Molecular Identification of Amphipod and Microsporidian Species

Species identification was performed using BLASTn searches [49 (link)] against NCBI GenBank and the Barcode of Life Data System (BOLD) [50 (link)]. Additionally, relevant primary literature with available genetic data for amphipods or microsporidians was consulted. Identification was positive, if our query sequence showed at least 98 % sequence identity with one or more reference sequences. A 98 % identity threshold (or 2 % accepted intraspecific variability) was chosen for the identification of amphipods and microsporidian lineages. For the latter, our strategy accounts for potential intragenomic variability present in some microsporidians at the SSU rRNA locus [51 (link)], whereas a 2 % threshold for CO1 is far below commonly observed values of interspecific variability in amphipods [52 (link)]. For the identification of clades (or potential cryptic species) within G. pulex the CO1-alignment of Lagrue et al. [53 (link)] was used. For visualization of the different host and parasite lineages, Neighbor-Joining trees were calculated with the program MEGA6 [54 (link)] under the pairwise-deletion option for the CO1- and SSU rDNA—alignment, respectively. Node support was calculated by 1000 bootstrap replicates. Trees were rooted with the respective outgroup sequence. Outgroups were omitted for the final visualization of trees.