The signal crayfish is not a single species: cryptic diversity and invasions in the Pacific Northwest range of Pacifastacus leniusculus

1. We used historical sources, morphology-based taxonomy and mtDNA sequence data to address questions about the signal crayfish Pacifastacus leniusculus. These included evaluating unrecognised cryptic diversity and investigating the extent to which P. leniusculus may have been introduced within its presumed native range in the Pacific Northwest region of North America. Our study builds and expands on Pacific Northwest phylogeographic knowledge, particularly related to patterns of glacial refugia for freshwater species. 2. Extensive collections (824 specimens) from British Columbia (Canada), Idaho, Nevada, Oregon and Washington (United States) were used to characterise P. leniusculus at the mitochondrial 16S rRNA gene. Genetic groups within the species were elucidated by phylogenetics and amova; evolutionary relationships within the most common and diverse group were investigated using a statistical parsimony haplotype network, a nested amova, and tests of isolation by distance. Morphological measurements were used to relate findings of molecular analyses to three historically recognised P. leniusculus subspecies and characterise cryptic diversity by morphology. 3. We found substantial cryptic diversity, with three groups highly distinct from P. leniusculus in discrete geographic regions: the Chehalis River glacial refugium, Central Oregon and the Okanagan Plateau. Disjunct distributions of P. leniusculus relative to these cryptic groups and known patterns of Pleistocene glaciation and landscape evolution cast doubt on whether P. leniusculus is native to some areas such as coastal drainages of northern Washington and southern British Columbia. Morphological traits previously used to characterise P. leniusculus subspecies still persist but may be incapable of distinguishing P. leniusculus from newly discovered cryptic groups. 4. Cryptic diversity found within P. leniusculus highlights the pressing need for a thorough investigation of the genus Pacifastacus using data based on more extensive gene and taxon sampling. It also warrants conservation attention, as introductions of P. leniusculus within the Pacific Northwest may carry risks of hybridisation and introgression for cryptic groups. Owing to high genetic diversity and limited dispersal capacity relative to more vagile organisms like freshwater fish, crayfish of the genus Pacifastacus offer powerful potential insights into the geological history and phylogeography of the Pacific Northwest region. Finally, by shedding light on the long-neglected native range of P. leniusculus, our results should also better inform our understanding of potential source populations for, and the ecology of, this important invasive species in regions including Europe, Japan and elsewhere in North America.