Aristeidis Parmakelis

The molecular evolution of four anti-malarial immune genes in the Anopheles gambiae species complex.

BackgroundIf the insect innate immune system is to be used as a potential blocking step in transmission of malaria, then it will require targeting one or a few genes with highest relevance and ease of manipulation. The problem is to identify and manipulate those of most importance to malaria infection without the risk of decreasing the mosquitos ability to stave off infections by microbes in general. Molecular evolution methodologies and concepts can help identify such genes. Within the setting of a comparative molecular population genetic and phylogenetic framework, involving six species of the Anopheles gambiae complex, we investigated whether a set of four pre-selected immunity genes (gambicin, NOS, Rel2 and FBN9) might have evolved under selection pressure imposed by the malaria parasite.ResultsWe document varying levels of polymorphism within and divergence between the species, in all four genes. Introgression and the sharing of ancestral polymorphisms, two processes that have been documented in the past, were verified in this study in all four studied genes. These processes appear to affect each gene in different ways and to different degrees. However, there is no evidence of positive selection acting on these genes.ConclusionConsidering the results presented here in concert with previous studies, genes that interact directly with the Plasmodium parasite, and play little or no role in defense against other microbes, are probably the most likely candidates for a specific adaptive response against P. falciparum. Furthermore, since it is hard to establish direct evidence linking the adaptation of any candidate gene to P. falciparum infection, a comparative framework allowing at least an indirect link should be provided. Such a framework could be achieved, if a similar approach like the one involved here, was applied to all other anopheline complexes that transmit P. falciparum malaria.

High Levels of Genetic Differentiation between Ugandan Glossina fuscipes fuscipes Populations Separated by Lake Kyoga

Background Glossina fuscipes fuscipes is the major vector of human African trypanosomiasis, commonly referred to as sleeping sickness, in Uganda. In western and eastern Africa, the disease has distinct clinical manifestations and is caused by two different parasites: Trypanosoma brucei rhodesiense and T. b. gambiense. Uganda is exceptional in that it harbors both parasites, which are separated by a narrow 160-km belt. This separation is puzzling considering there are no restrictions on the movement of people and animals across this region. Methodology and Results We investigated whether genetic heterogeneity of G. f. fuscipes vector populations can provide an explanation for this disjunct distribution of the Trypanosoma parasites. Therefore, we examined genetic structuring of G. f. fuscipes populations across Uganda using newly developed microsatellite markers, as well as mtDNA. Our data show that G. f. fuscipes populations are highly structured, with two clearly defined clusters that are separated by Lake Kyoga, located in central Uganda. Interestingly, we did not find a correlation between genetic heterogeneity and the type of Trypanosoma parasite transmitted. Conclusions The lack of a correlation between genetic structuring of G. f. fuscipes populations and the distribution of T. b. gambiense and T. b. rhodesiense indicates that it is unlikely that genetic heterogeneity of G. f. fuscipes populations explains the disjunct distribution of the parasites. These results have important epidemiological implications, suggesting that a fusion of the two disease distributions is unlikely to be prevented by an incompatibility between vector populations and parasite.

Molecular phylogeny of three Mesalina (Reptilia: Lacertidae) species (M. guttulata, M. brevirostris and M. bahaeldini) from North Africa and the Middle East: Another case of paraphyly?

Mesalina is a widespread lacertid genus occurring throughout the Saharo-Sindian region from North Africa to Pakistan. It has been through a series of taxonomic revisions, but the phylogenetic relationships among the species remain unclear. In this study we estimate the phylogeographic structure of M. guttulata across most of its distributional range and we evaluate the relationships between M. guttulata and the sympatric species M. brevirostris and M. bahaeldini using partial mitochondrial DNA (mtDNA) sequences (cyt b and 16S). M. guttulata and M. brevirostris represent species complexes, whereas M. bahaeldini considered before as M. guttulata is a recently described species with very restricted distribution. Here we present the first evidence that M. guttulata is a paraphyletic taxon with respect to M. bahaeldini, while M. brevirostris proves to be a polytypic species or even a species complex, confirming previous studies. Although mtDNA markers have several properties that make them suitable for phylogeographic studies, they are not free of difficulties. Phylogeographic inferences within and between closely related species can be mislead by introgression and retention of ancestral polymorphism (incomplete lineage sorting). However, the present distribution pattern, the estimated times of divergence and the significant variation in morphology within M. guttulata led us to accept that the paraphyletic pattern observed, is most likely due to inaccurate taxonomy. Our hypothesis is that what has hitherto been considered as intraspecific variation, actually reflects species-level variation. Furthermore, our biogeographic analyses and the estimated time of divergences suggest that the present distribution of M. guttulata was the result of several dispersal and vicariant events, which are associated with historical changes (climatic oscillations and paleogeographic barriers) of late Miocene and Pliocene period.

Animal Mitochondria, Positive Selection and Cyto-Nuclear Coevolution: Insights from Pulmonates

Pulmonate snails have remarkably high levels of mtDNA polymorphism within species and divergence between species, making them an interesting group for the study of mutation and selection on mitochondrial genomes. The availability of sequence data from most major lineages – collected largely for studies of phylogeography - provides an opportunity to perform several tests of selection that may provide general insights into the evolutionary forces that have produced this unusual pattern. Several protein coding mtDNA datasets of pulmonates were analyzed towards this direction. Two different methods for the detection of positive selection were used, one based on phylogeny, and the other on the McDonald-Kreitman test. The cyto-nuclear coevolution hypothesis, often implicated to account for the high levels of mtDNA divergence of some organisms, was also addressed by assessing the divergence pattern exhibited by a nuclear gene. The McDonald-Kreitman test indicated multiple signs of positive selection in the mtDNA genes, but was significantly biased when sequence divergence was high. The phylogenetic method identified five mtDNA datasets as affected by positive selection. In the nuclear gene, the McDonald-Kreitman test provided no significant results, whereas the phylogenetic method identified positive selection as likely present. Overall, our findings indicate that: 1) slim support for the cyto-nuclear coevolution hypothesis is present, 2) the elevated rates of mtDNA polymorphims and divergence in pulmonates do not appear to be due to pervasive positive selection, 3) more stringent tests show that spurious positive selection is uncovered when distant taxa are compared and 4) there are significant examples of positive selection acting in some cases, so it appears that mtDNA evolution in pulmonates can escape from strict deleterious evolution suggested by the Muller’s ratchet effect.

Patterns of Selection in Anti-Malarial Immune Genes in Malaria Vectors: Evidence for Adaptive Evolution in LRIM1 in Anopheles arabiensis

Background Co-evolution between Plasmodium species and its vectors may result in adaptive changes in genes that are crucial components of the vectors defense against the pathogen. By analyzing which genes show evidence of positive selection in malaria vectors, but not in closely related non-vectors, we can identify genes that are crucial for the mosquitos resistance against Plasmodium. Methodology/Principle Findings We investigated genetic variation of three anti-malarial genes; CEC1, GNBP-B1 and LRIM1, in both vector and non-vector species of the Anopheles gambiae complex. Whereas little protein differentiation was observed between species in CEC1 and GNBP-B1, McDonald-Kreitman and maximum likelihood tests of positive selection show that LRIM1 underwent adaptive evolution in a primary malaria vector; An. arabiensis. In particular, two adjacent codons show clear signs of adaptation by having accumulated three out of four replacement substitutions. Furthermore, our data indicate that this LRIM1 allele has introgressed from An. arabiensis into the other main malaria vector An. gambiae. Conclusions/Significance Although no evidence exists to link the adaptation of LRIM1 to P. falciparum infection, an adaptive response of a known anti-malarial gene in a primary malaria vector is intriguing, and may suggest that this gene could play a role in Plasmodium resistance in An. arabiensis. If so, our data also predicts that LRIM1 alleles in An. gambiae vary in their level of resistance against P. falciparum.

Molecular phylogeny of the Greek populations of the genus Ligidium (Isopoda, Oniscidea) using three mtDNA gene segments

The phylogeny of Greek populations of the terrestrial isopod genus Ligidium is reconstructed based on three mtDNA gene segments: 12S rRNA, 16S rRNA and COI. Two widely distributed European species, as well as three outgroups belonging to different isopod genera, were also included in the analyses. The samples used represent almost all Ligidium species known to occur in Greece, as well as several populations of unknown specific status plus some new records. Phylogenetic analyses of the combined data set were performed using Bayesian inference and maximum parsimony. The two main sister clades with good support indicate the sympatric differentiation of two lineages in southern continental Greece (Peloponnisos), where Ligidium populations exhibit a mosaic distribution of sibling species. The insular populations of the Aegean Islands show increased genetic divergence and form separate clades. The presence of a third lineage of Asiatic origin is strongly suggested by both the molecular phylogeny and morphology. The only presumably valid diagnostic morphological character exhibits only partial correspondence to well supported clades of the molecular phylogeny. Genetic differentiation between populations is very high, a fact that can be attributed to the strict ecological specialization of these animals that leads to increased levels of isolation even between populations that are in close proximity. As a consequence, Greek Ligidium populations, especially those present on islands, are unique genetic pools and extremely vulnerable to extinction.

Mitochondrial phylogeny and biogeographic history of the Greek endemic land-snail genus Codringtonia Kobelt 1898 (Gastropoda, Pulmonata, Helicidae).

The aim of this work was to infer the phylogeny of the Greek endemic land-snail genus Codringtonia Kobelt 1898, estimate the time frame of the radiation of the genus, and propose a biogeographic scenario that could explain the contemporary distribution of Codringtonia lineages. The study took place in the districts of Peloponnese, Central Greece and Epirus of mainland Greece. Sequence data originating from three mtDNA genes (COI, COII, and 16S rDNA) were used to infer the phylogeny of the eight nominal Codringtonia species. Furthermore, the radiation time-frame of extant Codringtonia species was estimated using a relaxed molecular clock analysis and mtDNA substitution rates of land snails. The phylogenetic analysis supported the existence of six Codringtonia lineages in Greece and indicated that one nominal species (Codringtonia neocrassa) might belong to a separate genus distantly related to Codringtonia. The time frame of differentiation of Codringtonia species was placed in the Late Miocene-Pleistocene epoch. The dispersal-vicariance analysis performed indicated that most probably Codringtonia exhibited a north-to-south spread with the ancestral area being that of central Greek mainland, accompanied with duplication (speciation) and vicariance events.

Microallopatry Caused Strong Diversification in Buthus scorpions (Scorpiones: Buthidae) in the Atlas Mountains (NW Africa)

The immense biodiversity of the Atlas Mountains in North Africa might be the result of high rates of microallopatry caused by mountain barriers surpassing 4000 meters leading to patchy habitat distributions. We test the influence of geographic structures on the phylogenetic patterns among Buthus scorpions using mtDNA sequences. We sampled 91 individuals of the genus Buthus from 51 locations scattered around the Atlas Mountains (Antiatlas, High Atlas, Middle Atlas and Jebel Sahro). We sequenced 452 bp of the Cytochrome Oxidase I gene which proved to be highly variable within and among Buthus species. Our phylogenetic analysis yielded 12 distinct genetic groups one of which comprised three subgroups mostly in accordance with the orographic structure of the mountain systems. Main clades overlap with each other, while subclades are distributed parapatrically. Geographic structures likely acted as long-term barriers among populations causing restriction of gene flow and allowing for strong genetic differentiation. Thus, genetic structure and geographical distribution of genetic (sub)clusters follow the classical theory of allopatric differentiation where distinct groups evolve without range overlap until reproductive isolation and ecological differentiation has built up. Philopatry and low dispersal ability of Buthus scorpions are the likely causes for the observed strong genetic differentiation at this small geographic scale.

Molecular systematics and historical biogeography of the green lizards (Lacerta) in Greece: Insights from mitochondrial and nuclear DNA

The green lizards of the genus Lacerta (Sauria, Lacertidae) comprise nine recognized species, which in Europe are mainly restricted to the southern peninsulas. Four of them (L. trilineata, L. viridis, L. bilineata and L. agilis) occur in Greece. The uncertainty of morphological diversification renders the taxonomic assignment into species and subspecies problematic. In this study sequence data derived from two mitochondrial (cytochrome b and 16S rRNA) genes and one nuclear (NKTR) gene were used to (a) evaluate the taxonomic status of the genus Lacerta in Greece with emphasis on L. trilineata group and (b) investigate the evolutionary history of the genus through the application of phylogenetic and phylogeographic analyses, using Gallotia and Timon as outgroups. The phylogenetic analyses revealed the existence of four major clades. The first clade corresponds to L. trilineata group, the second to L. media, the third to L. agilis and the fourth to a complex of L. viridis and L. bilineata. However, the produced phylogenetic relationships are not congruent with the current taxonomy, especially in the first clade in which L. trilineata appeared to be paraphyletic in regard to L. pamphylica. Six distinct lineages were inferred within L. trilineata, despite the current recognition of nine morphological subspecies, the genetic differentiation of which exceeds that of other Lacerta species, imposing a thorough taxonomic revision of the species. Our results suggested a rapid diversification of L. trilineata group during the late Miocene. We believe that the present distribution of the genus in Greece is the result of several dispersal and vicariant events that took place during the late Miocene and early Pliocene.

Peeking through the trapdoor: Historical biogeography of the Aegean endemic spider Cyrtocarenum Ausserer, 1871 with an estimation of mtDNA substitution rates for Mygalomorphae.

The Aegean region, located in the Eastern Mediterranean, is an area of rich biodiversity and endemism. Its position, geographical configuration and complex geological history have shaped the diversification history of many animal taxa. Mygalomorph spiders have drawn the attention of researchers, as excellent model systems for phylogeographical investigations. However, phylogeographic studies of spiders in the Aegean region are scarce. In this study, we focused on the phylogeography of the endemic ctenizid trap-door spider Cyrtocarenum Ausserer, 1871. The genus includes two morphologically described species: C. grajum (C.L. Koch, 1836) and C. cunicularium (Olivier, 1811). We sampled 60 specimens from the distributions of both species and analyzed four mitochondrial and two nuclear markers. Cyrtocarenum served as an example to demonstrate the importance of natural history traits in the inference of phylogeographic scenarios. The mtDNA substitution rates inferred for the genus are profoundly higher compared to araneomorph spiders and other arthropods, which seems tightly associated with their biology. We evaluate published mtDNA substitution rates followed in the literature for mygalomorph spiders and discuss potential pitfalls. Following gene tree (maximum likelihood, Bayesian inference) and species tree approaches ((*)BEAST), we reconstructed a time-calibrated phylogeny of the genus. These results, combined with a biogeographical ancestral-area analysis, helped build a biogeographic scenario that describes how the major palaeogeographic and palaeoclimatic events of the Aegean may have affected the distribution of Cyrtocarenum lineages. The diversification of the genus seems to have begun in the Middle Miocene in the present west Aegean area, while major phylogenetic events occurred at the Miocene-Pliocene boundary for C. cunicularium, probably related to the Messinian Salinity Crisis. Our results also demonstrate the clear molecular distinction of the two morphologically described species, but possible cryptic lineages may exist within C. cunicularium.