Hydrobiologia https://doi.org/10.1007/s10750-021-04576-z (0123456789().,-volV)(0123456789().,-volV) PRIMARY RESEARCH PAPER Metabarcoding meiofauna biodiversity assessment in four beaches of Northern Colombia: effects of sampling protocols and primer choice Lyda R. Castro . Rachel S. Meyer . Beth Shapiro . Sabrina Shirazi . Samuel Cutler . Ana M. Lagos . Sigmer Y. Quiroga Received: 31 July 2020 / Revised: 9 March 2021 / Accepted: 18 March 2021 Ó The Author(s), under exclusive licence to Springer Nature Switzerland AG 2021, corrected publication 2021 Abstract Environmental DNA (eDNA) metabar- metabarcodes and among sampling strategies, con- coding can enhance understanding of global biodiver- firming the sensitivity of the experiments to both sity by making it possible to study taxonomic groups parameters. Combining data from multiple barcodes that are difficult to sample. However, experimental and from multiple extraction protocols increased choices made when generating eDNA data can impact recovered meiofaunal taxonomic diversity. Future biodiversity surveys and must be carefully considered metabarcoding studies and meta-analyses should con- during study design. Here, we explored the impact of sider the effects of sampling protocols on biodiversity. DNA extraction protocol and metabarcode choice on Our results also highlight the need to continue to recovery of meiofauna DNA from sand. We extracted improve existing reference databases of morphologi- DNA from untreated sand and from sand treated with cal and molecular characterization of meiofauna, in either Ludox or MgCl2 and amplified DNA using the particular of the tropics, which are poorly represented 18S and CO1 metabarcodes. We found differences in in existing databases. species composition and richness both between Keywords DNA extraction Á Coastal biodiversity Á 18S Á CO1 Á Methodology Á Environmental DNA Handling editor: Ce´cile Fauvelot metabarcoding Á Biomonitoring Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ s10750-021-04576-z. L. R. Castro (&) A. M. Lagos Á S. Y. Quiroga Grupo de investigacio´n Evolucio´n, Sistema´tica y Ecologı´a Grupo de Investigacio´n en Manejo y Conservacio´nde Molecular, Universidad del Magdalena, Carrera 32 # Fauna, Flora y Ecosistemas Estrate´gicos Neotropicales 22-08, Santa Marta DTCH, Colombia MIKU, Universidad del Magdalena, Carrera32 # 22-08, e-mail: [email protected] Hangar B, Santa Marta DTCH, Colombia R. S. Meyer Á B. Shapiro Á S. Shirazi Á S. Cutler Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, USA B. Shapiro Howard Hughes Medical Institute, University of California Santa Cruz, Santa Cruz, CA 95064, USA 123 Hydrobiologia Introduction understand what biases emerge when cataloging meiofaunal diversity from choice of genetic locus, The meiofauna include all benthic animals and field collection and DNA extraction protocols, protozoa whose sizes range between macro- and metabarcoding library preparation assays, and bioin- microfauna. Meiofauna are defined as organisms that formatic approaches, all of which can impact results pass through a net with a mesh size of 500–1000 lm (Deiner et al., 2015; Clare et al., 2016; Leasi et al., and are held back by nets of 20–63 lm mesh size 2018; Fais et al., 2020). (Fenchel, 1978; Higgins & Thiel, 1988; Giere, 2009; DNA marker choice is a known challenge in Ptatscheck et al., 2020). Meiofauna are essential metabarcoding studies. The CO1 gene has been used members of the marine benthos, as they are basal in routinely in both barcoding and metabarcoding studies the trophic network and contribute to bioturbation of animal species (Hebert et al., 2003). Degenerate through recycling of nutrients and activation of primers aimed to amplify short fragments have been geochemical cycles (Bonaglia et al., 2014). This basal developed to broaden their taxonomic application and position and biological characteristics including high to optimize the amplification of degraded DNA from reproduction rates, short generation times, and the the environment (Vamos et al., 2017). In particular, absence of pelagic larval dispersion for some groups, primers developed by Leray (2013) target a 313 bp makes these organisms highly sensitive to environ- fragment that is commonly used in marine metazoan mental changes (Bongers & Ferris, 1999; Giere, metabarcoding, as is the combination mlCOIintF ? 2009), and therefore important data sources for jgHCO2198 primers developed by Geller et al. environmental impact and monitoring studies (Ken- (2013). Lobo (2013) developed degenerate primers nedy & Jacoby, 1999; Moreno et al., 2008; Zeppilli for the same region, and the combination ´ et al., 2015; Martınez et al., 2015). mlCOIintF ? LoboR1 was tested and recommended Many studies have attempted to estimate meioben- by Haenel et al. (2017) and Fais et al. (2020) for thic biodiversity worldwide, of which the vast major- meiofaunal metabarcoding assays. This combination ity focus on identifying species among the dominant was preferred by Chang et al. (2020) based on costs. meiofaunal groups using morpho-taxonomy (Ap- The 18S nuclear gene is also commonly used in peltans et al., 2012). Despite this work to characterize metabarcoding studies of marine metazoans, in par- the marine meiobenthos through traditional taxonomy, ticular the 18S V1-V2 region or the 18S V9 region several challenges remain, including the lack of (Van der Loos & Nijland, 2020), but has been specialist taxonomists and the fact that diagnostic suggested to have less power than CO1 to discriminate morphological features of these organisms are often species (Tang et al. 2012, Creer et al., 2010; Leray & lost after methodological treatment (Zeppilli et al., Knowlton, 2016). However, Fais et al. (2020) detected 2015). These challenges lead to potentially biased similar richness from CO1 and 18S, but different estimates of the richness and distribution of marine meiofauna composition in the assays. Because mark- benthos species. DNA metabarcoding approaches, in ers will often amplify different species, combinations which ‘‘barcode’’ primers are used to amplify all of two or more markers are recommended to result in species present in an environmental DNA (eDNA) more accurate estimates of species richness and sample that match the primer sequences, offer a composition (Alberdi et al., 2017). complementary approach to cataloging meiofaunal Experimental design can also impact what taxa are species of the marine benthos using morphology. recovered from environmental DNA samples. Many eDNA metabarcoding can be more efficient in meiofaunal studies undertake a preprocessing step estimating species richness than the morphotaxonomic prior to DNA extraction, which is thought to improve approach (Lejzerowicz et al., 2015), and can comple- DNA recovery of target taxa (Brannock et al., 2015). ment and expand existing approaches to assess overall The most commonly used preprocessing steps are the biodiversity (Fonseca et al., 2017). same as those used prior morphological determination While studies of meiofaunal communities using of meiofauna (Montagna et al., 2017; Vand der Loos & metabarcoding have shown the benefits of the tech- Nijland, 2020), and include separation via decanta- nique for this particular group, many challenges tion/flotation with either MgCl2 (Leasi et al., 2018)or remain. In particular, it would be useful to better Ludox solution (Fonseca et al., 2017; Faria et al., 123 Hydrobiologia 2018). Preprocessing is believed to facilitate recovery sand by washing the sample with isotonic MgCl2 of largely cellular DNA (Creer et al., 2010). Some solution and decanted by hand through 500 lm and studies extract DNA directly from sand or water 45 lm sieves before preserving it in a conical tube without preprocessing so as to specifically target with absolute ethanol. For pre-treatment no. 2, meio- extracellular DNA (Guardiola et al., 2015; Pearman fauna was floated with Ludox TM 50 (specific density et al., 2016), whereas other marine sediment studies 1.15 g/cm3, De Jonge & Bouwman, 1977), captured focus on total DNA (Lejzerowicz et al., 2015; on a 45 lm mesh sieve, washed with distilled H2O, Nascimento et al., 2018; Fais et al., 2020). Hajibabaei and stored again with absolute ethanol at - 20°C. et al. (2019) proposed that direct extraction of total Additionally, non-treated sand was preserved in DNA without pre-treatment reduces the probability of absolute ethanol for comparison to the pre-treatments. identifying many key species, such as benthic bio- All samples were centrifuged to eliminate the ethanol indicator taxa. Better understanding of the impact of and well homogenized using a mortar and a pestle preprocessing on taxonomic recovery will be useful, in before DNA extraction. particular as eDNA studies are increasingly used to target large diversity assemblages for ecological DNA extraction and metabarcoding analyses (Hermans et al., 2018). In this study we used eDNA metabarcoding to We performed DNA extractions using the Qiagen estimate intertidal meiofaunal biodiversity present in DNeasy PowerSoil Kit (Qiagen, Germantown, Mary- four sandy beaches of Northern Colombia. We land, USA) following the manufacturer’s protocol, performed DNA extraction from samples treated using which has been recommended in other meiofauna and two common pre-treatment protocols for meiofaunal metabarcoding studies using marine sediments (Ather- separation (MgCl2 and Ludox)