Clock genes exhibit substantial control over gene expression and ultimately life-histories using external cues such as photoperiod, and are thus likely to be critical for adaptation to shifting seasonal conditions and novel environments as species redistribute their ranges under climate change. Coding trinucleotide repeats (cTNRs) are found within several clock genes, and may be interesting targets of selection due to their containment within exonic regions and elevated mutation rates. Here, we conduct inter-specific characterization of the NR1D1 cTNR between Canada lynx and bobcat, and intra-specific spatial and environmental association analyses of neutral microsatellites and our functional cTNR marker, to investigate the role of selection on this locus in Canada lynx. We report signatures of divergent selection between lynx and bobcat, with the potential for hybrid-mediated gene flow in the area of range overlap. We also provide evidence that this locus is under selection across Canada lynx in eastern Canada, with both spatial and environmental variables significantly contributing to the explained variation, after controlling for neutral population structure. These results suggest that cTNRs may play an important role in the generation of functional diversity within some mammal species, and allow for contemporary rates of adaptation in wild populations in response to environmental change. We encourage continued investment into the study of cTNR markers to better understand their broader relevance to the evolution and adaptation of mammals.
Urban evolutionary biology is the study of rapid evolutionary change in response to humans and our use of lands to support city dwellers. Because cities are relatively modern additions to the natural world, research on urban evolution tends to focus on microevolutionary change that has happened across a few to many hundreds of generations. These questions still fall under the broad purview of evolutionary ecology. But the severity, rapidity, and replication of environmental changes that drive evolution in this context make it worthy of specific attention. Urban evolution provides the opportunity to study the earliest stages of evolution in a context that is scientifically interesting and societally important. The newness of urban populations and their proximity to natural populations also creates challenges when trying to detect population genetic change. In a From the Cover article in this issue of Molecular Ecology, Mueller et al. (2020) use whole genome resequencing data to address some of these challenges while exploring genetic changes associated with urbanization in 3 replicate urban-rural burrowing owl (Athene cunicularia) populations. Combining multiple approaches across these sample sites Mueller et al. find evidence for selection on genes whose function is related to synapses, neuron projections, brain connectivity, and cognitive function in general. That selection was parallel suggests brain processes were likely important for urban adaptation.
Corals show spatial acclimatisation to local environment conditions. However, the various cellular mechanisms involved in local acclimatisation and variable bleaching patterns in corals remain to be thoroughly understood. In this study, the modulation of a protein implicated in cellular heat stress tolerance, the Heat shock protein 70, was compared at both gene (hsp70) and protein (Hsp70) expression level in bleaching tolerant near-coast Acropora muricata colonies and bleaching susceptible reef colonies, in the lagoon of Belle Mare (Mauritius). The relative Hsp70 levels varied significantly between colonies from the two different locations, colonies having different health conditions and the year of collection. Before the bleaching event of 2016, near-coast colonies had higher basal levels of both Hsp70 gene and protein compared to reef colonies. During the bleaching event, the near-coast colonies did not bleach and had significantly higher relative levels of both Hsp70 gene and protein compared to bleached reef colonies. No significant genetic differentiation between the two studied coral populations was observed and all the colonies analysed were associated with Symbiodiniaceae of the genus Symbiodinium (Clade A) irrespective of location and sampling period. These findings provide further evidence of the involvement of Hsp70 in conferring bleaching tolerance to corals. Moreover, the consistent expression differences of Hsp70 gene and protein between the near-coast and reef coral populations in a natural setting indicate that the modulation of this Hsp is involved in local acclimatisation of corals to their environments.
We introduce a new pattern of population genetic structure in a host-parasite system that can arise after secondary contact of previously isolated populations. Due to different generation time and therefore different tempo of molecular evolution the host and parasite populations reach different degrees of genetic differentiation during their separation (e.g. in refugia). Consequently, during the secondary contact the host populations are able to re-establish a single panmictic population across the area of contact, while the parasite populations stop their dispersal at the secondary contact zone and create a narrow hybrid zone. From the host’s perspective, the parasite’s hybrid zone functions on a microevolutionary scale as a “parasite turnover zone”: while the hosts are passing from area A to area B, their parasites turn genetically from the area A genotypes to the area B genotypes. We demonstrate this novel pattern on a model composed of Apodemus mice and Polyplax lice by comparing maternally inherited markers (complete mitochondrial genomes, and complete genomes of vertically transmitted symbiont Legionella polyplacis) with SNPs derived from the louse genomic data. We discuss circumstances that may lead to this pattern and possible reasons why it has been overlooked in the studies on host-parasite population genetics.
With the growing anthropogenic pressure on marine ecosystems, the need for efficient monitoring of biodiversity grows stronger. DNA metabarcoding of bulk samples is increasingly implemented in ecosystem assessments and is more cost-efficient and less time-consuming than monitoring based on morphology. However, before raw sequences are obtained from bulk samples, a profound number of methodological choices must be made. Here, we critically review the recent methods used for metabarcoding of marine bulk samples (including benthic, plankton and diet samples) and indicate how potential biases can be introduced throughout sampling, pre-processing, DNA extraction, marker and primer selection, PCR amplification and sequencing. From a total of 64 studies evaluated, our recommendations for best practices include to (a) consider DESS as a fixative instead of ethanol, (b) use the DNeasy PowerSoil kit for any samples containing traces of sediment, (c) not limit the marker selection to COI only, but preferably include multiple markers for higher taxonomic resolution, (d) avoid touchdown PCR profiles, (e) use a fixed annealing temperature for each primer pair when comparing across studies or institutes, (f) use a minimum of 3 PCR replicates and (g) include both negative and positive controls. Although the implementation of DNA metabarcoding still faces several technical complexities, we foresee wide-ranging advances in the near future, including improved bioinformatics for taxonomic assignment, sequencing of longer fragments, and the use of whole-genome information. Despite the bulk of biases involved in metabarcoding of bulk samples, it is clear that DNA metabarcoding provides a valuable tool in ecosystem assessments.
Monitoring the genetic structure of pathogen populations may be an economical and sensitive approach to quantify the impact of control on transmission dynamics, highlighting the need for a better understanding of changes in population genetic parameters as transmission declines. Here we describe the first population genetic analysis of the major human malaria parasites, Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) populations following nationwide distribution of long-lasting insecticide treated nets (LLIN) in Papua New Guinea (PNG). Parasite isolates from pre- (2005-6) and post-LLIN (2010-2014) were genotyped using microsatellite markers. Despite parasite prevalence declining substantially (East Sepik: Pf=54.9-8.5%, Pv=35.7-5.6%, Madang: Pf=38.0-9.0%, Pv: 31.8-19.7%), genetically diverse and intermixing parasite populations remained. Pf diversity declined modestly post-LLIN relative to pre-LLIN (East Sepik: Rs = 7.1-6.4, He = 0.77-0.71; Madang: Rs= 8.2-6.1, He = 0.79-0.71). Unexpectedly, population structure present in pre-LLIN populations was lost post-LLIN, suggesting that more frequent human movement between provinces may have contributed to higher gene flow. Pv prevalence initially declined but increased again in one province, yet diversity remained high throughout the study period (East Sepik: Rs=11.4-9.3, He=0.83-0.80; Madang: Rs=12.2-14.5, He=0.85-0.88). Although genetic differentiation values increased between provinces over time, no significant population structure was observed at any time point. For both species, a decline in multiple infections and increasing clonal transmission and significant multilocus linkage disequilibrium (mLD) post-LLIN was a positive indicator of impact on the parasite population using microsatellite markers. These parameters may be useful adjuncts to traditional epidemiological tools in the early stages of transmission reduction.
Coral reefs are losing coral cover across the globe largely as a result of a rise in seawater temperatures that trigger coral bleaching and induce coral mortality. How coral reefs will respond to climate change will be a function of genetic variation and how it is partitioned among species. A critical initial step is to accurately delineate species and quantify their physiological potential to cope with heat stress. Cryptic species, morphologically indistinguishable but genetically different ones, typically harbor distinct physiological variation and respond differently to climatic changes. A dominant Caribbean reef builder severely affected by climate change is the mountainous star coral, Orbicella faveolata. Recently, Dziedzic et al. (2019) reported genetic variation in the physiological response to thermal stress in a single population of this species, suggesting that variation within populations will allow these corals to adapt to rising ocean temperatures. We reanalyzed their data and found multiple cryptic lineages rather than a single panmictic population, with only one of the lineages being heat-tolerant. Our finding of hidden lineages within a threatened species highlights the varying extinction risks faced by these independently evolving groups, especially when the prospects of survival under warmer oceans seem favorable for a few of them only.
Roe deer (Capreolus spp.) are a little odd. They are one of only a few placental mammals — and the only genus among even-toed ungulates — capable of putting embryonic development “on ice”, also known as embryonic diapause (Fig. 1). It would seem such an unusual trait is likely the product of natural selection, but a big question is, how does selection for important traits, such as diapause, interact with the historical demography of a species? In a ‘From the Cover’ article in this issue of Molecular Ecology, de Jong et al. (2020) demonstrate that selection is acting on genes associated with reproductive biology in roe deer, despite heightened genetic drift due to reduced effective population size through the Pleistocene.
Summer heat waves are the principal global driver of mortality in reef-building corals. Resilience-based genetic management may increase coral heat tolerance, but it is unclear how temperature responses are regulated at a genomic level and thus how corals may adapt to warming naturally or through selective breeding. Here we combine phenotypic, pedigree, and genomic marker data from colonies sourced from a warm reef on the Great Barrier Reef reproductively crossed with conspecific colonies from a cooler reef to produce combinations of warm and cool purebred and hybrid larvae and juveniles. Intra-population breeding created significantly greater genetic diversity across the coral genome and maintained diversity in key regions associated with heat tolerance and fitness. High-density genome-wide scans of single nucleotide polymorphisms (SNPs) identified alleles significantly associated with offspring reared at 27.5°C (87 – 2,224 loci), including loci putatively associated with proteins involved in responses to heat stress (cell membrane formation, metabolism, and immune responses). Underlying genetics explained 43% of PCoA variation in juvenile survival, growth, and bleaching responses at 27.5°C and 31°C between the multilocus genotypes. Genetic marker contribution to total variation in fitness traits (narrow-sense heritability) were high for survival but not for growth and bleaching in juveniles, with heritability of these traits influenced more at 31°C relative to 27.5°C. Using only a limited number of crosses, the mechanistic understanding presented here demonstrates that allele frequencies are affected by one generation of selective breeding, key information for the assessments of genetic intervention feasibility and modelling of reef futures.
The fire ant Solenopsis invicta exists in two alternate social forms: monogyne nests contain a single reproductive queen and polygyne nests contain multiple reproductive queens. This colony-level social polymorphism corresponds with individual differences in queen physiology, queen dispersal patterns, and worker discrimination behaviors, all evidently regulated by an inversion-based supergene that spans more than 13Mb of a “social chromosome,” contains over 400 protein-coding genes, and rarely undergoes recombination. The specific mechanisms by which this supergene influences expression of the many distinctive features that characterize the alternate forms remain almost wholly unknown. To advance our understanding of these mechanisms, we explore effects of social chromosome genotype and natal colony social form on gene expression in virgin queens sampled as they embarked on nuptial flights, using RNA-sequencing of two important tissues. We observe relatively minor effects of natal social form, that is, of the social/developmental environment, on gene expression profiles, but substantial effects of genotype, including i) supergene-associated gene upregulation, ii) allele-specific expression, and iii) pronounced extra-supergene trans-regulatory effects. These findings, along with observed spatial variation in differential and allele-specific expression within the supergene region, highlight the complex gene regulatory landscape that emerged following evolutionary divergence of the inversion-mediated Sb haplotype from its homolog that largely retained the ancestral gene order. The distinctive social chromosome-linked gene expression trajectories we document at the onset of a queen’s reproductive life expand the known record of relevant molecular correlates of a complex social polymorphism and point to putative genetic underpinnings of the alternate social syndromes.
Populations of ectothermic vertebrates are vulnerable to environmental pollution and climate change because certain chemicals and high temperature can cause sex reversal during their larval development (i.e. genetically female individuals develop male phenotype or vice versa), which may distort population sex ratios. However, we have troublingly little information on sex reversals in natural populations, due to unavailability of genetic sex markers. Here we developed a genetic sexing method based on sex-linked single nucleotide polymorphism loci to study the prevalence and fitness consequences of sex reversal in agile frogs (Rana dalmatina). Out of 125 juveniles raised in laboratory without exposure to sex-reversing stimuli, 6 showed male phenotype but female genotype according to our markers. These individuals exhibited several signs of poor physiological condition, suggesting stress-induced sex reversal and inferior fitness prospects. Among 162 adults from 11 wild populations in North-Central Hungary, 20% of phenotypic males had female genotype according to our markers. These individuals occurred more frequently in areas of anthropogenic land use; this association was attributable to agriculture and less strongly to urban land use. Female-to-male sex-reversed adults had similar body mass as normal males. We recorded no events of male-to-female sex reversal either in the lab or in the wild. These results support recent suspicions that sex reversal is widespread in nature, and suggest that human-induced environmental changes may contribute to its pervasiveness. Furthermore, our findings indicate that sex-reversal is associated with stress and poor health in early life, but sex-reversed individuals surviving to adulthood may participate in breeding.
Rapid shifts in environmental variables associated with elevational changes in montane ecosystems provide opportunities to test hypotheses regarding the effects of environmental heterogeneity on gene flow and genetic structure. In tropical mountains, spatial environmental heterogeneity combined with seasonal environmental stability is predicted to result in low dispersal across elevations. Few studies have investigated the genetic consequences of elevational environmental heterogeneity in tropical montane mammals. Here, we use a population genomics approach to test the hypothesis that mountain treeshrews (Tupaia montana) exhibit limited gene flow across elevational gradients and between two neighboring peaks within Kinabalu National Park (KNP) in Borneo. We sampled 83 individuals across elevations on Mt. Tambuyukon (MT) and Mt. Kinabalu (MK) and sequenced mitogenomes and 4,106 ultraconserved elements containing an average of 1.9 single nucleotide polymorphisms per locus. We detected high gene flow across elevations and between peaks. We found greater genetic differentiation on MT than MK despite its lower elevation and associated environmental variation. This implies that, contrary to our hypothesis, genetic structure in this system is not primarily shaped by elevation. We propose that this pattern may instead be the result of colonization history combined with restricted upslope gene flow on MT due to unique plant communities associated with its upper montane habitats. Our results serve as a foundation to identify and mitigate future effects of climate change on mountain treeshrews in KNP. Given predictions for 2100 CE, we predict that mountain treeshrews will maintain genetic connectivity in KNP, making it an important conservation stronghold.
Environmental DNA and metabarcoding have great potential for the biomonitoring of freshwater environments. However, successful application of metabarcoding to biodiversity monitoring requires universal primers with high taxonomic coverage that amplify highly-variable, short metabarcodes with high taxonomic resolution. Moreover, reliable and extensive reference databases are essential to match the outcome of metabarcoding analyses with the available taxonomy and biomonitoring indices. Benthic invertebrates, particularly insects, are key taxa for freshwater biomonitoring. Nevertheless, so far, no formal comparison has assessed primers for metabarcoding of freshwater macrobenthos. Here we combined in vitro and in silico analyses to test the performance of metabarcoding primers amplifying regions in the 18S rDNA (Euka02 metabarcode), 16S rDNA (Inse01), and COI (BF1_BR2-COI) genes, and developed an extensive database of benthic invertebrates of France and Europe, with a special focus on three key insect orders (Ephemeroptera, Plecoptera and Trichoptera). In vitro analyses on 1514 individuals, belonging to 578 different taxonomic units showed very different amplification rates across primer combinations. The Euka02 marker showed the highest universality, while the Inse01 marker showed excellent performance for the amplification of insects. The BF1_BR2-COI metabarcode showed the highest resolution, while the resolution of Euka02 was often limited. By combining in vitro data with GenBank information, we developed a curated database including sequences representing 822 genera. The heterogeneous performance of the different metabarcodes highlights the complexity of the identification of the best markers, and advocates for the integration of multiple metabarcodes for a more comprehensive and accurate understanding of ecological impacts on freshwater biodiversity.
Pacific lamprey (Entosphenus tridentatus) is a culturally important and imperiled anadromous fish with a parasitic ocean phase. Biological uncertainties challenge restoration efforts and life-history research is needed to explain observed trait variation and inform management actions. Using two new whole genome assemblies and genotypes from 7,716 single nucleotide polymorphism (SNP) loci in 518 individuals from across the species range, we identified four large regions of high genomic divergence (on chromosomes 01, 02, 04, and 22). We genotyped a subset of 302 broadly distributed SNPs in 2,145 individuals for genotype-by-phenotype trait associations for adult body size, sexual maturity, migration distance and timing, adult swimming ability, and larval growth. Body size traits were strongly associated with SNPs on chromosomes 02 and 04. Moderate associations also implicated SNPs on chromosome 01 as being associated with variation in female maturity. Using genotypic frequencies of candidate SNPs for female maturity and body size, we extrapolated a heterogeneous spatiotemporal distribution of these traits based on independent datasets of larval and adult collections. These maturity and body size results guide future studies to validate these predicted phenotypic distributions across the geographic range and elucidate factors driving regional optimization of these traits for fitness.
Monarch butterflies are known for their spectacular annual migration in eastern North America, with millions of monarchs flying up to 4,500 kilometers to overwintering sites in central Mexico. Monarchs also live west of the Rocky Mountains, where they travel shorter distances to overwinter along the Pacific Coast. It is often assumed that eastern and western monarchs form distinct evolutionary units, but genomic studies to support this notion are lacking. We used a tethered flight mill to show that migratory eastern monarchs have greater flight performance than western monarchs, consistent with their greater migratory distances. However, analyzing more than 20 million SNPs in 43 monarch genomes, we found no evidence for genomic differentiation between eastern and western monarchs. Genomic analysis also showed identical and low levels of genetic diversity, and demographic analyses indicated similar effective population sizes and ongoing gene flow between eastern and western monarchs. Gene expression analysis of a subset of candidate genes during active flight revealed differential gene expression related to non-muscular motor activity. Our results demonstrate that eastern and western monarchs maintain migratory differences despite ongoing gene flow, and suggest that migratory differences between eastern and western monarchs are not driven by select major-effects alleles. Instead, variation in migratory distance and destination may be driven by environmentally induced differential gene expression, or by many alleles of small effect.
Parallel evolution can occur through novel mutations, standing genetic variation, or adaptive introgression. Uncovering parallelism and introgressed populations can complicate management of threatened species, particularly as admixed populations are not generally considered under conservation legislations. We examined high coverage whole-genome sequences of 30 caribou (Rangifer tarandus) from across North America and Greenland, representing divergent intra-specific lineages, to investigate parallelism and levels of introgression contributing to the formation of ecotypes. Caribou are split into four subspecies and 11 extant conservation units, known as Designatable Units (DUs), in Canada. Using genomes from all four subspecies and six DUs, we undertake demographic reconstruction and confirm two previously inferred instances of parallel evolution in the woodland subspecies and uncover an additional instance of parallelism of the eastern migratory ecotype. Detailed investigations reveal introgression in the woodland subspecies, with introgressed regions found spread throughout the genomes encompassing both neutral and functional sites. Our comprehensive investigations using whole genomes highlight the difficulties in unequivocally demonstrating parallelism through adaptive introgression in non-model species with complex demographic histories, with standing variation and introgression both potentially involved. Additionally, the impact of parallelism and introgression on the designation of conservation units has not been widely considered, and the caribou designations will need amending in light of our results. Uncovering and decoupling parallelism and differential patterns of introgression will become prevalent with the availability of comprehensive genomic data from non-model species, and we highlight the need to incorporate this into conservation unit designations.
Accurate and testable species delimitation hypotheses are essential for measuring, surveying and managing biodiversity. Today, taxonomists often rely on mitochondrial DNA barcoding to complement morphological species delimitations. Although COI barcoding has largely proven successful in assisting identifications for most animal taxa, there are nevertheless numerous cases where mitochondrial barcodes do not necessarily reflect the species history. For instance, what is regarded as one single species can be associated with two distinct DNA barcodes, which can point either to cryptic diversity or to deep within-species mitochondrial divergences with no reproductive isolation. In contrast, two or more species can share barcodes, for instance due to mitochondrial introgression. These intrinsic limitations of mitochondrial DNA barcoding can only be addressed with nuclear genomic markers, which are expensive, labour intensive, poorly repeatable, and often require high-quality DNA. To overcome these limitations, we examined the use of ultraconserved nuclear genetic elements (UCEs) as a quick and robust genomic approach to address such problematic cases of species delimitation. This genomic method was assessed using six different bee species complexes suspected to harbour cryptic diversity, mitochondrial introgression, or mitochondrial paraphyly. The sequencing of UCEs recovered between 686 and 1860 homologous nuclear loci and provided explicit species delimitation hypotheses in all investigated species complexes. These results provide strong evidence for the suitability of UCEs as a fast method for species delimitation even in recently diverged lineages. Furthermore, this study provided the first conclusive evidence for both mitochondrial introgression among distinct species, and mitochondrial paraphyly within a single bee species.
Most new cryptic species are described using conventional tree- and distance-based species delimitation methods (SDMs), which rely on phylogenetic arrangements and measures of genetic divergence. However, although numerous factors such as spatial population structure and gene flow are known to confound phylogenetic and species delimitation inferences, the influence of these processes on species estimation is rarely evaluated. Using large amounts of exons, introns, and ultraconserved elements obtained using the FrogCap sequence-capture protocol, we compared conventional SDMs with more robust genomic analyses that assesses spatial population structure and gene flow to characterize species boundaries in a Southeast Asian frog complex (Pulchrana picturata). Our results showed that gene flow and introgression can produce phylogenetic patterns and levels of divergence that resemble distinct species (up to 10% divergent in mitochondrial DNA). Hybrid populations were inferred as independent (singleton) clades that were highly divergent from adjacent populations (7–10%) and unusually similar (<3%) to allopatric populations. Such anomalous patterns are not uncommon in Southeast Asian amphibians, which brings into question whether the high cryptic diversity observed in other amphibian groups reflect distinct cryptic species—or, instead, highly structured and admixed metapopulation lineages. Our results also provide an alternative explanation to the conundrum of divergent (sometimes non-sister) sympatric lineages―a pattern that has been celebrated as indicative of true cryptic speciation. Based on these findings, we recommend that species delimitation of continuously distributed “cryptic” groups should not rely solely on conventional SDMs but should necessarily examine spatial population structure and gene flow to avoid taxonomic inflation.