The rare Charitable Research Reserve is a 365+ hectare land reserve which was set aside in 2001 to preserve the cultural history and ecological integrity of the area, while providing opportunities for scientific research and public education within the context of an urbanized region. It is located at the confluence of the Speed and Grand Rivers in Cambridge, Ontario, Canada (43.381128, -80.357807), where the Carolinian and Northern Hardwood forests also meet. The reserve contains a diversity of habitats including existing and reclaimed agricultural lands, wetlands, floodplains, shrub thickets, limestone cliffs and alvars, cold-water creeks, and old growth forest. Due to these diverse habitats, as well as the organization’s mandate to facilitate scientific research, rare has been the site of a variety of innovative research studies, including studies on fern genetics (Henry et al. 2014), prairie community establishment (Harvey and MacDougall 2014), and pollination services (Woodcock et al. 2014). For the present study, we chose sampling sites to encompass a wide range of habitats within the area. In advance of the bioblitz, sampling was conducted within an alvar, reclaimed agricultural fields, forest edges, and a wetland (Fig. 1). Six additional sites were sampled for the bioblitz: terrestrial and aquatic sampling was performed at the Grand River, Blair Flats Wetland, and Cruickston Creek; terrestrial sampling was conducted at an alvar, a cedar stand, and a silver maple wetland (Fig. 1​​). The wetlands on the reserve are part of the Barrie's Lake Bauman Creek Wetland Complex, which has been classified as a Provincially Significant Wetland by the Ontario Ministry of Natural Resources. Much of the property is also considered locally significant by the Regional Municipality of Waterloo.

Figure 1.  

Map indicating habitat types and sampling sites for the 2015 biotic survey conducted at rare Charitable Research Reserve in Cambridge, Ontario, Canada.

Prior to this study, 2,246 species had been recorded at rare, including birds (231), mammals (37), insects (832), plants (836), mosses (63) and lichens (21) (Suppl. material 1​​​; note that 218 species still require confirmation, denoted by parentheses). These observations have come from a variety of sources including rare staff and advisors, citizen scientists, and academic researchers, some of whom have made notable contributions to this inventory, such as Woodcock et al. 2014 who added 150 species of pollinators. Others have provided observations of rarely encountered species, such as the rove beetles Xantholinus elegans (Olivier, 1795) (first record for North America) and Xantholinus linearis (Olivier, 1794) (previously known from the east coast of Canada, now present in Ontario) (Brunke and Majka 2010). The rare reserve also hosts several species that fall on the provincial or national lists of conservation concern: 1% of the total have a status ranging from special concern, threatened or endangered (15, 6 and 7 species, respectively), while 1.5% have an undefined status of rare or uncommon (15 and 19 species, respectively). Several of these species, such as the barn swallow [Hirundo rustica (Linnaeus, 1758)], which has been nationally listed as threatened since 2011, are the subjects of active recovery projects at rare. Similar to many species inventories, rare's list is evidently more complete for charismatic and well-studied taxa, such as birds, butterflies and vascular plants. The observations for these groups would be enabled by excellent field guides and the disproportionate interest of citizen scientists; the opposite would be the case for most invertebrate taxa, and rare's inventory reflects this. If it is assumed that the proportions of major taxonomic groups inhabiting this reserve resemble those for similar temperate sites (e.g., Great Smoky Mountains: Sharkey 2001) or the Canadian terrestrial and freshwater biota in general (Mosquin et al. 1995), this inventory is deficient in several major groups such as insects, arachnids, nematodes, fungi and lichens. The supporting data for most species entries are also lacking, such as specific locality, date or identifier of each observation. In addition, the bioblitz has highlighted errors in the inventory which may have been present due to the compilation of observations from a variety of sources including non-experts. Furthermore, nearly all observations are not supported by voucher specimens or images to permit verification (Bortolus 2008).

Two strategies were employed in an effort to maximize the diversity of organisms inventoried. The first was a comprehensive collecting scheme executed over a period of approximately four months (May to August 2015). It involved a variety of targeted taxa and techniques, but heavily favoured the collection of terrestrial arthropods by passive trapping. Four Malaise traps were set up in various habitats around the rare property (Fig. 1) and were serviced weekly. Three pitfall traps were set up in close proximity to each Malaise trap and serviced in parallel. From May 25-31 and July 6-12, 2015, 'standardized sampling' procedures developed by the Biodiversity Institute of Ontario were employed at three different sites at rare (Fig. 1). Standardized sampling includes 20 pitfall traps, 10 pan traps, three litter and/or soil samples for Berlese funnels, one flight intercept trap and one Malaise trap – all deployed for a total of seven days. Standardized sampling also includes 60 total minutes of sweep netting, which is performed in three sessions with four collectors sweep netting for five consecutive minutes simultaneously. Each session is pooled into one sample and preserved in 95% ethanol. Passive traps (pitfall, pan, intercept) were deployed with soapy water and serviced every two days, while the Malaise trap used 95% ethanol and ran for the duration of the collecting period. All specimens collected through this standardized sampling routine were consolidated into individual jars for each technique. From July 23 to Aug 13, 2015, at least two ultraviolet light traps were deployed for one night per week; each trap was left overnight using ethyl acetate as a killing agent. On August 5, 2015, several aquatic samples were collected using stable dip nets for surber sampling, deploying hand nets along the stream banks and selective turning of stones to recover macroinvertebrates hiding below. Then the sample was passed through a 50 µm mesh net to capture the smaller invertebrates. All specimens collected from these samples were preserved in 95% ethanol. This first strategy of comprehensive sampling over several months was conducted by technical staff, including undergraduate students, and completed on Aug 15, 2015.

The second strategy for surveying the reserve was a more concentrated effort and involved taxonomic experts – the execution of a bioblitz (Lundmark 2003). Termed the 'rare BioBlitz', the event involved 113 participants from 31 institutions and coincided with the 6th International Barcode of Life Conference (Adamowicz 2015​). Collection efforts at the bioblitz were focused on six sites selected based on habitat diversity, proximity, and potential species diversity (Fig. 1​​). Sampling efforts were concentrated between 1-3 pm and 9-11 pm and targeted taxa that fell within the expertise of the participants and in groups underrepresented in the rare inventory (e.g., spiders, parasitic wasps, mites, nematodes, fungi, and lichens). Various methods for collecting both terrestrial and aquatic arthropods were employed, including dip nets, seine nets, sweep netting, and plankton netting. Small teams surveyed the property and adjacent rivers for taxonomic groups not collected in the four previous months, including fungi, lichens, and vascular plants. Since the inventory is fairly complete for vascular plants (836 species), an effort was made to barcode all species of Blair Flats, a tall grass prairie site that is an active research site (e.g., Harvey and MacDougall 2014​). For these taxa, specimens were collected in the field into bags and processed afterward (e.g., pressed and dried onto herbarium sheets). Fungal specimens were collected and processed in a similar manner. Several taxa were targeted for sightings only, where no voucher specimens were collected; these groups included fish, birds, bats, herptiles and odonates (for bat sightings protocol, see Suppl. material 2​). All collected specimens were sorted and identified to the lowest taxonomic level possible by the appropriate expert, both on site and following the event from August 16-20, 2015. Vertebrates, plants, fungi and lichens were almost all identified to species, while most invertebrate specimens were assigned to order or family prior to analysis. Invertebrate specimens were either stored in 95% ethanol or pinned after collection. For all invertebrate, plant, and fungal taxa, voucher specimens were collected where possible and deposited in the Biodiversity Institute of Ontario's natural history collection (BIOUG) or herbarium (BIO-OAC) for permanent storage (Schilthuizen et al. 2015).

Both surveying strategies provided a large number of specimens that were sorted and prepared for subsequent DNA barcode analyses at the Canadian Centre for DNA barcoding (CCDB; www.ccdb.ca). A total of 25,287 specimens were sequenced from collection efforts from May to August 2015, followed by 3,502 specimens directly following the rare BioBlitz on Aug 16, 2015. Tissue samples were prepared in 96-well plate format and when necessary, the whole specimen proceeded through lysis and was recovered as voucher from the filter plate (Porco et al. 2010​). Tissue lysis and DNA extraction varied slightly for different taxa (Suppl. material 3), but followed standard CCDB procedures (Ivanova et al. 2006, deWaard et al. 2008, Ivanova et al. 2008, Ivanova et al. 2011, Fazekas et al. 2012​).

One or more standard DNA barcode markers were targeted for each major group of organisms: for animals, the mitochondrial gene cytochrome oxidase subunit 1 (COI) (Hebert et al. 2003); for plants, the plastid marker rbcL (Hollingsworth et al. 2009) and the nuclear ribosomal internal transcribed spacer 2 (ITS2) marker (Hollingsworth 2011, Schoch et al. 2012); and for fungi and lichens (where only the fungal component was targeted), the internal transcribed spacer (ITS) marker (Schoch et al. 2012). PCR amplification, cycle sequencing and sequence analysis followed typical CCDB protocols (Ivanova and Grainger 2006, Kuzmina and Ivanova 2011). The primer cocktails used for PCR and sequencing are detailed in Suppl. material 3​. The sequences were manually assembled and edited before upload to in the Barcode of Life Data Systems (BOLD, www.boldsystems.org) (Ratnasingham and Hebert 2007​). The DNA extracts for all specimens are stored in the DNA Archive of the CCDB where they are available for additional study.

For the sequences derived from animal specimens, the records were assigned operational taxonomic units (OTUs) called Barcode Index Numbers (BINs) by the Refined Single Linkage (RESL) algorithm implemented on BOLD (Ratnasingham and Hebert 2013). For the sequences that have at least 500 bp coverage of the barcode region, < 1% ambiguous bases, and no stop codon or contamination flags, the RESL algorithm calculates the number of clusters and their membership (see Ratnasingham and Hebert 2013​). The RESL algorithm runs weekly on all qualifying barcode sequences in BOLD, which as of August 2015, includes 5M specimens and 420K BINs. The BIN system is accessible through public, individual ‘BIN pages’ and permits rapid diversity assessments, even in the absence of taxonomic information. BINs show a high concordance with traditional taxonomic species names and can be used as a reliable proxy for species.

For each specimen that was assigned an existing BIN, the record received the existing identification of the BIN to the lowest level that did not have taxonomic conflict. For each specimen assigned a new BIN for BOLD, the sequence was queried through the BOLD Identification Engine (BOLD-ID Engine; http://www.boldsystems.org/index.php/IDS_OpenIdEngine). Identifications were applied based on sequence similarity (<15% for family, <5% for genus) if the query sequence fell within a monophyletic cluster of BINs assigned to this family or genus. For animal records that did not receive BINs (<500bp), the sequence was similarly queried through the BOLD-ID Engine, but used a <2% similarity cutoff for assignment to species, in addition to the genus and family thresholds. Following this, a neighbour-joining tree was constructed and examined for unexpected placements which might indicate overlooked contamination events or analytical error. Finally, specimens and images were inspected morphologically to check for errors and refine the assigned taxonomy where possible.