Large-scale Biodiversity Assessments Research Articles

DNA Barcoding for Species Identification of Moss-Dwelling Invertebrates: Performance of Nanopore Sequencing and Coverage in Reference Database

In view of the current biodiversity crisis and our need to preserve and improve ecosystem functioning, efficient means for characterizing and monitoring biodiversity are required. DNA barcoding, especially when coupled with new sequencing technologies, is a promising method that can, in principle, also be employed by taxonomic lay people. In this study we compare the performance of DNA barcoding by means of a third-generation sequencing technology, nanopore sequencing with classical Sanger sequencing, based on a sample of invertebrates collected from moss pads in a bog in Austria. We find that our nanopore sequencing pipeline generates DNA barcodes that are at least as good as barcodes generated with Sanger sequencing, with the MinION producing better results than the Flongle flowcell. We further find that while many arthropod taxa are well covered in the international reference DNA barcode database BOLD, this clearly is not the case for important taxa like mites and springtails, which hampers large-scale biodiversity assessments. Based on examples from our study we further highlight which factors might be responsible for ambiguous species identification based on BOLD and how this can, at least partly, be solved.

Using DNA metabarcoding to assess insect diversity in citrus orchards.

DNA metabarcoding is rapidly emerging as a cost-effective approach for large-scale biodiversity assessment and pest monitoring. The current study employed metabarcoding to assess insect diversity in citrus orchards in Ganzhou City, Jiangxi, China in both 2018 and 2019. Insects were sampled using Malaise traps deployed in three citrus orchards producing a total of 43 pooled monthly samples. The Malaise trap samples were sequenced following DNA metabarcoding workflow. Generated sequences were curated and analyzed using two cloud databases and analytical platforms, the barcode of life data system (BOLD) and multiplex barcode research and visualization environment (mBRAVE). These platforms assigned the sequences to 2,141 barcode index numbers (BINs), a species proxy. Most (63%) of the BINs were shared among the three sampling sites while BIN sharing between any two sites did not exceed 71%. Shannon diversity index (H') showed a similar pattern of BIN assortment at the three sampling sites. Beta diversity analysis by Jaccard similarity coefficient (J) and Bray-Curtis distance matrix (BC) revealed a high level of BIN similarity among the three sites (J = 0.67-0.68; BC = 0.19-0.20). Comparison of BIN records against all those on BOLD made it possible to identify 40% of the BINs to a species, 57% to a genus, 97% to a family and 99% to an order. BINs which received a species match on BOLD were placed in one of four categories based on this assignment: pest, parasitoid, predator, or pollinator. As this study provides the first baseline data on insect biodiversity in Chinese citrus plantations, it is a valuable resource for research in a broad range of areas such as pest management and monitoring beneficial insects in citrus gardens.

Repeated subsamples during DNA extraction reveal increased diversity estimates in DNA metabarcoding of Malaise traps.

With increased application of DNA metabarcoding in biodiversity assessment, various laboratory protocols have been optimized, and their further evaluation is subject of current research. Homogenization of bulk samples and subsequent DNA extraction from a subsample of destructed tissue is a common first stage of the metabarcoding process. This can either be conducted using sample material soaked in a storage fixative, e.g., ethanol (here referred to as "wet" treatment) or from dried individuals ("dry"). However, it remains uncertain if perfect mixing and equal distribution of DNA within the tube is ensured during homogenization and to what extent incomplete mixing and resulting variations in tissue composition affect diversity assessments if only a fraction of the destructed sample is processed in the downstream metabarcoding workflow. Here we investigated the efficiency of homogenization under wet and dry conditions and tested how variations in destructed tissue composition might affect diversity assessments of complex arthropod samples. We considered five time intervals of Malaise trap bulk samples and process nine different subsamples of homogenized tissue (20 mg each) in both treatments. Results indicate a more consistent diversity assessment from dried material, but at the cost of a higher processing time. Both approaches detected comparable OTU diversity and revealed similar taxa compositions in a single tissue extraction. With an increased number of tissue subsamples during DNA extraction, OTU diversity increased for both approaches, especially for highly diverse samples obtained during the summer. Here, particularly the detection of small and low-biomass taxa increased. The processing of multiple subsamples in the metabarcoding protocol can therefore be a helpful procedure to enhance diversity estimates and counteract taxonomic bias in biodiversity assessments. However, the process induces higher costs and time effort and the application in large-scale biodiversity assessment, e.g., in monitoring schemes needs to be considered on project-specific prospects.

Environmental DNA as a management tool for tracking artificial waterhole use in savanna ecosystems

Game parks are the last preserve of many large mammals, and in savanna ecosystems, management of surface waters poses a conservation challenge. In arid and semi-arid regions, water can be a scarce resource during dry seasons and drought. Artificial waterholes are common in parks and reserves across Africa, but can alter mammal community composition by favoring drought intolerant species, with consequences for disease dynamics, and population viability of drought-tolerant species. Analysis of waterborne environmental DNA (eDNA) is increasingly used to inform conservation of rare and invasive species, and conduct large-scale biodiversity assessments. To explore the reliability of eDNA as an indicator of mammal waterhole use in savannas, we compare eDNA metabarcoding and camera traps for documenting artificial waterhole use in the Kruger National Park, South Africa, a global hotspot for mammal diversity. We show that eDNA metabarcoding can recover the majority of mammal species detected by camera traps, including a number of endangered species, but DNA signatures of mammal visitation are temporally limited, with best performance when tracking water-dependent large bodied mammals visiting within two days of sampling. Our results highlight limitation of eDNA based monitoring in these systems, including the lack of long-term eDNA persistence in small and highly utilized waterholes, and variability in detection rates among species. However, we demonstrate that eDNA-based approaches can be used to track mammals of conservation concern, and reflect patterns of recent waterhole use and co-occurrence across water-dependent species, both of which are crucial for making evidence-based decisions regarding water management and provisioning.

A new European land systems representation accounting for landscape characteristics

ContextWhile land use change is the main driver of biodiversity loss, most biodiversity assessments either ignore it or use a simple land cover representation. Land cover representations lack the representation of land use and landscape characteristics relevant to biodiversity modeling.ObjectivesWe developed a comprehensive and high-resolution representation of European land systems on a 1-km2 grid integrating important land use and landscape characteristics.MethodsCombining the recent data on land cover and land use intensities, we applied an expert-based hierarchical classification approach and identified land systems that are common in Europe and meaningful for studying biodiversity. We tested the benefits of using this map as compared to land cover information to predict the distribution of bird species having different vulnerability to landscape and land use change.ResultsNext to landscapes dominated by one land cover, mosaic landscapes cover 14.5% of European terrestrial surface. When using the land system map, species distribution models demonstrate substantially higher predictive ability (up to 19% higher) as compared to models based on land cover maps. Our map consistently contributes more to the spatial distribution of the tested species than the use of land cover data (3.9 to 39.1% higher).ConclusionsA land systems classification including essential aspects of landscape and land management into a consistent classification can improve upon traditional land cover maps in large-scale biodiversity assessment. The classification balances data availability at continental scale with vital information needs for various ecological studies.

Can eDNA metabarcoding offer a catchment-based approach for biodiversity monitoring?

In previous studies eDNA metabarcoding has been demonstrated as a viable tool for catchment-level biodiversity sampling in rivers (Deiner et al. 2016). However, questions still remain over the appropriate sampling protocol for large spatial scale sampling. River reaches are composed of multiple habitats with species composition varying from one to the next (Costa and Melo 2007). Therefore, how many spatial replicates are needed to reliably represent the river network? Is the previously used approach to sample at every river confluence (Deiner et al. 2016) sufficient or is more needed? These questions were addressed using a case study in the headwaters of the Cound Brook, a tributary to the River Severn in Shropshire, UK. Two sub-catchments of the Cound Brook were used. One sub-catchment had a sample taken at the most downstream point before the confluence. Additionally, a sample at the upstream extent of the same sub-catchment was taken to estimate any correlation between the species found at the beginning of the river reach and at the end. Another sub-catchment also had the same up- and downstream sample design. However, in between was a systemic sampling regime every 500 m. This is to test if increasing the spatial resolution gave significantly different results to the sparser sampling regime. At each sample location, a 1 L water sample was sequentially filtered through membranes of three different mesh sizes: 5µm, 0.45µm and 0.2µm. Sequential filtering was performed because DNA resides in two forms in the environment (Civade et al. 2017), within whole cells (cellular DNA) and outside of cells (extracellular). The theory is that the coarser filters predominantly collect cellular DNA and the finer filters collect predominantly extracellular DNA of increasingly smaller fragment lengths. Consequently, sequential filtering could represent DNA degradation in the environment (Fig. 1). Also, Turner et al. 2014 suggested that the larger particles could determine very recent or local organisms. Therefore, we hypothesised that the DNA collected by the coarser filters would represent local diversity and the DNA collected by the finer filters would reflect biodiversity further upstream. Initial results suggest sequential filtering through the 5µm and 0.45µm filters caught detectable levels of eDNA where the 0.2µm did not catch enough to show up through gel electrophoresis. The relevance of the initial finding suggests that if we only used a 5µm filter the data collected at 0.45µm could have been discarded. Further investigations of any differences in species compositions between filters and the relationships to other sampling locations is still to be determined. This ongoing research is intended to determine the appropriate sampling protocol for a large-scale biodiversity assessment regime combining eDNA metabarcoding and species distribution modelling.

Wet grinding of invertebrate bulk samples – a scalable and cost-efficient protocol for metabarcoding and metagenomics

DNA metabarcoding is an efficient tool to characterize invertebrate species composition in environmental samples. Most metabarcoding protocols for invertebrate bulk samples start with sample homogenization, followed by DNA extraction, amplification of a specific marker region and sequencing on a high-throughput sequencer. Many of the above-mentioned laboratory steps have been verified thoroughly and best practice strategies exist. Giving the amount of research done to validate almost all laboratory steps of metabarcoding workflows it is surprising that there is no clear recommendation for the basis of almost all metabarcoding studies: the homogenization of samples itself. For homogenization, different devices are used that can be divided into two major categories: bead mills and blenders. While bead-mills accelerate small, hard particles in a closed container or tube to break down specimens into small tissue fragments, blenders work with a rapidly rotating blades that reliably slice specimens as well as other substrate of the sample. Both methods are currently used in metabarcoding studies and have downsides to consider. Bead-mills rely on single-use plastics and therefore produce a lot of waste and are expensive. In addition to that, processing times can go up to 30 minutes making them unsuitable for large scale studies. Blenders on the other hand offer the opportunity to be cleaned and can handle larger sample volumes in shorter time, with an increased risk of cross-contamination. We here aimed to develop a fast, robust, cheap and reliable sample homogenization protocol that overcomes the above-mentioned limitations of both methods, i.e. does not produce difficult to discard waste and avoid single-use plastics while reducing overall costs. We tested the performance of the new protocol using six sorted Malaise trap insect samples and six unsorted stream macroinvertebrate kick-net samples. We used 14 technical replicates of each sample and many negative controls per sample (Fig. 1) to quantify impacts of i) insufficient homogenization and ii) possible sources of cross-contamination. Our results show that homogenization is sufficient after 3 minutes of homogenization. Rinsing the blender with water is sufficient in most cases but leads to low read-numbers in some of the negative controls. These could be further reduced by rinsing the blender with self-made drain-safe sterilization solution based on bleach but far less corrosive than pure bleach. Our results suggest that rinsing 1-2 times for 20 seconds is sufficient to avoid any cross-contamination. The improvements of the protocol in terms of speed, ease of handling, overall reduction of costs as well as the documented reliability and robustness make it an important candidate for sample homogenization after sampling in particular for large-scale and regulatory metabarcoding biodiversity assessments and monitoring programs.

Metabarcoding reveals hidden species and improves identification of marine zooplankton communities in the North Sea

Abstract Although easily collected in large numbers, the subsequent processing and identification of zooplankton have usually been a barrier to large-scale biodiversity assessments. Therefore, DNA barcoding has been increasingly used by non-taxonomists to identify specimens. Here, we studied the community composition of zooplankton in the Belgian part of the North Sea over the course of 1 year. We identified zooplankton using both a traditional approach based on morphological characteristics and by metabarcoding of a 650 bp fragment of the V4-V5 region of the 18S rRNA gene using nanopore sequencing. Using long rDNA sequences, we were able to identify several taxa at the species level, across a broad taxonomic scale. Using both methods, we compared community composition and obtained diversity metrics. Diversity indices were not significantly correlated. Metabarcoding allowed for comparisons of diversity and community composition, but not all groups were successfully sequenced. Additionally, some disparities existed between relative abundances of the most abundant taxa based on traditional counts and those based on sequence reads. Overall, we conclude that for zooplankton samples, metabarcoding is capable of detecting taxa with a higher resolution, regardless of developmental stage of the organism. Combination of molecular and morphological methods results in the highest detection and identification levels of zooplankton.

Digitization and the Future of Natural History Collections

AbstractNatural history collections (NHCs) are the foundation of historical baselines for assessing anthropogenic impacts on biodiversity. Along these lines, the online mobilization of specimens via digitization—the conversion of specimen data into accessible digital content—has greatly expanded the use of NHC collections across a diversity of disciplines. We broaden the current vision of digitization (Digitization 1.0)—whereby specimens are digitized within NHCs—to include new approaches that rely on digitized products rather than the physical specimen (Digitization 2.0). Digitization 2.0 builds on the data, workflows, and infrastructure produced by Digitization 1.0 to create digital-only workflows that facilitate digitization, curation, and data links, thus returning value to physical specimens by creating new layers of annotation, empowering a global community, and developing automated approaches to advance biodiversity discovery and conservation. These efforts will transform large-scale biodiversity assessments to address fundamental questions including those pertaining to critical issues of global change.

Unlocking biodiversity and conservation studies in high-diversity environments using environmental DNA (eDNA): A test with Guianese freshwater fishes.

Determining the species compositions of local assemblages is a prerequisite to understanding how anthropogenic disturbances affect biodiversity. However, biodiversity measurements often remain incomplete due to the limited efficiency of sampling methods. This is particularly true in freshwater tropical environments that host rich fish assemblages, for which assessments are uncertain and often rely on destructive methods. Developing an efficient and nondestructive method to assess biodiversity in tropical freshwaters is highly important. In this study, we tested the efficiency of environmental DNA (eDNA) metabarcoding to assess the fish diversity of 39 Guianese sites. We compared the diversity and composition of assemblages obtained using traditional and metabarcoding methods. More than 7,000 individual fish belonging to 203 Guianese fish species were collected by traditional sampling methods, and ~17 million reads were produced by metabarcoding, among which ~8 million reads were assigned to 148 fish taxonomic units, including 132 fish species. The two methods detected a similar number of species at each site, but the species identities partially matched. The assemblage compositions from the different drainage basins were better discriminated using metabarcoding, revealing that while traditional methods provide a more complete but spatially limited inventory of fish assemblages, metabarcoding provides a more partial but spatially extensive inventory. eDNA metabarcoding can therefore be used for rapid and large-scale biodiversity assessments, while at a local scale, the two approaches are complementary and enable an understanding of realistic fish biodiversity.

Give me a sample of air and I will tell which species are found from your region: Molecular identification of fungi from airborne spore samples.

Fungi are a megadiverse group of organisms, they play major roles in ecosystem functioning and are important for human health, food production and nature conservation. Our knowledge on fungal diversity and fungal ecology is however still very limited, in part because surveying and identifying fungi is time demanding and requires expert knowledge. We present a method that allows anyone to generate a list of fungal species likely to occur in a region of interest, with minimal effort and without requiring taxonomical expertise. The method consists of using a cyclone sampler to acquire fungal spores directly from the air to an Eppendorf tube, and applying DNA barcoding with probabilistic species identification to generate a list of species from the sample. We tested the feasibility of the method by acquiring replicate air samples from different geographical regions within Finland. Our results show that air sampling is adequate for regional-level surveys, with samples collected >100km apart varying but samples collected <10km apart not varying in their species composition. The data show marked phenology, and thus obtaining a representative species list requires aerial sampling that covers the entire fruiting season. In sum, aerial sampling combined with probabilistic molecular species identification offers a highly effective method for generating a species list of air-dispersing fungi. The method presented here has the potential to revolutionize fungal surveys, as it provides a highly cost-efficient way to include fungi as a part of large-scale biodiversity assessments and monitoring programs.

Cryptic species as a window into the paradigm shift of the species concept.

The species concept is the cornerstone of biodiversity science, and any paradigm shift in the delimitation of species affects many research fields. Many biologists now are embracing a new "species" paradigm as separately evolving populations using different delimitation criteria. Individual criteria can emerge during different periods of speciation; some may never evolve. As such, a paradigm shift in the species concept relates to this inherent heterogeneity in the speciation process and species category-which is fundamentally overlooked in biodiversity research. Cryptic species fall within this paradigm shift: they are continuously being reported from diverse animal phyla but are poorly considered in current tests of ecological and evolutionary theory. The aim of this review is to integrate cryptic species in biodiversity science. In the first section, we address that the absence of morphological diversification is an evolutionary phenomenon, a "process" counterpart to the long-studied mechanisms of morphological diversification. In the next section regarding taxonomy, we show that molecular delimitation of cryptic species is heavily biased towards distance-based methods. We also stress the importance of formally naming of cryptic species for better integration into research fields that use species as units of analysis. Finally, we show that incorporating cryptic species leads to novel insights regarding biodiversity patterns and processes, including large-scale biodiversity assessments, geographic variation in species distribution and species coexistence. It is time for incorporating multicriteria species approaches aiming to understand speciation across space and taxa, thus allowing integration into biodiversity conservation while accommodating for species uncertainty.

Building capacity in biodiversity monitoring at the global scale

Human-driven global change is causing ongoing declines in biodiversity worldwide. In order to address these declines, decision-makers need accurate assessments of the status of and pressures on biodiversity. However, these are heavily constrained by incomplete and uneven spatial, temporal and taxonomic coverage. For instance, data from regions such as Europe and North America are currently used overwhelmingly for large-scale biodiversity assessments due to lesser availability of suitable data from other, more biodiversity-rich, regions. These data-poor regions are often those experiencing the strongest threats to biodiversity, however. There is therefore an urgent need to fill the existing gaps in global biodiversity monitoring. Here, we review current knowledge on best practice in capacity building for biodiversity monitoring and provide an overview of existing means to improve biodiversity data collection considering the different types of biodiversity monitoring data. Our review comprises insights from work in Africa, South America, Polar Regions and Europe; in government-funded, volunteer and citizen-based monitoring in terrestrial, freshwater and marine ecosystems. The key steps to effectively building capacity in biodiversity monitoring are: identifying monitoring questions and aims; identifying the key components, functions, and processes to monitor; identifying the most suitable monitoring methods for these elements, carrying out monitoring activities; managing the resultant data; and interpreting monitoring data. Additionally, biodiversity monitoring should use multiple approaches including extensive and intensive monitoring through volunteers and professional scientists but also harnessing new technologies. Finally, we call on the scientific community to share biodiversity monitoring data, knowledge and tools to ensure the accessibility, interoperability, and reporting of biodiversity data at a global scale.

High-throughput biodiversity analysis: Rapid assessment of species richness and ecological interactions of Chrysomelidae (Coleoptera) in the tropics.

Biodiversity assessment has been the focus of intense debate and conceptual and methodological advances in recent years. The cultural, academic and aesthetic impulses to recognise and catalogue the diversity in our surroundings, in this case of living objects, is furthermore propelled by the urgency of understanding that we may be responsible for a dramatic reduction of biodiversity, comparable in magnitude to geological mass extinctions. One of the most important advances in this attempt to characterise biodiversity has been incorporating DNA-based characters and molecular taxonomy tools to achieve faster and more efficient species delimitation and identification, even in hyperdiverse tropical biomes. In this assay we advocate for a broad understanding of Biodiversity as the inventory of species in a given environment, but also the diversity of their interactions, with both aspects being attainable using molecular markers and phylogenetic approaches. We exemplify the suitability and utility of this framework for large-scale biodiversity assessment with the results of our ongoing projects trying to characterise the communities of leaf beetles and their host plants in several tropical setups. Moreover, we propose that approaches similar to ours, establishing the inventories of two ecologically inter-related and species-rich groups of organisms, such as insect herbivores and their angiosperm host-plants, can serve as the foundational stone to anchor a comprehensive assessment of diversity, also in tropical environments, by subsequent addition of trophic levels.

Discovery of Highly Accurate Plant DNA Barcodes via Novel Iterative Methodologies

A DNA barcode is a short, variable segment of DNA used in species identification. A rapid and inexpensive molecular tool, DNA barcoding may allow for large-scale biodiversity assessments in the future, prompting logical and targeted conservation policies. However, a highly accurate DNA barcode for plants has not yet been found, hindering development of advanced DNA barcoding-based conservation paradigms and perpetuating loss of plant biodiversity. Previous attempts to develop a plant barcode have examined only small fractions of the plant genome. In this study, a more rigorous methodology was devised: whole plant chloroplast genomes were iteratively analyzed in successive 500 base pair segments, such that all potential barcodes contained within the chloroplast genome were assessed for their ability to distinguish plants. Moreover, an algorithm was constructed to optimize this novel process, yielding a 17000% increase in efficiency. Both non-optimized and optimized methods uncovered two 500 bp regions of DNA (out of the 2950 tested) that had unprecedented, near-perfect identification accuracy in a comprehensive sample set of whole chloroplast sequences. These DNA barcodes may enable larger biodiversity assessments, aiding plant species preservation efforts. And, our algorithm may facilitate discovery of barcodes for other kingdoms of life, expanding the reach of our methodology.

Automated DNA‐based plant identification for large‐scale biodiversity assessment

Rapid degradation of tropical forests urges to improve our efficiency in large-scale biodiversity assessment. DNA barcoding can assist greatly in this task, but commonly used phenetic approaches for DNA-based identifications rely on the existence of comprehensive reference databases, which are infeasible for hyperdiverse tropical ecosystems. Alternatively, phylogenetic methods are more robust to sparse taxon sampling but time-consuming, while multiple alignment of species-diagnostic, typically length-variable, markers can be problematic across divergent taxa. We advocate the combination of phylogenetic and phenetic methods for taxonomic assignment of DNA-barcode sequences against incomplete reference databases such as GenBank, and we developed a pipeline to implement this approach on large-scale plant diversity projects. The pipeline workflow includes several steps: database construction and curation, query sequence clustering, sequence retrieval, distance calculation, multiple alignment and phylogenetic inference. We describe the strategies used to establish these steps and the optimization of parameters to fit the selected psbA-trnH marker. We tested the pipeline using infertile plant samples and herbivore diet sequences from the highly threatened Nicaraguan seasonally dry forest and exploiting a valuable purpose-built resource: a partial local reference database of plant psbA-trnH. The selected methodology proved efficient and reliable for high-throughput taxonomic assignment, and our results corroborate the advantage of applying 'strict' tree-based criteria to avoid false positives. The pipeline tools are distributed as the scripts suite 'BAGpipe' (pipeline for Biodiversity Assessment using GenBank data), which can be readily adjusted to the purposes of other projects and applied to sequence-based identification for any marker or taxon.

Morphology, Molecular Genetics, and Bioacoustics Support Two New Sympatric Xenophrys Toads (Amphibia: Anura: Megophryidae) in Southeast China

Given their recent worldwide declines and extinctions, characterization of species-level diversity is of critical importance for large-scale biodiversity assessments and conservation of amphibians. This task is made difficult by the existence of cryptic species complexes, species groups comprising closely related and morphologically analogous species. The combination of morphology, genetic, and bioacoustic analyses permits robust and accurate species identification. Using these methods, we discovered two undescribed Xenophrys species, namely Xenophrys lini sp. nov. and Xenophrys cheni sp. nov. from the middle range of Luoxiao Mountains, southeast China. These two new species can be reliably distinguished from other known congeners by morphological and morphometric differences, distinctness in male advertisement calls, and substantial genetic distances (>3.6%) based on the mitochondrial 16s and 12s rRNA genes. The two new species, together with X. jinggangensis, are sympatric in the middle range of Luoxiao Mountains but may be isolated altitudinally and ecologically. Our study provides a first step to help resolve previously unrecognized cryptic biodiversity and provides insights into the understanding of Xenophrys diversification in the mountain complexes of southeast China.

A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents

IntroductionThe PCR-based analysis of homologous genes has become one of the most powerful approaches for species detection and identification, particularly with the recent availability of Next Generation Sequencing platforms (NGS) making it possible to identify species composition from a broad range of environmental samples. Identifying species from these samples relies on the ability to match sequences with reference barcodes for taxonomic identification. Unfortunately, most studies of environmental samples have targeted ribosomal markers, despite the fact that the mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most widely available sequence region in public reference libraries. This is largely because the available versatile (“universal”) COI primers target the 658 barcoding region, whose size is considered too large for many NGS applications. Moreover, traditional barcoding primers are known to be poorly conserved across some taxonomic groups.ResultsWe first design a new PCR primer within the highly variable mitochondrial COI region, the “mlCOIintF” primer. We then show that this newly designed forward primer combined with the “jgHCO2198” reverse primer to target a 313 bp fragment performs well across metazoan diversity, with higher success rates than versatile primer sets traditionally used for DNA barcoding (i.e. LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment coupled with an efficient bioinformatics pipeline can be used to characterize species diversity from environmental samples by pyrosequencing. We examine the gut contents of three species of planktivorous and benthivorous coral reef fish (family: Apogonidae and Holocentridae). After the removal of dubious COI sequences, we obtained a total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla from 16 fish guts. Of these, 52.5% matched a reference barcode (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment.ConclusionsThe molecular analysis of gut contents targeting the 313 COI fragment using the newly designed mlCOIintF primer in combination with the jgHCO2198 primer offers enormous promise for metazoan metabarcoding studies. We believe that this primer set will be a valuable asset for a range of applications from large-scale biodiversity assessments to food web studies.

Combining pitfall traps and soil samples to collect Collembola for site scale biodiversity assessments

Collembola are rarely included in landscape-level biodiversity assessments, large-scale surveys and monitoring projects because huge numbers of specimens would accumulate even in moderately sized programmes. Budgets are always limited, so sampling methods and identification need to be optimized. As no single sampling method collects all collembolan species equally well, we tested the efficiency of a combination of six pitfall traps and five soil subsamples in 30 oil seed rape fields in Eastern Austria. Work effort in man hours for sampling, sorting and identification was quantified for each method and related to the species richness of the collected fauna. Total identification effort was four times higher for the soil subsamples than the pitfall traps, however, soil samples also yielded more species (53 and 34, respectively). Out of the 70 species collected in total, an average of thirteen species per site was found in the pitfall samples, seventeen in the soil subsamples and 25 when combining the two methods. Using more than six pitfall samples alone would not have collected considerably more species. For the soil subsamples, still more species can be expected with the processing of more than five subsamples, but this would also result in higher costs. When including Collembola in large scale biodiversity assessments, surveys or monitoring projects, we therefore recommend combining the two methods. In combination, the identification of the catch from only two pitfalls and two soil subsamples already collected more than the average number of species in five soil subsamples or six pitfalls, respectively. Thus, combining the methods yielded a more complete picture of the collembolan community of a site than either method alone.

Cambios en el paisaje y prioridades de conservación: una perspectiva herpetofaunística mexicana a escalas local y regional

Few studies have demonstrated historical human impact on biodiversity at local and regional scales, largely due to lack of baseline information and long term monitoring for most taxa. In 1958 and 1959 researchers from the Museum of Vertebrate Zoology (MVZ) visited the Mexican state of Aguascalientes and increased its documented amphibian andreptile fauna from 21 to 30 species. Using MVZ collections, field notes, and landscape photographs taken during that expedition, we resurveyed those same localities in 2004 to document herpetofaunal changes coincident with greatly increased human activities. Despite its small area, Aguascalientes encompasses several biogeographic regions and the threat of local extinction at species' distributional limits has broader implications for regional biotas. New discoveries raise to 71 the number of species known for that state, but our comparisons suggest a gloomy future for amphibians and reptiles in Aguascalientes. Paradoxically, human impact is managed primarily at state and municipal levels, often devoid of locally relevant context. Our findings illustrate the conservation value of intensive small-scale studies, focused on the natural history of particular species and localities, as complements to large-scale biodiversity assessments on country wide and continental scales.