Identification of sex‐specific molecular markers using restriction site‐associated DNA sequencing

A major barrier to evolutionary studies of sex determination and sex chromosomes has been a lack of information on the types of sex‐determining mechanisms that occur among different species. This is particularly problematic in groups where most species lack visually heteromorphic sex chromosomes, such as fish, amphibians and reptiles, because cytogenetic analyses will fail to identify the sex chromosomes in these species. We describe the use of restriction site‐associated DNA (RAD) sequencing, or RAD‐seq, to identify sex‐specific molecular markers and subsequently determine whether a species has male or female heterogamety. To test the accuracy of this technique, we examined the lizard Anolis carolinensis. We performed RAD‐seq on seven male and ten female A. carolinensis and found one male‐specific molecular marker. Anolis carolinensis has previously been shown to possess male heterogamety and the recently published A. carolinensis genome facilitated the characterization of the sex‐specific RAD‐seq marker. We validated the male specificity of the new marker using PCR on additional individuals and also found that it is conserved in some other Anolis species. We discuss the utility of using RAD‐seq to identify sex‐determining mechanisms in other species with cryptic or homomorphic sex chromosomes and the implications for the evolution of male heterogamety in Anolis.     

Genomic characterization of sex‐identification markers in Sebastes carnatus and Sebastes chrysomelas rockfishes

Fish have evolved a variety of sex‐determining (SD) systems including male heterogamy (XY), female heterogamy (ZW) and environmental SD. Little is known about SD mechanisms of Sebastes rockfishes, a highly speciose genus of importance to evolutionary and conservation biology. Here, we characterize the sex determination system in the sympatrically distributed sister species Sebastes chrysomelas and Sebastes carnatus. To identify sex‐specific genotypic markers, double digest restriction site – associated DNA sequencing (ddRAD‐seq) of genomic DNA from 40 sexed individuals of both species was performed. Loci were filtered for presence in all of the individuals of one sex, absence in the other sex and no heterozygosity. Of the 74 965 loci present in all males, 33 male‐specific loci met the criteria in at least one species and 17 in both. Conversely, no female‐specific loci were detected, together providing evidence of an XY sex determination system in both species. When aligned to a draft reference genome from Sebastes aleutianus, 26 sex‐specific loci were interspersed among 1168 loci that were identical between sexes. The nascent Y chromosome averaged 5% divergence from the X chromosome and mapped to reference Sebastes genome scaffolds totalling 6.9Mbp in length. These scaffolds aligned to a single chromosome in three model fish genomes. Read coverage differences were also detected between sex‐specific and autosomal loci. A PCR‐RFLP assay validated the bioinformatic results and correctly identified sex of five additional individuals of known sex. A sex‐determining gene in other teleosts gonadal soma‐derived factor (gsdf) was present in the model fish chromosomes that spanned our sex‐specific markers.     

Identifying homomorphic sex chromosomes from wild‐caught adults with limited genomic resources

We demonstrate a genotyping‐by‐sequencing approach to identify homomorphic sex chromosomes and their homolog in a distantly related reference genome, based on noninvasive sampling of wild‐caught individuals, in the moor frog Rana arvalis. Double‐digest RADseq libraries were generated using buccal swabs from 30 males and 21 females from the same population. Search for sex‐limited markers from the unfiltered data set (411 446 RAD tags) was more successful than searches from a filtered data set (33 073 RAD tags) for markers showing sex differences in heterozygosity or in allele frequencies. Altogether, we obtained 292 putatively sex‐linked RAD loci, 98% of which point to male heterogamety. We could map 15 of them to the Xenopus tropicalis genome, all but one on chromosome pair 1, which seems regularly co‐opted for sex determination among amphibians. The most efficient mapping strategy was a three‐step hierarchical approach, where R. arvalis reads were first mapped to a low‐coverage genome of Rana temporaria (17 My divergence), then the R. temporaria scaffolds to the Nanorana parkeri genome (90 My divergence), and finally the N. parkeri scaffolds to the X. tropicalis genome (210 My). We validated our conclusions with PCR primers amplifying part of Dmrt1, a candidate sex determination gene mapping to chromosome 1: a sex‐diagnostic allele was present in all 30 males but in none of the 21 females. Our approach is likely to be productive in many situations where biological samples and/or genomic resources are limited.     

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.     

High‐throughput SNPs for all: genotyping‐in‐thousands

Understanding the genetic structure of species is essential for conservation. It is only with this information that managers, academics, user groups and land‐use planners can understand the spatial scale of migration and local adaptation, source‐sink dynamics and effective population size. Such information is essential for a multitude of applications including delineating management units, balancing management priorities, discovering cryptic species and implementing captive breeding programmes. Species can range from locally adapted by hundreds of metres (Pavey et al. 2010 ) to complete species panmixia (Côté et al. 2013 ). Even more remarkable is that this essential information can be obtained without fully sequenced or annotated genomes, but from mere (putatively) nonfunctional variants. First with allozymes, then microsatellites and now SNPs, this neutral genetic variation carries a wealth of information about migration and drift. For many of us, it may be somewhat difficult to remember our understanding of species conservation before the widespread usage of these useful tools. However most species on earth have yet to give us that ‘peek under the curtain’. With the current diversity on earth estimated to be nearly 9 million species (Mora et al. 2011 ), we have a long way to go for a comprehensive meta‐phylogeographic understanding. A method presented in this issue by Campbell and colleagues (Campbell et al. 2015 ) is a tool that will accelerate the pace in this area. Genotyping‐in‐thousands (GT‐seq) leverages recent advancements in sequencing technology to save many hours and dollars over previous methods to generate this important neutral genetic information.     

Homomorphic plant sex chromosomes are coming of age

Sex chromosomes are a very peculiar part of the genome that have evolved independently in many groups of animals and plants (Bull 1983 ). Major research efforts have so far been focused on large heteromorphic sex chromosomes in a few animal and plant species (Chibalina & Filatov 2011 ; Zhou & Bachtrog 2012 ; Bellott et al. 2014 ; Hough et al. 2014 ; Zhou et al. 2014 ), while homomorphic (cytologically indistinguishable) sex chromosomes have largely been neglected. However, this situation is starting to change. In this issue, Geraldes et al. ( 2015 ) describe a small (~100 kb long) sex‐determining region on the homomorphic sex chromosomes of poplars (Populus trichocarpa and related species, Fig.  1 ). All species in Populus and its sister genus Salix are dioecious, suggesting that dioecy and the sex chromosomes, if any, should be relatively old. Contrary to this expectation, Geraldes et al. ( 2015 ) demonstrate that the sex‐determining region in poplars is of very recent origin and probably evolved within the genus Populus only a few million years ago.     

Using RAD‐seq to develop sex‐linked markers in a haplodiplontic alga

For many taxa, including isomorphic haplodiplontic macroalgae, determining sex and ploidy is challenging, thereby limiting the scope of some population demographic and genetic studies. Here, we used double‐digest restriction site‐associated DNA sequencing (ddRAD‐seq) to identify sex‐linked molecular markers in the widespread red alga Agarophyton vermiculophyllum. In the ddRAD‐seq library, we included 10 female gametophytes, 10 male gametophytes, and 16 tetrasporophytes from one native and one non‐native site (N = 40 gametophytes and N = 32 tetrasporophytes total). We identified seven putatively female‐linked and 19 putatively male‐linked sequences. Four female‐ and eight male‐linked markers amplified in all three life cycle stages. Using one female‐ and one male‐linked marker that were sex‐specific, we developed a duplex PCR and tested the efficacy of this assay on a subset of thalli sampled at two sites in the non‐native range. We confirmed ploidy based on the visual observation of reproductive structures and previous microsatellite genotyping at 10 polymorphic loci. For 32 vegetative thalli, we were able to assign sex and confirm ploidy in these previously genotyped thalli. These markers will be integral to ongoing studies of A. vermiculophyllum invasion. We discuss the utility of RAD‐seq over other approaches previously used, such as RAPDs (random amplified polymorphic DNA), for future work designing sex‐linked markers in other haplodiplontic macroalgae for which genomes are lacking.     

Population genomics fits the bill: genetics of adaptive beak variation in Darwin's finches

Darwin's finches are an iconic case of adaptive radiation. The size and shape of their beaks are key adaptive traits related to trophic niche that vary among species and evolve rapidly when the food supply changes. Building on recent studies, a paper in this issue of Molecular Ecology (Chaves et al. 2016 ) investigates the genomic basis of beak size variation in sympatric populations of three species of ground finch (Geospiza) by performing a Genome‐wide association study using RAD‐seq data. The authors find that variation in a small number of markers can explain a substantial proportion of variation in beak size. Some of these markers are in genomic regions that have previously been implicated in beak size variation in Darwin's finches, whereas other markers have not, suggesting both conservation and divergence in the genetic basis of morphological evolution. Overall, the study confirms that loci of large effect are involved in beak size variation, which helps to explain the high heritability and rapid response to selection of this trait. The independent identification of regions containing HMGA2 and DLK1 loci in a GWAS makes them prime targets for functional studies. The study also shows that under the right conditions, RAD‐seq can be a viable alternative to genome sequencing for GWAS in wild vertebrate populations.     

Small‐ and large‐scale heterogeneity in genetic variation across the collard flycatcher genome: implications for estimating genetic diversity in nonmodel organisms

Population genetic studies in nonmodel organisms are often hampered by a lack of reference genomes that are essential for whole‐genome resequencing. In the light of this, genotyping methods have been developed to effectively eliminate the need for a reference genome, such as genotyping by sequencing or restriction site‐associated DNA sequencing (RAD‐seq). However, what remains relatively poorly studied is how accurately these methods capture both average and variation in genetic diversity across an organism's genome. In this issue of Molecular Ecology Resources, Dutoit et al. (2016) use whole‐genome resequencing data from the collard flycatcher to assess what factors drive heterogeneity in nucleotide diversity across the genome. Using these data, they then simulate how well different sequencing designs, including RAD sequencing, could capture most of the variation in genetic diversity. They conclude that for evolutionary and conservation‐related studies focused on the estimating genomic diversity, researchers should emphasize the number of loci analysed over the number of individuals sequenced.     

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 ).     

Next‐generation phylogenetics takes root

It has been a tumultuous 5 years in phylogeography and phylogenetics during which both fields have struggled to harness the power of next‐generation sequencing (NGS) (Ekblom & Galindo 2010 ; McCormack et al. 2012a ). Fortunately, several methodological approaches appear to be taking root. In this issue of Molecular Ecology, O'Neill et al. 2013 ) employ one such method – parallel tagged sequencing (PTS) – to elucidate the phylogeography of a tiger salamander (Ambystoma tigrinum) species complex. This study demonstrates a practical application of NGS on a scale appropriate (and not overkill) for most biologists interested in phylogeography (~100 loci for ~100 individuals), and their results highlight several analytical challenges that lie ahead for researchers employing NGS techniques.     

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.     

Positive range–abundance relationships in Indo‐Pacific bird communities

Reeve et al. (2016, Ecography, 39 , 990–997) recently reported negative range–abundance relationships in Indo‐Pacific bird communities and speculated that geographical isolation facilitates the evolution of broad‐niched, small‐ranged and abundant species. We tested this relationship using a large independent data set on range and abundance of birds across New Caledonia (over 4,000 bird census points for 17,300 km²). In contradiction to Reeve et al. (2016, Ecography, 39 , 990–997), we found clear evidence that range–abundance relationships are positive and endemic species have narrower habitat niches than wide‐range species. Our findings are likely valid also for other islands in the Indo‐Pacific.     

Opportunities and challenges of next‐generation sequencing applications in ecological epigenetics

Evolutionary theory posits that adaptation can result when populations harbour heritable phenotypic variation for traits that increase tolerance to local conditions. However, the actual mechanisms that underlie heritable phenotypic variation are not completely understood (Keller 2014 ). Recently, the potential role of epigenetic mechanisms in the process of adaptive evolution has been the subject of much debate (Pigliucci & Finkelman 2014 ). Studies of variation in DNA methylation in particular have shown that natural populations harbour high amounts of epigenetic variation, which can be inherited across generations and can cause heritable trait variation independently of genetic variation (Kilvitis et al. 2014 ). While we have made some progress addressing the importance of epigenetics in ecology and evolution using methylation‐sensitive AFLP (MS‐AFLP), this approach provides relatively few anonymous and dominant markers per individual. MS‐AFLP are difficult to link to functional genomic elements or phenotype and are difficult to compare directly to genetic variation, which has limited the insights drawn from studies of epigenetic variation in natural nonmodel populations (Schrey et al. 2013 ). In this issue, Platt et al. provide an example of a promising approach to address this problem by applying a reduced representation bisulphite sequencing (RRBS) approach based on next‐generation sequencing methods in an ecological context.     

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.     

Double‐digest RAD sequencing using Ion Proton semiconductor platform (ddRADseq‐ion) with nonmodel organisms

Research in evolutionary biology involving nonmodel organisms is rapidly shifting from using traditional molecular markers such as mtDNA and microsatellites to higher throughput SNP genotyping methodologies to address questions in population genetics, phylogenetics and genetic mapping. Restriction site associated DNA sequencing (RAD sequencing or RADseq) has become an established method for SNP genotyping on Illumina sequencing platforms. Here, we developed a protocol and adapters for double‐digest RAD sequencing for Ion Torrent (Life Technologies; Ion Proton, Ion PGM) semiconductor sequencing. We sequenced thirteen genomic libraries of three different nonmodel vertebrate species on Ion Proton with PI chips: Arctic charr Salvelinus alpinus, European whitefish Coregonus lavaretus and common lizard Zootoca vivipara. This resulted in ~962 million single‐end reads overall and a mean of ~74 million reads per library. We filtered the genomic data using Stacks, a bioinformatic tool to process RAD sequencing data. On average, we obtained ~11 000 polymorphic loci per library of 6–30 individuals. We validate our new method by technical and biological replication, by reconstructing phylogenetic relationships, and using a hybrid genetic cross to track genomic variants. Finally, we discuss the differences between using the different sequencing platforms in the context of RAD sequencing, assessing possible advantages and disadvantages. We show that our protocol can be used for Ion semiconductor sequencing platforms for the rapid and cost‐effective generation of variable and reproducible genetic markers.     

Marine island biogeography. Response to comment on ‘Island biogeography: patterns of marine shallow‐water organisms’

In this response we have incorporated data on gastropod and seaweed biodiversity referred to by Ávila et al. (2016, Journal of Biogeography, doi: 10.1111/jbi.12816 ) to allow an updated analysis on marine shallow‐water biogeography patterns. When compared to the biogeography patterns reported in Hachich et al. (2015, Journal of Biogeography, 42 , 1871–1882), we find (1) no differences in the patterns originally reported for reef fish or seaweeds, (2) minor differences in gastropod species–area and species–age patterns and (3) a significant difference for the gastropod species‐isolation pattern. In our original work, we reported that there was limited evidence that gastropod species richness was influenced by island isolation; however, our new analysis reveals a power‐model relationship between these variables. Thus, we are now able to conclude that gastropod species diversity, whose dispersal capacity is intermediate between seaweeds (lowest) and reef fish (highest), is also influenced by island isolation.     

Female‐only sex‐linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex‐reversed females (neomales). To provide molecular evidence for the proposed system, novel sex‐linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male‐specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female‐specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female‐specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female‐linked SCAR markers can be applied for rapid detection of prawn gender. These sex‐specific SCAR markers and sex‐associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).