Analysis of Leptospira isolates from mainland Portugal and the Azores islands

From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) (n=71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes (n=45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting (n=32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans (n=3), Leptospira kirschneri (n=8) and Leptospira borgpetersenii (n=21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed.     

Algae-based biotopes of the Azores (Portugal): spatial and seasonal variation

The increasing importance of coastal management created the need for a systematic classification and characterization of marine communities. Accurate quantitative methodologies for rocky shore algae-based biotope definition, were developed and tested on the Islands of São Miguel and Santa Maria (Azores). Shores of both islands were surveyed, covering all rocky substrate types. Biotopes were defined by assessing the associated habitat and species characteristics, using ANOSIM and SIMPER analysis, respectively. A total of ten biotopes were identified. Generally both islands’ biotopes are characterized by the same taxa/ecological categories, in summer and in winter. However, association between these taxa/ecological categories and the shore height at which they occur differs geographically and temporally. There is a generalized gradual succession of taxa/ecological categories from upper intertidal down to deepest subtidal, although geographical differences occur. Diversity is highest at the land–water interface and decreases towards both extremes (upper intertidal and deepest subtidal level). The strongest evidence of seasonal variation occurs at the upper intertidal. The methodology used proves to be effective in broad scale shoreline assessment of biological communities in warm-temperate coastal marine environments, and thus suitable for the purpose it was developed for. As a consequence it should be applied to the remaining islands of the Azorean archipelago as well as to other macaronesian islands, e.g. Madeira and the Canaries.     

Polymorphism of the APOE locus in the Azores Islands (Portugal)

Our aim in this study is to report on the polymorphism of the APOE gene in the Azores Islands (Portugal) to obtain a population baseline of the existing variation in this locus, known to be one of the genetic determinants of plasma lipid levels. One hundred twenty-six Azorean individuals were typed for the APOE polymorphism using standard PCR-RFLP. Allele frequencies obtained for APOE*2, APOE*3, and APOE*4 were 6.75%, 83.73%, and 9.52%, respectively. The APOE*3/*3 genotype presented the highest frequency (69.84%), and the APOE*4/*4 genotype had the lowest frequency (0.79%). Genotype frequencies were in conformity with Hardy-Weinberg expectations. The observed genotype and allele frequencies were similar to those reported for other Iberian samples. Furthermore, Nei's gene diversity (H = 0.2864 +/- 0.0351) was similar to that reported for samples from mainland Portugal. The data generated from this study will be of importance in the context of ongoing studies concerning the factors that influence lipid levels in the Azorean population.     

Peopling of the Azores Islands (Portugal): data from the Y chromosome

Nine Atlantic islands with approximately five and a half centuries of demographic history constitute the Portuguese archipelago of the Azores. Despite the recent peopling history of these islands, written records regarding the specific origin and relative proportions of the first settlers are scarce and incomplete. To gain insights into the history of the peopling of the Azores and to evaluate to what extent population imports described in historical sources left their marks on the genetic constitution of the present-day populations, we analyzed 11 Y-chromosome biallelic markers in a sample of 145 unrelated individuals of Azorean ancestry. The main results of this study indicate that the genetic profile of the Azorean male population shows high affinities with that of mainland Portugal, in accordance with the general knowledge, derived from historical sources, that the Portuguese were the major contributors to the Azorean founding population. Nevertheless, genetic traces of settlers from other origins also mentioned in historical records can still be found in the present-day population. Thus typically sub-Saharan male lineages were detected in the archipelago, in contrast to what has been described for mainland Portugal. Furthermore, compared to what has been described for the mainland Portugal population, our data support a stronger influence of people of Jewish origin, as detected by an increased frequency of lineages belonging to haplogroup J.     

Molecular systematics of the endemic Leptaxini (Gastropoda: Pulmonata) on the Azores islands

The Azorean representatives of the Leptaxini (Pulmonata) are single island endemics, where a high-spired shell distinguishes the monotypic genus Helixena from two slightly different low-spired forms within Leptaxis (azorica and caldeirarum type). We studied the evolutionary history of putative taxa and the three shell-types using 12 allozyme loci and sequences of nuclear (ITS-1 and ITS-2) and mitochondrial DNA (COI and 16S rRNA). While little variation was found in both ITS genes, allozyme and mtDNA divergence was among the highest reported for pulmonate land snails. Generally, phylogeographic patterns are indicative of allopatric differentiation via the successive colonization of (younger) islands, while a major role for adaptive evolution is not supported. The azorica shell-type is monophyletic and has no common history with other sympatric shell-types on the same islands. The (ambiguous) position of Helixena sanctaemariae makes Leptaxis paraphyletic on the Azores and possibly also the caldeirarum shell-type. Helixena can therefore not be distinguished as a separate genus on the Azores. Following a lineage-based concept, representatives on all (ancient) islands should be considered distinct species.     

Vegetation mapping and nature conservation: a case study in Terceira Island (Azores)

In order to emphasize the importance of vegetation mapping for nature conservation purposes a case study in Terceira island (Azores) is presented, in which the importance of the natural vegetation of the eastern slope of Santa Bárbara volcano (which is part of the Site of Community Importance of Santa Bárbara–Pico Alto) is evaluated through the elaboration of its vegetation map. Fourteen (14) different natural vegetation types were identified: grasslands (1 type), peat bogs (2 types), scrubs (2), forests (5), successional vegetation (3) and vegetation of rocky slopes (1). All communities are protected under the Habitat and Species Directive (EC/92/43) and most of them are endemic to the Azores Islands. This fact, together with the significant number of Azorean endemic taxa (18), Macaronesian endemic taxa (5) and species protected under the Habitat and Species Directive (7), gives this area an important conservation value that justifies future protection actions. Vegetation mapping is an important tool for the characterization, evaluation and implementation of managing plans of natural areas of the Azores islands. The use of a floristic-based classification, supported by multivariate analysis and structural data, is an efficient methodology for the construction of these maps. The data collected comprise an important set of information about the distribution and abundance of natural vegetation types and endemic and rare species. This information was not available until now and is indispensable for the elaboration of management plans of Special Zones for Conservation that will be part of the NATURA 2000 network.     

Detecting a hierarchical genetic population structure: the case study of the Fire Salamander (Salamandra salamandra) in Northern Italy

The multistep method here applied in studying the genetic structure of a low dispersal and philopatric species, such as the Fire Salamander Salamandra salamandra, was proved to be effective in identifying the hierarchical structure of populations living in broad‐leaved forest ecosystems in Northern Italy. In this study, 477 salamander larvae, collected in 28 sampling populations (SPs) in the Prealpine and in the foothill areas of Northern Italy, were genotyped at 16 specie‐specific microsatellites. SPs showed a significant overall genetic variation (Global F_ST = 0.032, P < 0.001). The genetic population structure was assessed by using STRUCTURE 2.3.4. We found two main genetic groups, one represented by SPs inhabiting the Prealpine belt, which maintain connections with those of the Eastern foothill lowland (PEF), and a second group with the SPs of the Western foothill lowland (WF). The two groups were significantly distinct with a Global F_ST of 0.010 (P < 0.001). While the first group showed a moderate structure, with only one divergent SP (Global F_ST = 0.006, P < 0.001), the second group proved more structured being divided in four clusters (Global F_ST = 0.017, P = 0.058). This genetic population structure should be due to the large conurbations and main roads that separate the WF group from the Prealpine belt and the Eastern foothill lowland. The adopted methods allowed the analysis of the genetic population structure of Fire Salamander from wide to local scale, identifying different degrees of genetic divergence of their populations derived from forest fragmentation induced by urban and infrastructure sprawl.     

Baseline Levels of Metals in Volcanic Soils of the Azores (Portugal)

Data on metal concentrations present in the soils of the Azores (Portugal) are scarce. The goal of this study was to measure the current levels of several metals in the top horizon of soils of two areas, distinguishable by their volcanic activity and physical characteristics, in order to establish some baseline concentrations of these elements. Soil samples were taken in similar ways from five sites in a volcanically active area and another five sites in an area without volcanic activity. Particle-size fractions, % organic matter, moisture content, pH, and major and trace elements compositions were measured. In general, the concentrations of trace metals in the soils from Santa Maria (inactive volcanism) were higher than those from Furnas (active volcanism), with the exception of Zn. The soils from Furnas, which have slightly lower pH and less % clay-silt than Santa Maria, will probably make such trace metals as Zn become more readily bioaccessible, and therefore pose a larger threat to living organisms inhabiting these soils.     

Testing the island rule: primates as a case study

The island rule states that after island colonization, larger animals tend to evolve reduced body sizes and smaller animals increased sizes. Recently, there has been disagreement about how often, if ever, this rule applies in nature, and much of this disagreement stems from differences in the statistical tests employed. This study shows, how different tests of the island rule assume different null hypotheses, and that these rely on quite different biological assumptions. Analysis and simulation are then used to quantify the biases in the tests. Many widely used tests are shown to yield false support for the island rule when island and mainland evolution are indistinguishable, and so a Monte Carlo permutation test is introduced that avoids this problem. It is further shown that tests based on independent contrasts lack power to detect the island rule under certain conditions. Finally, a complete reanalysis is presented of recent data from primates. When head-body length is used as the measure of body size, reports of the island rule are shown to stem from methodological artefacts. But when skull length or body mass are used, all tests agree that the island rule does hold in primates.     

Physiological responses of Pterocladia and Gelidium (Gelidiales,Rhodophyta) from the Azores,Portugal

Manometric studies were conducted on Pterocladia capillacea, Gelidium latifolium and Gelidium spinulosum from the Azores, Portugal to determine optimal values of temperature, light and salinity for growth. Physiological responses were considered in relation to vertical distribution patterns of these species commonly observed throughout the Azores. Optimal parameters for the growth of Pterocladia capillacea, Gelidium latifolium and G. spinulosum were 17 to 25 °C, a photon flux density between 200 and 300 µmol m^–2 s^–1 and salinities of 25 to 35.     

Understanding differences between phylogenetic and pedigree-derived mtDNA mutation rate: a model using families from the Azores Islands (Portugal)

We analyzed the control region of the mitochondrial DNA (mtDNA)from maternally related individuals originating from the AzoresIslands (Portugal) in order to estimate the mutation rate ofmtDNA and to gain insights into the process by which a new mutationarises and segregates into heteroplasmy. Length and/or pointheteroplasmies were found at least in one individual of 72%of the studied families. Eleven new point substitutions werefound, all of them in heteroplasmy, from which five appear tobe somatic mutations and six can be considered germinal, evidencingthe high frequency of somatic mutations in mtDNA in healthyyoung individuals. Different values of the mutation rate accordingto different assumptions were estimated. When considering allthe germinal mutations, the value of the mutation rate obtainedis one of the highest reported so far in family studies. However,when corrected for gender (assuming that the mutations presentin men have the same evolutionary weight of somatic mutationsbecause they will inevitably be lost) and for the probabilityof intraindividual fixation, the value for the mutation rateobtained for HVRI and HVRII (0.2415 mutations/site/Myr) wasin the upper end of the values provided by phylogenetic estimations.These results indicate that the discrepancy, that has been reportedpreviously, between the human mtDNA mutation rates observedalong evolutionary timescales and the estimations obtained usingfamily pedigrees can be minimized when corrections for genderproportions in newborn individuals and for the probability ofintraindividual fixation are introduced. The analyses performedsupport the hypothesis that (1) in a constant, tight bottleneckgenetic drift alone can explain different patterns of heteroplasmysegregation and (2) in neutral conditions, the destiny of anew mutation is strictly related to the initial proportion ofthe new variant. Another important point arising from the dataobtained is that, even in the absence of a paternal contributionof mtDNA, recombination may occur between mtDNA molecules presentin an individual, which is only observable if it occurs betweenmtDNA types that differ at two or more positions.     

Two new species of Aporhynchus (Cestoda: Trypanorhyncha) from deep water lanternsharks (Squaliformes: Etmopteridae) in the Azores, Portugal

New collections of cestodes from the spiral intestines of the lanternsharks Etmopterus spinax and Etmopterus pusillus off the island of Faial, in the Azores, Atlantic Ocean, have yielded 2 new species of trypanorhynchs belonging to Aporhynchus. Both species share the distinctive lack of all elements of the rhyncheal system that are characteristic of this genus. The identity of Aporhynchus norvegicus is clarified to allow it to be distinguished from A. menezesi n. sp., which also parasitizes E. spinax. This new species differs conspicuously from its congeners in that its mature and gravid proglottids are wider than long, rather than longer than wide, and also in its lack of spinitriches on the scolex. Aporhynchus pickeringae n. sp., the new species from E. pusillus , differs from all of its congeners except A. norvegicus in that it is a relatively delicate worm with relatively fewer testes. It also possesses fewer proglottids and a wider pedunculus scolecis than does A. norvegicus. Sections through the scolex of A. menezesi n. sp. support use of the term bothriate, rather than difossate, in reference to the scolex configuration of some trypanorhynchs. A key to the 4 species of Aporhynchus is provided.     

A new species of Ingolfiellidae (Peracarida, Amphipoda, Crustacea) from the Azores, Portugal

A new species of Ingolfiellid Amphipod Ingolfiella azorensis n. sp. is described from samples collected in a benthic survey off the Azores archipelago, Portugal. The new species is characterized by the lack of ocular lobes, a four-toothed dactylus on the first and second gnathopods, and having dissimilar claws on the third to seventh pereiopods. Individuals were obtained from shallow subtidal samples of sand. The new findings bring the number of Ingolfiellidae from Macaronesia up to six, equalling almost 20 % of the world’s known species, suggesting this area as a potential evolutionary centre for Ingolfiellidae.     

Management of drug resistance in the population: influenza as a case study

The rise of drug resistance remains a major impediment to the treatment of some diseases caused by fast-evolving pathogens that undergo genetic mutations. Models describing the within-host infectious dynamics suggest that the resistance is unlikely to emerge if the pathogen-specific immune responses are maintained above a certain threshold during therapy. However, emergence of resistance in the population involves both within-host and between-host infection mechanisms. Here, we employ a mathematical model to identify an effective treatment strategy for the management of drug resistance in the population. We show that, in the absence of pre-existing immunity, the population-wide spread of drug-resistant pathogen strains can be averted if a sizable portion of susceptible hosts is depleted before drugs are used on a large scale. The findings, based on simulations for influenza infection as a case study, suggest that the initial prevalence of the drug-sensitive strain under low pressure of drugs, followed by a timely implementation of intensive treatment, can minimize the total number of infections while preventing outbreaks of drug-resistant infections.     

The Linnean shortfall in oceanic island biogeography: a case study in the Azores

Aim Speciation processes on islands are still poorly understood. Previous studies based on the analysis of distribution data from checklists found that the flora of the Azores archipelago differs from other island floras in the exceptionally low number of radiations and the low number of single‐island endemics. The general mechanism(s) responsible for these apparently unique patterns remained unclear. One possible explanation for the distinctiveness of the Azorean endemic flora is the lack of a consistent and critical taxonomic framework for the floras of the Atlantic archipelagos. In this study, molecular variation within a range of Azorean endemic plant lineages was analysed to determine whether inadequacies in the current taxonomy of endemics might be an explanation for the unusual diversity patterns observed in the endemic flora of the Azores. Location Azores archipelago. Method Sixty‐nine populations of eight endemic species or subspecies belonging to five genetic lineages were sampled from all Azorean islands but one. Nuclear and plastid DNA regions were sequenced, and relationships among internal transcribed spacer (ITS) region ribotypes established using statistical parsimony. Results Molecular diversity patterns differ from current taxonomic groupings, with all lineages comprising previously overlooked genetic entities. Main conclusions Recognition as distinct taxa of the genetically distinct entities discovered in this study would drastically change the diversity patterns and make them more similar to those of other Atlantic archipelagos. The results serve to highlight that current knowledge of endemic diversity on oceanic islands may be far from complete, even in relatively well‐known groups such as angiosperms. This limitation is rarely considered in macroecological and evolutionary studies that make use of data from taxonomic checklists to draw inferences about oceanic island biogeographic processes.     

A combined approach using RAPD,ISSR and volatile analysis for the characterization of Thymus caespititius from Flores,Corvo and Graciosa islands (Azores,Portugal)

RAPDs and ISSRs were used as molecular markers and combined with the chemical analysis of the volatiles, aiming at characterizing Thymus caespititius from the islands of Corvo, Flores and Graciosa (Azores). Thirty-one individuals were analyzed using 17 RAPD and 11 ISSRs primers, which generated respectively 199 and 127 polymorphic scorable bands. Volatiles were isolated by distillation–extraction and analyzed by GC and GC–MS. Molecular data obtained from both markers discriminated assessments from Corvo and Flores which clustered separately from plants collected in Graciosa. Volatile data grouped plants in a similar way, giving rise to two groups, depending on their oil type; plants collected on Corvo and Flores yielded carvacrol-rich volatiles while those from Graciosa yielded both carvacrol and α-terpineol-rich oils.     

Immune response and virus population composition: HIV as a case study

Based on the current understanding of the immune response, we present what we believe to be a new model of intrahost virus dynamics. The model takes into account the relationship between virus replication rate and the level of antigen displayed by infected cells, and shows how the cell-directed immune response controls both virus load and virus replication rate. In contrast to conventional wisdom, it shows that the predominant virus variant does not necessarily have the highest replication rate. A strong immune response produces a selective advantage for latent viruses, whereas a deteriorating immune response invites in viruses of higher replication rates. The model is analysed in light of the well-studied HIV/AIDS disease progression, and shows how a wide range of major, seemingly unrelated issues in the study of HIV may be accounted for in a simple and unified manner.     

A geospatial approach to monitoring impervious surfaces in watersheds using Landsat data (the Mondego Basin,Portugal as a case study)

The urbanization of watersheds is a highly dynamic global phenomenon that must be monitored. With consequences for the environment, the population, and the economy, accurate products at adequate spatial and temporal resolutions are required and demanded by the science community and stakeholders alike. To address these needs, a new Impervious Surface Area (ISA) product was created for a Portuguese Watershed (Mondego river) from Landsat data (a combination of leaf-on multispectral bands, derived products, and NDVI time series), using Regression Tree Models (RTM). The product provides 30-m spatial resolution ISA estimates (0–100%) with a Mean Average Error (MAE) of 1.6% and Root Mean Square Error (RMSE) of 5.5%.A strategy to update the baseline product was tested in earlier imagery (2001 and 2007) for a subset of the watershed. Instead of updating the baseline product, the strategy seeks to identify stable training samples and remove those where change was detected in a time series of Change Vector Analysis (CVA). The stable samples were then used to create new ISA models using RTM. The updated maps were similar to the original product in terms of accuracy metrics (MAE: 2001: 2.6%; 2007:3.6%).The products and methodology offer a new perspective on the urban development of the watershed, at a scale previously unavailable. It can also be replicated elsewhere at a low cost, leveraging the growing Landsat data archive, and provide timely information on relevant land cover metrics to the scientific community and stakeholders.