Terry Bertozzi

Rise of the machines - recommendations for ecologists when using next generation sequencing for microsatellite development

Next generation sequencing is revolutionizing molecular ecology by simplifying the development of molecular genetic markers, including microsatellites. Here, we summarize the results of the large‐scale development of microsatellites for 54 nonmodel species using next generation sequencing and show that there are clear differences amongst plants, invertebrates and vertebrates for the number and proportion of motif types recovered that are able to be utilized as markers. We highlight that the heterogeneity within each group is very large. Despite this variation, we provide an indication of what number of sequences and consequent proportion of a 454 run are required for the development of 40 designable, unique microsatellite loci for a typical molecular ecological study. Finally, to address the challenges of choosing loci from the vast array of microsatellite loci typically available from partial genome runs (average for this study, 2341 loci), we provide a microsatellite development flowchart as a procedural guide for application once the results of a partial genome run are obtained.

Widespread horizontal transfer of retrotransposons

In higher organisms such as vertebrates, it is generally believed that lateral transfer of genetic information does not readily occur, with the exception of retroviral infection. However, horizontal transfer (HT) of protein coding repetitive elements is the simplest way to explain the patchy distribution of BovB, a long interspersed element (LINE) about 3.2 kb long, that has been found in ruminants, marsupials, squamates, monotremes, and African mammals. BovB sequences are a major component of some of these genomes. Here we show that HT of BovB is significantly more widespread than believed, and we demonstrate the existence of two plausible arthropod vectors, specifically reptile ticks. A phylogenetic tree built from BovB sequences from species in all of these groups does not conform to expected evolutionary relationships of the species, and our analysis indicates that at least nine HT events are required to explain the observed topology. Our results provide compelling evidence for HT of genetic material that has transformed vertebrate genomes.

Closing the mitochondrial circle on paraphyly of the Monogenea (Platyhelminthes) infers evolution in the diet of parasitic flatworms

Relationships between the three classes of Neodermata (parasitic Platyhelminthes) are much debated and restrict our understanding of the evolution of parasitism and contingent adaptations. The historic view of a sister relationship between Cestoda and Monogenea (Cercomeromorphae; larvae bearing posterior hooks) has been dismissed and the weight of evidence against monogenean monophyly has mounted. We present the nucleotide sequence of the complete mitochondrial (mt) genome of Benedenia seriolae (Monogenea: Monopisthocotylea: Capsalidae), the first complete non-gyrodactylid monopisthocotylean mt genome to be reported. We also include nucleotide sequence data for some mt protein coding genes for a second capsalid, Neobenedenia sp. Analyses of the new mt genomes with all available platyhelminth mt genomes provide new phylogenetic hypotheses, which strongly influence perspectives on the evolution of diet in the Neodermata. Our analyses do not support monogenean monophyly but confirm that the Digenea and Cestoda are each monophyletic and sister groups. Epithelial feeding monopisthocotyleans on fish hosts are basal in the Neodermata and represent the first shift to parasitism from free-living ancestors. The next evolutionary step in parasitism was a dietary change from epithelium to blood. The common ancestor of Digenea+Cestoda was monogenean-like and most likely sanguinivorous. From this ancestral condition, adult digeneans and cestodes independently evolved dietary specialisations to suit their diverse microhabitats in their final vertebrate hosts. These improved perspectives on relationships fundamentally enhance our understanding of the evolution of parasitism in the Neodermata and in particular, the evolution of diet.

Looks can deceive: Molecular phylogeny of a family of flatworm ectoparasites (Monogenea: Capsalidae) does not reflect current morphological classification

The morphological based taxonomy of highly derived parasite groups is likely to poorly reflect their evolutionary relationships. The taxonomy of the monogenean family Capsalidae, which comprises approximately 180 species of flatworm parasites that predominantly attach to external surfaces of chondrichthyan and teleost fishes, is based mainly on six morphological characters. The phylogenetic history of the family is largely unknown. We reconstructed the phylogenetic relationships of 47 species in 20 genera from eight of the nine subfamilies, from nucleotide sequences of three unlinked nuclear genes, 28S ribosomal RNA, Histone 3 and Elongation Factor 1 alpha. Our phylogeny was well corroborated, with 75% of branches receiving strong support from both Bayesian posterior probabilities and maximum likelihood bootstrap proportions and all nodes showed positive partitioned likelihood support for each of the three genes. We found that the family was monophyletic, with the Gyrodactylidae and Udonellidae forming the sister group. The Capsalinae was monophyletic, however, our data do not support monophyly for the Benedeniinae, Entobdellinae and Trochopodinae. Monophyly was supported for Capsala, Entobdella, Listrocephalos, Neobenedenia and Tristoma, but Benedenia and Neoentobdella were polyphyletic. Comparisons of the distribution of character states for the small number of morphological characters on the molecular phylogeny show a high frequency of apparent homoplasy. Consequently the current morphological classification shows little correspondence with the phylogenetic relationships within the family.

Anonymous nuclear loci in non-model organisms

MOTIVATION When working with non-model organisms, few if any species-specific markers are available for phylogenetic, phylogeographic and population studies. Therefore, researchers often try to adapt markers developed in distantly related taxa, resulting in poor amplification and ascertainment bias in their target taxa. Markers can be developed de novo and anonymous nuclear loci (ANL) are proving to be a boon for researchers seeking large numbers of fast-evolving, independent loci. However, the development of ANL can be laboratory intensive and expensive. A workflow is described to identify suitable low-copy anonymous loci from high-throughput shotgun sequences, dramatically reducing the cost and time required to develop these markers and produce robust multilocus datasets. RESULTS By successively removing repetitive and evolutionary conserved sequences from low coverage shotgun libraries, we were able to isolate thousands of potential ANL. Empirical testing of loci developed from two reptile taxa confirmed that our methodology yields markers with comparable amplification rates and nucleotide diversities to ANLs developed using other methodologies. Our approach capitalizes on next-generation sequencing technologies to enable the development of phylogenetic, phylogeographic and population markers for taxa lacking suitable genomic resources.

Host‐defence peptide profiles of the skin secretions of interspecific hybrid tree frogs and their parents, female Litoria splendida and male Litoria caerulea

Five healthy adult female first‐generation hybrid tree frogs were produced by interspecific breeding of closely related tree frogs Litoria splendida and L. caerulea in a cage containing large numbers of males and females of both species. Phylogenetic analysis of mitochondrial DNA sequences established the female parent to be L. splendida. The peptide profile of the hybrid frogs included the neuropeptide caerulein, four antibiotics of the caerin 1 family and several neuronal nitric oxide synthase inhibitors of the caerin 1 and 2 classes of peptides. The skin secretions of the hybrids contained some peptides common to only one parent, some produced by both parental species, and four peptides expressed by the hybrids but not the parental species.

Panmixia supports divergence with gene flow in Darwin's small ground finch, Geospiza fuliginosa, on Santa Cruz, Galapagos Islands

The divergence‐with‐gene‐flow model of speciation has a strong theoretical basis with a growing number of plausible examples in nature, but remains hotly debated. Darwin’s finches of the Galápagos Archipelago have played an important role in our understanding of speciation processes. Recent studies suggest that this group may also provide insights into speciation via divergence with gene flow. On the island of Santa Cruz, recent studies found evidence for adaptive divergence in Darwin’s small ground finch, Geospiza fuliginosa, between ecologically contrasting arid and humid zones. Despite the short geographical distance between these zones, strong disruptive selection during low rainfall periods is expected to generate and maintain adaptive divergence. Conversely, during high rainfall periods, when disruptive selection is predicted to be weakened, population divergence in adaptive traits is expected to break down. Because periods of low and high rainfall irregularly alternate, the geographical pattern of adaptive divergence can be assumed to break down and, importantly, regenerate in situ. Here, we use microsatellite allele frequency data to assess the genetic population structure of G. fuliginosa on Santa Cruz. We sample 21 sites and four ecological zones across the island. We reject hypotheses of population substructure linked to ecological and geographical differences among sites in favour of a single panmictic population. Panmixia implies high levels of gene flow within Santa Cruz, which favours selection over genetic drift as a valid process generating phenotypic divergence in G. fuliginosa on Santa Cruz. We discuss how our findings may support classic adaptation, phenotypic plasticity, matching habitat choice or any combination of these three processes.

LINEs between Species: Evolutionary Dynamics of LINE-1 Retrotransposons across the Eukaryotic Tree of Life

LINE-1 (L1) retrotransposons are dynamic elements. They have the potential to cause great genomic change because of their ability to ‘jump’ around the genome and amplify themselves, resulting in the duplication and rearrangement of regulatory DNA. Active L1, in particular, are often thought of as tightly constrained, homologous and ubiquitous elements with well-characterized domain organization. For the past 30 years, model organisms have been used to define L1s as 6–8 kb sequences containing a 5′-UTR, two open reading frames working harmoniously in cis, and a 3′-UTR with a polyA tail. In this study, we demonstrate the remarkable and overlooked diversity of L1s via a comprehensive phylogenetic analysis of elements from over 500 species from widely divergent branches of the tree of life. The rapid and recent growth of L1 elements in mammalian species is juxtaposed against the diverse lineages found in other metazoans and plants. In fact, some of these previously unexplored mammalian species (e.g. snub-nosed monkey, minke whale) exhibit L1 retrotranspositional ‘hyperactivity’ far surpassing that of human or mouse. In contrast, non-mammalian L1s have become so varied that the current classification system seems to inadequately capture their structural characteristics. Our findings illustrate how both long-term inherited evolutionary patterns and random bursts of activity in individual species can significantly alter genomes, highlighting the importance of L1 dynamics in eukaryotes.

Positive Selection in the N-Terminal Extramembrane Domain of Lung Surfactant Protein C (SP-C) in Marine Mammals

Maximum-likelihood models of codon and amino acid substitution were used to analyze the lung-specific surfactant protein C (SP-C) from terrestrial, semi-aquatic, and diving mammals to identify lineages and amino acid sites under positive selection. Site models used the nonsynonymous/synonymous rate ratio (ω) as an indicator of selection pressure. Mechanistic models used physicochemical distances between amino acid substitutions to specify nonsynonymous substitution rates. Site models strongly identified positive selection at different sites in the polar N-terminal extramembrane domain of SP-C in the three diving lineages: site 2 in the cetaceans (whales and dolphins), sites 7, 9, and 10 in the pinnipeds (seals and sea lions), and sites 2, 9, and 10 in the sirenians (dugongs and manatees). The only semi-aquatic contrast to indicate positive selection at site 10 was that including the polar bear, which had the largest body mass of the semi-aquatic species. Analysis of the biophysical properties that were influential in determining the amino acid substitutions showed that isoelectric point, chemical composition of the side chain, polarity, and hydrophobicity were the crucial determinants. Amino acid substitutions at these sites may lead to stronger binding of the N-terminal domain to the surfactant phospholipid film and to increased adsorption of the protein to the air-liquid interface. Both properties are advantageous for the repeated collapse and reinflation of the lung upon diving and resurfacing and may reflect adaptations to the high hydrostatic pressures experienced during diving.

Opsin transcripts of predatory diving beetles: a comparison of surface and subterranean photic niches.

The regressive evolution of eyes has long intrigued biologists yet the genetic underpinnings remain opaque. A system of discrete aquifers in arid Australia provides a powerful comparative means to explore trait regression at the genomic level. Multiple surface ancestors from two tribes of diving beetles (Dytiscidae) repeatedly invaded these calcrete aquifers and convergently evolved eye-less phenotypes. We use this system to assess transcription of opsin photoreceptor genes among the transcriptomes of two surface and three subterranean dytiscid species and test whether these genes have evolved under neutral predictions. Transcripts for UV, long-wavelength and ciliary-type opsins were identified from the surface beetle transcriptomes. Two subterranean beetles showed parallel loss of all opsin transcription, as expected under ‘neutral’ regressive evolution. The third species Limbodessus palmulaoides retained transcription of a long-wavelength opsin (lwop) orthologue, albeit in an aphotic environment. Tests of selection on lwop indicated no significant differences between transcripts derived from surface and subterranean habitats, with strong evidence for purifying selection acting on L. palmulaoides lwop. Retention of sequence integrity and the lack of evidence for neutral evolution raise the question of whether we have identified a novel pleiotropic role for lwop, or an incipient phase of pseudogene development.