Getting specific: making taxonomic and ecological sense of large sequencing data sets

Eukaryotic microbes comprise a diverse collection of phototrophic and heterotrophic creatures known to play fundamental roles in ecological processes. Some can be identified by light microscopy, generally the largest and with conspicuous shapes, while the smallest can be counted by epifluorescence microscopy or flow cytometry but remain largely unidentified. Microbial diversity studies greatly advanced with the analysis of phylogenetic markers sequenced from natural assemblages. Molecular surveys began in 1990 targeting marine bacterioplankton (Giovannoni et al. 1990 ) and first approached microbial eukaryotes in three studies published in 2001 (Díez et al. 2001 ; López‐García et al. 2001 ; Moon‐van der Staay et al. 2001 ). These seminal studies, based on cloning and Sanger sequencing the complete 18S rDNA, were critical for obtaining broad pictures of microbial diversity in contrasted habitats and for describing novel lineages by robust phylogenies, but were limited by the number of sequences obtained. So, inventories of species richness in a given sample and community comparisons through environmental gradients were very incomplete. These limitations have been overcome with the advent of high‐throughput sequencing (HTS) methods, initially 454‐pyrosequencing, today Illumina and soon others to come. In this issue of Molecular Ecology, Egge et al. ( 2015 ) show a nice example of the use of HTS to study the biodiversity and seasonal succession of a particularly important group of marine microbial eukaryotes, the haptophytes. Temporal changes were analysed first at the community level, then at the clade level, and finally at the lowest rank comparable to species. Interesting and useful ecological insights were obtained at each taxonomic scale. Haptophyte diversity differed along seasons in a systematic manner, with some species showing seasonal preferences and others being always present. Many of these species had no correspondence with known species, pointing out the high level of novelty in microbial assemblages, only accessible by molecular tools. Moreover, the number of species detected was limited, agreeing with a putative scenario of constrained evolutionary diversification in free‐living small eukaryotes. This study illustrates the potential of HTS to address ecological relevant questions in an accessible way by processing large data sets that, nonetheless, need to be treated with a fair understanding of their limitations.     

Why some parasites are widespread and abundant while others are local and rare?

Abundances and distributions of species are usually associated. This implies that as a species declines in abundance so does the number of sites it occupies. Conversely, when there is an increase in a species' range size, it is usually followed by an increase in population size (Gaston et al. 2000 ). This ecological phenomenon, also known as the abundance–occupancy relationship (AOR), is well documented in several species of animals and plants (Gaston et al. 2000 ) but has been little investigated in parasites. In this issue of Molecular Ecology, Drovetski et al. ( 2014 ) investigated the AOR in avian haemosporidians (vector‐borne blood parasites) using data from four well‐sampled bird communities. In support of the AOR, the research group found that the abundance of parasite cytochrome b lineages (a commonly used proxy for species identification within this group of parasites) was positively linked with the abundance of susceptible avian host species and that the most abundant haemospordian lineages were those with larger ranges. Drovetski et al. ( 2014 ) also found evidence for both hypotheses proposed to explain the AOR in parasites: the trade‐off hypothesis (TOH) and the niche‐breadth hypothesis (NBH). Interestingly, the main predictor of the AOR was the number of susceptible hosts (i.e. number of infected birds) and not the number of host species the parasites were able to exploit.     

Tending a complex microbiota requires major immune complexity

Animals maintain complex microbial communities within their guts that fill important roles in the health and development of the host. To what degree a host's genetic background influences the establishment and maintenance of its gut microbial communities is still an open question. We know from studies in mice and humans that external factors, such as diet and environmental sources of microbes, and host immune factors play an important role in shaping the microbial communities (Costello et al. 2012 ). In this issue of Molecular Ecology, Bolnick et al. ( 2014a ) sample the gut microbial community from 150 genetically diverse stickleback isolated from a single lake to provide evidence that another part of the adaptive immune response, the major histocompatibility complex class II (MHCII) receptors of antigen‐presenting cells, may play a role in shaping the gut microbiota of the threespine stickleback, Gasterosteus aculeatus (Bolnick et al. 2014a ). Bolnick et al. ( 2014a ) provide insight into natural, interindividual variation in the diversity of both stickleback MHCII alleles and their gut microbial communities and correlate changes in the diversity of MHCII receptor alleles with changes in the microbiota.     

Bacteriophage exclusion,a new defense system

The ability to withstand viral predation is critical for survival of most microbes. Accordingly, a plethora of phage resistance systems has been identified in bacterial genomes (Labrie et al, 2010 ), including restriction‐modification systems (R‐M) (Tock & Dryden, 2005 ), abortive infection (Abi) (Chopin et al, 2005 ), Argonaute‐based interference (Swarts et al, 2014 ), as well as clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) adaptive immune system (CRISPR‐Cas) (Barrangou & Marraffini, 2014 ; Van der Oost et al, 2014 ). Predictably, the dark matter of bacterial genomes contains a wealth of genetic gold. A study published in this issue of The EMBO Journal by Goldfarb et al ( 2015 ) unveils bacteriophage exclusion (BREX) as a novel, widespread bacteriophage resistance system that provides innate immunity against virulent and temperate phage in bacteria.     

Using RAD‐seq to recognize sex‐specific markers and sex chromosome systems

Next‐generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. 2010 ). Among the most impressive of these sequencing innovations is restriction site‐associated DNA sequencing or RAD‐seq (Baird et al. 2008 ; Andrews et al. 2016 ). RAD‐seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD‐seq data has been to identify sex‐specific genetic markers, markers found in one sex but not the other (Baxter et al. 2011 ; Gamble & Zarkower 2014 ). Sex‐specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon 1998 ; Mossman & Waser 1999 ), the management and breeding of endangered species (Taberlet et al. 1993 ; Griffiths & Tiwari 1995 ; Robertson et al. 2006 ) and sexing embryonic material (Hacker et al. 1995 ; Smith et al. 1999 ). Furthermore, sex‐specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank 2010 ; Gamble & Zarkower 2014 ). Thus, species with male‐specific markers have male heterogamety (XY) while species with female‐specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi ( 2016 ) illustrate the ease by which RAD‐seq data can generate sex‐specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD‐seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig.  1 ), Fowler & Buonaccorsi ( 2016 ) uncover shared sex‐specific markers and a conserved sex chromosome system.     

A tale of two lineages: unexpected,long‐term persistence of the amphibian‐killing fungus in Brazil

For the past 17 years, scientists have been compiling a list of amphibian species susceptible to infection by the amphibian‐killing chytrid fungus, Batrachochytrium dendrobatidis (Bd), all over the world, with >500 species infected on every continent except Antarctica (Olson et al. 2013 ). Where Bd has been found, the impacts on amphibians has been one of two types: either Bd arrives into a naïve amphibian population followed by a mass die‐off and population declines (e.g. Lips et al. 2006 ), or Bd is present at some moderate prevalence, usually infecting many species but at apparently nonlethal intensities for a long time. In this issue of Molecular Ecology, Rodriguez et al. ( 2014 ) discover that the Atlantic Coastal Forest of Brazil is home to two Bd lineages: the Global Pandemic Lineage (Bd‐GPL) – the strain responsible for mass die‐offs and population declines – and a lineage endemic to Brazil (Bd‐Bz). Even more surprising was that both lineages have been present in this area for the past 100 years, making these the oldest records of Bd infecting amphibians. The team also described a moderate but steady prevalence of ~20% across all sampled anuran families for over 100 years, indicating that Brazil has been in an enzootic disease state for over a century. Most amphibians were infected with Bd‐GPL, suggesting this lineage may be a better competitor than Bd‐Bz or may be replacing the Bd‐Bz lineage. Rodriguez et al. ( 2014 ) also detected likely hybridization of the two Bd lineages, as originally described by Schloegel et al. ( 2012 ).     

The discovery of Antarctic RNA viruses: a new game changer

Antarctic ecosystems are dominated by micro‐organisms, and viruses play particularly important roles in the food webs. Since the first report in 2009 (López‐Bueno et al. 2009 ), ‘omic’‐based studies have greatly enlightened our understanding of Antarctic aquatic microbial diversity and ecosystem function (Wilkins et al. 2013 ; Cavicchioli 2015 ). This has included the discovery of many new eukaryotic viruses (López‐Bueno et al. 2009 ), virophage predators of algal viruses (Yau et al. 2011 ), bacteria with resistance to phage (Lauro et al. 2011 ) and mechanisms of haloarchaeal evasion, defence and adaptation to viruses (Tschitschko et al. 2015 ). In this issue of Molecular Ecology, López‐Bueno et al. ( 2015 ) report the first discovery of RNA viruses from an Antarctic aquatic environment. High sequence coverage enabled genome variation to be assessed for four positive‐sense single‐stranded RNA viruses from the order Picornavirales. By examining the populations present in the water column and in the lake's catchment area, populations of ‘quasispecies’ were able to be linked to local environmental factors. In view of the importance of viruses in Antarctic ecosystems but lack of data describing them, this study represents a significant advance in the field.     

Disease defence through generations: leaf‐cutter ants and their symbiotic bacteria

Microbial ecology of animals is taking on significance in the modern dialogue for the biology of species. Similar to a nuclear genome, the entire bacterial assemblage maintains an ancestral signal of the host's evolution leading to cophylogeny between the host and the microbes they harbour (Brucker & Bordenstein 2012b). The stability of such associations is of great interest as they provide a means for species to acquire new traits and genetic diversity that their own genomes lack (McFall‐Ngai et al. 2013). The role of gut microbiota, for example, in host health and nutrition is widely recognized and a shared characteristic among animals. The role of bacteria colonizing the outside surfaces of animals is less well understood, but rather than random colonization, these microbes on skin, cuticles, scales and feathers in many cases provide benefits to the host. The symbiosis of leaf‐cutter ants, their fungus gardens and their microbiota is a fascinating and complex system. Whether culture‐independent bacterial diversity on the cuticle of leaf‐cutter ants is high or highly constrained by subcuticular gland secretions is one prominent question. In this issue of Molecular Ecology, Andersen et al. (2013) show that leaf‐cutting ants, Acromyrmex echinatior, maintain a dominant and colony‐specific bacterium called Pseudonocardia on their cuticles (the laterocervical plates in particular). This bacterium is involved in protecting the ants and their fungal gardens from disease. Other fungus‐gardening attine species as well as soil and vegetation can harbour Pseudonocardia. However, it was previously unknown how stable the bacterial strain–ant colony association was through the lifetime of the colony.     

Demystifying the RAD fad

We are writing in response to the population and phylogenomics meeting review by Andrews & Luikart ( 2014 ) entitled ‘Recent novel approaches for population genomics data analysis’. Restriction‐site‐associated DNA (RAD) sequencing has become a powerful and useful approach in molecular ecology, with several different published methods now available to molecular ecologists, none of which can be considered the best option in all situations. A&L report that the original RAD protocol of Miller et al. ( 2007 ) and Baird et al. ( 2008 ) is superior to all other RAD variants because putative PCR duplicates can be identified (see Baxter et al. 2011 ), thereby reducing the impact of PCR artefacts on allele frequency estimates (Andrews & Luikart 2014 ). In response, we (i) challenge the assertion that the original RAD protocol minimizes the impact of PCR artefacts relative to that of other RAD protocols, (ii) present additional biases in RADseq that are at least as important as PCR artefacts in selecting a RAD protocol and (iii) highlight the strengths and weaknesses of four different approaches to RADseq which are a representative sample of all RAD variants: the original RAD protocol (mbRAD, Miller et al. 2007 ; Baird et al. 2008 ), double digest RAD (ddRAD, Peterson et al. 2012 ), ezRAD (Toonen et al. 2013 ) and 2bRAD (Wang et al. 2012 ). With an understanding of the strengths and weaknesses of different RAD protocols, researchers can make a more informed decision when selecting a RAD protocol.     

Terra incognita of soil biodiversity: unseen invasions under our feet

Whilst cartographers of the 19th century endeavoured to chart the last unknown lands, the great challenge for biologists in the 21st century is to fill the gaps on the biodiversity map of the Earth. And one of the largest gaps concerns the biodiversity of soils, a terra incognita right under our feet. The study of soil biodiversity, and particularly the complex communities of small invertebrates, has suffered from a severe ‘taxonomic impediment’ (Decaëns 2010 ) leading to great uncertainties about total species richness, phylogenetic diversity, geographical structure, temporal dynamics of soil organisms, and consequently about their role on ecosystem function (Bardgett & van der Putten 2014 ). However, the revolution in high‐throughput sequencing is now revealing the hidden biodiversity of the soil with unprecedented detail (e.g. Arribas et al. 2016 ). In a noteworthy from the Cover article in this issue of Molecular Ecology, Cicconardi et al. ( 2017 ) apply these new tools to study soil communities of Collembola in three distant oceanic islands of volcanic origin, obtaining a striking result: only 38 of 70 species (54%) are exclusively found in a single island, with the remaining shared among islands or with other distant regions, suggesting a massive recent introduction of soil species, whose impact is entirely unknown.     

Metagenome skimming for phylogenetic community ecology: a new era in biodiversity research

It is now well recognized that considering species evolutionary history is crucial for understanding the processes driving community assembly (Cavender‐Bares et al. 2009 ). Considerable efforts have been made to integrate phylogenetics and community ecology into a single theoretical framework. Yet, assessing phylogenetic structure at the community scale remains a great challenge, in particular for poorly known organisms. While DNA metabarcoding is increasingly used for assessing taxonomic composition of complex communities from environmental samples, biases and limitations of this technique can preclude the retrieval of information on phylogenetic community structure. In this issue of Molecular Ecology, Andújar et al. (2015) demonstrate that shotgun sequencing of bulk samples of soil beetles and subsequent reconstruction of mitochondrial genomes can provide a solid phylogenetic framework to estimate species diversity and gain insights into the mechanisms underlying the spatial turnover of soil mesofaunal assemblages. This work highlights the enormous potential of ‘metagenome skimming’ not only for improving the current standards of DNA‐based biodiversity assessment but also for opening up the application of phylogenetic community ecology to hyperdiverse and poorly known biota, which was heretofore inconceivable.     

Host plant richness explains diversity of ectomycorrhizal fungi: Response to the comment of Tedersoo et al. (2014)

Exploring the relationships between the biodiversity of groups of interacting organisms yields insight into ecosystem stability and function (Hooper et al. 2000 ; Wardle 2006 ). We demonstrated positive relationships between host plant richness and ectomycorrhizal (EM) fungal diversity both in a field study in subtropical China (Gutianshan) and in a meta‐analysis of temperate and tropical studies (Gao et al. 2013 ). However, based on re‐evaluation of our data sets, Tedersoo et al. ( 2014 ) argue that the observed positive correlation between EM fungal richness and EM plant richness at Gutianshan and also in our metastudies was based mainly from (i) a sampling design with inconsistent species pool and (ii) poor data compilation for the meta‐analysis. Accordingly, we checked our data sets and repeated the analysis performed by Tedersoo et al. ( 2014 ). In contrast to Tedersoo et al. ( 2014 ), our re‐analysis still confirms a positive effect of plant richness on EM fungal diversity in Gutianshan, temperate and tropical ecosystems, respectively.     

A robust tool highlights the influence of bird migration on influenza A virus evolution

One of the fundamental unknowns in the field of influenza biology is a panoramic understanding of the role wild birds play in the global maintenance and spread of influenza A viruses. Wild aquatic birds are considered a reservoir host for all lowly pathogenic avian influenza A viruses (AIV) and thus serve as a potential source of zoonotic AIV, such as Australasian‐origin H5N1 responsible for morbidity and mortality in both poultry and humans, as well as genes that may contribute to the emergence of pandemic viruses. Years of broad, in‐depth wild bird AIV surveillance have helped to decipher key observations and ideas regarding AIV evolution and viral ecology including the trending of viral lineages, patterns of gene flow within and between migratory flyways and the role of geographic boundaries in shaping viral evolution (Bahl et al. 2009 ; Lam et al. 2012 ). While these generally ‘virus‐centric’ studies have ultimately advanced our broader understanding of AIV dynamics, recent studies have been more host‐focused, directed at determining the potential impact of host behaviour on AIV, specifically, the influence of bird migration upon AIV maintenance and transmission. A large number of surveillance studies have taken place in Alaska, United States—a region where several global flyways overlap—with the aim of detecting the introduction of novel, Australasian‐origin highly pathogenic H5N1 AIV into North America. By targeting bird species with known migration habits, long‐distance migrators were determined to be involved in the intercontinental movement of individual AIV gene segments, but not entire viruses, between the Australasian and North American flyways (Koehler et al. 2008 ; Pearce et al. 2010 ). Yet, bird movement is not solely limited to long‐distance migration, and the relationship of resident or nonmigratory and intermediate‐distance migrant populations with AIV ecology has only recently been explored by Hill et al. ( 2012 ) in this issue of Molecular Ecology. Applying a uniquely refined, multidimensional approach, Hill et al. validate the innovative use of stable isotope assays for qualifying migration status of wild mallards within the Pacific flyway. The authors reveal that AIV prevalence and diversity did not differ in wintering mallard ducks with different migration strategies, and while migrant mallards do indeed introduce AIV, these viruses do not circulate as the predominant viruses in resident birds. On the other hand, resident mallards from more temperate regions act as reservoirs, possibly contributing to the unseasonal circulation and extended transmission period of AIV. This study highlights the impact of animal behaviour on shaping viral evolution, and the unique observations made will help inform prospective AIV surveillance efforts in wild birds.     

Will reduced host connectivity curb the spread of a devastating epidemic?

The white‐nose syndrome (WNS), caused by the fungal pathogen Pseudogymnoascus destructans, is threatening the cave‐dwelling bat fauna of North America by killing individuals by the thousands in hibernacula each winter since its appearance in New York State less than ten years ago. Epidemiological models predict that WNS will reach the western coast of the USA by 2035, potentially eliminating most populations of susceptible bat species in its path (Frick et al. 2015; O'Regan et al. 2015). These models were built and validated using distributional data from the early years of the epidemic, which spread throughout eastern North America following a route driven by cave density and winter severity (Maher et al. 2012). In this issue of Molecular Ecology, Wilder et al. (2015) refine these findings by showing that connectivity among host populations, as assessed by population genetic markers, is crucial in determining the spread of the pathogen. Because host connectivity is much reduced in the hitherto disease free western half of North America, Wilder et al. make the reassuring prediction that the disease will spread more slowly west of the Great Plains.     

The diversity and function of intestinal microorganisms in four geographic Cephalcia chuxiongica (a pine defoliator) populations

Symbiotic microbiomes play important roles in hosts’ adaptation and evolution. Here, the gut bacterial communities in Cephalcia chuxiongica, a key pest of pines in China, were studied for the first time by using 16S rRNA amplicon sequencing. The composition of gut bacterial communities differed in different C. chuxiongica geographic populations but interestingly, the phylogeny and diversity of gut microbiota correlated with host geographic/genetic distance, that is the microbiota was more similar as the geographic/genetic distance decreased, and vice versa. The various microbes performed similar functions and showed functional complementation, in which most of identified KEGG pathways were shared by different populations with metabolism being the most dominant functional pathway and the function of major microbes associated with host dietary specialization (pine needles), such as cellulose degradation. In addition, some microbes also associated with host biological characteristics, such as Wolbachia with parthenogenesis and Serratia with the long-term larval diapause in C. chuxiongica. Therefore, the synergy of environmental and host factors shapes the structure of gut microbiota and gut microbiota play essential roles in host physiology and adaptation, suggesting some kind of symbiosis and coevolution. These results demonstrate the important contribution of gut microbiota and provide a sound foundation for developing control strategies for this pest.     

Don't throw the baby out with the bathwater: identifying and mapping paralogs in salmonids

Many eukaryotic genomes contain a large fraction of gene duplicates (or paralogs) as a result of ancient or recent whole‐genome duplications (Ohno 1970 ; Jaillon et al. 2004 ; Kellis et al. 2004 ). Identifying paralogs with NGS data is a pervasive problem in both ancient polyploids and neopolyploids. Likewise, paralogs are often treated as a nuisance that has to be detected and removed (Everett et al. 2012 ). In this issue of Molecular Ecology Resources, Waples et al. ( 2015 ) show that exclusion might not be necessary and how we may miss out on important genomic information in doing so. They present a novel statistical approach to detect paralogs based on the segregation of RAD loci in haploid offspring and test their method by constructing linkage maps with and without these duplicated loci in chum salmon, Oncorhynchus keta (Fig.  1 ). Their linkage map including the resolved paralogs shows that these are mostly located in the distal regions of several linkage groups. Particularly intriguing is their finding that these homoeologous regions appear impoverished in transposable elements (TE). Given the role that TE play in genome remodelling, it is noteworthy that these elements are of low abundance in regions showing residual tetrasomic inheritance. This raises the question whether re‐diploidization is constrained in these regions and whether they might have a role to play in salmonid speciation. This study provides an original approach to identifying duplicated loci in species with a pedigree, as well as providing a dense linkage map for chum salmon, and interesting insights into the retention of gene duplicates in an ancient polyploid.     

Enalikter is not an annelid: homology,autapomorphies and the interpretation of problematic fossils

A megacheiran arthropod, Enalikter aphson, was recently described by Siveter et al. (2014) from the mid‐Silurian (late Wenlock) of Herefordshire. Previously, megacheirans had only been recognized from the Cambrian. Struck et al. (2015) considered the body plan of Enalikter to be incompatible with this affinity, arguing that many of the arthropod features were either not present or misinterpreted. Instead, they compared Enalikter to polychaete annelids, identifying characters from numerous polychaete lineages which they considered to be present in Enalikter. A reply to this critique by Siveter et al. (2015) reaffirmed arthropod affinities for Enalikter by presenting additional evidence for key arthropod features, such as arthropodized appendages. Here, we augment Siveter et al. by critically addressing the putative annelid characters of Enalikter presented by Struck et al. and additionally explore the morphological and phylogenetic implications of their hypothesis. We conclude that similarities between Enalikter and polychaetes are superficial and that character combinations proposed by Struck et al. are not present in any annelid, living or extinct. This taxon highlights the importance of using a phylogenetic framework for interpreting fossils that present unusual morphologies, such that proposed shared characters are hypotheses of homology rather than merely phenotypic similarities. Crucially, we argue that autapomorphic characters of subgroups of large taxa (like families or classes within phyla) should not be used to diagnose problematic fossils.     

Coalescing molecular evolution and DNA barcoding

The DNA barcoding concept (Woese et al. 1990 ; Hebert et al. 2003 ) has considerably boosted taxonomy research by facilitating the identification of specimens and discovery of new species. Used alone or in combination with DNA metabarcoding on environmental samples (Taberlet et al. 2012 ), the approach is becoming a standard for basic and applied research in ecology, evolution and conservation across taxa, communities and ecosystems (Scheffers et al. 2012 ; Kress et al. 2015 ). However, DNA barcoding suffers from several shortcomings that still remain overlooked, especially when it comes to species delineation (Collins & Cruickshank 2012 ). In this issue of Molecular Ecology, Barley & Thomson ( 2016 ) demonstrate that the choice of models of sequence evolution has substantial impacts on inferred genetic distances, with a propensity of the widely used Kimura 2‐parameter model to lead to underestimated species richness. While DNA barcoding has been and will continue to be a powerful tool for specimen identification and preliminary taxonomic sorting, this work calls for a systematic assessment of substitution models fit on barcoding data used for species delineation and reopens the debate on the limitation of this approach.