taxalogue: a toolkit to create comprehensive CO1 reference databases.

Background: Taxonomic identification through DNA barcodes gained considerable traction through the invention of next-generation sequencing and DNA metabarcoding. Metabarcoding allows for the simultaneous identification of thousands of organisms from bulk samples with high taxonomic resolution. However, reliable identifications can only be achieved with comprehensive and curated reference databases. Therefore, custom reference databases are often created to meet the needs of specific research questions. Due to taxonomic inconsistencies, formatting issues, and technical difficulties, building a custom reference database requires tremendous effort. Here, we present taxalogue, an easy-to-use software for creating comprehensive and customized reference databases that provide clean and taxonomically harmonized records. In combination with extensive geographical filtering options, taxalogue opens up new possibilities for generating and testing evolutionary hypotheses. Methods: taxalogue collects DNA sequences from several online sources and combines them into a reference database. Taxonomic incongruencies between the different data sources can be harmonized according to available taxonomies. Dereplication and various filtering options are available regarding sequence quality or metadata information. taxalogue is implemented in the open-source Ruby programming language, and the source code is available at https://github.com/nwnoll/taxalogue. We benchmark four reference databases by sequence identity against eight queries from different localities and trapping devices. Subsamples from each reference database were used to compare how well another one is covered. Results: taxalogue produces reference databases with the best coverage at high identities for most tested queries, enabling more accurate, reliable predictions with higher certainty than the other benchmarked reference databases. Additionally, the performance of taxalogue is more consistent while providing good coverage for a variety of habitats, regions, and sampling methods. taxalogue simplifies the creation of reference databases and makes the process reproducible and transparent. Multiple available output formats for commonly used downstream applications facilitate the easy adoption of taxalogue in many different software pipelines. The resulting reference databases improve the taxonomic classification accuracy through high coverage of the query sequences at high identities.

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.

Emerging technologies revolutionise insect ecology and monitoring.

Insects are the most diverse group of animals on Earth, but their small size and high diversity have always made them challenging to study. Recent technological advances have the potential to revolutionise insect ecology and monitoring. We describe the state of the art of four technologies (computer vision, acoustic monitoring, radar, and molecular methods), and assess their advantages, current limitations, and future potential. We discuss how these technologies can adhere to modern standards of data curation and transparency, their implications for citizen science, and their potential for integration among different monitoring programmes and technologies. We argue that they provide unprecedented possibilities for insect ecology and monitoring, but it will be important to foster international standards via collaboration.

Pooling size sorted Malaise trap fractions to maximize taxon recovery with metabarcoding.

BACKGROUND: Small and rare specimens can remain undetected when metabarcoding is applied on bulk samples with a high specimen size heterogeneity. This is especially critical for Malaise trap samples, where most of the biodiversity is contributed by small taxa with low biomass. The separation of samples in different size fractions for downstream analysis is one possibility to increase detection of small and rare taxa. However, experiments systematically testing different size sorting approaches and subsequent proportional pooling of fractions are lacking, but would provide important information for the optimization of metabarcoding protocols. We set out to find a size sorting strategy for Malaise trap samples that maximizes taxonomic recovery but remains scalable and time efficient. METHODS: Three Malaise trap samples were sorted into four size classes using dry sieving. Each fraction was homogenized and lysed. The corresponding lysates were pooled to simulate unsorted samples. Pooling was additionally conducted in equal proportions and in four different proportions enriching the small size fraction of samples. DNA from the individual size classes as well as the pooled fractions was extracted and metabarcoded using the FwhF2 and Fol-degen-rev primer set. Additionally, alternative wet sieving strategies were explored. RESULTS: The small size fractions harboured the highest diversity and were best represented when pooling in favour of small specimens. Metabarcoding of unsorted samples decreases taxon recovery compared to size sorted samples. A size separation into only two fractions (below 4 mm and above) can double taxon recovery compared to not size sorting. However, increasing the sequencing depth 3- to 4-fold can also increase taxon recovery to levels comparable with size sorting, but remains biased towards biomass rich taxa in the sample. CONCLUSION: We demonstrate that size fractionation of Malaise trap bulk samples can increase taxon recovery. While results show distinct patterns, the lack of statistical support due to the limited number of samples processed is a limitation. Due to increased speed and lower risk of cross-contamination as well as specimen damage we recommend wet sieving and proportional pooling of the lysates in favour of the small size fraction (80-90% volume). However, for large-scale projects with time constraints, increasing sequencing depth is an alternative solution.

Agent-mediated spatial storage effect in heterogeneous habitat stabilizes competitive mouse lemur coexistence in Menabe Central, Western Madagascar.

BACKGROUND: Spatio-temporal distribution patterns of species in response to natural and anthropogenic drivers provide insight into the ecological processes that determine community composition. We investigated determinants of ecological structure in a species assemblage of 4 closely related primate species of the family Cheirogaleidae (Microcebus berthae, Microcebus murinus, Cheirogaleus medius, Mirza coquereli) in western Madagascar by extensive line transect surveys across spatial and temporal heterogeneities with the specific goal of elucidating the mechanisms stabilizing competitive coexistence of the two mouse lemur species (Microcebus spp.). RESULTS: Interspecific competition between the mouse lemurs was indicated by negative spatial associations in degraded habitat and by habitat partitioning along anthropogenic disturbance gradients during dry seasons with resource scarcity. In non-degraded habitat, intraguild predator M. coquereli, but not C. medius, was negatively associated with M. murinus on the population level, whereas its regional distribution overlapped spatially with that of M. berthae. The species' interspecific distribution pattern across spatial and temporal heterogeneities corresponded to predictions for agent-mediated coexistence and thus confirmed M. coquereli's stabilizing impact on the coexistence of mouse lemurs. CONCLUSIONS: Interspecific interactions contribute to ecological structure in this cheirogaleid assemblage and determinants vary across spatio-temporal heterogeneities. Coexistence of Microcebus spp. is stabilized by an agent-mediated spatial storage effect: M. coquereli creates refuges from competition for M. berthae in intact habitat, whereas anthropogenic environments provide M. murinus with an escape from resource competition and intraguild predation. Species persistence in the assemblage therefore depends on the conservation of habitat content and context that stabilizing mechanisms rely on. Our large-scale population level approach did not allow for considering all potential functional and stochastic drivers of ecological structure, a key limitation that accounts for the large proportion of unexplained variance in our models.

Distribution and Abundance of the World's Smallest Primate, Microcebus berthae, in Central Western Madagascar.

The distribution of most recently discovered or described lemur species remains poorly known, but many appear to have small geographical ranges, making them vulnerable to extinction. Research can contribute to future conservation actions on behalf of these species by providing accurate information on local distribution and abundance. The distribution of the world's smallest primate, the endangered Madame Berthe's mouse lemur (Microcebus berthae), is limited to the Menabe Central region of western Madagascar. This species was discovered in the 1990s, but many fundamental aspects of its ecology remain unknown. The aims of our study were therefore to determine the actual distribution of Microcebus berthae across the forests of this region, to estimate population density, and to examine the species' response to anthropogenic activities. We established 35 1-km line transects across Menabe Central, on which we surveyed mouse lemurs by distance sampling and live trapping. Microcebus berthae does not occur in all remaining forests of this small region and its population density is highly heterogeneous, both across its geographic range and locally. Within its area of occupancy, the population of Microcebus berthae not only was distributed according to spatial heterogeneities of the habitat, but also responded to anthropogenic disturbances and varied seasonally. Our results indicate that Microcebus berthae is susceptible to habitat degradation and avoids human environments spatially. As none of the forest remnants in which the species still occurs were officially protected until recently, immediate conservation actions should focus on effectively protecting Kirindy and Ambadira forests.