Tuesday, July 17, 2018

Temperature and mtDNA selection

Mitochondrial DNA (mtDNA) has traditionally been used in population genetic and biogeographic studies as a maternally-inherited and evolutionary-neutral genetic marker. However, it is now clear that polymorphisms within the mtDNA sequence are routinely non-neutral, and furthermore several studies have suggested that such mtDNA polymorphisms are also sensitive to thermal selection. 

A team of researchers from Japan, Australia, and the UK studied two naturally occurring mtDNA variants that are carried by fruit flies inhabiting the east coast of Australia. One of these variants is more common in the sub-tropical, northern part of the country, where temperatures are higher. The other fly variant is more common in the temperate, southern part, which tends to be colder. The colleagues collected flies from both sites and interbred them to get a series of populations with equally mixed genes. Each mixed population was subdivided into four subpopulations. Each of those wer maintained at different conditions, some at constant temperature(e.g. 19ºC and 25ºC respectively), some at fluctuating temperatures to simulate the thermal conditions at the two sites where the flies were collected. After three months, the researchers sequenced the mtDNA of flies from all these subpopulations.

In addition they also examined how the presence of bacteria such as Wolbachia, which commonly infects fruit flies, affects mtDNA selection. Some of the fly populations were treated with antibiotics to kill off any Wolbachia infections that they might be harboring.

The researchers found that in flies reared under warm laboratory conditions, one of the two mtDNA variants became more common than the other. The same mtDNA variant was also found to be widely prevalent in flies from the warmer northern parts of Australia. A similar pattern was observed with the other mtDNA variant in flies reared under cold laboratory conditions. However, researchers only observed this effect in populations where Wolbachia infections had been wiped out. Moreover, the variation patterns they observed in males didn't always match up with the ones in females.

The results show that temperature shapes how mtDNA varies in nature. They also suggest that additional factors, such as sex and infection with microbes, might also influence how the mitochondrial genome evolves in the wild. 

Our results strengthen the emerging view that intra-specific mtDNA variants are sensitive to selection, and suggest spatial distributions of mtDNA variants in natural populations of metazoans might reflect adaptation to climatic environments rather than within-population coalescence and diffusion of selectively-neutral haplotypes across populations.

Friday, July 13, 2018

Weekend reads

Back on track with the weekend read posts.  Maybe not as many as usual but still quite interesting to read.

Ecological and taxonomic knowledge is important for conservation and utilization of biodiversity. Biodiversity and ecology of fungi in Mediterranean ecosystems is poorly understood. Here, we examined the diversity and spatial distribution of fungi along an elevational gradient in a Mediterranean ecosystem, using DNA metabarcoding. This study provides novel information about diversity of all ecological and taxonomic groups of fungi along an elevational gradient in a Mediterranean ecosystem. Our analyses revealed that among all biotic and abiotic variables tested, host species identity is the main driver of the fungal richness and fungal community composition. Fungal richness was strongly associated with tree richness and peaked in Quercus-dominated habitats and Cistus-dominated habitats. The highest taxonomic richness of ectomycorrhizal fungi was observed under Quercus ilex, whereas the highest taxonomic richness of saprotrophs was found under Pinus. Our results suggest that the effect of plant diversity on fungal richness and community composition may override that of abiotic variables across environmental gradients.

Although insects dominate the terrestrial fauna, sampling constraints and the poor taxonomic knowledge of many groups have limited assessments of their diversity. Passive sampling techniques and DNA-based species assignments now make it possible to overcome these barriers. For example, Malaise traps collect specimens with minimal intervention while the Barcode Index Number (BIN) system automates taxonomic assignments. The present study employs Malaise traps and DNA barcoding to extend understanding of insect diversity in one of the least known zoogeographic regions, the Saharo-Arabian. Insects were collected at four sites in three countries (Egypt, Pakistan, Saudi Arabia) by deploying Malaise traps. The collected specimens were analyzed by sequencing 658 bp of cytochrome oxidase I (DNA barcode) and assigning BINs on the Barcode of Life Data Systems. The year-long deployment of a Malaise trap in Pakistan and briefer placements at two Egyptian sites and at one in Saudi Arabia collected 53,092 specimens. They belonged to 17 insect orders with Diptera and Hymenoptera dominating the catch. Barcode sequences were recovered from 44,432 (84%) of the specimens, revealing the occurrence of 3,682 BINs belonging to 254 families. Many of these taxa were uncommon as 25% of the families and 50% of the BINs from Pakistan were only present in one sample. Family and BIN counts varied significantly through the year, but diversity indices did not. Although more than 10,000 specimens were analyzed from each nation, just 2% of BINs were shared by Pakistan and Saudi Arabia, 4% by Egypt and Pakistan, and 7% by Egypt and Saudi Arabia. The present study demonstrates how the BIN system can circumvent the barriers imposed by limited access to taxonomic specialists and by the fact that many insect species in the Saharo-Arabian region are undescribed.

Fungal spores and mycelium fragments are particles which become and remain airborne and have been subjects of aerobiological studies. The presence and the abundance of taxa in aerobiological samples can be very variable and impaired by changeable climatic conditions. Because many fungi produce mycotoxins and both their mycelium fragments and spores are potential allergens, monitoring the presence of these taxa is of key importance. So far data on exposure and sensitization to fungal allergens are mainly based on the assessment of few, easily identifiable taxa and focused only on certain environments. The microscopic method used to analyze aerobiological samples and the inconspicuous fungal characters do not allow a in depth taxonomical identification. Here, we present a first assessment of fungal diversity from airborne samples using a DNA metabarcoding analysis. The nuclear ITS2 region was selected as barcode to catch fungal diversity in mixed airborne samples gathered during two weeks in four sites of North-Eastern and Central Italy. We assessed the taxonomic composition and diversity within and among the sampled sites and compared the molecular data with those obtained by traditional microscopy. The molecular analyses provide a tenfold more comprehensive determination of the taxa than the traditional morphological inspections. Our results prove that the metabarcoding analysis is a promising approach to increases quality and sensitivity of the aerobiological monitoring. The laboratory and bioinformatic workflow implemented here is now suitable for routine, high-throughput, regional analyses of airborne fungi.

Throughout the years, DNA barcoding has gained in importance in forensic entomology as it leads to fast and reliable species determination. High-quality results, however, can only be achieved with a comprehensive DNA barcode reference database at hand. In collaboration with the Bavarian State Criminal Police Office, we have initiated at the Bavarian State Collection of Zoology the establishment of a reference library containing arthropods of potential forensic relevance to be used for DNA barcoding applications. CO1-5P' DNA barcode sequences of hundreds of arthropods were obtained via DNA extraction, PCR and Sanger Sequencing, leading to the establishment of a database containing 502 high-quality sequences which provide coverage for 88 arthropod species. Furthermore, we demonstrate an application example of this library using it as a backbone to a high throughput sequencing analysis of arthropod bulk samples collected from human corpses, which enabled the identification of 31 different arthropod Barcode Index Numbers.

Environmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species-specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and "metabarcoding" have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real-time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high-throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species-specific surveys.

Thursday, July 12, 2018

How predictable is evolution?

Imagine 500 to 1,000 species of cichlids living in one of the African Great Lakes, one of the largest freshwater habitats in the world. The degree of complexity is unimaginable. Even the genealogical relationships between the cichlid species living in these lakes have only partially been resolved

For every two species of mammal there is one species of cichlid fish, which shows that biodiversity is distributed rather unevenly among animals. The question is why and to what extent can this be predicted? We know that a number of factors play a role in the evolution of this. One of them are ecological conditions, i.e. the number of different habitats and the similarity of ecological niches available. The demographic history of a population can also influence biodiversity. A higher level of genetic variation in a population is beneficial in a sense that it allows - given sufficient time - adaptation to more ecological niches. Quantifying all potential factors that contribute to biological diversity, even for only one group of animals, is not easy, not to mention that comparing mammals with a group of fish would be like comparing apples and oranges. 

A new study coming from the lab of my PhD supervisor, Axel Meyer shows some of the factors that contribute to recurrent patterns of diversity and similarity in cichlids. The colleagues aimed to determine factors that led to similar outcomes and thereby help to predict evolution. As any African Great Lake harbours an incredibly species diversity, the team studied a more simple system involving parallel species of Midas cichlids, which occur in two great lakes as well as in a chain of crater lakes in Nicaragua. 

The more similar the habitat of the crater lake is to that of the large source lake, the more similar the fish are to each other. Habitat seems to be more important than demographic criteria when it comes to predictability of diversity.  The data collected by the colleagues also shows that, compared to the source population, the morphology of all crater lake populations has diversified mostly in the same direction. Crater lake fish all very quickly evolved body shapes that are longer and more slender than those of their cousins from the bigger lakes. In addition body shapes of the crater lake populations correlate with the average depth of the lakes. It makes sense. The deeper a lake is, the more likely it is to provide various ecological niches, including in the deep open water. 

In summary parallel morphological divergence in allopatry and the propensity for diversification in sympatry across the entire Midas cichlid fish radiation is partly predictable and mostly driven by ecology.

Monday, July 9, 2018

(Post-) Weekend reads

Last Friday I was so invested in some data analysis that I forgot everything around me and that included my Friday blog post with weekend reading material. My apologies for that. Nevertheless, here are my weekly favourites for some throughout the week reading.

Efficient DNA extraction is fundamental to molecular studies. However, commercial kits are expensive when a large number of samples need to be processed. Here we present a simple, modular and adaptable DNA extraction ‘toolkit’ for the isolation of high purity DNA from multiple sample types (modular universal DNA extraction method or Mu-DNA). We compare the performance of our method to that of widely used commercial kits across a range of soil, stool, tissue and water samples. Mu-DNA produced DNA extractions of similar or higher yield and purity to that of the commercial kits. As a proof of principle, we carried out replicate fish metabarcoding of aquatic eDNA extractions, which confirmed that the species detection efficiency of our method is similar to that of the most frequently used commercial kit. Our results demonstrate the reliability of Mu-DNA along with its modular adaptability to challenging sample types and sample collection methods. Mu-DNA can substantially reduce the costs and increase the scope of experiments in molecular studies.

The monitoring of impacts of anthropic activities in marine environments, such as aquaculture, oil-drilling platforms or deep-sea mining, relies on Benthic Biotic Indices (BBI). Several indices have been formalised to reduce the multivariate composition data into a single continuous value that is ascribed to a discrete ecological quality status. Such composition data is traditionally obtained from macrofaunal inventories, which is time-consuming and expertise-demanding. Important efforts are ongoing towards using High-Throughput Sequencing of environmental DNA (eDNA metabarcoding) to replace or complement morpho-taxonomic surveys for routine biomonitoring. The computation of BBI from such composition data is usually being undertaken by practitioners with excel spreadsheets or through custom script. Hence, the updating of reference morpho-taxonomic tables and cross studies comparison could be hampered. Here we introduce the R package BBI for the computation of BBI from composition data, either obtained from traditional morpho-taxonomic inventories or from metabarcoding data. Its aim is to provide an open-source, transparent and centralised method to compute BBI for routine biomonitoring.

The degradation of freshwater ecosystems has become a common ecological and environmental problem globally. Owing to the complexity of biological communities, there remain tremendous technical challenges for investigating influence of environmental stressors (e.g. chemical pollution) on biological communities. High-throughput sequencing-based metabarcoding provides a powerful tool to reveal complex interactions between environments and biological communities. Among many technical issues, the clustering strategies for Operational Taxonomic Units (OTUs) which are crucial for assessing biodiversity of communities, may affect final conclusions. Here, we used zooplankton communities along an environmental pollution gradient in the Chaobai River in Northern China to test different clustering strategies, including non-clustering and clustering with varied thresholds. Our results showed that though the number of OTUs estimated by non-clustering strategies and clustering strategies with divergence thresholds of 99-97% largely varied, they were able to identify the same set of significant environmental and spatial variables responsible for geographical distributions of zooplankton communities. In addition, the ecological conclusions obtained by clustering thresholds of 99-97% were consistent with non-clustering strategies, where for all eight clustering scenarios we detected that species sorting predicted by environmental variables overrode dispersal as the dominant factor in structuring zooplankton communities. However, clustering with the divergence thresholds of <95% affected the environmental and spatial variables identified. We conclude that both newly developed non-clustering methods and traditional clustering methods with divergence thresholds ≥97% were reliable to reveal mechanisms of complex community-environment interactions, although different clustering strategies could lead to largely varied biodiversity estimates such as those for α-diversity.

Sediment bypass tunnels (SBTs) are guiding structures used to reduce sediment accumulation in reservoirs during high flows by transporting sediments to downstream reaches during operation. Previous studies monitoring the ecological effects of SBT operations on downstream reaches suggest a positive influence of SBTs on riverbed sediment conditions and macroinvertebrate communities based on traditional morphology-based surveys. Morphology-based macroinvertebrate assessments are costly and time-consuming, and the large number of morphologically cryptic, small-sized and undescribed species usually results in coarse taxonomic identification. Here, we used DNA metabarcoding analysis to assess the influence of SBT operations on macroinvertebrates downstream of SBT outlets by estimating species diversity and pairwise community dissimilarity between upstream and downstream locations in dam-fragmented rivers with operational SBTs in comparison to dam-fragmented (i.e., no SBTs) and free-flowing rivers (i.e., no dam). We found that macroinvertebrate community dissimilarity decreases with increasing operation time and frequency of SBTs. These factors of SBT operation influence changes in riverbed features, e.g. sediment relations, that subsequently effect the recovery of downstream macroinvertebrate communities to their respective upstream communities. Macroinvertebrate abundance using morphologically-identified specimens was positively correlated to read abundance using metabarcoding. This supports and reinforces the use of quantitative estimates for diversity analysis with metabarcoding data.

Metabarcoding is a popular application which warrants continued methods optimization. To maximize barcoding inferences, hierarchy-based sequence classification methods are increasingly common. We present methods for the construction and curation of a database designed for hierarchical classification of a 157 bp barcoding region of the arthropod cytochrome c oxidase subunit I (COI) locus. We produced a comprehensive arthropod COI amplicon dataset including annotated arthropod COI sequences and COI sequences extracted from arthropod whole mitochondrion genomes, the latter of which provided the only source of representation for Zoraptera, Callipodida and Holothyrida. The database contains extracted sequences of the target amplicon from all major arthropod clades, including all insect orders, all arthropod classes and Onychophora, Tardigrada and Mollusca outgroups. During curation, we extracted the COI region of interest from approximately 81 percent of the input sequences, corresponding to 73 percent of the genus-level diversity found in the input data. Further, our analysis revealed a high degree of sequence redundancy within the NCBI nucleotide database, with a mean of approximately 11 sequence entries per species in the input data. The curated, low-redundancy database is included in the Metaxa2 sequence classification software. Using this database with the Metaxa2 classifier, we performed a cross-validation analysis to characterize the relationship between the Metaxa2 reliability score, an estimate of classification confidence, and classification error probability. We used this analysis to select a reliability score threshold which minimized error. We then estimated classification sensitivity, false discovery rate and overclassification, the propensity to classify sequences from taxa not represented in the reference database. Our work will help researchers design and evaluate classification databases and conduct metabarcoding on arthropods and alternate taxa.

Honeydew produced from the excretion of plant-sucking insects (order Hemiptera) is a carbohydrate-rich material that is foraged by honey bees to integrate their diets. In this study, we used DNA extracted from honey as a source of environmental DNA to disclose its entomological signature determined by honeydew producing Hemiptera that was recovered not only from honeydew honey but also from blossom honey. We designed PCR primers that amplified a fragment of mitochondrial cytochrome c oxidase subunit 1 (COI) gene of Hemiptera species using DNA isolated from unifloral, polyfloral and honeydew honeys. Ion Torrent next generation sequencing metabarcoding data analysis assigned Hemiptera species using a customized bioinformatic pipeline. The forest honeydew honeys reported the presence of high abundance of Cinara pectinatae DNA, confirming their silver fir forest origin. In all other honeys, most of the sequenced reads were from the planthopper Metcalfa pruinosa for which it was possible to evaluate the frequency of different mitotypes. Aphids of other species were identified from honeys of different geographical and botanical origins. This unique entomological signature derived by environmental DNA contained in honey opens new applications for honey authentication and to disclose and monitor the ecology of plant-sucking insects in agricultural and forest landscapes.

Introduced species of mammals in New Zealand have had catastrophic effects on populations of diverse native species. Quantifying the diets of these omnivorous and predatory species is critical for understanding which native species are most impacted, and to prioritize which mammal species and locations should be targeted with control programmes. A variety of methods have been applied to quantify diet components in animals, including visual inspection of gut contents (Daniel 1973; Pierce and Boyle 1991), stable isotope analysis (Major et al. 2007; Carreon-Martinez and Heath 2010), and time-lapse video (Brown and Brown 1997; Dunlap and Pawlik 1996). Increasingly, DNA-based metabarcoding methods are being used (King et al. 2008; Soininen et al. 2009). These metabarcoding methods require a PCR step using primers that bind to highly conserved genomic regions (e.g. mitochondrial COI) to amplify specific regions for sequencing. This step introduces significant bias, primarily due to the lack of a universal primer set (King et al. 2008). Here we show that direct metagenomic sequencing using the Oxford Nanopore Minion allows rapid quantification of rat diets. Using a sample of rats collected from within 100km of Auckland, NZ, we show that these rats consume a wide variety of plant, invertebrate, vertebrate, and fungal taxa, with substantial differences in diet content between locales. We then show that, based on diet content alone, it is possible to pinpoint the sampling location of an individual rat within tens of kilometres. We expect that the rapidly increasing accuracy and throughput of nanopore-based sequencing, as well as increases in the species diversity of genomic databases, will soon allow rapid and unbiased assessments of animal diets in field settings.

Thursday, July 5, 2018

One decade of ZooKeys

One decade of ZooKeys - not bad at all. That means one decade of Open Access Taxonomy. Descriptions that are not hiding behind a paywall, a no-brainer if you ask me. How can we talk about democratization and equal access to information if a large part of the primary literature is still hidden to a substantial group of researchers simply because they or their institution can't afford a subscription. Especially for taxonomy that is pretty aggravating. So, my best wishes and congratulations to Pensoft (in particular Lubo) for this success story. 

The Pensoft blog has some more details. Here the first part:

So here we are, 10 years from that very first issue of ours published on a very special date – the 4th of July – and the result of a seemingly ordinary breakfast conversation between two respected entomologists, Prof Lyubomir Penev and Dr Terry Erwin, during the Entomological Society of America meeting in San Diego, USA, seven months earlier.

Then and there, under the California sun, an idea about a brand new taxonomic journal meant to revolutionise the scholarly publishing in zoology – in terms of both openness and technological innovation, was born. The rest, like they say, is history.

Ten years in, we stand as the most prolific open-access journal in zoology with a total of 4,103 published articles, 45 newly described animal families, 650 genera and 8977 species, authored by a total of 5,720 researchers coming from 131 different countries. We also take pride in having set an excellent example for the rest of the academic titles in Pensoft’s already extensive portfolio of open access journals.

Wednesday, July 4, 2018

Tour of Flanders video footage shows climate change impact on trees

Predicting how the timing of cyclic life history events, such as leafing and flowering, respond to climate change is of paramount importance due to the cascading impacts of vegetation phenology on species and ecosystem fitness. However, progress of this field is hampered by the relative scarcity, and geographic and phylogenetic bias, of longterm phenology datasets.

By analyzing nearly four decades of archive footage from the cycling Tour of Flanders, researchers from Ghent University have been able to detect climate change impacts on trees. Focusing on trees and shrubs growing around recognisable climbs and other landmarks along the route of this major annual road cycling race in Belgium, the colleagues looked at video footage from 1981 to 2016 obtained by a Flemish broadcaster. They visually estimated how many leaves and flowers were present on the day of the course (usually in early April) and linked their scores to climate data. 

They found that the trees had advanced the timing of leafing and flowering in response to recent temperature changes. Before 1990, almost no trees had grown leaves at the time of the spring race. After that year, more and more trees visible in the television footage, in particular magnolia, hawthorn, hornbeam and birch trees, were already in full leaf. These shifts were most strongly related to warmer average temperatures in the area, which have increased by 1.5°C since 1980.

Early-leafing trees can be good news for some species as they grow faster and produce more wood. However, their leaves also cast shadows. When trees flush earlier in the year, they shadow for a longer period of time, affecting other animals and plants, and even whole ecosystems. Some of the flowers growing under these trees may not be able to receive enough sunlight to bloom. As a result, insects can go without nectar and may struggle to find enough spots to sunbathe.

Phenology (the study of natural phenomena that recur periodically such as leafing and flowering) is mostly based on long-term observations and repeat photography, with data often being biased towards common species or geographical regions. In this study, archive footage allowed the researchers to use previously unexploited records of twelve tree species in the Flanders region in order to build long-term datasets of phenological responses.

Our method could also be used to collect data on other aspects important for ecological or evolutionary research, such as tree health, water levels in rivers and lakes, and the spread of invasive species. Only by compiling data from the past will we be able to predict the future effects of climate change on species and ecosystems.

Tuesday, July 3, 2018

Scale matters

Setophaga discolor - Credit: Julie Hart
Biodiversity is changing all around us and worldwide. Local species disappear and sometimes other species invade. Studying birds in the U.S. and worldwide, we show that patterns and implications of this ongoing change vary strongly with the scale.

A minor loss or gain of species richness or functional diversity at the local or county level can look like a major gain at the state or national level, and yet be a net loss when viewed at a global scale. Researchers at Yale University studied 50 years of data about nesting birds in North America and tracked biodiversity changes at different scales. They found significant differences in how much change had occurred, based upon how wide a geographic net they cast. In addition taxonomic diversity and functional diversity increased over all but the global scale. The larger the scale change in taxonomic diversity was higher than in functional diversity which suggests strong trait redundancy at those scales. Also, insectivorous birds (like the prairie warbler in the photo) showed the most drastic declines across all geographic scales, from local to continental.

Any reporting and interpretation of biodiversity change thus needs scale as a key qualifier. Better yet, researchers and practitioners of biodiversity science should adopt a multi-scale framework and consider all geographic scales simultaneously.