The Fellowship of the Ring Test: DNAqua-Net WG2 initiative to compare diatom metabarcoding protocols used in routine freshwater biomonitoring for standardisation

During the past decade genetic approaches have been developed to monitor biodiversity in aquatic ecosystems. These enable access to taxonomic and genetic information from biological communities using DNA from environmental samples (e.g. water, biofilm, soil) and methods based on high-throughput sequencing technologies, such as DNA metabarcoding. Within the context of the Water Framework Directive (WFD), such approaches could be applied to assess Biological Quality Elements (BQE). These are used as indicators of the ecological status of aquatic ecosystems as part of national monitoring programs of the european network of 110,000 surface water monitoring sites with 79.5% rivers and 11% lake sites (Charles et al. 2020). A high-throughput method has the potential to increase our spatio-temporal monitoring capacity and to accelerate the transfer of information to water managers with the aim to increase protection of aquatic ecosystems. Good progress has been made with developing DNA metabarcoding approaches for benthic diatom assemblages. Technological innovation and protocol optimization have allowed robust taxonomic (species) and genetic (OTU, ESV) information to be obtained from which diatom quality indices can be calculated to infer ecological status to rivers and lakes. Diatom DNA metabarcoding has been successfully applied for biomonitoring at the scale of national river monitoring networks in several countries around the world and can now be considered technically ready for routine application (e.g. Apothéloz-Perret-Gentil et al. 2017, Bailet et al. 2019, Mortágua et al. 2019, Vasselon et al. 2019, Kelly et al. 2020, Pérez-Burillo et al. 2020, Pissaridou et al. 2021). However, protocols and methods used by each laboratory still vary between and within countries, limiting their operational transferability and the ability to compare results. Thus, routine use of DNA metabarcoding for diatom biomonitoring requires standardization of all steps of the metabarcoding procedure, from the sampling to the final ecological status assessment in order to define good practices and standards. Following previous initiatives which resulted in a CEN technical report for biofilm sampling and preservation (CEN 2018), a set of experiments was initiated during the DNAqua-Net WG2 diatom workshop (Cyprus, 2019) to focus on DNA extraction and PCR amplification steps in order to evaluate: i) the transferability and reproducibility of a protocol between different laboratories; ii) the variability introduced by different protocols currently applied by the scientific community. 19 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using i) the same fixed protocol and ii) their own protocol. Experiments were performed by each participant on a set of standardized DNA and biofilm samples (river, lake, mock community). In order to specifically test the variability of DNA extraction and PCR amplification steps, all other steps of the metabarcoding process were fixed and the preparation of the Miseq sequencing was performed by only one laboratory. The variability within and between participants will be evaluated on DNA extracts quantity, taxonomic (genus, species) and genetic richness, community structure comparison and diatom quality index scores (IPS). We will also evaluate the variability introduced by different DNA extraction and PCR amplification protocols on diatom quality index scores and the final ecological status assessment. The results from this collaborative work will not serve to define “one protocol to rule them all”, but will provide valuable information to define guidelines and minimum requirements that should be considered when performing diatom metabarcoding for biomonitoring.

Applicability of diatom metabarcoding in the ecological status assessment of Hungarian lotic and soda pan habitats

Diatoms, eukaryotic algae with silica cell wall have been proved to be reliable bioindicators and are applied in ecological status assessment of aquatic ecosystems using indices calculated from pollution sensitivity and indicator values of species. Traditional method of identification of diatom species is based on morphological features of frustule that requires in-depth taxonomical knowledge and expertise. Metabarcoding that combines barcodes and high-throughput sequencing offers a promising alternative. In this pilot study we tested the applicability of metabarcoding of benthic diatom assemblages for ecological status assessment of water bodies in Hungary comparing its performance to that of morphology-based identification of species. Samples from 78 rivers and streams arranged along a trophic gradient and 14 soda pans representing unique types of habitats were investigated. Sequences of the barcode region of rbcL gene suggested by Vasselon et al. (2017) were acquired revealing 1135 diatom amplicon sequence variants (ASVs) of which 54% could be assigned at species level using Diat.barcode (Rimet et al. 2019) as reference database. Morphological investigation detected more species and intraspecific taxa in the lotic and lentic samples (413 and 78, respectively) than metabarcoding (190 and 75). Correspondence between taxa detected based on morphology and DNA sequences was relatively low (on average 24% in lotic and 26% in lentic samples) but considerably increased (on average 66% and 56%, respectively) when taking into account only the taxa reaching higher than 5% relative abundance in microscopy counting. The differences mainly derived from that a considerable portion (59% and 32%, respectively) of the morphologically identified taxa were not recorded in the reference database. Community structure of samples from both running and standing waters based on microscopy and DNA sequence analysis showed significant correlation as revealed using Mantel test. For rivers and streams diatom index IPS (Specific Pollution Sensitivity Index, (Coste in Cemagref, 1982) for soda pans indices H (Ziemann et al., 2001) and IBD (Indice Biologique Diatomées, Lenoir and Coste, 1996) were calculated from the results of the two aspects. Strong correlation was found between morphology- and sequence-based indices and in about half of the samples, the ecological status class obtained with the two methods coincided. Our results suggest that metabarcoding inheres a great opportunity and could be successfully applied in benthic diatom-based ecological status assessment of Hungarian waters after the reference database is supplemented with taxa frequently occurring in these habitats. References Cemagref, 1982. Etude des méthodes biologiques quantitativesd’appréciation de la qualité des eaux, Rapport Division Qualité des Eaux Lyon. Agence financière de Bassin Rhône-Méditerranée-Corse, Pierre-Bénite, France. Lenoir, A., Coste, M., 1996. Development of a practical diatom index of overall water quality applicable to the French National Water Board Network. Presented at the International symposium, Volksbildungsheim Grilhof Vill, AUT, 17-19 September 1995, Universität Innsbruck, pp. 29–43. Rimet, F., Gusev, E., Kahlert, M., Kelly, M.G., Kulikovskiy, M., Maltsev, Y., Mann, D.G., Pfannkuchen, M., Trobajo, R., Vasselon, V., Zimmermann, J., Bouchez, A., 2019. Diat.barcode, an open-access curated barcode library for diatoms. Sci. Rep. 9, 15116. Vasselon, V., Rimet, F., Tapolczai, K., Bouchez, A., 2017. Assessing ecological status with diatoms DNA metabarcoding: Scaling-up on a WFD monitoring network (Mayotte island, France). Ecol. Indic. 82, 1–12. Ziemann, H., Kies, L., Schulz, C.-J., 2001. Desalinization of running waters: III. Changes in the structure of diatom assemblages caused by a decreasing salt load and changing ion spectra in the river Wipper (Thuringia, Germany). Limnologica 31, 257–280.

Is the ecological status assessment result the same using genomic- and morphology-based benthic biotic indices?

Monitoring and assessment of benthic communities have been routinely undertaken using morphology-based benthic indices, among which AZTI’s Marine Biotic Index (AMBI) and multivariate-AMBI (M-AMBI), are the most successful worldwide (Borja et al., 2019). They are used officially in routine monitoring in many European countries, where they have been intercalibrated (European Commission, 2018). AMBI has been mirrored by metabarcoding, and the genomic version (gAMBI; Aylagas et al., 2014) has demonstrated to yield results comparable to the morphological version. However, we have so far failed to develop a reliable genomic version of M-AMBI (M-gAMBI), which includes gAMBI, richness and diversity (Aylagas et al., 2018). This is because richness and diversity present very different results in morphological and genomic analyses. Since the multivariate method needs reference conditions, these must be set specifically for M-gAMBI to make its results comparable to those obtained with M-AMBI. To this aim, we started annual surveys in 2018, in 22 sampling locations, in Basque estuaries and coast. We present the results from the first three years of these surveys and discuss the problems faced when developing genomic reference conditions for M-gAMBI. The findings are of paramount importance for managers, since any new method or modification of an existing assessment method, needs to demonstrate that the results obtained when assessing the status are similar to those morphological-based methods, already approved and intercalibrated. Aylagas, E., Á. Borja, N. Rodríguez-Ezpeleta, 2014. Environmental Status Assessment Using DNA Metabarcoding: Towards a Genetics Based Marine Biotic Index (gAMBI). PLoS ONE, 9: e90529. Aylagas, E., Á. Borja, I. Muxika, N. Rodríguez-Ezpeleta, 2018. Adapting metabarcoding-based benthic biomonitoring into routine marine ecological status assessment networks. Ecological Indicators, 95: 194-202. Borja, A., G. Chust, I. Muxika, 2019. Chapter Three - Forever young: The successful story of a marine biotic index. Advances in Marine Biology, 82: 93-127. European Commission, 2018. Commission Decision (EU) 2018/229 of 12 February 2018 establishing, pursuant to Directive 2000/60/EC of the European Parliament and of the Council, the values of the Member State monitoring system classifications as a result of the intercalibration exercise and repealing Commission Decision 2013/480/EU. Official Journal of the European Communities, L47: 1-91.

Ecological status assessment of the Samokovska river: 2018 case study

The Samokovska River, as the most important watercourse of the Kopaonik National Park, has been poorly investigated from algological and water quality aspects. This river is not covered by the monitoring program of the Serbian Environmental Protection Agency routine. Our research performed in October 2018 aimed to present an algal diversity, ecological status assessment, and negative anthropogenic impacts threatening this river. For ecological status assessment purposes, the diatom indices phytobenthos and physico-chemical parameters of water were used. The typology and the problem of reliable assessing of the ecological status were also discussed on the example of the Samokovska River.

Ecological Status Assessment of Transitional Waters

Transitional Waters are worldwide high valuable ecosystems that have undergone significant anthropogenic impacts. The ecological assessment is therefore of fundamental importance to protect, manage and restore these ecosystems. Numerous approaches can be used to understand the effects of human pressures, and, in case, the effectiveness of recovery plans. Eutrophication, climate change and morphological loss impacts can be assessed by means of aquatic vegetation, benthic fauna, and nekton. Moreover, before planning new infrastructures or interventions, predictive approaches and statistical analyses can provide indispensable tools for management policies.