How genomics can help biodiversity conservation.

The availability of public genomic resources can greatly assist biodiversity assessment, conservation, and restoration efforts by providing evidence for scientifically informed management decisions. Here we survey the main approaches and applications in biodiversity and conservation genomics, considering practical factors, such as cost, time, prerequisite skills, and current shortcomings of applications. Most approaches perform best in combination with reference genomes from the target species or closely related species. We review case studies to illustrate how reference genomes can facilitate biodiversity research and conservation across the tree of life. We conclude that the time is ripe to view reference genomes as fundamental resources and to integrate their use as a best practice in conservation genomics.

Threatened North African seagrass meadows have supported green turtle populations for millennia.

"Protect and restore ecosystems and biodiversity" is the second official aim of the current UN Ocean Decade (2021 to 2030) calling for the identification and protection of critical marine habitats. However, data to inform policy are often lacking altogether or confined to recent times, preventing the establishment of long-term baselines. The unique insights gained from combining bioarchaeology (palaeoproteomics, stable isotope analysis) with contemporary data (from satellite tracking) identified habitats which sea turtles have been using in the Eastern Mediterranean over five millennia. Specifically, our analysis of archaeological green turtle (Chelonia mydas) bones revealed that they likely foraged on the same North African seagrass meadows as their modern-day counterparts. Here, millennia-long foraging habitat fidelity has been directly demonstrated, highlighting the significance (and long-term dividends) of protecting these critical coastal habitats that are especially vulnerable to global warming. We highlight the potential for historical ecology to inform policy in safeguarding critical marine habitats.

Long-term stability in the circumpolar foraging range of a Southern Ocean predator between the eras of whaling and rapid climate change.

Assessing environmental changes in Southern Ocean ecosystems is difficult due to its remoteness and data sparsity. Monitoring marine predators that respond rapidly to environmental variation may enable us to track anthropogenic effects on ecosystems. Yet, many long-term datasets of marine predators are incomplete because they are spatially constrained and/or track ecosystems already modified by industrial fishing and whaling in the latter half of the 20th century. Here, we assess the contemporary offshore distribution of a wide-ranging marine predator, the southern right whale (SRW, Eubalaena australis), that forages on copepods and krill from ~30°S to the Antarctic ice edge (>60°S). We analyzed carbon and nitrogen isotope values of 1,002 skin samples from six genetically distinct SRW populations using a customized assignment approach that accounts for temporal and spatial variation in the Southern Ocean phytoplankton isoscape. Over the past three decades, SRWs increased their use of mid-latitude foraging grounds in the south Atlantic and southwest (SW) Indian oceans in the late austral summer and autumn and slightly increased their use of high-latitude (>60°S) foraging grounds in the SW Pacific, coincident with observed changes in prey distribution and abundance on a circumpolar scale. Comparing foraging assignments with whaling records since the 18th century showed remarkable stability in use of mid-latitude foraging areas. We attribute this consistency across four centuries to the physical stability of ocean fronts and resulting productivity in mid-latitude ecosystems of the Southern Ocean compared with polar regions that may be more influenced by recent climate change.

DNA methylation markers of age(ing) in non-model animals.

Inferring the chronological and biological age of individuals is fundamental to population ecology and our understanding of ageing itself, its evolution, and the biological processes that affect or even cause ageing. Epigenetic clocks based on DNA methylation (DNAm) at specific CpG sites show a strong correlation with chronological age in humans, and discrepancies between inferred and actual chronological age predict morbidity and mortality. Recently, a growing number of epigenetic clocks have been developed in non-model animals and we here review these studies. We also conduct a meta-analysis to assess the effects of different aspects of experimental protocol on the performance of epigenetic clocks for non-model animals. Two measures of performance are usually reported, the R2 of the association between the predicted and chronological age, and the mean/median absolute deviation (MAD) of estimated age from chronological age, and we argue that only the MAD reflects accuracy. R2 for epigenetic clocks based on the HorvathMammalMethylChip4 was higher and the MAD scaled to age range lower, compared with other DNAm quantification approaches. Scaled MAD tended to be lower among individuals in captive populations, and decreased with an increasing number of CpG sites. We conclude that epigenetic clocks can predict chronological age with relatively high accuracy, suggesting great potential in ecological epigenetics. We discuss general aspects of epigenetic clocks in the hope of stimulating further DNAm-based research on ageing, and perhaps more importantly, other key traits.

Seagrass ecosystem multifunctionality under the rise of a flagship marine megaherbivore.

Large grazers (megaherbivores) have a profound impact on ecosystem functioning. However, how ecosystem multifunctionality is affected by changes in megaherbivore populations remains poorly understood. Understanding the total impact on ecosystem multifunctionality requires an integrative ecosystem approach, which is especially challenging to obtain in marine systems. We assessed the effects of experimentally simulated grazing intensity scenarios on ecosystem functions and multifunctionality in a tropical Caribbean seagrass ecosystem. As a model, we selected a key marine megaherbivore, the green turtle, whose ecological role is rapidly unfolding in numerous foraging areas where populations are recovering through conservation after centuries of decline, with an increase in recorded overgrazing episodes. To quantify the effects, we employed a novel integrated index of seagrass ecosystem multifunctionality based upon multiple, well-recognized measures of seagrass ecosystem functions that reflect ecosystem services. Experiments revealed that intermediate turtle grazing resulted in the highest rates of nutrient cycling and carbon storage, while sediment stabilization, decomposition rates, epifauna richness, and fish biomass are highest in the absence of turtle grazing. In contrast, intense grazing resulted in disproportionally large effects on ecosystem functions and a collapse of multifunctionality. These results imply that (i) the return of a megaherbivore can exert strong effects on coastal ecosystem functions and multifunctionality, (ii) conservation efforts that are skewed toward megaherbivores, but ignore their key drivers like predators or habitat, will likely result in overgrazing-induced loss of multifunctionality, and (iii) the multifunctionality index shows great potential as a quantitative tool to assess ecosystem performance. Considerable and rapid alterations in megaherbivore abundance (both through extinction and conservation) cause an imbalance in ecosystem functioning and substantially alter or even compromise ecosystem services that help to negate global change effects. An integrative ecosystem approach in environmental management is urgently required to protect and enhance ecosystem multifunctionality.

Demographic changes in Pleistocene sea turtles were driven by past sea level fluctuations affecting feeding habitat availability.

Pleistocene environmental changes are generally assumed to have dramatically affected species' demography via changes in habitat availability, but this is challenging to investigate due to our limited knowledge of how Pleistocene ecosystems changed through time. Here, we tracked changes in shallow marine habitat availability resulting from Pleistocene sea level fluctuations throughout the last glacial cycle (120-14 thousand years ago; kya) and assessed correlations with past changes in genetic diversity inferred from genome-wide SNPs, obtained via ddRAD sequencing, in Caribbean hawksbill turtles, which feed in coral reefs commonly found in shallow tropical waters. We found sea level regression resulted in an average 75% reduction in shallow marine habitat availability during the last glacial cycle. Changes in shallow marine habitat availability correlated strongly with past changes in hawksbill turtle genetic diversity, which gradually declined to ~1/4th of present-day levels during the Last Glacial Maximum (LGM; 26-19 kya). Shallow marine habitat availability and genetic diversity rapidly increased after the LGM, signifying a population expansion in response to warming environmental conditions. Our results suggest a positive correlation between Pleistocene environmental changes, habitat availability and species' demography, and that demographic changes in hawksbill turtles were potentially driven by feeding habitat availability. However, we also identified challenges associated with disentangling the potential environmental drivers of past demographic changes, which highlights the need for integrative approaches. Our conclusions underline the role of habitat availability on species' demography and biodiversity, and that the consequences of ongoing habitat loss should not be underestimated.

Strong and lasting impacts of past global warming on baleen whales and their prey.

Global warming is affecting the population dynamics and trophic interactions across a wide range of ecosystems and habitats. Translating these real-time effects into their long-term consequences remains a challenge. The rapid and extreme warming period that occurred after the Last Glacial Maximum (LGM) during the Pleistocene-Holocene transition (7-12 thousand years ago) provides an opportunity to gain insights into the long-term responses of natural populations to periods with global warming. The effects of this post-LGM warming period have been assessed in many terrestrial taxa, whereas insights into the impacts of rapid global warming on marine taxa remain limited, especially for megafauna. In order to understand how large-scale climate fluctuations during the post-LGM affected baleen whales and their prey, we conducted an extensive, large-scale analysis of the long-term effects of the post-LGM warming on abundance and inter-ocean connectivity in eight baleen whale and seven prey (fish and invertebrates) species across the Southern and the North Atlantic Ocean; two ocean basins that differ in key oceanographic features. The analysis was based upon 7032 mitochondrial DNA sequences as well as genome-wide DNA sequence variation in 100 individuals. The estimated temporal changes in genetic diversity during the last 30,000 years indicated that most baleen whale populations underwent post-LGM expansions in both ocean basins. The increase in baleen whale abundance during the Holocene was associated with simultaneous changes in their prey and climate. Highly correlated, synchronized and exponential increases in abundance in both baleen whales and their prey in the Southern Ocean were indicative of a dramatic increase in ocean productivity. In contrast, the demographic fluctuations observed in baleen whales and their prey in the North Atlantic Ocean were subtle, varying across taxa and time. Perhaps most important was the observation that the ocean-wide expansions and decreases in abundance that were initiated by the post-LGM global warming, continued for millennia after global temperatures stabilized, reflecting persistent, long-lasting impacts of global warming on marine fauna.

The population genomic structure of green turtles (Chelonia mydas) suggests a warm-water corridor for tropical marine fauna between the Atlantic and Indian oceans during the last interglacial.

The occasional westward transport of warm water of the Agulhas Current, "Agulhas leakage", around southern Africa has been suggested to facilitate tropical marine connectivity between the Atlantic and Indian oceans, but the "Agulhas leakage" hypothesis does not explain the signatures of eastward gene flow observed in many tropical marine fauna. We investigated an alternative hypothesis: the establishment of a warm-water corridor during comparatively warm interglacial periods. The "warm-water corridor" hypothesis was investigated by studying the population genomic structure of Atlantic and Southwest Indian Ocean green turtles (N = 27) using 12,035 genome-wide single nucleotide polymorphisms (SNPs) obtained via ddRAD sequencing. Model-based and multivariate clustering suggested a hierarchical population structure with two main Atlantic and Southwest Indian Ocean clusters, and a Caribbean and East Atlantic sub-cluster nested within the Atlantic cluster. Coalescent-based model selection supported a model where Southwest Indian Ocean and Caribbean populations diverged from the East Atlantic population during the transition from the last interglacial period (130-115 thousand years ago; kya) to the last glacial period (115-90 kya). The onset of the last glaciation appeared to isolate Atlantic and Southwest Indian Ocean green turtles into three refugia, which subsequently came into secondary contact in the Caribbean and Southwest Indian Ocean when global temperatures increased after the Last Glacial Maximum. Our findings support the establishment of a warm-water corridor facilitating tropical marine connectivity between the Atlantic and Southwest Indian Ocean during warm interglacials.

Effects of parasites upon non-host predator avoidance behaviour in native and invasive gammarids.

The acanthocephalan parasite, Polymorphus minutus, manipulates its intermediate hosts' (gammarids) behaviour, presumably to facilitate its transmission to the definitive hosts. A fundamental question is whether this capability has evolved to target gammarids in general, or specifically sympatric gammarids. We assessed the responses to chemical cues from a non-host predator (the three-spined sticklebacks Gasterosteus aculeatus) in infected and non-infected gammarids; two native (Gammarus pulex and Gammarus fossarum), and one invasive (Echinogammarus berilloni) species, all sampled in the Paderborn Plateau (Germany). The level of predator avoidance was assessed by subjecting gammarids to choice experiments with the presence or absence of predator chemical cues. We did not detect any behavioural differences between uninfected and infected G. pulex and E. berilloni, but an elevated degree of predator avoidance in infected G. fossarum. Avoiding non-host predators may ultimately increase the probability of P. minutus' of predation by the definitive host. Our results suggested that P. minutus' ability to alter the host's behaviour may have evolved to specifically target sympatric gammarid host species. Uninfected gammarids did not appear to avoid the non-host predator chemical cues. Overall the results also opened the possibility that parasites may play a critical role in the success or failure of invasive species.

Return to the Sea, Get Huge, Beat Cancer: An Analysis of Cetacean Genomes Including an Assembly for the Humpback Whale (Megaptera novaeangliae).

Cetaceans are a clade of highly specialized aquatic mammals that include the largest animals that have ever lived. The largest whales can have ∼1,000× more cells than a human, with long lifespans, leaving them theoretically susceptible to cancer. However, large-bodied and long-lived animals do not suffer higher risks of cancer mortality than humans-an observation known as Peto's Paradox. To investigate the genomic bases of gigantism and other cetacean adaptations, we generated a de novo genome assembly for the humpback whale (Megaptera novaeangliae) and incorporated the genomes of ten cetacean species in a comparative analysis. We found further evidence that rorquals (family Balaenopteridae) radiated during the Miocene or earlier, and inferred that perturbations in abundance and/or the interocean connectivity of North Atlantic humpback whale populations likely occurred throughout the Pleistocene. Our comparative genomic results suggest that the evolution of cetacean gigantism was accompanied by strong selection on pathways that are directly linked to cancer. Large segmental duplications in whale genomes contained genes controlling the apoptotic pathway, and genes inferred to be under accelerated evolution and positive selection in cetaceans were enriched for biological processes such as cell cycle checkpoint, cell signaling, and proliferation. We also inferred positive selection on genes controlling the mammalian appendicular and cranial skeletal elements in the cetacean lineage, which are relevant to extensive anatomical changes during cetacean evolution. Genomic analyses shed light on the molecular mechanisms underlying cetacean traits, including gigantism, and will contribute to the development of future targets for human cancer therapies.

Decadal shift in foraging strategy of a migratory southern ocean predator.

Rapid anthropogenic environmental change is expected to impact a host of ecological parameters in Southern Ocean ecosystems. Of critical concern are the consequences of these changes on the range of species that show fidelity to migratory destinations, as philopatry is hypothesized to help or hinder adaptation to climate change depending on the circumstances. Many baleen whales show philopatry to feeding grounds and are also capital breeders that meet migratory and reproductive costs through seasonal energy intake. Southern right whales (Eubalaena australis, SRWs) are capital breeders that have a strong relationship between reproductive output and foraging success. The population dynamics of South Africa's population of SRWs are characterized by two distinct periods: the 1990s, a period of high calving rates; and the late 2010s, a period associated with lowered calving rates. Here we use analyses of stable carbon (δ13 C) and nitrogen (δ15 N) isotope values from SRW biopsy samples (n = 122) collected during these two distinct periods to investigate foraging ecology of the South African population of SRWs over a time period coincident with the demographic shift. We show that South African SRWs underwent a dramatic northward shift, and diversification, in foraging strategy from 1990s to 2010s. Bayesian mixing model results suggest that during the 1990s, South African SRWs foraged on prey isotopically similar to South Georgia/Islas Georgias del Sur krill. In contrast, in the 2010s, South African SRWs foraged on prey isotopically consistent with the waters of the Subtropical Convergence, Polar Front and Marion Island. We hypothesize that this shift represents a response to changes in preferred habitat or prey, for example, the decrease in abundance and southward range contraction of Antarctic krill. By linking reproductive decline to changing foraging strategies for the first time in SRWs, we show that altering foraging strategies may not be sufficient to adapt to a changing ocean.

Fin whale (Balaenoptera physalus) mitogenomics: A cautionary tale of defining sub-species from mitochondrial sequence monophyly.

The advent of massive parallel sequencing technologies has resulted in an increase of studies based upon complete mitochondrial genome DNA sequences that revisit the taxonomic status within and among species. Spatially distinct monophyly in such mitogenomic genealogies, i.e., the sharing of a recent common ancestor among con-specific samples collected in the same region has been viewed as evidence for subspecies. Several recent studies in cetaceans have employed this criterion to suggest subsequent intraspecific taxonomic revisions. We reason that employing intra-specific, spatially distinct monophyly at non-recombining, clonally inherited genomes is an unsatisfactory criterion for defining subspecies based upon theoretical (genetic drift) and practical (sampling effort) arguments. This point was illustrated by a re-analysis of a global mitogenomic assessment of fin whales, Balaenoptera physalus spp., published by Archer et al. (2013), which proposed to further subdivide the Northern Hemisphere fin whale subspecies, B. p. physalus. The proposed revision was based upon the detection of spatially distinct monophyly among North Atlantic and North Pacific fin whales in a genealogy based upon complete mitochondrial genome DNA sequences. The extended analysis conducted in this study (1676 mitochondrial control region, 162 complete mitochondrial genome DNA sequences and 20 microsatellite loci genotyped in 380 samples) revealed that the apparent monophyly among North Atlantic fin whales reported by Archer et al. (2013) to be due to low sample sizes. In conclusion, defining sub-species from monophyly (i.e., the absence of para- or polyphyly) can lead to erroneous conclusions due to relatively "trivial" aspects, such as sampling. Basic population genetic processes (i.e., genetic drift and migration) also affect the time to the most recent common ancestor and hence the probability that individuals in a sample are monophyletic.

Genomics meets applied ecology: Characterizing habitat quality for sloths in a tropical agroecosystem.

Understanding how habitat quality in heterogeneous landscapes governs the distribution and fitness of individuals is a fundamental aspect of ecology. While mean individual fitness is generally considered a key to assessing habitat quality, a comprehensive understanding of habitat quality in heterogeneous landscapes requires estimates of dispersal rates among habitat types. The increasing accessibility of genomic approaches, combined with field-based demographic methods, provides novel opportunities for incorporating dispersal estimation into assessments of habitat quality. In this study, we integrated genomic kinship approaches with field-based estimates of fitness components and approximate Bayesian computation (ABC) procedures to estimate habitat-specific dispersal rates and characterize habitat quality in two-toed sloths (Choloepus hoffmanni) occurring in a Costa Rican agricultural ecosystem. Field-based observations indicated that birth and survival rates were similar in a sparsely shaded cacao farm and adjacent cattle pasture-forest mosaic. Sloth density was threefold higher in pasture compared with cacao, whereas home range size and overlap were greater in cacao compared with pasture. Dispersal rates were similar between the two habitats, as estimated using ABC procedures applied to the spatial distribution of pairs of related individuals identified using 3,431 single nucleotide polymorphism and 11 microsatellite locus genotypes. Our results indicate that crops produced under a sparse overstorey can, in some cases, constitute lower-quality habitat than pasture-forest mosaics for sloths, perhaps because of differences in food resources or predator communities. Finally, our study demonstrates that integrating field-based demographic approaches with genomic methods can provide a powerful means for characterizing habitat quality for animal populations occurring in heterogeneous landscapes.

Shift of grey seal subspecies boundaries in response to climate, culling and conservation.

Identifying the processes that drive changes in the abundance and distribution of natural populations is a central theme in ecology and evolution. Many species of marine mammals have experienced dramatic changes in abundance and distribution due to climatic fluctuations and anthropogenic impacts. However, thanks to conservation efforts, some of these species have shown remarkable population recovery and are now recolonizing their former ranges. Here, we use zooarchaeological, demographic and genetic data to examine processes of colonization, local extinction and recolonization of the two northern European grey seal subspecies inhabiting the Baltic Sea and North Sea. The zooarchaeological and genetic data suggest that the two subspecies diverged shortly after the formation of the Baltic Sea approximately 4200 years bp, probably through a gradual shift to different breeding habitats and phenologies. By comparing genetic data from 19th century pre-extinction material with that from seals currently recolonizing their past range, we observed a marked spatiotemporal shift in subspecies boundaries, with increasing encroachment of North Sea seals on areas previously occupied by the Baltic Sea subspecies. Further, both demographic and genetic data indicate that the two subspecies have begun to overlap geographically and are hybridizing in a narrow contact zone. Our findings provide new insights into the processes of colonization, extinction and recolonization and have important implications for the management of grey seals across northern Europe.

Linking Genetic Kinship and Demographic Analyses to Characterize Dispersal: Methods and Application to Blanding's Turtle.

Characterizing how frequently, and at what life stages and spatial scales, dispersal occurs can be difficult, especially for species with cryptic juvenile periods and long reproductive life spans. Using a combination of mark-recapture information, microsatellite genetic data, and demographic simulations, we characterize natal and breeding dispersal patterns in the long-lived, slow-maturing, and endangered Blanding's turtle (Emydoidea blandingii), focusing on nesting females. We captured and genotyped 310 individual Blanding's turtles (including 220 nesting females) in a central Wisconsin population from 2010 to 2013, with additional information on movements among 3 focal nesting areas within this population available from carapace-marking conducted from 2001 to 2009. Mark-recapture analyses indicated that dispersal among the 3 focal nesting areas was infrequent (<0.03 annual probability). Dyads of females with inferred first-order relationships were more likely to be found within the same nesting area than split between areas, and the proportion of related dyads declined with increasing distance among nesting areas. The observed distribution of related dyads for nesting females was consistent with a probability of natal dispersal at first breeding between nearby nesting areas of approximately 0.1 based on demographic simulations. Our simulation-based estimates of infrequent female dispersal were corroborated by significant spatial genetic autocorrelation among nesting females at scales of <500 m. Nevertheless, a lack of spatial genetic autocorrelation among non-nesting turtles (males and females) suggested extensive local connectivity, possibly mediated by male movements or long-distance movements made by females between terrestrial nesting areas and aquatic habitats. We show here that coupling genetic and demographic information with simulations of individual-based population models can be an effective approach for untangling the contributions of natal and breeding dispersal to spatial ecology.

Inferring recent historic abundance from current genetic diversity.

Recent historic abundance is an elusive parameter of great importance for conserving endangered species and understanding the pre-anthropogenic state of the biosphere. The number of studies that have used population genetic theory to estimate recent historic abundance from contemporary levels of genetic diversity has grown rapidly over the last two decades. Such assessments often yield unexpectedly large estimates of historic abundance. We review the underlying theory and common practices of estimating recent historic abundance from contemporary genetic diversity, and critically evaluate the potential issues at various estimation steps. A general issue of mismatched spatio-temporal scales between the estimation itself and the objective of the estimation emerged from our assessment; genetic diversity-based estimates of recent historic abundance represent long-term averages, whereas the objective typically is an estimate of recent abundance for a specific population. Currently, the most promising approach to estimate the difference between recent historic and contemporary abundance requires that genetic data be collected from samples of similar spatial and temporal duration. Novel genome-enabled inference methods may be able to utilize additional information of dense genome-wide distributions of markers, such as of identity-by-descent tracts, to infer recent historic abundance from contemporary samples only.

PHFinder: assisted detection of point heteroplasmy in Sanger sequencing chromatograms.

Heteroplasmy is the presence of two or more organellar genomes (mitochondrial or plastid DNA) in an organism, tissue, cell or organelle. Heteroplasmy can be detected by visual inspection of Sanger sequencing chromatograms, where it appears as multiple peaks of fluorescence at a single nucleotide position. Visual inspection of chromatograms is both consuming and highly subjective, as heteroplasmy is difficult to differentiate from background noise. Few software solutions are available to automate the detection of point heteroplasmies, and those that are available are typically proprietary, lack customization or are unsuitable for automated heteroplasmy assessment in large datasets. Here, we present PHFinder, a Python-based, open-source tool to assist in the detection of point heteroplasmies in large numbers of Sanger chromatograms. PHFinder automatically identifies point heteroplasmies directly from the chromatogram trace data. The program was tested with Sanger sequencing data from 100 humpback whales (Megaptera novaeangliae) tissue samples with known heteroplasmies. PHFinder detected most (90%) of the known heteroplasmies thereby greatly reducing the amount of visual inspection required. PHFinder is flexible and enables explicit specification of key parameters to infer double peaks (i.e., heteroplasmies).

Wild pedigrees inform mutation rates and historic abundance in baleen whales.

Phylogeny-based estimates suggesting a low germline mutation rate (µ) in baleen whales have influenced research ranging from assessments of whaling impacts to evolutionary cancer biology. We estimated µ directly from pedigrees in four baleen whale species for both the mitochondrial control region and nuclear genome. The results suggest values higher than those obtained through phylogeny-based estimates and similar to pedigree-based values for primates and toothed whales. Applying our estimate of µ reduces previous genetic-based estimates of preexploitation whale abundance by 86% and suggests that µ cannot explain low cancer rates in gigantic mammals. Our study shows that it is feasible to estimate µ directly from pedigrees in natural populations, with wide-ranging implications for ecological and evolutionary research.

Reliability of genetic bottleneck tests for detecting recent population declines.

The identification of population bottlenecks is critical in conservation because populations that have experienced significant reductions in abundance are subject to a variety of genetic and demographic processes that can hasten extinction. Genetic bottleneck tests constitute an appealing and popular approach for determining if a population decline has occurred because they only require sampling at a single point in time, yet reflect demographic history over multiple generations. However, a review of the published literature indicates that, as typically applied, microsatellite-based bottleneck tests often do not detect bottlenecks in vertebrate populations known to have experienced declines. This observation was supported by simulations that revealed that bottleneck tests can have limited statistical power to detect bottlenecks largely as a result of limited sample sizes typically used in published studies. Moreover, commonly assumed values for mutation model parameters do not appear to encompass variation in microsatellite evolution observed in vertebrates and, on average, the proportion of multi-step mutations is underestimated by a factor of approximately two. As a result, bottleneck tests can have a higher probability of 'detecting' bottlenecks in stable populations than expected based on the nominal significance level. We provide recommendations that could add rigor to inferences drawn from future bottleneck tests and highlight new directions for the characterization of demographic history.

Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods.

BACKGROUND: Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. RESULTS: Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. CONCLUSION: Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments.