Multiscale climate change impacts on plant diversity in the Atacama Desert

Comprehending ecological dynamics requires not only knowledge of modern communities but also detailed reconstructions of ecosystem history. Ancient DNA (aDNA) metabarcoding allows biodiversity responses to major climatic change to be explored at different spatial and temporal scales. We extracted aDNA preserved in fossil rodent middens to reconstruct late Quaternary vegetation dynamics in the hyperarid Atacama Desert. By comparing our paleo‐informed millennial record with contemporary observations of interannual variations in diversity, we show local plant communities behave differentially at different timescales. In the interannual (years to decades) time frame, only annual herbaceous expand and contract their distributional ranges (emerging from persistent seed banks) in response to precipitation, whereas perennials distribution appears to be extraordinarily resilient. In contrast, at longer timescales (thousands of years) many perennial species were displaced up to 1,000 m downslope during pluvial events. Given ongoing and future natural and anthropogenically induced climate change, our results not only provide baselines for vegetation in the Atacama Desert, but also help to inform how these and other high mountain plant communities may respond to fluctuations of climate in the future.     

Toward accurate molecular identification of species in complex environmental samples: testing the performance of sequence filtering and clustering methods

Metabarcoding has the potential to become a rapid, sensitive, and effective approach for identifying species in complex environmental samples. Accurate molecular identification of species depends on the ability to generate operational taxonomic units (OTUs) that correspond to biological species. Due to the sometimes enormous estimates of biodiversity using this method, there is a great need to test the efficacy of data analysis methods used to derive OTUs. Here, we evaluate the performance of various methods for clustering length variable 18S amplicons from complex samples into OTUs using a mock community and a natural community of zooplankton species. We compare analytic procedures consisting of a combination of (1) stringent and relaxed data filtering, (2) singleton sequences included and removed, (3) three commonly used clustering algorithms (mothur, UCLUST, and UPARSE), and (4) three methods of treating alignment gaps when calculating sequence divergence. Depending on the combination of methods used, the number of OTUs varied by nearly two orders of magnitude for the mock community (60–5068 OTUs) and three orders of magnitude for the natural community (22–22191 OTUs). The use of relaxed filtering and the inclusion of singletons greatly inflated OTU numbers without increasing the ability to recover species. Our results also suggest that the method used to treat gaps when calculating sequence divergence can have a great impact on the number of OTUs. Our findings are particularly relevant to studies that cover taxonomically diverse species and employ markers such as rRNA genes in which length variation is extensive.     

两种分析方法在豹猫食物构成中的应用比较

豹猫(Prionailurus bengalensis)作为北京地区的顶级食肉动物,对于维持食物网结构和生态系统稳定性起到重要的生态作用。对于捕食动物食物构成研究,较为简便的方法是粪样内容物检视法,而粪样残余物DNA鉴定技术具有更为准确细致的优势,但也存在不足,探索不同方法的优势互补,将有助于提高技术应用成效。本研究利用DNA宏条形码技术与粪样内容物分析法,对采集自北京市4个自然保护区的71份豹猫粪样进行食物构成分析,比较两种分析方法的特点,了解豹猫的食物资源利用状况。结果显示,DNA宏条形码技术共鉴别出36种猎物,来自10目22科,4个保护区的豹猫食性具有显著差异,百花山、松山、云蒙山保护区的豹猫食物种类的出现比率均以小型哺乳类为主,其中对鼠类的捕食比例最高,对鸟类的捕食次之,而分布于云峰山保护区的豹猫对鸟类捕食比例最高,对鼠类的捕食次之。粪样内容物分析法鉴别出9类猎物,其中包括昆虫和植物两种DNA宏条形码技术未检出的食物,4个保护区的豹猫食物均以鼠类和鸟类为主,且最多检测出鼠类数量为3只、鸟类2只,次要食物则为植物和昆虫。两种方法均显示北社鼠(Niviventer confucia...     

Evaluation of 96-well high-throughput DNA extraction methods for 16S rRNA gene metabarcoding

Gaining meaningful insights into bacterial communities associated with animal hosts requires the provision of high-quality nucleic acids. Although many studies have compared DNA extraction methods for samples with low bacterial biomass (e.g. water) or specific PCR inhibitors (e.g. plants), DNA extraction bias in samples without inherent technical constraint (e.g. animal samples) has received little attention. Furthermore, there is an urgent need to identify a DNA extraction methods in a high-throughput format that decreases the cost and time for processing large numbers of samples. We here evaluated five DNA extraction protocols, using silica membrane-based spin columns and a 96-well microplate format and based on either mechanical or enzymatic lysis or a combination of both, using three bacterial mock communities and Illumina sequencing of the V4 region of the 16SrRNA gene. Our results showed that none of the DNA extraction methods fully eliminated bias associated with unequal lysis efficiencies. However, we identified a DNA extraction method with a lower bias for each mock community standard. Of these methods, those including an enzymatic lysis showed biases specific to some bacteria. Altogether, these results again demonstrate the importance of DNA extraction standardization to be able to compare the microbiome results of different samples. In this attempt, we advise for the use of the 96-well DNeasy Blood and Tissue kit (Qiagen) with a zirconia bead-beating procedure, which optimizes altogether the cost, handling time and bacteria-specific effects associated with enzymatic lysis.     

Cyanoseq: A database of cyanobacterial 16S rRNA gene sequences with curated taxonomy

Cyanobacteria are photosynthetic bacteria that occupy various habitats across the globe, playing critical roles in many of Earth's biogeochemical cycles both in both aquatic and terrestrial systems. Despite their well-known significance, their taxonomy remains problematic and is the subject of much research. Taxonomic issues of Cyanobacteria have consequently led to inaccurate curation within known reference databases, ultimately leading to problematic taxonomic assignment during diversity studies. Recent advances in sequencing technologies have increased our ability to characterize and understand microbial communities, leading to the generation of thousands of sequences that require taxonomic assignment. We herein propose CyanoSeq ( https://zenodo.org/record/7569105 ), a database of cyanobacterial 16S rRNA gene sequences with curated taxonomy. The taxonomy of CyanoSeq is based on the current state of cyanobacterial taxonomy, with ranks from the domain to genus level. Files are provided for use with common naive Bayes taxonomic classifiers, such as those included in DADA2 or the QIIME2 platform. Additionally, FASTA files are provided for creation of de novo phylogenetic trees with (near) full-length 16S rRNA gene sequences to determine the phylogenetic relationship of cyanobacterial strains and/or ASV/OTUs. The database currently consists of 5410 cyanobacterial 16S rRNA gene sequences along with 123 Chloroplast, Bacterial, and Vampirovibrionia (formally Melainabacteria) sequences.     

Deciphering the diet of a wandering spider (Phoneutria boliviensis; Araneae: Ctenidae) by DNA metabarcoding of gut contents

Arachnids are the most abundant land predators. Despite the importance of their functional roles as predators and the necessity to understand their diet for conservation, the trophic ecology of many arachnid species has not been sufficiently studied. In the case of the wandering spider, Phoneutria boliviensis F. O. Pickard‐Cambridge, 1897, only field and laboratory observational studies on their diet exist. By using a DNA metabarcoding approach, we compared the prey found in the gut content of males and females from three distant Colombian populations of P. boliviensis. By DNA metabarcoding of the cytochrome c oxidase subunit I (COI), we detected and identified 234 prey items (individual captured by the spider) belonging to 96 operational taxonomic units (OTUs), as prey for this wandering predator. Our results broaden the known diet of P. boliviensis with at least 75 prey taxa not previously registered in fieldwork or laboratory experimental trials. These results suggest that P. boliviensis feeds predominantly on invertebrates (Diptera, Lepidoptera, Coleoptera, and Orthoptera) and opportunistically on small squamates. Intersex and interpopulation differences were also observed. Assuming that prey preference does not vary between populations, these differences are likely associated with a higher local prey availability. Finally, we suggest that DNA metabarcoding can be used for evaluating subtle differences in the diet of distinct populations of P. boliviensis, particularly when predation records in the field cannot be established or quantified using direct observation.     

Environmental DNA metabarcoding primers for freshwater fish detection and quantification: In silico and in tanks

Environmental DNA (eDNA) techniques refer to utilizing the organisms’ DNA extracted from environment samples to genetically identify target species without capturing actual organisms. eDNA metabarcoding via high‐throughput sequencing can simultaneously detect multiple fish species from a single water sample, which is a powerful tool for the qualitative detection and quantitative estimates of multiple fish species. However, sequence counts obtained from eDNA metabarcoding may be influenced by many factors, of which primer bias is one of the foremost causes of methodological error. The performance of 18 primer pairs for COI, cytb, 12S rRNA, and 16S rRNA mitochondrial genes, which are all frequently used in fish eDNA metabarcoding, were evaluated in the current study. The ribosomal gene markers performed better than the protein‐coding gene markers during in silico screening, resulting in higher taxonomic coverage and appropriate barcode lengths. Four primer pairs—AcMDB07, MiFish‐U, Ve16S1, and Ve16S3—designed for various regions of the 12S and 16S rRNA genes were screened for tank metabarcoding in a case study targeting six freshwater fish species. The four primer pairs were able to accurately detect all six species in different tanks, while only MiFish‐U, Ve16S1, and Ve16S3 revealed a significant positive relationship between species biomass and read count for the pooled tank data. The positive relationship could not be found in all species within the tanks. Additionally, primer efficiency differed depending on the species while primer preferential species varied in different fish assemblages. This case study supports the potential for eDNA metabarcoding to assess species diversity in natural ecosystems and provides an alternative strategy to evaluate the performance of candidate primers before application of eDNA metabarcoding in natural ecosystems.