Species-specific markers for early detection of marine invertebrate invaders through eDNA methods: Gaps and priorities in GenBank as database example

Non-indigenous species (NIS) invasions are global phenomena that are the main drivers for ecosystem change and can often drastically affect the structure and function of novel ecosystems. Early detection is of prime importance for preventing the establishment and potential dispersal of NIS, making eradication and control more efficient and less costly since the species is present at very low densities. However, species at low density generally require intensive sampling efforts to be detected.An important shortcut to reduce sampling effort is to analyze environmental DNA (eDNA). Eukaryotic organisms leave traces of their presence in the environment where they live. In environments that are difficult to access, such as aquatic environments, DNA present in the water can be extracted and amplified using PCR target markers to identify the species that inhabit therein. Using species-specific markers, the presence of target species’ DNA from water samples can be detected using PCR and simple electrophoresis in agarose gel. This is an efficient and convenient approach when the target species is known that puts the focus on detection. However, a critical challenge of this methodology is the design of appropriate species-specific markers. They must be specific to one target species, without any cross-amplification even in close species. For this purpose, a DNA database with as many different species, genetic markers and haplotypes as possible is essential for genetic marker design. The principal aim of this work is to analyze the state-of-the-art of the databases, in this case GenBank, which is the most complete database in number of markers and species, in order to assess its utility in the design of species-specific primers for invasive species early detection.     

Use of environmental DNA to detect Eastern Sand Darter (Ammocrypta pellucida Putnam, 1863) in large Laurentian Great Lakes embayments

A single-species environmental DNA (eDNA) method was developed to sample for a small, benthic rare species, Eastern Sand Darter (Ammocrypta pellucida Putnam, 1863) in two large Lake Ontario embayments. Summer water sampling allowed for: (a) surveys of habitats (Wellers Bay) where traditional fish sampling gear could not be used; and, (b) a comparison between eDNA and seining-based detection probabilities at known occupied habitats (West Lake). In 2018, replicate (n = 3) 1 L water samples were collected from 90 Wellers Bay sites and 71 West Lake sites. A site-occupancy model, a hierarchical logistic regression model, was fitted to determine site occupancy, sample occupancy (presence of Eastern Sand Darter DNA in a water sample) and probability of detection (p) based on replicate quantitative polymerase chain reaction (qPCR) results for each water sample. Eastern Sand Darter was detected at 10 West Lake sites, but not from Wellers Bay. Mean site occupancy was 0.31 (0.12–0.70; 95% CLs), mean sample occupancy was 0.28 (0.09–0.58; 95% CLs), and mean detection probability in a subsample (i.e., successful qPCR amplification) given it was present was 0.40 (0.25–0.55; 95% CLs). While the eDNA method successfully detected Eastern Sand Darter from known occupied areas in West Lake, it was not more effective for assessing local site occupancy than traditional sampling methods, such as the seine.     

Predicting future community composition from random soil seed bank sampling – evidence from a drained lake bottom

Questions: What is the accuracy and reliability of the commonly used random soil sampling methodology for predicting seedling density, species richness and composition of the emerging seedling community? Location: Lake Kraenepoel, western Belgium. Methods: We compared density, species composition and observed and rarefactioned species richness of the seedling community emerging on a soft water lake bed exposed after drainage with the seedling community germinating in the laboratory from random soil samples in the same plots. Results: Seedling density did not differ between the two methods and there was a significant correlation between seedling density on the exposed lake bed and in the soil samples. This indicates that future seedling density can be reliably predicted based on soil sampling, in particular for the most abundant species. The most frequently occurring and abundant species among the seedlings in the soil samples were also the most frequent and abundant species germinating on the exposed lake bed. In contrast, species richness was much higher on the exposed lake bed than in the soil samples, and this difference was still significant for annual species after correction for differences in sampling intensity by rarefaction. We found no correlation between the number of species retrieved by the two methods. Although seedlings of rare and target species emerged on the lake bed, random soil sampling clearly failed to detect seeds of most of these species. Conclusions: Random soil sampling at a commonly used intensity and using the standard germination conditions can accurately predict future total seedling density and the density of the most abundant species. However, the method is not reliable for predicting the probability of establishment of populations of uncommon species. When executing a seed bank study, sampling intensity and germination conditions need to be adapted to the nature and the level of detail of the research question to be answered.     

Quantifying rotifer species richness in temperate lakes

1. Biodiversity assessments of lakes depend on the ability to identify the complement of species present, although the degree of sampling required is often uncertain. We utilise long‐term data to predict rotifer species richness in three habitats in three Polish lakes using rarefaction sampling methods. 2. Richness in littoral and psammon habitats did not saturate, even with up to 130 samples. Highest richness was observed in psammon habitat (119 species) in Lake Mikolajskie, followed by littoral habitat in Lakes ?uknajno (114 species) and Kuc (110 species). Littoral habitats in Lakes ?uknajno (56%) and Kuc (51%) had the most species not shared with other habitats in the same lake. 3. Species richness (Chao2) estimates ranged between 44 for pelagic and 135 for psammon habitat in Lake Mikolajskie, to 100 for psammon and 137 for littoral habitat in Lake Kuc, and 65 for pelagic and 162 for littoral habitat in Lake ?uknajno. Whole lake estimates were 167, 205 and 171 species, respectively, for these lakes, higher than the 150 to 160 species predicted by Dumont and Segers (Hydrobiologia, 1996, 341 , 125). 4. Using standardised sampling, richness was significantly higher in littoral than either pelagic or psammon habitats. Contrasts of standardised rarefaction curves revealed that richness in Lakes Kuc and Mikolajskie was described as well by littoral‐only or psammon‐only samples, respectively, as by those randomly drawn from across all habitats in the lake. 5. Species richness estimates for Lake Mikolajskie were highest in summer, followed by autumn and spring. Interannual estimates differed by up to 427%, nearly an order of magnitude greater than maximal seasonal variation of 70%. 6. Results indicate that much higher sampling intensity is required to establish species richness than is presently carried out in most lakes. Because many species can be detected only with very intensive sampling, conservation programmes must consider sampling intensity when designing studies.     

Estimating the influence of population density and dispersal behavior on the ability to detect and monitor Agrilus planipennis (Coleoptera: Buprestidae) populations

Emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), a phloem-feeding pest of ash (Fraxinus spp.) trees native to Asia, was first discovered in North America in 2002. Since then, A. planipennis has been found in 15 states and two Canadian provinces and has killed tens of millions of ash trees. Understanding the probability of detecting and accurately delineating low density populations of A. planipennis is a key component of effective management strategies. Here we approach this issue by 1) quantifying the efficiency of sampling nongirdled ash trees to detect new infestations of A. planipennis under varying population densities and 2) evaluating the likelihood of accurately determining the localized spread of discrete A. planipennis infestations. To estimate the probability a sampled tree would be detected as infested across a gradient of A. planipennis densities, we used A. planipennis larval density estimates collected during intensive surveys conducted in three recently infested sites with known origins. Results indicated the probability of detecting low density populations by sampling nongirdled trees was very low, even when detection tools were assumed to have three-fold higher detection probabilities than nongirdled trees. Using these results and an A. planipennis spread model, we explored the expected accuracy with which the spatial extent of an A. planipennis population could be determined. Model simulations indicated a poor ability to delineate the extent of the distribution of localized A. planipennis populations, particularly when a small proportion of the population was assumed to have a higher propensity for dispersal.     

A new approach to molecular biosurveillance of invasive species using DNA metabarcoding

Non‐indigenous species (NIS) reach every corner of the world, at times wreaking havoc on ecosystems and costing the global economy billions of dollars. A rapid and accurate biosurveillance tool tailored to a particular biogeographic region is needed to detect NIS when they are first introduced into an area as traditional detection methods are expensive and require specialized expertise. Metabarcoding of environmental and community DNA meets those biosurveillance requirements; a novel tool tailored to the Northwest Pacific Ocean is presented here using an approach that could revolutionize early detection of NIS. Eight newly designed genetic markers for multiple gene regions were implemented to meet the stringent taxonomic requirements for the detection of NIS across four major marine phyla. The tool was considered highly successful because it identified 12 known NIS in the study area and a further seven species representing potential new records. Overall community composition detected here was statistically different between substrate types; zooplankton sampling accounted for significantly higher species richness than filtered sea water in most cases, but this was dominated by mollusk and arthropod species. Both substrate types sampled were required to identify the wide taxonomic breadth of known NIS in the study area. Intensive sampling is known to be paramount for the detection of rare species, including new incursions of NIS, thus it is recommended to include diverse DNA sampling protocols based on species’ life‐history characteristics for broad detection capacity. Application of a metabarcoding‐based molecular biosurveillance tool optimized for biogeographic regions enables rapid and accurate early detection across a wide taxonomic range to allow quick implementation of eradication or control efforts and potentially mitigate some of the devastating effects of NIS worldwide.     

Trapping intensities for sampling ants in Australian rangelands

Abstract I investigated the relationship between species richness and composition of ant faunas, and sampling intensity in two regions with different long‐term histories of grazing intensity in mulga (Acacia aneura) woodlands in northern New South Wales. There were two aims: (i) to examine the relationship between sampling intensity and species richness and composition; and (ii) to explore the differences in ant assemblages from two regions of markedly different grazing intensity when sampled at different intensities (i.e. when a higher proportion of the local ant fauna were collected). Ants were sampled in pit traps (120‐mm diameter) at densities of two, four, six and nine pits per 100 m². Each sampling‐intensity treatment was replicated three times within each region. Pit traps filled with preservative were opened for 3 days. Species richness was higher with each successive increase in sampling intensity but was not different between regions for a given trapping intensity. There was no obvious asymptote of the curve relating trapping intensity to cumulative species richness suggesting that even greater trap densities than those used in the present study would be needed to collect most of the species of ants using a patch of ground over a few days. Spatial replication of a low‐intensity sampling design did not capture as many species as one higher‐intensity sampling array with the same total number of pit traps. This result can be explained by aggressive numerically dominant species of ants monopolizing access to a greater proportion of the traps in low‐density arrays. Ordination reveals that regions and sampling‐intensity treatments could be discriminated and that differences between regions with different grazing histories were less apparent with high‐intensity sampling arrays than they were with low‐intensity sampling arrays. This suggests that differences between locations in space (or potentially samples in time) could be exaggerated by incomplete sampling of the patch‐scale fauna. Comparison of the present study with other studies suggests that most studies to date have used sampling intensities that would not give a thorough assessment of the patch‐scale ground‐dwelling fauna if sampled only by pit traps. The implications of the results for programmes of ant monitoring in rangelands are discussed.     

Targeted and passive environmental DNA approaches outperform established methods for detection of quagga mussels,Dreissena rostriformis bugensis in flowing water

1. The early detection of invasive non‐native species (INNS) is important for informing management actions. Established monitoring methods require the collection or observation of specimens, which is unlikely at the beginning of an invasion when densities are likely to be low. Environmental DNA (eDNA) analysis is a highly promising technique for the detection of INNS—particularly during the early stages of an invasion.
2. Here, we compared the use of traditional kick‐net sampling with two eDNA approaches (targeted detection using both conventional and quantitative PCR and passive detection via metabarcoding with conserved primers) for detection of quagga mussel, Dreissena rostriformis bugensis, a high priority INNS, along a density gradient on the River Wraysbury, UK.
3. All three molecular tools outperformed traditional sampling in terms of detection. Conventional PCR and qPCR both had 100% detection rate in all samples and outperformed metabarcoding when the target species was at low densities. Additionally, quagga mussel DNA copy number (qPCR) and relative read count (metabarcoding) were significantly influenced by both mussel density and distance from source population, with distance being the most significant predictor.
4. Synthesis and application. All three molecular approaches were more sensitive than traditional kick‐net sampling for the detection of the quagga mussel in flowing water, and both qPCR and metabarcoding enabled estimates of relative abundance. Targeted approaches were more sensitive than metabarcoding, but metabarcoding has the advantage of providing information on the wider community and consequently the impacts of INNS.

     

The function and adaptive significance of tristyly in Pontederia cordata L. (Pontederiaceae)

Darwin proposed that the adaptive significance of tristyly is to promote insect-mediated pollination among floral morphs with anthers and stigmas at equivalent levels (legitimate pollination). Strong pollen trimorphism in Pontederia cordata enables an evaluation of this hypothesis. In an investigation of pollen flow patterns in different parts of the North American range of the species, legitimate pollination of some morphs was observed in all but one population investigated. However at only one site (Ft. McCoy, Florida) was Darwin's hypothesis confirmed for the three floral morphs. The long-styled morph most frequently exhibited legitimate pollination, the mid-styled morph was intermediate, and the short-styled morph rarely experienced legitimate pollination.
Temporal variation in pollen flow was observed during the flowering season of P. cordata . Total stigmatic pollen loads decreased as the density of inflorescences and activity of pollinators declined. In spite of variable total loads the legitimate component was relatively constant in the long- and mid-styled forms, while in the short-styled form this measure increased as the season progressed. Little variation was detected between stigmatic samples collected at different times during a single day.
Legitimate pollination may be inhibited by local foraging of pollinators and the spatial segregation of floral morphs. At Paugh Lake (Ontario) over 75% of all bumble bee flights are among the five nearest neighbours of a particular inflorescence. In the same population, there is a probability of over 70% that the nearest three neighbours of an inflorescence are of the same floral morph. Despite these influences, legitimate pollination occurs in P. cordata , suggesting substantial pollen carryover.     

Searching for a Needle in a Haystack: Evaluating Survey Methods for Non-indigenous Plant Species

The control and management of non-indigenous plant species (NIS) can be conceptually divided into three phases: inventory/survey, monitoring and management. Here we focus on phase one, determining which species are present and where they are located within the environment. Sampling for NIS is inherently time-consuming and thus costly. Many management areas are large and therefore can only be surveyed (partial observation of the total area by sampling) and not inventoried (total observation of area). Survey data should reflect the spatial distribution of the target species populations over the landscape. Such data can then be used in combination with environmental data, to create probability maps of target species occurrence for the entire area of interest. We used a GIS model to evaluate seven different survey methods for consistency and reliability of intersecting NIS species’ patches and producing samples which reflect the spatial distribution of the population, and which can be performed in a cost and time-efficient manner. The GIS model was developed to create NIS populations which were then sampled using the different survey methods, and the results recorded. To improve the applicability of the model, four patch sizes and levels of occurrence were used, along with random and weighted distribution patterns in relation to patch proximity to roads and trails. Grid and random points, and targeted (stratified continuous) transects (starting on a road or trail (rights of way (RoW)) and finishing 2 km from any RoW) methods provided the most consistent samples of the population. Logistically, point methods required an unrealistic distance and time commitment in comparison with transect methods. The importance of collecting information on the size of NIS patches was demonstrated as more small patches were intersected than larger ones when the area infested was held constant. Thus, if frequency of patches is used to explain the results of a survey then comparisons between species and methods are difficult to interpret thus leading to erroneous conclusions. However, use of percentage of area infested estimates provides for easier comparison between species and sample methods. The targeted transect method provided the most reliable, efficient and consistent sample with the expected spatial distribution.     

Density-dependent and frequency-dependent selection by bumblebees Bombus terrestris (L.) (Hymenoptera: Apidae)

The behaviour of bumblebee workers foraging on arrays of artificial flowers of two colour morphs was observed. Experiments were conducted on arrays of varying morph frequencies and at three different total flower densities. Bumblebees consistently showed a preference for the commonest colour morph, and this behaviour was not significantly affected by changing density. In contrast, frequency-independent preferences changed significantly with density. At low densities, there was a strong bias towards the more conspicuous colour, whereas at higher densities there was no overall colour bias. Flight distances between flowers decreased significantly at high density. Bumblebees also visited flowers of similar colours sequentially, but this behaviour was not density-dependent. It is suggested that as densities increase, there is an increased probability that bumblebees detect yellow flowers, which were probably less conspicuous compared with blue flowers, and that this might be caused by changes in flight speed with flight distance. Where there is a positive relationship between pollinator visitation and the relative fitness of a floral morph, the observed behaviour would induce positive frequency-dependent selection on a plant population with two corolla colour morphs on which the bumblebees were foraging, which would result in stabilizing selection for a single corolla colour, irrespective of density. There was no indication that rare colour morphs would be preferred at high density. The probability of different corolla colour morphs going to fixation would, however, be affected by density.     

Improving the Accuracy of Sampling Field Plots for Plant-Parasitic Nematodes

The validity of nematode data from field experiments depends largely on how well samples represent the nematode population. Data from an intensive sampling of three field plots before and after spring cultivation were used to compare eight simulated sampling schemes. Average deviation from the plot mean ranged from 10% to 34% before cultivation and from 7% to 16% after cultivation. Samples taken from only the plant row erred most before cultivation but were comparable to other schemes after cultivation. Several schemes achieved a 25% deviation or less in 90% of the sample simulations. Sampling a nematode population usually involves subsampling a composite bulk sample, however, and this increases error by an estimable amount. A random sample with 35 cores and four random subsamples estimated mean plot densities within 25% with probabilities ranging from 0.77 to 0.85. The probability of a sample-subsample combination coming within a specified percent error of the true mean can be extended cautiously to any field mean and variance more-or-less independent of species and area using formulae presented herein. The most economical method of increasing sample accuracy was to increase the number of soil cores.     

Lake Superior: an invasion coldspot?

Lake Superior receives a disproportionate number of ballast water discharges from transoceanic ships operating on the Laurentian Great Lakes. Although this provides dispersal opportunities for nonindigenous species (NIS), relatively few NIS were initially discovered in this lake prior to being recorded elsewhere in the basin. A lack of NIS records from this lake may be an artefact of sampling bias. We tested this hypothesis by sampling benthos and plankton from littoral and deepwater habitats around the perimeter of Lake Superior during June and August 2001. Using morphological analysis techniques, we identified a total of 230 invertebrate taxa representing planktonic, benthic and nektonic lifestyles. Five species with invasion histories in the lower Great Lakes, the bivalves Sphaerium corneum, Pisidium amnicum and P. moitessierianum, gastropod Potamopyrgus antipodarum and amphipod Echinogammarus ischnus, were identified for the first time in Lake Superior. In addition, records of expanded distributions within this lake are presented for the amphipod Gammarus fasciatus and oligochaetes Ripistes parasita and Vejdovskyella intermedia. Recently introduced NIS in Lake Superior were found near international ports, implicating shipping as the vector of their introduction. Intrinsic physical-chemical aspects of Lake Superior may account for the scarcity of NIS in this lake as compared to the lower Great Lakes.     

Sampling to determine density of arthropods in intensively grazed grasslands

Arthropod population field studies undertaken to understand the impacts and population dynamics of the target organism rely on sampling methods that provide accurate measurements of population density. Unsuitable methods may underestimate or provide widely variable measures of population density. The suitability of three vacuum sampling methods: a domestic vacuum cleaner, Vortis™, and blower-vac (G-vac), plus heat extraction of turves were compared for sampling common species in an intensively grazed irrigated dairy pasture. Each method used a different approach to sampling with the number of samples per method based on protocols that had been used in grasslands. Overall, for adults of the weevil Listronotus bonariensis, no method provided a consistently high mean density, while for adults of the weevil Sitona obsoletus, the vacuum cleaner and turf heat extraction methods gave generally higher mean densities. For predatory beetles and lacewings, heat extraction was the most effective for density measurements, while for ladybirds and spiders, the Vortis™ provided the best estimate of mean density. Increasing pasture dry matter (kg Dm/ha) generally had a significant negative effect on S. obsoletus density but not for L. bonariensis. While no method was consistently superior than another, the vacuum cleaner and heat extraction methods generally provided higher mean densities. The G-vac generally produced the lowest density estimates, but the sampling protocol meant a larger collection area per unit effort and therefore the probability of detection was higher compared with the three other methods. In conclusion, the study showed that the optimal sampling method depends on the taxa targeted, and more than one method may be required to measure density and diversity of species in both natural and modified grasslands.     

淀山湖鱼类生物多样性调查样本量的优化设计

样本量大小影响估算鱼类种群动态和鱼类群落特征的准确性和精确度.本文以淀山湖渔业资源调查为例,研究估算淀山湖鱼类群落特征所需的样本量大小.2010年7月—2011年6月研究期间每月进行一次淀山湖渔业资源调查,共捕获14科45种鱼类.利用重采样方法,估算淀山湖渔业资源调查最优调查频次和最佳站点数.结果表明: 在淀山湖渔业资源调查中,站点数和调查频次呈负相关关系;保持95%的探测率探测90%的物种,每年调查11和12次相应需要设置21和19个站点.Shannon多样性指数随着每次调查站点数的增多而变大,增长到一定程度之后逐渐趋于稳定.淀山湖鱼类群落的物种多样性调查最佳站点数为21.本文的研究方法和结果可以为其他类似研究提供借鉴.     

The positive correlation between avian species richness and human population density in Britain is not attributable to sampling bias

Aim  To assess whether spatial variation in sampling effort drives positive correlations between human population density and species richness.
Location  British 10 × 10 km squares.
Methods  We calculated three measures of species richness from atlas data of breeding birds in Britain: total species richness, species richness standardised for sampling effort, and the number of species only recorded in supplementary casual records in a manner not standardised for survey effort. We then assessed the form of the relationship between these richness estimates and human population density, both with and without taking spatial autocorrelation into account.
Results  Both total and standardised species richness exhibit similar species richness–human population density relationships; species richness generally increases with human population density, but decreases at the very highest densities. Supplementary species richness is very weakly correlated with human population density.
Main conclusions  In this example, sampling effort only slightly influences the form of species richness–human population density relationships. The positive correlation between species richness and human population density and any resultant conservation conflicts are thus not artefactual patterns generated by confounding human density and sampling effort.     

International study on Artemia LXIII. Field study of the Artemia urmiana (Günther, 1890) population in Lake Urmiah, Iran

Lake Urmiah is a large (total surface 4750–6100 km² in recent times) thalassohaline hypersaline lake (150–180 g l^–1 in the period 1994–1996), located in northwestern Iran. It is the habitat of the endemic Artemia urmiana. Over the period July 1994–January 1996 a sampling campaign was organized: 36 fixed sampling stations, distributed over the entire lake's area, were sampled weekly to determine water temperature, salinity and transparency. At each occasion a filter net was dragged over a distance of 400 m in the superficial water layer to assess the density and composition of the Artemia population. A more limited sampling campaign focused on the annual fluctuations in chlorophyll concentration and on the reproductive behaviour of the brine shrimp population. Several stages of brine shrimp survived during winter months (water temperature 3°C) at low densities. Compared to available data for the Great Salt Lake, USA, Lake Urmiah shows a low algal biomass and overall low Artemia density. The increasing grazing pressure of the developing brine shrimp population in spring seems to prevent the phytoplankton from reaching high blooming concentrations, and oviparity is the dominant reproductive mode throughout the reproductive season.     

Thyroid function, pituitary structure and serum lipids in Great Lakes coho salmon, Oncorhynchus kisutch Walbaum, 'jacks' compared with sexually immature spring salmon

Whereas spring salmon, Oncorhynchus kisutch , from Lake Ontario had no overt thyroid goiters, comparable Lake Erie spring salmon had a 44% goitre incidence. Goitre incidence in sexually precocious 'jacks' from Lakes Ontario and Michigan was 43–45% and 100% in 'jacks' from Lake Erie.
All fish displayed thyroid hyperplasia. The degree of hyperplasia (as indicated by thyroid follicle colloid content, thyroid follicle size and shape and thyroid follicle epithelial cell size) in Lake Ontario fish tended to be greater in the 'jacks' than in the spring fish; the reverse was true in Lake Erie fish. Serum total L-thyroxine (T4) concentrations in Lake Ontario spring fish were higher than in any other group. There were no differences in serum T, levels between the groups of 'jacks', nor were serum T, levels in 'jacks' and spring salmon from Lake Ontario significantly different. Serum total triiodo-thyronine (T) concentrations in Lake Ontario spring salmon were significantly higher than in all other groups and that in Lake Erie spring salmon greater than in all groups of 'jacks'. Serum T, levels in Lake Ontario 'jacks' were higher than in Lake Erie 'jacks'. Serum free fatty acid (FFA) and cholesterol levels showed significant interlake differences with lower levels present in Lake Erie salmon. Lake Erie 'jacks' appeared to have fewer and less well vacuolated pituitary gonadotropic basophils than 'jacks' from Lakes Michigan and Ontario. Putative thyrotrop cells appeared to be more numerous and more active in the Lake Erie spring salmon compared with Lake Ontario spring salmon. Prolactin, corticotrop and melanotrop cells appeared less active in spring salmon than in the 'jacks'.     

Real‐time PCR detection of Didemnum perlucidum (Monniot, 1983) and Didemnum vexillum (Kott, 2002) in an applied routine marine biosecurity context

Prevention and early detection are well recognized as the best strategies for minimizing the risks posed by nonindigenous species (NIS) that have the potential to become marine pests. Central to this is the ability to rapidly and accurately identify the presence of NIS, often from complex environmental samples like biofouling and ballast water. Molecular tools have been increasingly applied to assist with the identification of NIS and can prove particularly useful for taxonomically difficult groups like ascidians. In this study, we have developed real‐time PCR assays suited to the specific identification of the ascidians Didemnum perlucidum and Didemnum vexillum. Despite being recognized as important global pests, this is the first time specific molecular detection methods have been developed that can support the early identification and detection of these species from a broad range of environmental sample types. These fast, robust and high‐throughput assays represent powerful tools for routine marine biosecurity surveillance, as detection and confirmation of the early presence of species could assist in the timely establishment of emergency responses and control strategies. This study applied the developed assays to confirm the ability to detect Didemnid eDNA in water samples. While previous work has focused on detection of marine larvae from water samples, the development of real‐time PCR assays specifically aimed at detecting eDNA of sessile invertebrate species in the marine environment represents a world first and a significant step forwards in applied marine biosecurity surveillance. Demonstrated success in the detection of D. perlucidum eDNA from water samples at sites where it could not be visually identified suggests value in incorporating such assays into biosecurity survey designs targeting Didemnid species.     

Population density and age structure of the freshwater pearl mussel, Margaritifera margaritifera, in two Iberian rivers

1. The European freshwater pearl mussel, Margaritifera margaritifera (Bivalvia: Unionoida), is one of the most threatened mussels. The Iberian populations of this species are considered peripheral because their distinct characters such as growth rate and longevity, and require development of effective conservation strategies. 2. We assessed population density and age structure of pearl mussels in two Galician rivers (Eo and Masma in north‐west Spain). Four sampling sites were selected in each river to cover stretches of 100 m. The mean density of mussels in each of these sampling transects was estimated using the adaptive sampling technique, given that mussels occur at low densities and are highly aggregated in these rivers. 3. Age structure was inferred for each population using length–age keys. The empty shells encountered during sampling were used to determine the length of the specimens at different ages (years), together with length‐at‐age data from shells previously analysed for computing growth rates from the same rivers. Water samples from both rivers were analysed for typical physicochemical parameters. 4. Mean densities were very variable, even within the same river (from 0.27 to 6.55 m^?2 in the River Eo and from 0.98 to 2.38 m^?2 in the River Masma). Individuals in the 0‐ to 5‐year age class were scarce in both rivers. 5. Margaritifera margaritifera showed a preference for the strip of river bed within 1.5 m from the river bank and avoided sites at greater distances. The species also showed a preference for sites with more than 80% tree cover and avoided sites with <50% cover. 6. Iberian populations exhibit the highest growth rate, together with the lowest maximum age and maximum length known for M. margaritifera. Detailed knowledge about these peripheral Iberian populations will contribute to developing strategies for conservation and management of this endangered species.