The host-microbiome community is influenced by several host and environmental factors. In order to disentangle the individual effects of host and environment, we performed a laboratory experiment to ...assess the effects of the exposure to different water sources on the skin and gut microbiome of two amphibian species (Pelophylax perezi and Bufo spinosus). We observed that the bacterial communities greatly varied with water environment and host identity. Tadpoles of B. spinosus collected from a waterbody with poorer bacterial diversity exhibited a more diverse skin and gut microbiome after exposed to a richer water source. Tadpoles of P. perezi, originally collected from a richer water environment, exhibited less marked alterations in diversity patterns independently of the water source but showed alterations in gut composition. These results highlight that environment alterations, such as the water source, combined with the host effect, impact the microbiome of amphibian species in different ways; the population history (e.g., previous water environment and habitat) of the host species may also influence future alterations on tadpole microbiome.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Analysis of intra- and inter-population diversity has become important for defining the genetic status and distribution patterns of a species and a powerful tool for conservation programs, as high ...levels of inbreeding could lead into whole population extinction in few generations. Microsatellites (SSR) are commonly used in population studies but discovering highly variable regions across species' genomes requires demanding computation and laboratorial optimization. In this work, we combine next generation sequencing (NGS) with automatic computing to develop a genomic-oriented tool for characterizing SSRs at the population level. Herein, we describe a new Python pipeline, named Micro-Primers, designed to identify, and design PCR primers for amplification of SSR loci from a multi-individual microsatellite library. By combining commonly used programs for data cleaning and microsatellite mining, this pipeline easily generates, from a fastq file produced by high-throughput sequencing, standard information about the selected microsatellite loci, including the number of alleles in the population subset, and the melting temperature and respective PCR product of each primer set. Additionally, potential polymorphic loci can be identified based on the allele ranges observed in the population, to easily guide the selection of optimal markers for the species. Experimental results show that Micro-Primers significantly reduces processing time in comparison to manual analysis while keeping the same quality of the results. The elapsed times at each step can be longer depending on the number of sequences to analyze and, if not assisted, the selection of polymorphic loci from multiple individuals can represent a major bottleneck in population studies.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
DNA metabarcoding from the ethanol used to store macroinvertebrate bulk samples is a convenient methodological option in molecular biodiversity assessment and biomonitoring of aquatic ecosystems, as ...it preserves specimens and reduces problems associated with sample sorting. However, this method may be affected by errors and biases, which need to be thoroughly quantified before it can be mainstreamed into biomonitoring programmes. Here, we used 80 unsorted macroinvertebrate samples collected in Portugal under a Water Framework Directive monitoring programme, to compare community diversity and taxonomic composition metrics estimated through morphotaxonomy versus metabarcoding from storage ethanol using three markers (COI‐M19BR2, 16S‐Inse01 and 18S‐Euka02) and a multimarker approach. A preliminary in silico analysis showed that the three markers were adequate for the target taxa, with detection failures related primarily to the lack of adequate barcodes in public databases. Metabarcoding of ethanol samples retrieved far less taxa per site (alpha diversity) than morphotaxonomy, albeit with smaller differences for COI‐M19BR2 and the multimarker approach, while estimates of taxa turnover (beta diversity) among sites were similar across methods. Using generalized linear mixed models, we found that after controlling for differences in read coverage across samples, the probability of detection of a taxon was positively related to its proportional abundance, and negatively so to the presence of heavily sclerotized exoskeleton (e.g., Coleoptera). Overall, using our experimental protocol with different template dilutions, the COI marker showed the best performance, but we recommend the use of a multimarker approach to detect a wider range of taxa in freshwater macroinvertebrate samples. Further methodological development and optimization efforts are needed to reduce biases associated with body armouring and rarity in some macroinvertebrate taxa.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still ...required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Winter savory Satureja montana is a medicinal herb used in traditional gastronomy for seasoning meats and salads. This study reports a comparison between conventional (hydrodistillation, HD, and ...Soxhlet extraction, SE) and alternative (supercritical fluid extraction, SFE) extraction methods to assess the best option to obtain bioactive compounds. Two different types of extracts were tested, the volatile (SFE-90 bar, second separator vs HD) and the nonvolatile fractions (SFE-250 bar, first and second separator vs SE). The inhibitory activity over acetyl- and butyrylcholinesterase by S. montana extracts was assessed as a potential indicator for the control of Alzheimer’s disease. The supercritical nonvolatile fractions, which showed the highest content of (+)-catechin, chlorogenic, vanillic, and protocatechuic acids, also inhibited selectively and significantly butyrylcholinesterase, whereas the nonvolatile conventional extract did not affect this enzyme. Microbial susceptibility tests revealed the great potential of S. montana volatile supercritical fluid extract for the growth control and inactivation of Bacillus subtilis and Bacillus cereus, showing some activity against Botrytis spp. and Pyricularia oryzae. Although some studies were carried out on S. montana, the phytochemical analysis together with the biological properties, namely, the anticholinesterase and antimicrobial activities of the plant nonvolatile and volatile supercritical fluid extracts, are described herein for the first time.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring ...methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates.
In the present context of the ramping up of the global biodiversity crisis, improving our understanding on the genetic and biogeographic patterns of ill-known taxa is central to conservation ...planning. This is especially relevant for geographically isolated populations that suffer from little or no gene flow and an increased extinction risk. The few studies available on the molecular phylogeny and phylogeographic diversity of Frigatebirds have only focused on populations of three species,
Fregata magnificens
,
F. andrewsi
from Christmas Islands and
F. minor
in the Galapagos. To improve our knowledge on these tropical seabirds, our study aimed at assessing worldwide phylogeographic patterns and relationships among all five extant species of the genus
Fregata
. To accomplish it, we sampled museum specimens corresponding to 18 frigatebird populations spatially distributed by Brazil, Mexico, Ascension Islands, Cabo Verde and the Indo-Pacific region, and fresh samples from Cabo Verde, and amplified them for a mtDNA cytochrome b fragment. We complemented our dataset with previously available data representing a total of 36 populations in this study. Similar to the well-known endemic populations of the Galapagos and Christmas Island, the isolated ultraperipheral populations in the Atlantic were shown to be genetically divergent from their main populations for the three widespread species,
F. magnificens
,
F. ariel
and
F. minor
. We provide the first genetic data for
F. ariel
, whilst building upon the existing knowledge of the genetic patterns of
F. magnificens
,
F. aquila
and
F. minor
. Furthermore, our molecular data comes in support of most but not all the morphologically recognized frigatebird subspecies. This study provides important genetic insights into the evolutionary history of the genus
Fregata
and acts as a baseline for future molecular work and conservation efforts.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Freshwater macroinvertebrates provide valuable indicators for biomonitoring ecosystem change in relation to natural and anthropogenic drivers. DNA metabarcoding is an efficient approach for ...estimating such indicators, but its results may differ from morphotaxonomic approaches traditionally used in biomonitoring. Here we test the hypothesis that despite differences in the number and identity of taxa recorded, both approaches may retrieve comparable patterns of community change, and detect similar ecological gradients influencing such changes. We compared results obtained with morphological identification at family level of macroinvertebrates collected at 80 streams under a Water Framework Directive biomonitoring program in Portugal, with results obtained with metabarcoding from the ethanol preserving the bulk samples, using either single (COI-M19BR2, 16S-Inse01, 18S-Euka02) or multiple markers. Metabarcoding recorded less families and different communities compared to morphotaxonomy, but community sensitivities to disturbance estimated with the IASPT index were more similar across approaches. Spatial variation in local community metrics and the factors influencing such variation were significantly correlated between morphotaxonomy and metabarcoding. After reducing random noise in the dissimilarity matrices, the spatial variation in community composition was also significantly correlated across methods. A dominant gradient of community change was consistently retrieved, and all methods identified a largely similar set of anthropogenic stressors strongly influencing such gradient. Overall, results confirm our initial hypothesis, suggesting that morphotaxonomy and metabarcoding can estimate consistent spatial patterns of community variation and their main drivers. These results are encouraging for macroinvertebrate biomonitoring using metabarcoding approaches, suggesting that they can be intercalibrated with morphotaxonomic approaches to recover equivalent spatial and temporal gradients of ecological change.
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•We described freshwater macroinvertebrate communities using morphotaxonomy and metabarcoding.•Local community metrics differed significantly between morphotaxonomy and metabarcoding.•Patterns of spatial variation in local community metrics were correlated between approaches.•Dominant community gradients and their ecological drivers were consistently estimated.•Morphotaxonomy and metabarcoding can be intercalibrated to detect equivalent ecological changes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract The Corubal (Guinea-Bissau) is a wild but underexplored river in West Africa. This study underscores the potential of environmental DNA (eDNA) surveys to fill biodiversity knowledge gaps in ...the region. We filtered large water volumes at 11 sites along the watershed, amplified multiple molecular markers, and performed high PCR (polymerase chain reaction) replication and in-depth sequencing. We recorded 2589 amplicon sequence variants, with accumulation curves indicating the need for additional sampling to achieve a thorough survey. The taxonomic assignments were constrained by the scarcity of genomic resources. We recorded 125 species of aquatic and terrestrial vertebrates, including 21 new to the country, predominantly fish (61.9%). Surprisingly, crocodiles were not detected, despite their known presence. There were 11 imperiled species, two of which are Critically Endangered (Western chimpanzee and the mussel Pleiodon ovatus). Our findings support the conservation importance of the Corubal, provide a baseline for future monitoring, and highlight the challenges and opportunities of eDNA surveys in remote tropical rivers.
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