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

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DNA Barcoding Wild Flora in Pakistan’s Forests

DNA Barcoding Wild Flora in Pakistan’s Forests

DNA Barcoding Wild Flora in Pakistan’s Forests

Preserving voucher specimens and creating a virtual herbarium to understand and protect some of the oldest living trees on the planet.
Juniper Forest of Ziarat, Balochistan, Pakistan.
PHOTO CREDIT: Nazeer Ahmed
Balochistan, the largest province of Pakistan, is endowed with a variety of natural forests. Juniper (Juniperus excelsa), Pinus (Pinus gerardiana), wild Olive (Olea sp.) and mangroves are the predominant forest ecosystems of the province. The versatility of life forms in these forests support dynamic ecosystems and provide several important ‘ecosystem services’ like food, medicines, climate regulation, genetic resources, recreation facilities, etc. Biodiversity conservation, in the face of such benefits, becomes imperative. Comprehensive cataloguing of flora and fauna is, by all means, at the heart of such conservation endeavours.

The Juniper forest of Ziarat, Balochistan, declared a UNESCO Man and Biosphere Reserve, is considered one of the world’s largest compact forests of its kind spreading over an area of 100,000 ha. Being some of the oldest living trees on earth, they are termed “Living Fossils”. The Chilgoza (Pinus gerardiana), also known as the Chilgoza Pine, on the other hand, are listed as lower risk, near threatened forest. Anthropogenic interferences have further aggravated the situation in this ecosystem and a more focused study about their current status is needed.

Juniper Forest of Ziarat, Balochistan, Pakistan.
PHOTO CREDIT: Nazeer Ahmed

Fragmented studies exist attempting to document the associated flora of these forests; however, a more comprehensive approach is needed. The use of DNA barcoding techniques, duly augmented by classical taxonomy, is necessary for the creation of a reference library to inventory, assess, and describe the biodiversity of these forests. To fill this gap, a study was designed to provide a foundation for future biodiversity assessment and conservation efforts.

 

Funded by Pakistan Agricultural Research Council and Higher Education Commission of Pakistan, our research group at the Balochistan University of Information Technology, Engineering & Management Sciences, Quetta is expecting to barcode and acquire samples of approximately 1,000 wild plant species. 

To date, 730 samples of 525 different species have been collected and 29% (150 of 525) have been barcoded. Besides maintaining voucher specimens, a virtual herbarium will be made available to the global scientific community interested in the flora of these forest ecosystems.

Read more about Pakistan:

SMALL STEPS LEAD TO BIG INITIATIVES: PAKISTAN REAFFIRMS SUPPORT FOR IBOL BY LAUNCHING PAKBOL

From economically important insect species to plants to food security, Pakistani researchers are working to barcode all life in their country through a national initiative – PakBOL.

UNIVERSITY OF SINDH JAMSHORO BARCODES GRASSHOPPERS IN PAKISTAN’S THAR DESERT

Tracking the shift of non-pests to crop pests, a phenomenon accelerated by anthropogenic pressures in the Thar Desert.

Written by

Nazeer Ahmed

Nazeer Ahmed

Balochistan University of Information Technology, Quetta, Pakistan

April 7, 2019
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https://doi.org/10.21083/ibol.v9i1.5476

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Scat Raiders Unravel Animal-Plant Interactions in Lebanon Using DNA Barcoding Tools

Scat Raiders Unravel Animal-Plant Interactions in Lebanon Using DNA Barcoding Tools

Scat Raiders Unravel Animal-Plant Interactions in Lebanon Using DNA Barcoding Tools

Using DNA dietary analysis on Eastern Mediterranean wildlife to explore the role of animals in ecological restoration processes.
Plant collection in Ehden Nature Reserve – north Lebanon. PHOTO CREDIT: Saint Joseph University

Lebanon is considered a hotspot for biodiversity in the Mediterranean basin likely due to its geographic position at the transition of two major landmasses (that is Eurasia and Africa). The Lebanese territory is divided between mountainous slopes with fertile valleys separating the two mountain chains that run parallel with the sea and the steppe areas in the north-east. Deep canyons and numerous rivers characterize this mountainous landscape.

These geomorphological regions give rise to many bio-climatic zones and several habitat types that are home to more than 9,116 described species (4,486 for fauna and 4,630 for flora from which 91 are endemic). However, major taxonomic groups like insects and fungi are understudied and taxa are underrepresented within public data platforms. For example, according to the Barcode of Life Data System (BOLD), only 345 Lebanese specimens with sequences are published, forming 151 BINs and, of these records, only 108 have species names.

In September 2018, the Faculty of Science at Saint Joseph University of Beirut joined the iBOL Consortium providing us with the opportunity to unravel Lebanese biodiversity by DNA barcoding both small and large mammals as well as the main trees and shrubs used in reforestation programs. We will also target endemic plant species.

Animals are a crucial component for the resilience of forest ecosystems and an important factor in forest restoration projects as they promote the sustainability of reintroduced plants, as well as seed dispersal. However, we still need to identify the animals present in restored areas.

Animal scat collection. PHOTO CREDIT: Saint Joseph University

In addition, knowing what each animal eats and which plant seeds are being dispersed is crucial for reforestation schemes that promote wildlife and ensure ecosystem sustainability. The information needed to study the diets of animals can be found hidden in their scat which contains not only the animal’s DNA, but also what that animal has eaten. With the powerful technique of DNA metabarcoding, we now have the necessary tool to efficiently unravel the genetic information hidden in animal scat. The DNA sequences obtained from such material are identified by comparison to a reference library of animals and plants of the Eastern Mediterranean countries.

 

Constructing the Reference Library – DNA isolation. PHOTO CREDIT: Saint Joseph University
This reference library was prepared from leaves collected in the wild and from DNA isolated from dead animals found along roads or from private museums. Thus, we have generated sequences for 51 plants and 18 mammals. This study conducted in collaboration with the Smithsonian Conservation Biology Institute and the University of Otago is the first to employ a DNA dietary analysis on wildlife in the Eastern Mediterranean Region and explicitly considering the role of wildlife in ecological restoration processes. Our results will inform management strategies to help with the conservation efforts of these imperiled species.

Written by

Carole Saliba

Carole Saliba

Faculty of Science, Saint-Joseph University

Liliane Boukhdoud

Liliane Boukhdoud

Faculty of Science, Saint-Joseph University

Magda Bou Dagher Kharrat

Magda Bou Dagher Kharrat

Faculty of Science, Saint-Joseph University

April 7, 2019
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https://doi.org/10.21083/ibol.v9i1.5489

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HOW BIOSCAN IS INSPIRING THE NEXT GENERATION OF RESEARCHERS

They were enlightened by the idea of discovering new species and by the possibility of doing so using DNA barcoding tools.”

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Saving the Pangolin: Philippines’ Fight Against the Illegal Wildlife Trade

Saving the Pangolin: Philippines’ Fight Against the Illegal Wildlife Trade

Saving the Pangolin: Philippines’ Fight Against the Illegal Wildlife Trade

Governments, scientists, and enforcement agencies join forces to formally incorporate molecular identification of trafficked species within wildlife forensics.
The Palawan Pangolin, Manis culionensis.
PHOTO CREDIT: Renz Angelo Duco

On April 8, 2013, a Chinese-registered fishing vessel ran aground on Tubbataha Reef, a marine protected area southeast of the island province of Palawan, Philippines. When the ship was towed to port at Puerto Princesa City, it was found to contain 400 concealed boxes with more than 3,000 frozen pangolins. These specimens were initially thought to be the Palawan Pangolin (Manis culionensis), an IUCN-listed endangered species. This and many other pangolin species have been described as some of the most trafficked animals on Earth as they are priced for their ‘scales’ for supposed medicinal value as well as for their exotic meat, both of which fetch a high value in the Chinese market.

The Palawan Pangolin and many other Philippine endemic species are protected by the Philippine Wildlife Resources Conservation and Protection Act (Republic Act 9147), which prohibits the capture, sale and transport of threatened species. However, Philippine Wildlife Enforcement Officers (WEOs) are hindered from carrying out their duties because they are limited in their ability to correctly identify confiscated species, which is often based on morphology alone. More often, WEOs have to deal with specimens that are not intact (e.g. tissue, blood, bone, etc.), rendering a taxonomic identification impossible. This poses a significant challenge for WEOs who need to correctly identify confiscated specimens and prosecute poachers.

Going back to the Tubbataha case, the Department of Environment and Natural Resources (DENR) sought the help of the University of the Philippines Diliman, Institute of Biology (UPD-IB) through its DNA Barcoding Laboratory to identify the pangolin specimens. Adrian Luczon, the lead investigator for the molecular identification of the specimens, utilized the COI gene and two reference Manis culionensis samples. His team’s results demonstrated that the Tubbataha specimens actually belonged to another critically endangered species, the Sunda Pangolin (M. javanica) native to mainland Southeast Asia, Borneo, Java, Sumatra, and nearby islands. Despite the DNA barcoding results indicating the specimens to be from another species outside the Philippines, the trafficking of the Palawan Pangolin remains unabated. In fact, within the same year, several batches of confiscations involving these pangolins have taken place, which Luczon’s team identified as the Palawan Pangolin through DNA barcoding. Clearly, there was an urgent need to formally incorporate molecular identification of trafficked species within the wildlife forensics work in the Philippines.

In 2015, UPD-IB entered a collaboration with the DENR through its Biodiversity Management Bureau to establish the first Molecular Wildlife Forensics (WILDFORCE) Lab in the Philippines. Through this partnership, DENR provides samples of Philippine endemic species to populate the Philippine DNA barcode database. These samples are to be processed at the Biodiversity Research Laboratory, headed by Dr. Perry Ong, and the DNA Barcoding Laboratory of UPD-IB. Other specimens brought to the lab for proper identification through DNA barcoding include the Philippine Duck (Anas luzonica), the Philippine Tarsier (Tarsius syrichta), the Gray’s Monitor Lizard (Varanus olivaceus), and the Philippine Sailfin Lizard (Hydrosaurus pustulatus), among others.

In 2018, with financial support from the Japan Biodiversity Fund and endorsement from the Secretariat of the Convention on Biological Diversity, and in support of the Global Taxonomy Initiative, WILDFORCE was able to train 18 individuals among researchers from higher educational institutions (HEIs) and WEOs from regional DENR offices. The training aimed to capacitate these personnel on the basic principles of DNA barcoding and eventually allow them to set up their own labs. These efforts are envisioned to contribute to building a robust Philippine DNA barcode database and decentralize the processing of evidence towards the DENR regional offices and local HEIs.

Wildlife enforcement officers and researchers from higher educational institutions receive training on DNA barcoding.

PHOTO CREDIT: Adrian Luczon

The sad reality of illegal trafficking of endangered species, as manifested by the Tubbataha case, has prompted the Philippine government and various stakeholders to join forces to combat illegal wildlife trade. It is only through collective effort grounded in science that we can have a chance to protect biodiversity.

Written by

Ian Kendrich Fontanilla

Ian Kendrich Fontanilla

Institute of Biology, University of the Philippines, Diliman, Philippines

April 7, 2019
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https://doi.org/10.21083/ibol.v9i1.5490

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University of Sindh Jamshoro Barcodes Grasshoppers in Pakistan’s Thar Desert

University of Sindh Jamshoro Barcodes Grasshoppers in Pakistan’s Thar Desert

University of Sindh Jamshoro Barcodes Grasshoppers in Pakistan’s Thar Desert

Tracking the shift of non-pests to crop pests, a phenomenon accelerated by anthropogenic pressures in the Thar Desert.
The Thar Desert is considered the seventh largest desert in the world and the third largest in Asia. Although this desert is rich in unique biodiversity, efforts to explore and analyze its fauna and flora have been minimal. The desert harbours some important crop pests, particularly orthopterans, by providing them alternate host plants, overwintering space, and environments for reproduction. The region provides favourable soil and environmental conditions for the survival of Acridids (grasshoppers and locusts). In particular, it supports the reproduction, development, and outbreak of the desert locust; the gregarious phase of locusts results in attacks on neighbouring regions that cause severe loss to crops and forests.

Cattle grazing in the Thar region.
Photo credit: Ahmed Ali Samejo

Around 20,000 orthopterans have been described in the world including 1,750 from India, but the number of known species in Pakistan is merely 161. Our recent surveys of the Thar region have revealed 29 species of grasshoppers that are new to the country indicating the rich grasshopper diversity of this desert.

With expanding agricultural fields, overgrazing and desertification, and changing ecological conditions, biodiversity is also changing. These changes are pushing non-pests to become crop pests, a phenomenon that warrants further investigation using reliable identification methods. An effective, preventive management strategy of these pests relies on an improved knowledge of their biology and ecology, and on more efficient monitoring and control techniques. The Department of Zoology at the University of Sindh Jamshoro has taken initiative to document and understand the grasshopper fauna in the Thar Desert by coupling DNA barcoding with conventional taxonomy.

Field surveys in the Thar Desert with Kumar, Riffat, & Samejo (left to right).
PHOTO CREDIT: Ahmed Ali Samejo

With funding support from the Higher Education Commission (HEC) Pakistan, the department plans to develop a DNA barcode reference library for grasshoppers in the Thar Desert of Pakistan. Grasshopper collection and specimen identification is already in progress and, so far, 2,334 specimens have been identified to 22 species while the identity of 300 specimens is yet to be resolved. After the front-end processing (data-basing, imaging, tissue sampling) at the University of Sindh Jamshoro is complete, the identified specimens will be barcoded at the Centre for Biodiversity Genomics, University of Guelph.

This is the first effort towards understanding grasshopper diversity in the Thar using DNA methods and developing a reliable reference library for this important group of pest insects. The generated data will not only be used for the rapid identification of grasshoppers and locusts, it will also provide a useful tool for pest management and biodiversity conservation.

Written by

Riffat Sultana

Riffat Sultana

Department of Zoology, University of Sindh Jamshoro, Pakistan

April 7, 2019
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https://doi.org/10.21083/ibol.v9i1.5491

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Read more about Pakistan:

SMALL STEPS LEAD TO BIG INITIATIVES: PAKISTAN REAFFIRMS SUPPORT FOR IBOL BY LAUNCHING PAKBOL

From economically important insect species to plants to food security, Pakistani researchers are working to barcode all life in their country through a national initiative – PakBOL.

DNA BARCODING WILD FLORA IN PAKISTAN’S FORESTS

Preserving voucher specimens and creating a virtual herbarium to understand and protect some of the oldest living trees on the planet.

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