support as the sister clade to the other subtribes. The analyses clearly demonstrated the importance of exploring effects of alignment methods that may become particularly important in resolving polytomies and nodes with low support.

M.P.1.13 Novel mitochondrial tRNA LEU(CUN) transition and D4Z4 partial deletion in a patient with a facioscapulohumeral phenotype

M.P.1.13 Novel mitochondrial tRNA LEU(CUN) transition and D4Z4 partial deletion in a patient with a facioscapulohumeral phenotype

Geographic variation in the Japanese islands ofApis cerana japonicaand inA. ceranapopulations bordering its geographic range

Genetic variation among Apis cerana japonica isolates from Japan and Apis cerana isolates from the neighboring areas of Russia, South Korea, and Taiwan was determined from DNA sequences of the mitochondrial DNA non-coding region (between tRNA leu and COII). Three haplotypes were identified among 470 colonies samples at 47 Japanese sites. All isolates from the main Japanese Islands of Honshu, Shikoku, and Kyushu belonged to a single haplotype, a previously reported Japan 1 haplotype. Two new haplotypes were found on the far southern Japanese islands of Amami-Oshima and Tsushima (the Japan 3 and Japan 4 haplotypes, respectively). The A. cerana from Russia and South Korea were the Japan 1 isolate, the A. cerana from Taiwan was the previously known Taiwan haplotype. Our studies showed little genetic variation in the mtDNA of A. cerana japonica, indicating that this genomic region is of limited use for detecting genetic variation among closely related populations of A. cerana.

Detection of Low Levels of the Mitochondrial tRNALeu(UUR) 3243A>G Mutation in Blood Derived from Patients with Diabetes

Mutations in the human mitochondrial genome have been suspected to play a significant role in the etiological development of mitochondrial diabetes. Detection of the 3243A>G mutation in the mitochondrial transfer RNALeu(UUR) gene (MTTL1), especially at low heteroplasmy levels, is highly desirable since it facilitates the diagnosis and subsequent management of the disease. The proportions of mutant mitochondrial DNA (mtDNA) can vary between tissues and are usually significantly higher in muscle than in blood, but muscle biopsies from patients with diabetes are rarely available. Here, we describe a technique that can not only determine the presence of MTTL1 3243A>G, but can also estimate the percentage of mutant DNA. The technique is based on the use of the WAVE system for the high-performance liquid chromatography (HPLC)-mediated analysis of mutation-specific restriction fragments derived from mutant PCR amplicons. PCR amplicon restriction fragment analysis by HPLC (PARFAH) can also be used for the detection of other mutations. This PARFAH analytical approach led to the discovery of the 3243A>G mutation in blood samples from a series of patients who had initially been reported to lack the mutation, even though matrilineal relatives had been shown to harbor the mutation associated with maternally inherited diabetes and deafness (MIDD) or mitochondrial myopathy encephalopathy lactic acidosis stroke-like episodes (MELAS) phenotypes. We have established that the PARFAH method can reliably detect as little as 1% mutant DNA in a sample, which would normally be missed by commonly used gel electrophoresis or sequencing methods. The PARFAH method not only provides a sensitive, high-throughput, and cost-effective strategy for the detection of low levels of mtDNA mutations in peripheral tissues, but also facilitates the estimation of the percentage of mutant DNA in the sample. The fact that samples can be readily obtained from peripheral tissues in many cases will avoid the need for invasive muscle biopsies. Our ability to detect low levels of mtDNA mutations in blood samples of carriers will allow us to reassess the prevalence of the MTTL1 3243A>G mutation in patients with diabetes.

Phylogenetic position of a Pacific Northwest North American endemic cyanolichen, Nephroma occultum (Ascomycota, Peltigerales)

Nephroma occultum is a COSEWIC listed cyanolichen of “Special Concern”. It is endemic to old-growth cedar-hemlock forests in western North America. This is the first study to place N. occultum into a phylogenetic framework using nucleotide sequence and secondary structure data. It also addresses the phylogenetic relationship between N. occultum and N. arcticum. Analysis of fungal Internal Transcribed Spacer (ITS) ribosomal DNA (rDNA) produced four major clades. The cyanobacterial transfer RNALeu intron (tRNALeu) from N. occultum was separated from that of N. isidiosum and fell between the Eurasian and North American epiphytic taxa. Examination of length and complexity of the folded secondary structures revealed different trends in the ITS1 and ITS2 rRNA regions. Even though N. occultum is endemic to North America, it seems more closely related to South American temperate rainforest species than to the sympatric N. arcticum. Nephroma occultum is alone among the studied species of Peltigerales in having an exceptionally long ITS1 region, and a different tRNALeu intron DNA sequence of the photobiont suggesting association with a unique genotype of Nostoc. It may be argued that the fitness of N. occultum may be influenced by the complex ITS1 RNA structure, a unique photobiont genotype undergoing a genetic bottleneck, no sexual reproduction to generate variation, and the inability to associate with different photobionts to adapt to changing habitats.

Species diversification patterns in the Polynesian jumping spider genus Havaika Prószyński, 2001 (Araneae, Salticidae)

Hotspot archipelagoes provide exceptional models for the study of the evolutionary process, due to the effects of isolation and topographical diversity in inducing the formation of unique biotic assemblages. In this paper, we examine the evolutionary patterns exhibited by the jumping spider genus Havaika Prószyński, 2001 in the Polynesian islands of the Hawaiian and Marquesas chains. To date, systematic research on Havaika has been seriously limited by the poor taxonomic knowledge on the group, which was based on a handful of specimens that showed continuous variability and lacked clear-cut diagnostic characters. Here, we circumvent this problem by inferring a phylogeny based on DNA sequences of several fragments including both mitochondrial (protein coding cytochrome oxidase I, NAD1 dehydrogenase, ribosomal 16S, and tRNA leu) and nuclear (internal transcribed spacer 2) genes, and a statistical morphological analyses of a large sample of specimens. Results suggest that the Marquesan and Hawaiian Havaika may be the result of independent colonizations. Furthermore, data provide little support for the standard “progression rule” (evolution in the direction of older to younger islands) in Hawaiian Islands. This may be explained by a recent arrival of the group: age estimates of the different lineages suggest that Havaika colonized the Hawaiian Islands after most of the extant islands were already formed. The lack of clear-cut diagnostic characters among species may also be explained by the recent origin of the group since molecular data do not provide any evidence of hybridization among lineages. Quantitative morphological data coupled with the phylogenetic information allow us to reevaluate the current limitation of Havaika taxonomy. Molecular data support the existence of at least four different evolutionary lineages that are further morphologically diagnosable. However, genealogical relationships are better predicted by geographical affinity (i.e. island) than by morphological characters used in the original descriptions of the species. A pattern of size segregation linked to largely overlapping distributions of some of the species hints at a potential involvement of competition in generating morphological diversity. This study contributes to our understanding on the origin and shaping of the biodiversity of oceanic islands and sets the stage for more detailed studies on particular aspects of these previously overlooked spiders.

Simultaneous Detection and Quantification of Mitochondrial DNA Deletion(s), Depletion, and Over-Replication in Patients with Mitochondrial Disease

Heterogeneous clinical expression of mitochondrial DNA (mtDNA) disorders depends on both qualitative and quantitative changes in mtDNA. We developed a sensitive and effective method that simultaneously detects mtDNA deletion(s) and quantifies total mtDNA content. The percentage of deletions and mtDNA content of 19 patients with single or multiple deletions were analyzed by real-time quantitative polymerase chain reaction (real-time qPCR) using TaqMan probes specific for mtDNA (tRNA leu(UUR), ND4, ATPase8, and D-loop regions) and nuclear DNA (AIB1, beta-2-microglobulin, and beta-actin). The proportion of deletion mutants determined by real-time qPCR was consistent with that determined by Southern analysis. Most patients with mtDNA deletions also demonstrated compensatory mtDNA over-replication. Multiple mtDNA deletions that were not detectable by Southern analysis due to low percentage of each deletion molecule were readily detected and quantified by real-time qPCR. Furthermore, 12 patients with clinical features and abnormal biochemical/histopathological results consistent with mitochondrial respiratory chain disorders without identified mtDNA mutations had either substantially depleted or significantly over-replicated mtDNA content, supporting the diagnosis of mitochondrial disease. Our results demonstrate that both qualitative and quantitative analyses are important in molecular diagnosis of mitochondrial diseases. The presence of deletion(s) and mtDNA depletion or compensatory over-replication can be determined simultaneously by real-time qPCR.

Molecular identification of red tide-causing microalga Heterosigma akashiwo strains based on their chloroplast DNA sequences

: In order to establish genetic signatures for intraspecific identification, parts of the genes rbcL, rbcS and psbA and their intergenic spacer (IGS) regions were amplified by polymerase chain reaction (PCR) from the chloroplast genomes of Heterosigma akashiwo strains isolated from Japan. A transfer RNA-Leu gene trnL and a hypothetical gene cfxQ, which is related to ribulose bisphosphate carboxylase/oxygenase expression, were found in the upstream region of the rbcL gene and in the rbcS-psbA IGS region, respectively. All the gene-coding regions and the IGS regions between rbcL and rbcS showed the same sequences among the strains tested. In contrast, the rbcL upstream regions and the rbcS-cfxQ IGS regions showed some differences such as nucleotide substitutions, duplications and inversions between NIES-5 and the other strains. Based on these sequence data, five genetic signatures were established and their simple and rapid detection by means of strain-specific PCR primers and PCR-restriction fragment length polymorphism techniques was examined. The results suggested the usefulness of these genetic signatures and techniques for the discrimination of H. akashiwo populations.

Opening the black box: phylogenetics and morphological evolution of the Malagasy fossorial lizards of the subfamily “Scincinae”

The island of Madagascar harbors a highly endemic vertebrate fauna including a high diversity of lizards of the subfamily “Scincinae,” with about 57 species in eight genera. Since limb reduction seems to have been a common phenomenon during the evolution of Malagasy “scincines,” diagnosing evolutionary relationships based on morphology has been difficult. Phylogenetic analyses of multiple mitochondrial DNA sequences including the entire ND1, tRNA LEU, tRNA ILE, tRNA GLN genes, and fragments of the 12S and 16S rRNA and tRNA MET genes were conducted to test the monophyly of the largest genus Amphiglossus, and to evaluate the various formal and informal species groupings previously proposed for this species-rich group. A further objective was to determine the phylogenetic placements of the several greatly limb-reduced and limbless Malagasy “scincines” and ascertain whether any of these are derived from within the morphologically plesiomorphic Amphiglossus. As limb reduction in skinks is mostly associated with body elongation via an increase in the number of presacral vertebrae, we evaluate the pattern of evolution of the numbers of presacral vertebrae in the context of our phylogeny. We demonstrate that Amphiglossus as currently diagnosed is non-monophyletic, and the species fall into two major groups. One of these groups is a clade that contains the included species of the subgenus Amphiglossus ( Madascincus) among other species and is a member of a larger clade containing Paracontias and Pseudoacontias. In the second group, the nominate subgenus Amphiglossus ( Amphiglossus) forms several subclades within a larger clade that also contains Androngo crenni and Pygomeles braconnieri, and is sister to Voeltzkowia. All analyses provide strong support for the monophyly of Paracontias and Voeltzkowia. Based on the preferred phylogenetic hypothesis and weighted squared-change parsimony we show that the ancestor of the Malagasy clade was already elongated and had a moderately high number of presacral vertebrae (46–48), which is hypothesized to be the ancestral condition for the whole Malagasy “scincine” clade. We further demonstrate that both multiple increases and reductions of presacral vertebrae evolved in many clades of Malagasy “scincines” and that the use of presacral vertebrae as a major character to diagnose supraspecific units is dubious. Based on our results and published morphological evidence we consider Scelotes waterloti Angel, 1930 to be a junior synonym of Amphiglossus reticulatus (Kaudern, 1922).

Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: a single-step approach.

The A3243G mitochondrial tRNA leu(UUR) point mutation causes mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, the most common mitochondrial DNA (mtDNA) disorder, and is also found in patients with maternally inherited diabetes and deafness syndrome (MIDD). To correlate disease manifestation with mutation loads, it is necessary to measure the percentage of the A3243G mtDNA mutation. To reliably quantify low proportions of the mutant mtDNA, we developed a real-time amplification refractory mutation system quantitative PCR (ARMS-qPCR) assay. We validated the method with experimental samples containing known proportions of mutant A3243G mtDNA generated by mixing known amounts of cloned plasmid DNA containing either the wild-type or the mutant sequences. A correlation coefficient of 0.9995 between the expected and observed values for the proportions of mutant A3243G in the experimental samples was found. Evaluation of a total of 36 patient DNA samples demonstrated consistent results between PCR-restriction fragment length polymorphism (RFLP) analysis and real-time ARMS-qPCR. However, the latter method was much more sensitive for detecting low percentages of mutant heteroplasmy. Three samples contained allele-specific oligonucleotide-detectable but RFLP-undetectable mutations. The real-time ARMS-qPCR method provides rapid, reliable, one-step quantitative detection of heteroplasmic mutant mtDNA.

Ineffective hematopoiesis linked with a mitochondrial tRNA mutation (G3242A) in a patient with myelodysplastic syndrome

AbstractIn a patient with refractory anemia with excess blasts (RAEB), a somatic mutation of mitochondrial transfer RNALeu(UUR) was detected in bone marrow cells. Heteroduplex analysis indicated that 40% to 50% of mitochondrial DNA (mtDNA) molecules in the bone marrow (BM) carried the novel G3242A mutation. The proportion of mutant mtDNA was higher in CD34+ cells than in the unfractionated sample. Surprisingly, the mutation was not detectable by heteroduplex analysis in the peripheral blood (PB). However, PB CD34+ cells selected by immunomagnetic beads harbored the mutation with a proportion of approximately 50%. In hematopoietic colony assays, CD34+ cells from BM and PB yielded only colonies with wild-type mtDNA. These results indicate that the mtDNA mutation in CD34+ cells was associated with a maturation defect. Mitochondrial tRNA mutations impair mitochondrial protein synthesis, thereby causing dysfunction of the mitochondrial respiratory chain. We propose that this effect contributed to ineffective hematopoiesis in our patient.

New haplotypes for the non-coding region of mitochondrial DNA in cavity-nesting honey bees Apis koschevnikovi and Apis nuluensis

The cavity-nesting honeybees, Apis cerana, A. koschevnikovi and A. nuluensis, are distributed in Sabah, Borneo. We sequenced the non-coding intergenic region between the tRNA leu and COII mitochondrial genes of these three species. About 50 different haplotypes have been reported for the non-coding region of A. cerana, A. koschevnikovi and A. nigrocincta. This study detected new haplotypes in A. koschevnikovi and A. nuluensis, which we designated Sabah 1 and Sabah short, respectively. Sabah short is the first short sequence reported from Borneo.

Islands under the desert: molecular systematics and evolutionary origins of stygobitic water beetles (Coleoptera : Dytiscidae) from central Western Australia

Calcrete aquifers in the Yilgarn Craton of central Western Australia have recently been found to contain a rich invertebrate stygofauna, including the world's most diverse collection of stygobitic dytiscid water beetles. Our aim was to determine the evolutionary relationships between 11 species of stygobitic dytiscids and epigean species of the tribe Bidessini. Phylogenetic analyses of 1431 base pairs of mitochondrial DNA (mtDNA), including segments of cytochrome oxidase 1, 16S rRNA, tRNA leu and NADH dehydrogenase 1 genes, revealed that 10 of the 11 species form a natural group within the Bidessini and are most closely related to epigean taxa from the genera Allodessus Guignot, Boongurrus Larson, Limbodessus Guignot and Liodessus Guignot. The analyses support the morphological taxonomy of the stygobitic beetles at the species level, but generic level classification is not concordant with mtDNA lineages. Sympatric species of the large Tjirtudessus Watts & Humphreys and smaller Nirridessus Watts & Humphreys are more closely related to each other than either are to their congeners, suggesting a possible case of sympatric speciation. The analyses indicate that there have been multiple independent origins of stygobitic dytiscids and that origins correlate with the onset of aridity during the Miocene and also provide evidence that each calcrete aquifer may represent a 'subterranean island'.

Frequency of mitochondrial transfer RNA mutations and deletions in 225 patients presenting with respiratory chain deficiencies

OBJECTIVETo evaluate the frequency of pathogenic mtDNA transfer RNA mutations and deletions in biochemically demonstrable respiratory chain (RC) deficiencies in paediatric and adult patients.METHODSWe screened for deletions and sequenced mitochondrial...

Molecular characterization and population structure of the honeybees from the balearic islands (Spain)

Honeybees (Apis mellifera L.) were collected from 23 localities on the Balearic islands in the Mediterranean Sea. The mitochondrial genome (mtDNA) was surveyed for diagnostic restric- tion sites and characterized with DraI digestion of the tRNA leu -COII intergenic region. Both approaches demonstrated that honeybees bearing either African or west European haplotypes coexist on the Balearic islands. Two African and two west European haplotypes were found with different fre- quencies and distribution among the islands. Phylogenetic and population structure analyses support the clustering of these islands in two groups: Majorca-Minorca (Gymnesic) and Ibiza-Formentera (Pityusic) what corroborates the current biogeographical division of the Balearic organisms. These results partially agree with the observed distribution of African haplotypes in honeybee populations from other Mediterranean islands. The present distribution of genetic markers may reflect also the influ- ence of human movements, trade and settlements from prehistoric times. Apis mellifera / Balearic islands / mtDNA / biogeography