Gary R. Carvalho

Molecular genetics and the stock concept in fisheries

The concept of a sustainable yield (SY, Gulland, 1983; Lannan et al., 1989) has dominated fisheries management for almost 50 years. The central idea is that each stock has a harvestable surplus, and that fisheries that do not exceed this will not compromise the stock’s natural perpetuation. A basic assumption is that the fishery targets a unit stock with definable patterns of recruitment and mortality. Although it is difficult to reach agreement on what constitutes a stock (Gauldie, 1991), the notion of population units with varying degrees of temporal or spatial integrity stimulated a quest to characterize and identify such assemblages. It became apparent that few species form single homogeneous populations, but rather that fish species are often composed of discrete stocks, and that these stocks may react to harvesting more or less independently. It was the idea of independent responses of different stocks to exploitation that demanded information on stock structure. Hence, the stock concept was linked strongly, at least in theory, with the desire to balance the impacts of harvesting with efforts to ensure continued economic returns. What was much more difficult to achieve than the theory was: (i) how to actually recognize a stock, and (ii) how to translate stock structure data into fishery practices.

Loss of microsatellite diversity and low effective population size in an overexploited population of New Zealand snapper (Pagrus auratus)

Although the effects of overfishing on species diversity and abundance are well documented, threats to the genetic diversity of marine fish populations have so far been largely neglected. Indeed, there seems to be little cause for concern, as even “collapsed” stocks usually consist of several million individuals, whereas population genetics theory suggests that only very small populations suffer significant loss of genetic diversity. On the other hand, in many marine species the genetically effective population size (Ne), which determines the genetic properties of a population, may be orders of magnitude smaller than the census population size (N). Here, microsatellite analyses of a time series of archived scales demonstrated a significant decline in genetic diversity in a New Zealand snapper population during its exploitation history. Effective population sizes estimated both from the decline in heterozygosity and from temporal fluctuations in allele frequency were five orders of magnitude smaller than census population sizes from fishery data. If such low Ne/N ratios are commonplace in marine species, many exploited marine fish stocks may be in danger of losing genetic variability, potentially resulting in reduced adaptability, population persistence, and productivity.

SPECIATION IN ANCIENT CRYPTIC SPECIES COMPLEXES: EVIDENCE FROM THE MOLECULAR PHYLOGENY OF BRACHIONUS PLICATILIS (ROTIFERA)

Abstract Continental lake‐dwelling zooplanktonic organisms have long been considered cosmopolitan species with little geographic variation in spite of the isolation of their habitats. Evidence of morphological cohesiveness and high dispersal capabilities support this interpretation. However, this view has been challenged recently as many such species have been shown either to comprise cryptic species complexes or to exhibit marked population genetic differentiation and strong phylogeographic structuring at a regional scale. Here we investigate the molecular phylogeny of the cosmopolitan passively dispersing rotifer Brachionus plicatilis (Rotifera: Monogononta) species complex using nu‐cleotide sequence variation from both nuclear (ribosomal internal transcribed spacer 1, ITS1) and mitochondrial (cytochrome c oxidase subunit I, COI) genes. Analysis of rotifer resting eggs from 27 salt lakes in the Iberian Peninsula plus lakes from four continents revealed nine genetically divergent lineages. The high level of sequence divergence, absence of hybridization, and extensive sympatry observed support the specific status of these lineages. Sequence divergence estimates indicate that the B. plicatilis complex began diversifying many millions of years ago, yet has showed relatively high levels of morphological stasis. We discuss these results in relation to the ecology and genetics of aquatic invertebrates possessing dispersive resting propagules and address the apparent contradiction between zooplanktonic population structure and their morphological stasis.

Environmental DNA for wildlife biology and biodiversity monitoring

Extraction and identification of DNA from an environmental sample has proven noteworthy recently in detecting and monitoring not only common species, but also those that are endangered, invasive, or elusive. Particular attributes of so-called environmental DNA (eDNA) analysis render it a potent tool for elucidating mechanistic insights in ecological and evolutionary processes. Foremost among these is an improved ability to explore ecosystem-level processes, the generation of quantitative indices for analyses of species, community diversity, and dynamics, and novel opportunities through the use of time-serial samples and unprecedented sensitivity for detecting rare or difficult-to-sample taxa. Although technical challenges remain, here we examine the current frontiers of eDNA, outline key aspects requiring improvement, and suggest future developments and innovations for research.

Application of SNPs for population genetics of nonmodel organisms: new opportunities and challenges

Recent improvements in the speed, cost and accuracy of next generation sequencing are revolutionizing the discovery of single nucleotide polymorphisms (SNPs). SNPs are increasingly being used as an addition to the molecular ecology toolkit in nonmodel organisms, but their efficient use remains challenging. Here, we discuss common issues when employing SNP markers, including the high numbers of markers typically employed, the effects of ascertainment bias and the inclusion of nonneutral loci in a marker panel. We provide a critique of considerations specifically associated with the application and population genetic analysis of SNPs in nonmodel taxa, focusing specifically on some of the most commonly applied methods.

Ultrasequencing of the meiofaunal biosphere: practice, pitfalls and promises

Biodiversity assessment is the key to understanding the relationship between biodiversity and ecosystem functioning, but there is a well‐acknowledged biodiversity identification gap related to eukaryotic meiofaunal organisms. Meiofaunal identification is confounded by the small size of taxa, morphological convergence and intraspecific variation. However, the most important restricting factor in meiofaunal ecological research is the mismatch between diversity and the number of taxonomists that are able to simultaneously identify and catalogue meiofaunal diversity. Accordingly, a molecular operational taxonomic unit (MOTU)‐based approach has been advocated for en mass meiofaunal biodiversity assessment, but it has been restricted by the lack of throughput afforded by chain termination sequencing. Contemporary pyrosequencing offers a solution to this problem in the form of environmental metagenetic analyses, but this represents a novel field of biodiversity assessment. Here, we provide an overview of meiofaunal metagenetic analyses, ranging from sample preservation and DNA extraction to PCR, sequencing and the bioinformatic interrogation of multiple, independent samples using 454 Roche sequencing platforms. We report two examples of environmental metagenetic nuclear small subunit 18S (nSSU) analyses of marine and tropical rainforest habitats and provide critical appraisals of the level of putative recombinant DNA molecules (chimeras) in metagenetic data sets. Following stringent quality control measures, environmental metagenetic analyses achieve MOTU formation across the eukaryote domain of life at a fraction of the time and cost of traditional approaches. The effectiveness of Roche 454 sequencing brings substantial advantages to studies aiming to elucidate the molecular genetic richness of not only meiofaunal, but also all complex eukaryotic communities.

Single‐nucleotide polymorphism (SNP) discovery and applications of SNP genotyping in nonmodel organisms

A supplemental issue on the topic of single-nucleotidepolymorphism-enabled (SNP) research in nonmodel organisms is especially timely. In this issue, organisms with reference genomes are considered to be ‘model’; ‘nonmodel’ organisms are those whose genomes are yet to be sequenced. Advances in DNA sequencing and SNP genotyping have provided profound insights into the genetics of model organisms, but until recently, studies of nonmodel species lagged behind because of the scarcity of sequence and markers (see Fig. 1). In the past year, Tautz et al. (2010) and associated papers in supplemental issue of Molecular Ecology described a revolutionary transition from studies of ‘molecular ecology’ to studies of ‘ecological genomics’. Concurrently, Allendorf et al. (2010) grappled with placing the new-found wealth of sequence and SNP information into a ‘conservation genomics’ context. This revolution in molecular genetics studies would have been difficult to forecast a few years ago. Molecular genetic studies provide exceptional insight into relationships, migration and evolution of natural populations (Morin et al. 2004). During the origins of molecular ecology, in the 1960s and 1970s, it became clear that techniques such as allozyme electrophoresis would provide a basic framework for understanding species interactions and adaptation and for conserving natural genetic variability (Utter et al. 1966, 1974; Avise et al. 1975). Technical limitations at the time restricted both the ability to explore the dynamics of genetic diversity in species exhibiting low levels of variation as well as the direct analysis of adaptive variation in the wild. During the following years, innovators began to dream of potential applications for conservation and management of economically exploited species that included using molecular markers to determine the population-of-origin of migrating animals (see papers in Ryman & Utter 1987; Waples & Aebersold 1990), an important focus of many papers in this issue. Recent decades were punctuated by improvements in molecular and statistical techniques that produced an array of tools relevant to ecological and evolutionary studies such as assignment tests, estimates of effective population size, fine-scale population structure, kinship analyses (e.g. Helyar et al. this issue; Waples & Waples this issue) and genome-wide surveys based upon an ever increasing resolution of individuals and populations. The advantages of genotyping polymorphic SNPs with high-throughput assays have created much interest (Vignal et al. 2002; Brumfield et al. 2003; Morin et al. 2004; Schlötterer 2004). Until recently, however, the scarcity of available DNA sequence data for nonmodel species limited marker development. Further, because of comparatively low mutation rates, cross-species amplification of primers for SNP analyses did not yield the same results as for microsatellites. For example, Miller et al. (2010) tested the OvineSNP50 BeadChip, developed for domestic sheep, in two related ungulates and found only about 1% of the nearly 50 000 SNP loci to be polymorphic. Therefore, the SNP assays or probes developed for one species were not likely to be useful in others, even though primers may cross-amplify. The current supplemental issue contains 22 papers that underline the advantages of SNPs, advocate the need for SNP research in nonmodel organisms, and chart advances in discovery and applications. Although progress is apparent across a broad array of taxa, most papers presented here focus upon species of fish. This outcome, beyond the bias of the workshop organizers, may be in part because of the well-developed multinational collaborations that coordinate the sharing of DNA Correspondence: J. E. Seeb, Fax: (206) 543 5728; E-mail: jseeb@uw.edu

Temporal analysis of archived samples indicates marked genetic changes in declining North Sea cod (Gadus morhua)

Despite increasing evidence that current exploitation rates can contribute to shifts in life–history traits and the collapse of marine fish stocks, few empirical studies have investigated the likely evolutionary impacts. Here, we used DNA recovered from a temporal series of archived North Sea cod (Gadus morhua) otoliths, to investigate genetic diversity within the Flamborough Head population between 1954 and 1998, during which time the population underwent two successive declines. Microsatellite data indicated a significant reduction in genetic diversity between 1954 and 1970 (total number of alleles: 1954, 46; 1960, 42; 1970, 37), and a subsequent recovery between 1970 and 1998 (total number of alleles: 1970, 37; 1981, 42; 1998, 45). Furthermore, estimates of genetic differentiation (F and R) showed a significant divergence between 1998 and earlier samples. Data are consistent with a period of prolonged genetic drift, accompanied by a replacement of the Flamborough Head population through an increased effective migration rate that occurred during a period of high exploitation and appreciable demographic and phenotypic change. Other studies indicate that diversity at neutral microsatellite loci may be correlated with variability at selected genes, thus compromising a populations subsequent recovery and adaptive potential. Such effects are especially pertinent to North Sea cod, which are threatened by continuing exploitation and rising sea temperatures.

Power for detecting genetic divergence: differences between statistical methods and marker loci

Information on statistical power is critical when planning investigations and evaluating empirical data, but actual power estimates are rarely presented in population genetic studies. We used computer simulations to assess and evaluate power when testing for genetic differentiation at multiple loci through combining test statistics or P values obtained by four different statistical approaches, viz. Pearsons chi‐square, the log‐likelihood ratio G‐test, Fishers exact test, and an FST‐based permutation test. Factors considered in the comparisons include the number of samples, their size, and the number and type of genetic marker loci. It is shown that power for detecting divergence may be substantial for frequently used sample sizes and sets of markers, also at quite low levels of differentiation. The choice of statistical method may be critical, though. For multi‐allelic loci such as microsatellites, combining exact P values using Fishers method is robust and generally provides a high resolving power. In contrast, for few‐allele loci (e.g. allozymes and single nucleotide polymorphisms) and when making pairwise sample comparisons, this approach may yield a remarkably low power. In such situations chi‐square typically represents a better alternative. The G‐test without Williamss correction frequently tends to provide an unduly high proportion of false significances, and results from this test should be interpreted with great care. Our results are not confined to population genetic analyses but applicable to contingency testing in general.

Biocomplexity in a highly migratory pelagic marine fish, Atlantic herring

The existence of biologically differentiated populations has been credited with a major role in conferring sustainability and in buffering overall productivity of anadromous fish population complexes where evidence for spatial structure is uncontroversial. Here, we describe evidence of correlated genetic and life history (spawning season linked to spawning location) differentiation in an abundant and highly migratory pelagic fish, Atlantic herring, Clupea harengus, in the North Sea (NS) and adjacent areas. The existence of genetically and phenotypically diverse stocks in this region despite intense seasonal mixing strongly implicates natal homing in this species. Based on information from genetic markers and otolith morphology, we estimate the proportional contribution by NS, Skagerrak (SKG) and Kattegat and western Baltic (WBS) fish to mixed aggregations targeted by the NS fishery. We use these estimates to identify spatial and temporal differences in life history (migratory behaviour) and habitat use among genetically differentiated migratory populations that mix seasonally. Our study suggests the existence of more complex patterns of intraspecific diversity than was previously recognized. Sustainability may be compromised if such complex patterns are reduced through generalized management (e.g. area closures) that overlooks population differences in spatial use throughout the life cycle.