Double-blind validation of alternative wild bee identification techniques: DNA metabarcoding and in vivo determination in the field

Over the past few decades, several investigations around the globe have reported alarming declines in the abundance and diversity of bee species. The success of effective conservation strategies targeting these important pollinators relies heavily on accurate biodiversity assessments. The shortage of taxonomic experts and the escalation of the ongoing biodiversity crisis call for the development of alternative identification tools to implement efficient monitoring programs. The validation of such techniques is crucial to ensure that they provide results comparable to those of traditional morphotaxonomy. Here we performed two double-blind experiments to evaluate the accuracy of a pair of new techniques used for wild bee identification: DNA metabarcoding and in vivo identification in the field. The methods were tested on sets of wild bees from Germany and their results compared against evaluations done by panels of bee experts using traditional morphotaxonomy. On average the congruency of species identification between metabarcoding and morphotaxonomy was 88.98% across samples (N = 10), while in vivo identification and morphotaxonomy were 91.81% congruent (N = 7) for bees considered feasible for in vivo identification in the field. Traditional morphotaxonomy showed similar congruencies when compared to itself: 93.65% in the metabarcoding study and 92.96% in the in vivo study. Overall, these results support both new methods as viable alternatives to traditional microscopy-based assessment, with neither method being error-free. Metabarcoding provides a suitable option to analyze large numbers of specimens in the absence of highly trained taxonomic experts, while in vivo identification is recommended for repeated long-term monitoring, and when working in areas where the sampling of individuals could threaten local populations of endangered wild bee species. Further research is still needed to explore the potential of both techniques for conservation management and wildlife monitoring, as well as to overcome their current limitations as taxonomic tools

Selección natural en variantes genéticas de la proteína surfactante D en población altiplánica

Antropóloga físicaLos ambientes de altura representan un desafío para la capacidad de los seres humanos de sobrevivir y reproducirse debido a factores ambientales extremos, tales como las condiciones de hipoxia. En tres regiones del mundo las poblaciones humanas presentan características físicas que les permiten habitar permanentemente la altura: la meseta Tibetana-Qinghai (Asia Oriental), la meseta Semien (Etiopia) y el Altiplano Andino (Sudamérica). Los patrones genéticos que subyacen a estas características físicas son diferentes en cada población siendo un caso de convergencia evolutiva. Un estudio genómico encontró señales de selección positiva en la población altiplánica en la región del gen SFTPD, que codifica para la proteína surfactante D (SP-D) y diferencias significativas en las frecuencias alélicas de tres mutaciones no sinónimas (rs3088308,rs2243639,rs721917) entre esta población y grupos Guaraní (Valverde et al., 2015). Cambios en la conformación de esta proteína podría ser ventajosos en los ambientes de altura, debido a la importancia de la SP-D en la función respiratoria. En esta memoria, se caracterizaron las frecuencias alélicas, genotípicas y haplotípicas de las mutaciones mencionadas por Valverde et al., 2015, y de un conjunto de mutaciones intrónicas en la población altiplánica y en poblaciones que no habitan la altura. Se comparó una muestra de población Aymara de Puno con una muestra de población amerindia del sur de Chile que no habita la altura, y con muestras de siete poblaciones del 1000Genomes Project, utilizando análisis genéticos descriptivos: comparación de frecuencias alélicas, genotípicas y haplotípicas mediante la prueba exacta de Fisher, estimación de bloques haplotípicos y valores de Fst, construcción de dendrograma de Neighbour-Joining y Análisis de Componentes Principales. Los resultados de esta memoria indican que la población de Puno, las poblaciones amerindias del sur de Chile y la población mestiza de Lima (PEL) son muy similares en cuanto a frecuencias alélicas y haplotípicas. Las variantes encontradas en Puno son también frecuentes en varias poblaciones a nivel global, por lo que una relación entre este gen y el patrón adaptativo de las poblaciones que habitan el altiplano andino no parece probable. Los resultados de este estudio no apoyan la señal de selección natural encontrada por Valverde et al., 2015. Se encontró variabilidad interpoblacional en las frecuencias de los haplotipos de SFTPD, siendo algunos de estos característicos de ciertas regiones geográficas. En particular, se encontraron altos niveles de diferenciación genética al comparar las poblaciones occidentales con las población Han del sur de China (CHS) y con la población Japonesa de Tokyo (JPT), debido a la alta frecuencia de un haplotipo particular en estas poblaciones (>40%), el cual es muy raro en el resto del mundo (< 6%). Este haplotipo posee alelos raros de las tres mutaciones no sinónimas mencionadas, que han sido relacionados por estudios médicos a menores niveles de proteína surfactante D en sangre. Sin embargo, es más probable que esté panorama esté dado por eventos relacionados a expansiones territoriales en las poblaciones asiáticas, que por efecto de la selección natura

Updates to the checklist of the wild bee fauna of Luxembourg as inferred from revised natural history collection data and fieldwork

Museums and other institutions curating natural history collections (NHCs) are fundamental entities to many scientific disciplines, as they house data and reference material for varied research projects. As such, biological specimens preserved in NHCs represent accessible physical records of the living world's history. They provide useful information regarding the presence and distribution of different taxonomic groups through space and time. Despite the importance of museum biological specimens, their potential to answer scientific questions pertinent to the necessities of our current historical context is often under-explored.The currently known wild bee fauna of Luxembourg comprises 341 registered species distributed among 38 different genera. However, specimens stored in the archives of local NHCs represent an untapped resource to update taxonomic lists, including potentially overlooked findings relevant to the development of national conservation strategies.We re-investigated the wild bee collection of the Zoology Department of the National Museum of Natural History Luxembourg by using morphotaxonomy and DNA barcoding. The collection revision led to the discovery of four species so far not described for the country: Andrena lagopus (Latreille, 1809), Nomada furva (Panzer, 1798), Hoplitis papaveris (Latreille, 1799), and Sphecodes majalis (Pérez, 1903). Additionally, the presence of Nomada sexfasciata (Panzer, 1799), which inexplicably had been omitted by the most current species list, can be re-confirmed. Altogether, our findings increase the number of recorded wild bee species in Luxembourg to 346. Moreover, the results highlight the crucial role of NHCs as repositories of our knowledge of the natural world

A metabarcoding framework for wild bee assessment in Luxembourg

Wild bees are crucial organisms for terrestrial environments. Their ongoing decline could cause irreparable damage to ecosystem services vital to plant reproduction and human food production. The importance of taking swift action to prevent further declines is widely acknowledged, but the current deficit of reliable taxonomic information complicates the development of efficient conservation strategies targeting wild bees. DNA metabarcoding can help to improve this situation by providing rapid and standardized mass identification. This technique allows the analysis of large numbers of specimens without the need for specialized taxonomic knowledge by matching high-throughput sequencing reads against public DNA barcode reference libraries. However, the validation of this approach for wild bees requires the evaluation of potential error sources on a regional scale. Here we analyzed the effects of three potential error sources on a metabarcoding pipeline customized for the wild bee fauna of Luxembourg. In an in silico study, we checked the completeness of the BOLD reference library for 349 species found in the country, the correspondence between molecular and morphological species delimitation for these taxa, and the amplification efficiency of three commonly used metabarcoding primer pairs (mlCOlintF/HCO2198, LepF1/MLepF1-Rev and BF2/BR2). The detection power of the pipeline was evaluated based on the species recovery rates from mock communities of known composition under variable DNA concentration treatments. The reference barcode library evaluation results show that 97% of the species have at least a single barcode in BOLD Systems (minimal length 196 bp) and that 85% of species have ≥ 5 barcodes in the public domain. The mlCOlintF/HCO2198 target fragment presented the highest coverage (77.94% of the species with full barcode sequences), followed by the target fragments of LepF1/MLepF1-Rev (77.65%) and BF2/BR2 (68.48%). Only 60% of the morphospecies presented a complete coverage of the prominent Folmer region (658 bp). The in silico amplification efficiency analysis shows that the BF2/BR2 primer pair has the best-predicted amplification performance, but none of the primer combinations evaluated can be expected to efficiently amplify all local wild bee genera. Finally, all species detection rates in the mock communities, except for the sample with the most discrepant DNA concentrations, were above 97%, with no significant differences found among treatments. These results indicate that the detection capacity of the pipeline is robust enough to be used for the reliable assessment of local wild bee biodiversity, even if species from various size categories are pooled together. Primer bias has a major effect on species detection, which can be acknowledged with a preliminary assessment of primer-template mismatch and sophisticated methodological designs (e.g. mock community controls, replicates). Overall, the metabarcoding pipeline here described provides a suitable tool for quick and reliable taxonomic identification of the regional wild bee fauna to aid conservation initiatives in Luxembourg – and beyond

Supplementary material 4 from: Herrera-Mesías F, Bause C, Ogan S, Burger H, Ayasse M, Weigand AM, Eltz T (2022) Double-blind validation of alternative wild bee identification techniques: DNA metabarcoding and in vivo determination in the field. Journal of Hymenoptera Research 93: 189-214. https://doi.org/10.3897/jhr.93.86723

Supplementary file

Supplementary material 5 from: Herrera-Mesías F, Bause C, Ogan S, Burger H, Ayasse M, Weigand AM, Eltz T (2022) Double-blind validation of alternative wild bee identification techniques: DNA metabarcoding and in vivo determination in the field. Journal of Hymenoptera Research 93: 189-214. https://doi.org/10.3897/jhr.93.86723

Supplementary file

Supplementary material 1 from: Herrera-Mesías F, Bause C, Ogan S, Burger H, Ayasse M, Weigand AM, Eltz T (2022) Double-blind validation of alternative wild bee identification techniques: DNA metabarcoding and in vivo determination in the field. Journal of Hymenoptera Research 93: 189-214. https://doi.org/10.3897/jhr.93.86723

Supplementary file

National records of 3000 European bee and hoverfly species: A contribution to pollinator conservation

Pollinators play a crucial role in ecosystems globally, ensuring the seed production of most flowering plants. They are threatened by global changes and knowledge of their distribution at the national and continental levels is needed to implement efficient conservation actions, but this knowledge is still fragmented and/or difficult to access. As a step forward, we provide an updated list of around 3000 European bee and hoverfly species, reflecting their current distributional status at the national level (in the form of present, absent, regionally extinct, possibly extinct or non-native). This work was attainable by incorporating both published and unpublished data, as well as knowledge from a large set of taxonomists and ecologists in both groups. After providing the first National species lists for bees and hoverflies for many countries, we examine the current distributional patterns of these species and designate the countries with highest levels of species richness. We also show that many species are recorded in a single European country, highlighting the importance of articulating European and national conservation strategies. Finally, we discuss how the data provided here can be combined with future trait and Red List data to implement research that will further advance pollinator conservation