Orientador(es)
Resumo(s)
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.
Descrição
Palavras-chave
Benthic macroinvertebrates DNA extraction DNA metabarcoding Freshwater bioassessment Preservative ethanol Water framework directive
Contexto Educativo
Citação
Martins, Filipa M.S.; Galhardo, Mafalda; Filipe, Ana F.; Teixeira, Amílcar; Pinheiro, Paulo; Paupério, Joana; Alves, Paulo C.; Beja, Pedro (2019). Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring. Molecular Ecology Resources. ISSN 1755-098X. 19, p. 863-876