Phylogeny of ladybirds (Coleoptera: Coccinellidae): are the subfamilies monophyletic?

The Coccinellidae (ladybirds) is a highly speciose family of the Coleoptera. Ladybirds are well known because of their use as biocontrol agents, and are the subject of many ecological studies. However, little is known about phylogenetic relationships of the Coccinellidae, and a precise evolutionary framework is needed for the family. This paper provides the first phylogenetic reconstruction of the relationships within the Coccinellidae based on analysis of five genes: the 18S and 28S rRNA nuclear genes and the mitochondrial 12S, 16S rRNA and cytochrome oxidase subunit I (COI) genes. The phylogenetic relationships of 67 terminal taxa, representative of all the subfamilies of the Coccinellidae (61 species, 37 genera), and relevant outgroups, were reconstructed using multiple approaches, including Bayesian inference with partitioning strategies. The recovered phylogenies are congruent and show that the Coccinellinae is monophyletic but the Coccidulinae, Epilachninae, Scymninae and Chilocorinae are paraphyletic. The tribe Chilocorini is identified as the sister-group of the Coccinellinae for the first time.

Origin and number of founders in an introduced insular primate: estimation from nuclear genetic data.

Cynomolgus macaques (Macaca fascicularis) were introduced on the island of Mauritius between 400 and 500 years ago and underwent a strong population expansion after a probable initial founding event. However, in practice, little is known of the geographical origin of the individuals that colonized the island, on how many individuals were introduced, and of whether the following demographic expansion erased any signal of this putative bottleneck. In this study, we asked whether the current nuclear genome of the Mauritius population retained a signature that would allow us to answer these questions. Altogether, 21 polymorphic autosomal and sex-linked microsatellites were surveyed from 81 unrelated Mauritius individuals and 173 individuals from putative geographical sources in Southeast Asia: Java, the Philippines islands and the Indochinese peninsula. We found that (i) the Mauritius population was closer to different populations depending on the markers we used, which suggests a possible mixed origin with Java playing most probably a major role; and (ii) the level of diversity was lower than the other populations but there was no clear and consistent bottleneck signal using either summary statistics or full-likelihood methods. However, summary statistics strongly suggest that Mauritius is not at mutation-drift equilibrium and favours an expansion rather than a bottleneck. This suggests that on a short time scale, population decline followed by growth can be difficult to deduce from genetic data based on mutation-drift theory. We then used a simple Bayesian rejection algorithm to estimate the number of founders under different demographic models (exponential, logistic and logistic with lag) and pure genetic drift. This new method uses current population size estimates and expected heterozygosity of Mauritius and source population(s). Our results indicate that a simple exponential growth is unlikely and that, under the logistic models, the population may have expanded from an initial effective number of individuals of 10-15. The data are also consistent with a logistic growth with different lag values, indicating that we cannot exclude past population fluctuation.

Factors shaping genetic variation in the MHC of natural non-human primate populations.

Across a large distribution range, population-specific factors as well as pathogen-mediated selection may shape species genetic diversity in the major histocompatibility complex (MHC). We have studied genetic diversity and population differentiation in the MHC region of the Southeast Asian cynomolgus macaque (Macaca fascicularis fascicularis), a species with large and discontinuous range, in order to investigate the role of demography vs selection. Genetic variation was assessed at seven MHC microsatellites on 272 individuals from five populations (Indochina, Java, Borneo, Philippines, and Mauritius). A high genetic diversity was observed in all populations and the Philippines but also the Mauritius populations were the most genetically differentiated. The strength and extent of linkage disequilibrium (LD) (up to 4 Mb) varies across populations mainly because of demographic factors. In Indochina, the complete lack of LD could be the signature of ancient hybridization between cynomolgus and rhesus macaques in the Indochinese peninsula. With the additional support of seven autosomal microsatellites, tests for outlier loci based on intrapopulation diversity and interpopulation differentiation (using F-statistic) allowed to dissociate demographic from selective histories: (i) demographic history may itself explain levels of MHC variability in the Mauritius populations and (ii) positive selection could be responsible for the Philippines population differentiation, especially in the MHC class II region. Among various pathogens, Plasmodium knowlesi and Plasmodium coatneyi are two likely candidates to explain the higher frequency of some MHC haplotypes. Indeed, literature describes low parasitemia in the Philippines individuals, contrasting with fatal infections provoked by these parasites in other cynomolgus macaque populations.

Unbiased interpretation of haplotypes at duplicated microsatellites.

The Y-chromosome is a powerful tool for population geneticists to study human evolutionary history. Haploid and largely non-recombining, it should contain a simple record of past mutational events. However, this apparent simplicity is compromised by Y-linked duplicons, which make up approximately 35% of this chromosome; 25% of these duplicons are large inverted repeats (palindromes). For microsatellites lying in these palindromes, two loci cannot be easily distinguished due to PCR co-amplification, and this order misspecification of alleles generates an additional variance component. Due to this ambiguity, population geneticists have traditionally used an arbitrary method to assign the alleles (shorter allele to locus 1, larger allele to locus 2). Here, we simulate these posterior estimate distributions under three different novel allele assignment priors and compare this with the original method. We use a sample of 33 human populations, typed for duplicated microsatellites lying within palindrome P8, to illustrate our approach. We show that both intra- and inter-population statistics can be dramatically affected by order misspecification. Surprisingly, matrices of pairwise F-statistics or distance estimates appear far less sensitive to order misspecification and remain relatively unchanged under the priors considered, suggesting that these microsatellites can be considered as useful markers for population genetic studies using an appropriate data treatment. Duplicated microsatellites represent an attractive source of information to investigate the extensive structural polymorphism observed among human Y chromosomes, as well as processes of intra-chromosomal gene conversion acting between duplicons.

Multiplexed microsatellites for rapid identification and characterization of individuals and populations of Cercopithecidae.

Cross-amplification of 15 human microsatellites was performed successfully in cynomolgus (Macaca fascicularis) and rhesus (M. mulatta) macaques and 11 other Cercopithecidae species of biomedical and conservation relevance. To allow for quick, efficient, and high-throughput genotyping to assess intra- and interspecific genetic variation, we performed three multiplex sets, each comprised of five markers from different parts of the genome (i.e., autosomes, the MHC region, and the X-chromosome). These multiplex sets are likely to reveal allelic divergence between taxa, which could be used for their discrimination. Population studies on three regional populations of M. fascicularis and one of M. mulatta revealed that most of the loci, with the exception of one monomorphic locus, displayed polymorphisms (the expected heterozygosities were 0.48-0.91 for M. fascicularis, and 0.61-0.93 for M. mulatta), which makes them useful for population genetics. For the multiplex set M1, including the nonlinked autosomal markers, low probabilities of identity were observed: P(ID) values ranged from 8 x 10(-7) to 3 x 10(-5). This multiplex set is reliable for forensic applications, such as individual identification, parentage testing, and kinship analysis, in wild and captive populations.

Allele variation of DYS19 and Y-Alu insertion (YAP) polymorphisms in Basques: an insight into the peopling of Europe and the Mediterranean region.

Two Y-chromosome DNA polymorphisms, the DYS19 microsatellite and the YAP (at locus DYS287), were tested in males from two autochthonous Basque populations from France and northern Navarre (Spain). The results are compared to those obtained for the same genetic markers in 32 populations from Europe, northern Africa, and western Asia. The high predominance of the DYS19*11 (190-base-pair) allele in Basques indicates that their genetic diversity for microsatellite DYS19 is around half that observed in Europeans, North Africans, and western Asians. The Y-Alu insertion (YAP+) was not detected in the Basque samples. This study attempts to throw some light on the importance of historically recent migratory movements, the main corridors of gene flow, and demographic sizes and their variations in shaping gene frequency patterns in contemporary human populations, particularly in the Mediterranean region. Historical processes may have had more significant effects on the genetic make-up of current human populations than those of prehistoric times.

Microsatellite DNA markers from HLA region (D6S105, D6S265 and TNFa) in autochthonous Basques from Northern Navarre (Spain).

BACKGROUND: The extent of the genetic polymorphism of the HLA complex is becoming well characterized in Basque population and their subpopulations. This level of knowledge mainly concerns HLA class I loci. However, Basque population surveys dealing with HLA class II genes and/or microsatellites in the HLA region are still very scarce. AIM: The population genetics of three highly polymorphic short tandem repeat (STR) loci, D6S105, D6S265 and TNFa, from HLA region has been analysed in autochthonous (indigenous) Basques from Northern Navarre (Spain). The same blood samples have been typed for HLA class II genes from DQ/DR/DP regions and some findings from that information can be found therein. SUBJECTS AND METHODS: Blood samples were taken from 107 unrelated autochthonous Basques from Northern Navarre. The criterion used to define Northern Navarrese identity was that of three generations of Basque surnames and birthplaces. RESULTS: The main features observed in Navarrese Basques were the rather high frequencies of alleles D6S105*4 and D6S265*7. A novel allele has been detected at the D6S265 locus (13: 145 bp). The most frequent haplotype was D6S105*8-D6S265*4 with a highly significant linkage disequilibrium being presented. The high frequency of allele TNFa*1 in Basques is noteworthy and this characteristic is not shared by other European populations, where TNFa*1 is absent or shows negligible values. The multidimensional scaling analysis (MDS) for TNFa allele frequencies has shown a high variability among populations and that alleles TNFa*1 (F(ST) = 0.0615) and TNFa*12 (F(ST) = 0.0424) seem to have significant influence over the spatial population configuration. TNFa*2 showed the lowest FST value (0.0077) because of its conspicuous homogeneous distribution all over the European populations. CONCLUSIONS: Findings shown here on HLA microsatellites and their relationships with other HLA class I and class II genes in Basques can be helpful for those studies mainly addressed at detecting associations between HLA genes and diseases in the Basque area as a whole, and particularly in its autochthonous population, settled there since remote times.

Molecular variation of HLA class I genes in the Corsican population: approach to its origin.

Three human leucocyte antigen (HLA) class I loci (HLA-A, -B and -Cw) were typed for the first time at the DNA level in the Corsican population. This analysis was performed on 100 individuals of Corsican origin living in central Corsica (46 individuals) and in south-western Corsica (54 individuals). The genetic structure of these two subpopulations was analysed on the basis of the molecular polymorphisms of the HLA-A, -B and -Cw genes. A phylogenetic and a haplotypic analysis were performed. The genotypic analysis did not detect genetic differentiation. Examination of the allelic and haplotypic frequencies did, however, reveal some significant differences between the two zones. Similarities with the Sardinian population were found, and were particularly evident in the south-western sample. However, Corsica has probably been subject to greater West European influence, which can be seen in the Corte sample (Haute Corse).

Testing the reliability of noninvasive genetic sampling by comparing analyses of blood and fecal samples in Barbary macaques (Macaca sylvanus).

Genetic studies of wild animal populations are often hindered by difficulties in obtaining blood samples. Recent advances in molecular biology have allowed the use of noninvasive samples as sources of DNA (e.g., hair or feces), but such samples may provide low-quality DNA and prevent the determination of true genotypes in subsequent DNA analysis. We present a preliminary study aimed at assessing the reliability of using fecal samples for genotyping in Barbary macaques (Macaca sylvanus). The test was performed on samples of blood and feces from 11 captive animals, using three dinucleotide microsatellites. The CTAB DNA extraction method was found to be the most relevant for Barbary macaque feces, yielding successful amplification at all loci for 70% of PCRs. All the fecal samples tested gave correct genotypes at least once for each locus when referenced against blood-derived genotypes. An average of 18.3% of PCRs displayed spurious genotypes (false homozygous or false allele). The minimum theoretical probability required to obtain a 100% accurate genotype is 0.74, based on the criterion that a correct genotype is assessed only if it was observed at least twice. The observed probability of obtaining a correct genotype from three PCRs, based on our genotyping results, was greater (0.81 on average) than the minimum threshold. In conclusion, our comparison of blood and fecal samples showed that fecal sampling is a reliable tool for the further study of wild Barbary macaque populations.

West Mediterranean islands (Corsica, Balearic islands, Sardinia) and the Basque population: contribution of HLA class I molecular markers to their evolutionary history.

The genetic structure of Balearic islands (Corsica and Sardinia), situated on the same trans-Mediterranean maritime routes and having very similar histories, were compared and their position among the neighbouring Caucasian populations was inferred. For this purpose, three HLA loci (HLA-A, -B and -Cw) were typed at the DNA level in these populations and the allelic and haplotypic frequencies were estimated. Because previous studies have shown common genetic features in the Sardinians and Basques, HLA-Cw molecular typing was also performed in a sample of French Basques in order to establish the haplotypic structure of this population for a more accurate comparison with the three others. By its allelic composition, the Corsican population has an intermediate position between the two other islander populations. Its close relationship with the Sardinian population, however, was clearly revealed by the phylogenetic analysis which also suggests a proximity with eastern Mediterranean peoples, whereas the Balearic islands are more narrowly related to Spain and western Europe. Peculiarities were observed in the distributions of some common haplotypes in the populations of the islands that confirm the results of the phylogenetic analysis and could be related to their history. Noteworthy is the presence of the HLA-A30-Cw*0501-B18 haplotype at frequencies approximately 2% in Corsica and the Balearic islands, yet the estimated frequencies of this haplotype are much lower than in the Sardinian and Basque populations.

Evolution of an Alu DNA element of type Sx in the lineage of primates and the origin of an associated tetranucleotide microsatellite.

A 394-bp DNA fragment, which in human is on chromosome 6 near the MOG (myelin oligodendrocyte glycoprotein) gene and encompasses an Alu element and an associated tetranucleotide microsatellite, was sequenced from a large range of primate species to follow its evolutionary divergence and to understand the origin of the microsatellite. This Alu element is found at the same orthologous position in all primates sequenced, but the tetranucleotide repeat is present only in Catarrhini between the 3'-oligo(dA) of the Alu element and the 3' flanking direct repeat. Little intraspecific variation was found. Sequence identity values for this orthologous primate Alu averaged 90% (82-99%) with transitions comprising between 70% and 100% of the observed nucleotide substitutions. Although the insertion of the Alu element predates the separation of these species, the original sequence of the site of integration can still be identified. This identification of the direct repeats suggests an active role of the oligo(dA) of the Alu element in the origin of the tetranucleotide repeats. The microsatellite probably appeared after the insertion of the Alu element, early in the lineage leading to the common ancestor of the hominoids and the Old World monkeys.

Conservation and evolution of microsatellite loci in primate taxa.

Microsatellites are promising genetic markers for the study of demographic structure and phylogenetic history in populations. However, little information exists on the molecular nature of the repeats and their flanking sequences of a same microsatellite in a large range of species. In this study, we report polymorphism and consensus sequences of eight microsatellite loci using human primers in 20 primate species. The results show size polymorphism in almost all species and microsatellites. These loci are therefore useful markers for population genetic studies between populations of the same species. Insertion/deletion events are frequent in the flanking regions, the majority concerning several contiguous bases. This is in contrast with the more usual single base pair events in non-coding regions. The ranges of allele lengths in non-human primates often show no overlap with that of human, usually due to the deletion/insertion events in the flanking sequences, producing smaller allele lengths rather than smaller numbers of repeats. The use of length of PCR product will bias the inter-species interpretation reducing the number of observable alleles and treating as the same allele very divergent molecular sequences. Caution should be used when employing microsatellites in cross-species comparisons in which the species under study are separated by significant amounts of evolutionary time: in such cases allele comparison cannot be based on lengths alone.

Trans-speciation maintenance in the MHC region of a polymorphism which includes a polymorphic dinucleotide locus, and the de novo arisal of a polymorphic tetranucleotide microsatellite.

Alleles and the surrounding regions of DQCAR, a dinucleotide repeat tightly linked to HLA-DQB1, were sequenced in a range of primate species including man. Three polymorphic regions can usefully be defined in the description of these sequences: the dinucleotide GT repeat itself, the anonymous region 5' of this repeat, and a variable CTGT repeat in the 3' region. The 5' sequence displayed six alleles in the individuals studied. One of these alleles was invariably associated with substitutions in the GT repeat and absence of the CTGT repeat, the others with pure, polymorphic GT repeats and variation in the numbers of CTGT repeats. Haplotypes can be classified by the allele in the 5' region. Those carrying allele 1 were only found in man, those with allele 2 in man, chimpanzee and gorilla. The third haplotype (indicated by the presence of allele 3) was found in chimpanzee, gorilla and orang-utan, the fourth in chimpanzee and gibbon, the fifth in baboon, guenon and mangabey and the sixth in guenon and macaque. The alleles in the 5' region, but from different species, are thus often more similar than alleles from the same species, a phenomenon already shown for some HLA genes. This suggests that major histocompatibility sequences and surrounding sequences shared a correlated evolutionary history. The new polymorphic tetranucleotide microsatellite (CTGT, 3rd region) has possibly arisen de novo from the pre-existing dinucleotide GT. This study provides information not only on the molecular evolution of this particular microsatellite but also of the trans-speciation maintenance of polymorphism of its surrounding sequences.

A gene recently inactivated in human defines a new olfactory receptor family in mammals.

The olfactory receptor (OR) gene family constitutes one of the largest multigene families and is distributed among many chromosomal sites in the human genome. Four OR families have been defined in mammals. We previously demonstrated that a high fraction of human OR sequences have incurred deleterious mutations, thus reducing the repertoire of functional OR genes. In this study, we have characterized a new OR gene, 912-93, in primates. This gene is unique and it defines a new OR family. It localizes to human chromosome 11q11-12 and at syntenical sites in other hominoids. The sequence marks a previously unrecognized rearrangement of pericentromeric material from chromosome 11 to the centromeric region of gibbon chromosome 5. The human gene contains a nonsense point mutation in the region corresponding to the extracellular N-terminus of the receptor. This mutation is present in humans of various ethnic groups, but is absent in apes, suggesting that it probably appeared during the divergence of humans from other apes, <4 000 000-5 000 000 years ago. A second mutation, a frameshift at a different location, has occurred in the gorilla copy of this gene. These observations suggest that OR 912-93 has been recently silenced in human and gorilla, adding to a pool of OR pseudogenes whose growth may parallel a reduction in the sense of smell in primates.

Identification of Mycobacterium DNA in an Egyptian Pott's disease of 5,400 years old.

The antiquity of tuberculosis in the Old World is controversial because the morphology of the lesion in skeletal remains is non-specific. We report the recovery of a DNA fragment from a 5,400-year-old Predynastic Egyptian skeleton that exhibits a kyphotic, 'hunchback' spinal deformity consistent with Pott's disease and suggestive of tuberculous vertebral involvement. The recovered DNA fragment was sequenced and is consistent with an original Mycobacterium sequence. We cannot prove that it is M. tuberculosis, M. bovis or an ancient mycobacteria resembling the two current forms because the observed modifications in the sequence could be attributed to the antiquity of Mycobacterium and/or to the effects of Taq polymerase. This provides the most specific evidence for the antiquity of human Mycobacterium disease in the world.

Microsatellites in the HLA region: on overview.

Microsatellites are repeats of a DNA base motif (1-6 bp, mostly CA repeats) up to 100 times; they are distributed regularly all over the genome. Many of them are polymorphic and their high polymorphism is based upon a variable number of repeats. They are widely used for genetic mapping, linkage analysis, population genetics, evolutionary studies and in forensic medicine. Such markers have also been described in the HLA region since 1991, and a growing interest in their potential applications is being expressed. The aims of this review are: 1) to outline the presently available information from literature and molecular databases concerning 53 microsatellites in the HLA region (localization, type of repeat, number of alleles, heterozygosity, primers used for amplification); 2) to address the question of technical pitfalls when using such markers; 3) to discuss specific features such as their mutation rate (10 (-3) to 10 (-6), which is higher than that reported for HLA genes, and their linkage disequilibrium with HLA alleles; 4) to present an integrated map of microsatellites and genes of this region; and 5) to provide a synopsis of their different applications in HLA-related fields (disease studies, population genetics, recombination point studies, HLA region mapping, transplantation) along with perspectives for future use. Although some HLA region microsatellites have already been applied to the analysis of more than 10 diseases, it is now evident that their use in population genetics and the determination of genomic compatibility in bone marrow transplantation represent growing areas of application.

Microsatellite allelic homoplasy due to variable flanking sequences.

Microsatellite DNA sequences have become the dominant source of nuclear genetic markers for most applications. It is important to investigate the basis of variation between alleles and to know if current assumptions about the mechanisms of microsatellite mutation (that is to say, variations involving simple changes in the number of repeat) are correct. We have characterized, by DNA sequencing, the human alleles of a new highly informative (CA)n repeat localized approximately 20 kb centromeric to the HLA-B gene. Although 12 alleles were identified based on conventional length criteria, sequencing of the alleles demonstrated that differences between alleles were found to be more complex than previously assumed: A high degree of microsatellite variability is due to variation in the region immediately flanking the repeat. These data indicate that the mutational process which generates polymorphism in this region has involved not only simple changes in the number of dinucleotide CA repeats but also perturbations in the nonrepeated 5' and 3' flanking sequences. Three families of alleles (not visible from the overall length of the alleles), with presumably separate evolutionary histories, exist and can yield to homoplasy of size. Effectively, we can observe alleles of the same size with different internal structures which are separated by a significant amount of variation. Although allelic homoplasy for non-interrupted microsatellite loci has been suggested between different species, it has not been unequivocally demonstrated within species. A strong association is noted between alleles defined at the sequence level and HLA-B alleles. The observation of several families of alleles at the population level provides information about the evolutionary history and mutation processes of microsatellites and may have implications for the use of these markers in phylogenetic, linkage disequilibrium studies, and gene mapping.

Microsatellite variation in an introduced mouflon population.

As previous studies of genetic polymorphism in the mouflon (Ovis gmelini) have not provided any valuable markers for population studies, we tested the capacity of microsatellites to index the genetic diversity of a recently introduced mouflon population. Six pairs of bovine primer amplified microsatellites in mouflon, and all six were polymorphic. Furthermore, despite the low number of founders, five loci had a high gene diversity in this introduced population. Unlike other genetic markers, microsatellites could be powerful to study the genetic structure of mouflon populations.

New highly polymorphic microsatellite marker in linkage disequilibrium with HLA-B.

The difficulty of molecular typing of the HLA class I genes and the relevance of the genes of this region to disease susceptibility and transplantation have provided an impetus to develop useful typing markers. We have characterized by polymerase chain reaction analysis a new highly informative CA repeat localized approximately 25-kb centromeric to the gene HLA-B and 10-kb telomeric to the gene MICA. Twelve alleles defined by length were found in a sample of French Basques, with the PIC being 0.82. A detailed haplotype analysis was performed to investigate the association between this microsatellite and two others markers of the region (HLA-B gene and TNF region microsatellite). The 10 haplotypes with the highest estimated frequencies show evidence of a gametic association or linkage disequilibrium. A very strong association between the expressed HLA-B polymorphism and microsatellite alleles was also revealed in this sample and confirmed in the workshop cells lines of the Fourth Asia-Oceania Histocompatibility Workshop. This marker can be used in the fine mapping of this region and the association with some alleles of HLA-B may allow the replacement of HLA-B typing at least in a preliminary study. Moreover, these studies support the hypothesis of a high mutability for large alleles in microsatellite loci.