Relatedness dynamics and sex-biased dispersal in a seasonal cycle of corn mice from intensively managed agroecosystems.

The sex-biased dispersal and kinship dynamics are important factors shaping the spatial distribution of individuals and are key parameters affecting a variety of ecological and evolutionary processes. Here, we studied the spatial distribution of related individuals within a population of corn mice Calomys musculinus in a seasonal cycle to infer dispersal patterns. The sampling was carried out from spring 2005 to winter 2006 in field borders of intensively managed agroecosystems. Genotyping data from 346 individuals with 9 microsatellites showed spatial genetic structure was weak for males, but not for females. The results indicate a complex spatial kinship dynamic of related females across all seasons. Which, contrary to our expectations, dispersal distances decrease with the increase of the population abundance. Meanwhile, male dispersal distances were greater when population abundance increased and thus the availability of active females. Males disperse greater distances to mate and sire offspring with distant females as a possible inbreeding avoidance mechanism. This study shows that C. musculinus is capable of much greater scattering distances than previously reported and that dispersal occurs fluidly and without barriers across the agroecosystem. The indirect benefit of dispersal on individual fitness could be related to relaxing the competition in the natal area and increasing the mating rate. Our study highlights the value of combining genetic relatedness, fieldwork observations, and behavioral data to estimate dispersal at a fine geographical scale.

Effective Population Size Differences in Calomys musculinus, the Host of Junín Virus: Their Relationship with the Epidemiological History of Argentine Hemorrhagic Fever.

Argentine hemorrhagic fever (AHF) is a serious endemic disease in Argentina, produced by Junín virus, whose host is the Sigmodontinae rodent Calomys musculinus. Within the endemic area, human incidence and proportion of infected rodents remains high for 5-10 years after the first appearance of the disease (epidemic [E] zone) and then gradually declines to sporadic cases (historic [H] zone). We tested the hypothesis that host populations within the E zone are large and well connected by gene flow, facilitating the transmission and maintenance of the virus, whereas those in the H and nonendemic (NE) zones are small and isolated, with the opposite effect. We estimated parameters affected by levels of gene flow and population size in 14 populations of C. musculinus: population effective size (Ne), genetic variability, and mean relatedness. Our hypothesis was not supported: the lowest levels of variability and of Ne and the highest genetic relatedness among individuals were found in the H zone. Populations from the NE zone displayed opposite results, whereas those in the E zone showed intermediate values. If we consider that populations are first NE, then E, and finally H, a correlative decrease in Ne was observed. Chronically infected females have a low reproductive success. We propose that this would lower Ne because each cohort would originate from a fraction of females of the previous generation, and affect other factors such as proportion of individuals that develop acute infection, probability of viral transmission, and evolution of virulence, which would explain, at least partly, the changing incidence of AHF.

Thelastoma vanwaerebekei n. sp. (Oxyurida: Thelastomatidae) a parasite of Gymnetis litigiosa (Coleoptera: Scarabaeidae) from Uruguay.

Thelastoma vanwaerebekei n. sp. was extracted from the hindgut of Gymnetis litigiosa. The new species is included in the group of species with females having the excretory pore at the same level as the pharyngeal basal bulb (or slightly anteriorly), long tail length (approximately 30% of total body length) and males without spicules. Females of T. vanwaerebekei n. sp. differ from those of T. madecassum in tail shape (filiform and conical, respectively). They differ from T. basiri, T. imphalensis and T. dessetae in the morphology of the anterior region. In females, pharyngeal length is shorter than in T. depressum. In males, T. vanwaerebekei n. sp. differs from T. blabericola in the tail length, and from T. raoi in the number of caudal papillae. The partial 18S SSU and 28S LSU rRNA gene regions were amplified.

The disjunct pattern of the Neotropical harvestman Discocyrtus dilatatus (Gonyleptidae) explained by climate-driven range shifts in the Quaternary: Paleodistributional and molecular evidence.

The disjunct distribution of the harvestman Discocyrtus dilatatus (Opiliones, Gonyleptidae) is used as a case study to test the hypothesis of a trans-Chaco Pleistocene paleobridge during range expansion stages. This would have temporarily connected humid regions ('Mesopotamia' in northeastern Argentina, and the 'Yungas' in the northwest, NWA) in the subtropical and temperate South American lowlands. The present study combines two independent approaches: paleodistributional reconstruction, using the Species Distribution Modeling method MaxEnt and projection onto Quaternary paleoclimates (6 kya, 21 kya, 130 kya), and phylogeographic analyses based on the cytochrome oxidase subunit I molecular marker. Models predict a maximal shrinkage during the warm Last Interglacial (130 kya), and the rise of the hypothesized paleobridge in the Last Glacial Maximum (21 kya), revealing that cold-dry stages (not warm-humid ones, as supposed) enabled the range expansion of this species. The disjunction was formed in the mid-Holocene (6 kya) and is intensified under current conditions. The median-joining network shows that NWA haplotypes are peripherally related to different Mesopotamian lineages; haplotypes from Santa Fe and Córdoba Provinces consistently occupy central positions in the network. According to the dated phylogeny, Mesopotamia-NWA expansion events would have occurred in the last glacial period, in many cases closely associated to the Last Glacial Maximum, with most divergence events occurring shortly thereafter. Only two (out of nine) NWA haplotypes are shared with Mesopotamian localities. A single, presumably relictual NWA haplotype was found to have diverged much earlier, suggesting an ancient expansion event not recoverable by the paleodistributional models. Different measures of sequence statistics, genetic diversity, population structure and history of demographic changes are provided. This research offers the first available evidence for the historical origin of NWA disjunct populations of a Mesopotamian harvestman.

Molecular identification of two Culex (Culex) species of the neotropical region (Diptera: Culicidae).

Culex bidens and C. interfor, implicated in arbovirus transmission in Argentina, are sister species, only distinguishable by feature of the male genitalia; however, intermediate specimens of the species in sympatry have been found. Fourth-instar larvae and females of both species share apomorphic features, and this lack of clear distinction creates problems for specific identification. Geometric morphometric traits of these life stages also do not distinguish the species. The aim of the present study was to assess the taxonomic status of C. bidens and C. interfor using two mitochondrial genes and to determine the degree of their reproductive isolation using microsatellite loci. Sequences of the ND4 and COI genes were concatenated in a matrix of 993 nucleotides and used for phylogenetic and distance analyses. Bayesian and maximum parsimony inferences showed a well resolved and supported topology, enclosing sequences of individuals of C. bidens (0.83 BPP, 73 BSV) and C. interfor (0.98 BPP, 97 BSV) in a strong sister relationship. The mean K2P distance within C. bidens and C. interfor was 0.3% and 0.2%, respectively, and the interspecific variation was 2.3%. Bayesian clustering also showed two distinct mitochondrial lineages. All sequenced mosquitoes were successfully identified in accordance with the best close match algorithm. The low genetic distance values obtained indicate that the species diverged quite recently. Most morphologically intermediate specimens of C. bidens from Córdoba were heterozygous for the microsatellite locus GT51; the significant heterozygote excess observed suggests incomplete reproductive isolation. However, C. bidens and C. interfor should be considered good species: the ventral arm of the phallosome of the male genitalia and the ND4 and COI sequences are diagnostic characters.

Preferential host switching and its relation with Hantavirus diversification in South America.

In recent years, the notion of co-speciation between Hantavirus species and their hosts was discarded in favour of a more likely explanation: preferential host switching. However, the relative importance of this last process in shaping the evolutionary history of hantaviruses remains uncertain, given the present limited knowledge not only of virus-host relationships but also of the pathogen and reservoir phylogenies. In South America, more than 25 hantavirus genotypes were detected; several of them act as aetiological agents of hantavirus pulmonary syndrome (HPS). An understanding of the diversity of hantaviruses and of the processes underlying host switching is critical since human cases of HPS are almost exclusively the result of human-host interactions. In this study, we tested if preferential host switching is the main process driving hantavirus diversification in South America, by performing a co-phylogenetic analysis of the viruses and their primary hosts. We also suggest a new level of amino acid divergence to define virus species in the group. Our results indicate that preferential host switching would not be the main process driving virus diversification. The historical geographical proximity among rodent hosts emerges as an alternative hypothesis to be tested.

Morphological and molecular characterisation of the entomoparasitic nematode Hammerschmidtiella diesingi (Nematoda, Oxyurida, Thelastomatidae).

A population of Hammerschmidtiella diesingi was detected in specimens of Periplaneta americana collected in Córdoba city (Argentina) was studied. Nematodes were characterized based on morphological, morphometric and genetic (D2-D3 expansion segment) analyses. New data are provided, especially on particular morphological characters that were a matter of controversy in previous studies. H. diesingi is reported in Argentina for the first time.

Species delimitation in the continental forms of the genus Epicrates (Serpentes, Boidae) integrating phylogenetics and environmental niche models.

Until recently, the genus Epicrates (Boidae) presented only one continental species, Epicrates cenchria, distributed in Central and South America, but after a taxonomic revision using morphologic characters five species were recognized: E. cenchria, E. crassus, E. maurus, E. assisi, and E. alvarezi. We analyzed two independent data sets, environmental niche models and phylogeny based on molecular information, to explore species delimitation in the continental species of this genus. Our results indicated that the environmental requirements of the species are different; therefore there are not evidences of ecological interchangeability among them. There is a clear correlation between species distributions and the major biogeographic regions of Central and South America. Their overall distribution reveals that allopatry or parapatry is the general pattern. These evidences suggest that habitat isolation prevents or limits gene exchange among them. The phylogenetic reconstruction showed that the continental Epicrates are monophyletic, being E. alvarezi the sister species for the remaining two clades: E. crassus-E. assisi, and E. maurus-E. cenchria. The clade grouping the continental Epicrates is the sister taxon of the genus Eunectes and not of the Caribbean Epicrates clade, indicating that the genus is paraphyletic. There is a non-consistent pattern in niche evolution among continental Epicrates. On the contrary, a high variation and abrupt shifts in environmental variables are shown when ancestral character states were reconstructed on the sequence-based tree. The degree of genetic and ecological divergence among continental Epicrates and the phylogenetic analyses support the elevation to full species of E. cenchria, E. crassus, E. maurus, E. assisi, and E. alvarezi.

High genetic differentiation among populations of the small cavy Microcavia australis occupying different habitats.

The small cavy Microcavia australis, a social and fossorial rodent, inhabits a large distribution range in South American arid zones. The species is versatile in coping with the seasonal and spatial variability typical of these environments through changes in morphology, physiology, and behavior. In order to explore whether phenotypic variations are related to the evolutionary history of the species, we analyzed the levels of genetic variability and divergence among four populations that differ in climate and habitat characteristics, two belonging to highlands and the other two from lowlands. We sequenced the mitochondrial control region and used the Inter Simple Sequence Repeats technique to study variability in the noncoding nuclear genome. Results from both genetic markers were consistent. Variability levels were high for all populations, and even higher for lowland ones. Pairwise genetic differentiation varied greatly, all comparisons being statistically significant except for the two highland populations. Seventeen haplotypes were detected which displayed three clear lineages: two corresponding to each lowland population and one to those in the highlands. Levels of genetic differentiation between population pairs varied widely. Haplotypes showed a mean sequence divergence of 1.4% between lowland populations and 0.2% between highland ones, whereas divergence was around 9% when populations from different altitudes were compared. Results from BEAST analysis support extant hypotheses suggesting that lowland forms are clearly older than the highland group. The deep genetic divergence between lineages poses the need to search for new evidence for properly defining the taxonomic status of divergent populations of M. australis.

Morphological, genetic, and chromosomal variation at a small spatial scale within a mosaic hybrid zone of the grasshopper Dichroplus pratensis Bruner (Acrididae).

Hybrid zones are regions where genetically different populations meet and mate, resulting in offspring of mixed characteristics. In organisms with limited dispersal, such as melanopline grasshoppers, hybrid zones can occur at small spatial scales (i.e., <500 m). We assessed levels of morphological, chromosomal, and molecular variability in adult males of the grasshopper Dichroplus pratensis Bruner (N = 137 males, 188 females) collected at 12 sites within a mosaic hybrid zone in a heterogeneous environment in Sierra de la Ventana, Argentina. In this hybrid zone, 2 Robertsonian chromosomal races, polymorphic for different centric fusions, meet (the "Northern race" at low altitudes and the "Southern race" at higher altitudes), forming hybrids that show monobrachial homologies during meiosis. High morphometric variation in 6 traits was revealed among grasshoppers of both sexes, with male body size positively and significantly correlated with increasing altitude. Frequency of Robertsonian fusions characteristic of the Southern race increased significantly with altitude. Moreover, fusion frequencies covaried between samples. Considerable genetic variation was revealed by random amplification of polymorphic DNA markers, with heterozygosity ranging from 0.3477 to 0.3745. Insects from low-altitude and high-altitude populations showed significant genetic differentiation, as indicated by F(ST) values. The proposed model for D. pratensis, involving the generation and maintenance by chromosomal fusions, of gene complexes adaptive in different environments, could explain the observed clinal patterns within the contact zone.

Introduction of different lineages of Aedes aegypti in Argentina.

Based on sequence analysis of the mitochondrial gene ND4, we determined the presence in Argentina of 3 haplotypes representing different Aedes (Stegomyia) aegypti lineages previously identified in other countries of the Americas, Africa, and Asia. Haplotype 17, the most frequent in Argentina, was previously detected in Brazil. Haplotype 7, restricted in our study to the northwest of Argentina and Bolivia, was formerly found in low frequency in the USA, Brazil, Mexico, and Senegal. Also haplotype 11, belonging to a different haplogroup than the other two, was observed in the present study; it had been reported before in Africa and Asia, but not in the Americas. The coexistence of haplotypes belonging to divergent haplogroups supports the hypothesis of multiple introductions of the species in Argentina.

Genetic structure of Aedes aegypti in the city of Córdoba (Argentina), a recently reinfested area.

To understand the transmission of a vector-borne disease, knowledge of the magnitude of dispersal among vector populations is essential because of its influence on pathogen transfer. The principal vector of dengue, the most common arboviral disease in the world, is the mosquito Aedes aegypti (L.). This tropical and subtropical species is native to Africa but has dispersed worldwide since the XV century. In Argentina, the species was declared eradicated in 1963, but has reinfested the country in recent years. In the present work, we used RAPD-PCR markers to assess the levels of genetic variability and differentiation among populations of Ae. aegypti (the vector of dengue and yellow fever) in Córdoba, the second largest city in Argentina. We detected similar levels of genetic variability (He between 0.351-0.404) across samples and significant genetic differentiation between most population pairs within the city (F ST between 0.0013-0.0253). Genetic distances indicate that there are three distinct groups, formed predominantly by populations that are connected by, or near, main roads. This suggests that, in addition to other factors such as availability of oviposition sites or step-by-step migration, passive transport plays an important role in gene flow within the city.

Genetic structure of Aedes aegypti in the city of Córdoba (Argentina), a recently reinfested area

To understand the transmission of a vector-borne disease, knowledge of the magnitude of dispersal among vector populations is essential because of its influence on pathogen transfer. The principal vector of dengue, the most common arboviral disease in the world, is the mosquito Aedes aegypti (L.). This tropical and subtropical species is native to Africa but has dispersed worldwide since the XV century. In Argentina, the species was declared eradicated in 1963, but has reinfested the country in recent years. In the present work, we used RAPD-PCR markers to assess the levels of genetic variability and differentiation among populations of Ae. aegypti (the vector of dengue and yellow fever) in Córdoba, the second largest city in Argentina. We detected similar levels of genetic variability (He between 0.351-0.404) across samples and significant genetic differentiation between most population pairs within the city (F ST between 0.0013-0.0253). Genetic distances indicate that there are three distinct groups, formed predominantly by populations that are connected by, or near, main roads. This suggests that, in addition to other factors such as availability of oviposition sites or step-by-step migration, passive transport plays an important role in gene flow within the city.

Co-amplification of mitochondrial pseudogenes in Calomys musculinus (Rodentia, Cricetidae): a source of error in phylogeographic studies.

In a previous phylogeographic study of the rodent Calomys musculinus, 24 haplotypes of the mitochondrial DNA D-loop region were detected using the restriction fragment length polymorphism technique (PCR-RFLP). Seven percent of the individuals showed patterns in which the sum of the sizes of the restriction fragments exceeded the size of the original PCR product. In the present paper we analyze possible causes of these atypical haplotypes. PCR products were cloned, and two or three different clones from a single individual were detected by their RFLP patterns. Nine clones with different restriction patterns were selected for sequence analyses. A maximum parsimony phylogenetic analysis revealed two well-supported paraphyletic groups. One group comprised sequences showing low nucleotide divergence compared with the most common haplotypes detected in the phylogeographic study. The other group was basal to the three species of Calomys other than C. musculinus included in the study; the mutations in the short portion of the cytochrome b gene amplified corresponded to 12 amino acid substitutions. The results suggest that two independent insertions of mtDNA sequences into the nucleus occurred; these sequences would co-amplify in the PCR procedure. Identification of pseudogenes is crucial to obtain reliable reconstruction of the intraspecific genealogy in phylogeographic studies.

Isolation and characterization of microsatellite markers in Oligoryzomys longicaudatus (Muridae, Sigmodontinae, Oryzomini), the natural reservoir of genotype Andes hantavirus.

The rodent Oligoryzomys longicaudatus or long-tailed pygmy rice rat is the reservoir of the aetiological agent of the hantavirus pulmonary syndrome in southern Argentina and Chile. We characterize 11 polymorphic microsatellite loci which would be useful for studies on microgeographical population structure in the species. Amplification of these loci in 42 individuals from four natural populations revealed four to 21 alleles per locus, and values of observed heterozygosities ranging from 0.371 to 0.896. Cross-species amplifications showed that some of the primers designed may be useful for other species of the genus Oligoryzomys.

Laboratory evaluation of susceptibility of natural subpopulations of Aedes aegypti larvae to temephos.

Aedes aegypti showed the ability to develop resistance to different insecticides, including temephos, the most widely used larvicide. The objectives of this work were to 1) determine the resistance value of 4 natural subpopulations of Ae. aegypti, identified by their different haplotypes, to the insecticide temephos "Abate 1G (1%)"; 2) determine the lethal concentration (LC)50 and LC90 values by using the Rockefeller strain as control; and 3) estimate the resistance ratios. Mosquito samples were collected in Catamarca, Córdoba, and Posadas (Argentina) and in Yacuiba (Bolivia). Six insecticide concentrations were tested. The Rockefeller strain and the Posadas sample showed susceptibility to the diagnostic concentration (0.012 mg/liter), whereas the mortality in Catamarca was 87%. In the Yacuiba and Córdoba collections, mortality was 74% and 75%, respectively, indicating resistance. These results were coincident with those of the Probit analysis from which the highest resistance ratios were estimated for the last 2 subpopulations (5.2 and 4.9, respectively). Before this study, no information was available about the existence of resistance in natural populations of Ae. aegypti in the studied area.

Structural organization of the mitochondrial DNA control region in Aedes aegypti.

The complete A+T - rich region of Aedes aegypti mitochondrial DNA has been cloned and sequenced. In Argentinean populations of the species, a polymorphism in the length of the amplified fragment was observed. Nucleotide sequence comparison of the shortest and longest A+T - rich amplified fragments detected revealed the presence of 2 types of tandemly repeated blocks. The size variation observed in natural populations is mainly due to the presence of a variable number of a 181 bp tandem repeat unit, located toward the 12S rRNA gene end. The size of the longest A+T - rich region was of 2070 bp, representing the largest control sequence reported for any mosquito species. Few relevant short blocks of primary-sequence similarity conserved in the control region of mosquitoes and other insects were detected scattered throughout the whole region. Five putative stem-loop secondary structures were found, one of them flanked by conserved sequences described in other insects. Our results suggest that there are no universal models of structure-function relations in the control region of insect mtDNA. In addition, we identified a short A+T - rich variable segment in the Ae. aegyti control region that would be suitable for population genetic studies.

Haplotype diversity of the mitochondrial DNA D-loop region in Calomys musculinus (Rodentia, Muridae) detected by PCR-RFLP.

In order to contribute to knowledge of colonization patterns in the rodent Calomys musculinus, a natural reservoir of the virus producing Argentine hemorrhagic fever (AHF), we studied the haplotype diversity of the mitochondrial DNA D-loop region in five natural populations from central Argentina. Digestion with eight restriction enzymes (RsaI, MseI, Tsp509I, AluI, AciI, HaeIII, NlaIII, and AseI) revealed polymorphism in the 1300 bp fragment amplified by PCR. Twenty different composite haplotypes were detected. Hierarchical analyses indicated that almost all variation (94%) is contained within local populations. Haplotypes 1 and 2, shared by all populations, were the most frequent. Nonsignificant genetic differentiation was found among populations of the endemic and nonendemic areas of AHF: All locations sampled presented exclusive haplotypes in spite of their geographic proximity, which would support previous observations indicating restricted gene flow among C. musculinus populations.