An expanded phylogeny of the genus Pseudamnicola (Gastropoda; Truncatelloidea; Hydrobiidae) across the Mediterranean Basin.

The genus Pseudamnicola Paulucci, 1878, is commonly found throughout the Mediterranean region. The genus displays considerable levels of endemism, accompanied by notable systematic and taxonomic ambiguity. However, the application of molecular data has proven highly effective in clarifying taxonomy and unveiling the diversity of cryptic species within the genus. Therefore, we employed all cytochrome c oxidase subunit I sequence data available and generated new ones from Greece to infer the phylogeny of the genus throughout its Mediterranean range and estimate the divergence times as well as the ancestral area of diversification. Our phylogenetic and time-estimate analyses demonstrate that with 36 to 38 extant Pseudamnicola species and genetic divergences across species ranging from 0.5% to 11.9% on average, the genus underwent relatively recent diversification during late Miocene (6.53 Ma), and the primary speciation events occurred during Plio-Pleistocene. The Italian Peninsula and Islands and the Ionian Drainages as defined by the Freshwater Ecoregions of the World are the ancestral regions of the genus following two different dispersal routes. Our study contributes to deepening our understanding of Pseudamnicola phylogeny by using data from throughout its range for the first time. This phylogeny provides evidence and confirms previous studies that relatively recent habitat isolation, followed by founder and dispersal events, has been one of the primary reasons for the evolution of the genus Pseudamnicola in the Mediterranean basin.

Small Island Effects on the Thermal Biology of the Endemic Mediterranean Lizard Podarcis gaigeae.

Ectotherms are vastly affected by climatic conditions as they rely on external sources of heat to regulate their body temperature, and changes in their habitat thermal quality could seriously affect their overall biology. To overcome the problems of a thermally unfavorable habitat, lizards need to either adjust their thermoregulatory behavior or respond to directional selection and shift their preferred body temperatures. To assess the impact of habitat thermal quality on the thermoregulatory profile, we studied multiple islet and 'mainland' populations of the Skyros wall lizard Podarcis gaigeae, an endemic lacertid to Skyros Archipelago, Greece. We evaluated the effectiveness of thermoregulation (E) using the three main thermal parameters: body (Tb), operative (Te), and preferred (Tpref) temperatures. We first hypothesized that the spatial homogeneity, the scarcity of thermal shelters, and the exposure to higher winds on islets would result in more demanding climate conditions. Second, we anticipated that islet lizards would achieve higher E in response to the lower thermal quality therein. As hypothesized, thermal parameters differed between populations but not in the expected manner. Skyros 'mainland' habitats reached higher temperatures, had more intense fluctuations, and were of lower thermal quality. As a result, lizards showed higher accuracy, precision, and effectiveness of thermoregulation. Noteworthy, we found that lizards from different populations have shifted their thermal profile and preferred body temperatures to cope with the particular conditions prevailing in their habitats. The latter supports the labile view on the evolution of thermoregulation.

The evolutionary history of the relict scorpion family Iuridae of the eastern Mediterranean.

Iuridae is a family of scorpions that exhibits a highly complex biogeographic and taxonomic history. Iuridae taxa are mainly found in Turkey and Greece, whereas a single species is found in northern Iraq. Several taxonomic revisions have been conducted on this family that initially comprised two genera. The latest taxonomic review, based on morphological and anatomical features, raised the number of Iuridae genera to four, and the number of species to 14. Sequence data from three molecular markers (COX1, 16S rDNA, ITS1) originating from numerous Iuridae taxa were analyzed within a phylogenetic framework. Divergence time-estimate analyses, species delimitation approaches and estimation of ancestral areas were implemented in order to: (1) reconstruct the phylogenetic relationships of the Iuridae taxa, (2) evaluate the morphological classifications, and (3) obtain insights into the biogeographic history of the family in the East Mediterranean. The multi-locus phylogeny clearly confirms an ancient division into two clades, Calchinae and Iurinae. Ancient patterns of isolation and dispersal are revealed. Both subfamilies are largely confined to the Anatolian peninsula and its few coastal islands; only the most derived genus Iurus has dispersed westward to Crete and Peloponnese. Based on our findings, three new genera of Iurinae (Metaiurus, Anatoliurus, and Letoiurus) are established. The genus Neocalchas emerges as one of the most ancient scorpion clades, with divergence time about 27 mya.

Diversification within an oceanic Mediterranean island: Insights from a terrestrial isopod.

Understanding intra-island patterns of evolutionary divergence, including cases of cryptic diversity, is a crucial step towards deciphering speciation processes. Cyprus is an oceanic island isolated for at least 5.3 Mya from surrounding continental regions, while it remains unclear whether it was ever connected to the mainland, even during the Messinian Salinity Crisis. The terrestrial isopod species Armadillo officinalis, that is widespread across the Mediterranean, offers the opportunity to explore intra-island divergence patterns that might exhibit geographical structure related also to the region's known paleogeography. Genome-wide ddRADseq, as well as Sanger sequencing for four mitochondrial and three nuclear loci data were generated for this purpose. In total, 71 populations from Cyprus, neighbouring continental sites, i.e., Israel, Lebanon and Turkey, and other Mediterranean regions, i.e. Greece, Italy, and Tunisia, were included in the analysis. Phylogenetic reconstructions and population structure analyses support the existence of at least six genetically discrete groups across the study area. Five of these distinct genetic clades occur on Cyprus, four of which are endemic to the island and one is widely distributed along the circum-Mediterranean countries. The sixth clade is distributed in Israel. The closest evolutionary relationship of endemic Cypriot populations is with those from Israel, while the evolutionary clade that is present in countries all around the Mediterranean is very shallow. Cladochronological analyses date the origin of the species on the island at ∼6 Mya. Estimated f4 and D statistics as well as FST values indicate the genetic isolation between the populations sampled from Cyprus and surrounding continental areas, while there is evident gene flow among populations within the island. Species delimitation and population genetic metrics support the existence of three distinct taxonomic units across the study area, two of which occur on the island and correspond to the endemic clade and the widespread circum-Mediterranean one, respectively, while the third corresponds to Israel's clade. The islands' paleogeographic history and recent human activities seem to have shaped current patterns of genetic diversity in this group of species.

Hidden diversity revealed in the freshwater snails, Bythinella and Pseudamnicola, in the Island of Crete.

Crete with its complex geomorphological history is the island with the highest number of endemism observed in animal and plant taxa throughout the Aegean archipelago. While other groups of organisms within Crete are well-studied, the freshwater gastropod fauna still remains poorly investigated. Bythinella and Pseudamnicola, are 2 genera of freshwater springsnails, both present on the island, inhabiting springs and other freshwater habitats. Here, we conduct a comprehensive study on the distribution of the different genetic lineages of the 2 gastropod genera in order to assess the mode of their differentiation on the island and infer the actual number of species present in the island. Towards these aims, sequence data from the mitochondrial gene were used and analyzed within a phylogenetic framework. For Bythinella, our results strongly support at least 5 delineated Bythinella spp. inhabiting Crete, which correspond to the already described species from previous studies with the addition of a new one. Bythinella analyses reveal an old time-frame of differentiation with vicariant phenomena being more likely the main drivers shaping the present-day distribution of the genus' genetic lineages. For Pseudamnicola, our data indicate the presence of at least 2 delineated Pseudamnicola spp. with a differentiation more consistent to an isolation-by-distance pattern of a relatively recent origin. Dispersion processes followed by isolation of the populations and/or recent speciation, seem to be the underlying process for the current distribution of Pseudamnicola lineages.

The transcriptome of a "sleeping" invader: de novo assembly and annotation of the transcriptome of aestivating Cornu aspersum.

BACKGROUND: Cornu aspersum is a quite intriguing species from the point of view of ecology and evolution and its potential use in medical and environmental applications. It is a species of economic importance since it is farmed and used for culinary purposes. However, the genomic tools that would allow a thorough insight into the ecology, evolution, nutritional and medical properties of this highly adaptable organism, are missing. In this work, using next-generation sequencing (NGS) techniques we assessed a significant portion of the transcriptome of this non-model organism. RESULTS: Out of the 9445 de novo assembled contigs, 2886 (30.6%) returned significant hits and for 2261 (24%) of them Gene Ontology (GO) terms associated to the hits were retrieved. A high percentage of the contigs (69.4%) produced no BLASTx hits. The GO terms were grouped to reflect biological processes, molecular functions and cellular components. Certain GO terms were dominant in all groups. After scanning the assembled transcriptome for microsatellites (simple sequence repeats, SSRs), a total of 563 SSRs were recovered. Among the identified SSRs, trinucleotide repeats were the predominant followed by tetranucleotide and dinucleotide repeats. CONCLUSION: The annotation success of the transcriptome of C. aspersum was relatively low. This is probably due to the very limited number of annotated reference genomes existing for mollusc species, especially terrestrial ones. Several biological processes being active in the aestivating species were revealed through the association of the transcripts to enzymes relating to the pathways. The genomic tools provided herein will eventually aid in the study of the global genomic diversity of the species and the investigation of aspects of the ecology, evolution, behavior, nutritional and medical properties of this highly adaptable organism.

Dispersal ability determines the scaling properties of species abundance distributions: a case study using arthropods from the Azores.

Species abundance distributions (SAD) are central to the description of diversity and have played a major role in the development of theories of biodiversity and biogeography. However, most work on species abundance distributions has focused on one single spatial scale. Here we used data on arthropods to test predictions obtained with computer simulations on whether dispersal ability influences the rate of change of SADs as a function of sample size. To characterize the change of the shape of the SADs we use the moments of the distributions: the skewness and the raw moments. In agreement with computer simulations, low dispersal ability species generate a hump for intermediate abundance classes earlier than the distributions of high dispersal ability species. Importantly, when plotted as function of sample size, the raw moments of the SADs of arthropods have a power law pattern similar to that observed for the SAD of tropical tree species, thus we conjecture that this might be a general pattern in ecology. The existence of this pattern allows us to extrapolate the moments and thus reconstruct the SAD for larger sample sizes using a procedure borrowed from the field of image analysis based on scaled discrete Tchebichef moments and polynomials.

Myrtoessa hyas, a new valvatiform genus and a new species of the Hydrobiidae (Caenogastropoda, Truncatelloidea) from Greece.

A new to science valvatiform hydrobiid, Myrtoessa hyas Radea, gen. n. & sp. n., from southern Greece, is described and illustrated. The new genus is a tiny gastropod thriving in a stream and is differentiated from the other known European and circum-Mediterranean valvatiform hydrobiid genera by a unique combination of the male and female genitalia features i.e. penis long, flat, blunt, with wide wrinkled proximal part and narrow distal part with a sub-terminal eversible papilla on its left side, bursa copulatrix well-developed, pyriform, fully protruding from the posterior end of the albumen gland and two seminal receptacles respectively. The new monotypic and locally endemic genus is narrowly distributed and its single known population nearby a coastal bustling village is vulnerable to anthropogenic stressors.

New records and detailed distribution and abundance of selected arthropod species collected between 1999 and 2011 in Azorean native forests.

BACKGROUND: In this contribution we present detailed distribution and abundance data for arthropod species identified during the BALA - Biodiversity of Arthropods from the Laurisilva of the Azores (1999-2004) and BALA2 projects (2010-2011) from 18 native forest fragments in seven of the nine Azorean islands (all excluding Graciosa and Corvo islands, which have no native forest left). NEW INFORMATION: Of the total 286 species identified, 81% were captured between 1999 and 2000, a period during which only 39% of all the samples were collected. On average, arthropod richness for each island increased by 10% during the time frame of these projects. The classes Arachnida, Chilopoda and Diplopoda represent the most remarkable cases of new island records, with more than 30% of the records being novelties. This study stresses the need to expand the approaches applied in these projects to other habitats in the Azores, and more importantly to other less surveyed taxonomic groups (e.g. Diptera and Hymenoptera). These steps are fundamental for getting a more accurate assessment of biodiversity in the archipelago.

Comparative phylogeography of endemic Azorean arthropods.

BACKGROUND: For a remote oceanic archipelago of up to 8 Myr age, the Azores have a comparatively low level of endemism. We present an analysis of phylogeographic patterns of endemic Azorean island arthropods aimed at testing patterns of diversification in relation to the ontogeny of the archipelago, in order to distinguish between alternative models of evolutionary dynamics on islands. We collected individuals of six species (representing Araneae, Hemiptera and Coleoptera) from 16 forest fragments from 7 islands. Using three mtDNA markers, we analysed the distribution of genetic diversity within and between islands, inferred the differentiation time-frames and investigated the inter-island migration routes and colonization patterns. RESULTS: Each species exhibited very low levels of mtDNA divergence, both within and between islands. The two oldest islands were not strongly involved in the diffusion of genetic diversity within the archipelago. The most haplotype-rich islands varied according to species but the younger, central islands contributed the most to haplotype diversity. Colonization events both in concordance with and in contradiction to an inter-island progression rule were inferred, while a non-intuitive pattern of colonization from western to eastern islands was also inferred. CONCLUSIONS: The geological development of the Azores has followed a less tidy progression compared to classic hotspot archipelagos, and this is reflected in our findings. The study species appear to have been differentiating within the Azores for <2 Myr, a fraction of the apparent life span of the archipelago, which may indicate that extinction events linked to active volcanism have played an important role. Assuming that after each extinction event, colonization was initiated from a nearby island hosting derived haplotypes, the apparent age of species diversification in the archipelago would be moved closer to the present after each extinction-recolonization cycle. Exploiting these ideas, we propose a general model for future testing.

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.

The hydrobioid freshwater gastropods (Caenogastropoda, Truncatelloidea) of Greece: new records, taxonomic re-assessments using DNA sequence data and an update of the IUCN Red List Categories.

Hydrobioid freshwater gastropods were collected from mainland and insular Greece. Several threatened taxa, such as Graecoanatolica vegorriticola, Pseudamnicola negropontina, Pseudamnicola pieperi, Pseudobithynia eubooensis and Pseudoislamia balcanica, were recorded from new localities. Trichonia trichonica, which has been considered extinct from its type locality for the last twenty eight years, was re-discovered, whereas the presence of Daphniola exigua, G. vegorriticola, Marstoniopsis graeca, P. pieperi and Pseudobithynia trichonis in their type localities was verified. The taxonomic status of P. negropontina and the newly discovered populations of G. vegorriticola was elucidated using COI sequence data. The new data recorded during this survey indicate that the IUCN status of some Greek endemic hydrobioids needs to be updated.

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.

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.

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.

On the possible role of tRNA base modifications in the evolution of codon usage: queuosine and Drosophila.

The best documented selection-based hypothesis to explain unequal usage of codons is based on the relative abundance of isoaccepting tRNAs. In unicellular organisms the most used codons are optimally translated by the most abundant tRNAs. The chemical bonding energies are affected by modification of the four traditional bases, in particular in the first anti-codon corresponding to the third codon position. One nearly universal modification is queuosine (Q) for guanine (G) in tRNA(His), tRNA(Asp), tRNA(Asn), and tRNA(Tyr); this changes the optimal binding from codons ending in C to no preference or a slight preference for U-ending codons. Among species of Drosophila, codon usage is constant with the exception of the Drosophila willistoni lineage which has shifted primary usage from C-ending codons to U/T ending codons only for these four amino acids. In Drosophila melanogaster Q containing tRNAs only predominate in old adults. We asked the question whether in D. willistoni these Q containing tRNAs might predominate earlier in development. As a surrogate for levels of modification we studied the expression of the gene (tgt) coding for the enzyme that catalyzes the substitution of Q for G in different life stages of D. melanogaster, D. pseudoobscura, and D. willistoni. Unlike the other two species, the highest tgt expression in D. willistoni is in young females producing eggs. Because tRNAs laid down in eggs persist through the early stages of development, this implies that Q modification occurs earlier in development in D. willistoni than in other Drosophila.

Anopheles immune genes and amino acid sites evolving under the effect of positive selection.

BACKGROUND: It has long been the goal of vector biology to generate genetic knowledge that can be used to "manipulate" natural populations of vectors to eliminate or lessen disease burden. While long in coming, progress towards reaching this goal has been made. Aiming to increase our understanding regarding the interactions between Plasmodium and the Anopheles immune genes, we investigated the patterns of genetic diversity of four anti-Plasmodium genes in the Anopheles gambiae complex of species. METHODOLOGY/PRINCIPAL FINDINGS: Within a comparative phylogenetic and population genetics framework, the evolutionary history of four innate immunity genes within the An. gambiae complex (including the two most important human malaria vectors, An. gambiae and An. arabiensis) is reconstructed. The effect of natural selection in shaping the genes' diversity is examined. Introgression and retention of ancestral polymorphisms are relatively rare at all loci. Despite the potential confounding effects of these processes, we could identify sites that exhibited dN/dS ratios greater than 1. CONCLUSIONS/SIGNIFICANCE: In two of the studied genes, CLIPB14 and FBN8, several sites indicated evolution under positive selection, with CLIPB14 exhibiting the most consistent evidence. Considering only the sites that were consistently identified by all methods, two sites in CLIPB14 are adaptively driven. However, the analysis inferring the lineage -specific evolution of each gene was not in favor of any of the Anopheles lineages evolving under the constraints imposed by positive selection. Nevertheless, the loci and the specific amino acids that were identified as evolving under strong evolutionary pressure merit further investigation for their involvement in the Anopheles defense against microbes in general.

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.

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

BACKGROUND: If 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 mosquito's 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. RESULTS: We 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. CONCLUSION: Considering 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.