Understanding the impacts of widespread forest die-offs across France, Germany, and China

Understanding the impacts of widespread forest die-offs across France, Germany, and China

Research Article

Metabarcoding increases the taxonomic resolution and geographic scale at which researchers can assess the impacts of climate change on insect communities in forests

Entomologists studying tree dieback of Picea abies in Bavarian National Park.

PHOTO CREDIT: Heiner M. Elsner

Forests throughout the world are suffering from an increase in the frequency and severity of droughts as well as pathogen and insect infestations. These climate-exacerbated factors are leading to tree diebacks—the progressive death of tree branches—and subsequent large-scale forest die-offs, the effects of which are not well understood. Preliminary results from our study indicate that while the number of insect species along forest die-off gradients might not be affected, their composition is changing.

CLIMTREE is an international project funded by the Belmont Forum that assesses the impact of climate-induced forest die-off on invertebrate biodiversity in highland forests in France, Germany, and China. This project aims to better understand how tree mortality and associated changes in forest composition affect biodiversity and ecosystem functions.

Silver fir (Abies alba) dieback in the French eastern Pyrenees

PHOTO CREDIT: Carlos Lopez-Vaamonde

Our study measures changes in the insect communities along dieback and salvage-logging gradients of silver fir (Abies alba) forests in the French eastern and central Pyrenees, Norway, spruce (Picea abies) forests in the Bavarian Forest National Park, Germany, and Yunnan pine (Pinus yunnanensis) forests in Yunnan province, China. We examined patterns of variation in the species diversity of flying insects collected by Malaise traps. As these passive traps can collect a large number of specimens, we analyzed samples in bulk using DNA metabarcoding. This approach uses DNA barcode reference libraries to identify species from a mixed sample using high-throughput technologies that effectively provide large amounts of DNA sequence data.

Members of CLIMTREE assessing the level of decline of silver fir stands in Pays de Sault (French eastern Pyrenees)

PHOTO CREDIT: Carlos Lopez-Vaamonde

We also examined saproxylic beetles, a key functional group used as bioindicators in forest assessments of dead-wood availability. We collected these beetles using flight interception traps, and we also sampled specimens from natural history collections to build a DNA barcode reference library for the French saproxylic beetle fauna as a resource for future investigations.

The preliminary results from the 56 Malaise traps deployed in the French eastern and central Pyrenees have revealed more than 3,500 OTUs (Operational Taxonomic Units, a proxy for species) belonging to 18 insect orders, with considerable change in the compositions of the species detected along the dieback gradient as well as across a 4-month period. However, results to date do not suggest significant declines in species richness over the dieback gradient.

Rosalia alpina (Cerambycidae) is one of the 2,663 species of saproxylic beetles known to occur in France

PHOTO CREDIT: Carlos Lopez-Vaamonde

Polytrap flight interception trap

PHOTO CREDIT: Carl Moliard

Of the 55,571 saproxylic beetles collected by the flight interception traps, about 70% were morphologically identified to one of 284 species. Similar to the flying insects collected by Malaise traps, the diversity of beetles along dieback gradients did not decline. We are now trying to use a non-destructive metabarcoding approach that involves processing the collection media to determine whether we can uncover a comparable number of species with a morphology-based sorting approach. If results are similar, we will have a strong case for using this technique as a time-efficient alternative for biomonitoring moving forward.

Overall, there is an urgent need to obtain detailed baseline data on insect communities to quantify the impacts of climate change. By taking advantage of DNA metabarcoding approaches, our study is assessing biodiversity patterns at scales previously impossible and providing the data essential for evaluating future changes in insect communities. Our workflows are simple to use and provide an affordable, reliable, and repeatable method of assessing insect diversity in forests at a large geographical scale.

Written by

Lucas Sire

Lucas Sire

Institut de Recherche sur la Biologie de l’Insecte, Tours, France

Paul Schmidt Yañez

Paul Schmidt Yañez

Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany

Rodolphe Rougerie

Rodolphe Rougerie

Muséum National d’Histoire Naturelle, Paris, France

Christophe Bouget

Christophe Bouget

IRSTEA, Nogent-sur-Vernisson, France

Laurent Larrieu

Laurent Larrieu

Dynafor INRA & CRPF Occitanie, Toulouse, France

Douglas W. Yu

Douglas W. Yu

Kunming Institute of Zoology, Kunming, China

Michael T. Monaghan

Michael T. Monaghan

Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany

Institut Für Biologie, Freie Universität Berlin, Germany

Jörg Müller

Jörg Müller

Bavarian Forest National Park, Grafenau, Germany

Elisabeth A. Herniou

Elisabeth A. Herniou

Institut de Recherche sur la Biologie de l’Insecte, Tours, France

Carlos Lopez-Vaamonde

Carlos Lopez-Vaamonde

Institut de Recherche sur la Biologie de l’Insecte, Tours, France

INRA, Orléans, France

October 28, 2019
https://doi.org/10.21083/ibol.v9i1.5726

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Insects don’t talk, but new DNA-based technologies are helping to tell their stories

Insects don’t talk, but new DNA-based technologies are helping to tell their stories

Research Article

Researchers unlock clues about vertebrate diversity by examining arthropod samples
Malaise trap deployed in Carajás National Forest in Brazil. PHOTO CREDIT: Christina Lynggaard

The study of arthropod diversity often uses primers solely targeting arthropod DNA, ignoring those originating from non-arthropod sources. Yet, some arthropods (e.g. sand flies, blowflies, mosquitoes) feed on vertebrates and vertebrate remains. By specifically targeting these vertebrate-feeding arthropods, invertebrate derived DNA (iDNA) can be used to understand vertebrate diversity as well. Our study has found that by using samples collected for arthropod diversity research, we can tap into a rich information source of vertebrate diversity.

Arthropods are highly abundant and provide vital ecosystem services such as pollination, decomposition of organic matter, and they serve as a food source for other animals. Because of this, hundreds to thousands of arthropod samples are collected globally every year for monitoring programs, conservation efforts, and ecosystem assessments.

However, identifying arthropod species based on physical characteristics is difficult, requiring specific expertise and a significant amount of time. Due to this, molecular analyses, such as DNA metabarcoding, are being used to identify the arthropods present in bulk samples, speeding-up the otherwise time-demanding identification process by screening the contents of hundreds of samples simultaneously.

Bulk arthropod sample

PHOTO CREDIT: Christina Lynggaard

In this study, bulk arthropod samples were collected using pitfall and Malaise traps deployed at the Carajás National Forest in Brazil and in the Udzungwa Mountains in Tanzania. Our aim was to study the arthropod diversity in these areas by using metabarcoding primers targeting arthropod DNA. Nevertheless, we conducted an additional analysis targeting vertebrate DNA remains in the bulk samples by using metabarcoding vertebrate primers.

We were able to detect vertebrate DNA in 19 per cent of our 265 analyzed samples, identifying more than 30 vertebrate taxa including mammals, amphibians and birds. For example, we found South American tapir (Tapirus terrestris), chirinda screeching frog (Arthroleptis xenodactyloides), velvety free-tailed bat (Molossus molossus), Zanzibar bushbaby (Paragalago zanzibaricus) and honeyguide greenbul (Baeopogon indicator). The presence of some species was further confirmed through visual observations during the sample collections.

Some cases represent the first time those vertebrate species have been detected in the area. This could indicate that we are detecting vertebrates that live in different areas to where the arthropods were collected or that with this method it is possible to detect local animals that are difficult to observe.

Tapir footprint found in one of the Brazilian study sites during bulk arthropod sample collection confirmed the presence of some of the detected vertebrate taxa PHOTO CREDIT: Christina Lynggaard

Bushbaby (Paragalago zanzibaricus) found in the Udzungwa Mountains in the Tanzanian study site during bulk arthropod sample collection confirmed the presence of some of the detected vertebrate taxa

PHOTO CREDIT: mnielsenphotography

The sensitivity of new DNA-based technologies is changing the way we explore and understand the relationships between species. Approaches like DNA metabarcoding can not only tell us about the different types of insects in our environment, but also what they eat and where they have been. Our study is the first to not target a specific invertebrate group to detect vertebrate DNA but instead to use bulk arthropod samples. We encourage others to consider bulk samples during environmental monitoring not only as a source for arthropod diversity information, but one for vertebrate diversity as well.

This study is published in the eDNA Journal.

Written by

Christina Lynggaard

Christina Lynggaard

The GLOBE Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

Martin Nielsen

Martin Nielsen

The GLOBE Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

Luisa Santos-Bay

Luisa Santos-Bay

The GLOBE Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

NTNU University Museum, Norwegian University of Science and Technology (NTNU), Trondheim, Norway
Markus Gastauer

Markus Gastauer

Instituto Tecnológico Vale, Belém, Pará, Brazil

Guilherme Oliveira

Guilherme Oliveira

Instituto Tecnológico Vale, Belém, Pará, Brazil

Kristine Bohmann

Kristine Bohmann

The GLOBE Institute, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

University of East Anglia, Norwich Research Park, Norwich, UK

October 17, 2019
https://doi.org/10.21083/ibol.v9i1.5727

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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

Research Article

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
https://doi.org/10.21083/ibol.v9i1.5722

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

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

Research Article

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 communities1,2.

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.

For full details on the study, see Baloğlu et al. 2018.

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

Written by

Bilgenur Baloğlu

Bilgenur Baloğlu

Centre for Biodiversity Genomics, Guelph, ON, Canada

September 6, 2019
PDF
https://doi.org/10.21083/ibol.v9i1.5525

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Starving for data and more: what rangers and scientists stand to learn from one another in South Africa

Starving for data and more: what rangers and scientists stand to learn from one another in South Africa

Research Article

A one-year pilot biomonitoring program in Kruger National Park, South Africa – the Kruger Malaise Program – reignites rangers' energy about biodiversity conservation.

Silhouette of a giraffe in Kruger National Park, South Africa

PHOTO CREDIT: Michelle D’Souza

Insect biodiversity is understudied and often underappreciated. As evidence for large-scale insect declines emerge, there is an increasing need to address the extreme lack of data on the general ecology and population dynamics of most insect groups. Charismatic species, such as the iconic monarch butterflies (Danaus plexippus) of the Americas, are one of the few exceptions.

Closely related to the migratory Danaus plexippus, the non-migratory monarch species – Danaus chrysippus – is found in the warm climate of the African continent.
PHOTO CREDIT: Johandre van Rooyen

The caterpillars of the Emperor moth (Gonimbrasia belina) are just as iconic and societally relevant on the African continent. Locally referred to as ‘mopane worms’ after the mopane trees upon which they primarily feed, these insects have been a vital source of protein for generations. A mopane caterpillar contains on average 50 per cent protein1, a higher percentage than the average steak.

In recent years, mopane caterpillars have also provided an important source of income for many rural communities. It has been estimated that 9.5 billion caterpillars are harvested annually in Southern Africa’s 20,000 km2 of mopane forest. The ability to predict mopane caterpillar outbreaks in space and time becomes increasingly valuable, particularly for rural communities living along the borders of national parks, who rely heavily on natural resources to supplement their livelihood.

Mopane worm harvest in Kruger National Park, South Africa.
PHOTO CREDIT: Louise Swemmer

Local community members harvesting mopane worms in the Kruger.
PHOTO CREDIT: Louise Swemmer

Since 2010, permit-based harvesting projects have taken place in some South African National parks to share benefits and build positive relationships between the parks and their neighbouring communities. With the declining occurrence of mopane caterpillars outside of protected areas due to habitat change and over-harvesting, and the overall erratic nature of recent outbreaks, neighbouring communities risk losing an important source of food and income.

A better understanding of insect dynamics has the potential to inform the sustainable harvest of natural resources such as the mopane caterpillar, but it also tells us a lot more.

A pilot insect biomonitoring program in Kruger National Park, South Africa – the Kruger Malaise Program – is already demonstrating implications for natural resource harvesting, as well as agricultural pest and disease management. Perhaps even more significant, it has reignited energy in park rangers about biodiversity conservation.

One of 26 Malaise traps sampling insects in Kruger National Park with the Kruger Malaise Program.
PHOTO CREDIT: Ryan Rattray

The Kruger Malaise Program (KMP), a year-long monitoring effort, was undertaken in Kruger Park from May 2018 to June 2019. With the main goal of understanding insect diversity and seasonal variation, the program deployed 26 Malaise traps that sampled the flying insect community in all 22 sections of the park. Traps were set up within each section ranger’s property, and rangers were tasked with organizing and maintaining weekly sample collections. The samples were then retrieved in four large batches over the year by staff from the African Centre for DNA Barcoding (ACDB) in Johannesburg, South Africa, where they were packaged and shipped to the Centre for Biodiversity Genomics (CBG) in Guelph, Ontario, Canada for DNA barcode analysis. This program was only possible due to the collaborative efforts of park rangers and staff, researchers at the Savanna & Arid Research Unit in Skukuza, Kruger, and scientists at the ACDB and CBG.

The African Centre for DNA Barcoding (ACDB) team after collecting the last Malaise trap at the end of the KMP in June 2019: Zandisile Shongwe, Nolo Sello, Michelle van der Bank (ACDB Director), Ross Stewart, Jonathan Davies (top left to right), Johandre van Rooyen (bottom).
PHOTO CREDIT: Nolo Sello

With sampling now complete, analysis has begun in earnest. So far, more than 260,000 specimens have been processed, and 170,000 have been sequenced.  Preliminary results have delivered barcode coverage for 9,000 species including various agricultural pests (e.g., the olive fruit fly (Bactrocera oleae), and the rusty plum aphid (Hysteroneura setariae)) as well as several vector species known to transmit the bluetongue and African horse sickness viruses (e.g., Culicoides imicola) and West Nile Virus (Culex perexiguus). When compared against the DNA barcode database (BOLD Systems) of more than 600,000 species, almost half of the insect diversity uncovered by the program so far is only found in Kruger.

Based on species accumulation rates, it is likely that 25,000 species will be recorded in the park. This number represents more than half of the species previously reported from South Africa2, and quarter of those described in sub-Saharan Africa3.

Selection of specimens collected from the Kruger Malaise Program.
PHOTO CREDIT: CBG Imaging Lab

The Kruger Malaise Program reveals just how quickly DNA barcoding can provide in-depth and broad-scale information for regions where past research has largely been focused on particular taxonomic groups.  While one of the only comprehensive field guides for insects in South Africa contains 1,200 species – those that are ‘abundant, widespread, conspicuous, large or unusual’ – the Kruger Malaise program has largely uncovered the rare, small, inconspicuous, yet ecologically important, species.

In 2013, SANParks developed a biodiversity monitoring strategy but its activation has been very mixed across the 19 parks. Some began their monitoring efforts by focusing on rare species, while others used key indicator groups. But there have been no standardized techniques implemented across all parks, and there has been little monitoring of insects at a large scale, mainly because of the lack of taxonomic expertise. A program involving DNA technology makes large-scale biomonitoring of these national parks possible.

The KMP has been a huge success with the next steps set to fine tune logistics before its expansion to other parks and, ideally, to identify specific sites in Kruger for ongoing monitoring. The program also provided a test bed for TRACE (Tracking the Response of Arthropod Communities to Changing Environments), a major research theme within the 7-year, $180 million BIOSCAN program. Its success has demonstrated the feasibility of extending this work in other national parks within South Africa and on a global scale. In doing so, BIOSCAN will lay the foundation for a DNA-based global biodiversity observation system, similar to the monitoring systems that have been recording weather patterns since the 1800s. BIOSCAN has a grand vision, one that is necessary if we are to truly identify, understand, and manage the global decline in insects.

The park rangers and staff who managed the Malaise traps in Kruger National Park.
PHOTO CREDIT: Michelle D’Souza

But if you ask the people working in Kruger, the KMP was more than a biodiversity monitoring program. Most rangers start out as nature conservation and zoology students, but anti-poaching efforts are so time consuming that their roles have gone from biodiversity managers to single-species protectors. The KMP has not only sparked interest and reignited energy in the park rangers about their conservation work, it has engaged and valued the observational and experiential data that rangers have to offer, such as stories and strategies related to the mopane caterpillars.

In this way, the KMP has made a very big impact – and that is the true beauty of the program – its ability to spur interest in insect life, and the patterns and processes that define our world.

Please feel free to contact Michelle D’Souza, the KMP project manager, if you have any questions about the program: mdsouza@uoguelph.ca

References:

1. Glew RH, Jackson D, Sena L, VanderJagt DJ, Pastuszyn A and Millson M (1999) Gonimbrasia belina (Lepidoptera: Saturniidae): a Nutritional Food Source Rich in Protein, Fatty Acids, and Minerals. American Entomologist 45(4): 250–253

2. Scholtz CH and Chown SL (1995) Insects in southern Africa: how many species are there? South African Journal of Science 91:124–126

3. Miller SE and Rogo LM (2002) Challenges and opportunities in understanding and utilisation of African insect diversity. Cimbebasia 17:197–218

Written by

Michelle L. D'Souza

Michelle L. D'Souza

Centre for Biodiversity Genomics, Guelph, ON, Canada

Danny Govender

Danny Govender

General Manager: Savanna and Arid Research Unit, South Africa

June 12, 2019
PDF
https://doi.org/10.21083/ibol.v9i1.5471

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

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

Research Article

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

Katie M. McGee

Centre for Biodiversity Genomics, Guelph, ON, Canada

Mehrdad Hajibabaei

Mehrdad Hajibabaei

Centre for Biodiversity Genomics, Guelph, ON, Canada

May 23, 2019
PDF
https://doi.org/10.21083/ibol.v9i1.5472

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Resident or invasive species? Environmental DNA can provide reliable answers

Resident or invasive species? Environmental DNA can provide reliable answers

Research Article

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
PDF
https://doi.org/10.21083/ibol.v9i1.5474

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Diagnosing a rare human disease in Mexico’s lowlands

Diagnosing a rare human disease in Mexico’s lowlands

Research Article

The first multidisciplinary study using DNA barcodes as a medical diagnostic tool forms a unique collaborative group comprised of medical practitioners and scientists.

Scanning electron micrograph of the anterior part of Lagochilascaris minor.
PHOTO CREDIT: Manuel Elías-Gutiérrez

Roundworms (Nematoda) are one of the most diverse groups of invertebrates. Lagochilascaris minor, a parasitic nematode often found in wild cats such as jaguars and pumas, as well as in domestic cats and dogs, has only rarely been known to infect humans. However, a recent case in the Yucatan Peninsula has brought about a very unique collaboration between medical practitioners and scientists.

Lagochilascaris minor has been reported in the Americas, commonly in South America where the eggs have been found in public parks1. However, identifying L. minor with conventional methods can be misleading as they resemble other ascaridoid eggs.

When a 23-year old man from a village in the forests of southern Quintana Roo state (Yucatan Peninsula) came into the local hospital in July 2016, physicians were surprised to find that a parasite had destroyed the mastoid apophysis, the lateral sinus, and part of the cerebellum. After a radical mastoidectomy and medical treatment for 63 days, the patient made a full recovery. After removal, the parasite was identified as L. minor using DNA barcodes and morphology. While the method of infection is uncertain, it is thought to be through direct exposure to the eggs or through the consumption of uncooked meat from wild animals.

Coronal computerized tomography scan of the human patient. Arrow indicates destruction of the left mastoid bone.
PHOTO CREDIT: Hospital General de Chetumal, Mexico
While it has been a challenge to successful identify Nematoda using genetic markers in the past, high-quality DNA barcode sequences were obtained using semi-degenerate primers designed for micro-crustaceans2. A comparison with 81 ascaridoids obtained from the Barcode of Life Data System (BOLD) revealed its position in a unique clade, most closely related to Baylisascaris procyonis.

This is the first multidisciplinary study involving DNA barcodes as a diagnostic medical tool in a human patient. This field of research can be promising because we can get a precise identification of the parasites in any stage of their life cycle. Diagnosis using DNA barcoding will allow the recognition of the infection parameters, transmission, and more precise epidemiology of parasites. With this information, we can not only diagnose the disease but also prevent it by finding the infectious stages, the intermediate hosts, or the vectors in the environment.

All sequences of Lagochilascaris are in the public project ‘NECHE Lagochilascaris from Yucatan’ in BOLD. This study is published in the Journal of Parasitology.

References:

  1. de Moura MQ, Jeske S, Gallina T, Borsuk S, Berne MEA, Villela MM (2012) First report of Lagochilascaris (Nematoda: Ascarididae) eggs in a public park in Southern Brazil. Veterinary Parasitology 184(2-4): 359-361. https://doi.org/10.1016/j.vetpar.2011.09.019
  2. Prosser SW, Velarde-Aguilar MG, Leon-Regagnon V, Hebert PD (2013) Advancing nematode barcoding: A primer cocktail for the cytochrome c oxidase subunit I gene from vertebrate parasitic nematodes. Molecular Ecology Resources 13(6): 1108-1115. https://doi.org/10.1111/1755-0998.12082

Written by

David González-Solís

David González-Solís

El Colegio de la Frontera Sur, Chetumal, México

Manuel Elías-Gutiérrez

Manuel Elías-Gutiérrez

El Colegio de la Frontera Sur, Chetumal, México

Jenny Alejandra Prado-Bernal

Clínica Carranza, Chetumal, México

Miguel Alfredo García-de la Cruz

Hospital General Dr. Manuel Gea González, Ciudad de México, México

April 30, 2019
PDF
https://doi.org/10.21083/ibol.v9i1.5475

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