The perils of plenty: what are we going to do with all these genes?

This new century's biology promises more of everything--more genes, more organisms, more species and, in short, more data. The flood of data challenges us to find better and quicker ways to summarize and analyse. Here, we present preliminary results and proofs of concept from three of our research projects that are motivated by our search for solutions to the perils of plenty. First, we discuss how models of evolution can accommodate change to better reflect the dynamics of sequence diversity, particularly when it is becoming a lot easier to obtain sequences at different times and across intervals where the probability of new mutations contributing to this diversity is high. Second, we describe our work on the use of a single locus for species delimitation; this research targets the new DNA-barcoding approach that aims to catalogue the entirety of life. We have developed a single-locus test based on the coalescent that tests the null hypothesis of panmixis. Finally, we discuss new sequencing technologies, the types of data available and the efficacy of alignment-free methods to estimate pairwise distances for phylogenetic analyses.     

Quantifying sequence proportions in a DNA‐based diet study using Ion Torrent amplicon sequencing: which counts count?

A goal of many environmental DNA barcoding studies is to infer quantitative information about relative abundances of different taxa based on sequence read proportions generated by high‐throughput sequencing. However, potential biases associated with this approach are only beginning to be examined. We sequenced DNA amplified from faeces (scats) of captive harbour seals (Phoca vitulina) to investigate whether sequence counts could be used to quantify the seals’ diet. Seals were fed fish in fixed proportions, a chordate‐specific mitochondrial 16S marker was amplified from scat DNA and amplicons sequenced using an Ion Torrent PGM?. For a given set of bioinformatic parameters, there was generally low variability between scat samples in proportions of prey species sequences recovered. However, proportions varied substantially depending on sequencing direction, level of quality filtering (due to differences in sequence quality between species) and minimum read length considered. Short primer tags used to identify individual samples also influenced species proportions. In addition, there were complex interactions between factors; for example, the effect of quality filtering was influenced by the primer tag and sequencing direction. Resequencing of a subset of samples revealed some, but not all, biases were consistent between runs. Less stringent data filtering (based on quality scores or read length) generally produced more consistent proportional data, but overall proportions of sequences were very different than dietary mass proportions, indicating additional technical or biological biases are present. Our findings highlight that quantitative interpretations of sequence proportions generated via high‐throughput sequencing will require careful experimental design and thoughtful data analysis.     

Ecometagenetics confirm high tropical rainforest nematode diversity

The general patterns of increasing biodiversity from the poles to the equator have been well documented for large terrestrial organisms such as plants and vertebrates but are largely unknown for microbiota. In contrast to macrobiota, microbiota have long been assumed to exhibit cosmopolitan, random distributions and a lack of spatial patterns. To evaluate the assumption, we conducted a survey of nematode diversity within the soil, litter and canopy habitats of the humid lowland tropical rainforest of Costa Rica using an ultrasequencing ecometagenetic approach at a species-equivalent taxonomic level. Our data indicate that both richness and diversity of nematode communities in the tropical rainforests of Costa Rica are high and exceed observed values from temperate ecosystems. The majority of nematode species were unknown to science, providing evidence for the presence of highly endemic (not cosmopolitan) species of still completely undiscovered biodiversity. Most importantly, the greater taxonomic resolution used here allowed us to reveal predictable habitat associations for specific taxa and thus gain insights into their nonrandom distribution patterns.     

Does complete plastid genome sequencing improve species discrimination and phylogenetic resolution in Araucaria?

Obtaining accurate phylogenies and effective species discrimination using a small standardized set of plastid genes is challenging in evolutionarily young lineages. Complete plastid genome sequencing offers an increasingly easy‐to‐access source of characters that helps address this. The usefulness of this approach, however, depends on the extent to which plastid haplotypes track morphological species boundaries. We have tested the power of complete plastid genomes to discriminate among multiple accessions of 11 of 13 New Caledonian Araucaria species, an evolutionarily young lineage where the standard DNA barcoding approach has so far failed and phylogenetic relationships have remained elusive. Additionally, 11 nuclear gene regions were Sanger sequenced for all accessions to ascertain the success of species discrimination using a moderate number of nuclear genes. Overall, fewer than half of the New Caledonian Araucaria species with multiple accessions were monophyletic in the plastid or nuclear trees. However, the plastid data retrieved a phylogeny with a higher resolution compared to any previously published tree of this clade and supported the monophyly of about twice as many species and nodes compared to the nuclear data set. Modest gains in discrimination thus are possible, but using complete plastid genomes or a small number of nuclear genes in DNA barcoding may not substantially raise species discriminatory power in many evolutionarily young lineages. The big challenge therefore remains to develop techniques that allow routine access to large numbers of nuclear markers scaleable to thousands of individuals from phylogenetically disparate sample sets.     

Phylogeny, phylogeography, phylobetadiversity and the molecular analysis of biological communities

There has been much recent interest and progress in the characterization of community structure and community assembly processes through the application of phylogenetic methods. To date most focus has been on groups of taxa for which some relevant detail of their ecology is known, for which community composition is reasonably easily quantified and where the temporal scale is such that speciation is not likely to feature. Here, we explore how we might apply a molecular genetic approach to investigate community structure and assembly at broad taxonomic and geographical scales, where we have little knowledge of species ecology, where community composition is not easily quantified, and where speciation is likely to be of some importance. We explore these ideas using the class Collembola as a focal group. Gathering molecular evidence for cryptic diversity suggests that the ubiquity of many species of Collembola across the landscape may belie greater community complexity than would otherwise be assumed. However, this morphologically cryptic species-level diversity poses a challenge for attempts to characterize diversity both within and among local species assemblages. Recent developments in high throughput parallel sequencing technology, combined with mtDNA barcoding, provide an advance that can bring together the fields of phylogenetic and phylogeographic analysis to bear on this problem. Such an approach could be standardized for analyses at any geographical scale for a range of taxonomic groups to quantify the formation and composition of species assemblages.     

基于环境DNA技术的辽东湾真核微藻群落结构特征

以18S r DNA V4区作为目标基因,利用自行设计的真核微藻鉴定引物V4(F/R),结合高通量测序技术,对辽东湾2014年四季海水中真核微藻多样性进行了检测。结果发现,辽东湾海域注释到种的真核微藻有136种,41%的种类在中国海域未见报道,其中自养型占60%、异养型占10%、混合营养型占30%。研究对丰富中国海域微藻名录和外来海洋微藻背景数据库具有较大意义。     

Identifying fern gametophytes using DNA sequences

Identification of fern gametophytes is generally hampered by low morphological complexity. Here we explore an alternative: DNA‐based identification. We obtained a plastid rbcL sequence from a sterile gametophyte of unknown origin (cultivated for more than 30 years) and employed blast to determine its affinities. Using this approach, we identified the gametophyte as Osmunda regalis. To evaluate the robustness of this determination, and the usefulness of rbcL in differentiating among species, we conducted a phylogenetic analysis of osmundaceous fern sequences. Based on our results, it is evident that DNA‐based identification has considerable potential in exploring the ecology of fern gametophytes.     

An emergent science on the brink of irrelevance: a review of the past 8 years of DNA barcoding

DNA barcoding has become a well-funded, global enterprise since its proposition as a technique for species identification, delimitation and discovery in 2003. However, the rapid development of next generation sequencing (NGS) has the potential to render DNA barcoding irrelevant because of the speed with which it generates large volumes of genomic data. To avoid obsolescence, the DNA barcoding movement must adapt to use this new technology. This review examines the DNA barcoding enterprise, its continued resistance to improvement and the implications of this on the future of the discipline. We present the consistent failure of DNA barcoding to recognize its limitations and evolve its methodologies, reducing the usefulness of the data produced by the movement and throwing into doubt its ability to embrace NGS.