Lineage Identification Affects Estimates of Evolutionary Mode in Marine Snails.

In order to study evolutionary pattern and process, we need to be able to accurately identify species and the evolutionary lineages from which they are derived. Determining the concordance between genetic and morphological variation of living populations, and then directly comparing extant and fossil morphological data, provides a robust approach for improving our identification of lineages through time. We investigate genetic and shell morphological variation in extant species of Penion marine snails from New Zealand, and extend this analysis into deep time using fossils. We find that genetic and morphological variation identify similar patterns and support most currently recognized extant species. However, some taxonomic over-splitting is detected due to shell size being a poor trait for species delimitation, and we identify incorrect assignment of some fossil specimens. We infer that a single evolutionary lineage (Penion sulcatus) has existed for 22 myr, with most aspects of shell shape and shell size evolving under a random walk. However, by removing samples previously classified as the extinct species P. marwicki, we instead detect morphological stasis for one axis of shell shape variation. This result demonstrates how lineage identification can change our perception of evolutionary pattern and process. [Genotyping by sequencing; geometric morphometrics; morphological evolution; Neogastropoda; phenotype; speciation; stasis.].

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.

Genomic characterisation of Felis catus papillomavirus type 5 with proposed classification within a new papillomavirus genus.

Four Felis catus papillomavirus (FcaPV) types have been fully sequenced from domestic cats. Of these, FcaPV-2 and -3 are thought to cause feline viral plaques and Bowenoid in situ carcinomas. Two short sequences of DNA from a previously unreported PV type were amplified from a feline viral plaque using consensus PCR primers. DNA was then extracted from a swab of the lesion and two sets of 'outward facing' primers were designed using the short sequences to amplify the entire 7600bp genome of the novel PV. The PV was designated FcaPV-5 and contained putative coding regions that were predicted to produce five early proteins and two late ones. The ORF L1 showed over 65% similarity to that of FcaPV-3 and -4. Assignment to a genus was difficult as the PV was over 60% similar to PV types from 4 different genera. However, due to the ORF L1 similarity of FcaPV-3, -4, and -5, the shared host species of all three PVs, and the similar lesions associated with FcaPV-3 and -5, it is proposed all three PVs are classified within a new genus. FcaPV-5 is the third PV type to be associated with feline viral plaques. The plaque that contained FcaPV-5 showed unusual histological features including hyperplasia and PV-induced cell changes in sebaceous glands and deep within hair follicles. While additional study of further lesions of this type is required, it is possible that FcaPV-5 may be able to infect a broader range of cells than other PV types.

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.

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.

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.

Genomic characterisation of Felis catus papillomavirus 4, a novel papillomavirus detected in the oral cavity of a domestic cat.

Three papillomaviruses (PVs) from the domestic cat have been fully sequenced so far including Felis domesticus PV-1 (FdPV-1), FdPV-2, and a recently described Felis catus PV-3 (FcaPV-4). In the current article, we describe the full genomic sequence of a fourth PV from the domestic cat. This PV was amplified from the oral cavity of a cat with severe gingivitis. However, the aetiological involvement of FcaPV-4 in development of lesions observed in this cat remains uncertain. The complete genome of the novel virus comprised 7,616 bp and was predicted to encode five early (E1, E2, E4, E6 and E7) and two late (L1 and L2) genes, with the organisation typical for PVs. The L1 showed 65.1 % nucleotide sequence identity to L1 of FcaPV-3 and approximately 60 % identity to L1 of canine tau-papillomaviruses CPV-2 and CPV-7. The novel virus clustered with FcaPV-3, CPV-2 and CPV-7 on a phylogenetic tree constructed from a concatenated alignment of 3,013 bp from E1, E2, L1 and L2. Based on the genomic and phylogenetic data, we propose that the novel virus is classified as a distinct species within the same genus as FcaPV-3. We also propose that both viruses are classified within the genus Taupapillomavirus, although this classification may need to be re-visited after more tau-PV genomes become available.

Genomic characterization of Felis catus papillomavirus-3: a novel papillomavirus detected in a feline Bowenoid in situ carcinoma.

There is increasing evidence that papillomaviruses (PVs) may cause skin cancer in cats. Neoplasms most frequently contain Felis domesticus PV type 2 (FdPV-2) DNA, but other PV DNA sequences have also been detected suggesting multiple PVs could cause disease. One of these sequences, FdPV-MY2, was previously detected in 5 of a series of 70 feline skin cancers. The aim was to determine the genome sequence of this PV. Using the circular nature of PV DNA, 'outward facing' primers specific for FdPV-MY2 were designed and amplified a 7300 bp length of DNA from a feline Bowenoid in situ carcinoma (BISC) that showed microscopic evidence of a viral etiology and tested positive for FdPV-MY2 DNA. The PCR product was sequenced using next generation sequencing technology. The full genomic sequence of the virus, comprising 7583 bp, was assembled and analyzed. As this is the third PV from a domestic cat, the virus was designated Felis catus PV type 3 (FcaPV-3). Consistent with other PVs, the putative coding regions of FcaPV-3 were predicted to produce 6 early proteins and 2 late ones. Classification was difficult as the virus contained over 60% nucleotide similarity within the ORF L1 with PVs from 3 different genera. However, based on phylogenetic analysis of ORF L1, FcaPV-3 was most closely related to the tau-PVs CPV-2 and CPV-7. As FcaPV-3 has over 60% nucleotide similarity with the ORF L1 of both tau-PVs, it is proposed that FcaPV-3 is classified in the genus Taupapillomavirus and is the first non-canine PV in this genus.

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.

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.