Novel molecular resources for single-specimen barcoding of enigmatic crustacean y-larvae.

Despite discovery more than 100years ago and documented global occurrence from shallow waters to the deep sea, the life cycle of the enigmatic crustacean y-larvae isincompletely understood and adult forms remain unknown. To date, only 2 of the 17 formally described species, all based on larval stages, have been investigated using an integrative taxonomic approach. This approach provided descriptions of the morphology of the naupliar and cyprid stages, and made use of exuvial voucher material and DNA barcodes. To improve our knowledge about the evolutionary history and ecological importance of y-larvae, we developed a novel protocol that maximises the amount of morpho-ecological and molecular data that can be harvested from single larval specimens. This includes single-specimen DNA barcoding and daily imaging of y-nauplii reared in culture dishes, mounting of the last naupliar exuviae on a slide as a reference voucher, live imaging of the y-cyprid instar that follows, and fixation, DNA extraction, amplification and sequencing of the y-cyprid specimen. Through development and testing of a suite of new primers for both nuclear and mitochondrial protein-coding and ribosomal genes, we showcase how new sequence data can be used to estimate the phylogeny of Facetotecta. We expect that our novel procedure will help to unravel the complex systematics of y-larvae and show how these fascinating larval forms have evolved. Moreover, we posit that our protocols should work on larval specimens from a diverse array of moulting marine invertebrate taxa.

Geometric morphometric analysis of morphologic disparity, intraspecific variation and ontogenetic allometry of beyrichitine ammonoids.

Beyrichitine ammonoids of NV Nevada reveal a high taxonomic diversity of Anisian (Middle Triassic). This diversity is, however, in contrast to their relatively low morphologic disparity. Depending on the exact definition, morphologic disparity of a data set is a direct consequence of the sum of all ontogenetic changes. In the past, however, the interplay of both morphological processes has only rarely been addressed. Using geometric morphometric methods, this study aims at a quantification of allometric processes and the morphologic disparity of beyrichitine ammonoids. The multivariate statistical analysis revealed that morphologic disparity, intraspecific variation respectively, within and between the studied species seems to be the result of deviations in the ontogenetic allometric growth pattern (i.e. heterochrony). During deposition of the studied stratigraphic sequence, a general progressive pedomorphism (juvenilization) was observed. The intraspecific variability pattern coincides with the total morphologic disparity of the analyzed species, which suggests that intraspecific variability facilitated morphologic disparity. The comparison of ontogenetic allometric patterns and changes in intraspecific variation and morphologic disparity are likely to refine our understanding of the intrinsic factors influencing the speciation of this group.

Effects of low pH on the coral reef cryptic invertebrate communities near CO2 vents in Papua New Guinea.

Small cryptic invertebrates (the cryptofauna) are extremely abundant, ecologically important, and species rich on coral reefs. Ongoing ocean acidification is likely to have both direct effects on the biology of these organisms, as well as indirect effects through cascading impacts on their habitats and trophic relationships. Naturally acidified habitats have been important model systems for studying these complex interactions because entire communities that are adapted to these environmental conditions can be analyzed. However, few studies have examined the cryptofauna because they are difficult to census quantitatively in topographically complex habitats and are challenging to identify. We addressed these challenges by using Autonomous Reef Monitoring Structures (ARMS) for sampling reef-dwelling invertebrates >2 mm in size and by using DNA barcoding for taxonomic identifications. The study took place in Papua New Guinea at two reef localities, each with three sites at varying distances from carbon dioxide seeps, thereby sampling across a natural gradient in acidification. We observed sharp overall declines in both the abundance (34-56%) and diversity (42-45%) of organisms in ARMS under the lowest pH conditions sampled (7.64-7.75). However, the overall abundance of gastropods increased slightly in lower pH conditions, and crustacean and gastropod families exhibited varying patterns. There was also variability in response between the two localities, despite their close proximity, as one control pH site displayed unusually low diversity and abundances for all invertebrate groups. The data illustrate the complexity of responses of the reef fauna to pH conditions, and the role of additional factors that influence the diversity and abundance of cryptic reef invertebrates.

Phylogenetic Signal Dissection of Heterogeneous 28S and 16S rRNA Genes in Spinicaudata (Branchiopoda, Diplostraca).

It is still a challenge to reconstruct the deep phylogenetic relationships within spinicaudatans, and there are several different competing hypotheses regarding the interrelationships among Eocyzicidae, Cyzicidae s. s., Leptestheriidae, and Limnadiidae of the Suborder Spinicaudata. In order to explore the source of the inconsistencies, we focus on the sequence variation and the structure model of two rRNA genes based on extensive taxa sampling. The comparative sequence analysis revealed heterogeneity across species and the existence of conserved motifs in all spinicaudatan species. The level of intraspecific heterogeneity differed among species, which suggested that some species might have undergone a relaxed concerted evolution with respect to the 28S rRNA gene. The Bayesian analyses were performed on nuclear (28S rRNA, EF1α) and mitochondrial (16S rRNA, COI) genes. Further, we investigated compositional heterogeneity between lineages and assessed the potential for phylogenetic noise compared to signal in the combined data set. Reducing the non-phylogenetic signals and application of optimal rRNA model recovered a topology congruent with inference from the transcriptome data, whereby Limnadiidae was placed as a sister group to Leptestheriidae + Eocyzicidae with high support (topology I). Tests of alternative hypotheses provided implicit support for four competing topologies, and topology I was the best.

A New Species of Pseudostrandesia Savatenalinton and Martens, 2009 (Ostracoda, Crustacea) Collected from Two Pet Shops in Central Japan: an Alien Species?

An undescribed species of freshwater ostracod belonging to the genus Pseudostrandesia Savatenalinton and Martens, 2009 was collected from two pet shops in the Kanto region of central Japan. This species, herein named Pseudostrandesia tenebrarum sp. nov., is similar to four species previously reported from Southeast Asia, but can be distinguished by carapace and appendage features. It is the second species of the genus for which males are known. Of the nine previously described species in the genus, one is exclusively known from Turkey, and the others are found in Southeast Asia and the vicinity, one of which is also recorded in India and east China. There are two scenarios to explain the existence of Pseudostrandesia tenebrarum sp. nov. in pet shops in Japan: it is either native to Japan but has yet to be discovered in its natural habitat, or it is an alien species, perhaps unwittingly imported with plants or fish for the pet trade. We review the likelihood of both scenarios, and conclude that although there is insufficient evidence to be sure, it is potentially an alien species in Japan. The most likely origin is Southeast Asia, as evidenced by its close morphological resemblance to particular Southeast Asian species. Juveniles as well as adults were recovered, indicating that this species is reproducing in the pet trade, supporting the notion that it has invasive potential to areas outside of its natural range. The description and report of this species highlights a possible introduction of an alien species to Japan, and facilitates further monitoring.

Review of the southern African Leptestheriidae (Branchiopoda: Spinicaudata) I: redescription of Leptestheria brevirostris Barnard, 1924 with comments on diagnostic characters.

We present the first records of Leptestheria brevirostris since its discovery in Namibia by Barnard in 1924. Our records come from Botswana and South Africa, and present significant range extensions. We redescribe L. brevirostris according to modern standards and present the first description of the male. We also discovered that L. brevirostris is likely a rock pool specialist, specific to sandstone and gneiss outcrops; this is the first record of a rock pool specialist clam shrimp from Africa and the second rock pool specialist described for Leptestheriidae. Finally, we depict and discuss the validity and usefulness of diagnostic characters for Leptestheria species in southern Africa, especially the usefulness of carapace interval ornamentation.

Three new species of Heteromysis (Crustacea: Mysida) from coral reef aquaria in Florida and Central Europe.

Three new species of the genus Heteromysis S.I. Smith, 1873 (tribus Heteromysini), are described from a rich stock of mysids obtained on request from the international community of professional aquarium keepers. The 18S rDNA and COI sequences of the three species were distinct from each other and also from other sequences published in DNA databases. Heteromysis (Olivemysis) schoenbrunnensis sp. nov. is morphologically characterized within the subgenus Olivemysis based on the structure of the first and second antennae, male pleopods, uropods, and telson. Heteromysis (Heteromysis) gulfarii sp. nov. is outstanding within the subgenus Heteromysis by sexually dimorphic modified setae on the antennular trunk. These setae are non-dimorphic in the very similar Heteromysis (Heteromysis) korntalensis sp. nov. Both species H. gulfarii and to a lesser degree also H. korntalensis show modified eyes, subquadrate in dorsal view, eyestalks anteriorly tapering in lateral view; small, well-developed cornea implanted laterally on modified eyestalk. Apart from eye structure H. gulfarii and H. korntalensis clearly fall morphologically within the nominotypical subgenus Heteromysis.

Heterocypris spadix sp. nov. (Crustacea: Ostracoda: Cypridoidea) from Japan, with Information on Its Reproductive Mode.

We describe the cypridoidean ostracod Heterocypris spadix sp. nov. from brackish water on Okinawa Island, Japan. The species closely resembles Heterocypris salina (Brady, 1868) but differs in that (1) the marginal infolds on valves are less developed, (2) the tubercles on the anterior margin of the right valve are completely covered by the selvage and invisible in inner view, and (3) the calcified inner lamella on the ventral margin of the left and right valves is scarcely evident in inner view, as the ventral margins of the valves bend inwardly. We determined partial sequences for the cytochrome c oxidase subunit I (COI; cox1) and 18S rRNA genes in H. spadix for future DNA barcoding and phylogenetic analyses. Our sample contained only females. A breeding experiment revealed that H. spadix females reproduce parthenogenetically. Another experiment showed that H. spadix has low tolerance to desiccation, with all individuals at 25°C dying between 1-2 hours after removal from water. We amplified and sequenced a partial 16S rRNA sequence for the endosymbiotic bacterium Cardinium from H. spadix. Infection by Cardinium may be related to the parthenogenetic reproductive mode we observed in H. spadix.

Comments on "Is emCopytus/em Skogsberg, 1939 (Crustacea: Ostracoda) a neocytherideid? With description of a new family and two new species" by Coimbra, Bergue amp; Ramos (2020).

Most recently, Coimbra et al. (2020) published an article in this journal (Zootaxa, 4729 (2): 177-194) questioning the taxonomic position of the genus Copytus Skogsberg, 1939 (Crustacea, Ostracoda) along with proposing it as the type genus of their new family (Copytidae Coimbra et al., 2020), and erecting two new species that were listed by previous authors as Copytus sp. 1 and Copytus sp. 2. The main diagnostic characteristics of their new proposed family (and the genus Copytus) are the hinge type and muscle scars on the carapace and/or valves (see lines 6-8 from the bottom, p. 179 in Coimbra et al. 2020). They also underlined that (p. 179) "...this study is based exclusively on the morphology of the animals' hard parts". While the authors considered another genus (Neocopytus) proposed by Külköylüoglu, Colin Kiliç (2007) of the family Neocytherididae as invalid, they interestingly transferred some species of Neocopytus to Copytus as species of their new family (Coimbra et al. 2020). Herein, my point with the comments listed below is to clarify that, when possible, both soft and hard parts should be considered in taxonomy, and such an integrated approach clearly indicates that Neocopytus is a valid and taxonomically useful genus.

Triassic clam shrimps ("Conchostraca"; Branchiopoda: Diplostraca) from Mallorca: Taxonomic description and interregional comparisons.

We describe four upper Lower Triassic to lower Middle Triassic clam shrimp-bearing intervals from Mallorca, which include the clam shrimp species Hornestheria sp. aff. Hornestheria sollingensis and several other forms of carapace valve morphology: Hornestheria? Morphotype 1, Hornestheria? Morphotype 2, and other undetermined carapace valves. All of this material was obtained from red-bed units cropping out in the Serra de Tramuntana mountains of Mallorca (western Mediterranean). Except for a few morphologically similar carapace valves of Middle Triassic age from China, Hornestheria is known only from the type locality of its type species, Hornestheria sollingensis Kozur et Lepper, in the Solling Formation (Middle Buntsandstein Subgroup) in the German part of the Central European Basin. According to its original definition, the larval carapace valve of Hornestheria Kozur et Lepper is characterized by a radial sculpture, but this characteristic apparently is only variably developed. Due to both a limited number of previously known occurrences of Hornestheria and its poorly known carapace valve morphology, open nomenclature is applied to the taxonomy herein. The studied specimens were freshly collected from outcrop sections composed of greyish-green to greyish-red laminated claystones and siltstones that accumulated in a fluvial facies. The clam shrimp specimens are accompanied by remains of insects and fishes, invertebrate and tetrapod ichnofossils, and micro-/macroplant remains, all of which either have been described by previous workers or are currently part of a separate study.

Three nuclear protein-coding genes corroborate a recent phylogenomic model of the Branchiopoda (Crustacea) and provide estimates of the divergence times of the major branchiopodan taxa.

The class Branchiopoda (Crustacea) shows great diversity in morphology and lifestyle among its constituent higher-level taxa: Anostraca, Notostraca, Laevicaudata, Spinicaudata, Cyclestherida and Cladocera. The phylogenetic relationships among these taxa have long been controversial. We sequenced three orthologous nuclear genes that encode the catalytic subunit of DNA polymerase delta and the largest and second-largest subunits of RNA polymerase II in the expectation that the amino acid sequences encoded by these genes might be effective in clarifying branchiopod phylogeny and estimating the times of divergence of the major branchiopodan taxa. The results of phylogenetic analyses based on these amino acid sequences support the monophyly of Branchiopoda and provide strong molecular evidence in support of the following phylogenetic relationships: (Anostraca, (Notostraca, (Laevicaudata, (Spinicaudata, (Cyclestherida, Cladocera))))). Within Cladocera, comparison of the nucleotide sequences of these same genes shows Ctenopoda to be the sister group of Haplopoda + Anomopoda. Three statistical tests based on the present amino acid sequence data-the approximately unbiased test, Kishino-Hasegawa test and weighted Shimodaira-Hasegawa test-tend to refute most of the previous molecular phylogenetic studies on Branchiopoda, which have placed Notostraca differently than here; however, our results corroborate those of one recent phylogenomic study, thus confirming the effectiveness of these three genes to investigate relationships among branchiopod higher taxa. Divergence time estimates calibrated on the basis of fossil evidence suggest that the first divergence of extant branchiopods occurred about 534 Ma during the early Cambrian period and that diversification within the extant branchiopod lineages started in or after the late Permian.

A Crab Is Not a Fish: Unique Aspects of the Crustacean Endocrine System and Considerations for Endocrine Toxicology.

Crustaceans-and arthropods in general-exhibit many unique aspects to their physiology. These include the requirement to moult (ecdysis) in order to grow and reproduce, the ability to change color, and multiple strategies for sexual differentiation. Accordingly, the endocrine regulation of these processes involves hormones, receptors, and enzymes that differ from those utilized by vertebrates and other non-arthropod invertebrates. As a result, environmental chemicals known to disrupt endocrine processes in vertebrates are often not endocrine disruptors in crustaceans; while, chemicals that disrupt endocrine processes in crustaceans are often not endocrine disruptors in vertebrates. In this review, we present an overview of the evolution of the endocrine system of crustaceans, highlight endocrine endpoints known to be a target of disruption by chemicals, and identify other components of endocrine signaling that may prove to be targets of disruption. This review highlights that crustaceans need to be evaluated for endocrine disruption with consideration of their unique endocrine system and not with consideration of the endocrine system of vertebrates.

Convergent evolution of optic lobe neuropil in Pancrustacea.

A prevailing opinion since 1926 has been that optic lobe organization in malacostracan crustaceans and insects reflects a corresponding organization in their common ancestor. Support for this refers to malacostracans and insects both possessing three, in some instances four, nested retinotopic neuropils beneath their compound eyes. Historically, the rationale for claiming homology of malacostracan and insect optic lobes referred to those commonalities, and to comparable arrangements of neurons. However, recent molecular phylogenetics has firmly established that Malacostraca belong to Multicrustacea, whereas Hexapoda and its related taxa Cephalocarida, Branchiopoda, and Remipedia belong to the phyletically distinct clade Allotriocarida. Insects are more closely related to remipedes than are either to malacostracans. Reconciling neuroanatomy with molecular phylogenies has been complicated by studies showing that the midbrains of remipedes share many attributes with the midbrains of malacostracans. Here we review the organization of the optic lobes in Malacostraca and Insecta to inquire which of their characters correspond genealogically across Pancrustacea and which characters do not. We demonstrate that neuroanatomical characters pertaining to the third optic lobe neuropil, called the lobula complex, may indicate convergent evolution. Distinctions of the malacostracan and insect lobula complexes are sufficient to align neuroanatomical descriptions of the pancrustacean optic lobes within the constraints of molecular-based phylogenies.

A New Species of Ostracod (Crustacea) Associated with a Feather Star: First Report of Ostracoda from Crinoidea.

We describe Obesostoma crinophilum sp. nov. (Ostracoda: Podocopida: Paradoxostomatidae) obtained from the body surface of the feather star Antedon serrata A. H. Clark, 1908 (Crinoidea: Comatulida: Antedonidae). This is the first report of Ostracoda associated with Crinoidea. None of the highly specialized appendages and/or carapace that are related to a commensal lifestyle were observed in O. crinophilum sp. nov. Therefore, the relationship between O. crinophilum sp. nov. and A. serrata must be transient rather than obligatory. However, O. crinophilum sp. nov. has a more developed hook-like distal claw on the antenna in comparison with four previously known Obesostoma species. The relatively well-developed distal claw of the antenna in O. crinophilum sp. nov. should indicate its intimate association with feather stars, though the feeding habit is still unknown.

Using larval barcoding to estimate stomatopod species richness at Lizard Island, Australia for conservation monitoring.

Stomatopods (Crustacea, Stomatopoda) are well studied for their aggressive behavior and unique visual system as well as their commercial importance in Asian and European countries. Like many crustaceans, stomatopods undergo indirect development, passing though several larval stages before reaching maturity. Adult stomatopods can be difficult to catch due to their inaccessible habitats and cryptic coloration. By sampling larvae from the planktonic community, less effort is required to obtain accurate measures of species richness within a region. Stomatopod larvae were collected between 2006 and 2015 from the waters around the Lizard Island reef platform in Eastern Australia. Cytochrome oxidase I (COI) mitochondrial DNA sequences were generated from each larval sample and compared to a database of COI sequences tied to adult specimens. Of the 20 species collected from Lizard Island as adults which have COI data available, 18 species were identified from larval sampling. One additional species identified from larval samples, Busquilla plantei, was previously unknown from Lizard Island. Nine larval OTUs were found not to match any published adult sequences. Sampling larval stomatopod populations provides a comparable picture of the adult population to benthic sampling methods and may include species richness beyond what is measurable by sampling adult populations.

Molecular and Morphological Diversity of Heterodesmus Brady and Its Phylogenetic Position within Cypridinidae (Ostracoda).

Ostracod genus Heterodesmus Brady, 1866 is known thus far to contain only three species: H. adamsii Brady, 1866; H. apriculus Hiruta, 1992; and H. naviformis (Poulsen, 1962). This genus has been recorded from the Sea of Japan, and the coastal areas of Thailand and Vietnam. The main generic character is the presence of antero-dorsal and postero-dorsal tube-like processes on the rostrum on both valves. The three species mostly differ in the shell lateral projections. Their relationship and the position of Heterodesmus within family Cypridinidae are poorly understood, partly due to the lack of publication of DNA data so far. We study Heterodesmus collected from several localities in the Northwest Pacific, namely Tsushima and Iki Islands in Japan and Maemul Island in Korea. Besides morphological characters, we also use two mitochondrial markers (16S rRNA and mtCOI) and three nuclear regions (18S rRNA, 28S rRNA, and internal transcribed spacer - ITS) in the samples to detect the biodiversity of this genus. Our phylogenetic tree based on molecular data coupled with morphology reveals the presence of two species, H. adamsii and H. apriculus. We report on their morphological variability, molecular diversity, and phylogenetic position within Cypridinidae based on 16S, 28S and 18S rRNAs, and provide a taxonomic key for all living genera of this family. For the first time, we give an overview of the intrageneric and intrafamily DNA distances of the above markers for the entire subclass Myodocopa.

Population structure of Callichirus major (Say 1818) (Crustacea: Callianassidae) and conservation considerations at Southeast coast of São Paulo, Brazil.

Globally, there is limited knowledge about management and conservation of Callianassidae family. Their catches seem to be recently increasing in several parts of the world and evidences raise concern on the need of specialized literature focused on populations monitoring. At Southeast of Brazil, the species Callichirus major (Say 1818), known as the ghost shrimp, is frequently captured for use as bait in sport fishing and, according to previous studies, the overexploitation could threaten the population. The aim of this study was evaluated over a year this harmful human pressure on the species C. major in two beaches contiguous but susceptible to different anthropogenic impacts due to the existence of legislation on one beach that prohibits the capture of this crustacean. The population of C. major presented where the prohibition regulation does not exist a density almost three times lower when compared to the beach that prohibits the capture, with individuals with smaller carapace oval area and a shorter reproductive period. This paper alert to some evidence of C. major structure population disturbance resulting from its exploitation at Southeast of Brazil. Recommendations were also addressed to conservation management considering the species' ecological importance.

"Unicorn from Hades", a new genus of Mysidae (Malacostraca: Mysida) from the Mariana Trench, with a systematic analysis of the deep-sea mysids.

Since the 19th century, oceanic explorations have confirmed that the hadal zone (water depth > 6000 m) is not lifeless, but contains many fascinating organisms. Amongst them are the Mysida, which is a group of crustaceans found in many deep-sea trenches. Based on morphological observations and molecular phylogenetic analyses of an undescribed taxon within the subfamily Erythropinae, a new genus of deep-sea mysids, Xenomysis gen. n., is described from the Challenger Deep in the Mariana Trench. The new genus is not assigned to any of the tribes within the Erythropinae, as our analyses do not support the current classification scheme of Erythropinae. The result of a molecular-clock analysis with fossil calibration reveals that several groups of Mysida have independently colonized deep water habitats in different geological periods, from Triassic to Cretaceous. In addition, ancestral state reconstruction analyses indicate the degenerate eyes in both Mysidae and Petalophthalmidae is a result of parallel evolution, and the reduction of compound eyes to both "single fused eyeplate" and "two separate eyeplates" occurred multiple times independently in the evolution of Erythropinae.

Scratching the surface of subterranean biodiversity: Molecular analysis reveals a diverse and previously unknown fauna of Parabathynellidae (Crustacea: Bathynellacea) from the Pilbara, Western Australia.

Like other crustacean families, the Parabathynellidae is a poorly studied subterranean and aquatic (stygobiontic) group in Australia, with many regions of available habitat having not yet been surveyed. Here we used a combined approach of molecular species delimitation methods, applied to mitochondrial and nuclear genetic data, to identify putative new species from material obtained from remote subterranean habitats in the Pilbara region of Western Australia. Based on collections from these new localities, we delineated a minimum of eight and up to 24 putative new species using a consensus from a range of molecular delineation methods and additional evidence. When we placed our new putative species into the broader phylogenetic framework of Australian Parabathynellidae, they grouped with two known genera and also within one new and distinct Pilbara-only clade. These new species significantly expand the known diversity of Parabathynellidae in that they represent a 22% increase to the 109 currently recognised species globally. Our investigations showed that sampling at new localities can yield extraordinary levels of new species diversity, with the majority of species showing likely restricted endemic geographical ranges. These findings represent only a small sample from a region comprising less than 2.5% of the Australian continent.

Increased performance of DNA metabarcoding of macroinvertebrates by taxonomic sorting.

DNA-based identification through the use of metabarcoding has been proposed as the next step in the monitoring of biological communities, such as those assessed under the Water Framework Directive (WFD). Advances have been made in the field of metabarcoding, but challenges remain when using complex samples. Uneven biomass distributions, preferential amplification and reference database deficiencies can all lead to discrepancies between morphological and DNA-based taxa lists. The effects of different taxonomic groups on these issues remain understudied. By metabarcoding WFD monitoring samples, we analyzed six different taxonomic groups of freshwater organisms, both separately and combined. Identifications based on metabarcoding data were compared directly to morphological assessments performed under the WFD. The diversity of taxa for both morphological and DNA-based assessments was similar, although large differences were observed in some samples. The overlap between the two taxon lists was 56.8% on average across all taxa, and was highest for Crustacea, Heteroptera, and Coleoptera, and lowest for Annelida and Mollusca. Taxonomic sorting in six basic groups before DNA extraction and amplification improved taxon recovery by 46.5%. The impact on ecological quality ratio (EQR) scoring was considerable when replacing morphology with DNA-based identifications, but there was a high correlation when only replacing a single taxonomic group with molecular data. Different taxonomic groups provide their own challenges and benefits. Some groups might benefit from a more consistent and robust method of identification. Others present difficulties in molecular processing, due to uneven biomass distributions, large genetic diversity or shortcomings of the reference database. Sorting samples into basic taxonomic groups that require little taxonomic knowledge greatly improves the recovery of taxa with metabarcoding. Current standards for EQR monitoring may not be easily replaced completely with molecular strategies, but the effectiveness of molecular methods opens up the way for a paradigm shift in biomonitoring.