High-throughput sequencing (HTS) strategies became an economically accessible and commonplace way of generating data for non-model organisms, mainly thanks to the development of reduced-representation genome sequencing techniques (Davey et al., 2011). Nevertheless, those data are often generated to address rather specific biological problems and come in different kinds, depending on project goals and available budget, ranging from whole genome sequencing to targeting specific genome regions with restriction site-associated DNA sequencing (RADseq; Andrews et al., 2016). Despite becoming popular amongst population ecologists, evolutionary geneticists or conservationists, the breadth and technical specifics of the HTS data pose a barrier to many alpha taxonomists to adopt and merge with those in their toolboxes used to study organismal diversity. Consequently, HTS data are accumulating in public repositories but are seldom mined for goals other than those originally intended. Applicability of HTS data is wide and their adoption not only allows researchers to formulate and test a new generation of biological questions, but mainly to address old questions with unprecedentedly rich data rigour (Rokas, 2016). The field of cestode integrative taxonomy has already seen advances from estimating single gene phylogenies to broader genome-scale phylogenetic inferences, mostly mitogenomics, achieved using genome skimming techniques (Brabec et al., 2016, Trevisan et al., 2019, 2021), but the use of HTS data in cestode taxonomy remains uncommon.

Proteocephalus longicollis (Zeder, 1800) is a common and geographically widespread cestode found across the Holarctic region. Numerous nominal species of Proteocephalus were originally described from various genera of salmonid fishes including Coregonus, Oncorhynchus, Salmo, Salvelinus and Thymallus, foregrounding tapeworms’ differences in size and shape of proglottids, and the number of testes. However, these differences were later considered a host-related intraspecific variation because all tapeworms invariably shared species-specific characteristics of presumed taxonomic value, i.e., a similar shape of the scolex and positions of suckers, the shape and size of the vestigial apical sucker, the presence of a large cirrus-sac of similar shape and its relative size (ratio of the cirrus-sac length to the proglottid width), and a well-developed ring-like vaginal sphincter. As a result, most of the species have been synonymised with P. longicollis based on the lack of distinctive morphological characters (Scholz and Hanzelová, 1998, Hanzelová and Scholz, 1999). Proteocephalus longicollis circulates between two aquatic hosts to complete its life cycle: copepod crustaceans as intermediate and salmonids as definitive hosts. Similar to other proteocephalids, P. longicollis does not possess a motile free-swimming ciliated larva (coracidium) and successful transmission thus relies on activity of the intermediate host, copepods, which ingest eggs passively floating in the water (Scholz, 1999).

Together with more than a dozen other species of Proteocephalus from Holarctic freshwater teleosts, P. longicollis forms a group known as the Proteocephalus-aggregate de Chambrier, Zehnder, Vaucher, and Mariaux, 2004, a clade of unresolved relationships amongst its members continuously recovered by multiple studies based on various fragments of nuclear ribosomal or mitochondrial (mt) genes (Zehnder and Mariaux, 1999, Škeříková et al., 2001, Scholz et al., 2007, Scholz et al., 2019, de Chambrier et al., 2015). So far, all the analyses had P. longicollis represented with limited data, mostly a single representative, and rarely a few representatives from a limited geographical range. Parasites of European whitefish (Coregonus lavaretus L. species complex, C. pollan Thompson, 1835) and North American lake whitefish (Coregonus clupeaformis Mitchill, 1818) have never been directly compared, and specimens from the other genera of salmonid hosts remained molecularly uncharacterised. The species hypothesis, so far based exclusively on the morphology of P. longicollis, slender worms with elongate proglottids and euryxenous host specificity, has thus never been tested with molecular data.

Here, we demonstrate a strategy of mining useful data from various sources of HTS data to supplement alpha-taxonomy studies of otherwise opportunistically collected parasites. We reconstruct the interrelationships of the Proteocephalus-aggregate, a group of common Holarctic fish cestodes, by filtering novel sequence data from various genomic and transcriptomic projects representing hundreds of parasite individuals from European whitefish and other salmonid fish hosts.