Metabarcoding has been used in a range of ecological applications such as taxonomic assignment, dietary analysis and the analysis of environmental DNA. However, after a decade of use in these applications there is little consensus on the extent to which proportions of reads generated corresponds to the original proportions of species in a community. To quantify our current understanding, we conducted a structured review and meta‐analysis. The analysis suggests that a weak quantitative relationship may exist between the biomass and sequences produced (slope = 0.52 ± 0.34, p < 0.01), albeit with a large degree of uncertainty. None of the tested moderators, sequencing platform type, the number of species used in a trial or the source of DNA, were able to explain the variance. Our current understanding of the factors affecting the quantitative performance of metabarcoding is still limited: additional research is required before metabarcoding can be confidently utilized for quantitative applications. Until then, we advocate the inclusion of mock communities when metabarcoding as this facilitates direct assessment of the quantitative ability of any given study.

Otolith microchemistry can provide valuable information about stock structure and mixing patterns when the magnitude of environmental differences among areas is greater than the cumulative influence of any vital effects. Here, the current understanding of the underlying mechanisms governing element incorporation into the otolith is reviewed. Hard and soft acid and base (HSAB) theory is employed to explore the differences in chemical behaviours, distributions and affinities between elements. Hard acid cations (e.g. Mg(2+) , Li(+) and Ba(2+) ) tend to be less physiologically influenced and accepted more readily into the otolith crystal lattice but are relatively homogeneous in seawater. Soft acid cations (e.g. Zn(2+) and Cu(2+) ) on the other hand, exhibit more varied distributions in seawater, but are more likely to be bound to blood proteins and less available for uptake into the otolith. The factors influencing the geographical distribution of elements in the sea, and their incorporation into the otoliths of marine fishes are reviewed. Particular emphasis is placed on examining physiological processes, including gonad development, on the uptake of elements commonly used in population studies, notably Sr. Finally, case studies are presented that either directly or indirectly compare population structuring or movements inferred by otolith elemental fingerprints with the patterns indicated by additional, alternative proxies. The main obstacle currently limiting the application of otolith elemental microchemistry to infer movements of marine fishes appears to lie in the largely homogeneous distribution of those elements most reliably measured in the otolith. Evolving technologies will improve the discriminatory power of otolith chemistry by allowing measurement of spatially explicit, low level elements; however, for the time being, the combination of otolith minor and trace element fingerprints with alternative proxies and stable isotopic ratios can greatly extend the scope of migration studies. Among the otolith elements that routinely occur above instrument detection limits, Ba, Mn and Li were deemed the most likely to prove reliable geographic markers in marine species.

Summary1. Trace element concentrations in fish earstones ('otoliths') are widely used to discriminate spatially discrete populations or individuals of marine fish, based on a commonly held assumption that physiological influences on otolith composition are minor, and thus variations in otolith elemental chemistry primarily reflect changes in ambient water chemistry. 2. We carried out a long-term (1-year) experiment, serially sampling seawater, blood plasma and otoliths of mature and immature European plaice (Pleuronectes platessa L.) to test relationships between otolith chemistry and environmental and physiological variables. 3. Seasonal variations in otolith elemental composition did not track seawater concentrations, but instead reflected physiological controls on metal transport and biokinetics, which are likely moderated by ambient temperature. The influence of physiological factors on otolith composition was particularly evident in Sr/Ca ratios, the most widely used elemental marker in applied otolith microchemistry studies. Reproduction also triggered specific variations in otolith and blood plasma metal chemistry, especially Zn/Ca ratios in female fish, which could potentially serve as retrospective spawning indicators. 4. The influence of physiology on the trace metal composition of otoliths may explain the success of microchemical stock discrimination in relatively homogenous marine environments, but could complicate alternative uses for trace element compositions in biominerals of higher organisms.

Most studies that infer geographic distributions of fish using otolith microchemistry assume that environmental factors (e.g. temperature, salinity) outweigh intrinsic effects (e.g. size, condition); however, this assumption has not been rigorously tested, particularly in marine fish. Here, we report the results of a long-term experimental study of European plaice Pleuronectes platessa L. and explore relationships between blood plasma and ambient water chemistry over a 12 mo reproductive cycle. Overall, blood plasma was found to be highly regulated, with few elements exhibiting strong, if any, correlation with ambient concentrations. This sets a first order limit on the sensitivity of otolith chemistry to fluctuations in ambient seawater chemistry. The observed temporal, ontogenetic and sex-specific variations in blood plasma elemental concentrations indicated significant physiological influences on elemental uptake and processing mechanisms. Physiological variables exerted relatively strong influences on the uptake and regulation of the softer, more thiophilic elements (Mn, Cu, Zn, Se and Pb), as well as Sr and Ca. By contrast, seasonal and sex-related variations were relatively minor among the hard acid metal ions (Li+, Mg2+, K+, Rb+, Ba2+). Overall, plasma elemental concentrations covaried most strongly and consistently with plasma protein concentrations. For this exclusively marine species, seasonal changes in physiology governed intra-annual variations in blood chemistry and, by implication, also regulate ion availability to the otolith. Based on these observations, we recommend that sex and age should be controlled for in future experimental designs using otolith microchemistry to infer stock structure or migration patterns

Data from plaice, Pleuronectes platessa L., tagged with electronic data storage tags, were used to test whether these fishes exhibited migration route and spawning area fidelity in successive spawning seasons. Depth and temperature data were recorded for each fish over 365-512 days in the central North Sea and this information was used to reconstruct movements based on tidal locations. We discovered highly directed seasonal migrations from the winter spawning area south of a major topographical feature, Dogger Bank Tail End, to summer feeding grounds 250 km to the north in deep, cold, thermally stratified water. Our results show synchronous timing of migration, repeated pre-and post-spawning migration routes and 100% spawning area fidelity, including two individuals that returned to within 20 km of their previous season's spawning location. This is the first study to provide a complete reconstruction of annual migrations by individual fishes, showing strong homing behaviour along consistent migration routes.

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although depleted throughout the european continental shelf, the relatively high density of thornback rays raja clavata, in the thames estuary (uk) makes it an important stock centre and potential focus for species management. to describe spatial and temporal distribution, 197 thornback rays were tagged with electronic data storage tags (dsts) and released in the thames estuary in october 1999 and 2000, and 100 rays tagged with conventional tags in 2000. fifty-one per cent of dsts and 48% of conventional tags were returned. fishery-independent estimates of position (‘geolocations’) between the time of release and recapture using the tidal location method were possible using 75 individual data records of between 31 and 423 days. ninety-six per cent of rays were recaptured within the thames estuary. the rays were located in water of 20–35 m depth during the autumn and winter, then migrated into shallower water (<20 m depth) during the spring. fishery-independent analysis of distribution demonstrated that the rays were more widely distributed in the southern north sea during the autumn and winter. the range contracted in spring, when the fish moved into the inner thames estuary. no gross behaviour differences were observed between males and females. displacement and dispersion coefficients calculated from geolocation data demonstrated clear annual cycles, indicative of migration. these movements were not apparent from the mark–recapture data, a fishery-related effect. the extent of migration as determined from experiments with dsts was approximately three times greater than that suggested by conventional tagging data alone.

Summary 1.Migration is widespread among marine fishes, yet little is known about variation in the migration of individuals within localities, and the consequences for spatial population structure. We tested the hypothesis that variation in the migratory behaviour among plaice ( Pleuronectes platessa L.) in the North Sea could be explained by largescale differences in the speed and directions of the tidal streams, which the fish use as a transport mechanism. 2. We released 752 mature female plaice tagged with electronic data storage tags at eight locations with contrasting tidal flow properties between December 1993 and September 1999. 3. The experiment yielded 20 403 days of data from 145 plaice. Individual tags recorded depth and temperature for up to 512 days. The position of each fish was determined at intervals throughout the liberty period using the tidal location method. 4. The results show that the fish were segregated into three discrete feeding aggregations during the summer non-breeding season. Two clusters were in warm, thermally mixed water in the eastern and western North Sea, respectively, and one was in deeper, cold, thermally stratified water to the north. 5. In the winter spawning period, fish from all three aggregations mixed together in the southern North Sea, and fish from the eastern and northern subunits spawned in the south-eastern North Sea. The only fish that left the North Sea were western subunit plaice that visited spawning grounds in the eastern English Channel. 6. Our results re-affirmed the major role of the tidal streams in the southern North Sea in structuring plaice dispersion, both by providing transport and guidance and by delimiting the extent of distribution due to thermal stratification during the summer. However, plaice from the northern North Sea did not use tidal stream transport. 7. These results confirm the prediction that large-scale variation in migration behaviour can be explained in part by the tidal guidance and transport mechanisms available. They have also revealed features of spatial dynamics not observed previously from a century of conventional tagging experiments and illustrate how the study of individual fish can successfully define the migratory characteristics of populations.