Abstract
Metabarcoding of plant DNA recovered from environmental samples, termed environmental DNA (eDNA), has been used to detect invasive species, track biodiversity changes, and reconstruct past ecosystems. The P6 loop of the trnL intron is the most widely utilised gene region for metabarcoding plants due to the short fragment length and subsequent ease of recovery from degraded DNA, which is characteristic of environmental samples. However, the taxonomic resolution for this gene region is limited, often precluding species level identification. Additionally, targeting gene regions using universal primers can bias results as some taxa will amplify more effectively than others. To increase the ability of DNA metabarcoding to better resolve flowering plant species (angiosperms) within environmental samples, and reduce bias in amplification, we developed a multi-gene targeted capture method that simultaneously targets 20 chloroplast gene regions in a single assay across all flowering plant species. Using this approach, we effectively recovered multiple chloroplast gene regions for three species within artificial DNA mixtures down to 0.001 ng/μL of DNA. We tested the detection level of this approach, successfully recovering target genes for 10 flowering plant species. Finally, we applied this approach to sediment samples containing unknown compositions of eDNA and confidently detected plant species that were later verified with observation data. Targeting multiple chloroplast gene regions in environmental samples, enabled species-level information to be recovered from complex DNA mixtures. Thus, the method developed here, confers an improved level of data on community composition, which can be used to better understand flowering plant assemblages in environmental samples.
Highlights
Environmental DNA is a rapidly growing field of research and has been applied extensively to monitor site-based vegetation change over periods of hundreds to thousands of years using samples from soil cores (Willerslev et al, 2003, 2014; Parducci et al, 2013; Zimmermann et al, 2017; Del Carmen Gomez Cabrera et al, 2019)
There was no difference in target gene region recovery between the wider and restricted reference databases (P = 0.85), there was an interactive effect between species and concentration (P < 0.05) where the percentage of target regions recovered for Z. marina started decreasing at higher concentrations than the other two species
Six of the 10 species placed in the mixture were recovered at species level resolution (A. marina, Disphyma crassifolium, Frankenia pauciflora, Parapholis incurva, Samolus repens, and Wilsonia humilis) and the other 4 were recovered at genus level (Posidonia australis, Sarcocornia quinqueflora, Tecticornia halocnemoides, and Z. muelleri)
Summary
Environmental DNA (eDNA) is a rapidly growing field of research and has been applied extensively to monitor site-based vegetation change over periods of hundreds to thousands of years using samples from soil cores (Willerslev et al, 2003, 2014; Parducci et al, 2013; Zimmermann et al, 2017; Del Carmen Gomez Cabrera et al, 2019). The variable region most often used to recover plant DNA in environmental samples is the P6 loop of the chloroplast trnL (UAA) intron (Taberlet et al, 2006) This gene region was adopted due to being short enough to amplify DNA in environmental samples (10–143 bp), whilst still possessing enough discriminatory information to distinguish many groups of plant taxa. Primers can preferentially bind to certain taxa, creating an unreliable representation of the vegetation community and biasing results (Pedersen et al, 2015) Recovering this short section of the trnL gene region does not ensure resolution to species level (Lamb et al, 2016), especially when compared to other plant barcodes such as rbcL and matK (Hollingsworth et al, 2011; Fahner et al, 2016). These shortcomings highlight that a more effective approach is needed to fully utilise plant DNA from environmental samples and obtain detailed information on plant community composition
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