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.     

Somatic mutations as a useful tool for studying clonal dynamics in trees

The seemingly eternal cycles of clonal growth in many tree species, with members of Populus (aspen, poplars, cottonwoods and the like) featuring most prominently, provoke a number of questions on the interface between ecology, genetics and forestry. In this issue, two groups present their approaches to clonal dynamics ( Ally et al. 2008 and Mock et al. 2008 ), using microsatellite (or simple sequence repeat, SSR) variation in P. tremuloides. Ally et al. developed and applied a model for using microsatellites to estimate clone age and infer other community characteristics. Mock et al. used fewer microsatellites but in more individuals, to examine clone size and distribution across the landscape.     

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.     

Standing and flowing: the complex origins of adaptive variation

A population faced with a new selection pressure can only adapt if appropriate genetic variation is available. This genetic variation might come from new mutations or from gene exchange with other populations or species, or it might already segregate in the population as standing genetic variation (which might itself have arisen from either mutation or gene flow). Understanding the relative importance of these sources of adaptive variation is a fundamental issue in evolutionary genetics (Orr & Betancourt 2001 ; Barrett & Schluter 2008 ; Gladyshev et al. 2008 ) and has practical implications for conservation, plant and animal breeding, biological control and infectious disease prevention (e.g. Robertson 1960 ; Soulé & Wilcox 1980 ; Prentis et al. 2008 ; Pennings 2012 ). In this issue of Molecular Ecology, Roesti et al. ( 2014 ) make an important contribution to this longstanding debate.     

Population genomic analyses reveal possible drivers of population divergence

Recent advances in sequencing technology and efficiency enable new and improved methods to investigate how populations diverge and species evolve. Fungi have relatively small and simple genomes and can often be cultured in the laboratory. Fungal populations can thus be sequenced for a relatively low cost, which makes them ideal for population genomic analyses. In several recent population genomic studies, wild populations of fungal model organisms and human pathogens have been analysed, for example Neurospora crassa (Ellison et al. 2011 ), Saccharomyces uvarum (Almeida et al. 2014 ), Coccidioides spp. (Neafsey et al. 2010 ) and Cryptococcus gatti (Engelthaler et al. 2014 ). In this issue of Molecular Ecology, Branco et al. ( 2015 ) apply population genomic tools to understand population divergence and adaptation in a symbiotic (mycorrhizal) fungus. This study exemplifies the possibilities of diving deeper into the genomic features involved in population divergence and speciation, also for nonmodel organisms, and how molecular and analytical tools will improve our understanding of the patterns and mechanisms that underlie adaptation to habitats, population divergence and dispersal limitation of fungi.     

It's a family act: the geminin triplets take center stage in motile ciliogenesis

The balance between proliferation and differentiation is a fundamental aspect of multicellular life. Perhaps nowhere is this delicate balance more palpable than in the multiciliated cells (MCCs) that line the respiratory tract, the ependyma, and the oviduct. These cells contain dozens to hundreds of motile cilia that beat in a concerted fashion to generate directed fluid flow over the tissue surface. Although MCCs have exited the cell cycle, remarkably, they retain the ability to duplicate their centrioles and to mature those centrioles into ciliary basal bodies—two features, which are known to be normally under strict cell cycle control (Firat‐Karalar & Stearns, 2014 ). How post‐mitotic MCCs retain this ability, remains unclear. In the past several months, four research articles, including one from Terré et al in this issue of The EMBO Journal, have described a vital role for the geminin coiled‐coil domain‐containing protein (Gemc1) in the MCC gene expression program in multiple tissues and organisms, that bring us closer to understanding this question (Kyrousi et al, 2015 ; Zhou et al, 2015 ; Arbi et al, 2016 ; Terré et al, 2016 ).     

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 origin of the non‐recombining region of sex chromosomes in Carica and Vasconcellea

Carica and Vasconcellea are two closely related sister genera in the family Caricaceae, and were once classified as two sections under Carica. Sex chromosomes have been found in papaya and originated approximately 2–3 million years ago. The objectives of this study were to determine whether sex chromosomes have evolved in Vasconcellea. Six X/Y gene pairs were cloned, sequenced and analyzed from three dioecious, one trioecious and one monoecious species of Vasconcellea. The isolation of distinctive X and Y alleles in dioecious and trioecious species of Vasconcellea demonstrated that sex chromosomes have evolved in this genus. Phylogenetic analyses indicated a monophyletic relationship between the X/Y alleles of Carica and those of Vasconcellea. Distinctive clusters of X/Y alleles were documented in V. parviflora and V. pulchra for all available gene sequences, and in V. goudatinana and V. cardinamarcensis for some X/Y alleles. The X and Y alleles within each species shared most single nucleotide polymorphism haplotypes that differed from other species. Limited evidence of gene conversion was documented among the X/Y alleles of some species, but was not sufficient to cause the evolutionary patterns reported herein. The Carica and Vasconcellea sex chromosomes may have originated from the same autosomes bearing the X allelic form that still exist in the monoecious species V. monoica, and have evolved independently after the speciation event that separated Carica from Vasconcellea. Within Vasconcellea, sex chromosomes have evolved at the species level, at least for some species.     

ANOLIS SEX CHROMOSOMES ARE DERIVED FROM A SINGLE ANCESTRAL PAIR

To explain the frequency and distribution of heteromorphic sex chromosomes in the lizard genus Anolis, we compared the relative roles of sex chromosome conservation versus turnover of sex‐determining mechanisms. We used model‐based comparative methods to reconstruct karyotype evolution and the presence of heteromorphic sex chromosomes onto a newly generated Anolis phylogeny. We found that heteromorphic sex chromosomes evolved multiple times in the genus. Fluorescent in situ hybridization (FISH) of repetitive DNA showed variable rates of Y chromosome degeneration among Anolis species and identified previously undetected, homomorphic sex chromosomes in two species. We confirmed homology of sex chromosomes in the genus by performing FISH of an X‐linked bacterial artificial chromosome (BAC) and quantitative PCR of X‐linked genes in multiple Anolis species sampled across the phylogeny. Taken together, these results are consistent with long‐term conservation of sex chromosomes in the group. Our results pave the way to address additional questions related to Anolis sex chromosome evolution and describe a conceptual framework that can be used to evaluate the origins and evolution of heteromorphic sex chromosomes in other clades.     

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.     

Conserved ZZ/ZW sex chromosomes in Caribbean croaking geckos (Aristelliger: Sphaerodactylidae)

Current understanding of sex chromosome evolution is largely dependent on species with highly degenerated, heteromorphic sex chromosomes, but by studying species with recently evolved or morphologically indistinct sex chromosomes we can greatly increase our understanding of sex chromosome origins, degeneration and turnover. Here, we examine sex chromosome evolution and stability in the gecko genus Aristelliger. We used RADseq to identify sex‐specific markers and show that four Aristelliger species, spanning the phylogenetic breadth of the genus, share a conserved ZZ/ZW system syntenic with avian chromosome 2. These conserved sex chromosomes contrast with many other gecko sex chromosome systems by showing a degree of stability among a group known for its dynamic sex‐determining mechanisms. Cytogenetic data from A. expectatus revealed homomorphic sex chromosomes with an accumulation of repetitive elements on the W chromosome. Taken together, the large number of female‐specific A. praesignis RAD markers and the accumulation of repetitive DNA on the A. expectatus W karyotype suggest that the Z and W chromosomes are highly differentiated despite their overall morphological similarity. We discuss this paradoxical situation and suggest that it may, in fact, be common in many animal species.     

Is colour polymorphism advantageous to populations and species?

I am writing in response to an article by Bolton, Rollins and Griffith (2015) entitled ‘The danger within: the role of genetic, behavioural and ecological factors in population persistence of colour polymorphic species’ that was recently published as an Opinion under the NEWS AND VIEWS section in Molecular Ecology. Bolton et al. (Molecular Ecology, 2015, 24 , 2907) argue that colour polymorphism may reduce population fitness and increase extinction risk and emphasize that this is contrary to predictions put forward by Forsman et al. (Ecology, 89 , 2008, 34) and Wennersten & Forsman (Biological Reviews 87 , 2012, 756) that the existence of multiple colour morphs with co‐adapted gene complexes and associated trait values may increase the ecological and evolutionary success of polymorphic populations and species. Bolton et al. (Molecular Ecology, 2015, 24 , 2907) further state that there is no clear evidence from studies of ‘true polymorphic species’ that polymorphism promotes population persistence. In response, I (i) challenge their classifications of polymorphisms and revisit the traditional definitions recognizing the dynamic nature of polymorphisms, (ii) review empirical studies that have examined whether and how polymorphism is associated with extinction risk, (iii) discuss the roles of trait correlations between colour pattern and other phenotypic dimensions for population fitness and (iv) highlight that the causes and mechanisms that influence the composition and maintenance of polymorphisms are different from the consequences of the polymorphic condition and how it may impact on aspects of ecological success and long‐term persistence of populations and species.     

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.     

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.     

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.     

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 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 ).     

Mock communities highlight the diversity of host‐associated eukaryotes

Host‐associated microbes are ubiquitous. Every multicellular eukaryote, and even many unicellular eukaryotes (protists), hosts a diverse community of microbes. High‐throughput sequencing (HTS) tools have illuminated the vast diversity of host‐associated microbes and shown that they have widespread influence on host biology, ecology and evolution (McFall‐Ngai et al. 2013 ). Bacteria receive most of the attention, but protists are also important components of microbial communities associated with humans (Parfrey et al. 2011 ) and other hosts. As HTS tools are increasingly used to study eukaryotes, the presence of numerous and diverse host‐associated eukaryotes is emerging as a common theme across ecosystems. Indeed, HTS studies demonstrate that host‐associated lineages account for between 2 and 12% of overall eukaryotic sequences detected in soil, marine and freshwater data sets, with much higher relative abundances observed in some samples (Ramirez et al. 2014 ; Simon et al. 2015 ; de Vargas et al. 2015 ). Previous studies in soil detected large numbers of predominantly parasitic lineages such as Apicomplexa, but did not delve into their origin [e.g. (Ramirez et al. 2014 )]. In this issue of Molecular Ecology, Geisen et al. ( 2015 ) use mock communities to show that many of the eukaryotic organisms detected by environmental sequencing in soils are potentially associated with animal hosts rather than free‐living. By isolating the host‐associated fraction of soil microbial communities, Geisen and colleagues help explain the surprisingly high diversity of parasitic eukaryotic lineages often detected in soil/terrestrial studies using high‐throughput sequencing (HTS) and reinforce the ubiquity of these host‐associated microbes. It is clear that we can no longer assume that organisms detected in bulk environmental sequencing are free‐living, but instead need to design studies that specifically enumerate the diversity and function of host‐associated eukaryotes. Doing so will allow the field to determine the role host‐associated eukaryotes play in soils and other environments and to evaluate hypotheses on assembly of host‐associated communities, disease ecology and more.     

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.