Metabarcoding reveals seasonal and spatial patterns of arthropod community assemblages in two contrasting habitats: Desert and oasis of the Baja California Peninsula,Mexico

Aim

Desert springs or oases are the only permanent mesic environments in highly water-limited arid regions. Oases have immense cultural, evolutionary and ecological importance for people and a high number of endemic and relic species. Nevertheless, they are also highly vulnerable ecosystems, with invasive species, overexploitation and climate change being the primary threats. We used the arthropod communities' spatiotemporal diversity and distribution patterns as a proxy to understand biodiversity dynamics in two geographically close but ecologically contrasting and highly threatened ecosystems: deserts and oases.

Location

Baja California Peninsula, Mexico.

Methods

Arthropod communities at five oases and surrounding desert scrub areas were sampled in two seasons. Using DNA metabarcoding and traditional taxonomic surveys, we tried to identify what biotic and abiotic characteristics of the habitat are important drivers of arthropod diversity and how these characteristics can change across spatial and temporal scales.

Results

Over 6200 individuals representing 23 orders were collected. In oasis samples, the community composition fluctuated more in space (i.e. among sites) than in time (i.e. seasons). Thus, seasonal changes did not affect oasis community diversity and composition, but the dissimilarity among sites increased with geographic distance. Moreover, anthropic activities negatively correlated with arthropod diversity in oases. On the other hand, the season, geography (e.g. latitude) and biotic characteristics of the habitat (e.g. sampled scrub species) significantly affected the diversity and composition of the desert arthropod communities.

Main Conclusions

Neutral dynamics (e.g. historical climatic events, dispersal limitation and spatial component) and human impact significantly influenced the biodiversity patterns of each oasis. In contrast, the habitat's seasonal variation and biotic characteristics were the most important variables influencing the diversity of the desert communities. Baja California oases harbour distinct invertebrate communities; therefore, each oasis should be conserved individually to preserve these unique assemblages.     

基于环境DNA技术的赤水河秋季鱼类多样性分布特征

赤水河是长江上游少有的仍保持自然流态的大型一级支流,是长江鱼类重要的繁衍场和珍稀物种的保护地,摸清其鱼类多样性现状及鱼类群落结构特征对赤水河水生态恢复评估极为重要。于2021年9月对赤水河流域开展了鱼类多样性、分布及其特征调查,全流域共设置52个采样点,采用环境DNA技术采集并研究了赤水河鱼类的组成及其分布。结果显示通过环境DNA方法共调查到鱼类6目18科62属77种,包含16种长江特有鱼类。以鲤形目为主,占总数的87.87%。赤水河鱼类食性以杂食性和肉食性鱼类为主,群落结构上,处于下层水环境鱼类较多;赤水河鱼类优势种为宽鳍鱲(Y=0.205)、西昌华吸鳅(Y=0.085)、麦穗鱼(Y=0.068)、乌苏拟鲿(Y=0.033)、云南光唇鱼(Y=0.027);赤水河上游和下游鱼类群落(P<0.01)和Shannon-Wiener指数差异均显著(P<0.05)。海拔、流速、pH、电导率和温度是影响赤水河鱼类多样性的主要环境因素。为环境DNA技术在赤水河鱼类多样性调查中的应用提供了探索性研究,将有助于赤水河生物多样性的保护。     

环境DNA在两栖动物监测中的应用研究进展

两栖动物是我国受威胁程度最高的动物类群,加强两栖动物资源调查和多样性监测,是开展两栖动物保护和濒危物种拯救行动的关键性基础工作。传统的两栖动物监测主要以形态学和声学为基础,耗时费力,且难以发现一些隐蔽性较强的稀有物种。基于环境DNA(environmental DNA, eDNA)的调查方法以其快速、灵敏、高效、无创等独特优势,为两栖动物多样性监测及保护提供了新的工具。综述了eDNA在两栖动物多样性监测、外来入侵和珍稀濒危物种调查、物种丰度或生物量估测等研究领域的应用进展,分析了两栖动物eDNA产生、扩散、迁移和降解的动态变化特征及其关键影响因子,探讨了eDNA应用于两栖动物监测研究的局限性并提出了优化建议,同时对未来的研究方向进行了展望,以充分挖掘eDNA在两栖动物监测中的应用潜力,为两栖动物多样性保护和管理提供新的思路。     

A robust approach to estimate relative phytoplankton cell abundances from metagenomes

Phytoplankton account for >45% of global primary production, and have an enormous impact on aquatic food webs and on the entire Earth System. Their members are found among prokaryotes (cyanobacteria) and multiple eukaryotic lineages containing chloroplasts. Genetic surveys of phytoplankton communities generally consist of PCR amplification of bacterial (16S), nuclear (18S) and/or chloroplastic (16S) rRNA marker genes from DNA extracted from environmental samples. However, our appreciation of phytoplankton abundance or biomass is limited by PCR-amplification biases, rRNA gene copy number variations across taxa, and the fact that rRNA genes do not provide insights into metabolic traits such as photosynthesis. Here, we targeted the photosynthetic gene psbO from metagenomes to circumvent these limitations: the method is PCR-free, and the gene is universally and exclusively present in photosynthetic prokaryotes and eukaryotes, mainly in one copy per genome. We applied and validated this new strategy with the size-fractionated marine samples collected by Tara Oceans, and showed improved correlations with flow cytometry and microscopy than when based on rRNA genes. Furthermore, we revealed unexpected features of the ecology of these ecosystems, such as the high abundance of picocyanobacterial aggregates and symbionts in the ocean, and the decrease in relative abundance of phototrophs towards the larger size classes of marine dinoflagellates. To facilitate the incorporation of psbO in molecular-based surveys, we compiled a curated database of >18,000 unique sequences. Overall, psbO appears to be a promising new gene marker for molecular-based evaluations of entire phytoplankton communities.     

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.     

Disentangling the assembly mechanisms of ant cuticular bacterial communities of two Amazonian ant species sharing a common arboreal nest

Bacteria living on the cuticle of ants are generally studied for their protective role against pathogens, especially in the clade of fungus‐growing ants. However, little is known regarding the diversity of cuticular bacteria in other ant host species, as well as the mechanisms leading to the composition of these communities. Here, we used 16S rRNA gene amplicon sequencing to study the influence of host species, species interactions and the pool of bacteria from the environment on the assembly of cuticular bacterial communities on two phylogenetically distant Amazonian ant species that frequently nest together inside the roots system of epiphytic plants, Camponotus femoratus and Crematogaster levior. Our results show that (a) the vast majority of the bacterial community on the cuticle is shared with the nest, suggesting that most bacteria on the cuticle are acquired through environmental acquisition, (b) 5.2% and 2.0% of operational taxonomic units (OTUs) are respectively specific to Ca. femoratus and Cr. levior, probably representing their respective core cuticular bacterial community, and (c) 3.6% of OTUs are shared between the two ant species. Additionally, mass spectrometry metabolomics analysis of metabolites on the cuticle of ants, which excludes the detection of cuticular hydrocarbons produced by the host, were conducted to evaluate correlations among bacterial OTUs and m/z ion mass. Although some positive and negative correlations are found, the cuticular chemical composition was weakly species‐specific, suggesting that cuticular bacterial communities are prominently environmentally acquired. Overall, our results suggest the environment is the dominant source of bacteria found on the cuticle of ants.     

Reconstructing marine plankton food web interactions using DNA metabarcoding

Knowledge of zooplankton in situ diet is critical for accurate assessment of marine ecosystem function and structure, but due to methodological constraints, there is still a limited understanding of ecological networks in marine ecosystems. Here, we used DNA‐metabarcoding to study trophic interactions, with the aim to unveil the natural diet of zooplankton species under temporal variation of food resources. Several target consumers, including copepods and cladocerans, were investigated by sequencing 16S rRNA and 18S rRNA genes to identify prokaryote and eukaryote potential prey present in their guts. During the spring phytoplankton bloom, we found a dominance of diatom and dinoflagellate trophic links to copepods. During the summer period, zooplankton including cladocerans showed a more diverse diet dominated by cyanobacteria and heterotrophic prey. Our study suggests that copepods present trophic plasticity, changing their natural diet over seasons, and adapting their feeding strategies to the available prey spectrum, with some species being more selective. We did not find a large overlap of prey consumed by copepods and cladocerans, based on prey diversity found in their guts, suggesting that they occupy different roles in the trophic web. This study represents the first molecular approach to investigate several zooplankton–prey associations under seasonal variation, and highlights how, unlike other techniques, the diversity coverage is high when using DNA, allowing the possibility to detect a wide range of trophic interactions in plankton communities.     

Keeping it cool: Soil sample cold pack storage and DNA shipment up to 1 month does not impact metabarcoding results

With the advances of sequencing tools, the fields of environmental microbiology and soil ecology have been transformed. Today, the unculturable majority of soil microbes can be sequenced. Although these tools give us tremendous power and open many doors to answer important questions, we must understand how sample processing may impact our results and interpretations. Here, we test the impacts of four soil storage methods on downstream amplicon metabarcoding and qPCR analyses for fungi and bacteria. We further investigate the impact of thaw time on extracted DNA to determine a safe length of time during which this can occur with minimal impact on study results. Overall, we find that storage using standard cold packs with subsequent storage at ?20°C is little different than immediate storage in liquid nitrogen, suggesting that the historical and current method is adequate. We further find evidence that storage at room temperature or with aid of RNAlater can lead to changes in community composition and in the case of RNAlater, lower gene copies. We therefore advise against these storage methods for metabarcoding analyses. Finally, we show that over 1 month, DNA extract thaw time does not impact diversity or qPCR metrics. We hope that this work will help researchers working with soil bacteria and fungi make informed decisions about soil storage and transport to ensure repeatability and accuracy of results and interpretations.     

Application of plant metabarcoding to identify diverse honeybee pollen forage along an urban–agricultural gradient

Understanding animal foraging ecology requires large sample sizes spanning broad environmental and temporal gradients. For pollinators, this has been hampered by the laborious nature of morphologically identifying pollen. Identifying pollen from urban environments is particularly difficult due to the presence of diverse ornamental species associated with consumer horticulture. Metagenetic pollen analysis represents a potential solution to this issue. Building upon prior laboratory and bioinformatic methods, we applied quantitative multilocus metabarcoding to characterize the foraging ecology of honeybee colonies situated in urban, suburban, mixed suburban–agricultural and rural agricultural sites in central Ohio, USA. In cross‐validating a subset of our metabarcoding results using microscopic palynology, we find strong concordance between the molecular and microscopic methods. Our results suggest that forage from the agricultural site exhibited decreased taxonomic diversity and temporal turnover relative to the urban and suburban sites, though the generalization of this observation will require replication across additional sites and cities. Our work demonstrates the power of honeybees as environmental samplers of floral community composition at large spatial scales, aiding in the distinction of taxa characteristically associated with urban or agricultural land use from those distributed ubiquitously across the sampled landscapes. Observed patterns of high forage diversity and compositional turnover in our more urban sites are likely reflective of the fine‐grain heterogeneity and high beta diversity of urban floral landscapes at the scale of honeybee foraging. This provides guidance for future studies investigating how relationships between urbanization and measures of pollinator health are mediated by variation in floral resource dynamics across landscapes.     

Meta-Fish-Lib: A generalised,dynamic DNA reference library pipeline for metabarcoding of fishes

The accuracy and reliability of DNA metabarcoding analyses depend on the breadth and quality of the reference libraries that underpin them. However, there are limited options available to obtain and curate the huge volumes of sequence data that are available on public repositories such as NCBI and BOLD. Here, we provide a pipeline to download, clean and annotate mitochondrial DNA sequence data for a given list of fish species. Features of this pipeline include (a) support for multiple metabarcode markers; (b) searches on species synonyms and taxonomic name validation; (c) phylogeny assisted quality control for identification and removal of misannotated sequences; (d) automatically generated coverage reports for each new GenBank release update; and (e) citable, versioned DOIs. As an example we provide a ready-to-use curated reference library for the marine and freshwater fishes of the U.K. To augment this reference library for environmental DNA metabarcoding specifically, we generated 241 new MiFish-12S sequences for 88 U.K. marine species, and make available new primer sets useful for sequencing these. This brings the coverage of common U.K. species for the MiFish-12S fragment to 93%, opening new avenues for scaling up fish metabarcoding across wide spatial gradients. The Meta-Fish-Lib reference library and pipeline is hosted at https://github.com/genner-lab/meta-fish-lib .