The larvae of Chinese Hydropsychidae (Insecta: Trichoptera): Delimiting species boundaries using morphology and DNA sequences

While the utility of using hydropsychid (Trichoptera: Hydropsychidae) caddisfly larvae for freshwater biomonitoring has been demonstrated, the major impediment to this implementation has been the lack of species-level larval descriptions and illustrations. Because conventional approaches to associating undescribed larvae with adults have been slow and problematic, a rapid and more reliable molecular protocol is proposed, which also integrates morphology. Male adults were identified before the DNA sequence analyses were employed. These established morpho-species boundaries were then mapped on phylograms constructed from two independent gene fragments: mitochondrial COI and nuclear 28S ribosomal DNA (the D2 region). Species boundaries were confirmed if they were monophyletic on both molecular phylograms. Larval associations were made with reference to the phylogenetic analyses under two criteria: sequence identity across both genes or nested placement within a reference species boundary.; Three hundred and seventy-six individuals of hydropsychid caddisflies and thirty-one outgroup taxa were amplified for D2 and COI sequences. These DNA markers have each provided sufficient nucleotide change to differentiate most hydropsychid species, with ambiguity in only a few species that could be eventually resolved with additional sequences and specimens. As a result, twenty larval species were successfully associated across both DNA markers. Additionally, a number of species were associated with one of the two markers while no sufficient sequences were obtained for the alternative marker. Future associations are expected to be made at a faster pace while the DNA dataset of D2 and COI for Chinese hydropsychids accumulates. Twenty-three larval species, including several ones with uncertain associations, were described and illustrated. Larval keys to the genera of Chinese Hydropsychidae and to the subgenera of the genus Hydropsyche sensu lato were provided.; Associating larvae and adults of hydropsychids using morphology and DNA sequences appears to be promising for both reliability and speed. This molecular approach is also expected to be helpful in many problems related to polyphenism across insect groups, such as identification of various life stages, metamorphs, and sexual and seasonal morphs.