The Trichoptera (caddisflies) represent one of the major orders of aquatic insects. Combined with Ephemeroptera (mayflies) and Plecoptera (stoneflies), caddisflies are routinely used in the assessment of ecosystem health (Lenat Reference Lenat1988; Sheffield et al. Reference Sheffield, Dewaard, Morse and Rasmussen2019). With the acceleration of anthropogenic climate change, assessing biodiversity is an increasing priority (Parmesan Reference Parmesan2006).

Protoptila coloma Ross (Trichoptera: Glossosomatidae) was first described in the mid-twentieth century (Ross Reference Ross1941). The adult male of this species is only 3 mm long. It belongs to the Protoptilinae (Ross Reference Ross1963), which is the only subfamily of the Glossosomatidae found in the Neotropics (Robertson and Holzenthal Reference Robertson and Holzenthal2013). The distribution of Protoptila spp. ranges from Canada to South America. Currently, only four species of Protoptila are recorded in Canada, of which only P. tenebrosa is previously known from British Columbia, Canada. However, the genus Protoptila has been recorded to have much greater diversity in the Neotropics (Wiggins Reference Wiggins1996; Blahnik et al. Reference Blahnik, Aguirre and Armitage2023). Protoptila coloma, which is closely related to P. tenebrosa (Ross Reference Ross1941), has been previously recorded in California, Colorado, Idaho, Montana, Nevada, Oregon, Utah, Washington, and Wyoming, United States of America (Ross Reference Ross1941; Zack et al. Reference Zack, Ruiter, Strenge and Landolt2006; Ruiter et al. Reference Ruiter, Baumann and Flint2014). More generally, Sheffield et al. (Reference Sheffield, Dewaard, Morse and Rasmussen2019) estimate a further 8–15 species of Glossosomatidae remain unrecorded in Canada.

The use of DNA barcoding is a well-established approach for identifying insects, often to the species level, and allows for the rapid assessment of biodiversity (Hebert et al. Reference Hebert, Cywinska, Ball and DeWaard2003; Zhou et al. Reference Zhou, Adamowicz, Jacobus, DeWalt and Hebert2009, Reference Zhou, Jacobus, DeWalt, Adamowicz and Hebert2010). The Trichoptera have been extensively identified through DNA barcoding in combination with morphology-based taxonomy (Zhou et al. Reference Zhou, Adamowicz, Jacobus, DeWalt and Hebert2009, Reference Zhou, Robinson, Geraci, Parker, Flint and Etnier2011; Ruiter et al. Reference Ruiter, Boyle and Zhou2013), which makes this group particularly amenable to the barcoding methodology. Generally, a 2% difference in the cytochrome c oxidase subunit 1 mitochondrial gene (CO1) sequence among caddisflies supports the separation of species within the order. The Barcode of Life Database (BOLD) is routinely used for such assessments (Zhou et al. Reference Zhou, Adamowicz, Jacobus, DeWalt and Hebert2009; Ruiter et al. Reference Ruiter, Boyle and Zhou2013; Ratnasingham and Hebert Reference Ratnasingham and Hebert2007, Reference Ratnasingham and Hebert2013).

The Stellako River, located west of Prince George, British Columbia is in the traditional territory of the Stellat’en First Nation, and it connects Francois Lake (Neda Bun) to Fraser Lake (Nee Tai Bun). The river is ecologically and culturally important and has been impacted by past economic activities (Rajala Reference Rajala2010). Every fall, sockeye salmon migrate into the Stellako to spawn, where they have been harvested by the Stellat’en First Nation for thousands of years (www.stellaten.ca). The Stellako River sockeye population is closely monitored by both the Stellat’en First Nation and Fisheries and Oceans Canada. In addition, the Stellako River and Stellako Lodge on its banks are world-renowned among fly anglers for the rainbow trout that inhabit the river year-round. With the Stellako being a culturally and economically important river, the rainbow trout population is closely monitored and studied by the BC Ministry of Water, Land and Resource Stewardship (Hagen et al. Reference Hagen, Spendlow, Pillipow and Gantner2020).

Juvenile sockeye salmon and rainbow trout in the Stellako rely on aquatic insects as a food source. However, no work has been done on the biodiversity of the aquatic insects in the river. A thorough understanding of the river’s macroinvertebrate community is important for informing the substantial and longstanding conservation management efforts along its course (Rajala Reference Rajala2010; Sanderson et al. Reference Sanderson, Picketts, Déry, Fell, Baker, Lee-Johnson and Auger2015).

We captured insects using SLAM Malaise traps (BugDorm BT1004; MegaView Science Co. Ltd., Taichung, Taiwan) hung from trees along the edge of the Stellako River from May 2023 until September 2023. Malaise traps were set up at the level of the high-water mark. Trap bottles contained 95% (v/v) ethanol, and traps were set at three locations: Glenannan Bridge, 54° 0′ 33.86″ N, 125° 0′ 15.83″ W; Cabin Pool, 54° 0′ 40.27″ N, 124° 59′ 47.40″ W; and Millionaires Pool, 54° 1′ 5.99″ N, 124° 58′ 7.44″ W. Bottles were collected every two weeks. The specimens were sorted based on morphology, and several individuals of each morphotype were submitted for sequencing by the Canadian Centre for DNA Barcoding at the University of Guelph (Guelph, Ontario, Canada). The DNA barcode sequences can be accessed in a BOLD data set (dx.doi.org/10.5883/DS-STELLAKO), and specimens were vouchered at the Royal BC Museum, Victoria, British Columbia.

To determine distribution of P. coloma, the following databases were consulted: www.trichoptera.org, https://trichopt.app.clemson.edu/welcome.php, BOLD (http://www.boldsystems.org); Electronic Atlas of the Wildlife of British Columbia (http://ibis.geog.ubc.ca/biodiversity/efauna/); Natureserve (http://www.natureserve.org/); Canadensys (http://www.canadensys.net/), Global Biodiversity Information Facility (http://www.gbif.org/); and the Royal BC Museum collections (http://search-collections.royalbcmuseum.bc.ca/Entomology). In addition, the following primary sources were also consulted: Ross Reference Ross1963; Nimmo Reference Nimmo1974, Reference Nimmo1977; Schmid Reference Schmid1982; Wymer and Morse Reference Wymer and Morse2000; Robertson and Holzenthal Reference Robertson and Holzenthal2013; and Genco and Morse Reference Genco and Morse2017.

Initial species identification was based on the 650-bp sequence in the CO1 5′ region using the bioinformatic tools within BOLD, followed by phylogenetic analysis using Molecular Evolutionary Genetics Analysis (MEGA), version 11.0, software to set up neighbour-joining trees based on the Kimura-2 model (Tamura et al. Reference Tamura, Stecher and Kumar2021; Supplementary material, data 1–4). To place our specimens’ DNA sequences in context, publicly available DNA sequences were obtained from BOLD for P. coloma and P. tenebrosa of specimens with barcodes from the nearest geographical location to our specimens.

We captured 25 Protoptila spp. adults, of which four were sequenced. The DNA sequence analyses revealed that these four specimens (STRBC123-24, STRBC121-24, STRBC109-24, and STRBC120-24) were P. coloma (Fig. 1). Based on the CO1 tree analysis of the DNA barcodes, our specimens cluster closely with P. coloma from Washington State and Montana but separately from P. tenebrosa. Protoptila tenebrosa has been identified previously in British Columbia, Alberta, and Manitoba, Canada; however, DNA barcodes for P. tenebrosa in Canada are available only from Manitoba. Our analyses showed at least 4% barcode divergence between our specimens and those of P. tenebrosa, supporting the hypothesis that our specimens belong to P. coloma and that the specimens collected during the present study represent the first record of P. coloma in Canada. This was also confirmed by comparing the male genitalia of our specimens to published keys for P. coloma (Ross Reference Ross1941; Fig. 2).

Figure 1. Phylogenetic tree of Protoptila coloma from the Stellako River, British Columbia (STRBC123-24, STRBC121-24, STRBC109-24, and STRBC120-24), Montana (MPGT622-19 and MPGT536-16), and Washington State (DRCAD372-10 and DRCAD371-10), and P. tenebrosa (CUCAD756-08 and CUCAD751-08) from Manitoba.

Figure 2. Male genitalia (lateral aspect) of Protoptila coloma that were collected from the Stellako River, British Columbia.

Protoptila coloma is known to exist in many western states of the United States of the America: California, Colorado, Idaho, Montana, Nevada, Oregon, Utah, Washington, and Wyoming. Currently, only four Protoptila species are known in Canada: P. tenebrosa, P. erotica (Ross), P. lega (Ross) and P. maculata (Hagen), of which only P. tenebrosa had previously been reported for British Columbia. Our finding adds a fifth Protoptila species to Canada and a second to British Columbia.

The low number of Canadian Protoptila species is in stark contrast with the neotropics, where much smaller geographic regions such as Panama and Costa Rica contain 15 and 19 Protoptila spp., respectively (Blahnik et al. Reference Blahnik, Aguirre and Armitage2023). The lack of diversity of Protoptila spp. in Canada may be due to Canada’s northern geography. The genus prefers warmer streams (Wiggins Reference Wiggins1996). However, bioassessment surveys most often use only the larval stage and morphology-based taxonomy, and caddisfly species identification is almost entirely based on the adult stage (Wiggins Reference Wiggins2004). It is possible that surveys to sample macroinvertebrates often inadvertently select for the larger caddisfly species because they are easier to collect and identify than extremely small Protoptila spp. The larval cases of Protoptila spp. are only 4 mm (Wiggins Reference Wiggins1996). Such small species can be missed in surveys because they are physically harder to collect and identify, leading to less focus on Protoptila spp. and other small trichopteran taxa in Canada. Focused collection efforts in undersurveyed regions, in combination with the use of the substantial reference resources available for the Trichoptera, including highly comprehensive DNA barcode databases such as IBOL and BOLD (Zhou et al. Reference Zhou, Frandsen, Holzenthal, Beet, Bennett and Blahnik2016), could significantly increase our understanding of Canadian aquatic invertebrate fauna.