Florian Leese

Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol

Metabarcoding is an emerging genetic tool to rapidly assess biodiversity in ecosystems. It involves high-throughput sequencing of a standard gene from an environmental sample and comparison to a reference database. However, no consensus has emerged regarding laboratory pipelines to screen species diversity and infer species abundances from environmental samples. In particular, the effect of primer bias and the detection limit for specimens with a low biomass has not been systematically examined, when processing samples in bulk. We developed and tested a DNA metabarcoding protocol that utilises the standard cytochrome c oxidase subunit I (COI) barcoding fragment to detect freshwater macroinvertebrate taxa. DNA was extracted in bulk, amplified in a single PCR step, and purified, and the libraries were directly sequenced in two independent MiSeq runs (300-bp paired-end reads). Specifically, we assessed the influence of specimen biomass on sequence read abundance by sequencing 31 specimens of a stonefly species with known haplotypes spanning three orders of magnitude in biomass (experiment I). Then, we tested the recovery of 52 different freshwater invertebrate taxa of similar biomass using the same standard barcoding primers (experiment II). Each experiment was replicated ten times to maximise statistical power. The results of both experiments were consistent across replicates. We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads. Furthermore, we reliably recovered 83% of the 52 taxa used to test primer bias. However, sequence abundance varied by four orders of magnitudes between taxa despite the use of similar amounts of biomass. Our metabarcoding approach yielded reliable results for high-throughput assessments. However, the results indicated that primer efficiency is highly species-specific, which would prevent straightforward assessments of species abundance and biomass in a sample. Thus, PCR-based metabarcoding assessments of biodiversity should rely on presence-absence metrics.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 August 2009-30 September 2009

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross‐tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

Genome-wide analysis of tandem repeats in Daphnia pulex - a comparative approach

BackgroundDNA tandem repeats (TRs) are not just popular molecular markers, but are also important genomic elements from an evolutionary and functional perspective. For various genomes, the densities of short TR types were shown to differ strongly among different taxa and genomic regions. In this study we analysed the TR characteristics in the genomes of Daphnia pulex and 11 other eukaryotic species. Characteristics of TRs in different genomic regions and among different strands are compared in details for D. pulex and the two model insects Apis mellifera and Drosophila melanogaster.ResultsProfound differences in TR characteristics were found among all 12 genomes compared in this study. In D. pulex, the genomic density of TRs was low compared to the arthropod species D. melanogaster and A. mellifera. For these three species, very few common features in repeat type usage, density distribution, and length characteristics were observed in the genomes and in different genomic regions. In introns and coding regions an unexpectedly high strandedness was observed for several repeat motifs. In D. pulex, the density of TRs was highest in introns, a rare feature in animals. In coding regions, the density of TRs with unit sizes 7-50 bp were more than three times as high as for 1-6 bp repeats.ConclusionsTRs in the genome of D. pulex show several notable features, which distinguish it from the other genomes. Altogether, the highly non-random distribution of TRs among genomes, genomic regions and even among different DNA-stands raises many questions concerning their functional and evolutionary importance. The high density of TRs with a unit size longer than 6 bp found in non-coding and coding regions underpins the importance to include longer TR units in comparative analyses.

Predator-induced defences in Daphnia pulex: Selection and evaluation of internal reference genes for gene expression studies with real-time PCR

BackgroundThe planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research. One aspect that has sustained interest in the study system is the ability of D. pulex to develop inducible defence structures when exposed to predators, such as the phantom midge larvae Chaoborus. The available draft genome sequence for D. pulex is accelerating research to identify genes that confer plastic phenotypes that are regularly cued by environmental stimuli. Yet for quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data.ResultsIn this study, we tested six candidate reference genes for normalizing transcription levels of D. pulex genes; alpha tubulin (aTub), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box binding protein (Tbp) syntaxin 16 (Stx16), X-box binding protein 1 (Xbp1) and CAPON, a protein associated with the neuronal nitric oxide synthase, were selected on the basis of an earlier study and from microarray studies. One additional gene, a matrix metalloproteinase (MMP), was tested to validate its transcriptional response to Chaoborus, which was earlier observed in a microarray study. The transcription profiles of these seven genes were assessed by qRT-PCR from RNA of juvenile D. pulex that showed induced defences in comparison to untreated control animals. We tested the individual suitability of genes for expression normalization using the programs geNorm, NormFinder and BestKeeper. Intriguingly, Xbp1, Tbp, CAPON and Stx16 were selected as ideal reference genes. Analyses on the relative expression level using the software REST showed that both classical housekeeping candidate genes (aTub and GAPDH) were significantly downregulated, whereas the MMP gene was shown to be significantly upregulated, as predicted. aTub is a particularly ill suited reference gene because five copies are found in the D. pulex genome sequence. When applying aTub for expression normalization Xbp1 and Tbp are falsely reported as significantly upregulated.ConclusionsOur results suggest that the genes Xbp1, Tbp, CAPON and Stx16 are suitable reference genes for accurate normalization in qRT-PCR studies using Chaoborus-induced D. pulex specimens. Furthermore, our study underscores the importance of verifying the expression stability of putative reference genes for normalization of expression levels.

Ecological genomics: steps towards unraveling the genetic basis of inducible defenses in Daphnia

Little is known about the genetic mechanisms underlying inducible defenses. Recently, the genome of Daphnia pulex, a model organism for defense studies, has been sequenced. Building on the genome information, recent preliminary studies in BMC Developmental Biology and BMC Molecular Biology have assessed gene response profiles in Daphnia under predation pressure. We review the significance of the findings and highlight future research perspectives.See research articles http://www.biomedcentral.com/1471-2164/10/527, http://www.biomedcentral.com/1471-2105/6/45, http://www.biomedcentral.com/1471-213X/10/45

STAMP: Extensions to the STADEN sequence analysis package for high throughput interactive microsatellite marker design

BackgroundMicrosatellites (MSs) are DNA markers with high analytical power, which are widely used in population genetics, genetic mapping, and forensic studies. Currently available software solutions for high-throughput MS design (i) have shortcomings in detecting and distinguishing imperfect and perfect MSs, (ii) lack often necessary interactive design steps, and (iii) do not allow for the development of primers for multiplex amplifications. We present a set of new tools implemented as extensions to the STADEN package, which provides the backbone functionality for flexible sequence analysis workflows. The possibility to assemble overlapping reads into unique contigs (provided by the base functionality of the STADEN package) is important to avoid developing redundant markers, a feature missing from most other similar tools.ResultsOur extensions to the STADEN package provide the following functionality to facilitate microsatellite (and also minisatellite) marker design: The new modules (i) integrate the state-of-the-art tandem repeat detection and analysis software PHOBOS into workflows, (ii) provide two separate repeat detection steps – with different search criteria – one for masking repetitive regions during assembly of sequencing reads and the other for designing repeat-flanking primers for MS candidate loci, (iii) incorporate the widely used primer design program PRIMER 3 into STADEN workflows, enabling the interactive design and visualization of flanking primers for microsatellites, and (iv) provide the functionality to find optimal locus- and primer pair combinations for multiplex primer design. Furthermore, our extensions include a module for storing analysis results in an SQLite database, providing a transparent solution for data access from within as well as from outside of the STADEN Package.ConclusionThe STADEN package is enhanced by our modules into a highly flexible, high-throughput, interactive tool for conventional and multiplex microsatellite marker design. It gives the user detailed control over the workflow, enabling flexible combinations of manual and automated analysis steps. The software is available under the OpenBSD License [1, 2]. The high efficiency of our automated marker design workflow has been confirmed in three microsatellite development projects.

The utility of fast evolving molecular markers for studying speciation in the Antarctic benthos

The Southern Ocean is surprisingly rich in species that coexist in one of the most extreme environments on Earth yet the processes leading to speciation in this ecosystem are not well understood. To remedy this, tools that measure the genetic connectedness within a species are needed. Although useful for phylogenetic purposes, the readily available mitochondrial markers (e.g. 16S, COI) suffer from numerous shortcomings for population genetics. Therefore, molecular markers are needed that are sufficiently variable, unlinked, biparentally inherited, and distributed over the whole genome. We argue that microsatellites are suitable markers that have not been widely used in exploratory studies due to their difficult initial set-up. Working with the Ceratoserolis trilobitoides species complex (Isopoda), we demonstrate that using a novel protocol many microsatellites can be identified quickly. An increased availability of these highly sensitive markers will be useful for studies addressing the origin of species in the Southern Ocean and their response to future climate change.

Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate.

Identification and characterization of microsatellites from the Antarctic isopod Ceratoserolis trilobitoides: nuclear evidence for cryptic species

We report the successful isolation of 10 polymorphic microsatellite markers for one species of the marine isopod species complex Ceratoserolis trilobitoides (Eights, 1833) from the Southern Ocean. The number of alleles per locus ranged from 6 to 30 for the 148 specimens analysed and heterozygosity ranged from 0.34 to 0.98. Seven microsatellites amplified successfully in a cryptic sister species, which is restricted to the Antarctic Peninsula. This novel marker set provides the opportunity to study and monitor population structure, demography and gene flow patterns for a benthic model taxon in a region that is now subject to rapid climate change.