Research paperCan high-throughput sequencing detect macroinvertebrate diversity for routine monitoring of an urban river?
Introduction
Freshwater biological monitoring has traditionally been based on mostly family level identification of macroinvertebrates using morphological characteristics (Barbour et al., 1999, Rosenberg and Resh, 1993). Currently, well-established protocols exist in many countries to sample macroinvertebrate family biodiversity and compare it with environmental objectives set in policy (Barbour et al., 1992, Bonada et al., 2006, King and Richardson, 2003, Metcalfe, 1989, Metzeling et al., 2006). However, DNA technologies that allow rapid species-level identification can change how macroinvertebrate biodiversity is used to assess freshwater ecosystems (Baird and Hajibabaei, 2012, Carew et al., 2013, Hajibabaei et al., 2011, Jackson et al., 2014, Pfrender et al., 2010). DNA based approaches, like DNA barcoding, can recognise more species compared to morphological identification (Hebert et al., 2003), as morphologically cryptic, immature taxa or those that represent little known groups can easily be distinguished in samples (Pilgrim et al., 2011, Shackleton and Rees, 2015, Sweeney et al., 2011).
By identifying macroinvertebrate species, species-specific responses to pollutants and environmental characteristics can be used as bioindicators (Baird and Hajibabaei, 2012). This is especially useful when species within families vary in environmental responses (Bailey et al., 2001, Carew et al., 2011, Lenat and Resh, 2001, Resh and Unzicker, 1975). For example, Chironomidae species can vary greatly in their response to different environmental stressors, and show specific responses to certain pollutants (Carew et al., 2007, Marshall et al., 2010, Pettigrove and Hoffmann, 2005b, Sharley et al., 2008). By considering these groups only at higher levels of taxonomic resolution, valuable environmental signal is lost. There is greater sensitivity in distinguishing between impacting factors where species level identification using DNA barcoding are performed (Jackson et al., 2014, Sweeney et al., 2011). Species level identifications could provide new sensitive and diagnostic monitoring tools for environmental condition in freshwater systems.
While substantial effort and resources are needed to individually DNA barcode macroinvertebrate species, high-throughput DNA sequencing (HTS) transforms DNA barcoding into a cost-effective tool for routine identification (Baird and Hajibabaei, 2012, Carew et al., 2013, Stein et al., 2014). Bulk DNA extraction and simultaneous processing of multiple samples enables rapid and accurate detection of species. However, successfully identifying species detected with HTS relies on DNA barcoding libraries with comprehensive coverage of accurately identified species (Pfrender et al., 2010, Shackleton and Rees, 2015, Webb et al., 2012). When comprehensive DNA barcoding libraries are available, species detection using HTS and subsequent identification is relatively straightforward (Carew et al., 2013, Hajibabaei et al., 2011, Hajibabaei et al., 2012). However, it is more difficult when DNA barcoding libraries are incomplete, as PCR artefacts can be confused with new species if there are no DNA barcodes from voucher specimens available (Creer et al., 2010, Tedersoo et al., 2010). DNA barcoding libraries need to be developed alongside HTS to turn this approach into a practical tool (Ekrem et al., 2007, Vivien et al., 2015, Zimmermann et al., 2014).
A challenge in using HTS in routine assessment is potential bias in detecting taxa. In multi-family HTS analysis of invertebrate samples or ‘environmental barcoding’, around 90% of species in samples are detected (Gibson et al., 2014, Hajibabaei et al., 2011, Hajibabaei et al., 2012). However, some taxonomic groups might be overrepresented in the fraction of species not detected. Moreover, additional taxa can also be detected through HTS through amplification of egg masses, tissue fragments, gut content or environmental DNA present in invertebrate samples (Gibson et al., 2014, Hajibabaei et al., 2011). While taxa originating from these sources could be included in environmental assessments as they often reflect resident invertebrates, others might need to be excluded, such as tissues transported aerially into a site. These potential biases need to be examined by comparing the results of an HTS-based approach with those obtained using morphological approaches.
In this study, we test how HTS performs in routine assessment of sites along an urban river system. These systems are likely to benefit from species-level identification, as macroinvertebrate communities in urban environments can lack high family diversity and be dominated by a few speciose groups with diverse environmental responses, such as the Chironomidae and Oligochaeta (Beavan et al., 2001, Carew et al., 2007, Rosenberg, 1992, Vivien et al., 2015). Furthermore, factors responsible for deterioration of aquatic and riparian habitats in urban river systems can be difficult to identify because of multiple impacting factors, such as invasion by exotic species, changes to hydrology, and increased water temperatures, turbidity and contamination (Ellis and Hvitved-Jacobsen, 1996, Marsalek, 1998, Paul and Meyer, 2001, Pettigrove and Hoffmann, 2005b). If species-specific stressor responses could be included in assessments, they may assist in identifying the dominant factors responsible for environmental degradation.
We test HTS in routine assessments of environmental condition by first applying three sets of DNA barcoding primers to bulk-extracted macroinvertebrate samples, and examine their ability to detect species within morphologically identified families. We compare family diversity metrics calculated from morphologically- and HTS- derived data sets, and examine how HTS-derived data compares to existing protocols and objectives for routine assessment. We then explore species diversity within families and whether species level identification based on DNA barcodes provides additional information. Finally, we draw on a database of Chironomidae species distributions along environmental gradients to determine if species composition is diagnostic of pollution levels at sites.
Section snippets
Study location
The Maribyrnong River is located in the Greater Melbourne Area, Victoria, Australia (Fig. 1). It is ∼180 km long flowing south from the Great Dividing Range to the Yarra Estuary. The upper reaches are in rural areas, while areas surrounding the lower reaches are mostly urbanised with some industrialised areas. While the majority of riparian vegetation along the upper and middle reaches has been preserved, many tributaries are affected by urbanisation and development that has reduced riparian
Macroinvertebrate family diversity
Family level macroinvertebrate diversity was low in samples from the Maribyrnong River, Arundel Creek and Taylors Creek (Table 1). Morphological examination revealed a total of 38 families at the study sites. The highest family diversity (20 families) was found at the most upstream site (MAR1 SYD), while the lowest diversity (9 families) was found at the Taylors Creek site (TAY BP).
Bulk DNA extraction of these macroinvertebrate samples produced three amplicons for HTS (Table 2, Appendix C in
Discussion
High throughput DNA sequencing can change the way macroinvertebrates are used in routine biological monitoring by allowing rapid and cost-effective assessment of species biodiversity. Our study shows that species biodiversity in macroinvertebrate samples from urban rivers can be identified with DNA barcodes, which are readily detected by bulk sequencing of samples using HTS. This study supports the finding that HTS is a suitable method for assessing species biodiversity in macroinvertebrate
Conclusions
High throughput DNA sequencing technologies promise to improve biological monitoring by increasing speed and taxonomic resolution. The low cost of this approach will make this technology attractive to water managers, but continued efforts to refine HTS approaches and DNA barcoding of aquatic macroinvertebrates will be required.
Acknowledgements
The authors thank Katherine Jeppe, Daniel MacMahon and Cameron Amos for their assistance with collection of macroinvertebrates and sediments. We acknowledge Gavin Rose, Pei Zhang and Anh Duyen Bui for sediment and water pesticide analysis. We also thank Michael Shackleton, David Cartwright and John Dean for the use of their DNA barcode information. We also thank Rahul Rane for his advice in the laboratory and on data analysis, and Isabel Valenzuela for assistance with primer selection. This
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2021, Ecological IndicatorsCitation Excerpt :This does, however, come at the cost of increasing the number of undetected taxa due to primer bias and primer-template mismatches. While Carew et al. (2018) were able to recover 85% of families known to be in their samples, in the present study the average number of families recovered ranged from 70.3% to 84.3% in the BoBW dataset with a range from 38.1% to 100% of families known to occur in the samples were recovered. It should be noted that many of the species used in the present study have since been added to GenBank, and it is thus possible that these percentages will increase if analyses are performed on updated libraries.