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Multiple-stressor effects on stream invertebrates: DNA barcoding reveals contrasting responses of cryptic mayfly species
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November 2015
... Marshall et al., 2006), and have typically remained relatively localised in geographic scope (Townsend et al., 1997;Harding, 2003;Thompson and Townsend, 2003). By contrast, the recent development and application of genetic barcoding approaches promises to enhance taxonomic resolution (Macher et al., 2016) and facilitate rapid and replicated community-wide assessment of freshwater biodiversity over broader ranges of spatial scales (Lim et al., 2016;Macher et al., 2018;Coble et al., 2019). ...
... Environmental detection approaches are particularly well suited for analysis of water samples (Dowle et al., 2016). While eDNA approaches may struggle to detect all rare taxa in freshwater systems (see Roussel et al., 2015), this environmental sampling approach nevertheless carries the major advantage of readily identifying lineages to species level (Macher et al., 2016). Additionally, eDNA approaches can easily reveal previously unrecognised taxa, thereby potentially increasing power to resolve biologically meaningful (e.g. ...
... The current study highlights the potential of eDNA for resolving major ecological shifts that may potentially be overlooked based on conventional taxonomic sampling of stream invertebrates. The molecular differentiation of morphologically similar but ecologically distinct taxa may be particularly important in this regard (Macher et al., 2016). For instance, many previous analyses of New Zealand stream ecology have identified the ecologically important mayfly Deleatidium only to genus level (Townsend et al., 1997;Nyström et al., 2003;Thompson and Townsend, 2003). ...
... Besides the overall potential for misidentifications [8,[11][12][13], particularly the morphological classification of early fish life stages and immature aquatic invertebrates with insufficient diagnostic characters is challenging, time consuming, and therefore considered to be costly [14][15][16][17]. This results in severe problems in species determination, with cryptic species or lineages remaining undetected [17][18][19]. Hence, such "problematic" organisms are usually identified only to coarser taxonomic levels, i.e. to genus, family or order, or are even excluded [13,17]. ...
... Hence, such "problematic" organisms are usually identified only to coarser taxonomic levels, i.e. to genus, family or order, or are even excluded [13,17]. Information based on higher-level taxonomy can be sufficient in standard bioassessments [20,21], but valuable information about species-specific ecological requirements and stressor tolerances may remain unnoticed [19,22]. This may in turn lead to potentially inaccurate water quality assessments and mismanagement of freshwater ecosystems [23,24]. ...
... In most standard water bioassessments, many organisms are determined to higher levels such as genera or family only, in order to minimize processing time-and hence maximize cost efficiency [44,45]. As in some aquatic invertebrate taxa even closely related species can vary substantially in their ecological tolerance and respond different to environmental disturbances, the consequence of this traditional approach is a potential information loss, which may moreover result in inaccurate water quality evaluations [13,19,24]. ...
Although aquatic macroinvertebrates and freshwater fishes are important indicators for freshwater quality assessments, the morphological identification to species-level is often impossible and thus especially in many invertebrate taxa not mandatory during Water Framework Directive monitoring, a pragmatism that potentially leads to information loss. Here, we focus on the freshwater fauna of the River Sieg (Germany) to test congruence and additional value in taxa detection and taxonomic resolution of DNA barcoding vs. morphology-based identification in monitoring routines. Prior generated morphological identifications of juvenile fishes and aquatic macroinvertebrates were directly compared to species assignments using the identification engine of the Barcode of Life Data System. In 18% of the invertebrates morphology allowed only assignments to higher systematic entities, but DNA barcoding lead to species-level assignment. Dissimilarities between the two approaches occurred in 7% of the invertebrates and in 1% of the fishes. The 18 fish species were assigned to 20 molecular barcode index numbers, the 104 aquatic invertebrate taxa to 113 molecular entities. Although the cost-benefit analysis of both methods showed that DNA barcoding is still more expensive (5.30–8.60€ per sample) and time consuming (12.5h), the results emphasize the potential to increase taxonomic resolution and gain a more complete profile of biodiversity, especially in invertebrates. The provided reference DNA barcodes help building the foundation for metabarcoding approaches, which provide faster sample processing and more cost-efficient ecological status determination.
... Monitoring and quantifying changes in ecosystem community structure are important for assessing the effects of human impacts on all aquatic ecosystems. While awareness of the importance of evaluating loss of biodiversity in freshwater ecosystems is growing (e.g., Mueller et al. 2013, Macher et al. 2016, saline lakes have received far less attention, possibly due to the perception that they are less common than freshwater lakes, or that their biological constituents and food webs are relatively simple (Hammer 1986, Belovsky et al. 2011. However, the effect of anthropogenic pressures on saline lakes may be even more acute because of the specialization of organisms adapted to high salt environments. ...
... The barcodes presented here clearly distinguish the most abundant brine flies associated with the GSL ecosystem. Molecular analyses, such as DNA metabarcoding, provide a complementary approach to morphological surveys, leading to enhanced resolution of macroinvertebrate ecosystem assessments (Macher et al. 2016). In a comparison of morphological and genetic species identification from 5 study sites in California, Jackson et al. (2014) found a substantial increase in macroinvertebrate biodiversity in streams when morphological identification was augmented with barcoding. ...
Great Salt Lake (GSL) is the center of a valuable wetland ecosystem in the Great Basin of North America. The lake is an important site for millions of migratory birds that feed on two principle invertebrates, brine shrimp and brine flies (Diptera: Ephydridae). In spite of their ecological and economic importance, there are no published genetic studies of either resident GSL invertebrates. The family Ephydridae (shore flies and brine flies) is one of the largest in the order Diptera, with nearly 2000 described species. Members of this family are prominent in a variety of aquatic environments and are of particular interest because of their adaptation to a number of marginal habitats. These include hot springs, oil ponds, highly saline lakes, and inland alkaline pools and marshes. This report provides cytochrome c oxidase (COI) DNA barcodes for five species of GSL shore flies, distributed among five genera and three subfamilies. The phylogenetic content of these DNA sequences is explored by comparing a molecular phylogeny to those based on morphological features. Over the past decade, urbanization and inflow diversion have reduced the surface area of GSL by nearly 50%, with unknown consequences for the ecosystem. This study establishes a genetic framework for assessing changes in GSL invertebrate diversity that will be important in monitoring the effects of anthropogenic and climate pressures on this important natural resource.
... Ståhls and Savolainen 2008;Lucentini et al. 2011). This low taxonomic resolution hampers our ability to detect environmental impacts, because even closely-related species can vary in their tolerance to a given stressor and, thus, respond differently to changes in their environment (Resh and Unzicker 1975;Macher et al. 2016a). If this is accompanied by misidentifications, the detection of biological impairment is further obscured (Lenat and Resh 2001;Haase et al. 2010;Sweeney et al. 2011). ...
... Rutschmann et al. 2014;Vitecek et al. 2017). This is problematic for bioassessments as taxa of the same genus can exhibit differing responses to environmental stressors (Macher et al. 2016a). A particularly problematic group in this regard is the mayfly family Baetidae (e.g. ...
Most stream bioassessment and biodiversity surveys are currently based on morphological identification of communities. However, DNA metabarcoding is emerging as a fast and cost-effective alternative for species identification. We compared both methods in a survey of benthic macroinvertebrate communities across 36 stream sites in northern Finland. We identified 291 taxa of which 62% were identified only by DNA metabarcoding. DNA metabarcoding produced extensive species level inventories for groups (Oligochaeta, Chironomidae, Simuliidae, Limoniidae and Limnephilidae), for which morphological identification was not feasible due to the high level of expertise needed. Metabarcoding also provided more insightful taxonomic information on the occurrence of three red-listed vulnerable or data deficient species, the discovery of two likely cryptic and potentially new species to Finland and species information of insect genera at an early larval stage that could not be separated morphologically. However, it systematically failed to reliably detect the occurrence of gastropods that were easily identified morphologically. The impact of mining on community structure could only be shown using DNA metabarcoding data which suggests that the finer taxonomic detail can improve detection of subtle impacts. Both methods generally exhibited similar strength of community-environment relationships, but DNA metabarcoding showed better performance with presence/absence data than with relative DNA sequence abundances. Our results suggest that DNA metabarcoding holds a promise for future anthropogenic impact assessments, although, in our case, the performance did not improve much from the morphological species identification. The key advantage of DNA metabarcoding lies in efficient biodiversity surveys, taxonomical studies and applications in conservation biology.
... Different studies on genetic variation, often associating morphological and geographic data, revealed the existence of cryptic species in several groups of animals and plants worldwide, in diverse types of habitats (e.g., Dawson & Jacobs, 2001;Feulner et al., 2006;G omez et al., 2002;Grundt et al., 2006;Hebert et al., 2004;de Rezende Dias et al., 2018;Vrijenhoek et al., 1994). The advent of molecular techniques and analyses has brought about increased awareness of the importance of DNA data in insect taxonomy, which has proven very useful in the discovery of cryptic species (e.g., Bickford et al., 2007;Cardoni et al., 2015;Cook et al., 2008;Fujita et al., 2012;Hebert et al., 2003;Hendrich et al., 2015;Macher et al., 2016;Ossa-L opez et al., 2017;Petit & Excoffier, 2009;Silveira et al., 2016). ...
... Insects are one of the most well represented groups in the cryptic species literature, being the description and recognition of them of great implication for human health (e.g., Anopheles malaria-transmitting mosquitoes), pest management (different species have variable pesticide resistance), and studies of coevolution and species interaction (Bickford et al., 2007). More recently, several studies using molecular tools have drawn attention to the existence of cryptic species complexes in Ephemeroptera (e.g., Gill et al., 2016;Macher et al., 2016;Ossa-L opez et al., 2017;Pereira-da-Conceicoa et al., 2012;Rutschmann et al., 2014;Williams et al., 2006), especially using information from the mtDNA cytochrome C oxidase subunit I (COI) gene. However, the within-species genetic variation remains largely unexplored in South American mayflies. ...
Leptohyphodes inanis (Pictet) is an enigmatic species with a rare trait among leptohyphid males – large and divided compound eyes. In addition, the color of its upper portion is variable across – but not within – populations. However, the geographic variation of this trait and its relation to gene flow across populations remain unknown. Here, we analyzed individuals across Southeastern Brazil (19º to 24ºS and 40º to 48º W) to (i) assess genetic (COI) and eye color variation, and (ii) evaluate if L. inanis is a single species, by combining Bayesian phylogenetic analyses (including two other leptohyphid genera – Tricorythopsis and Tricorythodes) and species delimitation methods: ABGD and mPTP. To further investigate within-species variation in phenotypic traits, we evaluated quantitative and qualitative morphological traits of 1,252 individuals. We found that genetic variation in L. inanis is largely unrelated to eye color, and that pairwise genetic divergences in COI mtDNA are remarkably higher (up to 30.7%) than previously found in other mayfly lineages. L. inanis was recovered as monophyletic, although results suggest it includes three to seven cryptic species, each one related to mountain ranges across Southeastern Brazil. Furthermore, we found no genetic variation among individuals of the same drainage basin, suggesting that populations might be largely isolated from one another. Because morphological traits traditionally used in Ephemeroptera taxonomy were ineffective in distinguishing the cryptic species, we propose L. inanis to be a species complex.
... DNA-based methods for species identification known as DNA barcoding and DNA metabarcoding allow more comprehensive assessments of aquatic communities (Hajibabaei et al., 2011;Taberlet et al., 2012;Elbrecht et al., 2017;Leese et al., 2016Leese et al., , 2018. It has been also demonstrated that non-invasive approaches relying on DNA and cells from filtered water samples can be effective in detecting relevant taxa (e.g., Hajibabaei et al., 2012;Majaneva et al., 2018;Zizka et al., 2019). ...
... Several such cases have been published where DNA sequences have helped revealing hidden species level diversity in odonates (Mitchell & Samways, 2005;Damm, Schierwater & Hadrys, 2010;López-Estrada et al., 2020;Vega-Sánchez, Mendoza-Cuenca & Gonzalez-Rodriguez, 2020). Neglecting the presence of cryptic diversity does not only lead to underestimation of factual biodiversity, but also can impair indicator species approaches as different taxa react differently to stressors (Zettler et al., 2013;Macher et al., 2016). But also the opposite-low or absent mitochondrial divergence between species-is expected to occur in large DNA barcode datasets. ...
Background. Dragonflies and damselflies (Odonata) are important components in biomonitoring due to their amphibiotic lifecycle and specific habitat requirements. They are charismatic and popular insects, but can be challenging to identify despite large size and often distinct coloration, especially the immature stages. DNA-based assessment tools rely on validated DNA barcode reference libraries evaluated in a supraregional context to minimize taxonomic incongruence and identification mismatches.
Methods. This study reports on findings from the analysis of the most comprehensive DNA barcode dataset for Central European Odonata to date, with 103 out of 145 recorded European species included and publicly deposited in the Barcode of Life Data System (BOLD). The complete dataset includes 697 specimens (548 adults, 108 larvae) from 274 localities in 16 countries with a geographic emphasis on Central Europe. We used BOLD to generate sequence divergence metrics and to examine the taxonomic composition of the DNA barcode clusters within the dataset and in comparison with all data on BOLD.
Results. Over 88% of the species included can be readily identified using their DNA barcodes and the reference dataset provided. Considering the complete European dataset, unambiguous identification is hampered in 12 species due to weak mitochondrial differentiation and partial haplotype sharing. However, considering the known species distributions only two groups of five species possibly co-occur, leading to an unambiguous identification of more than 95% of the analysed Odonata via DNA barcoding in real applications. The cases of small interspecific genetic distances and the observed deep intraspecific variation in Cordulia aenea (Linnaeus, 1758) are discussed in detail and the corresponding taxa in the public reference database highlighted. They should be considered in future applications of DNA barcoding and metabarcoding and represent interesting evolutionary biological questions, which call for in depth analyses of the involved taxa throughout their distribution ranges.
... This is particularly true in freshwater systems, where an increase in the number of genetic studies has revealed both new species and, interestingly, many indistinctive morphologically cryptic species (Baker et al. 2004, Bickford et al. 2007, Weiss et al. 2014, Katouzian et al. 2016. The presence of sympatric cryptic taxa poses a range of interesting ecological questions; such as, what are the mechanism(s) that allow cryptic species of the same complex to co-exist in the same locality (Gabald on et al. 2017) and do cryptic species show differential responses to environmental factors (Ortells et al. 2003, Macher et al. 2016a. In fact, studies have suggested that differences in physiological responses among cryptic taxa might limit their spatial and temporal distributions (Ortells et al. 2003, Xiang et al. 2011. ...
... To answer this question, further ecological studies need to be conducted at a fine spatial scale and an effort should be focused to explore the mechanisms responsible for co-occurrence of cryptic species of both species complexes studied. 4. Finally, precise knowledge of the distributions and tolerances of cryptic species is crucial for developing comprehensive biomonitoring programs and biodiversity assessments (Macher et al. 2016a). ...
Knowledge of cryptic species distributions and their relationships with environmental factors may be extremely valuable for biodiversity conservation. In freshwater ecosystems, morphologically cryptic species often have different geographic distributions that can overlap to varying extents. The importance of differential species responses to environmental conditions in determining their spatial distributions is, however, unclear. Here, we evaluated the importance of species responses to environmental drivers, in particular the physicochemical factors, in the spatial distributions and environmental relationships of two shrimp species complexes Caridina indistincta Calman, 1926 and Paratya australiensis Kemp, 1917. We analyzed shrimp specimens from 89 sites in 17 catchments across South‐East Queensland by sequencing a fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) to identify individuals. Furthermore, although there is evidence that morphologically the cryptic species of these shrimps differ very little, we made detailed morphological assessments to combine with molecular data, hoping to be able to distinguish among the species more easily and cheaply for future studies. There were significant morphological differences among the three cryptic species of the C. indistincta species complex, specifically in carapace length (cl), number of dorsal teeth (nDt), number of ventral teeth (nVt), teeth posterior to orbital margin (TPOM), and the calculated ratios A/rl, and rl/cl, while the two lineages of the P. australiensis species complex differed in the number of dorsal teeth (nDt) and the calculated ratios of A/rl, rl/cl, and rh/ch. To determine the importance of species responses in explaining the spatial distribution of cryptic species based on the species‐variables relationships, a redundancy analysis (RDA) was used to summarize these relationships. This ordination analysis showed distinct differences among cryptic species in their correlation with water quality variables and elevation. C. indistincta sp. B and C. indistincta sp. D were significantly associated with elevation and dissolved oxygen range, respectively. As well, P. australiensis lineages 4 and 6 were significantly correlated with elevation and conductivity, respectively. Overall, our results demonstrated the advantage of using analyses of biotic and abiotic variables as a valid approach for defining species responses to abiotic factors in cryptic species of atyid freshwater shrimps.
... Accordingly, classification 52 is often performed only to a higher taxonomic level. However, the species within a higher taxonomic group may exhibit 53 diverse responses to stress (Macher et al. 2015), and these differences can go unnoticed in studies with low taxonomic 54 resolution. Misidentification, low comparability, and limited taxonomic resolution for difficult groups, such as 55 chironomids, can lead to inaccurate assessments and potentially to the mismanagement of stream ecosystems (Stein et 56 al. 2013). ...
... DNA metabarcoding can provide much more accurate taxonomic identification than 241 morphology-based methods, and can even be used to detect cryptic species (Elbrecht & Leese 2015). This increase in 242 accuracy provides an opportunity to investigate potential differences in ecological preferences and detect stressors 243 based on indicator taxa when larval morphology alone is not sufficient (Macher et al. 2015). This valuable additional 244 information could be integrated into future assessment techniques by refining and expanding the taxa lists for expected 245 reference conditions. ...
1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification.
2) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Specimens were morphologically classified, following standardized protocols, to the lowest taxonomic level for which identification was feasible in the routine national monitoring.
3) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of specimens per taxon (proxy for biomass) were significantly correlated in each sample, although adjusted R2 were low. With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately equal priced per sample to morphology-based identification.
4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve similar assessment results as those of current protocols for morphological identification. Thus, metabarcoding represents a feasible and reliable method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems with current laboratory protocols and reference databases.
... Over the past decade, the use of short standardized genetic markers for species identification, so-called DNA barcoding [1,2], has proven effective in biodiversity assessments and taxonomic revisions [3][4][5][6][7][8][9]. DNA barcodes have also been used efficiently to detect hidden species diversity in Ichneumonidae [10]. ...
DNA barcoding is an effective modern tool in taxonomy, evolutionary biology, and biodiversity research. Many new species have been discovered and described with DNA barcodes as part of their diagnostic features. We combined morphological examination and molecular species delimitation of the mitochondrial cytochrome c oxidase 1 (COI) gene using the automatic barcode gap discovery (ABGD) to investigate species boundaries. The genus Prochas Walkley (Hymenoptera, Ichneumonidae, Campopleginae) was first reported from China and is new for the Oriental and Eastern Palearctic regions. Using an integrative taxonomy method, two new species P. rugipunctata sp. nov. and P. striata sp. nov. are hereby described and illustrated. A key to the world species and a distribution map are provided.
... Recently, eDNA metabarcoding methods are non-invasive, cost-efficient, and rapid surveys. Besides, their finer taxonomic resolution of diatoms provides more information about environmental responses (Macher et al., 2016). ...
... DNA barcoding has also enabled pre-and post-mortem analyses of invertebrate diets by amplifying COI from regurgitates (Waldner & Traugott, 2012) and gut contents (Blankenship & Yayanos, 2005). Other ecological applications have included examining stream invertebrate abundance in connection with nutrient and sediment levels (Macher et al., 2016). Further, biodiversity assessments can make use of "DNA metabarcoding" to identify multiple species from bulk samples of organisms or environmental samples (Taberlet et al., 2012). ...
DNA barcoding is commonly used for species identification. Despite this, there has not been a comprehensive assessment of the utility of DNA barcoding in crayfishes (Decapoda: Astacidea). Here we examined the extent to which local barcoding gaps (used for species identification) and global barcoding gaps (used for species discovery) exist among crayfishes, and whether global gaps met a previously suggested 10× threshold (mean interspecific difference being 10× larger than mean intra specific difference). We examined barcoding gaps using publicly available mitochondrial COI sequence data from the National Center for Biotechnology Information's nucleotide database. We created two versions of the COI datasets used for downstream analyses: one focused on the number of unique haplotypes (NH) per species, and another that focused on total number of sequences (NS; i.e., including redundant haplotypes) per species. A total of 81 species were included, with 58 species and five genera from the family Cambaridae and 23 species from three genera from the family Parastacidae. Local barcoding gaps were present in only 30 species (20 Cambaridae and 10 Parastacidae species). We detected global barcoding gaps in only four genera (Cambarus, Cherax, Euastacus, and Tenuibranchiurus), which were all below (4.2× to 5.2×) the previously suggested 10× threshold. We propose that a ~5× threshold would be a more appropriate working hypothesis for species discovery. While the NH and NS datasets yielded largely similar results, there were some discrepant inferences. To understand why some species lacked a local barcoding gap, we performed species delimitation analyses for each genus using the NH dataset. These results suggest that current taxonomy in crayfishes may be inadequate for the majority of examined species, and that even species with local barcoding gaps present may be in need of taxonomic revisions. Currently, the utility of DNA barcoding for species identification and discovery in crayfish is quite limited, and caution should be exercised when mitochondrial‐based approaches are used in place of taxonomic expertise. Assessment of the evidence for local and global barcoding gaps is important for understanding the reliability of molecular species identification and discovery, but outcomes are dependent on the current state of taxonomy. As this improves (e.g., via resolving species complexes, possibly elevating some subspecies to the species‐level status, and redressing specimen misidentifications in natural history and other collections), so too will the utility of DNA barcoding.
... For instance, as indicated in freshwater ecosystems, the identification at species level as well as the identification of OTUs and even haplotypes can enhance our capacity to infer community dynamics (Laini et al., 2020(Laini et al., , 2023. Moreover, genetic characterization of organisms can contribute to identification of key taxa and species with complex diagnostic characters and facilitate the early detection of alien species in freshwater systems (Macher et al., 2016;Blackman et al., 2022). On the other side, DNA metabarcoding to date prevents the assessment of quantitative data (e.g., abundance, biomass) mainly because of species-specific primer efficiency (Elbrecht and Leese, 2015;Takahashi et al., 2023). ...
Saline and hypersaline wetlands account for almost half of the volume of inland water globally. They provide pivotal habitat for a vast range of species, including crucial ecosystem services for humans such as carbon sink storage and extractive resource reservoirs. Despite their importance, effective ecological assessment is in its infancy compared to current conventional surveys carried out in freshwater ecosystems. The integration of environmental DNA (eDNA) analysis and traditional techniques has the potential to transform biomonitoring processes, particularly in remote and understudied saline environments. In this context, this preliminary study aims to explore the potential of eDNA coupled with conventional approaches by targeting five hypersaline lakes at Rottnest Island (Wadjemup) in Western Australia. We focused on the invertebrate community, a widely accepted key ecological indicator to assess the conservational status in rivers and lakes. The combination of metabarcoding with morphology-based taxonomic analysis described 16 taxa belonging to the orders Anostraca, Diptera, Isopoda, and Coleoptera. DNA-based diversity assessment revealed more taxa at higher taxonomic resolution than the morphology-based taxonomic analysis. However, certain taxa (i.e., Ephydridae, Stratyiomidae, Ceratopogonidae) were only identified via net surveying. Overall, our results indicate that great potential resides in combining conventional net-based surveys with novel eDNA approaches in saline and hypersaline lakes. Indeed, urgent and effective conservational frameworks are required to contrast the enormous pressure that these ecosystems are increasingly facing. Further investigations at larger spatial-temporal scales will allow consolidation of robust, reliable, and affordable biomonitoring frameworks in the underexplored world of saline wetlands.
... Species richness and abundance of sensitive and tolerant taxa have been the most commonly used metrics to determine the ecological impacts of macroinvertebrates below dams (Martínez et al., 2013). Using biotic indicators to detect the effects of dams on the macroinvertebrate community requires a profound knowledge of the species identities because even closely related species may have different tolerances to environmental stressors (Macher et al., 2016;Mezgebu, 2022). However, accurate identification of freshwater macroinvertebrates to the species level can be difficult, especially for larval or subadult specimens, and often results in low taxonomic resolution or misidentification (Haase et al., 2010;Sweeney et al., 2011). ...
... For most species there is a threshold of salinity beyond which they are extirpated (Hart et al. 1991;Kefford et al. 2006). Salinity tolerances vary greatly, however, between and within all taxonomic levels (James et al. 2003;Dunlop et al. 2005;Kefford et al. 2012;Cañedo-Argüelles 2013;Dunlop et al. 2015;Castillo et al. 2018), and probably also at a subspecies level, as is the case with other stressors (Dunlop et al. 2005;Macher et al. 2016). For example, Ephemeroptera (the mayflies) is a sensitive order, but certain families, and especially certain species of mayflies, tolerate brackish water quite well ). ...
Coalbed methane (CBM) is a type of natural gas produced from coal beds, and its extraction brings massive quantities of water from coal formations to the surface. CBM produced water is elevated in salinity and sodicity and can also contain heavy metals, trace elements, and organic compounds, all of which can be harmful to aquatic life. Discharge of produced water directly into streams is permitted in some CBM basins and has been occurring in the semi-arid Raton Basin of southern Colorado since the 1990s. Field studies assessing the impacts of this type of discharge on stream ecosystems have been few and have yielded equivocal results, and none have been conducted in the Raton Basin. The effects of the surface discharge of CBM produced water on the health of small headwater streams in a 30,000-acre State Wildlife Area in the Purgatoire River watershed of Las Animas County, Colorado were studied. Ten contaminated streams (below discharge points) and six comparable reference streams (having no discharge) were sampled and analyzed for differences in macroinvertebrate community structure and water quality. Non-metric multidimensional scaling ordinations showed significant separation in both water quality and community structure between the two stream types. Based on their concentrations and published regulatory/safe levels, the water quality parameters of concern in the produced water streams were determined to be: alkalinity, conductivity, chloride, pH, fluoride, aluminum, iron, temperature, dissolved oxygen, ammonia, and the sodium adsorption ratio (SAR). Reduced calcium and magnesium were also of concern. The biodiversity metrics Taxa Richness, EPT Richness, and Shannon-Wiener Diversity were all significantly lower in the produced water streams than the natural streams. Also, the Top 5 Taxa Percent was significantly higher, indicating lower diversity due to unevenness. The Colorado Macroinvertebrate Multimetric Index (MMI) did not differ between the two stream types, however. Stoneflies and oligochaetes were significantly reduced in both taxa richness and relative abundance in the produced water streams. Mayflies and caddisflies showed significantly decreased richness but unchanged relative abundance levels, due to certain tolerant taxa proliferating in the produced water streams. The variables showing the strongest correlation to biodiversity and community composition were calcium, SAR, and magnesium, with calcium appearing to have a protective effect on the communities. Though CBM produced water may not be as deleterious to aquatic life as other oil and gas produced waters, and although it is not regulated by EPA effluent guidelines, the present study shows that CBM produced water discharge can have significant and possibly long-lasting effects on small intermittent/ephemeral receiving streams.
... Many of these studies use the first approach and calculate species' ecological niches using the environmental factors in combination with data on species' occurrence. Virtually all these studies imply that cryptic species differ in their ecological niches (Eisenring et al., 2016;Fišer et al., 2015;Macher et al., 2016). This approach, however, cannot reliably tell apart the species' ecological differentiation from the differences rooted in biogeography and depends on many assumptions, including unlimited dispersal (Warren et al., 2014). ...
The advent of genetic methods has led to the discovery of an increasing number of species that previously could not be distinguished from each other on basis of morphological characteristics. Even though we observe an exponential growth of publications on cryptic species, cryptic species are rarely considered in ecotoxicology. Thus, particularly the question of ecological differentiation and sensitivity of closely related cryptic species is rarely addressed. Tackling this question, however, is of key importance for evolutionary ecology, conservation biology and, in particular, regulatory ecotoxicology. At the same time, the use of species with (known or unknown) cryptic diversity might be a reason for the lack of reproducibility of ecotoxicological experiments and implies a false extrapolation of the findings. Our critical review includes a database and literature search, where we investigated how many of the species most frequently used in ecotoxicological assessments show evidence for cryptic diversity. We found a high proportion of reports indicating overlooked species diversity, especially in invertebrates. In terrestrial and aquatic realms, at least 67% and 54% of commonly used species were identified as cryptic species complexes, respectively. The issue is less prominent in vertebrates, where we found evidence for cryptic species complexes in 27% of aquatic and 6.7% of terrestrial vertebrates. We further exemplified why different evolutionary histories may significantly determine cryptic species' ecology and sensitivity to pollutants. This in turn may have a major impact on the results of ecotoxicological tests and, consequently, the outcome of environmental risk assessments. Finally, we provide a brief guideline on how to practically deal with cryptic diversity in ecotoxicological studies in general and its implementation in risk assessment procedures in particular.
... These studies could incorporate locally relevant abiotic and biotic factors (e.g. UV light or microbial activity; see Strickler et al., 2015) to provide temporal caveats for species assemblages generated from eDNA detections (Macher et al., 2016). An a priori understanding of these caveats would help improve the accuracy of eDNA survey techniques because they could account for rapid DNA degradation with either more frequent sampling (see Krehenwinkel et al., 2018), or targeting of shorter amplicons (see Saito & Doi, 2021 This approach maximized the amount of recoverable taxa because ribosomal genes show less taxonomic bias (Deagle et al., 2014), while the reference libraries developed for CO1 are more extensive and offer higher taxonomic resolution (Elbrecht et al., 2019). ...
In the face of global biodiversity declines, surveys of beneficial and antagonistic arthropod diversity as well as the ecological services that they provide are increasingly important in both natural and agro-ecosystems. Conventional survey methods used to monitor these communities often require extensive taxonomic expertise and are time-intensive, potentially limiting their application in industries such as agriculture, where arthropods often play a critical role in productivity (e.g. pollinators, pests and predators). Environmental DNA (eDNA) metabarcoding of a novel substrate, crop flowers, may offer an accurate and high throughput alternative to aid in the detection of these managed and unmanaged taxa. Here, we compared the arthropod communities detected with eDNA metabarcoding of flowers, from an agricultural species (Persea americana-'Hass' avocado), with two conventional survey techniques: digital video recording (DVR) devices and pan traps. In total, 80 eDNA flower samples, 96 h of DVRs and 48 pan trap samples were collected. Across the three methods, 49 arthropod families were identified, of which 12 were unique to the eDNA dataset. Environmental DNA metabarcoding from flowers revealed potential arthropod pollinators, as well as plant pests and parasites. Alpha diversity levels did not differ across the three survey methods although taxonomic composition varied significantly, with only 12% of arthropod families found to be common across all three methods. eDNA metabarcoding of flowers has the potential to revolutionize the way arthropod communities are monitored in natural and agro-ecosystems, potentially detecting the response of pollinators and pests to climate change, diseases, habitat loss and other disturbances.
... For example, the response of aquatic invertebrate juveniles to drying in intermittent water bodies differs among congeneric species within the true fly genus Polypedilum (Diptera, Gusev et al. 2014) and the stonefly genus Taeniopteryx (Plecoptera, López-Rodríguez & de Figueroa 2006). Moreover, cryptic species and congeners within species complexes can differ markedly in their tolerance of environmental stressors (Sturmbauer et al. 1999;Eisenring et al. 2016;Macher et al. 2016). Methods that provide species-level or even intraspecific information for whole communities could thus advance understanding of metacommunity processes (Tesson & Edelaar 2013;Gounand et al. 2018;Gauthier et al. 2020a). ...
Most empirical metacommunity studies rely solely on morphological identification of taxa, precluding the species-level identification of several biotic groups, which can influence the characterization of metacommunities. DNA metabarcoding enables inference of species and even intraspecific diversity from community samples but has rarely been used to infer metacommunity structure. Here, we combined morphology and metabarcoding to improve the characterization of an insect metacommunity at different identification levels. We included measures of taxonomic, functional and phylogenetic richness, and we evaluated drivers affecting metacommunity structure (i.e., environmental filtering and dispersal). Communities were sampled from an area that included nine perennial, two near-perennial and two intermittent sites in a river network characterized by high hydrological variability. We identified organisms to a mixed (family to species) taxonomic level using morphology, and to operational taxonomic unit (OTU) and haplotype levels using metabarcoding of the mitochondrial cytochrome c oxidase gene. Diptera and Ephemeroptera showed the greatest increases in taxonomic and phylogenetic richness but not biological trait richness with increasing taxonomic resolution. The joint effect of environmental filtering and dispersal was more important than their individual effects in shaping metacommunity structure at all identification levels. Mixed-level and OTU-level identification were more effective than family and haplotype in characterizing the drivers of metacommunity structure. We demonstrate that the greater taxonomic resolution enabled by metabarcoding could improve understanding of metacommunities within river networks, thus enhancing our capacity to predict ecological responses in ecosystems adapting to global change.
... For biological monitoring and the assessment of the ecological state of aquatic habitats (e.g. within the European Water Framework Directive 2000/60/EC by the European Union), benthic macroinvertebrates are commonly used as they show high species diversity and distinct sensitivity to water and habitat quality (Hering et al. 2004;Birk et al. 2012;Macher et al. 2016;Elbrecht and Leese 2017;Kuntke et al. 2020). Within the group of benthic macroinvertebrates, odonates, comprising dragonflies and damselflies are very suitable as bioindicator organisms, due to their well-known species-specific habitat preferences. ...
Monitoring of odonates has become an important instrument for ecological status assessment of (semi-)aquatic habitats. Besides information on presence and abundance, knowledge about a species´ autochthony at the surveyed waterbody is a significant information within the assessment process. Here, the finding of exuviae represents the ultimate proof of successful reproduction. Although feasible for most odonate species, morphological identification of exuviae is often time consuming, as it relies on small, fragile structures. To facilitate species identification of exuviae, a DNA barcoding approach was developed, including (1) non-destructive extraction of DNA using whole exuviae or their tracheal tubes, and (2) primer systems for long (< 600 bp) and short (< 200 bp) CO1 fragments. A total of 85 exuviae from 33 species were analysed and compared to results of morphological identification. Additionally, factors potentially influencing DNA quality and quantity, as well as PCR and sequencing success were investigated. Eighty-two exuviae matched the morphologically identified genus, and 60 matched at species level. Of the 33 species present in the data set, 82% could be identified to species level via DNA barcoding. The results show how DNA-based approaches can support fast and accurate species identification and therefore enhance monitoring of an ecologically important taxonomic group, with high relevance for conservation and habitat restoration. Moreover, the use of exuviae as DNA resource once more shows that non-invasive sampling offers great potential for molecular species identification, which is essential when studying rare and endangered species.
Implications for insect conservation
Our results show how molecular tools, here DNA barcoding of odonate exuviae, can support species monitoring without the need of catching individuals, harming, or even killing them. Obtaining DNA from non-invasive sources can thus be a direct advantage to the conservation of insects, especially when dealing with rare and endangered species and/or populations. Using the example of odonates as bioindicator organisms for aquatic and semi-aquatic habitats, we highlight the importance of non-invasive genetic approaches for population studies and monitoring of insect species and/or species communities for ecosystem assessments and conservation management.
... Chironomidae are also widely adopted as useful bioindicators in aquatic ecosystems because they are diverse in ecological traits and sensitive to environmental variables (Nicacio & Juen, 2015;Porinchu & MacDonald, 2003). Species-level identification of aquatic biota has been advocated in freshwater bioassessments because congeneric species can differ substantially in their biological traits (Krosch et al., 2015;Macher et al., 2016). However, chironomids are often identified as coarse taxonomic groups in ecological and paleolimnological studies (Beermann et al., 2018;Van Hardenbroek et al., 2011). ...
The main aim of this study was to curate a COI barcode library of Chironomidae from the Tibetan Plateau (TP) as an essential supplement to the public database. Another aim is to evaluate the current status of the public database of Chironomidae in aspects of taxonomic coverage, geographic representation, barcode quality, and efficiency for molecular identification, the Tibetan Plateau, China. In this study, 512 individuals of Chironomidae from the TP were identified based on morphological taxonomy and barcode analysis. The metadata of public records of Chironomidae were downloaded from the BOLD, and the quality of the public barcodes was ranked using the BAGS program. The reliability of the public library for molecular identification was evaluated with the newly curated library using the BLAST method. The newly curated library comprised 159 barcode species of 54 genera, of which 58.4% of species were likely new to science. There were great gaps in the taxonomic coverage and geographic representation in the public database, and only 29.18% of barcodes were identified at the species level. The quality of the public database was of concern, with only 20% of species being determined as concordant between BINs and morphological species. The accuracy of molecular identification using the public database was poor, and about 50% of matched barcodes could be correctly identified at the species level at the identity threshold of 97%. Based on these data, some recommendations are included here for improving barcoding studies on Chironomidae. The species richness of Chironomidae from the TP is much higher than ever recorded. Barcodes from more taxonomic groups and geographic regions are urgently needed to fill the great gap in the current public database of Chironomidae. Users should take caution when public databases are adopted as reference libraries for the taxonomic assignment.
... Exploratory DNA barcoding studies revealed the presence of a great number of previously overlooked morphologically cryptic species (Fi ser et al., 2018;Johnson et al., 2008;Kane et al., 2008;Katouzian et al., 2016;Nakano and Spencer, 2007;Pfenninger and Schwenk, 2007;Weiss et al., 2014). Furthermore, an increasing number of studies focusing on the ecology of cryptic species have revealed that there can be significant differences in their ecological demands as well as their resilience against stressors (Feckler et al., 2012;Fi ser et al., 2018;Macher et al., 2016), underlining the importance of correct species identification (Bickford et al., 2007;Fi ser et al., 2018;Pfenninger and Schwenk, 2007). For a long time, DNA-based species delineation relied only on single genetic markers, which can mislead species diversity estimates (Flot, 2015;Fontaneto et al., 2015;Padial et al., 2010;Pante et al., 2015). ...
DNA sequence information has revealed many morphologically cryptic species worldwide. For animals, DNA‐based assessments of species diversity usually rely on the mitochondrial cytochrome c oxidase subunit I (COI) gene. However, a growing amount of evidence indicate that mitochondrial markers alone can lead to misleading species diversity estimates due to mito‐nuclear discordance. Therefore, reports of putative species based solely on mitochondrial DNA should be verified by other methods, especially in cases where COI sequences are identical for different morphospecies or where divergence within the same morphospecies is high. Freshwater amphipods are particularly interesting in this context because numerous putative cryptic species have been reported. Here, we investigated the species status of the numerous mitochondrial molecular operational taxonomic units (MOTUs) found within Echinogammarus sicilianus. We used an integrative approach combining DNA barcoding with mate selection observations, detailed morphometrics and genome‐wide double digest restriction site‐associated DNA sequencing (ddRAD‐seq). Within a relatively small sampling area, we detected twelve COI MOTUs (divergence = 1.8–20.3%), co‐occurring in syntopy at two‐thirds of the investigated sites. We found that pair formation was random and there was extensive nuclear gene flow among the ten MOTUs co‐occurring within the same river stretch. The four most common MOTUs were also indistinguishable with respect to functional morphology. Therefore, the evidence best fits the hypothesis of a single, yet genetically diverse, species within the main river system. The only two MOTUs sampled outside the focal area were genetically distinct at the nuclear level and may represent distinct species. Our study reveals that COI‐based species delimitation can significantly overestimate species diversity, highlighting the importance of integrative taxonomy for species validation, especially in hyperdiverse complexes with syntopically occurring mitochondrial MOTUs.
... A low value of He will endanger the survival of a species or population, low value of genetic diversity also reflects a smaller population in the ecosystem (Ribeiro and Lopes, 2013), there are several causes of decreased genetic diversity of marine invertebrates such as overexploitation. Anthropogenic pollution, oil pollution, global warming, and tourism activities (Macher et al., 2016). High genetic diversity can be designed to estimate population size, because population size affects genetic diversity (Cao et al., 2018), differences between many orders of magnitude variation in population size and a much narrower distribution of diversity levels, related selection plays an important role both in Overall genetic diversity of a species as well as in the variation of diversity within the genome, genetic diversity also seems to be predictable from the life history of a species (Ellegren and Galtier, 2016). ...
Mangrove forests play an important role in macrozoobenthos ecology, assisting them in foraging, sheltering, and reproduction. In order to better understanding the genetic diversity and population genetic structure of macrozoobenthos, the mitochondrial 16S subunit (16S rRNA) gene was used. Present study aimed to determine genetic diversity and community structure of macrozoobenthos in the North Sumatra and Aceh mangrove forests, Indonesia. The reliaprep gDNA tissue miniprep system kit was used to extract 50 samples. To determine the PCR product's molecular weight, UVITEX software was employed. Genetic polymorphism was examined with GenAlex version 6.502, and each community underwent principal coordinates analysis (PCoA). MVSP 3.2.2 software and the unweighted pair group method with arithmetic mean (UPGMA) were used to construct the dendrogram. The 16S rRNA gene revealed a band size in the range of 370-500 bp. The PCoA showed that approximately all individual macrozoobenthos from each community structure were localized in the same quadrant. Genetic variation was varied by 3% between populations and 97.5% within individuals according to the analysis of molecular variance (AMOVA), with the dendrogram classifying the populations into two major clusters. Dendrogram analysis showed low genetic differentiation between macrozoobenthos populations of North Sumatra and the Aceh mangroves, implying the low ability of individuals in a population to adapt. The high polymorphic information content (PIC) value (0.886) reported was consistent with the number of alleles and size of the population's heterozygosity value. Present findings provide important information that will assist in formulation of mangrove conservation and restoration approaches.
... This can be a laborious practice, slowing the delivery of timely results to bioassessment programs. Moreover, misidentifications of closely related taxa with differing tolerances to environmental stressors could have consequences for bioassessment, introducing error and potentially compromising study conclusions (Macher et al., 2016). Thus, there is an unmet need to produce rapid, robust bioassessment data to improve river management. ...
... As indicated by the results from metabarcoding peDNA, these species rich groups could potentially contain sensitive taxa missed by morphological identification. Studies that rely on a coarse taxonomic resolution may fail to detect responses on the species or OTU level to stressors (Macher et al., 2016;Beermann et al., 2018) and since many biomonitoring programs are based on higher-level taxonomic identification (e.g., genus and family), anthropogenic impacts may be overlooked (Beermann et al., 2018). ...
The botanical compound rotenone is extensively used to eradicate populations and reduce the negative impacts of freshwater invasive animals. The method is controversial as non-target organisms often are negatively affected, but these effects are highly variable among taxa and may be difficult to monitor on species-level as many invertebrates are challenging or costly to identify using morphology. We investigated the effect of rotenone treatment on freshwater invertebrates in two Nordic lakes using both traditional morphological identification and DNA metabarcoding of the preservative ethanol DNA (peDNA). We used a before-after-control-impact design in assessing the effect of the rotenone treatment, with two treated and two control lakes, all sampled immediately before the rotenone treatment, immediately after and 1 year after the treatment. The two methods of identification gave different results: The peDNA based method detected 333 taxa, while 90 taxa were recorded by morphological identification. Twenty-eight taxa were in common for both methods. Both traditional morphological identification and identification using metabarcoding of peDNA showed a significant effect of the rotenone treatment on invertebrate community composition expressed as Bray-Curtis dissimilarities. Using the results from peDNA, the number of taxa decreased just after the treatment and increased again 1 year after the treatment in the treated lakes, indicating a short-term effect of the treatment and a subsequent recovery of the invertebrate community. We conclude that DNA metabarcoding of preservative ethanol has a promising potential to record effects of anthropogenic stressors such as rotenone treatment. However, as peDNA based metabarcoding missed several taxa, and detected DNA from species not living in the actual sampling sites, the method needs further improvement and should perhaps be used in combination with morphological-based identification until sufficiently refined.
... Species richness and abundance of sensitive and tolerant taxa have been the most commonly used metrics to determine the ecological impacts of macroinvertebrates below dams (Martínez et al., 2013). Using biotic indicators to detect the effects of dams on the macroinvertebrate community requires a profound knowledge of the species identities because even closely related species may have different tolerances to environmental stressors (Macher et al., 2016;Mezgebu, 2022). However, accurate identification of freshwater macroinvertebrates to the species level can be difficult, especially for larval or subadult specimens, and often results in low taxonomic resolution or misidentification (Haase et al., 2010;Sweeney et al., 2011). ...
The Ruzizi River, the outlet of Lake Kivu in the Albertine Rift, flows into Lake Tanganyika and is important for hydropower generation and irrigation. The impacts of 2 dams in the Ruzizi River on macroinvertebrate community composition and diversity were surveyed every 3 months from December 2015 to October 2017. Macroinvertebrate samples were collected at sites upstream and downstream and additionally at two sites further downstream of the dams, in both comparatively pristine and highly disturbed areas. Several indices (Shannon-Wiener index, Simpson index, Pielou’s evenness, Rare Family Prevalence, and Average Score Per Taxa) were used to determine the alpha diversity and evenness of macroinvertebrates at the family level. Our results showed little to no immediate effect of the dams on macroinvertebrate diversity. Macroinvertebrate composition differed slightly below the dams compared to upstream. Communities near Dam II had slightly higher diversity compared to Dam I, probably because the vicinity to Lake Kivu has an immediate effect on diversity upstream of the first dam and likely because Dam II is 30 years younger than Dam I. This study suggests the importance of using species-level indices to better understand the ecological impacts of dams on macroinvertebrate diversity of tropical rivers with low species diversity.
... Exploratory DNA barcoding studies revealed the presence of a great number of previously overlooked morphologically cryptic species [12][13][14][15][16][17][18]. Further, an increasing number of studies focusing on the ecology of cryptic species have revealed that there can be significant differences in their ecological demands as well as their resilience against stressors [18][19][20], underlining the importance of correct species identification [12,18,21]. For a long time, DNA-based species delineation relied only on single genetic markers which can lead to wrong species diversity estimates [22][23][24][25]. ...
Background
The emergence of DNA taxonomy sparked a paradigm shift in biodiversity assessments and revealed the existence of many morphologically cryptic species in all ecosystems worldwide. For animals, DNA-based assessments of species diversity usually rely on the mitochondrial cytochrome c oxidase subunit I (COI) gene. However, an increasing number of studies report patterns of mito-nuclear discordance originating for example from shared ancestral polymorphisms or introgression, leading to an over- or underestimation of true species diversity. Therefore, reports of mitochondrial lineages should be verified by other methods. Freshwater amphipods are ideal model organisms since putative cryptic species have been reported in many of the nominal taxa. In this study, we investigated the species status of the numerous mitochondrial molecular operational taxonomic units (MOTUs) found within the Mediterranean freshwater amphipod Echinogammarus sicilianus on Sicily. For validation, we used an integrative taxonomic approach combining DNA barcoding with behavioural data, detailed morphometrics and genome-wide double digest restriction site-associated DNA sequencing (ddRAD-seq).
Results
Within a relatively small sampling area (approx. 200 km²), we detected 12 divergent COI MOTUs, with genetic distances ranging from 1.8% to 20.3% and a mean distance of 11%. At 17 of the 25 sampling sites up to six MOTUs occurred in syntopy. The four most common MOTUs were analysed with high resolution morphometric data (49 measurements per specimen), but we found no clear evidence of morphological differentiation among them. We also observed a high level of MOTU intermixing (38%) in precopulatory pairs. Similarly, ten of the mitochondrial MOTUs were not supported by the genome-wide ddRAD-seq data. However, for the two remaining mitochondrial MOTUs found at sites outside of the main river stretch, ddRAD-seq and COI data were congruent, indicating that those MOTUs might indeed represent cryptic species.
Conclusions
Our study on the amphipod E. sicilianus reveals another prominent example of mito-nuclear discordance indicating that species delimitation based solely on mitochondrial markers can significantly overestimate species diversity. The study highlights that integrative taxonomy approaches are important for validating species status, especially in hyperdiverse species complexes, where many mitochondrial MOTUs occur in close proximity or even syntopy.
... Exploratory DNA barcoding studies revealed the presence of a great number of previously overlooked morphologically cryptic species [12][13][14][15][16][17][18]. Further, an increasing number of studies focusing on the ecology of cryptic species have revealed that there can be significant differences in their ecological demands as well as their resilience against stressors [18][19][20], underlining the importance of correct species identification [12,18,21]. For a long time, DNA-based species delineation relied only on single genetic markers which can lead to wrong species diversity estimates [22][23][24][25]. ...
Background
The emergence of DNA taxonomy sparked a paradigm shift in biodiversity assessments and revealed the existence of many morphologically cryptic species in all ecosystems worldwide. For animals, DNA-based assessments of species diversity usually rely on the mitochondrial cytochrome c oxidase subunit I (COI) gene. However, an increasing number of studies report patterns of mito-nuclear discordance originating for example from shared ancestral polymorphisms or introgression, leading to an over- or underestimation of true species diversity. Therefore, reports of mitochondrial lineages should be verified by other methods. Freshwater amphipods are ideal model organisms since putative cryptic species have been reported in many of the nominal taxa. In this study, we investigated the species status of the numerous mitochondrial molecular operational taxonomic units (MOTUs) found within the Mediterranean freshwater amphipod Echinogammarus sicilianus on Sicily. For validation, we used an integrative taxonomic approach combining DNA barcoding with behavioural data, detailed morphometrics and genome-wide double digest restriction site-associated DNA sequencing (ddRAD-seq).
Results
Within a relatively small sampling area (approx. 200 km²), we detected 12 divergent COI MOTUs, with genetic distances ranging from 1.8% to 20.3% and a mean distance of 11%. At 17 of the 25 sampling sites up to six MOTUs occurred in syntopy. The four most common MOTUs were analysed with high resolution morphometric data (49 measurements per specimen), but we found no clear evidence of morphological differentiation among them. We also observed a high level of MOTU intermixing (38%) in precopulatory pairs. Similarly, ten of the mitochondrial MOTUs were not supported by the genome-wide ddRAD-seq data. However, for the two remaining mitochondrial MOTUs found at sites outside of the main river stretch, ddRAD-seq and COI data were congruent, indicating that those MOTUs might indeed represent cryptic species.
Conclusions
Our study on the amphipod E. sicilianus reveals another prominent example of mito-nuclear discordance indicating that species delimitation based solely on mitochondrial markers can significantly overestimate species diversity. The study highlights that integrative taxonomy approaches are important for validating species status, especially in hyperdiverse species complexes, where many mitochondrial MOTUs occur in close proximity or even syntopy.
... In our previous studies, we found two-fold differences in sensitivities to pyrethroids across clades (B, C, and D) even within non-resistant, wild-type H. azteca (Weston et al., 2013;Major et al., 2018). Similar findings have been reported for clades of the cryptic mayfly species, Deleatidium, in response to agricultural stressors (Macher et al., 2016). Taken together, these findings suggest the potential for different detoxification capacity and thus toxicokinetics within species complexes, though studies with the H. azteca complex are absent. ...
Recent studies demonstrated pyrethroid resistance associated with voltage-gated sodium channel mutations in populations of the epibenthic amphipod, Hyalella azteca. Resistant populations were able to tolerate and bioconcentrate pyrethroids at concentrations significantly higher than toxic levels for non-resistant populations. In conjunction with elevated bioconcentration potential, environmental alteration particularly as a result of global climate change is anticipated to significantly alter abiotic parameters including temperature and salinity. These changes are expected to influence uptake and biotransformation of contaminants. Thus, the aims of the current study were a) to examine the bioconcentration potential of permethrin in two pyrethroid-resistant clades of H. azteca and b) assess the influence of temperature and salinity changes on toxicokinetic parameters. Two pyrethroid-resistant clades of H. azteca were exposed to ¹⁴C-permethrin at three salinities (0.2, 1.0 and 6.0 practical salinity units (PSU)) and temperatures (18, 23 and 28 °C). Tests were conducted for up to 36 h and uptake, elimination and biotransformation rates were calculated. Both populations demonstrated bioconcentration factors (BCFs) between five and seven times greater than published data for non-resistant H. azteca, with significant differences between clades. Calculated BCF values were comparable to field populations of resistant H. azteca, emphasizing the potential for elevated pyrethroid bioconcentration in the natural environment and increased exposure for predators consuming pyrethroid-resistant aquatic invertebrates. Alterations to temperature and salinity had no statistically significant effect on uptake or parent compound half-life in either population, though biotransformation was elevated at higher temperatures in both populations. Salinity had a variable effect between the two populations, with lower BCF values at 1.0 PSU in clade D H. azteca and greater BCFs at 6.0 PSU in clade C H. azteca. This is the first study to demonstrate the potential for future climate scenarios to influence toxicokinetics in pyrethroid-resistant aquatic organisms.
... The finer taxonomic resolution achieved with genetic techniques will improve our ability to identify natural/anthropogenic impacts on biotic communities, because more precise species-specific sensitivity scores can be obtained for different stressors. For instance, cryptic species of the mayfly genus Deleatidium identified with genetic techniques showed contrasting responses to nutrient and sediments levels in New Zealand rivers (Macher et al., 2016). Furthermore, haplotypes (a measure of the relatedness among individuals of the same species, which can be inferred from metabarcoding data; Elbrecht et al., 2018) could be used to characterize dispersal patterns of organisms and to detect genetic 'bottlenecks' (in which population size and thus genetic diversity decline sharply due an event such as a drought) in IRES, thus informing management actions taken to conserve target species. ...
... Previous research has shown that MOTU-based approaches can provide better resolution in impact assessments, such as with undescribed cryptic diversity demonstrating contrasting responses to stressors (Macher et al., 2016), or reference databases being unable to identify all the encountered molecular variation (Beermann et al., 2018). Several studies have shown that MOTU-based assessment methods can accurately predict stressor impact on water systems (e.g., Andújar et al., 2018;Li et al., 2018). ...
Freshwater habitats are under stress from agricultural land use, most notably the influx of neonicotinoid pesticides and increased nutrient pressure from fertilizer. Traditional studies investigating the effects of stressors on freshwater systems are often limited to a narrow range of taxa, depending heavily on morphological expertise. Additionally, disentanglement of multiple simultaneous stressors can be difficult in field studies, whereas controlled laboratory conditions do not accurately reflect natural conditions and food webs. To overcome these drawbacks, we investigated the impacts of two agricultural stressors (the neonicotinoid insecticide thiacloprid and fertilizer) in full-factorial design in a semi-natural research site, using environmental DNA sampling to study three different taxonomic groups representing three trophic levels: bacteria (decomposers), phytoplankton (primary producers), and chironomids (consumers). The results show considerable impact of both stressors across trophic levels, with an additive effect of fertilizer and thiacloprid on community composition at all levels. These findings suggest that agricultural stressors affect the entire food web, either directly or through cascade reactions. They are also consistent with morphological assessments that were performed in the same study site, even at a lower number of replicates. The study presented shows that the use of multi-marker environmental DNA provides a more comprehensive assessment of stressor impacts across multiple trophic levels, at a higher taxonomic resolution than traditional surveys. Additionally, many putative novel bio-indicators for both agricultural stressors were discovered. We encourage further investigations into stressors impacts at different trophic levels, which will lead to more effective monitoring and management of freshwater systems.
... Macroinvertebrates are a group of major interest for biomonitoring, particularly in river ecosystems where they are an important component of environmental assessment (Seymour et al., 2016) and indicators of functional change (Heino et al., 2004). Several recent studies have shown stark differences in data from eDNA methods and traditional macroinvertebrate sampling; however, the general biodiversity assessment patterns (e.g., assessment scores, inter-community variation) are often found to be similar (Macher et al., 2016). A major advantage of eDNA sampling over traditional macroinvertebrate sampling is the ability to sample water from any type of freshwater environment, including lakes, as well as both deep and fast-flowing rivers. ...
Aim: The aim of this chapter is to introduce the concepts and applications of environmental DNA (eDNA) for species detection and biomonitoring of freshwater ecosystems. Environmental assessment of inland waters is currently undergoing a revolution due to the increased utilization of eDNA and major advancements in molecular techniques. Several aspects of ecology and conservation biology across the academic, private and government sectors are already utilizing eDNA-based approaches with more applications being rapidly developed. Therefore, this chapter disseminates current fundamental understanding of the dynamics and applications of eDNA in freshwater environments.
Main concepts covered: Environmental DNA (eDNA) is DNA extracted from environmental samples without targeting a particular organism or group of organisms. Within the realm of inland waters, eDNA samples typically include water, sediment or biofilm samples, though there is potential for other environmental sources. The ecology of eDNA (e.g., transport, degradation rate, molecular state) greatly affects the detectability of eDNA, as well as the interpretation of the results drawn from eDNA-based assessment. In particular, understanding the difference between eDNA detection dynamics in lotic (e.g., rivers and streams) versus lentic (e.g., ponds and lakes) environments is key to understanding and applying eDNA-derived information.
Main methods covered: A major characteristic of eDNA-based research is the non-targeted aspect of species and community detection. It is therefore paramount that an understanding of the sampling and DNA extraction methods are outlined, and that concerns regarding potential inhibition (false negatives) and contamination sources (false positives) are addressed. The use of eDNA also requires additional experimental design considerations, particularly regarding replication and spatial resolution, due to the need to cross validate findings and the increased complexity of the data created compared to traditional taxonomic-based approaches.
Currently, eDNA-based research can be divided into two main groups, population- and community-based analyzes. Population-based analyzes rely primarily on single-targeted (i.e., species) methods such as qPCR, which are lower cost and easier for smaller institutes or individuals to independently implement. Community-based approaches rely largely on high throughput sequencing (HTS) and require additional molecular and bioinformatics specialization and support, but result in greater potential for data generation and analytical power, given the proper study design.
Future eDNA work will include applying PCR free-based methods to population analyzes and combining multi-dimensional environmental data for environmental community analyzes. Other advances in eDNA research may look to assess the transcriptional profiles of eDNA samples to assess functional community diversity.
Conclusion/Outlook: This chapter provides an overview of current molecular and eDNA-based approaches for inland water assessment. There are many aspects of eDNA that are still largely unknown, but the ability to apply standardized non-invasive sampling with high throughput data is hard to ignore in the modern age.
... This can be a laborious practice, slowing the delivery of timely results to bioassessment programs. Moreover, misidentifications of closely related taxa with differing tolerances to environmental stressors could have consequences for bioassessment, introducing error and potentially compromising study conclusions (Macher et al., 2016). Thus, there is an unmet need to produce rapid, robust bioassessment data to improve river management. ...
River bioassessment programs require robust methods to accurately observe the status of aquatic biodiversity, as it is well understood that physico-chemical monitoring alone is not sufficient to support current policy and management objectives. Whereas traditional microscopy-based identification of organisms can be expensive and laborious, direct sampling of environmental DNA, coupled with high-throughput sequencing technologies, now provides a rapid and accurate alternative to support bioassessment analysis and interpretation. Here, we discuss the various insights that can be generated from eDNA, illustrate how river monitoring can benefit from eDNA approaches, knowledge gaps, and how this can support bioassessment from local to national and international scales.
... When using molecular methods for species identification and delimitation, generally from the variation in the cytochrome c oxidase subunit I (COI) gene (widely applied for freshwater animals [13][14][15][16]), most studies involve evaluation of genetic distances between populations and application of species delimitation algorithms such as jMOTU, GMYC, and ABGD, which diagnose the difference between intra-and inter-specific variability by statistical criteria [17][18][19]. Although these methods have been widely tested in aquatic taxa [5,[20][21][22][23][24], only a few studies use them in a comparative setting, i.e., simultaneously on several co-distributed freshwater species from the same area. ...
Comparative phylogeography has become a powerful approach in exploring hidden or cryptic diversity within widespread species and understanding how historical and biogeographical factors shape the modern patterns of their distribution. Most comparative phylogeographic studies so far focus on terrestrial and vertebrate taxa, while aquatic invertebrates (and especially freshwater invertebrates) remain unstudied. In this article, we explore and compare the patterns of molecular diversity and phylogeographic structure of four widespread freshwater copepod crustaceans in Eu-ropean water bodies: the harpacticoids Attheyella crassa, Canthocamptus staphylinus and Nitokra hiber-nica, and the cyclopoid Eucyclops serrulatus, using sequence data from mtDNA COI and nuclear ITS/18S rRNA genes. The three taxa A. crassa, C. staphylinus and E. serrulatus each consist of deeply diverged clusters and are deemed to represent complexes of species with largely (but not completely) non-overlapping distributions, while in N. hibernica only little differentiation was found, which may however reflect the geographically more restricted sampling. However, the geographical patterns of subdivision differ. The divisions in A. crassa and E. serrulatus follow an east-west pattern in Northern Europe whereas that in C. staphylinus has more of a north-south pattern, with a distinct Fennoscandian clade. The deep mitochondrial splits among populations of A. crassa, C. staphylinus and E. serrulatus (model-corrected distances 26-36%) suggest that divergence of the lin-eages predate the Pleistocene glaciations. This study provides an insight into cryptic diversity and biogeographic distribution of freshwater copepods.
... However, since many organisms respond to environmental characteristics and their changes, it is clear that the number of bioindicator candidates is potentially extremely high-thousands, at least-which complicates their selection. Choosing the "right" bioindicator is by no means trivial because its use will affect policy and management decisions, and undoubtedly, using too many bioindicators may generate contrasting results and be confusing (e.g., [3]). What bioindicator should be used is a sensitive matter that too often leads to partisan arguments, as well as to attempts to "sell" a given organism as a bioindicator because raising its public image in terms of "usefulness" would support its protection. ...
Bats show responses to anthropogenic stressors linked to changes in other ecosystem components such as insects, and as K-selected mammals, exhibit fast population declines. This speciose, widespread mammal group shows an impressive trophic diversity and provides key ecosystem services. For these and other reasons, bats might act as suitable bioindicators in many environmental contexts. However, few studies have explicitly tested this potential, and in some cases, stating that bats are useful bioindicators more closely resembles a slogan to support conservation than a well-grounded piece of scientific evidence. Here, we review the available information and highlight the limitations that arise in using bats as bioindicators. Based on the limited number of studies available, the use of bats as bioindicators is highly promising and warrants further investigation in specific contexts such as river quality, urbanisation, farming practices, forestry, bioaccumulation, and climate change. Whether bats may also serve as surrogate taxa remains a controversial yet highly interesting matter. Some limitations to using bats as bioindicators include taxonomical issues, sampling problems, difficulties in associating responses with specific stressors, and geographically biased or delayed responses. Overall, we urge the scientific community to test bat responses to specific stressors in selected ecosystem types and develop research networks to explore the geographic consistency of such responses. The high cost of sampling equipment (ultrasound detectors) is being greatly reduced by technological advances, and the legal obligation to monitor bat populations already existing in many countries such as those in the EU offers an important opportunity to accomplish two objectives (conservation and bioindication) with one action.
... Nevertheless, original ESVs list lacks this complete species information, and not all ESVs will obtain accurate and effective taxonomic annotation. Coarse taxonomic resolution is insufficient to reveal specific responses of benthic macroinvertebrates to stressors (Macher et al., 2016); thus, higher taxonomic resolution is indispensable for the assessments of status and trends in assemblages of benthic invertebrates (Jones, 2008), such as life history and functional diversity. In this study, after bioinformatic processing and purposefully screening, assigned ESVs were less than expected. ...
Aim
High‐throughput pipelines supported by eDNA metabarcoding have been applied in various freshwater ecosystems. Both eDNA in ethanol (EtOH) samples (ES‐eDNA) and in water samples (WS‐eDNA) can provide comprehensive classification lists with good taxonomic resolution and coverage for determining freshwater biodiversity and biomonitoring. But, the advantages of ES‐eDNA metabarcoding over WS‐eDNA metabarcoding remain unclear for routine assessments of diversity of benthic macroinvertebrates in streams.
Location
Qiantang River Basin, China.
Methods
Here, we compared ES‐eDNA and WS‐eDNA metabarcoding to evaluate the performance of two eDNA workflows in determining biodiversity and recovery of damaged macroinvertebrate communities. All eDNA samples from the environment and bulk specimen of macroinvertebrates were processed into available molecular operational taxonomic units (MOTUs) and identified to the level of genus.
Results
WS‐eDNA detected more exact sequence variants (ESVs) (formerly referred to as operational taxonomic units; OTUs), than did ES‐eDNA (2,866 vs. 2,406), but fewer macroinvertebrate ESVs (381 vs. 481). Among sampling sites, the two eDNA workflows exhibited relatively large dissimilarity on inferred community composition (p < .001). Furthermore, ES‐eDNA metabarcoding exhibited more consistent with morphological identification approaches than did WS‐eDNA metabarcoding (24.24% vs. 17.63%, p = .002), especially for species identified by traditional morphology (morphotaxa).
Main conclusions
Based on the attributes of ES‐eDNA and WS‐eDNA, it is suggested that ES‐eDNA metabarcoding performs better than does WS‐eDNA metabarcoding in detecting local biodiversity and was consistent with morphological results, while WS‐eDNA was more suitable for exploring biodiversity patterns on a broad scale, as it is the easiest and most convenient way to collect samples. Results of this study suggest ES‐eDNA metabarcoding could be an option in building molecular measurement biomonitoring programme based on EtOH sample used for preserving biological samples.
... Macroinvertebrates have been frequently utilized to monitor the water quality worldwide (Chandler, 1970;Helson & Willians, 2013), especially for those water bodies (e.g., rivers and lakes) affected by anthropogenic activities (Wang et al., 2006;Zhang et al., 2011). However, these monitoring functions depend, to a large extent, on the accuracy and precision of species/ specimen identification (Frézal & Leblois, 2008;Macher et al., 2016). ...
Macroinvertebrates have been recognized as key ecological indicators of aquatic environment and are the most commonly used approaches for water quality assessment. However, species identification of macroinvertebrates (especially of aquatic insects) proves to be very difficult due to the lack of taxonomic expertise in some regions and can become time‐consuming. In this study, we evaluated the feasibility of DNA barcoding for the classification of benthic macroinvertebrates and investigated the genetic differentiation in seven orders (Insecta: Ephemeroptera, Plecoptera, Trichoptera, Diptera, Hemiptera, Coleoptera, and Odonata) from four large transboundary rivers of northwest China and further explored its potential application to biodiversity assessment. A total of 1,144 COI sequences, belonging to 176 species, 112 genera, and 53 families were obtained and analyzed. The barcoding gap analysis showed that COI gene fragment yielded significant intra‐ and interspecific divergences and obvious barcoding gaps. NJ phylogenetic trees showed that all species group into monophyletic species clusters whether from the same population or not, except two species (Polypedilum. laetum and Polypedilum. bullum). The distance‐based (ABGD) and tree‐based (PTP and MPTP) methods were utilized for grouping specimens into Operational Taxonomic Units (OTUs) and delimiting species. The ABGD, PTP, and MPTP analysis were divided into 177 (p = .0599), 197, and 195 OTUs, respectively. The BIN analysis generated 186 different BINs. Overall, our study showed that DNA barcoding offers an effective framework for macroinvertebrate species identification and sheds new light on the biodiversity assessment of local macroinvertebrates. Also, the construction of DNA barcode reference library of benthic macroinvertebrates in Eurasian transboundary rivers provides a solid backup for bioassessment studies of freshwater habitats using modern high‐throughput technologies in the near future.
... Given the importance of fine sediment as a stressor and the lack of long-term datasets both nationally and internationally, we did not want to compromise the large spatial and multi-year temporal nature of our study. Moreover, two earlier regional-scale surveys that used data from Southland waterways collected with a 'snapshot' approach (Wagenhoff et al. 2011;Macher et al. 2016) had already included nutrients as a stressor. ...
Macroinvertebrate communities in running waters are commonly used as bioindicators of fine sediment pollution, but few studies evaluate impacts across multiple years. We used a 5-year dataset from 46 rivers in Southland, New Zealand to investigate the consistency of the relationship between deposited fine sediment and stream macroinvertebrates across three categories of agricultural land-use intensity (low, medium, and high). We also compared the performance of four widely used invertebrate stream health metrics and their recently developed sediment-specific counterparts. Linear and non-linear regressions were fitted and effect sizes were interpreted to identify biologically meaningful relationships (r ² ≥ 0.1). Sites within medium-intensity catchments showed the greatest number of such relationships (29 of 40 cases), compared to low- (8) or high-intensity catchments (23). Invertebrate metrics responded more frequently, and mostly negatively, to increasing sediment in medium- and high-intensity catchments. Overall, sediment-specific metrics performed better than their widely used counterparts. Our findings show that land-use intensity influences the multi-year dynamics of deposited fine sediment and the corresponding stream invertebrate responses. These temporal dynamics can be substantial and should be considered in future stream biomonitoring efforts.
... Por otro lado, la mayor parte de índices biológicos basados en macroinvertebrados utilizan el nivel taxonómico de familia, debido a su mayor simplicidad de identificación y a que cada familia tiene tolerancias ambientales específicas. Una identificación taxonómica a nivel de especie permitiría trabajar con métricas que podrían ser más sensibles a perturbaciones antrópicas (Bonada et al., 2006;Macher et al., 2016), pero también a perturbaciones naturales como la temporalidad del flujo en los RTs. Además, su identificación a nivel de especie no siempre es posible, ya que en ocasiones los taxones aún no están descritos en las guías taxonómicas estandarizadas (Sweeney et al., 2011). ...
... Even undescribed and morphologically cryptic species can be distinguished with this approach (113), which is unlikely to be possible with deep learning. This is of great importance as morphologically similar species can have distinct ecological preferences (114), and thus, distinguishing them unambiguously is important for monitoring, ecosystem assessment, and conservation biology. However, mass sequencing-based molecular methods cannot provide precise abundance or biomass estimates and assign sequences to individual specimens (12). ...
Most animal species on Earth are insects, and recent reports suggest that their abundance is in drastic decline. Although these reports come from a wide range of insect taxa and regions, the evidence to assess the extent of the phenomenon is sparse. Insect populations are challenging to study, and most monitoring methods are labor intensive and inefficient. Advances in computer vision and deep learning provide potential new solutions to this global challenge. Cameras and other sensors can effectively, continuously, and noninvasively perform entomological observations throughout diurnal and seasonal cycles. The physical appearance of specimens can also be captured by automated imaging in the laboratory. When trained on these data, deep learning models can provide estimates of insect abundance, biomass, and diversity. Further, deep learning models can quantify variation in phenotypic traits, behavior, and interactions. Here, we connect recent developments in deep learning and computer vision to the urgent demand for more cost-efficient monitoring of insects and other invertebrates. We present examples of sensor-based monitoring of insects. We show how deep learning tools can be applied to exceptionally large datasets to derive ecological information and discuss the challenges that lie ahead for the implementation of such solutions in entomology. We identify four focal areas, which will facilitate this transformation: 1) validation of image-based taxonomic identification; 2) generation of sufficient training data; 3) development of public, curated reference databases; and 4) solutions to integrate deep learning and molecular tools.
... It can possibly be further improved by adding further variability, yet too many degenerate bases also limit amplification success (see Table S2 for primers not working). The increased resolution for ecologically important taxa such as chironomids and oligochaetes can be of immense relevance for biomonitoring (Beermann et al., 2018;Macher et al., 2016;Milošević et al., 2013;Vivien et al., 2020;Vivien et al., 2015Vivien et al., , 2016. They dominated eDNA signals here and were often site-specific. ...
DNA metabarcoding of freshwater communities typically relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase I (COI) gene with degenerate primers. The advantage of COI is its taxonomic resolution and the availability of an extensive reference database. However, when universal primers are used on environmental DNA (eDNA) isolated from water, benthic invertebrate read and OTU numbers are typically “watered down,” that is, under represented, compared to whole specimen “bulk samples” due to greater co‐amplification of abundant nontarget taxa (e.g., fungi, algae, and bacteria). Because benthic stream invertebrate taxa are of prime importance for regulatory biomonitoring, more effective ways to capture their diversity via eDNA isolated from water are important. In this study, we aimed to improve benthic invertebrate assessment from eDNA by minimizing nontarget amplification. Therefore, we generated eDNA data using universal primers BF2/BR2 on samples collected throughout 15 months from a German Long‐Term Ecological Research site (Rhine‐Main‐Observatory, Kinzig River) to identify most abundant nontarget taxa. Based on these data, we designed a new reverse primer (EPTDr2n) with 3’‐specificity toward benthic invertebrate taxa and validated its specificity in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. We then performed in situ tests using 20 Kinzig River eDNA samples. We found that the percentage of target reads was much higher for the new primer combination compared to two universal benthic invertebrate primer pairs, BF2/BR2 and fwhF2/fwhR2n (99.6% versus 25.89% and 39.04%, respectively). Likewise, the number of detected benthic invertebrate species was substantially higher (305 versus 113 and 185) and exceeded the number of 153 species identified by expert taxonomists at nearby sites across two decades of sampling. While few taxa, such as flatworms, were not detected, we show that the optimized primer avoids the nontarget amplification bias and thus significantly improves benthic invertebrate detection from eDNA.
... species complex, supporting the existence of six geographically relevant species from our study (with a seventh in Asia). Recent evidence from the study of other mayfly species found fine-scale ecological differences among cryptic species detected with molecular methods (Leys et al., 2016;Macher et al., 2016), lending support to the ecological and evolutionary significance of these and other DNA-based findings. Another widespread species, Baetis harrisoni, was also found to consist of several cryptic species (Pereirada-Conceicoa et al., 2012). ...
The study of processes driving diversification requires a fully sampled and well resolved phylogeny. Multilocus approaches to the study of recent diversification provide a powerful means to study the evolutionary process, but their application remains restricted because multiple unlinked loci with suitable variation for phylogenetic or coalescent analysis are not available for most non-model taxa. Here we identify novel, putative single-copy nuclear DNA (nDNA) phylogenetic markers to study the colonization and diversification of an aquatic insect species complex, Cloeon dipterum L. 1761 (Ephemeroptera: Baetidae), in Macaronesia. Whole-genome sequencing data from one member of the species complex were used to identify 59 nDNA loci (32,213 base pairs), followed by Sanger sequencing of 29 individuals sampled from 13 islands of three Macaronesian archipelagos. Multispecies coalescent analyses established six putative species. Three island species formed a monophyletic clade, with one species occurring on the Azores, Europe and North America. Ancestral state reconstruction indicated at least two colonization events from the mainland (Canaries, Azores) and one within the archipelago (between Madeira and the Canaries). Random subsets of the 59 loci showed a positive linear relationship between number of loci and node support. In contrast, node support in the multispecies coalescent tree was negatively correlated with mean number of phylogenetically informative sites per locus, suggesting a complex relationship between tree resolution and marker variability. Our approach highlights the value of combining coalescent-based phylogeography, species delimitation, and phylogenetic reconstruction to resolve recent diversification events in an archipelago species complex.
As the gentle ripples of our aquatic realms caress the shores of scientific discovery, there lies a mesmerizing world waiting to be unveiled—a realm teeming with life, complexity, and ecological marvels. Within this realm, where the seemingly small and insignificant hold the keys to vast ecological mysteries, Aquatic Invertebrates and Ecosystem Research (AIER) stands as a beacon, illuminating the depths of our understanding. Edited by a consortium of passionate experts, AIER serves as the sacred codex of aquatic invertebrate science, offering a sanctuary for the dissemination of high-quality, original research that spans the breadth of this captivating field. From taxonomy and systematics to climate change impacts and socio-ecological systems, every page of AIER echoes with the symphony of scientific inquiry, inviting readers to embark on a journey of exploration and enlightenment. At the heart of AIER's mission lies a commitment to unraveling the intricate tapestry of aquatic ecosystems—a mission that resonates with the ethos of the publisher. With unwavering dedication, AIER strives to publish papers that delve into the nuances of ecology and behavior, the intricacies of physiological adaptations, and the profound implications of biodiversity conservation.
The site of study is wadi El Harrach (north-central Algeria), located in the capital of Algeria. The samples were tested by means of physicochemical analysis, carried out in situ and in the laboratory. This study aims to study the effect of water quality on the distribution of macroinvertebrates. According to SEQ-Water standards, we were able to highlight a spatial differentiation according to the different alterations in the physicochemical quality of the water. Thus, the river was divided into three sectors: Unpolluted sector: It has a biological capacity to harbor a strong taxonomic diversity in which the electrical conductivity values are around 529 µS cm−1 in H2. As for dissolved oxygen, it varies between 7 and 9 mg l−1. In these stations, we have an important diversity of macroinvertebrates, mostly the pollution-sensitive taxon (Plecoptera). Moderately polluted sector: Their biological aptitude to harbor the macrofauna is weak compared to the first stations. The electrical conductivity reached 3560 µS cm−1 in H4. These values are explained by the releases from the thermal station in the Hammam Melouene village. Polluted sector: The degree of pollution is variable between these stations. In this sector, we have collected an abundance of the pollution-tolerant taxon as an example of the Chironomus genus. In this study, the degrees of mineralization and oxidizable materials are the most relevant alterations in the assessment of the water quality of the El Harrach River and the identification of the types of pollution.
Gammarus is the largest genus of aquatic crustaceans in the family Gammaridea, with more than 200 species worldwide. These crustaceans are very important components of the food chain in running waters. Therefore, their population changes can affect all communities, especially the health of fish stocks, and disturb the balance of the ecosystem. Due to their small size, high abundance and sensitivity to environmental conditions, they are suitable as indicator species for evaluation. This study investigated the genetic diversity of the genus Gammarus between different populations in Golestan National Park. After sampling, the common identification keys were used to identify the basic morphology and the COI mitochondrial gene sequence was used to conduct molecular studies. Genealogical relationships were determined by maximum likelihood (ML) method. The genetic distance in the populations and some indices of genetic diversity (number of haplotypes (h), haplotype diversity (Hd) and nucleotide diversity (Pi)) were calculated. Based on the results of the genealogical trees, the samples were placed in two separate clades (western and central) and separated from the gene bank samples with high bootstrap. The results of molecular variance analysis showed that the diversity between populations (72.49) is more than the diversity within populations (27.51) and the value of Fst index (0.68) shows the significant genetic difference between populations and the limitation of gene flow. Haplotype diversity is equal to 0.94, the highest and lowest of which is related to Khandushan River (1.00) and Golestan Spring (.40). Also, among the identified haplotypes, 17 haplotypes had only one individual, which indicates the high diversity of Gammarus populations in Golestan National Park. Also, G. komareki was identified as a dominant species complex with many hidden species in the region that have not yet been accurately identified.
This survey reports the spatial distribution of gastropods belonging to Caenogastropoda, Architaenioglossa, Littorinimorpha, Cycloneritida and Hygrophila orders, and malacostracans from Amphipoda and Mysida orders in the lower sector of the Danube River, Romania, using DNA barcoding based on the cytochrome C oxidase I (COI) gene sequence. Sampling was performed for eight locations of Danube Delta branches and Bechet area during three consecutive years (2019–2021). Molecular identification of sixteen gastropods and twelve crustacean individuals was confirmed to the species level, providing the first molecular identification of gastropods from the Lower Danube sector. Phylogenetic analysis showed that species of gastropods and crustaceans clustered in monophyletic groups. Among gastropods, Microcolpia daudebartii acicularis, Viviparus viviparus, Bithynia tentaculata, Physa fontinalis, Ampullaceana lagotis and Planorbarius corneus were identified in Chilia and Sulina branches; and the Bechet area was populated by Holandriana holandrii, Theodoxus transversalis and Gyraulus parvus. The amphipods and mysids were present along the three main Danube branches. The calculated density of these species revealed an abundant community of crustacean Chelicorophium robustum on Sulina branch, and Dikerogammarus haemobaphes and D. villosus in extended areas of the Danube Delta. The presence of these invertebrates along Danube River was reported in relation to the sediment type and water depth.
Accurate quantification of biodiversity can be demanding and expensive. Although environmental DNA (eDNA) metabarcoding can facilitate biodiversity assessments through non‐invasive, cost‐efficient and rapid surveys, the approach struggles to outperform traditional morphological approaches in providing reliable quantitative estimates for surveyed species (e.g. abundance and biomass).
We present an integrated methodology for improving biodiversity surveys that pairs eDNA metabarcoding with morphological data, following a series of taxonomic and geographical filters. We demonstrate its power by applying it to a new spatiotemporal dataset generated on an Iberian‐wide distributed aquatic mesocosm infrastructure that spans a wide biogeographical gradient.
By building upon the strengths that these two approaches offer, our framework improved taxonomic resolution for 30% of the taxa and enabled species’ traits (e.g. body size) and abundance to be assigned to 85% of the taxa in hybrid datasets.
These results indicate that eDNA‐based assessments can complement, but not always replace, conventional approaches. Integrating conventional and modern eDNA metabarcoding approaches, already available in the ecologist's toolbox, will greatly enhance biodiversity assessments.
The study aimed to define the classes in the Heavy Metals Section of the Regional Environmental Ranking System based on long-term data (1997-2017). When distinguishing water quality classes, the following factors were taken into account: background content of heavy metals, content of heavy metals in water bodies exposed to different levels and character of anthropogenic pollution, and response of biological communities to toxic pollution of their natural habitats. According to the proposed classification, the non-contaminated water of Сlass 1 contains the following: Cd<0.2, Cu<2.5, Zn<4, Pb<3, Cr<0.5, and Ni<0.5 μg dm-3. Class 2 water is characterized by Cd<0.5, Cu<6, Zn<6, Pb<7, Cr<1, and Ni<2 μg dm-3 content. Moderately polluted water of Class 3 contains Cd<3, Cu<10, Cr<10, Ni<10, Zn<20, and Pb<20 μg dm-3. Concentrations of all heavy metals increase proportionally and exceed 30-100 μg dm-3 in the most polluted water of Class 6. The proposed methodological approach assesses not only the local content of heavy metals in water bodies of Kazakhstan but also the degree of toxic pollution of their vast catchment basins. The proposed methods are applicable to other arid regions with similar physical and climatic conditions.
The Water Framework Directive – WFD (2000/60/EC) is the cornerstone of European Union water policy. Its main objective is to protect and enhance the status of aquatic ecosystems and promote sustainable water use across Europe. The WFD summarises much of the European experience of pollution, water quality and ecosystem management, and it represents a new and compressive way of source-to-sink thinking, where the primary goals are to achieve the desired quality of the water resources and to ensure that there is enough clean water for different use. This way of thinking is widely accepted and the WFD is one of the most substantial and ambitious pieces of European environmental legislation to date. However, twenty years after the WFD was introduced, achieving its objectives remains a challenge, with 60% of EU surface waters failing the good ecological status in 2018. There is growing concern that the objective of good status, or higher, in all EU waters (i.e. over 111.000 surface waters and over 13.000 groundwaters) by 2027 is a long way from being achieved in many countries. Since sustainable water management will play an important role towards allowing mankind to adopt to its altered environment, more than ever management of this vital resource requires a truly integrated approach, taking account of the environmental, social, economic and health dimensions. Integrated water resource management is never easy to achieve, but successful examples demonstrate that real progress can be made. In this paper we discuss how science and water policy can incorporate innovations and adapt to the newly identified challenges (as biodiversity and springs ecosystems) facing European waters.
To assess and manage human impacts on freshwater ecosystems is a central challenge in the present era of biodiversity loss. Macroinvertebrates are an ideal group for Environmental Impacts Assessment (EIA). However, generating accurate macroinvertebrate inventories is difficult and error-prone based on larval morphology. Here, DNA metabarcoding provides new opportunities. Its potential to accurately identify bulk invertebrates on species level has been demonstrated in several case studies. However, DNA based identification is often limited by primer bias, potentially leading to taxa in the sample remaining undetected. Thus, the success of DNA metabarcoding as an emerging technique for EIA critically relies on carefully evaluated primers. We used the R package PrimerMiner to obtain and process cytochrome c oxidase I (COI) sequence data for the 15 globally most relevant freshwater invertebrate groups in EIAs. Using these sequence alignments, we developed four primers combinations optimized for freshwater macrozoobenthos. All primers were evaluated by sequencing ten mock community samples each consisting of 52 freshwater invertebrate taxa. Additionally popular metabarcoding primers from the literature and the here developed primers were tested in silico against the 15 relevant invertebrate groups. The developed primers varied in amplification efficiency and the amount of detected taxa, yet all retrieved more taxa than standard ‘Folmer’ barcoding primers. Two new primer combinations showed more consistent amplification than a previously tested ribosomal marker (16S) and detected all 42 insect taxa present in the mock community samples. In silico evaluation revealed critical design flaws in some commonly used primers from the literature. We demonstrate a reliable strategy to develop optimized primers using the tool PrimerMiner. The developed primers detected almost all taxa present in the mock samples, and we argue that high base degeneracy is necessary to decrease primer bias as confirmed by experimental results and in silico primer evaluation. We further demonstrate that not all primers currently used in metabarcoding studies are likely not suitable for amplification of insect and freshwater taxa. Thus careful primer evaluation and more region / ecosystem specific primers might be needed, before DNA metabarcoding can be used for routine EIA of freshwater ecosystems.
Environmental DNA metabarcoding has become a popular tool for the assessment of freshwater biodiversity, but it is largely unclear how sampling time and location influence the assessment of communities. Abiotic factors in rivers can change on small spatial and temporal scale and might greatly influence eDNA metabarcoding results. In this study, we sampled three German rivers at four locations per sampling site: 1. Left river side, surface water 2. Right river side, surface water, 3. Left side, close to the riverbed, 4. Right side, close to the riverbed. For the rivers Ruhr and Möhne, sampling was conducted three times in spring, each sampling one week apart. The Ruhr was again sampled in autumn and the Gillbach was sampled in winter. Sequencing on an Illumina MiSeq with COI primers Bf2/BR2 revealed diverse communities (6493 Operational taxonomic units, OTUs), which largely differed between rivers. Communities changed significantly over time in the Ruhr, but not in the Möhne. Sampling location influenced recovered communities in the Möhne and in the Ruhr in autumn. Our results have important implications for future eDNA studies, which should take into account that not all eDNA in rivers is everywhere and not at all times.
Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.
The assessment of biological samples is critical for measuring the ‘health’ of New Zealand aquatic environments. Analysis of these samples commonly requires species identification and enumeration, which usually involves microscopy or microbiological methods. These techniques can be time-consuming, laborious, and are dependent on taxonomic expertise. Recent advances in molecular methods provide promising tools for assessing environmental samples. A range of molecular techniques are now used in New Zealand including: fluorescent in situ hybridisation; automated ribosomal intergenic spacer analysis; quantitative polymerase chain reaction; and, most recently, next-generation sequencing. The organisms (or targets) and environments monitored are equally diverse, ranging from cyanobacteria, rotifers and invasive fish in lakes, to macroinvertebrates, and biofilm communities in rivers, to bacteria, micro- and macro-algae and invertebrates in marine ecosystems. Despite research and validation demonstrating their potential, the application of these tools by monitoring agencies has been limited. Legislative requirements, costs, and a reluctance to change methodologies, are the most likely reasons for this. This review examines molecular tools that have been previously or are currently used for monitoring aquatic environments in New Zealand, and explores how these, and new techniques, may be applied in the future.
Metabarcoding is an emerging genetic tool to rapidly assess biodiversity in ecosystems. It involves high-throughput sequencing of a standard gene from an environmental sample and comparison to a reference database. However, no consensus has emerged regarding laboratory pipelines to screen species diversity and infer species abundances from environmental samples. In particular, the effect of primer bias and the detection limit for specimens with a low biomass has not been systematically examined, when processing samples in bulk. We developed and tested a DNA metabarcoding protocol that utilises the standard cytochrome c oxidase subunit I (COI) barcoding fragment to detect freshwater macroinvertebrate taxa. DNA was extracted in bulk, amplified in a single PCR step, and purified, and the libraries were directly sequenced in two independent MiSeq runs (300-bp paired-end reads). Specifically, we assessed the influence of specimen biomass on sequence read abundance by sequencing 31 specimens of a stonefly species with known haplotypes spanning three orders of magnitude in biomass (experiment I). Then, we tested the recovery of 52 different freshwater invertebrate taxa of similar biomass using the same standard barcoding primers (experiment II). Each experiment was replicated ten times to maximise statistical power. The results of both experiments were consistent across replicates. We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads. Furthermore, we reliably recovered 83% of the 52 taxa used to test primer bias. However, sequence abundance varied by four orders of magnitudes between taxa despite the use of similar amounts of biomass. Our metabarcoding approach yielded reliable results for high-throughput assessments. However, the results indicated that primer efficiency is highly species-specific, which would prevent straightforward assessments of species abundance and biomass in a sample. Thus, PCR-based metabarcoding assessments of biodiversity should rely on presence-absence metrics.
1.Measures of organic matter processing have been proposed as indices of stream ecosystem function to complement structural measures (water quality, taxonomic composition) in stream health monitoring. However, to be meaningful for ecosystem management, functional responses must be informed by knowledge of multiple stressors in the face of global change.
2.We manipulated nutrient concentrations (two levels), fine sediment (two levels) and water temperature (eight levels, 0–6°C above ambient) in 128 streamside mesocosms to determine the individual and combined effects of these stressors on decomposition rates of fresh and pre-dried leaf packs and cotton strips.
3.Raised temperature affected all seven studied decomposition measures. Leaf biomass loss rates were linearly positively affected while strength loss rates of leaves and cotton strips showed non-linear, weakly unimodal responses. Respiration of fresh leaves was positively affected by warming but respiration of dried leaves showed the opposite pattern. Nutrient enrichment enhanced dried leaf biomass loss and respiration, and cotton tensile strength loss. Sediment addition increased loss of fresh leaf strength and biomass, but decreased dried leaf respiration.
4.Interactions among stressors occurred in three cases. The positive effect of raised temperature on biomass loss of dried leaves was weaker in mesocosms with added sediment, the negative effect of sediment on dried leaf respiration annulled the positive effect of nutrients, and cotton strength loss increased strongly under enriched nutrients but less so when sediment was added.
5.Synthesis and applications. Sediment greatly increased decay rates of fresh (living) leaves, but not of dried leaves or cotton strips. Leaf condition can strongly affect decomposition processes, with important consequences for streams that naturally receive freshly fallen leaves and have high sediment loads. Non-linear responses of decomposition to rising temperature highlight how a warming climate may alter organic matter processing in complex ways. Our finding that dried leaf biomass loss responded positively to nutrient enrichment but not sediment addition, whilst fresh leaf biomass loss responded positively to sediment addition but not nutrient enrichment, suggests the two measures may offer complementary information in the functional assessment of multiple stressors in streams.
Ecosystem diversity, species richness and genetic diversity are the three major facets of biodiversity which deserve equal attention for conservation. However, genetic diversity is significant in identification of unique populations for conservation purpose. Aspects of DNA barcoding, loss of genetic diversity in cryptic species and examples of molecular phylogenetic and molecular phylogeographic studies on aquatic insects of headwaters from major biogeographic realms are briefly reviewed in the light of prioritization of taxa and habitats for conservation. Special emphasis is laid on identification of evolutionary significant units for effective conservation in the context of global climate change. Current methodologies for identifying potential loss of intraspecific genetic diversity are also highlighted with suggestions on future research priorities.
Over the past two decades there have been major increases in dairy production in New Zealand. This increase in intensity has required increased use of external inputs, in particular fertilizer, feed, and water. Intensified dairy farming thus incurs considerable environmental externalities: impacts that are not paid for directly by the dairy farmer. These externalities are left for the wider New Zealand populace to deal with, both economically and environmentally. This is counter-intuitive given the dairy industry itself relies on a 'clean green' image to maximize returns. This is the first nationwide assessment of some of the environmental costs of the recent increase of dairy intensification in New Zealand. Significant costs arise from nitrate contamination of drinking water, nutrient pollution to lakes, soil compaction, and greenhouse gas emissions. At the higher end, the estimated cost of some environmental externalities surpasses the 2012 dairy export revenue of NZ5 billion. For the dairy industry to accurately report on its profitability and maintain its sustainable marketing label, these external costs should be reported. This assessment is in fact extremely conservative as many impacts have not been valued, thus, the total negative external impact of intensified dairying is probably grossly underestimated.
Phenotypically similar species coexisting in extreme environments like sulfidic water are subject to two opposing eco-evolutionary processes: those favoring similarity of environment-specific traits, and those promoting differences of traits related to resource use. The former group of processes includes ecological filtering and convergent or parallel evolution, the latter competitive exclusion, character displacement and divergent evolution. We used a unique eco-evolutionary study system composed of two independent pairs of coexisting amphipod species (genus Niphargus) from the sulfidic caves Movile in Romania and Frasassi in Italy to study the relative contribution and interaction of both processes. We looked at the shape of the multifunctional ventral channel as a trait ostensibly related to oxygenation and sulfide detoxification, and at body size as a resource-related trait. Phylogenetic analysis suggests that the sulfidic caves were colonized separately by ancestors of each species. Species within pairs were more dissimilar in their morphology than expected according to a null model based on regional species pool. This might indicate competitive interactions shaping the morphology of these amphipod species. Moreover, our results suggest that the shape of the ventral channel is not subject to long-term convergent selection or to the process of environmental filtering, and as such probably does not play a role in sulfide tolerance. Nevertheless, the ancestral conditions reconstructed using the comparative method tended to be more similar than null-model expectations. This shift in patterns may reflect a temporal hierarchy of eco-evolutionary processes, in which initial environmental filtering became later on superseded by character displacement or other competition-driven divergent evolutionary processes.
Land use change due to intensifying agricultural practices has profound effects on streams and rivers worldwide, and has been identified as an important source of fine sediment inputs to streams affecting both habitats and biota. Although the effects of sedimentation on stream organisms have been widely studied, manipulative field experiments involving fish and conducted at the reach scale are lacking. Further, it is unknown whether experimentally removing fine sediment from streams can reverse the negative impacts of sedimentation.
Two farmland streams were manipulated for 2 months, by adding or removing fine sediment (using a powerful water blaster) in 50‐m reaches twice (initially and 1 month later). Each stream contained one sediment addition, one removal and one control reach separated by buffer zones. Streams were sampled on five occasions, and water physicochemistry, invertebrate community variables and fish densities (native species and introduced brown trout), diet and condition were determined.
Multivariate benthic invertebrate community composition changed in response to the sediment manipulations. Total taxon richness, density of the caddis fly larva A oteapsyche spp. and brown trout density responded negatively to sediment addition and positively to its removal. A similar trend, although not statistically significant, was observed for native fish density. Trout condition was also poorer in sediment addition reaches than in removal or control reaches.
Our study provides experimental evidence from real streams that increased levels of deposited fine sediment can not only affect stream habitats and invertebrate communities, but also fish communities including trout density and condition. Further, sediment removal can reverse these negative effects, at least temporarily. These findings should help decision makers develop mitigation plans for current farming practices and improved best management systems for the future.
The Canary Islands and Madeira are reportedly home to seven recognised species of baetid mayflies (Ephemeroptera, Baetidae), two of which also occur on the European mainland. Their species status remains unsure, and loss of habitat suggests they are of conservation concern.
We applied morphological characters and a general mixed Yule‐coalescent (gmyc) model analysis of the cytochrome c oxidase subunit 1 ( cox1 ) gene to delineate putative species within morphologically cryptic species groups Baetis ( Rhodobaetis ) and Cloeon dipterum s.l. We used a three‐gene mitochondrial data set (1450 base pairs) to infer phylogenetic relationships and a molecular clock calibrated using island geological ages to infer colonisation history.
Genetic and morphological evidence indicated the presence of 12 putative species, 11 of which were endemic to the islands. Only Baetis atlanticus , on Madeira, also occurs on the European mainland. Two lineages ( B. pseudorhodani s.l. and B. canariensis s.l.) appear to have arisen in the past 15 million years (mya) and diversified in parallel throughout the Canary Islands. Within the canariensis lineage, sister species occur on the island of Gran Canaria and in North Africa.
Pronounced island endemism contradicts previous taxonomic work, which reported a depauperate fauna that included several mainland species. Recent diversification among islands and a close link to North Africa suggest a complex evolutionary history. Owing to their small population size and ongoing habitat alteration, several of these island endemics are among the most endangered aquatic insects in Europe.
A programme of research is introduced on the effects on stream ecosystems of land‐use change from native forest to pasture, and subsequent re‐afforestation with exotic pine plantations. The results detailed in the 11 accompanying papers conclude that land‐use change has had strong effects on many key physical, chemical, and biological aspects of stream ecosystems in and adjacent to Whatawhata Research Centre, near Hamilton, New Zealand. Six of these papers compare streams, draining catchments with similar topography, geology, and soils, to investigate land‐use effects on stream lighting, water quality, channel morphology, benthic and hyporheic habitat and faunas, fish, and food webs. Two papers investigate hypotheses on the influence of shade and crayfish using streamside mesocosms. Finally, three papers use the experimental sites to evaluate methods for studying crayfish populations, stream carbon pathways, and a stream temperature model. These studies provide a comprehensive description of land‐use effects on physical, chemical, and biological characteristics of streams and contribute to the basis for future long‐term ecological research on the effects on streams of sustainable land management practices.
Taxonomic identification of benthic macroinvertebrates is critical to protocols used to assess the
biological integrity of aquatic ecosystems. The time, expense, and inherent error rate of species-level
morphological identifications has necessitated use of genus- or family-level identifications in most large,
statewide bioassessment programs. Use of coarse-scale taxonomy can obscure signal about biological
condition, particularly if the range of species tolerances is large within genera or families. We hypothesized
that integration of deoxyribonucleic acid (DNA) barcodes (partial cytochrome c oxidase subunit I
sequences) into bioassessment protocols would provide greater discriminatory ability than genus-level
identifications and that this increased specificity could lead to more sensitive assessments of water quality
and habitat. Analysis of DNA barcodes from larval specimens of Ephemeroptera, Plecoptera, and
Trichoptera (EPT) taxa collected as part of Maryland’s Biological Stream Survey (MBSS) revealed ,2 to 33
as many DNA-barcode groups or molecular operational taxonomic units (mOTUs) as morphologically
identified genera. As expected, geographic distributions for several mOTUs were tighter than for the
parent genus, but few mOTUs showed closer associations with water-quality variables or physical-habitat
features than did the genus in which they belonged. The need for improved protocols for the consistent
generation of DNA barcodes is discussed.
Species-level identifications are difficult or impossible for many larval aquatic macroinvertebrates. We described the taxonomic composition of macroinvertebrate communities from 5 coastal streams in 3 neighboring catchments in southern California. We compared taxonomic identifications based on deoxyribonucleic acid (DNA) barcoding (cytochrome c oxidase subunit I [COI]) with morphological identifications of the same specimens. We examined 5870 individuals, and barcodes with sequence lengths >350 base pairs (bp) for 91% of those specimens. We used the naturally occurring gaps in divergence frequencies for each order (usually 2% level of genetic divergence) to delimit putative species for all taxonomic groups except Simulium (3%) and Baetis (1%). We identified 200 species across these 5 streams. We identified 104 more species via barcodes than via morphology (200 vs 96, a 108% increase). Richness increases were greatest for Chironomidae (60 more species), Ephemeroptera (10 species), Acari (10 species), and Trichoptera (6 species). Forty-five percent of the genera/species identified morphologically represented >2 species. Many (86) species identified with barcodes were represented by only 1 or 2 specimens and were found at only 1 stream. Thus, species rarity (either spatially or numerically) appears to be a common characteristic of these streams. Barcoding increased total richness at each site by 12 to 40 taxa over morphology alone, and increased the difference between reference and impact sites in terms of lost taxa. These results suggest that macroinvertebrate biodiversity in streams has been underestimated substantially in the past, as has the biodiversity lost in response to environmental stress. The potential of DNA barcoding will not be fully realized until we can assign traits, such as habitat preference, ecological function, and pollution tolerance, at the species level.
Molecular genetic methods continuously uncover cryptic lineages harboured by various species. However, from an applied perspective, it remains unclear whether and to which extent such a genetic diversity affects biological traits (e.g. ecological, behavioural and physiological characteristics) and environmental management.
We assessed potential deviations regarding the trait ‘environmental stress tolerance’ using individuals from five field populations of each of two cryptic lineages (called A and B ) comprised under the nominal species G ammarus fossarum . We used ammonia as a chemical stressor while assessing the feeding rate on leaf discs as a measure of sublethal response. In this context, we established a restriction fragment length polymorphism assay to allow a rapid identification of the lineages.
We observed a biologically meaningful and statistically significant twofold higher overall tolerance of one cryptic lineage, lineage B , over the other. Confounding factors that may have the potential to influence the test results, such as life stage, sex, season of collection, parasitism, physiological status of organisms and upstream land‐use patterns of the river catchments, were either controlled for or displayed only minor deviations between lineages.
Synthesis and applications . The trait differences observed in the present study seem to be mainly explained by the considerable genetic differentiation between cryptic lineages of one nominal species. Although traits other than tolerance have been minimally investigated in this context, this study indicates implications in the reliability and quality of environmental monitoring and management if cryptic lineage complexes are ignored.
Aquatic insects are widely used as indicator taxa to assess the ecological state of streams and
to evaluate the success of stream restoration projects. Information on intraspecific genetic diversity
and population connectivity is often lacking for such indicator taxa. However, these parameters are of
critical importance for restoration plans and conservation management because: 1) species sometimes
consist of several cryptic species and 2) species can recolonize only those restored habitats within a
reachable distance from their source populations. Gene flow generally cannot be observed directly,
and molecular markers provide a reasonable alternative to assess the dispersal potential and evaluate
species’ genetic diversity. We investigated the genetic diversity and dispersal potential of the predatory
stonefly Dinocras cephalotes using 323 specimens from 29 populations in the Sauerland, a low mountain
range in Germany. We used a 658 base pair (bp) fragment of the mitochondrial cytochrome c
oxidase subunit I gene (COI) and found 2 distinct and diverse haplotype groups, which were shared
across most populations. The groups were separated by a minimum intraspecific p-distance of 4.3%,
suggesting historic isolation and possible presence of cryptic species. However, complementing analyses
of the nuclear Wingless gene and 3 newly developed microsatellite markers clearly showed that
individuals from both COI haplotype groups are interbreeding, and therefore, D. cephalotes is considered
a single valid species. Population comparisons indicated high connectivity among all populations,
with only a few individual populations showing signatures of isolation. Based on the molecular data,
we conclude that dispersal is primarily achieved by the adult females of D. cephalotes.
Taxonomic sufficiency (TS) has been proposed for assessing community composition and environmental impacts as a way to balance the need to indicate the biology of the organisms present with time and effort needed for species identification. TS has been applied most often to marine and freshwater macroinvertebrates, but tests of its usefulness are lacking for other freshwater groups. We analyzed the effects of taxonomic resolution, functional groupings, and data transformation on multivariate community patterns in periphyton, macrophytes, macroinvertebrates, and fishes, and on the quantification of biodiversity and environmental gradients. The applicability of TS differed strongly among taxonomic groups, depending on the average taxonomic breadth of the species sets. Numerical data resolution had more pronounced effects on community patterns than taxonomic resolution. Richness was strongly affected by data aggregation, but diversity indices were statistically reliable up to order level. Taxonomic aggregation had no significant influence on ability to detect environmental gradients. Functional surrogates based on biological traits, such as feeding type, reproductive strategy, and trophic state, were strongly correlated (ρ = 0.64–0.85) with taxonomic community composition. However, environmental correlations were generally lower with data aggregated to functional traits rather than to species. TS was universally applicable within taxonomic groups for different habitats in one biogeographic region. Aggregation to family or order was suitable for quantifying biodiversity and environmental gradients, but multivariate community analyses required finer resolution in fishes and macrophytes than in periphyton and macroinvertebrates. Sampling effort in environmental-impact studies and monitoring programs would be better invested in quantitative data and number of spatial and temporal replicates than in taxonomic detail.
Four approaches to or levels of identifying macroinvertebrates (amateur/family, expert entomologist/genus, expert entomologist/genus–species, and DNA barcoding/species) were used to assess community structure and water quality in White Clay Creek, Pennsylvania, USA. Macroinvertebrates were collected in March 2008 from 2 riffle sites 3.9 km apart on the same stream. The downstream site was known to be degraded by land and water use. About 98% of the 1617 specimens used for analysis, including small, immature, and damaged specimens, were successfully barcoded (sequenced) for the mitochondrial cytochrome c oxidase subunit I gene. A criterion of 2 to 4% genetic divergence provided good separation of presumptive species. Barcodes increased the taxonomic inventory across the 2 sites by 475% (124 taxa) relative to the amateur level, and 125% (83 taxa) and 70% (62 taxa) relative to the expert genus and species levels, respectively. Barcoding revealed species not currently described in larval taxonomic keys, including multiple (2–11) coexisting congeneric species. That 150 species were revealed by barcoding samples collected on the same date and in the same habitat was unprecedented, as was the fact that 60 cm2 of stream bottom supported an average of 248 to 347 individuals representing 55 to 68 species. Most barcode species were rare, with 42% represented by ≤2 individuals. Across all species, 43 of 89 barcode species were unique to upstream site 11 and 60 of 107 were unique to downstream site 12. In terms of water-quality assessment, most of the 17 metrics studied changed significantly (α = 0.05) when taxonomy changed from family to genus–species (79% and 93% for sites 11 and 12, respectively), and many also changed when taxonomy changed from genus to species (59 and 65% for sites 11 and 12, respectively). The proportion of metrics able to detect a difference (α = 0.05) between sites 11 and 12 increased with improved taxonomic resolution (36, 47, 65, and 76% for family, genus, genus–species, and barcode, respectively). The results revealed a pollution-tolerance gap because barcoding pushed larval taxonomy beyond the available pollution-tolerance data. Regardless, the combined morphological and molecular approach provides a finer resolution for evaluating environmental change associated with both natural and anthropogenic processes. The ability to distinguish larvae at the species level through barcoding finally puts biodiversity assessments for aquatic communities in terms comparable to those used for terrestrial ecosystems where estimates of biodiversity for plants and animals are never quantified at the level of genus or family. We conclude that DNA barcodes of stream macroinvertebrates will improve descriptions of community structure and water quality for both ecological and bioassessment purposes.
