Beyond Bioindicators:

Next-Generation Approaches to Environmental Biomonitoring

Jennifer Spall1, Shadi Shokralla1, Donald Baird2, Mehrdad Hajibabaei1 1Biodiversity Institute of Ontario, Department of Integrative Biology, University of Guelph, Guelph, Canada

2Environment Canada @ Canadian Rivers Institute, Department of Biology, University of New Brunswick, Fredericton, Canada

Workflow

Introduction

Sampling

Results

References Acknowledgements

Discussion

Bioindicator species such as benthic macroinvertebrates are commonly tracked to infer the health of ecosystems, but inaccurate and labour-intensive morphological identification is a bottleneck for implementing effective biomonitoring programs. The environmental barcoding approach using next-generation sequencing of the COI barcode region has proved effective for identification of macrobenthic organisms commonly used in biomonitoring. More recently, by selecting different marker genes and habitats to gain a comprehensive picture of biota, we are moving towards the development of biodiversity fingerprints from NGS data. This framework can be applied for ecosystem health assessment on an unprecedented scale both taxonomically and ecologically, compared to traditional low bandwidth biomonitoring approaches based on small numbers of bioindicator taxa. Here we employ this system to evaluate the health status of the Humber River, which flows through the heart of Canada's largest city, Toronto. We have collected benthic, water and soil samples for 13 sites along the river along an urbanization gradient moving from sparsely populated agricultural land to a dense urban setting. Data output will be compared to data collected by the local river conservation authority allowing us to develop a framework by which an accurate and consistent DNA-based environmental monitoring approach can be implemented for practical large-scale environmental assessment.

Morphological assessment is currently the most common method for identification and measurement of habitat biodiversity (Bonada et al., 2006). This approach is extremely time-consuming and tedious as well as highly inaccurate beyond the family level (Bonada et al., 2006). The development of molecular techniques such as DNA barcoding provided a much needed alternative to morphological identification and led to the establishment of large-scale projects such as the Barcode of Life (Marshall, 2005; Hajibabaei et al., 2007). However, traditional molecular approaches tackle specimens one-by-one and remain inadequate with regards to complex environmental samples such as larval benthic specimens. These samples often contain hundreds if not thousands of small individuals. Although Sanger sequencing has provided a stable method for the development of large sequence libraries such as DNA barcode reference libraries, the number of individuals in an environmental sample is beyond the scope of its ability (Hajibabaei et al., 2011). The advances in next generation sequencing technologies have provided the ability to read millions of DNA sequences in parallel, making them best suited for large-scale biodiversity analyses on environmental samples for biomonitoring applications. The sequencing firepower of NGS platforms allows for building DNA-based ecological footprints using a comprehensive biodiversity sampling unit. This involves habitats such as soil and water, transforming biodiversity analysis to an information-rich endeavour that can be implemented across space and time.

Bonada, N., Prat, N., Resh, V. H., & Statzner, B. (2006). Developments in aquatic insect biomonitoring: a comparative analysis of recent approaches. Annual review of entomology, 51, 495-523. Hajibabaei M., Singer G.A.C., Clare E.L., Hebert P.D.N. (2007). Design and Applicability of DNA Arrays and DNA Barcodes in Biodiversity Monitoring. BMC Biology, 5(24). Hajibabaei, M., Shokralla, S., Zhou, X., Singer, G.A.C., & Baird, D.J. (2011). Environmental Barcoding: A Next-Generation Sequencing Approach for Biomonitoring Applications Using River Benthos. PLoS ONE, 6, e17497. Marshall E. (2005). Will DNA Bar Codes Breathe Life Into Classification? Science, 307, 1037-1037.

N=878

Morphological Identification

Sanger DNA Barcoding and Environmental Barcoding of

Benthic Community

Sanger DNA Barcodes

454 E-Barcodes

Environmental Barcoding Profile of Benthic Community

jspall@uoguelph.ca

WATER

1L from approximately

midstream

200ml 800ml

Chemical Analysis

E-Barcode (COI, ITS, 16S, rbcL,

28S)

Filter, DNA Extraction

SOIL

4 samples from each of upstream,

midstream and downstream

3 samples 1 sample

DNA Extraction

Chemical Analysis

E-Barcode (COI, ITS, 16S,

rbcL, 28S)

BENTHOS

Kick-net transects

4/13 sites 13/13 sites

Morphological ID, DNA

Extraction from each

individual

Combine individuals,

one DNA Extraction

per Site

Sanger Sequence

each individual

(COI)

E-Barcode (COI)

Funding for this project has been generously provided by the Natural Sciences and Engineering Research Council of Canada, Environment Canada and Genome Canada through the Ontario Genomics Institute. We would like to thank the Toronto and Region Conservation Authority for their help navigating and sampling the Humber River. Thank you also to the Biodiversity Institute of Ontario and the Departments of Integrative Biology and Laboratory Services at the University of Guelph. This project would not be possible without help from all the members of the Hajibabaei lab or the dedicated lab work of Melissa Braschel.

Sample sites

Stream

Vegetation

Lakes

Saturated Soil

Transport

Industrial

Buildings and Structures

Legend