Comparison of different genetic distances to test isolation by distance between populations.

Studying isolation by distance can provide useful demographic information. To analyze isolation by distance from molecular data, one can use some kind of genetic distance or coalescent simulations. Molecular markers can often display technical caveats, such as PCR-based amplification failures (null alleles, allelic dropouts). These problems can alter population parameter inferences that can be extracted from molecular data. In this simulation study, we analyze the behavior of different genetic distances in Island (null hypothesis) and stepping stone models displaying varying neighborhood sizes. Impact of null alleles of increasing frequency is also studied. In stepping stone models without null alleles, the best statistic to detect isolation by distance in most situations is the chord distance DCSE. Nevertheless, for markers with genetic diversities HS<0.4-0.5, all statistics tend to display the same statistical power. Marginal sub-populations behave as smaller neighborhoods. Metapopulations composed of small sub-population numbers thus display smaller neighborhood sizes. When null alleles are introduced, the power of detection of isolation by distance is significantly reduced and DCSE remains the most powerful genetic distance. We also show that the proportion of null allelic states interact with the slope of the regression of FST/(1-FST) as a function of geographic distance. This can have important consequences on inferences that can be made from such data. Nevertheless, Chapuis and Estoup's FreeNA correction for null alleles provides very good results in most situations. We finally use our conclusions for reanalyzing and reinterpreting some published data sets.

Reproduction in Leishmania: A focus on genetic exchange.

One key process of the life cycle of pathogens is their mode of reproduction. Indeed, this fundamental biological process conditions the multiplication and the transmission of genes and thus the propagation of diseases in the environment. Reproductive strategies of protozoan parasites have been a subject of debate for many years, principally due to the difficulty in making direct observations of sexual reproduction (i.e. genetic recombination). Traditionally, these parasites were considered as characterized by a preeminent clonal structure. Nevertheless, with the development of elaborate culture experiments, population genetics and evolutionary and population genomics, several studies suggested that most of these pathogens were also characterized by constitutive genetic recombination events. In this opinion, we focused on Leishmania parasites, pathogens responsible of leishmaniases, a major public health issue. We first discuss the evolutionary advantages of a mixed mating reproductive strategy, then we review the evidence of genetic exchange, and finally we detail available tools to detect naturally occurring genetic recombination in Leishmania parasites and more generally in protozoan parasites.

A primer for Leishmania population genetic studies.

Leishmaniases remain a major public health problem. Despite the development of elaborate experimental techniques and sophisticated statistical tools, how these parasites evolve, adapt themselves to new environmental compartments and hosts, and develop resistance to new drugs remains unclear. Leishmania parasites constitute a complex model from a biological, ecological, and epidemiological point of view but also with respect to their genetics and phylogenetics. With this in view, we seek to outline the criteria, caveats, and confounding factors to be considered for Leishmania population genetic studies. We examine how the taxonomic complexity, heterozygosity, intraspecific and interspecific recombination, aneuploidy, and ameiotic recombination of Leishmania intersect with population genetic studies of this parasite.

Genetic comparison of Glossina tachinoides populations in three river basins of the Upper West Region of Ghana and implications for tsetse control.

Tsetse flies are the cyclical vectors of African animal trypanosomosis (AAT) and human African trypanosomosis (HAT). In March 2010, the Government of Ghana initiated a large scale integrated tsetse eradication campaign in the Upper West Region (UWR) (≈18,000 km(2)) under the umbrella of the Pan-African Tsetse and Trypanosomosis Eradication Campaign (PATTEC). We investigated the structuring of Glossina tachinoides populations within and between the three main river basins of the target area in the UWR. Out of a total sample of 884 flies, a sub-sample of 266 was genotyped at nine microsatellite loci. The significance of the different hierarchical levels was tested using Yang's parameters estimated with Weir and Cockerham's method. A significant effect of traps within groups (pooling traps no more than 3 km distant from each other), of groups within river basins and of river basins within the whole target area was observed. Isolation by distance between traps was highly significant. A local density of 0.48-0.61 flies/m(2) was estimated and a dispersal distance that approximated 11 m per generation [CI 9, 17]. No significant sex-biased dispersal was detected. Dispersal distances of G. tachinoides in the UWR were relatively low, possibly as a result of the fragmentation of the habitat and the seasonality of the Kulpawn and Sissili rivers. Moreover, very high fly population densities were observed in the sample sites, which potentially reduces dispersal at constant habitat saturation, because the probability that migrants can established is reduced (density dependent dispersal). However, the observed spatial dispersal was deemed sufficient for a G. tachinoides-cleared area to be reinvaded from neighboring populations in adjacent river basins. These data corroborate results from other population genetics studies in West Africa, which indicate that G. tachinoides populations from different river basins cannot be considered isolated.

First clinical case of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis in a domestic cat from French Guiana.

We report the first case of natural infection of a domestic female cat (Felis catus) by Leishmania (Viannia) braziliensis in French Guiana. The infected animal had a cutaneous ulcer on the nose and nodules of different sizes in the ears. The diagnosis was confirmed by molecular analysis of cutaneous samples that detected the presence of Leishmania parasites and allowed identifying the Leishmania (Viannia) braziliensis species. The discovery of a cat infected by L. (V.) braziliensis suggests the possibility that cats could be potential secondary reservoirs of Leishmania parasites in French Guiana. Thus, it would be important to investigate the possible epidemiological role of domestic cats in domestic foci of Leishmania in this region.

Strong genetic structure in Cameroonian populations of Bulinus truncatus (Gastropoda: Planorbidae), intermediate host of Schistosoma haematobium.

In order to evaluate the snail host contribution on the variability of mollusk/schistosome compatibility, the genetic structure of seven Cameroonian populations of the schistosome vector, Bulinus truncatus, was studied using four variable microsatellite loci. A substantial polymorphism mainly distributed among populations was observed. No heterozygous genotype was scored, confirming the high level of selfing rate occurring in B. truncatus populations. Contemporaneous samples were highly and significantly differentiated with a marginally significant correlation with geographical distances (P-value=0.069). The different sites sampled seemed to rarely exchange migrants with very small Nm (∼0.22 or below). The data also suggest that B. truncatus subpopulations might be composed of very small and isolated units at much smaller surfaces than what was investigated. Even if more data (in particular more loci) will be needed to confirm these issues, they suggest that restricted gene flow plays an important role in maintaining differentiation among snail populations in the transmission foci, potentially leading to specific adaptation between each B. truncatus population and its local Schistosoma haematobium population.

Swift sympatric adaptation of a species of cattle tick to a new deer host in New Caledonia.

The occurrence and frequency of sympatric speciation in natural systems continue to be hotly debated issues in evolutionary biology. This might reflect the timescale over which evolution occurs resulting in there being few compelling observations of the phenomenon (lake fishes, phytophagous insects and Island trees). Despite predictions, few examples of sympatric speciation have been recorded in animal parasites, at least widely accepted as such. Here we show that, in New Caledonia, the monophasic (exploiting one individual host per generation) cattle tick Rhipicephalus microplus has evolved in contact with two sympatric host species into two differentiated genetic pools: on the cattle, its original host and on rusa deer, a new host for this tick. This sympatric isolation has occurred over a relative short period of time (i.e. less than 244 tick generations) as a consequence of differential selection pressure imposed by hosts. It is most likely that this phenomenon has occurred in many other places across the globe where this tick has come in contact with different host species in sympatry with cattle.

A battery of 12 microsatellite markers for genetic analysis of the Leishmania (Viannia) guyanensis complex.

We used 12 microsatellite markers developed for Leishmania braziliensis to genotype 28 strains of the main species of the Leishmania guyanensis complex (i.e. L. guyanensis and L. panamensis) collected in Ecuador and Peru. The important heterozygote deficits observed in these populations are similar with the previous data obtained in L. braziliensis and raise again the debate on the reproductive mode of these protozoan parasites. The data showed genetic polymorphism and geographical differentiation giving information on population structure of the L. guyanensis complex. Regarding the two species, this study enhances again the debate on the taxonomic status of the different isolates belonging to L. guyanensis s.l. since the results showed substantial heterogeneity within this species complex. In conclusion, this study increases the number of available microsatellite loci for L. guyanensis species complex and raises fundamental biological questions. It confirms that microsatellite markers constitute good tools for population genetic studies on parasites of this complex.

Population structuring of the tsetse Glossina tachinoides resulting from landscape fragmentation in the Mouhoun River basin, Burkina Faso.

The impact of landscape fragmentation resulting from human- and climate-mediated factors on the structure of a population of Glossina tachinoides Westwood (Diptera: Glossinidae) in the Mouhoun River basin, Burkina Faso, was investigated. Allele frequencies at five microsatellite loci were compared in four populations. The average distance between samples was 72 km. The sampling points traversed an ecological cline in terms of rainfall and riverine forest ecotype, along a river loop that enlarged from upstream to downstream. Microsatellite DNA demonstrated no structuring among the groups studied (F(ST) = 0.015, P = 0.07), which is contrary to findings pertaining to Glossina palpalis gambiensis Vanderplank in the same geographical area. The populations of G. tachinoides showed complete panmixia (F(IS) = 0, P = 0.5 for the whole sample) and no genetic differentiation among populations or global positioning system trap locations. This is in line with the results of dispersal studies which indicated higher diffusion coefficients for G. tachinoides than for G. p. gambiensis. The impact of these findings is discussed within the framework of control campaigns currently promoted by the Pan African Tsetse and Trypanosomosis Eradication Campaign.

Hydrographic network structure and population genetic differentiation in a vector of fasciolosis, Galba truncatula.

We report a preliminary analysis on the relationships between drainage basin structure and genetic structure of populations of the European vector of fasciolosis, Galba truncatula. In the study area, 251 snails belonging to 12 populations were collected along different ditches of a same river network. Each snail was genotyped at six variable microsatellite loci. Our results show that all sample sites are characterized by a low level of polymorphism and a very high and significant heterozygote deficiency. Our data reveal a significant genetic differentiation, even at a small scale, and failed to delimit clear patterns of isolation by euclidian distance. Our study shows that genetic differentiation significantly increases with hydrographic distance along the streams (p<0.002), in consistence with the hypothesis that dispersion along the stream is dependent on the direction of water flow. This study shows that relationships can exist between the organization of the hydrological network and population biology of a disease vector, which has strong potential applications to drainage network management issues.

Apparent high recombination rates in clonal parasitic organisms due to inappropriate sampling design.

Sampling design is of primary importance for empirical studies, in particular, population genetics. For parasitic organisms, a rather frequent way of sampling individuals from local populations is to collect and genotype only one randomly chosen parasite (or isolate) per host individual (or subpopulation), although each host (subpopulation) harbors a set of parasites belonging to the same species (that is, an infrapopulation). Here, we investigate, using simulations, the consequences of such sampling design regarding the estimates of linkage disequilibrium and departure from the Hardy-Weinberg expectations (H-WE) in clonal parasites with an acyclic life cycle. We show that collecting and genotyping only one individual pathogen per host individual (or per subpopulation) and pooling them to form one 'artificial' subpopulation may generate strongly misleading patterns of genetic variations that may lead to false conclusions regarding their reproduction mode. In particular, we show that when subpopulations (or infrapopulations) are genetically differentiated, (i) the level of linkage disequilibrium is significantly reduced and (ii) the departure from the H-WE is strongly modified, sometimes giving a forged picture of a strongly recombining organism despite high levels of clonal reproduction.

Population genetic structure of wild and farmed rusa deer (Cervus timorensis russa) in New-Caledonia inferred from polymorphic microsatellite loci.

Historical records indicate that 12 rusa deer (Cervus timorensis russa) were introduced in New-Caledonia during the 1870s. We used eight polymorphic microsatellite DNA loci to assess the genetic differentiation and diversity of farmed and wild deer populations. Past genetic bottlenecks were detected in both sub-populations, although higher genetic diversity was maintained in farmed populations, probably due to the regular introduction of reproducers from wild populations and from other farms. The genetic structure of farmed and wild populations differed significantly. There was a significant isolation by distance for wild populations, whereas farmed populations were significantly differentiated between farms independently from their geographical proximity. Wild rusa deer consisted of small populations (with effective population sizes ranging between 7 and 19 individuals depending on the methods used), with a low parent-offspring dispersion range (0.20-2.02 km). Genetic tools and direct observations provided congruent estimates of dispersion and population sizes. We discuss the relevance of our results for management purposes.

Population sizes and dispersal pattern of tsetse flies: rolling on the river?

The West African trypanosomoses are mostly transmitted by riverine species of tsetse fly. In this study, we estimate the dispersal and population size of tsetse populations located along the Mouhoun river in Burkina Faso where tsetse habitats are experiencing increasing fragmentation caused by human encroachment. Dispersal estimated through direct (mark and recapture) and indirect (genetic isolation by distance) methods appeared consistent with one another. In these fragmented landscapes, tsetse flies displayed localized, small subpopulations with relatively short effective dispersal. We discuss how such information is crucial for designing optimal strategies for eliminating this threat. To estimate ecological parameters of wild animal populations, the genetic measures are both a cost- and time-effective alternative to mark-release-recapture. They can be applied to other vector-borne diseases of medical and/or economic importance.

The impact of clonalty on parasite population genetic structure.

In this paper, we briefly review the consequences of clonal reproduction on the apportionment of genetic diversity in parasite populations. We distinguish three kinds of parasite lifecycle where clonal reproduction occurs. The consequences of this mode of reproduction for the different kinds of parasite life-cycles are described. We here particularly focus on clonal diploids.

PERMANENT GENETIC RESOURCES: A set of 12 microsatellite loci for genetic studies of Leishmania braziliensis.

Twelve microsatellite loci of Leishmania braziliensis were examined, nine of which were developed in this work. Fifty-six Leishmania braziliensis were genotyped with these microsatellite loci. The 12 loci studied were polymorphic with the number of alleles ranging from five to 19, with a mean of 9.7 ± 4.1 and the observed heterozygosity averaging 0.425 ± 0.202. The important heterozygote deficits we observed (F(IS)  = 0.41, P value = 0.004) appear incompatible with the heterozygote excess expected in clonal diploids. This last result could revive the clonality/sexuality debate regarding Leishmania. This work validates the potential use of these microsatellites for population genetics analysis.

On the power of some binomial modifications of the Bonferroni multiple test.

Widely used in testing statistical hypotheses, the Bonferroni multiple test has a rather low power that entails a high risk to accept falsely the overall null hypothesis and therefore to not detect really existing effects. We suggest that when the partial test statistics are statistically independent, it is possible to reduce this risk by using binomial modifications of the Bonferroni test. Instead of rejecting the null hypothesis when at least one of n partial null hypotheses is rejected at a very high level of significance (say, 0.005 in the case of n = 10), as it is prescribed by the Bonferroni test, the binomial tests recommend to reject the null hypothesis when at least k partial null hypotheses (say, k = [n/2]) are rejected at much lower level (up to 30-50%). We show that the power of such binomial tests is essentially higher as compared with the power of the original Bonferroni and some modified Bonferroni tests. In addition, such an approach allows us to combine tests for which the results are known only for a fixed significance level. The paper contains tables and a computer program which allow to determine (retrieve from a table or to compute) the necessary binomial test parameters, i.e. either the partial significance level (when k is fixed) or the value of k (when the partial significance level is fixed).

Asexual reproduction: genetics and evolutionary aspects.

Reproduction is essential to all organisms if they are to contribute to the next generation. There are various means and ways of achieving this goal. This review focuses on the role of asexual reproduction for eukaryotic organisms and how its integration in a life cycle can influence their population genetics and evolution. An important question for evolutionary biologists as to why some organisms reproduce sexually, as opposed to asexually, is addressed. We also discuss the economic and medical importance of asexual organisms.

The tsetse fly Glossina palpalis palpalis is composed of several genetically differentiated small populations in the sleeping sickness focus of Bonon, Côte d'Ivoire.

Glossina palpalis is the main vector of human African trypanosomosis (HAT, or sleeping sickness) that dramatically affects human health in sub-Saharan Africa. Because of the implications of genetic structuring of vector populations for the design and efficacy of control campaigns, G. palpalis palpalis in the most active focus of sleeping sickness in Côte d'Ivoire was studied to determine whether this taxon is genetically structured. High and statistically significant levels of within population heterozygote deficiencies were found at each of the five microsatellite loci in two temporally separated samples. Neither null alleles, short allele dominance, nor trap locations could fully explain these deviations from random mating, but a clustering within each of the two samples into different genetic sub-populations (Wahlund effect) was strongly suggested. These different genetic groups, which could display differences in infection rates and trypanosome identity, were composed of small numbers of individuals that were captured together, leading to the observed Wahlund effect. Implications of this population structure on tsetse control are discussed.

Geographical variations in infectivity and susceptibility in the host-parasite system Schistosoma mansoni/Biomphalaria glabrata: no evidence for local adaptation.

We investigated local adaptation in the spatially structured natural Biomphalaria glabrata/Schistosoma mansoni host-parasite system in the marshy forest focus of Guadeloupe using cross-transplantation experiments. We demonstrated strong and highly significant variations in susceptibility/infectivity of host and parasite populations, respectively, but found no evidence of local adaptation neither for S. mansoni nor for B. glabrata. Environmental as well as genetic factors are discussed to explain susceptibility/infectivity variations between both host and parasite populations. The absence of local adaptation is discussed in relation to the metapopulation dynamics of both host and parasite, in particular their relative rates of dispersal at the scale under scrutiny. Our study constitutes the first cross-transplantation experiment concerning this host-parasite system of which both hosts and parasites came directly from the wild, excluding laboratory generations and experimental host passages.

"Clonal" population structure of the malaria agent Plasmodium falciparum in high-infection regions.

The population genetic structure of Plasmodium falciparum, the agent of malignant malaria, has been shown to be predominantly "clonal" (i.e., highly inbred) in regions of low infectivity; in high-infectivity regions, it is often thought to be panmictic, or nearly so, although there is little supporting evidence for this. The matter can be settled by investigating the parasite's genetic makeup in the midgut oocysts of the mosquito vector, where the products of meiosis can directly be observed. The developmental stages of P. falciparum are haploid, except in the oocysts of infected mosquito vectors, where two gametes fuse, diploidy occurs, and meiosis ensues. We have investigated genetic polymorphisms at seven microsatellite loci located on five chromosomes by assaying 613 oocysts in 145 mosquitoes sampled from 11 localities of Kenya, where malignant malaria is perennial and intense. There is considerable allelic variation, 16.3 +/- 2.1 alleles per locus, and considerable inbreeding, approximately 50% on the average. The inbreeding is caused by selfing (approximately 25%) and nonrandom genotype distribution of oocysts among mosquito guts (35%). The observed frequency of heterozygotes is 0.43 +/- 0.03; the expected frequency, assuming random mating, is 0.80 +/- 0.05. Linkage disequilibrium is statistically significant for all 21 pairwise comparisons between loci, even though 19 comparisons are between loci in different chromosomes, which is consistent with strong deviation from panmixia and the consequent reproduction of genomes as clones, without recombination between gene loci. This is of considerable evolutionary significance and of epidemiological consequence, concerning the spread of multilocus drug and vaccine resistance.