Synchronised monitoring of plant and insect diversity: a case study using automated Malaise traps and DNA-based methods.

The occurrence and distribution of insects and their possible associations with plant species are largely unknown in Germany and baseline data to monitor future trends are urgently needed. Using newly-designed automated Malaise trap multi-samplers, the occurrence of insect species and their potential associations with plants was monitored synchronously at two contrasting field sites in Germany: an urban botanical garden and a forest research station. Taxa were identified by metabarcoding of the insects and the plant traces present in the preservative ethanol of the Malaise trap samples. For comparison, a botanical survey was conducted in the vicinity of the traps. Across both sites, we identified a total of 1290 exact sequence variants (ESVs) assigned to Insecta, of which 205 are known to be pollinators. In the botanical garden, we detected the occurrence of 128 plant taxa, of which 41 also had one of their known insect pollinator species detected. Insect species richness was highest in May, mainly attributed to an increase in Diptera. These results present a case study of the applicability of automated sampling and DNA-based methods to monitor the timings of flowering and corresponding activity of plant-visiting insects.

More complex than you think: Taxonomic and temporal patterns of plant-pollinator networks of caraway (Carum carvi L.).

Caraway (Carum carvi L.) is a crop species that is gaining in importance in Europe, especially as a condiment and medicinal plant. Here, we present the plant-pollinator network of caraway in a central European agricultural landscape, focusing on two diverse potential pollinator taxa, Diptera: Brachycera (= true flies) and Hymenoptera (sawflies, bees, and wasps). We specifically studied qualitative differences in interactions between the two insect taxa as well as the intraday and intraseasonal variability of the network. Insect and pollen plant species determination was done via morphological identification and DNA (meta)barcoding. In total, 121 species representing 33 families of Hymenoptera and Brachycera were found to carry caraway pollen. These taxa included many nonhoneybee and nonhoverfly species, showing a wide taxonomic breadth of potential pollinators and a higher network complexity than previously anticipated. There are distinct qualitative differences between Brachycera and Hymenoptera networks, suggesting complementary roles of both taxa in the pollination of native and crop plants. Strong intraday differences in potential pollinator diversity make it necessary to collect insects and pollen at different times of the day to compile complete plant-pollinator networks. Intraseasonal analyses of the plant-pollinator network of caraway show the potential of caraway as an important food source for insect species with an activity peak in late summer.

Development and Testing of the A1 Volumetric Air Sampler, an Automatic Pollen Trap Suitable for Long-Term Monitoring of eDNA Pollen Diversity.

Airborne pollen surveys provide information on various aspects of biodiversity and human health monitoring. Such surveys are typically conducted using the Burkard Multi-Vial Cyclone Sampler, but have to be technically optimized for eDNA barcoding. We here developed and tested a new airborne pollen trap, especially suitable for autonomous eDNA-metabarcoding analyses, called the A1 volumetric air sampler. The trap can sample pollen in 24 different tubes with flexible intervals, allowing it to operate independently in the field for a certain amount of time. We compared the efficiency of the new A1 volumetric air sampler with another automated volumetric spore trap, the Burkard Multi-Vial Cyclone Sampler, which features shorter and fewer sampling intervals to evaluate the comparability of ambient pollen concentrations. In a sterile laboratory environment, we compared trap performances between the automated volumetric air samplers by using pure dry pollen of three species-Fagus sylvatica, Helianthus annuus and Zea mays-which differ both by exine ornamentation and pollen size. The traps had a standard suction flow rate of 16.5 L/min, and we counted the inhaled pollen microscopically after a predefined time interval. Our results showed that though we put three different pollen types in the same container, both the traps inhaled all the pollens in a statistically significant manner irrespective of their size. We found that, on average, both traps inhaled equal an number of pollens for each species. We did not detect any cross-contamination between tubes. We concluded that the A1 volumetric air sampler has the potential to be used for longer and more flexible sampling intervals in the wild, suitable for autonomous monitoring of eDNA pollen diversity.

Continuous inbreeding affects genetic variation, phenology, and reproductive strategy in ex situ cultivated Digitalis lutea.

PREMISE: Ex situ cultivation is important for plant conservation, but cultivation in small populations may result in genetic changes by drift, inbreeding, or unconscious selection. Repeated inbreeding potentially influences not only plant fitness, but also floral traits and interactions with pollinators, which has not yet been studied in an ex situ context. METHODS: We studied the molecular genetic variation of Digitalis lutea from a botanic garden population cultivated for 30 years, a frozen seed bank conserving the original genetic structure, and two current wild populations including the source population. In a common garden, we studied the effects of experimental inbreeding and between-population crosses on performance, reproductive traits, and flower visitation of plants from the garden and a wild population. RESULTS: Significant genetic differentiation was found between the garden population and the wild population from which the seeds had originally been gathered. After experimental selfing, inbreeding depression was only found for germination and leaf size of plants from the wild population, indicating a history of inbreeding in the smaller garden population. Moreover, garden plants flowered earlier and had floral traits related to selfing, whereas wild plants had traits related to attracting pollinators. Bumblebees visited more flowers of outbred than inbred plants and of wild than garden plants. CONCLUSIONS: Our case study suggests that high levels of inbreeding during ex situ cultivation can influence reproductive traits and thus interactions with pollinators. Together with the effects of genetic erosion and unconscious selection, these changes may affect the success of reintroductions into natural habitats.

Testing the effect of pollen exine rupture on metabarcoding with Illumina sequencing.

Pollen metabarcoding has received much attention recently for its potential to increase taxonomic resolution of the identifications of pollen grains necessary for various public health, ecological and environmental inquiry. However, methodologies implemented are widely varied across studies confounding comparisons and casting uncertainty on the reliability of results. In this study, we investigated part of the methodology, the effects of level of exine rupture and lysis incubation time, on the performance of DNA extraction and Illumina sequencing. We examined 15 species of plants from 12 families with pollen that varies in size, shape, and aperture number to evaluate effort necessary for exine rupture. Then created mock communities of 14 of the species from DNA extractions at 4 levels of exine rupture (0, 33, 67, and 100%) and two levels of increased lysis incubation time without exine rupture (2 or 24 hours). Quantities of these DNA extractions displayed a positive correlation between increased rupture and DNA yield, however increasing time of lysis incubation was associated with decreased DNA yield. Illumina sequencing was performed with these artificial community treatments with three common plant DNA barcode regions (rbcL, ITS1, ITS2) with two different primer pairings for ITS2 and rbcL. We found decreased performance in treatments with 0% or 100% exine rupture compared to 33% and 67% rupture, based on deviation from expected proportions and species retrieval, and increased lysis incubation was found to be detrimental to results.

Plant DNA barcoding necessitates marker-specific efforts to establish more comprehensive reference databases.

The problem of low species-level identification rates in plants by DNA barcoding is exacerbated by the fact that reference databases are far from being comprehensive. We investigate the impact of increased sampling depth on identification success by analyzing the efficacy of established plant barcode marker sequences (rbcL, matK, trnL-trnF, psbA-trnH, ITS). Adding sequences of the same species to the reference database led to an increase in correct species assignment of +10.9% for rbcL and +19.0% for ITS. Simultaneously, erroneous identification dropped from ∼40% to ∼12.5%. Despite its evolutionary constraints, ITS showed the highest identification rate and identification gain by increased sampling effort, which makes it a very suitable marker in the planning phase of a barcode study. The limited sequence availability of trnL-trnF is problematic for an otherwise very promising plastid plant barcoding marker. Future developments in machine learning algorithms have the potential to give new impetus to plant barcoding, but are dependent on extensive reference databases. We expect that our results will be incorporated into future plans for the development of DNA barcoding reference databases and will lead to these being developed with greater depth and taxonomic coverage.

Repositories for Taxonomic Data: Where We Are and What is Missing.

Natural history collections are leading successful large-scale projects of specimen digitization (images, metadata, DNA barcodes), thereby transforming taxonomy into a big data science. Yet, little effort has been directed towards safeguarding and subsequently mobilizing the considerable amount of original data generated during the process of naming 15,000-20,000 species every year. From the perspective of alpha-taxonomists, we provide a review of the properties and diversity of taxonomic data, assess their volume and use, and establish criteria for optimizing data repositories. We surveyed 4113 alpha-taxonomic studies in representative journals for 2002, 2010, and 2018, and found an increasing yet comparatively limited use of molecular data in species diagnosis and description. In 2018, of the 2661 papers published in specialized taxonomic journals, molecular data were widely used in mycology (94%), regularly in vertebrates (53%), but rarely in botany (15%) and entomology (10%). Images play an important role in taxonomic research on all taxa, with photographs used in >80% and drawings in 58% of the surveyed papers. The use of omics (high-throughput) approaches or 3D documentation is still rare. Improved archiving strategies for metabarcoding consensus reads, genome and transcriptome assemblies, and chemical and metabolomic data could help to mobilize the wealth of high-throughput data for alpha-taxonomy. Because long-term-ideally perpetual-data storage is of particular importance for taxonomy, energy footprint reduction via less storage-demanding formats is a priority if their information content suffices for the purpose of taxonomic studies. Whereas taxonomic assignments are quasifacts for most biological disciplines, they remain hypotheses pertaining to evolutionary relatedness of individuals for alpha-taxonomy. For this reason, an improved reuse of taxonomic data, including machine-learning-based species identification and delimitation pipelines, requires a cyberspecimen approach-linking data via unique specimen identifiers, and thereby making them findable, accessible, interoperable, and reusable for taxonomic research. This poses both qualitative challenges to adapt the existing infrastructure of data centers to a specimen-centered concept and quantitative challenges to host and connect an estimated $ \le $2 million images produced per year by alpha-taxonomic studies, plus many millions of images from digitization campaigns. Of the 30,000-40,000 taxonomists globally, many are thought to be nonprofessionals, and capturing the data for online storage and reuse therefore requires low-complexity submission workflows and cost-free repository use. Expert taxonomists are the main stakeholders able to identify and formalize the needs of the discipline; their expertise is needed to implement the envisioned virtual collections of cyberspecimens. [Big data; cyberspecimen; new species; omics; repositories; specimen identifier; taxonomy; taxonomic data.].

BCdatabaser: on-the-fly reference database creation for (meta-)barcoding.

SUMMARY: DNA barcoding and meta-barcoding have become irreplaceable in research and applications, where identification of taxa alone or within a mixture, respectively, becomes relevant. Pioneering studies were in the microbiological context, yet nowadays also plants and animals become targeted. Given the variety of markers used, formatting requirements for classifiers and constant growth of primary databases, there is a need for dedicated reference database creation. We developed a web and command-line interface to generate such on-the-fly for any applicable marker and taxonomic group with optional filtering, formatting and restriction specific for (meta-)barcoding purposes. Also, databases optionally receive a DOI, making them well-documented with meta-data, publicly sharable and citable. AVAILABILITY AND IMPLEMENTATION: source code: https://www.github.com/molbiodiv/bcdatabaser, webservice: https://bcdatabaser.molecular.eco, documentation: https://molbiodiv.github.io/bcdatabaser.

Open Data in Global Environmental Research: The Belmont Forum's Open Data Survey.

This paper presents the findings of the Belmont Forum's survey on Open Data which targeted the global environmental research and data infrastructure community. It highlights users' perceptions of the term "open data", expectations of infrastructure functionalities, and barriers and enablers for the sharing of data. A wide range of good practice examples was pointed out by the respondents which demonstrates a substantial uptake of data sharing through e-infrastructures and a further need for enhancement and consolidation. Among all policy responses, funder policies seem to be the most important motivator. This supports the conclusion that stronger mandates will strengthen the case for data sharing.

Does origin always matter? Evaluating the influence of nonlocal seed provenances for ecological restoration purposes in a widespread and outcrossing plant species.

For restoration purposes, nature conservation generally enforces the use of local seed material based on the "local-is-best" (LIB) approach. However, in some cases recommendations to refrain from this approach have been made. Here we test if a common widespread species with no obvious signs of local adaptation may be a candidate species for abandoning LIB during restoration. Using 10 microsatellite markers we compared population genetic patterns of the generalist species Daucus carota in indigenous and formerly restored sites (nonlocal seed provenances). Gene diversity overall ranged between H e = 0.67 and 0.86 and showed no significant differences between the two groups. Hierarchical AMOVA and principal component analysis revealed very high genetic population admixture and negligible differentiation between indigenous and restored sites (F CT = 0.002). Moreover, differentiation between groups was caused by only one outlier population, where inbreeding effects are presumed. We therefore conclude that the introduction of nonlocal seed provenances in the course of landscape restoration did not jeopardize regional species persistence by contributing to inbreeding or outbreeding depressions, or any measurable adverse population genetic effect. On the basis of these results, we see no obvious objections to the current practice to use the 10-fold cheaper, nonlocal seed material of D. carota for restoration projects.

Metabarcoding vs. morphological identification to assess diatom diversity in environmental studies.

Diatoms are frequently used for water quality assessments; however, identification to species level is difficult, time-consuming and needs in-depth knowledge of the organisms under investigation, as nonhomoplastic species-specific morphological characters are scarce. We here investigate how identification methods based on DNA (metabarcoding using NGS platforms) perform in comparison to morphological diatom identification and propose a workflow to optimize diatom fresh water quality assessments. Diatom diversity at seven different sites along the course of the river system Odra and Lusatian Neisse from the source to the mouth is analysed with DNA and morphological methods, which are compared. The NGS technology almost always leads to a higher number of identified taxa (270 via NGS vs. 103 by light microscopy LM), whose presence could subsequently be verified by LM. The sequence-based approach allows for a much more graduated insight into the taxonomic diversity of the environmental samples. Taxa retrieval varies considerably throughout the river system, depending on species occurrences and the taxonomic depth of the reference databases. Mostly rare taxa from oligotrophic parts of the river systems are less well represented in the reference database used. A workflow for DNA-based NGS diatom identification is presented. 28 000 diatom sequences were evaluated. Our findings provide evidence that metabarcoding of diatoms via NGS sequencing of the V4 region (18S) has a great potential for water quality assessments and could complement and maybe even improve the identification via light microscopy.

The Global Genome Biodiversity Network (GGBN) Data Portal.

The Global Genome Biodiversity Network (GGBN) was formed in 2011 with the principal aim of making high-quality well-documented and vouchered collections that store DNA or tissue samples of biodiversity, discoverable for research through a networked community of biodiversity repositories. This is achieved through the GGBN Data Portal (http://data.ggbn.org), which links globally distributed databases and bridges the gap between biodiversity repositories, sequence databases and research results. Advances in DNA extraction techniques combined with next-generation sequencing technologies provide new tools for genome sequencing. Many ambitious genome sequencing projects with the potential to revolutionize biodiversity research consider access to adequate samples to be a major bottleneck in their workflow. This is linked not only to accelerating biodiversity loss and demands to improve conservation efforts but also to a lack of standardized methods for providing access to genomic samples. Biodiversity biobank-holding institutions urgently need to set a standard of collaboration towards excellence in collections stewardship, information access and sharing and responsible and ethical use of such collections. GGBN meets these needs by enabling and supporting accessibility and the efficient coordinated expansion of biodiversity biobanks worldwide.

The DNA bank network: the start from a german initiative.

The explicit aim of the DNA Bank Network is to close the divide between biological specimen collections and molecular sequence databases. It provides a technically optimized DNA and tissue collection service facility in the interest of all biological research, with access to well-documented DNA-containing samples and voucher specimens as well as to corresponding molecular data stored in public sequence databases. The Network enables scientists to (i) query and order DNA samples of organisms collected from natural habitats via a shared Web portal, (ii) store DNA samples for reference under optimal conditions after project completion or data publication, (iii) obtain DNA material to conduct new studies or to extend and complement previous investigations, and (iv) support good scientific practice as the deposition of DNA samples and related specimens facilitates the verification of published results.

Incongruent plastid and nuclear DNA phylogenies reveal ancient intergeneric hybridization in Pilosella hawkweeds (Hieracium, Cichorieae, Asteraceae).

Phylogenetic relationships for Hieracium subgen. Pilosella were inferred from chloroplast (trnT-trnL, matK) and nuclear (ITS) sequence data. Chloroplast markers revealed the existence of two divergent haplotype groups within the subgenus that did not correspond to presumed relationships. Furthermore, chloroplast haplotypes of the genera Hispidella and Andryala nested each within one of these groups. In contrast, ITS data were generally in accord with morphology and other evidence and were therefore assumed to reflect the true phylogeny. They revealed a sister relationship between Pilosella and Hispidella and a joint clade of Hieracium subgenera Hieracium and Chionoracium (Stenotheca) while genus Andryala represented a third major lineage of the final ingroup cluster. Detailed analysis of trnT-trnL character state evolution along the ITS tree suggested two intergeneric hybridization events between ancestral lineages that resulted in cytoplasmic transfer (from Hieracium/Chionoracium to Pilosella, and from the introgressed Pilosella lineage to Andryala). These chloroplast capture events, the first of which involved a now extinct haplotype, are the most likely explanation for the observed incongruencies between plastid and nuclear DNA markers.