Ancient Mitogenomes Reveal the Evolutionary History and Biogeography of Sloths

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Evolutionary Analysis of Mitogenomes from Parasitic and Free-Living Flatworms

Mitochondrial genomes (mitogenomes) are useful and relatively accessible sources of molecular data to explore and understand the evolutionary history and relationships of eukaryotic organisms across diverse taxonomic levels. The availability of complete mitogenomes from Platyhelminthes is limited; of the 40 or so published most are from parasitic flatworms (Neodermata). Here, we present the mitogenomes of two free-living flatworms (Tricladida): the complete genome of the freshwater species Crenobia alpina (Planariidae) and a nearly complete genome of the land planarian Obama sp. (Geoplanidae). Moreover, we have reanotated the published mitogenome of the species Dugesia japonica (Dugesiidae). This contribution almost doubles the total number of mtDNAs published for Tricladida, a species-rich group including model organisms and economically important invasive species. We took the opportunity to conduct comparative mitogenomic analyses between available free-living and selected parasitic flatworms in order to gain insights into the putative effect of life cycle on nucleotide composition through mutation and natural selection. Unexpectedly, we did not find any molecular hallmark of a selective relaxation in mitogenomes of parasitic flatworms; on the contrary, three out of the four studied free-living triclad mitogenomes exhibit higher A+T content and selective relaxation levels. Additionally, we provide new and valuable molecular data to develop markers for future phylogenetic studies on planariids and geoplanids.     

Chloroplast DNA intraspecific phylogeography of plants from the Pacific Northwest of North America

Molecular studies of plants from the Pacific Northwest of North America suggest a recurrent pattern of genetic differentiation and geographic structuring. In each of five angiosperms and one fern species representing diverse life histories, cpDNA data indicate two clades of populations that are geographically structured. A northern group comprises populations from Alaska to central or southern Oregon, whereas populations from central Oregon southward to northern California form a southern group. In several of these species, a few populations having southern genotypes may have survived in glacial refugia further north in the Olympic Peninsula, Queen Charlotte Islands, and Prince of Wales Island. Allozyme data reveal a similar pattern of differentiation in several other plants from the Pacific Northwest. North-south partitioning of genotypes has also been reported for several animal species from this region. On a broader geographic scale, northsouth partitioning of genotypes has also been observed in other plants from western North America having a variety of geographic distributions. Some species also display a reduction of genetic variability in the northern portion of their range compared to the south. The data suggest strongly that past glaciation profoundly influenced the genetic architecture of the flora and fauna of the Pacific Northwest. Two alternative hypotheses are advanced to explain the geographic structuring of genotypes. First, past glaciation may have created discontinuities in the geographic distributions of plant species, with populations surviving in several well-isolated northern and southern refugia. Following glaciation, migration of genetically differentiated, once-isolated populations resulted in the formation of a continuous geographic distribution with a major genetic discontinuity. Alternatively, plants survived and subsequently migrated northward from a southern refugium, and a genotype became fixed in one or a few populations at the leading edge of recolonization. Subsequent long-distance dispersal from this leading edge resulted in a relatively uniform northern genotype that differs from the southern genotype(s). Whatever the underlying mechanism, Pleistocence glaciation may have molded the intraspecific genetic architecture of both plants and animals from the Pacific Northwest in a geographically similar manner. Future studies should seek to obtain a comprehensive phylogeography for regions that includes a diversity of both plants and animals.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

A new xanthone-containing Micarea from Northwest Europe and the Pacific Northwest of North America

Abstract:Micarea xanthonica sp. nov. is described from the western British Isles, westernmost Norway and the Pacific Northwest of North America. It belongs to the M. prasina group, but differs from related species in possessing xanthones in the thallus.     

Ancient DNA from South-East Europe Reveals Different Events during Early and Middle Neolithic Influencing the European Genetic Heritage

The importance of the process of Neolithization for the genetic make-up of European populations has been hotly debated, with shifting hypotheses from a demic diffusion (DD) to a cultural diffusion (CD) model. In this regard, ancient DNA data from the Balkan Peninsula, which is an important source of information to assess the process of Neolithization in Europe, is however missing. In the present study we show genetic information on ancient populations of the South-East of Europe. We assessed mtDNA from ten sites from the current territory of Romania, spanning a time-period from the Early Neolithic to the Late Bronze Age. mtDNA data from Early Neolithic farmers of the Starčevo Criş culture in Romania (Cârcea, Gura Baciului and Negrileşti sites), confirm their genetic relationship with those of the LBK culture (Linienbandkeramik Kultur) in Central Europe, and they show little genetic continuity with modern European populations. On the other hand, populations of the Middle-Late Neolithic (Boian, Zau and Gumelniţa cultures), supposedly a second wave of Neolithic migration from Anatolia, had a much stronger effect on the genetic heritage of the European populations. In contrast, we find a smaller contribution of Late Bronze Age migrations to the genetic composition of Europeans. Based on these findings, we propose that permeation of mtDNA lineages from a second wave of Middle-Late Neolithic migration from North-West Anatolia into the Balkan Peninsula and Central Europe represent an important contribution to the genetic shift between Early and Late Neolithic populations in Europe, and consequently to the genetic make-up of modern European populations.     

An Occurrence of Shepheardella Siddal (Foraminiferida) from the West Coast of North America

SYNOPSIS. An occurrence of the allogromid genus Shepheardella is here reported for the first time from the western hemisphere. The foraminifer is briefly redescribed and illustrated.     

Southeast Asian Mitochondrial DNA Analysis Reveals Genetic Continuity of Ancient Mongoloid Migrations

Human mitochondrial DNAs (mtDNAs) from 153 independent samples encompassing seven Asian populations were surveyed for sequence variation using the polymerase chain reaction (PCR), restriction endonuclease analysis and oligonucleotide hybridization. All Asian populations were found to share two ancient AluI/DdeI polymorphisms at nps 10394 and 10397 and to be genetically similar indicating that they share a common ancestry. The greatest mtDNA diversity and the highest frequency of mtDNAs with HpaI/HincII morph 1 were observed in the Vietnamese suggesting a Southern Mongoloid origin of Asians. Remnants of the founding populations of Papua New Guinea (PNG) were found in Malaysia, and a marked frequency cline for the COII/tRNA(Lys) intergenic deletion was observed along coastal Asia. Phylogenetic analysis indicates that both insertion and deletion mutations in the COII/tRNA(Lys) region have occurred more than once.     

Genome Analysis of a Transmissible Lineage of Pseudomonas aeruginosa Reveals Pathoadaptive Mutations and Distinct Evolutionary Paths of Hypermutators

Genome sequencing of bacterial pathogens has advanced our understanding of their evolution, epidemiology, and response to antibiotic therapy. However, we still have only a limited knowledge of the molecular changes in in vivo evolving bacterial populations in relation to long-term, chronic infections. For example, it remains unclear what genes are mutated to facilitate the establishment of long-term existence in the human host environment, and in which way acquisition of a hypermutator phenotype with enhanced rates of spontaneous mutations influences the evolutionary trajectory of the pathogen. Here we perform a retrospective study of the DK2 clone type of P. aeruginosa isolated from Danish patients suffering from cystic fibrosis (CF), and analyze the genomes of 55 bacterial isolates collected from 21 infected individuals over 38 years. Our phylogenetic analysis of 8,530 mutations in the DK2 genomes shows that the ancestral DK2 clone type spread among CF patients through several independent transmission events. Subsequent to transmission, sub-lineages evolved independently for years in separate hosts, creating a unique possibility to study parallel evolution and identification of genes targeted by mutations to optimize pathogen fitness (pathoadaptive mutations). These genes were related to antibiotic resistance, the cell envelope, or regulatory functions, and we find that the prevalence of pathoadaptive mutations correlates with evolutionary success of co-evolving sub-lineages.The long-term co-existence of both normal and hypermutator populations enabled comparative investigations of the mutation dynamics in homopolymeric sequences in which hypermutators are particularly prone to mutations. We find a positive exponential correlation between the length of the homopolymer and its likelihood to acquire mutations and identify two homopolymer-containing genes preferentially mutated in hypermutators. This homopolymer facilitated differential mutagenesis provides a novel genome-wide perspective on the different evolutionary trajectories of hypermutators, which may help explain their emergence in CF infections.     

Ancient Mitochondrial DNA Reveals the Origin of Sus scrofa from Rebun Island, Japan

The Kabukai A site (5 to 8C A.D.) of the Okhotsk cultural area is on Rebun Island, a small island near the coast, north–northwest of Hokkaido, Japan. Specimens of Sus scrofa, called the Sakhalin pig, were discovered in five cultural layers at the Kabukai A site. Ancient DNA was extracted from the remains of 42 Sakhalin pig bones. Thirty-nine nucleotide sequences of the 574-bp mitochondrial DNA control region, estimated to have originated from at least 21 individuals, were amplified and analyzed phylogenetically. Nine distinct haplotypes (A1, A2, A3, B1, B2, C1, C2, D1, and D2) from this site were classified into four haplotype groups (A, B, C, and D) by parsimonious network analysis. Phylogenetic analysis of 9 ancient and 55 modern haplotypes indicated that the population of Sakhalin pigs at the Kabukai A site belonged to two distinct clusters; haplotype groups A and B formed a cluster comprised only of themselves, and haplotype groups C and D belonged to the cluster of one of the two genetic groups of Japanese wild boars uniquely distributed in the western part of Japan, including one northeast Mongolian wild boar. Analysis of the haplotype distribution among three archaeological sites and their historical transitions among the five layers reflecting the cultural periods at the Kabukai A site suggests that the Sakhalin pig populations were introduced from Sakhalin island and the Amur River basin in the northeastern Eurasian continent together with some cultural influences. Received: 18 April 2000 / Accepted: 24 November 2000     

The First Dinosaur from Washington State and a Review of Pacific Coast Dinosaurs from North America

We describe the first diagnostic dinosaur fossil from Washington State. The specimen, which consists of a proximal left femur, was recovered from the shallow marine rocks of the Upper Cretaceous (Campanian) Cedar District Formation (Nanaimo Group) and is interpreted as pertaining to a large theropod on the basis of its hollow medullary cavity and proximally placed fourth trochanter. The Washington theropod represents one of the northernmost occurrences of a Mesozoic dinosaur on the west coast of the United States and one of only a handful from the Pacific coast of Laramidia during the Cretaceous. Its isolated nature and preservation in marine rocks suggest that the element was washed in from a nearby fluvial system. If the femur pertains to a tyrannosauroid, which seems likely given its size and the widespread occurrence of the group across Laramidia during Late Cretaceous times, then it would represent an earlier occurrence of large body size than previously recognized (complete femur length estimated at 1.2 meters). Uncertainty surrounding the latitude of deposition of the Nanaimo Group (i.e., the Baja-British Columbia hypothesis) precludes assigning the Washington theropod to either of the putative northern or southern biogeographic provinces of Laramidia.     

An Evolutionary Analysis of Antigen Processing and Presentation across Different Timescales Reveals Pervasive Selection

The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage.We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes. Thus, we used evolutionary information to generate experimentally-testable hypotheses and to provide a list of sites to prioritize in follow-up analyses.     

Arrival of Paleo-Indians to the Southern Cone of South America: New Clues from Mitogenomes

With analyses of entire mitogenomes, studies of Native American mitochondrial DNA (mtDNA) variation have entered the final phase of phylogenetic refinement: the dissection of the founding haplogroups into clades that arose in America during and after human arrival and spread. Ages and geographic distributions of these clades could provide novel clues on the colonization processes of the different regions of the double continent. As for the Southern Cone of South America, this approach has recently allowed the identification of two local clades (D1g and D1j) whose age estimates agree with the dating of the earliest archaeological sites in South America, indicating that Paleo-Indians might have reached that region from Beringia in less than 2000 years. In this study, we sequenced 46 mitogenomes belonging to two additional clades, termed B2i2 (former B2l) and C1b13, which were recently identified on the basis of mtDNA control-region data and whose geographical distributions appear to be restricted to Chile and Argentina. We confirm that their mutational motifs most likely arose in the Southern Cone region. However, the age estimate for B2i2 and C1b13 (11–13,000 years) appears to be younger than those of other local clades. The difference could reflect the different evolutionary origins of the distinct South American-specific sub-haplogroups, with some being already present, at different times and locations, at the very front of the expansion wave in South America, and others originating later in situ, when the tribalization process had already begun. A delayed origin of a few thousand years in one of the locally derived populations, possibly in the central part of Chile, would have limited the geographical and ethnic diffusion of B2i2 and explain the present-day occurrence that appears to be mainly confined to the Tehuelche and Araucanian-speaking groups.     

Whole-Genome Sequencing Analysis from the Chikungunya Virus Caribbean Outbreak Reveals Novel Evolutionary Genomic Elements

Background

Chikungunya virus (CHIKV), an alphavirus and member of the Togaviridae family, is capable of causing severe febrile disease in humans. In December of 2013 the Asian Lineage of CHIKV spread from the Old World to the Americas, spreading rapidly throughout the New World. Given this new emergence in naïve populations we studied the viral genetic diversity present in infected individuals to understand how CHIKV may have evolved during this continuing outbreak.

Methodology/Principle Findings

We used deep-sequencing technologies coupled with well-established bioinformatics pipelines to characterize the minority variants and diversity present in CHIKV infected individuals from Guadeloupe and Martinique, two islands in the center of the epidemic. We observed changes in the consensus sequence as well as a diverse range of minority variants present at various levels in the population. Furthermore, we found that overall diversity was dramatically reduced after single passages in cell lines. Finally, we constructed an infectious clone from this outbreak and identified a novel 3’ untranslated region (UTR) structure, not previously found in nature, that led to increased replication in insect cells.

Conclusions/Significance

Here we preformed an intrahost quasispecies analysis of the new CHIKV outbreak in the Caribbean. We identified novel variants present in infected individuals, as well as a new 3’UTR structure, suggesting that CHIKV has rapidly evolved in a short period of time once it entered this naïve population. These studies highlight the need to continue viral diversity surveillance over time as this epidemic evolves in order to understand the evolutionary potential of CHIKV.     

Genetic characterization of Zostera asiatica on the Pacific Coast of North America

We gathered sequence information from the nuclear 5.8S rDNA gene and associated internal transcribed spacers, ITS-1 and ITS-2 (5.8S rDNA/ITS), and the chloroplast maturase K (matK) gene, from Zostera samples collected from subtidal habitats in Monterey and Santa Barbara (Isla Vista) bays, California, to test the hypothesis that these plants are conspecific with Z. asiatica Miki of Asia. Sequences from approximately 520 base pairs of the nuclear 5.8S rDNA/ITS obtained from the subtidal Monterey and Isla Vista Zostera samples were identical to homologous sequences obtained from Z. marina collected from intertidal habitats in Japan, Alaska, Oregon and California. Similarly, sequences from the matK gene from the subtidal Zostera samples were identical to matK sequences obtained from Z. marina collected from intertidal habitats in Japan, Alaska, Oregon and California, but differed from Z. asiatica sequences accessioned into GenBank. This suggests the subtidal plants are conspecific with Z. marina, not Z. asiatica. However, we found that herbarium samples accessioned into the Kyoto University Herbarium, determined to be Z. asiatica, yielded 5.8S rDNA/ITS sequences consistent with either Z. japonica, in two cases, or Z. marina, in one case. Similar results were observed for the chloroplast matK gene; we found haplotypes that were inconsistent with published matK sequences from Z. asiatica collected from Japan. These results underscore the need for closer examination of the relationship between Z. marina along the Pacific Coast of North America, and Z. asiatica of Asia, for the retention and verification of specimens examined in scientific studies, and for assessment of the usefulness of morphological characters in the determination of taxonomic relationships within Zosteraceae.     

Molecular Analysis of Dehalococcoides 16S Ribosomal DNA from Chloroethene-Contaminated Sites throughout North America and Europe

The environmental distribution of Dehalococcoides group organisms and their association with chloroethene-contaminated sites were examined. Samples from 24 chloroethene-dechlorinating sites scattered throughout North America and Europe were tested for the presence of members of the Dehalococcoides group by using a PCR assay developed to detect Dehalococcoides 16S rRNA gene (rDNA) sequences. Sequences identified by sequence analysis as sequences of members of the Dehalococcoides group were detected at 21 sites. Full dechlorination of chloroethenes to ethene occurred at these sites. Dehalococcoides sequences were not detected in samples from three sites at which partial dechlorination of chloroethenes occurred, where dechlorination appeared to stop at 1,2-cis-dichloroethene. Phylogenetic analysis of the 16S rDNA amplicons confirmed that Dehalococcoides sequences formed a unique 16S rDNA group. These 16S rDNA sequences were divided into three subgroups based on specific base substitution patterns in variable regions 2 and 6 of the Dehalococcoides 16S rDNA sequence. Analyses also demonstrated that specific base substitution patterns were signature patterns. The specific base substitutions distinguished the three sequence subgroups phylogenetically. These results demonstrated that members of the Dehalococcoides group are widely distributed in nature and can be found in a variety of geological formations and in different climatic zones. Furthermore, the association of these organisms with full dechlorination of chloroethenes suggests that they are promising candidates for engineered bioremediation and may be important contributors to natural attenuation of chloroethenes.     

Ancient DNA Clarifies the Evolutionary History of American Late Pleistocene Equids

Hippidions are past members of the equid lineage which appeared in the South American fossil record around 2.5 Ma but then became extinct during the great late Pleistocene megafaunal extinction. According to fossil records and numerous dental, cranial, and postcranial characters, Hippidion and Equus lineages were expected to cluster in two distinct phylogenetic groups that diverged at least 10 MY, long before the emergence of the first Equus. However, the first DNA sequence information retrieved from Hippidion fossils supported a striking different phylogeny, with hippidions nesting inside a paraphyletic group of Equus. This result indicated either that the currently accepted phylogenetic tree of equids was incorrect regarding the timing of the evolutionary split between Hippidion and Equus or that the taxonomic identification of the hippidion fossils used for DNA analysis needed to be reexamined (and attributed to another extinct South American member of the equid lineage). The most likely candidate for the latter explanation is Equus (Amerhippus) neogeus. Here, we show by retrieving new ancient mtDNA sequences that hippidions and Equus (Amerhippus) neogeus were members of two distinct lineages. Furthermore, using a rigorous phylogenetic approach, we demonstrate that while formerly the largest equid from Southern America, Equus (Amerhippus) was just a member of the species Equus caballus. This new data increases the known phenotypic plasticity of horses and consequently casts doubt on the taxonomic validity of the subgenus Equus (Amerhippus).     

Molecular Analysis of an Allozyme Cline: Alcohol Dehydrogenase in Drosophila Melanogaster on the East Coast of North America

Clines may either be selectively maintained or be the by-product of nonadaptive processes related to population structure and history. Drosophila melanogaster populations on the east coast of North America show a latitudinal cline in the frequencies of two common electrophoretically distinguishable alleles at the alcohol dehydrogenase locus (Adh), designated Adh-S and Adh-F. This cline may either be adaptive or an artifact of a possible recent dual founding of North American D. melanogaster populations in which frequencies of Adh alleles differed between founder populations. By means of a high resolution restriction-mapping technique, we studied the distribution of 113 haplotypes derived from 44 polymorphic DNA markers within the Adh region in 1533 individuals from 25 populations throughout the cline. We found significant clinal differentiation at the polymorphisms determining the mobility-difference causing amino acid replacement between Adh-F and Adh-S alleles. Hitchhiking was limited, despite extensive linkage disequilibrium, and other sites did not vary clinally. Such a pattern of differentiation implies that selection is responsible for the cline. To investigate whether selection acts only on the Adh-F/S site, we performed a ``selective equivalence'''' test under the assumption that all variability within the specified allelic class is selectively neutral. This revealed selective equivalence among Adh-S-bearing haplotypes, whose frequencies showed no differentiation throughout the cline, implying high levels of frequency-homogenizing gene flow. Geographical heterogeneity among Adh-F-bearing haplotypes implied the action of selection on one or more additional variants in linkage disequilibrium with Adh-F. In a further study of a subset of the data (n = 1076 from 18 populations), we found a combined insertion/deletion polymorphism, designated &1, located in the 5'' adult intron and in linkage disequilibrium with Adh-F, to show more marked clinal variation than Adh-F/S. Although the unequivocal identification of the precise target(s) of selection requires further study, we suggest that clinal selection may be acting epistatically on the Adh-F/S and &1 polymorphisms.