Revolutionizing fish monitoring in Alpine rivers

Revolutionizing fish monitoring in Alpine rivers

Revolutionizing fish monitoring in Alpine rivers

Environmental DNA provides high resolution data on Alpine fish populations and facilitates their management.
The river Inn (Kufstein, Tyrol, Austria) as an example of epipotamal habitats under anthropogenic influence. PHOTO CREDIT: Bettina Thalinger

Many fish communities in Alpine rivers are impacted by anthropogenically altered riverbeds, making effective biomonitoring critical for the long-term sustainability and health of river systems. Traditional surveys require fish to be temporarily captured, an approach that can only be done outside of protected periods and at low water flow. A new method of detecting fish DNA in the environment has the potential to revolutionize monitoring efforts. Our study applied environmental DNA (eDNA) analyses in dynamic Alpine habitats to successfully detect species even at low population densities and to obtain information on the whole fish species community at a regional scale.

The river Inn upstream of a hydropower plant in Tyrol, Austria. PHOTO CREDIT: Bettina Thalinger
Hydropower plants, transverse structures, and artificially straightened riverbeds are just some examples of how Alpine rivers and the landscapes around them are being altered for the benefit of neighbouring communities. These changes exert negative influences on fish species and make population management challenging. For instance, straightened river segments lack areas with low flow rates, which can be crucial for spawning and refuge seeking during the annual spring and summer high waters. Hence, detailed knowledge on the species present and how they are distributed along rivers is essential for their management, which also needs to incorporate sport fishing and artificial stocking in many places.
Traditionally, fish populations in Alpine rivers are assessed via electrofishing, but this method can only be applied during short periods outside of spawning times and when water levels and turbidity are low enough to effectively see and capture fish. Additionally, the considerable time and people necessary for electrofishing campaigns means only a small number of locations can be assessed, which are then taken as a representation for the whole river.
Water sampling for eDNA analysis in the river Rissbach (Hinterriß, Tyrol, Austria). PHOTO CREDIT: Bettina Thalinger

Environmental DNA (eDNA) – genetic material obtained directly from environmental samples (soil, sediment, water, etc.) without any obvious signs of biological source material1 – has the potential to revolutionize fish monitoring in rivers as it enables the cost-effective analysis of multiple water samples from different locations within river networks. It allows us to easily pinpoint the upstream distribution limits of species and the changes in their longitudinal distribution. Although Alpine rivers are characterized by a comparably low species diversity, low population densities coupled with high water flow fluctuations represent a significant challenge for the broad-scale application of eDNA-based fish monitoring. Our team, scientists based at the University of Innsbruck and freshwater ecologists from the ARGE Limnologie, is examining what is needed for successful eDNA-based fish detections in Alpine rivers through the Austrian Research Promotion Agency (FFG) project “eDNA-AlpFish”.

First, our team defined detection limits for distinct flow situations in the summer and winter months. We were able to confirm the high sensitivity of our species-specific eDNA amplification approach by detecting signals from fish at low numbers and at a considerable downstream distance and water flow. For example, we detected signals stemming from ~50 individuals (200 g) of Eurasian minnow (Phoxinus phoxinus) at a downstream distance of 550 m and at a discharge of 0.25 m³/s. Under similar circumstances, a detection via electrofishing is uncertain as usually a shorter river stretch is sampled and not all present fish individuals can be caught.

High water flow during summer due to snowmelt (top) in comparison to low winter flow (bottom). In both situations, fish were placed in cages in the river Melach (Tyrol, Austria) for downstream eDNA detection. PHOTO CREDIT: Bettina Thalinger

In the next step, we evaluated the performance of these molecular approaches in direct comparison to electrofishing by sampling water from an Alpine river and its tributaries in the epirhithral zone. Whilst water samples from the whole river network could be obtained in a day, quantitative electrofishing was only possible at three sites within this timeframe. Additionally, the molecular analysis enabled the detection of bullhead (Cottus gobio) upstream of the point where low population levels made catches via electrofishing impossible.

Samples were also taken at epipotamal locations along the river Inn where the fish community is more diverse, and they were analysed with DNA metabarcoding. This approach permits the simultaneous assessment of the whole fish community and led to the detection of all the species known to occur in these waters. Additionally, we also obtained eDNA signals stemming from the surrounding tributaries in these samples.

The river Inn (Kufstein, Tyrol, Austria) as an example of epipotamal habitats under anthropogenic influence. PHOTO CREDIT: Bettina Thalinger

The results of the eDNA-AlpFish project will inform sampling strategy and data interpretation of future projects and, overall, highlight the massive potential of eDNA for large-scale fish monitoring in Alpine rivers.

References:

1. Thomsen, P. F., & Willerslev, E. (2015). Environmental DNA – An emerging tool in conservation for monitoring past and present biodiversity. Biological Conservation. doi: 10.1016/j.biocon.2014.11.019

Written by

Bettina Thalinger

Bettina Thalinger

Department of Ecology, University of Innsbruck, Austria

Centre for Biodiversity Genomics, University of Guelph, Canada
Yannick Pütz

Yannick Pütz

Department of Ecology, University of Innsbruck, Austria

Dominik Kirschner

Dominik Kirschner

Department of Ecology, University of Innsbruck, Austria

Christian Moritz

Christian Moritz

ARGE Limnologie GesmbH, Innsbruck, Austria

Richard Schwarzenberger

Richard Schwarzenberger

ARGE Limnologie GesmbH, Innsbruck, Austria

Josef Wanzenböck

Josef Wanzenböck

Research Department for Limnology, Mondsee, University of Innsbruck, Austria

Michael Traugott

Michael Traugott

Department of Ecology, University of Innsbruck, Austria

November 26, 2019
https://doi.org/10.21083/ibol.v9i1.5798

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The celebrities of the microcosmos aren’t always easy to find: detecting tardigrades in environmental DNA

The celebrities of the microcosmos aren’t always easy to find: detecting tardigrades in environmental DNA

The celebrities of the microcosmos aren’t always easy to find: detecting tardigrades in environmental DNA

The hidden diversity of tardigrades is being uncovered in Norwegian forests using DNA barcoding and metabarcoding

Scanning electron microscopy image of Diploechiniscus oihonnae

PHOTO CREDIT: Lasse Topstad

Found across every continent on Earth, to now potentially living on our moon, tardigrades are some of the most resilient microorganisms we know of. But despite our fascination with these microscopic water bears, there is still much to discover. Our study is exploring the applicability of using environmental DNA to facilitate the examination of tardigrade diversity.

The popular narrative that tardigrades can withstand anything – from -272 degrees Celsius to as high as 150 degrees Celsius, 6,000 times the atmospheric pressure, extreme radiation, and vacuum – has earned them celebrity status of the microcosmos. However, tardigrades are more than just superstars. They constitute their own phylum of life, ranked at the same taxonomic level as arthropods (insects and spiders), and currently hold around 1,270 described species. Many of these species fulfill ecologically important roles related to the breakdown of organic material in the soil. Other species are found in freshwater streams, sediments, mosses, lichens, and leaf-litter, occurring in most ecosystems throughout the world. As with other tiny taxa, telling tardigrade species apart can be challenging. Confident identifications of many species depend on the presence of both adult specimens and eggs. Additionally, tardigrade taxonomy is traditionally based on a limited set of morphological traits. This has resulted in several complex species groups, comprising morphologically inseparable, but genetically distinct species.

The claws of one of the species in the Macrobiotus hufelandi group. These species are often inseparable based on morphology, but clearly distinct species based on the COI gene.

PHOTO CREDIT: Lasse Topstad

DNA barcodes offer a solution to these impediments by generating unique genetic characteristics for each of these species. In recent years, there has been an increase in the use of molecular tools on tardigrades, but currently, only a small portion of the known species have barcodes deposited in public databases. Such reference sequences are essential if tardigrades are to be included in large-scale biomonitoring methods such as metabarcoding of environmental DNA (eDNA). Our study is the first to compare the applicability of eDNA-based metabarcoding of tardigrade diversity with morphologically identified communities.

Collection of lichen samples during fieldwork in Southern Norway PHOTO CREDIT: Torbjørn Ekrem

We extracted tardigrades and eggs from samples of moss, lichens, and leaf-litter and identified them using morphology. The 3,788 recorded tardigrade specimens and eggs were identified as 40 morphologically distinct species, of which 24 were successfully sequenced for the gene cytochrome c oxidase I (COI). These were represented by 151 successfully sequenced individuals. Interestingly, the barcodes revealed 32 genetically distinct linages among the 24 morpho-species, showing high levels of hidden diversity.

Figure 1. Overlap in species recovery by the different methods.

Next, we extracted eDNA from the same environmental samples and sequenced two fragments of the COI marker and one fragment of the 18S marker using the Illumina MiSeq next-generation sequencing platform. This method recovered 57 species of tardigrades compared to the 40 species detected by conventional methods. Mostly, the two methods identified the same species (Figure 1), yet, metabarcoding detected cryptic species elusive to morphological identification. This indicates that metabarcoding of eDNA successfully captures tardigrade diversity.

However, the credibility of such records needs to be evaluated thoroughly. While the COI marker distinguishes well between tardigrade species, the 18S marker might not be as useful as there is not sufficient sequence variation between species (a so-called barcode gap). Furthermore, the 18S marker detected Acutuncus antarcticus in two of the samples, a species endemic to Antarctica. This species is likely not found in Norway and highlights the danger of blindly trusting marker-based identifications without carefully evaluating taxonomic assignments and possibilities of contamination.

Our findings were dependent on our barcode reference library of locally sampled species and the use of multiple markers. As only a small portion of tardigrade species are deposited with reference sequences in public databases, both the COI and 18S markers are limited in their ability to detect species of tardigrades as most sequences will go unmatched. We demonstrate that metabarcoding is applicable for large-scale biomonitoring of tardigrades, but highlight the need for better reference libraries for tardigrade species.

Aknowledgements:

This research is part of a Master thesis at the NTNU University Museum and the project ‘Tardigrades in Norwegian Forests’ funded by the Norwegian Taxonomy Initiative and NorBOL. Special thanks to Roberto Guidetti at University of Modena and Reggio Emilia for his supervision during my stay in Italy.

Written by

Lasse Topstad

Lasse Topstad

Norwegian University of Science and Technology University Museum, Department of Natural History

September 18, 2019
doi: 10.21083/ibol.v9i1.5722

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Resident or invasive species? Environmental DNA can provide reliable answers

Resident or invasive species? Environmental DNA can provide reliable answers

Resident or invasive species? Environmental DNA can provide reliable answers

Environmental DNA can be successfully applied to identify vertebrates in a tropical lake improving our capacity to map and monitor species.
Panoramic view of Bacalar Lake including the 40-m deep Esmeralda sinkhole. PHOTO CREDIT: Manuel Elías-Gutiérrez

Monitoring life within large bodies of water – those species that should and shouldn’t live there – can be very expensive and time consuming. To overcome these limitations, efforts in many temperate regions employ methods that use environmental DNA (eDNA), enabling effective and targeted detection of invasive and resident endangered species.

Our study is the first to demonstrate that eDNA-based monitoring can be successfully applied to target the whole fish community in a tropical freshwater system and its adjacent wetlands.

Between 1980 -1990, eDNA was the term introduced to define particulate DNA and it was used to detect and describe microbial communities in marine sediments and phytoplankton communities in the water column1. However, eDNA is presently defined as the genetic material left behind by eukaryotic organisms in the environment, reflecting a rise in the use of eDNA for the detection of vertebrate and invertebrate species in aquatic systems1. The popularity of using eDNA has increased following the development of next-generation sequencing, advances in quantitative PCR (qPCR), and the growth of DNA barcodes libraries such as the Barcode of Life Data System (BOLD), providing a quicker and more taxonomically comprehensive tool for biodiversity assessments.

 

South end of lake Bacalar with the sinkhole Cenote Azul.
PHOTO CREDIT: Manuel Elías-Gutiérrez

Lake Bacalar is the largest epicontinental habitat in Mexico’s Yucatan Peninsula, and it is renowned for its striking blue color, clarity of the water, and for the world’s largest occurrence of living stromatolites, a calcareous mound built up of layers of lime-secreting cyanobacteria. Due to the presence of sediments derived from karst limestone, it represents the world’s largest fresh groundwater-feed ecosystem. The northern part of Lake Bacalar is connected to a complex system of lagoons and the southern part has an indirect connection to the sea via a wetland system that connects with Hondo River and enters Chetumal Bay. This river has been heavily impacted by the discharge of organic waste and pesticides, by vegetation clearing, and by the introduction of invasive fish such as tilapia and the Amazon sailfin catfish (Pterygoplichthys pardalis) 2-4, first detected in 2013 4. The Amazon sailfin catfish is a serious threat to the fragile stromatolite ecosystem due to its burrowing habits and competition with local fish. The impact of declining water quality and the rise of invasive species on the native fish fauna needs to be carefully monitored in aid of conservation efforts of Lake Bacalar.

A team of researchers from the Instituto Tecnológico de Chetumal and El Colegio de la Frontera Sur sampled eight localities in December 2015, and January and April 2016. After each of 14 sampling events, water and sediment samples were immediately placed on ice before transportation to the lab in Chetumal. To minimize eDNA degradation, we filtered water samples within seven hours of collection. All filters and sediments were stored at -18°C before being transported on ice from Chetumal to the Centre for Biodiversity Genomics in Guelph, Canada, where DNA extraction was undertaken.

 

Water sampling between stromatolites.
PHOTO CREDIT: Miguel Valadez

We sequenced short fragments (<200 bp) of the cytochrome c oxidase I (COI) gene on Ion Torrent PGM or S5 platforms. In total, we recovered eDNA sequences from 75 species of vertebrates including 47 fishes, 15 birds, seven mammals, five reptiles, and one amphibian. Although all species are known from this region, six fish species represent new records for the study area, while two require verification (Vieja fenestrata and Cyprinodon beltrani /simus), because their presence is unlikely in this ecosystem. While there were species (two birds, two mammals, one reptile) only detected from sediments, water samples recovered a much higher diversity (52 species), indicating better eDNA preservation in the slightly alkaline Bacalar water.  Because DNA from the Amazon sailfin catfish was not detected, we used a mock eDNA experiment that confirmed our methods were effective.

Interesting findings include the detection of rare species, such as an anteater Tamandua mexicana, which was detected by both PGM and S5 instruments from a river sample (Juan Sarabia), and migratory birds, such as warbler Oreothlypis peregrina known to overwinter in the Yucatan Peninsula.

Docks in front of Bacalar town
PHOTO CREDIT: Miguel Valadez

Our study indicates that eDNA can be successfully applied to monitor vertebrates in a tropical oligotrophic lake as well as more eutrophic (higher primary production) wetlands and can aid conservation and monitoring programs in tropical areas by improving our capacity to map occurrence records for resident and invasive species.

Our next step is to convince Mexican and international stakeholders to implement these methodologies and establish a permanent biomonitoring system for this and other pristine freshwater ecosystems found in Yucatan Peninsula. This work is necessary to detect effects of climate change, declining water quality, and the increasing tourism activities in this region.

References:

1. Díaz-Ferguson EE, Moyer GR (2014) History, applications, methodological issues and perspectives for the use of environmental DNA (eDNA) in marine and freshwater environments. Revista de Biología Tropical 62: 1273-1284. DOI: 10.15517/RBT.V62I4.13231

2. Wakida-Kusunoki AT, Luis Enrique Amador-del Ángel (2011) Aspectos biológicos del pleco invasor Pterygoplichthys pardalis (Teleostei : Loricariidae) en el río Palizada, Campeche, México. Revista Mexicana de Biodiversidad 82: 870-878

3. Alfaro REM, Fisher JP, Courtenay W, Ramírez Martínez C, Orbe-Mendoza A, Escalera Gallardo C, et al. (2009) Armored catfish (Loricariidae) trinational risk assessment guidlines for aquatic alien invasive species. Test cases for the snakeheads (Channidae) and armored catfishes (Loricariidae) in North American inland waters. Montreal, Canada: Commission for Environmental Cooperation. pp. 25-49.

4. Schmitter-Soto JJ, Quintana R, Valdéz-Moreno ME, Herrera-Pavón RL, Esselman PC (2015) Armoured catfish (Pterygoplichthys pardalis) in the Hondo River basin, Mexico-Belize. Mesoamericana 19: 9-19.

Written by

Natalia V. Ivanova

Natalia V. Ivanova

Centre for Biodiversity Genomics, Guelph, ON, Canada

Martha Valdez-Moreno

Martha Valdez-Moreno

El Colegio de la Frontera Sur, Unidad Chetumal, Chetumal, Mexico

Manuel Elías-Gutiérrez

Manuel Elías-Gutiérrez

El Colegio de la Frontera Sur, Unidad Chetumal, Chetumal, Mexico

May 15, 2019
doi: 10.21083/ibol.v9i1.5474

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DNA Barcoding and Genomics in the Megadiverse Amazon Altitude Fields

DNA Barcoding and Genomics in the Megadiverse Amazon Altitude Fields

DNA BARCODING AND GENOMICS IN THE MEGADIVERSE AMAZON ALTITUDE FIELDS

Scientists are contributing to the most profound molecular representation of biodiversity in any Brazilian environment.
Entrance of a ferruginous cave

The entrance of a ferruginous cave at the Bocaina mountain in the Carajás region, Pará State, Brazil. PHOTO CREDIT: João Marcos Rosa.

Industrial activities in the Brazilian Amazon are highly regulated by governmental agencies. However, the lack of knowledge about megadiverse areas is a problem for the establishment of best conservation practices; this is the case for mining operations in the eastern Amazonian Carajás, a region comprised of a mosaic of national parks, indigenous peoples’ conservation areas, and nature reserves. Of particular interest are the ferruginous altitude fields known as the Canga. Our floristic survey described the presence of 1,094 species from just over 200 previously known1. The lack of biodiversity data is even more significant for the ferruginous caves where only around 10 invertebrate species are identified to the species level. Unfortunately, this is typical for the Amazon basin.

To provide reliable scientific data that contributes to the implementation of best conservation practices, Instituto Tecnológico Vale is developing DNA barcode reference libraries for the flora, cave invertebrates, and bats of the region, and providing deeper genomic references for species that are endangered or difficult to identify. To achieve this goal, we established the necessary infrastructure to conduct DNA sequencing using Sanger, Illumina, and PacBio technologies, coupled with high-performance computing, artificial intelligence algorithms, and highly trained personnel.

 

Botanists collecting samples

Botanists collecting samples in a temporary lake in ferruginous altitude fields in the Carajás region, Pará State, Brazil.
PHOTO CREDIT: João Marcos Rosa

To date, over 8,575 barcodes for 3,548 specimens of plants and invertebrates have been produced, while a large number of species remain to be identified by morphological attributes. Morphological specimen determination is conducted by in-house specialists, as well as by an extensive network of specialists in universities and museums across Brazil and abroad.

For this purpose, nuclear and chloroplast or mitochondrial markers as well as low coverage to whole genome sequencing or restriction site-associated DNA sequencing (RADSeq) are being employed to unravel the vast genetic diversity of the biota of Carajás2-4. For several endemic plants, such as species of Asteraceae, Melastomataceae5, Convolvulaceae6, and Isoetaceae2, diversity analyses, based on next-generation sequencing, aim to characterize the genetic variability among and within populations, as well as the identification of markers under selective pressure. These methods also contribute to the understanding of population structure and the process of gene flow between populations affected by natural factors and industrial operations. Models of environmental distribution, including parameters sensitive to climate change, were determined for several taxonomic groups, including plants and bats7.

Specimens of the pinheirinho-da-canga (Paepalanthus fasciculoides) highly adapted to inhabit the canga (ancient ferruginous rock outcrops) at the altitude fields in the Carajás region, Pará State, Brazil.
PHOTO CREDIT: João Marcos Rosa

We are also establishing eDNA methods, as well as metagenomics and metaproteomics data for environmental monitoring of ferruginous fields phytophysiognomies, areas under rehabilitation processes, and caves8. Together these data constitute the most profound molecular representation of any environment in Brazil. We have contributed a total of 3,072 specimens to the Barcode of Life Data System (BOLD) comprising 398 genera (291 new) in addition to the 408 different genera collected through the national effort for angiosperms in Brazil. We have also provided 571 cave fauna specimens. It is important to highlight that all of these data generated are being provided to the public and its use will be critical to the conservation of such a unique collection of species.

References:

1. Brazil Flora Group (2018) Growing knowledge: an overview of Seed Plant diversity in Brazil. Rodriguésia 66(4): 1085–1113. http://dx.doi.org/10.1590/2175-7860201566411

2. Nunes GL, Oliveira RRM, Guimarães JTF, Giulietti AM, Caldeira C, Vasconcelos S, et al., (2018) Quillworts from the Amazon: A multidisciplinary populational study on Isoetes serracarajensis and Isoetes cangae. PLoS ONE 13(8): e0201417. https://doi.org/10.1371/journal.pone.0201417

3. Ramalho AJ, Zappi DC, Nunes GL, Watanabe MTC, Vasconcelos S, Dias MC, Jaffé R, Prous X, Giannini TC, Oliveira G and Giulietti AM (2018) Blind testing: DNA barcoding sheds light upon the identity of plant fragments as a subsidy for cave conservation. Frontiers in Plant Science 9:1052. https://doi.org/10.3389/fpls.2018.01052

4. Oliveira RRMO, Vasconcelos S, Pires ES, Pietrobon T, Prous X and Oliveira G (2019) Complete mitochondrial genomes of three troglophile cave spiders (Mesabolivar, pholcidae), Mitochondrial DNA Part B 4(1): 251–252. https://doi.org/10.1080/23802359.2018.1547139

5. Carvalho CdS, Lanes ECM, Silva AR, Caldeira CF, Carvalho-Filho N, Gastauer M, Imperatriz-Fonseca VL, Nascimento W, Oliveira G, Siqueira JO, Viana PL, Jaffe R (2019) Habitat loss does not always entail negative genetic consequences. bioRxiv 528430. https://doi.org/10.1101/528430

6. Lanes ÉC, Pope NS, Alves R, Carvalho Filho NM, Giannini TC, Giulietti AM, Imperatriz-Fonseca VL, Monteiro W, Oliveira G, Silva AR, Siqueira JO, Souza-Filho PW, Vasconcelos S and Jaffé R (2018) Landscape genomic conservation assessment of a narrow-endemic and a widespread morning glory from Amazonian Savannas. Frontiers in Plant Science 9:532. https://doi.org/10.3389/fpls.2018.00532

7. Costa WF, Ribeiro M, Saraiva AM, Imperatriz-Fonseca VL, Giannini TC (2018) Bat diversity in Carajás National Forest (Eastern Amazon) and potential impacts on ecosystem services under climate change. Biological Conservation 218: 200–210. https://doi.org/10.1016/j.biocon.2017.12.034

8. Gastauer M, Vero MPO, de Souza KP, Pires ES, Alves R, Caldeira CF, Ramos SJ, Oliveira G (2019) A metagenomic survey of soil microbial communities along a rehabilitation chronosequence after iron ore mining. Scientific Data 6:190008. https://doi.org/10.1038/sdata.2019.8

Written by

Guilherme Oliveira

Guilherme Oliveira

Environmental Genomics Group, Instituto Tecnológico Vale, Belém, Brazil

Gisele Nunes Lopes

Gisele Nunes Lopes

Environmental Genomics Group, Instituto Tecnológico Vale, Belém, Brazil

Rafael Valadares

Rafael Valadares

Environmental Genomics Group, Instituto Tecnológico Vale, Belém, Brazil

Ronnie Alves

Ronnie Alves

Environmental Genomics Group, Instituto Tecnológico Vale, Belém, Brazil

Santelmo Vasconcelos

Santelmo Vasconcelos

Environmental Genomics Group, Instituto Tecnológico Vale, Belém, Brazil

April 7, 2019
doi: 10.21083/ibol.v9i1.5498

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