Simon F. K. Hills

Index-free de novo assembly and deconvolution of mixed mitochondrial genomes

Second-generation sequencing technology has allowed a very large increase in sequencing throughput. In order to make use of this high throughput, we have developed a pipeline for sequencing and de novo assembly of multiple mitochondrial genomes without the costs of indexing. Simulation studies on a mixture of diverse animal mitochondrial genomes showed that mitochondrial genomes could be reassembled from a high coverage of short (35 nt) reads, such as those generated by a second-generation Illumina Genome Analyzer. We then assessed this experimentally with long-range polymerase chain reaction products from mitochondria of a human, a rat, a bird, a frog, an insect, and a mollusc. Comparison with reference genomes was used for deconvolution of the assembled contigs rather than for mapping of sequence reads. As proof of concept, we report the complete mollusc mitochondrial genome of an olive shell (Amalda northlandica). It has a very unusual putative control region, which contains a structure that would probably only be detectable by next-generation sequencing. The general approach has considerable potential, especially when combined with indexed sequencing of different groups of genomes.

Molecular support for Pleistocene persistence of the continental Antarctic moss Bryum argenteum

Abstract We examined sequence variation of ITS and phy2 for Bryum argenteum from Antarctica, sub-Antarctic, New Zealand and Australia to understand better taxonomic delimitations and resolve relationships between these geographic regions. Bryum argenteum has been recorded as two species, B. argenteum and B. subrotundifolium, in all four regions with the latter now referred to as B. argenteum var. muticum. We found disagreement between taxon delimitations (based on morphology) and molecular markers. All continental Antarctic specimens consistently formed a monophyletic sister group that consisted of both morphologically identified B. argenteum varieties, separate to all non-Antarctic specimens (also consisting of both varieties). We suggest, contrary to previous records, that all continental Antarctic (Victoria Land) populations are referable to B. argenteum var. muticum, while sub-Antarctic, Australian and New Zealand populations included here are B. argenteum var. argenteum. Additionally, since there was less genetic diversity within Victoria Land, Antarctica, than observed between non-Antarctic samples, we suggest that this is, in part, due to a potentially lower rate of DNA substitution and isolation in northern and southern refugia within Victoria Land since the Pleistocene.

Canine parvoviruses in New Zealand form a monophyletic group distinct from the viruses circulating in other parts of the world

Canine parvovirus 2 (CPV-2) is a well-recognized cause of acute haemorrhagic enteritis in dogs worldwide. The aim of the current study was to identify which CPV-2 subtypes circulate among dogs in New Zealand, and to investigate the evolutionary patterns of contemporary CPV-2 viruses. Faecal samples were collected from 79 dogs with suspected CPV-2 infection over the period of 13 months, and tested for the presence of CPV-2 DNA by PCR. Of 70 positive samples, 69 were subtyped as CPV-2a and one as CPV-2. A majority of CPV-2 positive samples were collected from unvaccinated or not-fully vaccinated puppies ≤6 months of age. The haplotype network produced from New Zealand CPV-2 sequences showed no structure when assessed based on location, vaccination status or age of the animals sampled. International haplotype network indicated that, unlike CPV-2 from other countries, the population of CPV-2 in New Zealand appeared to be monophyletic.

A phylogeny of Southern Hemisphere whelks (Gastropoda: Buccinulidae) and concordance with the fossil record

Under current marine snail taxonomy, the majority of whelks from the Southern Hemisphere (Buccinulidae) are hypothesised to represent a monophyletic clade that has evolved independently from Northern Hemisphere taxa (Buccinidae). Phylogenetic analysis of mitochondrial genomic and nuclear ribosomal DNA sequence data indicates that Southern Hemisphere taxa are not monophyletic, and results suggest that dispersal across the equator has occurred in both directions. New Zealand buccinulid whelks, noted for their high endemic diversity, are also found to not be monophyletic. Using independent fossil calibrations, estimated genetic divergence dates show remarkable concordance with the fossil record of the Penion and Kelletia. The divergence dates and the geographic distribution of the genera through time implies that some benthic marine snails are capable of dispersal over long distances, despite varied developmental strategies. Phylogenetic results also indicate that one species, P. benthicolus belongs in Antarctoneptunea.

Intergenerational mutation rate does not equal long-term evolutionary substitution rate

Recently in PNAS, Langergraber et al. (1) presented interesting findings regarding body size and generation times in chimpanzees and gorillas. The authors then combined these data with recent whole-genome estimates of human mutation rate per generation to recalibrate previous estimates of divergence times in great apes and humans. The authors’ divergence estimates are older than previous findings, which reduce the conflict of previous estimates with some contentious older fossil hominins. It is important to have accurate estimations of generation time, but the authors were quick to apply new intergenerational mutation rates to estimates of divergences millions of years ago, without consideration of the issues this has raised in the past (see ref. 2).

DNA and morphology unite two species and 10 million year old fossils.

Species definition and delimitation is a non-trivial problem in evolutionary biology that is particularly problematic for fossil organisms. This is especially true when considering the continuity of past and present species, because species defined in the fossil record are not necessarily equivalent to species defined in the living fauna. Correctly assigned fossil species are critical for sensitive downstream analysis (e.g., diversification studies and molecular-clock calibration). The marine snail genus Alcithoe exemplifies many of the problems with species identification. The paucity of objective diagnostic characters, prevalence of morphological convergence between species and considerable variability within species that are observed in Alcithoe are typical of a broad range of fossilised organisms. Using a synthesis of molecular and morphometric approaches we show that two taxa currently recognised as distinct are morphological variants of a single species. Furthermore, we validate the fossil record for one of these morphotypes by finding a concordance between the palaeontological record and divergence time of the lineage inferred using molecular-clock analysis. This work demonstrates the utility of living species represented in the fossil record as candidates for molecular-clock calibration, as the veracity of fossil species assignment can be more rigorously tested.

Sticky Genomes: Using NGS Evidence to Test Hybrid Speciation Hypotheses.

Hypotheses of hybrid origin are common. Here we use next generation sequencing to test a hybrid hypothesis for a non-model insect with a large genome. We compared a putative hybrid triploid stick insect species (Acanthoxyla geisovii) with its putative paternal diploid taxon (Clitarchus hookeri), a relationship that provides clear predictions for the relative genetic diversity within each genome. The parental taxon is expected to have comparatively low allelic diversity that is nested within the diversity of the hybrid daughter genome. The scale of genome sequencing required was conveniently achieved by extracting mRNA and sequencing cDNA to examine expressed allelic diversity. This allowed us to test hybrid-progenitor relationships among non-model organisms with large genomes and different ploidy levels. Examination of thousands of independent loci avoids potential problems produced by the silencing of parts of one or other of the parental genomes, a phenomenon sometimes associated with the process of stabilisation of a hybrid genome. Transcript assembles were assessed for evidence of paralogs and/or alternative splice variants before proceeding. Comparison of transcript assemblies was not an appropriate measure of genetic variability, but by mapping reads back to clusters derived from each species we determined levels of allelic diversity. We found greater cDNA sequence diversity among alleles in the putative hybrid species (Acanthoxyla geisovii) than the non-hybrid. The allelic diversity within the putative paternal species (Clitachus hookeri) nested within the hybrid-daughter genome, supports the current view of a hybrid-progenitor relationship for these stick insect species. Next generation sequencing technology provides opportunities for testing evolutionary hypotheses with non-model organisms, including, as here, genomes that are large due to polyploidy.

Evolutionary lineages of marine snails identified using molecular phylogenetics and geometric morphometric analysis of shells

The relationship between morphology and inheritance is of perennial interest in evolutionary biology and palaeontology. Using three marine snail genera Penion, Antarctoneptunea and Kelletia, we investigate whether systematics based on shell morphology accurately reflect evolutionary lineages indicated by molecular phylogenetics. Members of these gastropod genera have been a taxonomic challenge due to substantial variation in shell morphology, conservative radular and soft tissue morphology, few known ecological differences, and geographical overlap between numerous species. Sampling all sixteen putative taxa identified across the three genera, we infer mitochondrial and nuclear ribosomal DNA phylogenetic relationships within the group, and compare this to variation in adult shell shape and size. Results of phylogenetic analysis indicate that each genus is monophyletic, although the status of some phylogenetically derived and likely more recently evolved taxa within Penion is uncertain. The recently described species P. lineatus is supported by genetic evidence. Morphology, captured using geometric morphometric analysis, distinguishes the genera and matches the molecular phylogeny, although using the same dataset, species and phylogenetic subclades are not identified with high accuracy. Overall, despite abundant variation, we find that shell morphology accurately reflects genus-level classification and the corresponding deep phylogenetic splits identified in this group of marine snails.

Genome statistics and phylogenetic reconstructions for Southern Hemisphere whelks (Gastropoda: Buccinulidae)

This data article provides genome statistics, phylogenetic networks and trees for a phylogenetic study of Southern Hemisphere Buccinulidae marine snails [1]. We present alternative phylogenetic reconstructions using mitochondrial genomic and 45S nuclear ribosomal cassette DNA sequence data, as well as trees based on short-length DNA sequence data. We also investigate the proportion of variable sites per sequence length for a set of mitochondrial and nuclear ribosomal genes, in order to examine the phylogenetic information provided by different DNA markers. Sequence alignment files used for phylogenetic reconstructions in the main text and this article are provided here.

Genetic characterization of equid herpesvirus type 1 from cases of abortion in Poland

Equid herpesvirus type 1 (EHV-1) is a common viral infection associated with varied clinical outcomes including respiratory disease, abortion and neurological disease. We have characterized EHV-1 sequences (n = 38) obtained from cases of equine abortion in Poland between 1999 and 2016, based on sequencing of PCR products from open reading frames (ORF) 30 and 68 of the EHV-1 genome. The majority (81.6%) of sequences were not classified into any of the previously described groups based on the ORF68 sequence. The remaining sequences belonged to ORF68 group III (7.9%) or IV (10.5%). A haplotype network analysis did not show any obvious structure within networks of local Polish sequences, nor within a global network of 215 EHV-1 sequences when these networks were coloured based on the geographical origin of viruses or date of detection. Our data suggest that ORF68 does not provide a reliable molecular marker for epidemiological studies of EHV-1, at least in a global sense. Its usefulness to aid local investigations of individual outbreaks remains to be established. All but two Polish EHV-1 sequences belonged to the ORF30 N752 genotype. The two ORF30 D752 viruses were obtained from abortion cases in 2009 and 2010. Hence, abortion cases that occurred in Poland between 1999 and 2016 were caused predominantly by EHV-1 with the ORF30 N752 genotype, with no indication of an increase in the prevalence of the ORF30 D752 variant.