Reconstructing the diet of an elusive wood grouse (western capercaillies) using metagenomics

by Physilia Chua | Nov 12, 2019 | Illuminations

Reconstructing the diet of an elusive wood grouse (western capercaillies) using metagenomics

Environmental DNA provides a non-invasive and simple means of biomonitoring

By Physilia Chua

Spotted! A male capercaillie displaying its magnificent tail feathers

PHOTO CREDIT: Per Gätzschmann

Gone are the days when researchers needed to spend countless hours observing an animal in the wild to understand its behaviour and ecology. As we demonstrate with our study, valuable data can be gathered by simply examining faecal samples with powerful metagenomics approaches.

The need for data that effectively informs biological conservation is intensifying as the rate of biodiversity loss increases. Traditionally, scientists have endured long hours in the field, often hiding uncomfortably in bushes or traversing dangerous and hard-to-reach places, all for the purpose of observing elusive animals.

Searching for capercaillies in the midst of a snow storm in the Norwegian boreal forests

Photo credit: Physilia Chua

With the advent of next-generation sequencing (NGS) technologies – those that effectively provide large amounts of DNA sequence data – it is now possible to obtain a wealth of ecological information from just a single faecal sample. The ease of collecting such samples circumvents some of the challenges of studying animals otherwise hard to find.

One such NGS approach is metagenomics shotgun sequencing (MSS), which determines the nucleotide composition of large amounts of random DNA molecules recovered from complex samples of DNA from various sources. This method makes it possible to simultaneously retrieve information about the host’s diet, microbiome, gut parasites, as well as the population structure of the species¹. While it has vast potential for conservation biology, few studies have utilised MSS to reconstruct the diet of animals, and none have done so for herbivorous birds.

A typical day out in the fields high above the Arctic Circle in Tromsø, Dividalen National Park, Norway

Photo credit: Physilia Chua

The western capercaillie (Tetrao urogallus), or wood grouse, is an emblematic species which can be found in the coniferous forest of Eurasia. Highly susceptible to the increased levels of habitat destruction and fragmentation, their declining population has placed them on the International Union for Conservation of Nature (IUCN) Red-list throughout most of western and central Europe². By studying the wood grouse’s diet, we could gain clues about the resources it requires and the other species it interacts with in its habitat, informing better conservation strategies. By observing the animal and morphologically identifying plant remains from their faecal samples, it was determined that the capercaillie’s diet consists of mostly pine needles in the winter, and Vaccinium species in the summer³.

A pile of capercaillie scat

Photo credit: Physilia Chua

Capercaillie’s favourite food? Pine needles (left) and Vaccinium sp. (right)

Photo credit: Physilia Chua

However, preliminary results from our study show promising signs that the capercaillie’s diet is more diverse than once thought. Other than plants, we have also discovered parasitoid wasps and several species of mites, which could have been accidental ingestion while feeding or preening. And with the use of metagenomics, there is also the possibility of obtaining more detailed quantitative information about its diet that can be used to inform habitat management choices. Their gut microbiome, intestinal parasites, and population genetics are also currently being analysed. Unexpectedly, we were also able to detect the presence of plant-pathogenic fungus and nematodes from their faecal samples, providing some interesting ecological insights about the capercaillie’s habitat. Even though our research is still in its infancy, by using metagenomics shotgun sequencing on faecal samples, our initial study has already yielded a wealth of data. There is truly an untapped potential for its application in conservation biology and biomonitoring, which should be further explored.

The road less travelled might lead to unexpected discoveries

Photo credit: Physilia Chua

AcknowledgementS:

I thank my supervisors Kristine Bohmann, Sanne Boessenkool, and Inger Greve Alsos for their guidance in every step of this research, without whom this study would not have been possible. I am deeply grateful to Torbjørn Ekrem for his invaluable support both in and outside of fieldwork. I am indebted to my collaborators Kat Bruce and Alex Crampton-Platt for taking me into their team at NatureMetrics and making bioinformatics look so easy. Lastly, my sincere gratitude to the members of the eDNA group at the Section for Evolutionary Genomics, University of Copenhagen, and also to my fellow Plant.ID ESRs for keeping me in the right headspace. This project is part of the H2020 MSCA-ITN-ETN Plant.ID network and has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 765000.

References:

1. Srivathsan A, Sha JCM, Vogler A, Meier R (2015) Comparing the effectiveness of metagenomics and metabarcoding for diet analysis of a leaf-feeding monkey (Pygathrix nemaeus). Molecular Ecology Resources 15(2): 250–261. https://doi.org/10.1111/1755-0998.12302 2. Storch I (2000) Grouse: status survey and conservation action plan 2000-2004. IUCN/SSC Action Plans for the Conservation of Biological Diversity. Retrieved from http://www.iucn.org/dbtw-wpd/edocs/2000-031.pdf 3. Picozzi N, Moss R, Catt DC (1996) Capercaillie habitat, diet and management in a Sitka spruce plantation in central Scotland. International Journal of Agriculture and Forestry. 69(4): 373 – 388. https://doi.org/10.1093/forestry/69.4.373

Written by

Physilia Chua

Department of Biology, University of Copenhagen, Copenhagen, Denmark.

November 12, 2019

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https://doi.org/10.21083/ibol.v9i1.5725

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Discovering a resilient and hyperdiverse midge fly fauna in a Singaporean swamp forest

by Bilgenur Baloğlu | Sep 6, 2019 | Research

Discovering a resilient and hyperdiverse midge fly fauna in a Singaporean swamp forest

Researchers uncover a highly unique and diverse chironomid community in a Singaporean swamp forest highlighting the importance of these ecosystems and the power of Next-Generation Sequencing for biomonitoring efforts.

Nee Soon Swamp Forest, Singapore

PHOTO CREDIT: Wang Luan Keng

Benthic macroinvertebrates – those animals that live at the bottoms of water bodies – are abundant, diverse, relatively immobile, and responsive to environmental stresses, and these traits make them ideal indicators of the quality of aquatic ecosystems. Our study demonstrates the utility of Next-Generation Sequencing (NGS) platforms as an efficient and rapid tool for monitoring efforts.

In freshwater ecosystems, non-biting midges (Diptera: Chironomidae) often constitute the majority of diversity and biomass with different chironomid species varying in their sensitivity to environmental changes. But, when monitoring these habitats, chironomids are either ignored entirely or not studied at a species-level because morphological assessments are expensive and laborious, and the identification literature is based on adults while larvae are most often collected.

Chironomid adults collected from Nee Soon Swamp Forest. Different chironomid species vary in their sensitivity to environmental parameters. PHOTO CREDIT: Bilgenur Baloglu

The solution? NGS platforms. They allow for fast and effective species-level assessments of large-scale samples at low cost (less than $0.40 USD/specimen). Moreover, there is a high congruence between molecular and morphological identification, enabling a detailed examination of the composition of taxonomically complex communities^1,2. Freshwater swamp forests – the forested wetlands occurring along rivers and lakes – are home to various endemic and endangered species with 33% of birds and 45% of mammals either threatened or endangered on the IUCN Red List³, and with most of the insect fauna unknown. These ecosystems are under threat worldwide from habitat destruction, pollution, and climate crisis. Most of the world’s tropical swamp forests are found in Southeast Asia’s Indo-Malayan region collectively occupying more than 13 million ha⁴ among many geographically separated peninsulas and islands. Nee Soon swamp forest is the largest remnant (90 ha) of its kind in Singapore and thus of high national conservation value.

Bilge Baloglu sampling water DNA from Singapore’s largest swamp forest remnant.
PHOTO CREDIT: Dickson Ng

We generated DNA barcodes using NGS to study chironomids among the natural swamp forest Nee Soon and three adjacent man-made reservoirs. We wanted to understand the effects of urbanization and to know whether the chironomid fauna of Nee Soon is resistant to, that is, minimally impacted by, the adjacent reservoirs. We sampled >14,000 chironomid specimens (both adults and larvae) as part of a freshwater quality monitoring program, and quantified species richness and compositional changes using NGS and DNA barcoding.

Our study showed that Singapore’s biggest swamp forest remnant maintains a rich and largely unique fauna of about 350 species. The minimal species overlap between sites indicated that the Nee Soon swamp forest is resistant against the invasion of species from surrounding artificial reservoirs.

These findings suggest that even small or fragmented swamp forests can be suitable habitats for chironomids, shedding light on many other swamp forests in Southeast Asia that collectively occupy a much larger area and that are threatened by destruction for oil palm plantations and paper pulp production. Overall, our study exposes the enormous power of NGS and DNA barcoding in ecological research to study ecosystem health, biological diversity, and habitat conservation.

References:

1. Brodin Y, Ejdung G, Strandberg J, Lyrholm T (2013) Improving environmental and biodiversity monitoring in the Baltic Sea using DNA barcoding of Chironomidae (Diptera). Molecular Ecology Resources 13:996–1004.

2. Montagna M, Mereghetti V, Lencioni V, Rossaro B (2016) Integrated taxonomy and DNA barcoding of alpine midges (Diptera: Chironomidae). PLoS One 11:e0149673

3. Posa MR (2011) Peat swamp forest avifauna of Central Kalimantan, Indonesia: Effects of habitat loss and degradation. Biological Conservation 144(10):2548-2556.

4. Hooijer A, Page S, Canadell JG, Silvius M, Kwadijk J, Wösten H, Jauhiainen J (2010) Current and future CO2 emissions from drained peatlands in Southeast Asia. Biogeosciences 7:1505–1514

For full details, please refer to the publication in Frontiers in Zoology.

Written by

Bilgenur Baloğlu

Centre for Biodiversity Genomics, Guelph, ON, Canada

September 6, 2019

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doi: 10.21083/ibol.v9i1.5525

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The deep connection between soil microbes and trees: DNA metabarcoding and reforestation

by Katie McGee and Mehrdad Hajibabaei | May 23, 2019 | Research

The deep connection between soil microbes and trees: DNA metabarcoding and reforestation

Forest restoration can be better facilitated by considering the diversity and biomass of soil microbiomes

Aerial view of Laguna del Lagarto Lodge and primary forest, Costa Rica.

PHOTO CREDIT: Fritz Fucik

Tropical deforestation has contributed to the atmospheric rise in greenhouse gas levels, negative impacts on nutrient cycles, and declines in biodiversity. While forest restoration schemes are being implemented, the success of such efforts needs to be better evaluated. Our study demonstrates that soil microbial communities can guide the selection of key tree species important for local forest restoration processes and, ultimately, the global recovery of tropical forests.

2-year old logging road amongst Costa Rican primary forest with no vegetation re-growth due to severe soil degradation and compaction.

PHOTO CREDIT: Katie M. McGee

Tropical forests only comprise 7–10% of the Earth’s land surface but contain 20% of the planet’s carbon within the first three metres of soil. They also exchange more carbon dioxide (CO₂) with the atmosphere than any other terrestrial ecosystem. Such characteristics make tropical areas critical for terrestrial primary productivity and global nutrient cycling. Yet, these important ecosystems are continually under threat from human-driven land-use practices.

Deforestation activities across the tropics contribute to the increase of atmospheric CO₂ at levels comparable to fossil fuels¹. If tropical deforestation were a country, it would be the third largest contributor of CO₂ emissions (behind China and the United States), producing more than the European Union². One of the main contributing factors, often ignored, is the large release of CO₂ from the soil when forests are clear-cut; this occurs due to alterations in the respiration maintenance processes of soil microbes that result in a rapid release of the massive stock of soil carbon that has accumulated over time. Moreover, the soil in areas facing extraction-based land-use strategies have been so degraded that the capacity to recover and sustain biological productivity, and to capture and store carbon is significantly reduced.

Source Graph: Seymour and Busch (2016), Source Data: Busch and Engelmann (2015)

To remediate these consequences, restoration attempts have been implemented throughout the tropics. However, the success of these efforts is largely explored by studying charismatic organisms, such as birds, or by assessing plant biomass, with substantially less focus on soil dynamics. As soil microbes are key components in biogeochemical and nutrient cycling processes, it is thought that certain tree species and their affiliated soil microorganisms may help to serve as a principal pathway to ameliorate degraded soils. Many tropical trees can convert or ‘fix’ atmospheric nitrogen (N₂) into ammonium through specialized root microbial symbionts. This conversion is critical to the growth and development of plants and soil microbes, yet the influence that N-fixing trees can have on the soil organisms in their immediate vicinity is still unclear.

The use of DNA-based identification techniques has significantly advanced research on soil microbial communities. Since the 1980s, popular methods have involved Terminal Restriction Fragment Length Polymorphism techniques and Sanger sequencing. However, all of these methods are time consuming, costly, and involve laborious processes. The more recent development of DNA metabarcoding has allowed us to rapidly and comprehensively characterize soil biotic communities.

DNA metabarcoding is a method that combines traditional marker gene surveys – targeting particular organisms using standardized PCR primers for specific gene regions – with next-generation sequencing. By comparing obtained DNA sequences to a standard reference library of known organisms, taxa present in an environmental sample such as soil can be identified with high confidence. This allows us to address ecological questions linked to environmental impact and biomonitoring in a more efficient manner.

Pentaclethra macroloba and its soil microbiome shown to effectively support forest restoration in northern Costa Rica.

PHOTO CREDIT: Katie M. McGee

Using DNA metabarcoding, our study investigated individual plant effects of the soil collected around two types of trees, Pentaclethra macroloba (Gavilán; nitrogen-fixing) and Dipteryx panamensis (Almendro; non-nitrogen-fixing), in Costa Rica’s northern region. We wanted to examine differences in the soil bacterial and fungal community composition.

We found that each plant species contained a unique soil microbial community, and that the nitrogen-fixing tree, Pentaclethra, supported soil microbes and microbial biomass at levels similar to those measured in primary forests. This indicates their importance for the recovery of soils to a pre-disturbed state. In comparison to the non-N-fixer Dipteryx, Pentaclethra stimulates a soil microbial community that is more efficient in storing soil carbon into biomass, as opposed to carbon loss via aforementioned respiration maintenance processes. These effects appeared to be associated with the amount of soil ammonium that the Pentaclethra-soil is able to provide to the surrounding soil.

Our results indicate the importance of this N-fixing tree in building back up carbon storage as biomass in the soil as well as promoting plant and soil microbial growth. As such, we suggest the use of Pentaclethra and its associated soil microbiome as an important ecosystem restoration tool in facilitating early regeneration of secondary forests.

Our method of using soil microbes, characterized by DNA metabarcoding, is a novel approach that can be applied globally to guide regeneration efforts that most effectively improve the quality and fertility of degraded soils as well as inform restoration ecology and the policy surrounding it.

References:

1. Seymour F and Busch J (2016) Why forests? Why now? The science, economics, and politics of tropical forests and climate change. Center for Global Development. Washington, DC, USA. ISBN: 978-1-933286-85-3

2. Busch J and Engelmann J (2015) The Future of Forests: Emissions from Deforestation With and Without Carbon Pricing Policies, 2015– 2050. CGD Working Paper 411. Center for Global Development. Washington, DC, USA.

Written by

Katie M. McGee

Centre for Biodiversity Genomics, Guelph, ON, Canada

Mehrdad Hajibabaei

Centre for Biodiversity Genomics, Guelph, ON, Canada

May 23, 2019

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doi: 10.21083/ibol.v9i1.5472

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Resident or invasive species? Environmental DNA can provide reliable answers

by Natalia Ivanova, Manuel Elías-Gutiérrez and Martha Valdez-Moreno | May 15, 2019 | Research

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 2013⁴. 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

Centre for Biodiversity Genomics, Guelph, ON, Canada

Martha Valdez-Moreno

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

Manuel Elías-Gutiérrez

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

May 15, 2019

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doi: 10.21083/ibol.v9i1.5474

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