Phylogenetic assessment of the earthworm Aporrectodea caliginosa species complex (Oligochaeta: Lumbricidae) based on mitochondrial and nuclear DNA sequences.

The Aporrectodea caliginosa species complex includes the most abundant earthworms in grasslands and agricultural ecosystems of the Paleartic region. Historically this complex consisted of the following taxa: A. caliginosa s.s.Savigny, 1826, A. trapezoides Dugés (1828), A. tuberculata (Eisen, 1874), and A. nocturna Evans (1946). These four taxa are morphologically very similar and difficult to differentiate because of their morphological variability. Consequently, their taxonomic status and their phylogenetic relationships have been a matter of discussion for more than a century. To study these questions, we sequenced the COII (686 bp), 12S (362 bp), 16S (1200 bp), ND1 (917 bp), and tRNAs(Asn-Asp-Val-Leu-Ala-Ser-Leu) (402 bp) mitochondrial and 28S (809 bp) nuclear gene regions for 85 European earthworms from 27 different localities belonging to the A. caliginosa species complex and four outgroup taxa. DNA sequences were analyzed using maximum parsimony, maximum likelihood, and Bayesian approaches of phylogenetic inference. The resulting trees were combined with morphological, ecological, and genomic evidence to test species boundaries (i.e., integrative approach). Our molecular analyses showed that A. caliginosa s.s. and A. tuberculata form a sister clade to A. trapezoides, A. longa, and A. nocturna, which indicates that A. longa is part of the A. caliginosa species complex. We confirm the species status of all these taxa and identify two hitherto unrecognized Aporrectodea species in Corsica (France). Moreover our analyses also showed the presence of highly divergent lineages within A. caliginosa, A. trapezoides, and A. longa, suggesting the existence of cryptic diversity within these taxa.

Patterns, Mechanisms and Genetics of Speciation in Reptiles and Amphibians.

In this contribution, the aspects of reptile and amphibian speciation that emerged from research performed over the past decade are reviewed. First, this study assesses how patterns and processes of speciation depend on knowing the taxonomy of the group in question, and discuss how integrative taxonomy has contributed to speciation research in these groups. This study then reviews the research on different aspects of speciation in reptiles and amphibians, including biogeography and climatic niches, ecological speciation, the relationship between speciation rates and phenotypic traits, and genetics and genomics. Further, several case studies of speciation in reptiles and amphibians that exemplify many of these themes are discussed. These include studies of integrative taxonomy and biogeography in South American lizards, ecological speciation in European salamanders, speciation and phenotypic evolution in frogs and lizards. The final case study combines genomics and biogeography in tortoises. The field of amphibian and reptile speciation research has steadily moved forward from the assessment of geographic and ecological aspects, to incorporating other dimensions of speciation, such as genetic mechanisms and evolutionary forces. A higher degree of integration among all these dimensions emerges as a goal for future research.

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.

Habitat use in eight populations of Sceloporus grammicus (Squamata: Phrynosomatidae) from the Mexican Plateau.

Studies on habitat use have often helped explain observed variation in morphology, behavior and reproductive characteristics among populations within a single species. Here we analyze morphological and ecological characteristics of individuals from the Sceloporus grammicus species complex from 7 different localities (CER, El Cerezo; PAC, Pachuca; HUI, Huichapan; EZA, Emiliano Zapata; SMR, San Miguel Regla; LMJ, La Mojonera; and LMZ, La Manzana) in the state of Hidalgo, and one locality (Cahuacán) in the State of Mexico. A canonical correspondence analysis (CCA) showed that females from PAC, EZA, LMZ, HUI, SMR and CAH populations use similar microhabitats characterized mostly by bare soil, in females from LMJ and CER use microhabitats characterized primarily by vegetation and rocks. Females were observed using 12 different types of perches. With regard to perch height use, the CCA showed that females from PAC, LMJ, LMZ, SMR, CER and CAH populations were correlated with height to nearest perch (HNP), in the rest of the females were not related to any perch use variable. In contrast, the CCA showed that males from PAC, LMJ and CAH were characterized by microhabitats with higher vegetal coverage, while males from LMZ and CER used microhabitats composed of bare soil, but males from HUI and SMR populations used microhabitats composed chiefly of bare soil and rocks. With respect to perch height use, the CCA showed that males from PAC, LMJ, EZA and LMZ were correlated with distance to the nearest perch, but the rest of the males were not correlated with any perch use variables. Males were observed in 9 different perch types. The males were larger than the females in all morphological variables analyzed. Moreover, in both sexes the snout-vent length is positively correlated with all morphological variables, and although both the slope and ordinate of the origin of all morphological variables were larger in males than females, the analysis of covariance indicated that there is no increase in the morphological variables with increasing SVL between sexes. Our results suggest that variation in habitat use and morphology among populations is an adaptive response (phenotypic plasticity) to the environmental conditions where these populations of Sceloporus grammicus occur.

Delimiting species: comparing methods for Mendelian characters using lizards of the Sceloporus grammicus (Squamata: Phrynosomatidae) complex.

Species form the fundamental units of analysis in many areas of biology and, therefore, rigorous delimitation of this unit is important to a broad array of researchers. Recently, many new empirical methods have been proposed to delimit species in nature, and a large literature exists on the theoretical merit and superiority of each method. However, few empirical studies actually compare the results of these methods applied in the same study system. We used a large allozyme and chromosome dataset to apply a number of genetic-distance, character-based, and tree-based methods to a well-studied, data-rich system: the Sceloporus grammicus lizard complex of central Mexico. We hypothesized species boundaries under a general lineage or evolutionary species conceptual framework in an a priori fashion using mapped restriction-site data (mitochondrial DNA and nuclear rDNA), allozymes, and morphology. We then compared the ability of different methods to recover the "hypothesized evolutionary species" (HES). Highton's genetic-distance method and a tree-based method consistently recovered all four HES, although sometimes with weak support. With two exceptions, other methods recovered the same HES, but additional groups were weakly delimited and nested within the HES. Given the apparent recent divergence of some of the chromosome races and distinct populations in this complex, these are encouraging results. We emphasize the value of specifying testable criteria as clearly as possible and testing these with methods that make use of different properties of a single dataset.

Short report: Phylogenetic relationships of the anthropophilic Plasmodium falciparum malaria vectors in Africa.

Malaria kills more than one million people a year, and understanding the historical association between its most notorious causative agent, Plasmodium falciparum, and its mosquito vectors is important in fighting the disease. We present a phylogenetic analysis of a number of species within the mosquito subgenus Cellia based on a selection of mitochondrial and nuclear genes. Although some of these relationships have been estimated in other studies, generally few species were included and/or statistical support at many nodes was low. Here we include two additional species of anthropophilic P. falciparum malaria vectors and reanalyze these relationships using a Bayesian method that allows us to simultaneously incorporate different models of evolution. We report data that indicate a paraphyletic relationship between five anthropophilic African mosquito vectors. Such a relationship suggests that these species can serve as independent natural experiments for anopheline immunologic responses to regular, prolonged contact with P. falciparum.

Female preference for sympatric vs. allopatric male throat color morphs in the mesquite lizard (Sceloporus grammicus) species complex.

Color polymorphic sexual signals are often associated with alternative reproductive behaviors within populations, and the number, frequency, or type of morphs present often vary among populations. When these differences lead to assortative mating by population, the study of such polymorphic taxa may shed light on speciation mechanisms. We studied two populations of a lizard with polymorphic throat color, an important sexual signal. Males in one population exhibit orange, yellow, or blue throats; whereas males in the other exhibit orange, yellow, or white throats. We assessed female behavior when choosing between allopatric and sympatric males. We asked whether females discriminated more when the allopatric male was of an unfamiliar morph than when the allopatric male was similar in coloration to the sympatric male. We found that female rejection of allopatric males relative to sympatric males was more pronounced when males in a pair were more different in throat color. Our findings may help illuminate how behavioral responses to color morph differences between populations with polymorphic sexual signals contribute to reproductive isolation.

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.

Exploring the origin and degree of genetic isolation of Anopheles gambiae from the islands of São Tomé and Príncipe, potential sites for testing transgenic-based vector control.

The evolutionary processes at play between island and mainland populations of the malaria mosquito vector Anopheles gambiae sensu stricto are of great interest as islands may be suitable sites for preliminary application of transgenic-based vector control strategies. São Tomé and Príncipe, located off the West African coast, have received such attention in recent years. This study investigates the degree of isolation of An. gambiae s.s. populations between these islands and the mainland based on mitochondrial and ribosomal DNA molecular data. We identify possible continental localities from which these island populations derived. For these purposes, we used F ST values, haplotype networks, and nested clade analysis to estimate migration rates and patterns. Haplotypes from both markers are geographically widespread across the African continent. Results indicate that the populations from São Tomé and Príncipe are relatively isolated from continental African populations, suggesting they are promising sites for test releases of transgenic individuals. These island populations are possibly derived from two separate continental migrations. This result is discussed in the context of the history of the African slave trade with respect to São Tomé and Príncipe.

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 vector's 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 mosquito's 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.

Genetic divergence of Connecticut Melanoplus femurrubrum populations.

We surveyed Melanoplus femurrubrum populations within the state of Connecticut for genetic diversity at multiple genetic markers, including three mitochondrial [cytochrome oxidase subunit 1 (COI), reduced form of nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2), and AT rich] and one nuclear [internal transcribed spacers of the ribosomal DNA cluster (ITS1)] gene regions. All markers were variable, and the AT-rich gene showed the highest sequence divergence. Analysis of molecular variance (AMOVA), fixation index (Fst) analysis, and phylogeographic patterns showed little divergence between northern and southern regions. Estimates of genetic diversity (pi) showed higher mitochondrial diversity in the northern region but nearly equal diversity for the ITS1 gene. This study shows for the first time in Melanoplus genetic variation for the ND2, AT rich, and ITS genes within a small geographic area. Our methods and results should be useful for other researchers interested in conducting population-level studies on closely related species.

The impact of species concept on biodiversity studies.

Species are defined using a variety of different operational techniques. While discussion of the various methodologies has previously been restricted mostly to taxonomists, the demarcation of species is also crucial for conservation biology. Unfortunately, different methods of diagnosing species can arrive at different entities. Most prominently, it is widely thought that use of a phylogenetic species concept may lead to recognition of a far greater number of much less inclusive units. As a result, studies of the same group of organisms can produce not only different species identities but also different species range and number of individuals. To assess the impact of different definitions on conservation issues, we collected instances from the literature where a group of organisms was categorized both under phylogenetic and nonphylogenetic concepts. Our results show a marked difference, with surveys based on a phylogenetic species concept showing more species (48%) and an associated decrease in population size and range. We discuss the serious consequences of this trend for conservation, including an apparent change in the number of endangered species, potential political fallout, and the difficulty of deciding what should be conserved.