Howard A. Ross

Testing the Reliability of Genetic Methods of Species Identification via Simulation

Although genetic methods of species identification, especially DNA barcoding, are strongly debated, tests of these methods have been restricted to a few empirical cases for pragmatic reasons. Here we use simulation to test the performance of methods based on sequence comparison (BLAST and genetic distance) and tree topology over a wide range of evolutionary scenarios. Sequences were simulated on a range of gene trees spanning almost three orders of magnitude in tree depth and in coalescent depth; that is, deep or shallow trees with deep or shallow coalescences. When the querys conspecific sequences were included in the reference alignment, the rate of positive identification was related to the degree to which different species were genetically differentiated. The BLAST, distance, and liberal tree-based methods returned higher rates of correct identification than did the strict tree-based requirement that the query was within, but not sister to, a single-species clade. Under this more conservative approach, ambiguous outcomes occurred in inverse proportion to the number of reference sequences per species. When the querys conspecific sequences were not in the reference alignment, only the strict tree-based approach was relatively immune to making false-positive identifications. Thresholds affected the rates at which false-positive identifications were made when the querys species was unrepresented in the reference alignment but did not otherwise influence outcomes. A conservative approach using the strict tree-based method should be used initially in large-scale identification systems, with effort made to maximize sequence sampling within species. Once the genetic variation within a taxonomic group is well characterized and the taxonomy resolved, then the choice of method used should be dictated by considerations of computational efficiency. The requirement for extensive genetic sampling may render these techniques inappropriate in some circumstances.

Immune-Mediated Positive Selection Drives Human Immunodeficiency Virus Type 1 Molecular Variation and Predicts Disease Duration

ABSTRACT Using likelihood-based evolutionary methods, we demonstrate that the broad genetic diversity of human immunodeficiency virus type 1 (HIV-1) in an infected individual is a consequence of site-specific positive selection for diversity, a likely consequence of immune recognition. In particular, the extent of positive selection appears to be a good predictor of disease duration. Positively selected sites along HIV-1 partial env sequences are numerous but not distributed uniformly. In a sample of eight patients studied longitudinally, the proportion of sites per sample under positive selection was a statistically significant predictor of disease duration. Among long-term progressors, positive selection persisted at sites over time and appears to be associated with helper T-cell epitopes. In contrast, sites under positive selection shifted from one longitudinal sample to the next in short-term progressors. Our study is consistent with the hypothesis that a broad and persistent immunologic response is associated with a slower rate of disease progression. In contrast, narrow, shifting immune responses characterize short-term progressors.

Species delimitation – a geneious plugin for the exploration of species boundaries

Species Delimitation is a plugin to the Geneious software to support the exploration of species boundaries in a gene tree. The user assigns taxa to putative species and the plugin computes statistics relating to the probability of the observed monophyly or exclusivity having occurred by chance in a coalescent process. It also assesses the within and between species genetic distances to infer the probability with which members of a putative species might be identified successfully with tree‐based methods.

Mosaic structure of human coronavirus NL63, one thousand years of evolution.

Abstract Before the SARS outbreak only two human coronaviruses (HCoV) were known: HCoV-OC43 and HCoV-229E. With the discovery of SARS-CoV in 2003, a third family member was identified. Soon thereafter, we described the fourth human coronavirus (HCoV-NL63), a virus that has spread worldwide and is associated with croup in children. We report here the complete genome sequence of two HCoV-NL63 clinical isolates, designated Amsterdam 57 and Amsterdam 496. The genomes are 27,538 and 27,550 nucleotides long, respectively, and share the same genome organization. We identified two variable regions, one within the 1a and one within the S gene, whereas the 1b and N genes were most conserved. Phylogenetic analysis revealed that HCoV-NL63 genomes have a mosaic structure with multiple recombination sites. Additionally, employing three different algorithms, we assessed the evolutionary rate for the S gene of group Ib coronaviruses to be ∼3×10−4 substitutions per site per year. Using this evolutionary rate we determined that HCoV-NL63 diverged in the 11th century from its closest relative HCoV-229E.

Epidemiology, Genetic Diversity, and Evolution of Endemic Feline Immunodeficiency Virus in a Population of Wild Cougars

ABSTRACT Within the large body of research on retroviruses, the distribution and evolution of endemic retroviruses in natural host populations have so far received little attention. In this study, the epidemiology, genetic diversity, and molecular evolution of feline immunodeficiency virus specific to cougars (FIVpco) was examined using blood samples collected over several years from a free-ranging cougar population in the western United States. The virus prevalence was 58% in this population (n = 52) and increased significantly with host age. Based on phylogenetic analysis of fragments of envelope (env) and polymerase (pol) genes, two genetically distinct lineages of FIVpco were found to cooccur in the population but not in the same individuals. Within each of the virus lineages, geographically nearby isolates formed monophyletic clusters of closely related viruses. Sequence diversity for env within a host rarely exceeded 1%, and the evolution of this gene was dominated by purifying selection. For both pol and env, our data indicate mean rates of molecular evolution of 1 to 3% per 10 years. These results support the premise that FIVpco is well adapted to its cougar host and provide a basis for comparing lentivirus evolution in endemic and epidemic infections in natural hosts.

Latitude, elevation and the tempo of molecular evolution in mammals

Faster rates of microevolution have been recorded for plants and marine foraminifera occupying warmer low latitude environments relative to those occurring at higher latitudes. By contrast, because this rate heterogeneity has been attributed to a relationship between thermal habit and mutagenesis via a body temperature linkage, it has been assumed that microevolution in mammals should not also vary systematically with environmental temperature. However, this assumption has not previously been empirically examined. In this study, we tested for a thermally mediated influence on the tempo of microevolution among mammals using a comprehensive global dataset that included 260 mammal species, from 10 orders and 29 families. In contrast to theoretical predictions, we found that substitution rates in the cytochrome b gene have been substantially faster for species living in warmer latitudes and elevations relative to sister species living in cooler habitats. These results could not be attributed to factors otherwise thought to influence rates of microevolution, such as body mass differentials or genetic drift. Instead, the results indicate that the tempo of microevolution among mammals is either responding directly to the thermal environment or indirectly via an ecological mechanism such as the ‘Red Queen’ effect.

Pyrosequencing reveals regional differences in fruit‐associated fungal communities

We know relatively little of the distribution of microbial communities generally. Significant work has examined a range of bacterial communities, but the distribution of microbial eukaryotes is less well characterized. Humans have an ancient association with grape vines (Vitis vinifera) and have been making wine since the dawn of civilization, and fungi drive this natural process. While the molecular biology of certain fungi naturally associated with vines and wines is well characterized, complementary investigations into the ecology of fungi associated with fruiting plants is largely lacking. DNA sequencing technologies allow the direct estimation of microbial diversity from a given sample, avoiding culture-based biases. Here, we use deep community pyrosequencing approaches, targeted at the 26S rRNA gene, to examine the richness and composition of fungal communities associated with grapevines and test for geographical community structure among four major regions in New Zealand (NZ). We find over 200 taxa using this approach, which is 10-fold more than previously recovered using culture-based methods. Our analyses allow us to reject the null hypothesis of homogeneity in fungal species richness and community composition across NZ and reveal significant differences between major areas.

Feline Lentivirus Evolution in Cross-Species Infection Reveals Extensive G-to-A Mutation and Selection on Key Residues in the Viral Polymerase

ABSTRACT Factors that restrict a virus from establishing productive infection in a new host species are important to understand because cross-species transmission events are often associated with emergent viral diseases. To determine the evolutionary pressures on viruses in new host species, we evaluated the molecular evolution of a feline immunodeficiency virus derived from a wild cougar, Puma concolor, during infection of domestic cats. Analyses were based on the coding portion of genome sequences recovered at intervals over 37 weeks of infection of six cats inoculated by either intravenous or oral-nasal routes. All cats inoculated intravenously, but only one inoculated orally-nasally, became persistently viremic. There were notable accumulations of lethal errors and predominance of G-to-A alterations throughout the genome, which were marked in the viral polymerase gene, pol. Viral structural (env and gag) and accessory (vif and orfA) genes evolved neutrally or were under purifying selection. However, sites under positive selection were identified in reverse transcriptase that involved residues in the nucleotide binding pocket or those contacting the RNA-DNA duplex. The findings of extensive G-to-A alterations in this cross-species infection are consistent with the recently described editing of host cytidine deaminase on lentivirus genomes. Additionally, we demonstrate that the primary site of hypermutation is the viral pol gene and the dominant selective force acting on this feline immunodeficiency virus as it replicates in a new host species is on key residues of the virus polymerase.

www.DNA-surveillance: applied molecular taxonomy for species conservation and discovery

guished using molecular methods in addition to morphological data, but molecular methods cannot realistically be the first or only appropriate method, as has been proposed [4]. Furthermore, the genetic diversity of individuals within and among populations begs the question posed by Lipscomb et al. [3]: what are the appropriate gene sequences for objective taxonomic assessments, and the appropriate genetic/ molecular dissimilarity to define ‘taxa’? We have known for almost a decade that the biodiversity crisis is resulting in the renewed relevance of natural-history collections [7], and the wider use of morphological ‘data’ contained in such collections has been repeatedly validated [8,9]. Perhaps this DNA versus morphology debate is largely one of each camp attempting to get ‘there’ first by proving greater relevance in the competition for limited funds. However, there is, and must be, room for both approaches. References 1 Mallet, J. and Willmott, K. (2003) Taxonomy: renaissance or Tower of Babel? Trends Ecol. Evol. 18, 57–59 2 Seberg, O. et al. (2003) Shortcuts in systematics? A commentary on DNA-based taxonomy. Trends Ecol. Evol. 18, 63–65 3 Lipscomb, D. et al. (2003) The intellectual content of taxonomy: a comment on DNA in taxonomy. Trends Ecol. Evol. 18, 65–67 4 Tautz, D. et al. (2003) A plea for DNA taxonomy. Trends Ecol. Evol. 18, 70–74 5 Schaub, M. and Dunn, C.P. (2002) vPlants: a virtual herbarium of the Chicago region. First Monday 7 http://www.firstmonday.dk/issues/ issue7_5/schaub/index.html 6 Soberón, J.M. et al. (2000) The use of specimen-label databases for conservation purposes: an example using Mexican papilionid and pierid butterflies. Biodiv. Conserv. 9, 1441–1466 7 Alberch, P. (1993) Museums, collections and biodiversity inventories. Trends Ecol. Evol. 8, 372–375 8 Butler, D. (1998) Museum research comes off the list of endangered species. Nature 394, 115–119 9 Winker, K. (1999) How to bring collections data into the net. Nature 401, 524

Evaluating a multigene environmental DNA approach for biodiversity assessment

BackgroundThere is an increasing demand for rapid biodiversity assessment tools that have a broad taxonomic coverage. Here we evaluate a suite of environmental DNA (eDNA) markers coupled with next generation sequencing (NGS) that span the tree of life, comparing them with traditional biodiversity monitoring tools within ten 20×20 meter plots along a 700 meter elevational gradient.ResultsFrom six eDNA datasets (one from each of 16S, 18S, ITS, trnL and two from COI) we identified sequences from 109 NCBI taxonomy-defined phyla or equivalent, ranging from 31 to 60 for a given eDNA marker. Estimates of alpha and gamma diversity were sensitive to the number of sequence reads, whereas beta diversity estimates were less sensitive. The average within-plot beta diversity was lower than between plots for all markers. The soil beta diversity of COI and 18S markers showed the strongest response to the elevational variation of the eDNA markers (COI: r=0.49, p<0.001; 18S: r=0.48, p<0.001). Furthermore pairwise beta diversities for these two markers were strongly correlated with those calculated from traditional vegetation and invertebrate biodiversity measures.ConclusionsUsing a soil-based eDNA approach, we demonstrate that standard phylogenetic markers are capable of recovering sequences from a broad diversity of eukaryotes, in addition to prokaryotes by 16S. The COI and 18S eDNA markers are the best proxies for aboveground biodiversity based on the high correlation between the pairwise beta diversities of these markers and those obtained using traditional methods.