Genetic load: genomic estimates and applications in non-model animals.

Genetic variation, which is generated by mutation, recombination and gene flow, can reduce the mean fitness of a population, both now and in the future. This 'genetic load' has been estimated in a wide range of animal taxa using various approaches. Advances in genome sequencing and computational techniques now enable us to estimate the genetic load in populations and individuals without direct fitness estimates. Here, we review the classic and contemporary literature of genetic load. We describe approaches to quantify the genetic load in whole-genome sequence data based on evolutionary conservation and annotations. We show that splitting the load into its two components - the realized load (or expressed load) and the masked load (or inbreeding load) - can improve our understanding of the population genetics of deleterious mutations.

Tracing Eastern Wolf Origins From Whole-Genome Data in Context of Extensive Hybridization.

Southeastern Canada is inhabited by an amalgam of hybridizing wolf-like canids, raising fundamental questions regarding their taxonomy, origins, and timing of hybridization events. Eastern wolves (Canis lycaon), specifically, have been the subject of significant controversy, being viewed as either a distinct taxonomic entity of conservation concern or a recent hybrid of coyotes (C. latrans) and grey wolves (C. lupus). Mitochondrial DNA analyses show some evidence of eastern wolves being North American evolved canids. In contrast, nuclear genome studies indicate eastern wolves are best described as a hybrid entity, but with unclear timing of hybridization events. To test hypotheses related to these competing findings we sequenced whole genomes of 25 individuals, representative of extant Canadian wolf-like canid types of known origin and levels of contemporary hybridization. Here we present data describing eastern wolves as a distinct taxonomic entity that evolved separately from grey wolves for the past ∼67,000 years with an admixture event with coyotes ∼37,000 years ago. We show that Great Lakes wolves originated as a product of admixture between grey wolves and eastern wolves after the last glaciation (∼8,000 years ago) while eastern coyotes originated as a product of admixture between "western" coyotes and eastern wolves during the last century. Eastern wolf nuclear genomes appear shaped by historical and contemporary gene flow with grey wolves and coyotes, yet evolutionary uniqueness remains among eastern wolves currently inhabiting a restricted range in southeastern Canada.

Relaxation of Natural Selection in the Evolution of the Giant Lungfish Genomes.

Nonadaptive hypotheses on the evolution of eukaryotic genome size predict an expansion when the process of purifying selection becomes weak. Accordingly, species with huge genomes, such as lungfish, are expected to show a genome-wide relaxation signature of selection compared with other organisms. However, few studies have empirically tested this prediction using genomic data in a comparative framework. Here, we show that 1) the newly assembled transcriptome of the Australian lungfish, Neoceratodus forsteri, is characterized by an excess of pervasive transcription, or transcriptional leakage, possibly due to suboptimal transcriptional control, and 2) a significant relaxation signature in coding genes in lungfish species compared with other vertebrates. Based on these observations, we propose that the largest known animal genomes evolved in a nearly neutral scenario where genome expansion is less efficiently constrained.

Unravelling the mystery of endemic versus translocated populations of the endangered Australian lungfish (Neoceratodus forsteri).

The Australian lungfish is a primitive and endangered representative of the subclass Dipnoi. The distribution of this species is limited to south-east Queensland, with some populations considered endemic and others possibly descending from translocations in the late nineteenth century shortly after European discovery. Attempts to resolve the historical distribution of this species have met with conflicting results based on descriptive genetic studies. Understanding if all populations are endemic or some are the result of, or influenced by, translocation events, has implications for conservation management. In this work, we analysed the genetic variation at three types of markers (mtDNA genomes, 11 STRs and 5196 nuclear SNPs) using the approximate Bayesian computation (ABC) algorithm to compare several demographic models. We postulated different contributions of Mary River and Burnett River gene pools into the Brisbane River and North Pine River populations, related to documented translocation events. We ran the analysis for each marker type separately, and we also estimated the posterior probabilities of the models combining the markers. Nuclear SNPs have the highest power to correctly identify the true model among the simulated datasets (where the model was known), but different marker types typically provided similar answers. The most supported demographic model able to explain the real dataset implies that an endemic gene pool is still present in the Brisbane and North Pine Rivers and coexists with the gene pools derived from past documented translocation events. These results support the view that ABC modelling can be useful to reconstruct complex historical translocation events with contemporary implications, and will inform ongoing conservation efforts for the endangered and iconic Australian lungfish.

Evidence of backcross inviability and mitochondrial DNA paternal leakage in sea turtle hybrids.

Hybridization is known to be part of many species' evolutionary history. Sea turtles have a fascinating hybridization system in which species separated by as much as 43 million years are still capable of hybridizing. Indeed, the largest nesting populations in Brazil of loggerheads (Caretta caretta) and hawksbills (Eretmochelys imbricata) have a high incidence of hybrids between these two species. A third species, olive ridleys (Lepidochelys olivacea), is also known to hybridize although at a smaller scale. Here, we used restriction site-associated DNA sequencing (RAD-Seq) markers, mitogenomes, and satellite-telemetry to investigate the patterns of hybridization and introgression in the Brazilian sea turtle population and their relationship with the migratory behaviours between feeding and nesting aggregations. We also explicitly test if the mixing of two divergent genomes in sea turtle hybrids causes mitochondrial paternal leakage. We developed a new species-specific PCR-assay capable of detecting mitochondrial DNA (mtDNA) inheritance from both parental species and performed ultra-deep sequencing to estimate the abundance of each mtDNA type. Our results show that all adult hybrids are first generation (F1) and most display a loggerhead migratory behaviour. We detected paternal leakage in F1 hybrids and different proportions of mitochondria from maternal and paternal species. Although previous studies showed no significant fitness decrease in hatchlings, our results support genetically-related hybrid breakdown possibly caused by cytonuclear incompatibility. Further research on hybrids from other populations in addition to Brazil and between different species will show if backcross inviability and mitochondrial paternal leakage is observed across sea turtle species.

Fitness consequences and ancestry loss in the Apennine brown bear after a simulated genetic rescue intervention.

Reduction in population size, with its predicted effects on population fitness, is the most alarming anthropogenic impact on endangered species. By introducing compatible individuals, genetic rescue (GR) is a promising but debated approach for reducing the genetic load unmasked by inbreeding and for restoring the fitness of declining populations. Although GR can improve genetic diversity and fitness, it can also produce loss of ancestry, hampering local adaptation, or replace with introduced variants the unique genetic pools evolved in endemic groups. We used forward genetic simulations based on empirical genomic data to assess fitness benefits and loss of ancestry risks of GR in the Apennine brown bear (Ursus arctos marsicanus). There are approximately 50 individuals of this isolated subspecies, and they have lower genetic diversity and higher inbreeding than other European brown bears, and GR has been suggested to reduce extinction risks. We compared 10 GR scenarios in which the number and genetic characteristics of migrants varied with a non-GR scenario of simple demographic increase due to nongenetic factors. The introduction of 5 individuals of higher fitness or lower levels of deleterious mutations than the target Apennine brown bear from a larger European brown bear population produced a rapid 10-20% increase in fitness in the subspecies and up to 22.4% loss of ancestry over 30 generations. Without a contemporary demographic increase, fitness started to decline again after a few generations. Doubling the population size without GR gradually increased fitness to a comparable level, but without losing ancestry, thus resulting in the best strategy for the Apennine brown bear conservation. Our results highlight the importance for management of endangered species of realistic forward simulations grounded in empirical whole-genome data.

Consecuencias en la aptitud y pérdida de ascendencia del oso pardo de los Apeninos después de un rescate genético simulado Resumen La reducción del tamaño poblacional, con los previsibles efectos sobre su aptitud, es el impacto antropogénico más alarmante sobre las especies amenazadas. Mediante la introducción de individuos compatibles, el rescate genético (RG) es una estrategia prometedora para reducir la carga genética revelada por la endogamia y restaurar la aptitud de las poblaciones en declive, aunque todavía se debate la eficiencia de esta. Aunque el RG puede mejorar la diversidad genética y la aptitud, también puede producir pérdida de ascendencia, lo que puede dificultar la adaptación local, o sustituir con variantes introducidas por los migrantes los acervos genéticos únicos que han evolucionado en grupos endémicos. En este trabajo realizamos simulaciones genéticas a futuro basadas en datos genómicos empíricos para evaluar los beneficios del RG en términos de aptitud y los riesgos de la pérdida de ascendencia en el oso pardo de los Apeninos (Ursus arctos marsicanus). Quedan aproximadamente 50 individuos de esta subespecie aislada que cuentan con una menor diversidad genética y un mayor nivel de endogamia comparado con otros osos pardos europeos y se ha sugerido que el RG podria reducir el riesgo de extinción de esta población. Comparamos 10 escenarios de RG en los que variaban el número y las características genéticas de los osos migrantes con un escenario sin RG con aumento demográfico causado por factores no genéticos. La introducción de 5 individuos procedentes de una población europea de oso pardo con mayor aptitud o niveles menores de mutaciones deletéreas que el oso pardo de los Apeninos produjo un rápido aumento de la aptitud del 10-20% en la subespecie y hasta un 22.4% de pérdida de ascendencia durante 30 generaciones. En las simulaciones sin un aumento demográfico, la aptitud empezó a disminuir de nuevo después de unas pocas generaciones. La duplicación del tamaño de la población sin RG aumentó gradualmente la aptitud hasta un nivel comparable al de algunos escenarios de RG, pero sin pérdida de ascendencia, por lo que parece ser la mejor estrategia para la conservación del oso pardo de los Apeninos. Nuestros resultados resaltan la importancia que tienen las simulaciones realistas a futuro basadas en datos empíricos del genoma completo para la gestión de especies amenazadas.

A high-quality reference genome for the critically endangered Aeolian wall lizard, Podarcis raffonei.

The Aeolian wall lizard, Podarcis raffonei, is an endangered species endemic to the Aeolian archipelago, Italy, where it is present only in 3 tiny islets and a narrow promontory of a larger island. Because of the extremely limited area of occupancy, severe population fragmentation and observed decline, it has been classified as Critically Endangered by the International Union for the Conservation of Nature (IUCN). Using Pacific Biosciences (PacBio) High Fidelity (HiFi) long-read sequencing, Bionano optical mapping and Arima chromatin conformation capture sequencing (Hi-C), we produced a high-quality, chromosome-scale reference genome for the Aeolian wall lizard, including Z and W sexual chromosomes. The final assembly spans 1.51 Gb across 28 scaffolds with a contig N50 of 61.4 Mb, a scaffold N50 of 93.6 Mb, and a BUSCO completeness score of 97.3%. This genome constitutes a valuable resource for the species to guide potential conservation efforts and more generally for the squamate reptiles that are underrepresented in terms of available high-quality genomic resources.

Population Dynamics and Structural Effects at Short and Long Range Support the Hypothesis of the Selective Advantage of the G614 SARS-CoV-2 Spike Variant.

SARS-CoV-2 epidemics quickly propagated worldwide, sorting virus genomic variants in newly established propagules of infections. Stochasticity in transmission within and between countries or an actual selective advantage could explain the global high frequency reached by some genomic variants. Using statistical analyses, demographic reconstructions, and molecular dynamics simulations, we show that the globally invasive G614 spike variant 1) underwent a significant demographic expansion in most countries explained neither by stochastic effects nor by overrepresentation in clinical samples, 2) increases the spike S1/S2 furin-like site conformational plasticity (short-range effect), and 3) modifies the internal motion of the receptor-binding domain affecting its cross-connection with other functional domains (long-range effect). Our results support the hypothesis of a selective advantage at the basis of the spread of the G614 variant, which we suggest may be due to structural modification of the spike protein at the S1/S2 proteolytic site, and provide structural information to guide the design of variant-specific drugs.

Population structure, genomic diversity and demographic history of Komodo dragons inferred from whole-genome sequencing.

Population and conservation genetics studies have greatly benefited from the development of new techniques and bioinformatic tools associated with next-generation sequencing. Analysis of extensive data sets from whole-genome sequencing of even a few individuals allows the detection of patterns of fine-scale population structure and detailed reconstruction of demographic dynamics through time. In this study, we investigated the population structure, genomic diversity and demographic history of the Komodo dragon (Varanus komodoensis), the world's largest lizard, by sequencing the whole genomes of 24 individuals from the five main Indonesian islands comprising the entire range of the species. Three main genomic groups were observed. The populations of the Island of Komodo and the northern coast of Flores, in particular, were identified as two distinct conservation units. Degrees of genomic divergence among island populations were interpreted as a result of changes in sea level affecting connectivity across islands. Demographic inference suggested that Komodo dragons probably experienced a relatively steep population decline over the last million years, reaching a relatively stable Ne during the Saalian glacial cycle (400-150 thousand years ago) followed by a rapid Ne decrease. Genomic diversity of Komodo dragons was similar to that found in endangered or already extinct reptile species. Overall, this study provides an example of how whole-genome analysis of a few individuals per population can help define population structure and intraspecific demographic dynamics. This is particularly important when applying population genomics data to conservation of rare or elusive endangered species.

Divergence and hybridization in sea turtles: Inferences from genome data show evidence of ancient gene flow between species.

Reconstructing past events of hybridization and population size changes are required to understand speciation mechanisms and current patterns of genetic diversity, and ultimately contribute to species' conservation. Sea turtles are ancient species currently facing anthropogenic threats including climate change, fisheries, and illegal hunting. Five of the seven extant sea turtle species are known to currently hybridize, especially along the Brazilian coast where some populations can have ~32%-42% of hybrids. Although frequently observed today, it is not clear what role hybridization plays in the evolutionary diversification of this group of reptiles. In this study, we generated whole genome resequencing data of the five globally distributed sea turtle species to estimate a calibrated phylogeny and the population size dynamics, and to understand the role of hybridization in shaping the genomes of these ancient species. Our results reveal discordant species divergence dates between mitochondrial and nuclear genomes, with a high frequency of conflicting trees throughout the nuclear genome suggesting that some sea turtle species frequently hybridized in the past. The reconstruction of the species' demography showed a general decline in effective population sizes with no signs of recovery, except for the leatherback sea turtle. Furthermore, we discuss the influence of reference bias in our estimates. We show long-lasting ancestral gene flow events within Chelonioidea that continued for millions of years after initial divergence. Speciation with gene flow is a common pattern in marine species, and it raises questions whether current hybridization events should be considered as a part of these species' evolutionary history or a conservation issue.

Survival and divergence in a small group: The extraordinary genomic history of the endangered Apennine brown bear stragglers.

About 100 km east of Rome, in the central Apennine Mountains, a critically endangered population of ∼50 brown bears live in complete isolation. Mating outside this population is prevented by several 100 km of bear-free territories. We exploited this natural experiment to better understand the gene and genomic consequences of surviving at extremely small population size. We found that brown bear populations in Europe lost connectivity since Neolithic times, when farming communities expanded and forest burning was used for land clearance. In central Italy, this resulted in a 40-fold population decline. The overall genomic impact of this decline included the complete loss of variation in the mitochondrial genome and along long stretches of the nuclear genome. Several private and deleterious amino acid changes were fixed by random drift; predicted effects include energy deficit, muscle weakness, anomalies in cranial and skeletal development, and reduced aggressiveness. Despite this extreme loss of diversity, Apennine bear genomes show nonrandom peaks of high variation, possibly maintained by balancing selection, at genomic regions significantly enriched for genes associated with immune and olfactory systems. Challenging the paradigm of increased extinction risk in small populations, we suggest that random fixation of deleterious alleles (i) can be an important driver of divergence in isolation, (ii) can be tolerated when balancing selection prevents random loss of variation at important genes, and (iii) is followed by or results directly in favorable behavioral changes.

Phylogenomic proof of Recurrent Demipolyploidization and Evolutionary Stalling of the "Triploid Bridge" in Arundo (Poaceae).

Polyploidization is a frequent phenomenon in plants, which entails the increase from one generation to the next by multiples of the haploid number of chromosomes. While tetraploidization is arguably the most common and stable outcome of polyploidization, over evolutionary time triploids often constitute only a transient phase, or a "triploid bridge", between diploid and tetraploid levels. In this study, we reconstructed in a robust phylogenomic and statistical framework the evolutionary history of polyploidization in Arundo, a small genus from the Poaceae family with promising biomass, bioenergy and phytoremediation species. Through the obtainment of 10 novel leaf transcriptomes for Arundo and outgroup species, our results prove that recurrent demiduplication has likely been a major driver of evolution in this species-poor genus. Molecular dating further demonstrates that the species originating by demiduplication stalled in the "triploid bridge" for evolutionary times in the order of millions of years without undergoing tetratploidization. Nevertheless, we found signatures of molecular evolution highlighting some of the processes that accompanied the genus radiation. Our results clarify the complex nature of Arundo evolution and are valuable for future gene functional validation as well as reverse and comparative genomics efforts in the Arundo genus and other Arundinoideae.

Computer simulations: tools for population and evolutionary genetics.

Computer simulations are excellent tools for understanding the evolutionary and genetic consequences of complex processes whose interactions cannot be analytically predicted. Simulations have traditionally been used in population genetics by a fairly small community with programming expertise, but the recent availability of dozens of sophisticated, customizable software packages for simulation now makes simulation an accessible option for researchers in many fields. The in silico genetic data produced by simulations, along with greater availability of population-genomics data, are transforming genetic epidemiology, anthropology, evolutionary and population genetics and conservation. In this Review of the state-of-the-art of simulation software, we identify applications of simulations, evaluate simulator capabilities, provide a guide for their use and summarize future directions.

Decreased Nucleotide and Expression Diversity and Modified Coexpression Patterns Characterize Domestication in the Common Bean.

Using RNA sequencing technology and de novo transcriptome assembly, we compared representative sets of wild and domesticated accessions of common bean (Phaseolus vulgaris) from Mesoamerica. RNA was extracted at the first true-leaf stage, and de novo assembly was used to develop a reference transcriptome; the final data set consists of ∼190,000 single nucleotide polymorphisms from 27,243 contigs in expressed genomic regions. A drastic reduction in nucleotide diversity (∼60%) is evident for the domesticated form, compared with the wild form, and almost 50% of the contigs that are polymorphic were brought to fixation by domestication. In parallel, the effects of domestication decreased the diversity of gene expression (18%). While the coexpression networks for the wild and domesticated accessions demonstrate similar seminal network properties, they show distinct community structures that are enriched for different molecular functions. After simulating the demographic dynamics during domestication, we found that 9% of the genes were actively selected during domestication. We also show that selection induced a further reduction in the diversity of gene expression (26%) and was associated with 5-fold enrichment of differentially expressed genes. While there is substantial evidence of positive selection associated with domestication, in a few cases, this selection has increased the nucleotide diversity in the domesticated pool at target loci associated with abiotic stress responses, flowering time, and morphology.

Bayesian inferences suggest that Amazon Yunga Natives diverged from Andeans less than 5000 ybp: implications for South American prehistory.

BACKGROUND: Archaeology reports millenary cultural contacts between Peruvian Coast-Andes and the Amazon Yunga, a rainforest transitional region between Andes and Lower Amazonia. To clarify the relationships between cultural and biological evolution of these populations, in particular between Amazon Yungas and Andeans, we used DNA-sequence data, a model-based Bayesian approach and several statistical validations to infer a set of demographic parameters. RESULTS: We found that the genetic diversity of the Shimaa (an Amazon Yunga population) is a subset of that of Quechuas from Central-Andes. Using the Isolation-with-Migration population genetics model, we inferred that the Shimaa ancestors were a small subgroup that split less than 5300 years ago (after the development of complex societies) from an ancestral Andean population. After the split, the most plausible scenario compatible with our results is that the ancestors of Shimaas moved toward the Peruvian Amazon Yunga and incorporated the culture and language of some of their neighbors, but not a substantial amount of their genes. We validated our results using Approximate Bayesian Computations, posterior predictive tests and the analysis of pseudo-observed datasets. CONCLUSIONS: We presented a case study in which model-based Bayesian approaches, combined with necessary statistical validations, shed light into the prehistoric demographic relationship between Andeans and a population from the Amazon Yunga. Our results offer a testable model for the peopling of this large transitional environmental region between the Andes and the Lower Amazonia. However, studies on larger samples and involving more populations of these regions are necessary to confirm if the predominant Andean biological origin of the Shimaas is the rule, and not the exception.

Geography has more influence than language on maternal genetic structure of various northeastern Thai ethnicities.

Several literatures have shown the influence of geographic and linguistic factors in shaping genetic variation patterns, but their relative impact, if any, in the very heterogeneous northeastern region of Thailand has not yet been studied. This area, called Isan, is geographically structured in two wide basins, the Sakon Nakorn Basin and the Korat Basin, serving today as home to diverse ethnicities encompassing two different linguistic families, that is, the Austro-Asiatic; Suay (Kui), Mon, Chaobon (Nyahkur), So and Khmer, and the Tai-Kadai; Saek, Nyaw, Phu Tai, Kaleung and Lao Isan. In this study, we evaluated the relative role of geographic distance and barriers as well as linguistic differences as possible causes affecting the maternal genetic distances among northeastern Thai ethnicities. A 596-bp segment of the hypervariable region I mitochondrial DNA was utilized to elucidate the genetic structure and biological affinity from 433 individuals. Different statistical analyses agreed in suggesting that most ethnic groups in the Sakon Nakorn Basin are closely related. Mantel test revealed that genetic distances were highly associated to geographic (r = 0.445, P<0.01) but not to linguistic (r = 0.001, P>0.01) distances. Three evolutionary models were compared by Approximate Bayesian Computation. The posterior probability of the scenario, which assumed an initial population divergence possibly related to reduced gene flow among basins, was equal or higher than 0.87. All analyses exhibited concordant results supporting that geography was the most relevant factor in determining the maternal genetic structure of northeastern Thai populations.

Genetic variation and population structure in the endangered Hermann's tortoise: the roles of geography and human-mediated processes.

The Hermann's tortoise (Testudo hermanni) is an endangered land tortoise distributed in disjoint populations across Mediterranean Europe. We investigated its genetic variation by typing 1 mitochondrial locus and 9 nuclear microsatellites in approximately 300 individuals from 22 localities. Our goal was to understand the relative impact of natural and human-mediated processes in shaping the genetic structure and to identify the genetic priorities for the conservation of this species. We found that 1) all geographic areas are highly differentiated, mainly as a function of their distance but with a clear genetic discontinuity (F st values larger than 0.4) between the Eastern and the Western subspecies; 2) the contact zone between subspecies is located farthest to the west than previously believed, and it probably coincides with the delta of the largest Italian river; 3) extinction events due to climatic conditions in the Upper Palaeolithic and subsequent human-mediated translocations in the Neolithic possibly explain the unexpected similarity among Spain, Sicily, and Corsica. For conservation purposes, the large majority of genetic pools appears native although hybridization among subspecies, related to extensive 20th century trade of tortoises across Europe, is observed in Spain and some Italian samples. Most populations do not seem at immediate risk of low genetic variation, except the French population, which has very low nuclear genetic diversity (heterozygosity = 0.25) and where 50 out of 51 sampled animals shared the same mitochondrial sequence. In general, restocking and reintroduction plans should carefully consider the genetic background of the individuals.

High variance in reproductive success generates a false signature of a genetic bottleneck in populations of constant size: a simulation study.

BACKGROUND: Demographic bottlenecks can severely reduce the genetic variation of a population or a species. Establishing whether low genetic variation is caused by a bottleneck or a constantly low effective number of individuals is important to understand a species' ecology and evolution, and it has implications for conservation management. Recent studies have evaluated the power of several statistical methods developed to identify bottlenecks. However, the false positive rate, i.e. the rate with which a bottleneck signal is misidentified in demographically stable populations, has received little attention. We analyse this type of error (type I) in forward computer simulations of stable populations having greater than Poisson variance in reproductive success (i.e., variance in family sizes). The assumption of Poisson variance underlies bottleneck tests, yet it is commonly violated in species with high fecundity. RESULTS: With large variance in reproductive success (Vk ≥ 40, corresponding to a ratio between effective and census size smaller than 0.1), tests based on allele frequencies, allelic sizes, and DNA sequence polymorphisms (heterozygosity excess, M-ratio, and Tajima's D test) tend to show erroneous signals of a bottleneck. Similarly, strong evidence of population decline is erroneously detected when ancestral and current population sizes are estimated with the model based method MSVAR. CONCLUSIONS: Our results suggest caution when interpreting the results of bottleneck tests in species showing high variance in reproductive success. Particularly in species with high fecundity, computer simulations are recommended to confirm the occurrence of a population bottleneck.

The number of markers and samples needed for detecting bottlenecks under realistic scenarios, with and without recovery: a simulation-based study.

Detecting bottlenecks is a common task in molecular ecology. While several bottleneck detection method sexist, evaluations of their power have focused only on severe bottlenecks (e.g. to Ne ~10). As a component of a recent review, Peery et al. (2012) analysed the power of two approaches, the M-ratio and heterozygote excess tests, to detect moderate bottlenecks (e.g. to Ne ~100),which is realistic for many conservation situations. In this Comment, we address three important points relevant to but not considered in Peery et al. Under moderate bottleneck scenarios, we test the (i) relative advantage of sampling more markers vs. more individuals, (ii) potential power to detect the bottleneck when utilizing dozens of microsatellites (a realistic possibility for contemporary studies) and (iii) reduction in power when post bottle neck recovery has occurred. For the realistic situations examined,we show that (i) doubling the number of loci shows equal or better power than tripling the number of individuals,(ii) increasing the number of markers (up to 100) results in continued additive gains in power, and (iii)recovery after a moderate amount of time or gradual change in size reduces power, by up to one-half. Our results provide a practical supplement to Peery et al. and encourage the continued use of bottleneck detection methods in the genomic age, but also emphasize that the power under different sampling schemes should be estimated,using simulation modelling, as a routine component of molecular ecology studies.