Cryptic connectivity between hyporheic and riparian zones via winged aquatic insects revealed by DNA barcoding

How subsurface hyporheic zone (HZ) of rivers is connected to riparian zones remains largely unknown. We collected benthic macroinvertebrates and adult aquatic insects for six years, including those at 30-cm depth in the HZ to identify insect taxa having a high level of habitat affinity for HZ (HZ taxa). Adults of HZ taxa were identified with the aid of CO1 gene barcoding, and the relative abundance of HZ taxa in the riparian zone was quantified. In addition to the previously known stonefly Alloperla ishikariana, three species of stonefly Leuctridae and one caddisfly species of Philopotamidae were identified as HZ taxa. At the annual scale, HZ taxa accounted for approximately 38% of the total aquatic insects in the family of stoneflies (Plecoptera), caddisflies (Trichoptera), and mayflies (Ephemeroptera) in abundance and 26% of biomass, with their seasonal peaks in early spring and mid-summer (60% in abundance and 47% in biomass). Few individuals of HZ taxa were found in benthic samples (<0.1%), and hyporheic samples quantified more but erroneously estimated relative abundances of adult HZ taxa, with Leuctridae and Philopotamidae being substantially underrepresented relative to their adult abundance. Cryptic biological connectivity between subsurface and riparian zones via winged HZ-taxa adults is substantial. DNA-based species identification combined with community surveys of adult HZ-taxa complementarily used with benthic data can substantially improve the effectiveness of biomonitoring programs and outcomes of habitat conservation based on more complete picture of ecosystem health.