The butterfly effect: geographic patterns of DNA barcode variation in subtropical Lepidoptera

The butterfly effect: geographic patterns of DNA barcode variation in subtropical Lepidoptera

The butterfly effect: geographic patterns of DNA barcode variation in subtropical Lepidoptera

Forest dynamics, spatial distribution patterns, and sampling scale are associated with mitochondrial DNA variation in Argentinian butterflies

Andean forests in northwestern Argentina.
PHOTO CREDIT: Ezequiel Núñez Bustos

Argentina harbours more than 1,200 species of butterflies, most of them found in two biodiversity hotspots and priority areas for conservation: the Atlantic Forest and the Andean forests1.

Figure 1: Sampling localities in northwestern Argentina (NWA, black squares) and northeastern Argentina (NEA, white triangles)2. The Atlantic Forest (dark blue) extends along the Brazilian coast and reaches its southernmost portion in NEA, while the Central Andean forests (red) descend from southern Peru and reach NWA. The distribution of eight other ecoregions indicated.

Despite their current isolation (Figure 1), these two areas have been cyclically and transiently connected in the past, promoting the interchange of flora and fauna, and resulting in a pattern of disjunctly co-distributed taxa. While the historical relationship between these allopatric forests and its evolutionary effects on shared fauna has been the subject of recent (and ongoing) research, studies have been concentrated mostly on vertebrates.

This study explores the butterflies of the Atlantic Forest and the Andean forests providing new insights into both the diversification patterns in southern South America and the impact of increasing the geographic and taxonomic scale of sampling on DNA barcoding performance in the region.

Atlantic Forest in northeastern Argentina. PHOTO CREDIT: Ezequiel Núñez Bustos

In 2017, we assembled and analyzed a DNA barcode reference library for 417 species from northeastern Argentina (NEA)2, focusing on the Atlantic Forest and covering around one-third of the butterfly fauna of the country. To expand the geographic and taxonomic distribution of this library, we generated DNA barcodes for 213 butterfly species from northwestern Argentina (NWA) with a focus on the Andean forests.

We then used these libraries to examine three themes, outlined below.

1.The effectiveness of DNA barcodes for species discrimination and identification

 

The mean intraspecific distance for the butterflies of NWA was 0.29%, while mean interspecific distance among congeneric species was 7.24% (Figure 2). More importantly, mean distance to the nearest neighbour (7.56%) was nearly 13 times larger than the mean distance to the furthest conspecific (0.60%), resulting in a distinct barcode gap for all but two species represented by two or more individuals (Figure 2).

Genetic distance or sequence variation in the COI sequences within and between species was estimated using the Kimura-2-parameter (K2P) model of nucleotide substitution

Substitution models describe the process of genetic variation through fixed mutations, constituting the foundation of evolutionary analysis at the molecular level.

Arenas M (2015) Trends in substitution models of molecular evolution. Frontiers in Genetics 6(319). 

Figure 2: Frequency histogram of COI sequence distances within species (orange) and among congeneric species (blue) of butterflies in NWA. The inset graph shows the barcode gap analysis for species represented by two or more COI sequences, where each dot represents a specimen. Red dots correspond to individuals with a maximum intraspecific distance higher than the distance to the nearest heterospecific. The vertical dashed line shows the 95th percentile of all intraspecific distances (2.02%), while the horizontal line corresponds to the lower 5% of all congeneric distances (3.36%).

Consistently, sequence-based specimen identification simulations showed that this library is extremely effective in the identification of the butterflies of NWA, exceeding a 98% success rate regardless of the identification criteria implemented.

We then used different clustering algorithms to assess the presence of cryptic species. Overall, these methods generated between 1.4–9.9% more Molecular Operational Taxonomic Units (MOTUs) than the number of reference species, suggesting that the butterfly diversity of NWA might be higher than currently recognized.

Figure 3: Taxonomic coverage of the complete DNA barcode reference library for the butterflies of Argentina. Dark shading indicates the proportion of species covered within each family based on the total known for the country.

Merging the NWA and NEA databases resulted in a DNA barcode reference library for nearly 500 butterfly species, covering ~40% of the butterfly fauna of Argentina (Figure 3) and representing 549 barcode clusters (BINs) on BOLD (170 of which are new to the platform).

2.The impact of increasing the spatial and taxonomic coverage on DNA barcoding performance

When we compared the two reference libraries, we found that the barcode gap was significantly narrower in the NEA than in the NWA library (Figure 4). This is most likely associated with the higher geographic and taxonomic coverage of the former, since expanding the spatial scale of sampling is expected to not only increase intraspecific variation as a result of isolation by distance but also reduce interspecific divergences as more closely related species appear.

Figure 4: Maximum intraspecific distance (blue) and minimum interspecific distance (red) for the three datasets. Note the different scales.

When we tried to identify specimens from NWA by using the reference library of NEA, a considerably high proportion of individuals representing shared species between these regions could not be identified or resulted in an ambiguous identification, even when we allowed a maximum intraspecific distance of as high as 2% in the identification procedure. This was due to the existence of deep intraspecific divergences between conspecifics from northeastern and northwestern Argentina, two regions separated on average by more than 1,000 km.

At the same time, however, we observed that the effect of increasing the geographic (and taxonomic) scale was more profound on the minimum interspecific distances than on the maximum intraspecific distances. Therefore, it is possible that butterfly species in NEA are also naturally more variable than in NWA based on our current sampling. While specimens from NWA came almost exclusively from the montane forest on the east slope of the Andes, the sampling in NEA covered not only larger geographic distances but also a more heterogeneous landscape, characterized by the existence of different ecoregions (Figure 1) and physical barriers such as river, specifically the Paraná-Paraguay River axis. Regardless, our results show that both large geographic distances and increased taxonomic coverage can affect DNA barcoding identification performance, especially when using a local library to identify the fauna from another distant region.

As expected, the maximum intraspecific distance was significantly higher and minimum interspecific distance was significantly lower in the complete database (NEA + NWA) than within the NWA and NEA libraries alone (Figure 4). However, the logical and anticipated reduction in the barcode gap did not have, in this case, a significant impact on the identification performance of DNA barcodes, which were able to correctly identify ~99% of the individuals. This reflects the importance of increasing the spatial and taxonomic coverage of DNA barcode libraries to improve identification success, and of considering the use of a local database to identify regional fauna when a more comprehensive COI database is not available.

Doxocopa cyane burmeisteri
Doxocopa cyane burmeisteri
Parides erithalion erlaces

Parides erithalion erlaces

Pteronymia ozia tanampaya

Pteronymia ozia tanampaya

Butterfly species from the Andean forests. PHOTO CREDIT: Ezequiel Núñez Bustos

3.Geographic patterns of intraspecific variation across Argentina

A total of 135 butterfly species are shared between the databases of NEA and NWA. Mean intraspecific distance for these species was significantly higher between regions (1.02%) than within them (NEA, mean 0.35%; NWA, mean 0.33%), especially for a subset of 43 species that showed particularly deeper distance (mean 2.43%) between NEA and NWA.

We then focused only on the 85 species that are present in both the Atlantic Forest and the Andean forests (Figure 5), 27 of which have a disjunct distribution between forests, being absent from intermediate ecoregions, while the remaining 57 have a continuous range across northern Argentina.

Figure 5: Proportion of shared species between NEA and NWA that occur in both forests. The spatial distribution pattern (disjunct vs continuous) and the percentage of species with a deep intraspecific divergence between forest populations indicated.

We found that mean intraspecific distance between forest populations was significantly higher for the disjunctly distributed species (1.65%) than for species with continuous ranges (0.78%), showing that spatial distribution patterns have an influence on the level of intraspecific variation. Moreover, the proportion of species showing the deep divergence between populations from the Atlantic Forest and the Andean forests was notably higher among species with fragmented distributions (nearly 50%) than for species with continuous ranges (less than 30%) (Figure 5).

Lastly, based on standard molecular rates and COI sequence divergence, all diversification events between forest populations were dated to the last two million years, a time period when the currently isolated Atlantic Forest and Andean forests experienced multiple transient connections across the open vegetation corridor, a diagonal of more open and drier savanna-like environments (Caatinga, Cerrado and Chaco) that isolates the Atlantic Forest from the Andean forests (and the adjacent Amazonia) (Figure 1). These past connections were promoted mainly by Pleistocene climatic changes and habitat shifts.

Catonephele numilia neogermanica

Catonephele numilia neogermanica

Callicore hydaspes

Callicore hydaspes

Doxocopa agathina vacuna

Doxocopa agathina vacuna

Butterfly species from the Atlantic Forest.
PHOTO CREDIT: Ezequiel Núñez Bustos

Conclusions

Our study has not only expanded the DNA barcode reference library for the butterflies of Argentina, but it also constitutes, to our knowledge, the first multi-species assessment of the historical relationship between the currently isolated Atlantic Forest and Andean forests using butterfly species as model organisms.

Importantly, our research supports the fact that, even in the era of genomic data, large-scale analyses of mitochondrial DNA variation are still extremely useful for evolutionary studies, as they unveil spatial diversification patterns and highlight cases that deserve further investigation.

ACKNOWLEDGEMENTS:

We thank our colleagues from the Museo Argentino de Ciencias Naturales and the staff at the Centre for Biodiversity Genomics (CBG) for their help during different stages of this ongoing investigation. We also thank Michelle D’Souza for her helpful comments and suggestions that improved this contribution. This project is supported by Richard Lounsbery Foundation, the CBG, the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), the Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación, Fundación Williams, Fundación Bosques Nativos Argentinos and Fundación Temaiken. For granting the permits and transit guides, we thank the Offices of Fauna of the Argentinian provinces in which fieldwork was conducted, the Administración de Parques Nacionales, and the Ministerio de Ambiente y Desarrollo Sostenible from Argentina.

References:

1. Klimaitis J, Núñez Bustos E, Klimaitis C, Güller R (2018) Mariposas-Butterflies-Argentina. Guía de Identificación-Identification Guide. Vazquez Mazzini Editores. Buenos Aires. pp. 327.

2. Lavinia P, Núñez Bustos E, Kopuchian C, Lijtmaer D, García N, Hebert P, Tubaro P (2017) Barcoding the butterflies of southern South America: Species delimitation efficacy, cryptic diversity and geographic patterns of divergence. PLOS ONE 12(10), e0186845. https://dx.doi.org/10.1371/journal.pone.0186845

Written by

Natalí Attiná

Natalí Attiná

Ezequiel Núñez Bustos

Ezequiel Núñez Bustos

Darío A. Lijtmaer

Darío A. Lijtmaer

Pablo L. Tubaro

Pablo L. Tubaro

Pablo D. Lavinia

Pablo D. Lavinia

Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” (MACN–CONICET)

July 31, 2020

doi: 10.21083/ibol.v10i1.6256  

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Hunting for a water mite neotype in southern Norway

Hunting for a water mite neotype in southern Norway

Hunting for a water mite neotype in southern Norway

Scientists rediscover lost specimens of water mite in Norway 120 years after they were first described

A stream near the church of Vanse at Lista in southern Norwaythe type locality of Lebertia porosa Thor, 1900.

PHOTO CREDIT: Torbjørn Ekrem

Did you know that scientists can assess natural water quality by monitoring the diversity of aquatic invertebrates? Freshwater insect and arachnid populations are often important indicators of environmental change. This is evident in particularly species-rich groups, such as water mites and biting or non-biting midges, which have great potential for monitoring water quality. The problem is only that they are too difficult and time consuming to identify in routine water quality assessments. This hurdle can be overcome with DNA metabarcoding, but only if a good reference barcode library is available.

Elisabeth Stur of the Norwegian University of Science and Technology (NTNU) University Museum, along with her team, have been doing summer fieldwork for the Water Mites and Midges in southern Norway (Water M&M) project. One of the many goals for this year’s fieldwork was not only to contribute to the reference barcode library, but also to sample the type locality of the water mite Lebertia porosa, described 120 years ago by Sig Thor, a Norwegian priest and acarologist.

The Great Lakes
Phaenopsectra flavipes (Diptera: Chironomidae) with water mite larvae attached. PHOTO CREDIT: Aina Mærk Aspaas, NTNU University Museum

Barcode data indicate that there are at least six cryptic genetic lineages within this species, but it is unknown which of these applies to the nominal species. Since the original type material is lost, re-sampling L. porosa from its type locality is important in designating a neotype that most likely belongs to the species described by Thor in 1900. This way, researchers can stabilize the definition of the L. porosa species name, such that potential new species could be described. This species delineation is part of a MSc. project by Valentina Tyukosova at NTNU: Integrative taxonomy and species delimitation in the Lebertia porosa species complex (Acari, Parasitengona: Hydrachnidia).

The type locality of L. porosa was vaguely described in Thor’s original publication as a “stream near the church of Vanse”. After studying maps of the surrounding area, researchers learned that this church still stands, and were able to locate two nearby streams.

Now they wondered, would these streams still be in good condition 120 years later? As the team of researchers approached what they thought might be the stream in June 2020, they were pleased to see running, clear water under the bridge. Next mystery: could the streams hold a population of L. porosa 120 years after first collection? They found that yes, the waters could, and the water mite populations were bountiful!

The Great Lakes DNA Barcoding Project team

Water mites from the type locality of Lebertia porosa Thor, 1900.
PHOTO CREDIT: Torbjørn Ekrem

Stur and her team are now looking forward to getting these critters under the compound microscope. Using DNA analysis, they hope to identify which barcode clusters they match with, potentially revealing the nominal species of L. porosa. We’re sure that Sig Thor would be thrilled to learn that his identified species is still thriving, 120 years later.

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GBOL III: Dark Taxa

GBOL III: Dark Taxa

GBOL III: Dark Taxa

Researchers launch new BIOSCAN project that aims to illuminate thousands of new insect species on Germany's doorstep.

A “pixelated” Diptera.

Currently, around 1.4 million species of animals are known. For tropical regions, many species are still unknown, with estimates of global biodiversity ranging from five to 30 or even 100 million species. More recent studies suggest that there are about 10 million species on our planet. In contrast to the tropics, the Central European fauna is considered to be very well studied. However, specialists have mostly concentrated on less diverse and easy-to-study organisms, neglecting the species-rich, often taxonomically difficult groups, like many Diptera and Hymenoptera. This led to a mismatch between high species numbers and a small number of researchers, often referred to as the ‘taxonomic impediment’. This is most prominent for the megadiverse faunas of tropical regions. Less known is that this also applies, to some extent, for countries with a long history of taxonomic research like Germany, covering 200 or more years. For example, for the compilation of the German checklist of Hymenoptera, 32 specialists were available for 247 species of digger wasps (Crabronidae), while for parasitoid wasps of the family Ichneumonidae one specialist had to deal with 3,332 species.

In Germany, about 48,000 species of animals have been documented, including about 33,300 species of insects. In little-studied groups such as insects and arachnids, preliminary results of earlier DNA barcoding initiatives indicate the presence of thousands of species that are still awaiting discovery. Among the groups with a particularly large suspected number of unknown species are the Diptera (flies) and the Hymenoptera (in particular, the parasitoid wasps). With almost 10,000 known species each, these two insect orders account for two-thirds of the German insect fauna, underlining their importance.

Bar Graph depicting availability of taxonomic expertise for major insects orders in Germany.

“Dark taxa” are, as a rule, small-sized and rich in species, and have therefore been largely ignored by taxonomists. This is reflected by the number of undescribed species in these taxa, combined with a low chance to get specimens identified by specialists.

The insight that there are not only a few but many unknown species in Germany is a result of the earlier German Barcode of Life projects GBOL I and II, both supported by the Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF) and the Bavarian Ministry of Science (project Barcoding Fauna Bavarica). The projects aimed at making all German species reliably, quickly and inexpensively identifiable by DNA barcodes. Since the first project was launched about ten years ago, more than 25,000 animal species have been barcoded, in collaboration with national and international partners. Among them are mostly well-known groups such as butterflies, moths, beetles, grasshoppers, spiders, bees and wasps.

Two scatterplots demonstrating relationship between body size (top) and species richness (bottom) in German Diptera

Relationship between body size (top) and species richness (bottom) in German Diptera1

Despite their popularity, these groups represent only a fraction of the total inventory of German insects. In Germany there are 170 butterfly species, 81 dragonfly and damselfly species, 87 species of grasshoppers, katydids and crickets, and 580 species of ground beetles, all of which are well-studied. Taken together, these 918 species stand for only a small fraction (2.8%) of the German insect fauna. They are morphologically well identifiable, have manageable species numbers, can easily be monitored during daytime and are therefore regarded as relevant in nature conservation and often used for monitoring species diversity. Conversely, however, this means that the vast majority of the native species diversity has been largely ignored in nature conservation and in general and applied research.

Circular tree depicting Nematocera (midges) and Brachycera (flies)
A circular neighbour‐joining tree for the two suborders of flies, Nematocera and Brachycera1. Each line in the tree corresponds to a distinct Barcode Index Number (BIN). Whereas for two of the “big four” insect orders, the Lepidoptera and Coleoptera, the number of German species are very precisely known, the numbers for the Diptera and Hymenoptera must rely on rough estimates. 

This applies in particular to the Diptera (flies). The observation that estimates of the number of species of native Diptera have been far too low was not only a result of the DNA barcoding projects at the ZSM, but became clear in a recent study by Paul Hebert and his team2. In this large-scale study, DNA barcodes of about one million insects were analyzed. Based on this study, Canada’s gall midges alone are estimated to include about 16,000 species, suggesting the existence of at least two million species on earth. That would be more species of gall midges worldwide than all previously described animal species combined.

The little-known or unknown species, referred to as ‘dark taxa’, are the subject of another BMBF-funded DNA barcoding project that is being carried out at the ZSM in collaboration with other German natural history museums and institutions. The project focuses on Diptera and Hymenoptera (in particular, parasitoid wasps), each with a large proportion of ‘dark taxa’. The new project, funded by a grant of 5.3 million Euro, starts July 1st 2020, with 12 PhD students at three major natural history institutions in Bonn (Zoological Research Museum Alexander Koenig), Munich (SNSB – Zoologische Staatssammlung München) and Stuttgart (State Museum of Natural History Stuttgart), to address a range of questions related to the taxonomy of German ‘dark taxa’, targeting selected groups of Diptera and parasitoid Hymenoptera.

Detailed photo of a Eulophidae specimen
Yellow Mymaridae specimen

Small parasitoid wasps of the families Eulophidae (top) and Mymaridae (bottom), both group with possibly hundreds of new species in Germany that still await discovery.

Among the major aims of GBOL III is assessing of the performance of DNA barcoding for species identification of ‘dark taxa’, and assessing the species detection ability of DNA barcodes in mass samples that are obtained from metabarcoding studies. Other aims of the project include the development of a platform for managing OTU-based taxonomic data, developing a pipeline for reliable and fast barcoding of small and poor-quality samples, and training of the next generation of taxonomists.

GBOL III is designed to make an important contribution to the global BIOSCAN initiative of the Centre for Biodiversity Genomics. It helps to lay the foundations for a global biomonitoring system to record the biodiversity of our planet on a large geographical scale in times of rising temperatures, increasing weather extremes and receding ice, and to track its changes as a result of global environmental changes.

References:

1. Morinière J, Balke M, Doczkal D, Geiger MF, Hardulak LA, Haszprunar G, Hausmann A, Hendrich L, Regalado L, Rulik B, Schmidt S, Wägele J, Hebert PDN (2019) A DNA barcode library for 5,200 German flies and midges (Insecta: Diptera) and its implications for metabarcoding‐based biomonitoring. Molecular Ecology Resources 19: 900–928. https://doi.org/10.1111/1755-0998.13022

2. Hebert PDN, Ratnasingham S, Zakharov EV, Telfer AC, Levesque-Beaudin V, Milton MA, Pedersen S, Jannetta P, deWaard JR (2016) Counting animal species with DNA barcodes: Canadian insects. Philosophical Transactions of the Royal Society B: Biological Sciences 371: 20150333. https://doi.org/10.1098/rstb.2015.0333

Written by

Axel Hausmann

Axel Hausmann

SNSB - Zoologische Staatssammlung München, Munich, Germany

Lars Krogmann

Lars Krogmann

State Museum of Natural History Stuttgart, Stuttgart, Germany

Ralph S. Peters

Ralph S. Peters

Zoological Research Museum Alexander Koenig, Bonn, Germany

Vera Rduch

Vera Rduch

Zoological Research Museum Alexander Koenig, Bonn, Germany

Stefan Schmidt

Stefan Schmidt

SNSB - Zoologische Staatssammlung München, Munich, Germany

July 10, 2020

doi: 10.21083/ibol.v10i1.6242

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Spring into action: Life in Earth’s rarest soils under threat

Spring into action: Life in Earth’s rarest soils under threat

Spring into action: Life in Earth’s rarest soils under threat

Springtails (Collembola) in the Antarctic indicate that unique soil biodiversity in the region faces biotic homogenization due to increased human activity

Invasive springtail (Collembola) in sub-Antarctic soil.

PHOTO CREDIT: Laura Phillips

Efforts to understand and protect Earth’s biodiversity have largely overlooked life present in the soil. However, soil biodiversity is critical to ecosystem health, playing an integral role in nutrient cycling, carbon storage, water filtration, and food production1.

The Antarctic, once a pristine wilderness, is undergoing rapid environmental change and increased human pressures. Yet the true diversity, uniqueness, and vulnerability of its soil communities are only beginning to be revealed. As tourism and science expeditions bring ever more passengers and cargo to Antarctica, human-induced transport of species (‘biological invasion’) – both into and throughout the region – presents a growing threat to soil biodiversity, as highlighted recently in our study.

The Antarctic terrestrial biome near McMurdo station. PHOTO CREDIT: Helena Baird

In the Antarctic region, which broadly covers approximately 30% of the planet’s surface, soil organisms represent the vast majority of terrestrial life. Antarctic soil organisms, which include nematodes, mites, springtails, fungi, and tardigrades, have eked out a largely isolated existence over millions of years in fragmented patches of ice-free land on the continent, or on the sub-Antarctic islands.

On the Antarctic continent, ice-free soil represents <1% of the total land area and can be found on mountain tops, scree slopes, valleys, and the coast. The sub-Antarctic islands encircle the continent, located in the frigid waters of the Southern Ocean and each is separated by up to thousands of kilometres of open ocean. Antarctic soil species are therefore highly isolated and possess unique adaptations to their harsh environment. They are at risk of being outcompeted, or ultimately even replaced, by invasive species as the climate warms2.

LEFT: Helena Baird and colleagues busy with sub-Antarctic fieldwork on Marion Island while king penguins watch in the background. RIGHT: Soil sampling with the use of a soil core – an undisturbed cylindrical sample.
PHOTO CREDIT: Charlene Janion-Scheepers

In our study, we used DNA barcoding to investigate the spread of an invasive springtail species, known to adversely affect native soil species, across the sub-Antarctic islands. Identification of numerous divergent barcode sequences revealed that the invasive has been introduced to Antarctica several times.

By comparing barcodes from Antarctic specimens in BOLD to those found elsewhere in the world, we could identify genetic lineages shared across countries which aligned with known shipping routes to the Southern Ocean, highlighting the utility of molecular tools in tracking invasion. For example, a shared barcode haplotype between Norway and the sub-Antarctic island of South Georgia accords directly with Norway’s long history of whaling on this island. That a well-known invasive species has been introduced on multiple occasions to such a remote region emphasises the importance of ongoing biosecurity monitoring, even for invasive species that have already established, since multiple invasions can introduce more genetic resilience and enable the invasive species to spread.

Research vessel as it approaches Possession island, Crozet archipelago, one of the sub-Antarctic islands in the region. PHOTO CREDIT: Helena Baird

Charlene Janion-Scheepers (left) and Helena Baird (right) sort soil species at sea using Berlese funnels.
PHOTO CREDIT: Steven Chown

Our study also explored the consequences of intra-regional human transport on native soil species. Antarctic soil organisms are typically highly endemic, even to local patches within the region. This raises the concern that increased human traffic throughout Antarctica could transport and ultimately homogenise soil populations, altering the region’s unique biogeography. Using genome-wide SNPs, we showed that a widespread native springtail species is indeed so distinct between sub-Antarctic islands that it is likely in the process of speciating. Clearly, future exchange of individuals among islands could possibly disrupt this biodiversity process, diluting the specific adaptations each population has evolved over millennia. Potential evolutionary consequences include a decrease in the fitness of island-specific populations, lineage extinction, or the loss of biodiversity by ‘reverse speciation’3.

Regardless of the outcome, the threat of disrupting biodiversity processes among these unique and fragile islands emphasises the importance of biosecurity for ships travelling throughout the Southern Ocean, particularly when passengers embark and disembark at multiple locations.

Taking a break to enjoy the view in the sub-Antarctic. PHOTO CREDIT: Helena Baird

One of the main hurdles to accurately predicting future changes to soil communities is a lack of basic biodiversity knowledge. In the Antarctic, molecular work such as metabarcoding continues to reveal far more soil diversity – most of which is locally endemic – than previously recognised4,5. This situation echoes worldwide, with biodiversity and biogeography patterns constantly revised as we probe the soil biome deeper. Fortunately, schemes such as the Global Soil Biodiversity Initiative are bringing fragile soil ecosystems under the spotlight, where we will be better served to protect them.

References:

1. Bardgett RD & van der Putten WH (2014) Belowground biodiversity and ecosystem functioning. Nature, 515. 2. Janion-Scheepers C, Phillips L, Sgrò CM, Duffy GA, Hallas R & Chown SL (2018) Basal resistance enhances warming tolerance of alien over indigenous species across latitude. Proceedings of the National Academy of Sciences, 115. 3. Seehausen O (2006) Conservation: losing biodiversity by reverse speciation. Current Biology, 16. 4. Czechowski P, Clarke LJ, Cooper A & Stevens MI (2016) A primer to metabarcoding surveys of Antarctic terrestrial biodiversity. Antarctic Science, 1-13. 5. Velasco-Castrillón A, McInnes SJ, Schultz MB, Arróniz-Crespo M, D’Haese CA, Gibson JAE, . . . Stevens MI (2015) Mitochondrial DNA analyses reveal widespread tardigrade diversity in Antarctica. Invertebrate Systematics, 29.

Read the complete manuscript in Evolutionary Applications.

Read more news about the Antarctic:

NEW AUSTRALIAN PROGRAM TO SECURE FUTURE FOR ANTARCTICA’S ENVIRONMENT AND BIODIVERSITY

The Australian Government has awarded $36 million to a new research program led by Monash University, Securing Antarctica’s Environmental Future (SAEF).

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Judge a caterpillar by what they eat, not where they’re found

Judge a caterpillar by what they eat, not where they’re found

Judge a caterpillar by what they eat, not where they’re found

Gut content analysis of Peruvian caterpillars reveals new insights into host-plant relationships and the methods used to examine species interactions key to BIOSCAN

The original primary rainforest surrounding Panguana station, Peru, Dept Huánuco, western Amazonia.

PHOTO CREDIT: K. Wothe

Understanding species and their associations with each other and with the environment – a key aspect of synecological research – is of great importance. For example, data on insect-host plant relationships can aid investigations into food webs and extrapolations of global species numbers as well as inform forestry, agriculture, and conservation practices.

Supported by the Bavarian Ministry of Science (‘SNSB-Innovativ’), our recent pilot study examined the gut contents of Peruvian caterpillars demonstrating the potential for gathering large-scale data on species interactions when applying DNA barcoding and high-throughput sequencing technologies. We obtained 130 caterpillars (moth larvae) by canopy fogging at the Panguana research station, an area of tropical primary forest in western Amazonia. DNA barcode analysis resulted in 119 successfully sequenced larvae, more than half of which matched with moth reference sequences on BOLD. Surprisingly high biodiversity was uncovered from our modest sample – 92 BINs or species proxies. The trees from which caterpillars were collected were also identified, both by morphology and DNA barcoding.

Panguana research station, Peru, Dept Huánuco, western Amazonia, with the characteristic Lupuna tree (Ceiba pentandra, Malvaceae) in the background.

PHOTO CREDIT: J. Diller

Knowing the tree and larva identity is not enough to conclude a host-plant relationship, particularly in a dense tropical rainforest. Caterpillars may in fact be feeding on the epiphytes, lianas, lichens, algae, fungi, or mosses associated with trees (i.e., alternative feeding), and sometimes larvae may have been fogged down from neighbouring trees. To confirm a direct insect-host plant relationship, we partnered with the company Advanced Identification Methods (AIM) to design a high-throughput sequencing (HTS) protocol with plant markers (rbcL, psbA) that would enable the identification of plant matter from the gut contents of ten larvae. Results revealed only two matches between the fogged tree and larval gut content which suggests a rather high percentage of alternative feeding. In three cases, the gut content clearly indicated feeding on lianas and neighbouring trees. Interestingly, the analysis of four larvae resulted in the putative presence of Bryophyta, suggesting moss-feeding in Lepidoptera, a phenomenon rarely observed. Potential contamination (for example, through the diffusion of plant DNA into the alcohol of the bulk sample) has yet to be ruled out, work which is currently being validated in a subsequent project investigating the gut contents of an additional 190 larvae.

Automeris denticulata (Conte, 1906) (Saturniidae): Larva (left), selected from canopy fogging bulk samples of a Poulsenia (Moraceae) tree at the Panguana station, identified by its COI barcode; Adult (right), collected at the Panguana station.

PHOTO CREDIT: Mei-Yu Chen & Dr. R. Mörtter

Our approach of combined canopy fogging, DNA-based identification, and gut content analysis resulted in two key findings. First, a significant portion of both insect and plant taxa can be identified even in highly diverse, tropical regions – more than 97% to a family level and about 80% to a species or genus level. Secondly, we can successfully confirm or reject the hypothesis that caterpillars feed on the trees where they are collected by identifying their diets through an HTS protocol on gut contents. Importantly, the taxonomic resolution of animal and plant identifications will increase with further investments into DNA reference libraries. We recommend specimen de-contamination (e.g. by bleaching) and/or isolated storage of the target taxa rather than bulk storage to improve the reliability of gut content analysis.

Urania leilus (Linnaeus, 1758) (Uraniidae): Larva (left), selected from canopy fogging bulk samples of an Oxandra polyantha (Annonaceae) tree at the Panguana station, identified by its COI barcode; Adult (right).

PHOTO CREDIT: Mei-Yu Chen & Dr. J. Diller

The techniques employed in our pilot have immense potential for unveiling trophic interactions in tropical regions at a very large scale as they are fast and cost-effective. The latter is enabled, in part, by the availability of target specimens in the by-catch of other studies. For example, our efforts fogging 150 trees in a separate project assessing the biodiversity of ants have resulted in 1,200 lepidopteran larvae. Subsequent aspects of the workflow, from selecting the larvae from bulk samples, tissue sampling, photography, and databasing, required 10–20 minutes per larva and can be performed with relatively low expertise. The costs for subsequent lab work, i.e. identification of larvae and their gut contents, currently amount to 20–25 € per larva and these costs will soon drop considerably. In contrast, traditional approaches involving the searching and rearing of larvae, and the identification of hatched adults by experts is massively time and resource consuming.

Providing reliable data on trophic interactions is one of the major goals of the BIOSCAN program, one that will be a powerful tool for investigating food webs, for determining the amplitude of alternative or multiple feeding sources, and for the study of phagism (monophagy versus polyphagy), thus gaining data for extrapolations of global species numbers. These data will also be particularly important for pest management in forestry, and agriculture, and for conservation purposes.

Overcoming the current lack of knowledge is a major challenge, particularly in ecoregions with megadiverse faunas and floras. Yet, its success is imperative for humanity considering the unprecedented biodiversity losses we currently face. In this context, the recently launched BIOSCAN with its focus on revealing species interactions will embolden an important plan for the international research community to come together in understanding nature and conserving it for a sustainable future.

 

Read the complete manuscript in PLoS ONE.

Written by

Axel Hausmann

Axel Hausmann

Juliane Diller

Juliane Diller

Amelie Höcherl

Amelie Höcherl

SNSB – Staatliche Naturwissenschaftliche Sammlungen Bayerns - Zoologische Staatssammlung München, Munich, Germany

May 6, 2020
https://doi.org/10.21083/ibol.v10i1.6133 

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Discovering ten new species of Paramyia Williston (Diptera: Milichiidae) in North America using DNA barcoding

Discovering ten new species of Paramyia Williston (Diptera: Milichiidae) in North America using DNA barcoding

Discovering ten new species of Paramyia Williston (Diptera: Milichiidae) in North America using DNA barcoding

Making the process of species identification more efficient by focusing morphological efforts using DNA-based tools

Flowering plant visited by Paramyia sp.
PHOTO CREDIT: Steve A. Marshall

The process of discovering and describing new species – the job of taxonomists – is time-consuming. To tackle the challenge, one must become an expert on a specific group in order to notice those rarities among the masses. This is without counting the added challenges of cryptic groups where the external morphology is of limited help as individuals often appear to belong to the same species despite being genetically distinct. In many cases, the taxonomist has to dissect hundreds of specimens to detect slight differences in their genitalia which are usually unique for each species. DNA barcoding can greatly assist any taxonomist by speeding up this laborious identification process, particularly with small flies like Paramyia Williston (Diptera: Milichiidae).

Flowering plant visited by Paramyia sp.

PHOTO CREDIT: Steve A. Marshall

Paramyia, a kleptoparasitic genus well represented worldwide, provides a perfect example of joining DNA barcoding and traditional taxonomy. Paramyia is a small genus, with under 30 described species, of tiny blackish flies, usually under 2mm long, with very similar external morphology. In the Nearctic, it was only represented by one species, P. nitens Loew. With that knowledge in mind, no particular attention was given to specimens collected in that geographic range. That is until multiple Barcode Index Numbers (BIN) were found on the Barcode of Life Data System (BOLD) under the same species name. This strongly indicated that multiple undescribed species may have been placed under one species – P. nitens. A closer look at their genitalia revealed this to be true, and so a revision of the genus was then needed.

Paramyia sp. displaying kleptoparasitism, that is, feeding on the captured prey (stink bug) of another predator (spider)

PHOTO CREDIT: Steve A. Marshall

As with any revision, I first acquired multiple loans from large museum collections to compare and study the many diverse and variable specimens from a specific geographic range, in this case, North America. Then, I studied the morphology of these specimens in-depth to detect variation between those grouped together based on their similarities (i.e, morphs) and dissected the genitalia to confirm if they were indeed new species. With a genus like Paramyia, most helpful characters to differentiate between the species are genitalic, which means that good dissection skills are essential. The skill needed to dissect the genitalia of such small flies is comparable to performing surgery on a baby’s tooth. Important to note, there are no morphology characters to split the females of most species apart.

This is where DNA barcoding comes in handy. I sequenced specimens from my different morphs, and then dissect males grouped in the same BIN to verify the correspondence between the BIN and the species concept. When the molecular and the morphological analysis align perfectly, females can get correctly associates with their male counterpart, which would have otherwise been impossible. Hence, the species description can be more complete and the sequences are available to be used by other researchers to correctly identified that group, e.g. in monitoring programs. I applied this process in the Nearctic revision of Paramyia and described 10 new species! Future revisions tackling the remaining geographic regions can build from this work.

Comparative morphology between the new species P. pseudonitens and P. brevikeraia with a body profile, frontal head and genitalia photos (top to bottom)

PHOTO CREDIT: Valerie Levesque-Beaudin

The taxonomic impediment coupled with the current rate of species extinction is making the job of the taxonomist increasingly more difficult and yet, there’s an urgent need to record species before they disappear. As this study demonstrates, by quickly sorting specimens based on morphology and sequencing representative of each group, the number of undescribed species can be assessed and the amount of dissection needed to make such a discovery can be managed. The focus can then be on the morphology and genitalia of the different BINs, hence speeding up the process of species identification.

For full details, please refer to the publication in Zootaxa.

Written by

Valerie Levesque-Beaudin

Valerie Levesque-Beaudin

Taxonomic Specialist – Diptera, Centre for Biodiversity Genomics

February 27, 2020
https://doi.org/10.21083/ibol.v10i1.6081

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Revealing the widespread adulteration of commercial herbal medicines and food supplements

Revealing the widespread adulteration of commercial herbal medicines and food supplements

Revealing the widespread adulteration of commercial herbal medicines and food supplements

DNA barcoding and metabarcoding have substantially contributed to the authentication of herbal products and they hold the key to validated, DNA-based diagnostics for ingredient identification
Commercial herbal products available for the consumers outside of the regulated/controlled marketing chains in an agri-food market in Romania. PHOTO CREDIT: Paula Paraschiva Sosoi

The World Health Organization has estimated that up to 80% of the world’s population, especially in developing countries, rely on herbal medicinal products as a primary source of healthcare. Yet, these herbal products (HPs) – widely sold in food stores and supermarkets, as well as over the internet – are poorly regulated. By gathering and analyzing a dataset of a little less than 6,000 commercial HPs across 37 countries, this study found undeclared contaminant, substitute, and filler species, or none of the labelled species in more than a quarter of products thus highlighting the large-scale presence of herbal product adulteration across the globe.

Shelves with herbal products in a supermarket

PHOTO CREDIT: Mihael Cristin Ichim

Along with the tremendous increase in demand for both plant raw materials and processed HPs in the global marketplace, there has been growing scientific evidence of accidental contamination or intentional adulteration of commercial HPs. Irrespective of the authentication method used, the traditional pharmacopoeial (e.g., macroscopic and microscopic examination, chemical methods) or emerging DNA-based tools (e.g., DNA barcoding and metabarcoding), numerous studies have identified adulterants when the contents of commercial HPs were tested against their labelled ingredient species. But the scale of this phenomenon is unknown.

To understand whether herbal adulteration is a global phenomenon, I examined peer-reviewed scientific journals for studies on HP authentication that were based on detection methods using species-specific DNA sequences. By analyzing 5,957 commercial HPs, sold in 37 countries scattered across all continents, results revealed 27% of products contained undeclared contaminant, substitute, and filler species, or none of the labelled species. It is unclear whether these cases are accidental, or intentional and economically motivated.

Figure 1: The proportion of adulterated products (black) among continents. 

The proportion of adulterated products varied significantly among continents, as high as 79% in Australia and as low as 23% in Asia (Figure 1). Of the countries with at least 100 products successfully authenticated and reported, the highest percentage of adulterated commercial HPs was found in Brazil (68%), followed distantly by Taiwan (32%), India (31%), and others (Figure 2).

Figure 2: The proportion of adulterated products among countries with at least 100 products successfully authenticated and reported.

All the commercial herbal products examined in this study were authenticated with DNA-based methods. These gradually advanced from classical molecular methods (e.g., Restriction Fragment Length Polymorphism) in early 2000 to DNA barcoding and metabarcoding in more recent years. The latter DNA barcoding methods were used to authenticate the majority of the reported commercial HPs, illustrating their growing utility as a tool to quantify rates of adulteration.

DNA barcoding – which makes use of short, standardized regions of the genome as species ‘barcodes’ – is a targeted approach suitable for the authentication of raw plant materials and the testing of single-ingredient HPs. Metabarcoding – a combination of high-throughput sequencing and DNA barcoding – enables untargeted, simultaneous multi-taxa identification by using DNA from different origins that can be extracted from more complex mixtures.

Besides the indisputable analytical advantages brought to herbal authentication, DNA barcoding and metabarcoding also have limitations. These are mainly derived from their high sensitivity to any amplifiable DNA extracted from the herbal product. Air-borne biological particles, such as pollen grains from neighbouring plants, can be deposited on the target plant and, when harvested, can be amplified, identified, and reported as adulterants of the finished herbal products rather than an accidental contamination. The ever-increasing analytical sensitivity of high-throughput DNA sequencing likely means that the proportion of adulterated HPs is expected to significantly increase.

It is important that the sensitivity of DNA-based authentication methods be optimized and effectively used to manage contaminated and adulterated HPs as they pose a significant risk for both the environment and human health. For example, often among the adulterants detected in commercial HPs are plant and animal species protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). More relevant to human health, HPs are often used in combination with conventional drugs. As HPs contain many pharmacologically active ingredients, the risk of adverse drug reactions – due to herb-drug interactions – increases when unknown, unlabeled plant ingredients are present in adulterated herbs taken with prescribed medications. Chronic, acute, and sometimes even lethal adverse health effects have been reported after the use of adulterated herbal products.

An accurate estimation of the adulteration of the HPs commercially available in the global marketplace is of paramount importance to all involved in their value chain, from growers, collectors, and producers, to pharmacists and medical practitioners, and finally, to the consumer.

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

Written by

Mihael Cristin Ichim

Mihael Cristin Ichim

“Stejarul” Research Centre for Biological Sciences, National Institute of Research and Development for Biological Sciences, Piatra Neamt, Romania

February 10, 2020
https://doi.org/10.21083/ibol.v10i1.5928

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Assessing herbarium material with novel molecular techniques reveals a wealth of new data from old treasure troves

Assessing herbarium material with novel molecular techniques reveals a wealth of new data from old treasure troves

Assessing herbarium material with novel molecular techniques reveals a wealth of new data from old treasure troves

Using large-scale genome skimming to build a resilient resource for the future
Hamersley Range, Pilbara, Western Australia PHOTO CREDIT: Stephen van Leeuwen

Herbaria are valuable sources of extensive curated plant material that are important reference specimens for plant identification. These plant materials are now also accessible to genetic studies because of advances in high-throughput, next-generation sequencing (NGS) methods.

In our study, we conducted a large-scale applied assessment of one such NGS approach – genome skimming – and its ability to recover plastid and ribosomal genome sequences from a broad taxonomic sampling of herbarium material for the Western Australian flora. We sequenced 672 samples covering 21 families, 142 genera, and 530 named and proposed named species, and explored the impact of sample age, DNA concentration and quality, read depth and fragment length on plastid assembly error.

We demonstrate that herbaria are a valuable source of plant material for building a comprehensive DNA sequence database which serves various applications from modernizing plant surveys to improving the resolution of plant phylogenies.

Gastrolobium grandiflorum, Pilbara, Western Australia PHOTO CREDIT: Stephen van Leeuwen

Genome skimming1 was effective at producing genomic information at large scale. Substantial sequence information on the chloroplast genome was obtained from 96.1% of samples, and complete or near-complete sequences of the nuclear ribosomal RNA gene repeat were obtained from 93.3% of samples.

Eucalyptus kingsmillii, Pilbara, Western Australia PHOTO CREDIT: Stephen van Leeuwen
Grevillea wickhamii, Pilbara, Western Australia PHOTO CREDIT: Stephen van Leeuwen

We extracted sequences for plastid markers rbcL and matK – the core DNA barcode regions – from 96.4% and 93.3% of samples, respectively. Read quality and DNA fragment length had significant effects on sequencing outcomes and error correction of reads proved essential. Assembly problems were specific to certain taxa with low GC and high repeat content (e.g. Goodenia, Scaevola, Cyperus, Bulbostylis, Fimbristylis), suggesting the influence of biological rather than technical factors. The structure of related genomes was needed to guide the assembly of repeats that exceeded the read length. DNA-based matching proved highly effective and showed that the efficacy for species identification declined in the following order: total chloroplast DNA >> ribosomal DNA > matK >> rbcL.

Ptilotus rotundifolius, Pilbara, Western Australia PHOTO CREDIT: Stephen van Leeuwen

Our success is important as it demonstrates that herbaria can be used as a source of plant material for building a comprehensive DNA sequence database. These data form the basis of development of a molecular identification system for the Western Australian flora. This will enable identification of specimens throughout the year (e.g., non-flowering times) and for hard-to-identify species (e.g., those with constrained or reduced morphological characters) or for specimens where only fragments of non-diagnostic material are available. The availability of this technology will modernize plant surveys by reducing constraints on survey effort through moderating sampling timing restrictions and seasonal effects, as well as enabling rapid verifiable identification. It will also have practical applications in a wide range of ecological contexts using eDNA metabarcoding, such as gut and scat analysis of animals to determine dietary preferences of threatened species and livestock, and checking the integrity of seed collections for seed banking and use in land restoration/revegetation programs. Other potential uses of extensive plastid sequence data, beyond species identification, include improving the resolution of plant phylogenies and studies on the evolution of plastid genome function, including understanding adaptive changes.

References:

1. Straub S, Parks M, Weitemier K, Fishbein M, Cronn R, Liston A (2012) Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics. American Journal of Botany 99(2), 349-364. https://dx.doi.org/10.3732/ajb.1100335

For full details, please refer to the publication in BMC Plant Methods.

Written by

Paul Nevill

Paul Nevill

Curtin University, School of Molecular and Life Sciences, ARC Centre for Mine Site Restoration

& Trace and Environmental DNA (TrEnD) Lab, Perth, Western Australia

February 4, 2020
https://doi.org/10.21083/ibol.v10i1.5934 

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