Marcos Pérez-Losada

Pathogen typing in the genomics era: MLST and the future of molecular epidemiology

Multi-locus sequence typing (MLST) is a high-resolution genetic typing approach to identify species and strains of pathogens impacting human health, agriculture (animals and plants), and biosafety. In this review, we outline the general concepts behind MLST, molecular approaches for obtaining MLST data, analytical approaches for MLST data, and the contributions MLST studies have made in a wide variety of areas. We then look at the future of MLST and their relative strengths and weaknesses with respect to whole genome sequence typing approaches that are moving into the research arena at an ever-increasing pace. Throughout the paper, we provide exemplar references of these various aspects of MLST. The literature is simply too vast to make this review comprehensive, nevertheless, we have attempted to include enough references in a variety of key areas to introduce the reader to the broad applications and complications of MLST data.

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.

Testing hypotheses of population structuring in the Northeast Atlantic Ocean and Mediterranean Sea using the common cuttlefish Sepia officinalis

Population structuring in species inhabiting marine environments such as the Northeast Atlantic Ocean (NEA) and Mediterranean Sea (MS) has usually been explained based on past and present physical barriers to gene flow and isolation by distance (IBD). Here, we examined the relative importance of these factors on population structuring of the common cuttlefish Sepia officinalis by using methods of phylogenetic inference and hypothesis testing coupled with coalescent and classical population genetic parameter estimation. Individuals from 10 Atlantic and 15 Mediterranean sites were sequenced for 659 bp of the mitochondrial COI gene (259 sequences). IBD seems to be the main factor driving present and past genetic structuring of Sepia populations across the NEA–MS, both at large and small geographical scales. Such an evolutionary process agrees well with some of the biological features characterizing this cuttlefish species (short migrations, nektobenthic habit, benthic eggs hatching directly to benthic juveniles). Despite the many barriers to migration/gene flow suggested in the NEA–MS region, genetic population fragmentation due to past isolation of water masses (Pleistocene; 0.56 million years ago) and/or present‐day oceanographic currents was only detected between the Aegean‐Ionian and western Mediterranean Seas. Restricted gene flow associated with the Almería‐Oran hydrographic front was also suggested between southern and eastern Spanish populations. Distinct population boundaries could not be clearly determined, except for the Aegean‐Ionian stock. Two Atlantic and five Mediterranean samples showed evidence of current decline in genetic diversity, which may indicate over‐exploitation of Sepia in both marine regions.

Pleistocene glaciation leaves deep signature on the freshwater crab Aegla alacalufi in Chilean Patagonia

Quaternary glacial cycles have played an important role in shaping the biodiversity in temperate regions. This is well documented in Northern Hemisphere, but much less understood for Southern Hemisphere. We used mitochondrial DNA and nuclear elongation factor 1α intron sequences to examine the Pleistocene glacial impacts on the phylogeographical pattern of the freshwater crab Aegla alacalufi in Chilean Patagonia. Phylogenetic analyses, which separated the glaciated populations on eastern continent into a north group (seven populations) and a south group (one population), revealed a shallow phylogenetic structure in the north group but a deep one in the non‐glaciated populations on western islands, indicating the significant influence of glaciation on these populations. Phylogenies also identified the Yaldad population on Chiloé Island as a potentially unrecognized new species. The non‐glaciated populations showed higher among population genetic divergence than the glaciated ones, but lower population genetic diversity was not detected in the latter. The two glaciated groups, which diverged from the non‐glaciated populations at ~96 800–29 500 years ago and ~104 200–73 800 years ago, respectively, seem to have different glacial refugia. Unexpectedly, the non‐glaciated islands did not serve as refugia for them. Demographic expansion was detected in the glaciated north group, with a constant population increase after the last glacial maximum. Nested clade analyses suggest a possible colonization from western islands to eastern continent. After arriving on the continent and surviving the last glacial period there, populations likely have expanded from high to low altitude, following the flood of melting ice. Aegla alacalufi genetic diversity has been primarily affected by Pleistocene glaciation and minimally by drainage isolation.

Comparing Phylogenetic Codivergence between Polyomaviruses and Their Hosts

ABSTRACT Seventy-two full genomes corresponding to nine mammalian (67 strains) and two avian (5 strains) polyomavirus species were analyzed using maximum likelihood and Bayesian methods of phylogenetic inference. Our fully resolved and well-supported (bootstrap proportions > 90%; posterior probabilities = 1.0) trees separate the bird polyomaviruses (avian polyomavirus and goose hemorrhagic polyomavirus) from the mammalian polyomaviruses, which supports the idea of spitting the genus into two subgenera. Such a split is also consistent with the different viral life strategies of each group. Simian (simian virus 40, simian agent 12 [Sa12], and lymphotropic polyomavirus) and rodent (hamster polyomavirus, mouse polyomavirus, and murine pneumotropic polyomavirus [MPtV]) polyomaviruses did not form monophyletic groups. Using our best hypothesis of polyomavirus evolutionary relationships and established host phylogenies, we performed a cophylogenetic reconciliation analysis of codivergence. Our analyses generated six optimal cophylogenetic scenarios of coevolution, including 12 codivergence events (P< 0.01), suggesting that Polyomaviridae coevolved with their avian and mammal hosts. As individual lineages, our analyses showed evidence of host switching in four terminal branches leading to MPtV, bovine polyomavirus, Sa12, and BK virus, suggesting a combination of vertical and horizontal transfer in the evolutionary history of the polyomaviruses.

Recombination in viruses: Mechanisms, methods of study, and evolutionary consequences

Abstract Recombination is a pervasive process generating diversity in most viruses. It joins variants that arise independently within the same molecule, creating new opportunities for viruses to overcome selective pressures and to adapt to new environments and hosts. Consequently, the analysis of viral recombination attracts the interest of clinicians, epidemiologists, molecular biologists and evolutionary biologists. In this review we present an overview of three major areas related to viral recombination: (i) the molecular mechanisms that underlie recombination in model viruses, including DNA-viruses (Herpesvirus) and RNA-viruses (Human Influenza Virus and Human Immunodeficiency Virus), (ii) the analytical procedures to detect recombination in viral sequences and to determine the recombination breakpoints, along with the conceptual and methodological tools currently used and a brief overview of the impact of new sequencing technologies on the detection of recombination, and (iii) the major areas in the evolutionary analysis of viral populations on which recombination has an impact. These include the evaluation of selective pressures acting on viral populations, the application of evolutionary reconstructions in the characterization of centralized genes for vaccine design, and the evaluation of linkage disequilibrium and population structure.

Conservation phylogenetics of Chilean freshwater crabs Aegla (Anomura, Aeglidae): assigning priorities for aquatic habitat protection

Recently, a molecular phylogeny based on four mtDNA gene regions has been estimated for 17 species and subspecies of Aegla freshwater crabs from Chile. With this phylogenetic hypothesis and information on geographic distribution, environmental conditions, habitat requirements, and population abundance, Chilean aeglids were assessed for conservation status based on the criteria included in the IUCN Red List Categories (2001). Aegla concepcionensis and Aegla expansa qualify as ‘‘Extinct in the Wild’’ and nine other taxa fall within the threatened category: three as ‘‘Critically Endangered’’ (A. laevis laevis, A. papudo and A. spectabilis) and six as ‘‘Vulnerable’’ (A. alacalufi, A. bahamondei, A. cholchol, A. hueicollensis, A. laevis talcahuano and A. manni). Six hydrographic regions within temperate Chile were ranked for conservation priority using species richness, and phylogenetic and genetic

Composition, taxonomy and functional diversity of the oropharynx microbiome in individuals with schizophrenia and controls

The role of the human microbiome in schizophrenia remains largely unexplored. The microbiome has been shown to alter brain development and modulate behavior and cognition in animals through gut-brain connections, and research in humans suggests that it may be a modulating factor in many disorders. This study reports findings from a shotgun metagenomic analysis of the oropharyngeal microbiome in 16 individuals with schizophrenia and 16 controls. High-level differences were evident at both the phylum and genus levels, with Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria dominating both schizophrenia patients and controls, and Ascomycota being more abundant in schizophrenia patients than controls. Controls were richer in species but less even in their distributions, i.e., dominated by fewer species, as opposed to schizophrenia patients. Lactic acid bacteria were relatively more abundant in schizophrenia, including species of Lactobacilli and Bifidobacterium, which have been shown to modulate chronic inflammation. We also found Eubacterium halii, a lactate-utilizing species. Functionally, the microbiome of schizophrenia patients was characterized by an increased number of metabolic pathways related to metabolite transport systems including siderophores, glutamate, and vitamin B12. In contrast, carbohydrate and lipid pathways and energy metabolism were abundant in controls. These findings suggest that the oropharyngeal microbiome in individuals with schizophrenia is significantly different compared to controls, and that particular microbial species and metabolic pathways differentiate both groups. Confirmation of these findings in larger and more diverse samples, e.g., gut microbiome, will contribute to elucidating potential links between schizophrenia and the human microbiota.

The evolution of HIV: Inferences using phylogenetics

Abstract Molecular phylogenetics has revolutionized the study of not only evolution but also disparate fields such as genomics, bioinformatics, epidemiology, ecology, microbiology, molecular biology and biochemistry. Particularly significant are its achievements in population genetics as a result of the development of coalescent theory, which have contributed to more accurate model-based parameter estimation and explicit hypothesis testing. The study of the evolution of many microorganisms, and HIV in particular, have benefited from these new methodologies. HIV is well suited for such sophisticated population analyses because of its large population sizes, short generation times, high substitution rates and relatively small genomes. All these factors make HIV an ideal and fascinating model to study molecular evolution in real time. Here we review the significant advances made in HIV evolution through the application of phylogenetic approaches. We first examine the relative roles of mutation and recombination on the molecular evolution of HIV and its adaptive response to drug therapy and tissue allocation. We then review some of the fundamental questions in HIV evolution in relation to its origin and diversification and describe some of the insights gained using phylogenies. Finally, we show how phylogenetic analysis has advanced our knowledge of HIV dynamics (i.e., phylodynamics).

Conservation assessment of southern South American freshwater ecoregions on the basis of the distribution and genetic diversity of crabs from the genus Aegla.

We assessed the conservation priority of 18 freshwater ecoregions in southern South America on the basis of Aegla (genus of freshwater crabs) genetic diversity and distribution. Geographical distributions for 66 Aegla species were taken from the literature and plotted against ecoregions and main river basins of southern South America. Species richness and number of threatened and endemic species were calculated for each area. To assess taxonomic and phylogenetic diversity, we generated a molecular phylogeny based on DNA sequences for one nuclear (28S) and 4 mitochondrial (12S, 16S, COI, and COII) genes. All species richness and phylogenetic methods agreed, to a large extent, in their rankings of the importance of conservation areas, as indicated by the Spearmans rank correlation coefficient (p < 0.01); nonetheless, some of the lowest correlations were observed between taxonomic and phylogenetic diversity indices. The 5 ecoregions of the Laguna dos Patos Basin (Eastern Brazil), Central Chile, South Brazilian Coast, Chilean Lakes, and Subtropical Potamic Axis (northern Argentina and southern Uruguay and Paraguay) had the highest biodiversity scores. Conservation of these regions will preserve the largest number of species and the greatest amount of genetic diversity within the South American freshwater Aegla fauna. Biodiversity across rivers and within areas was heterogeneously distributed in the ecoregions of Upper Paraná, Ribeira do Iguape, Upper Uruguay, and South Brazilian Coast (i.e., one river showed significantly more biodiversity than any other river from the same ecoregion), but homogeneously distributed in the other ecoregions. Hence, conservation plans in the former regions will potentially require less effort than plans in the latter regions.