Molecular characterisation of the Microsporidia of the amphipod Gammarus duebeni across its natural range revealed hidden diversity, wide-ranging prevalence and potential for co-evolution.

Microsporidia comprise an unusual group of intracellular, eukaryotic parasites that exhibit ubiquitous distribution throughout the animal kingdom. We analysed the small subunit ribosomal gene (SSUrDNA) using PCR and sequencing and screened 894 Gammarus duebeni (Crustacea, Amphipoda) specimens from 35 European marine and freshwater populations. We discovered considerable hidden microsporidian diversity. Blast searches, sequence analysis and phylogenetic analysis revealed intraspecific sequence variants of known species Dictyocoela duebenum,Dictyocoela muelleri, Pleistophora mulleri and Nosema granulosis. For seven SSUrDNA sequences, we did not find corresponding GenBank entries; they likely represent new species, provisionally classified within the genus Microsporidium. Phylogenetic reconstructions revealed their position as polyphyletic, thereby lending support to the hypothesis of an early microsporidian radiation within this host group. Nevertheless, four of the presumptive novel species formed a discrete and well-supported subclade in the phylogenetic analysis. The respective host specimens were collected from disjunct freshwater sites in Wales, Ireland and Brittany (France) and may represent a new, G. duebeni-specific, microsporidian genus. At the population level, our screening showed that parasitism through Microsporidia is the rule rather than the exception in G. duebeni. We found Microsporidia in 91% of sampled G. duebeni populations. This finding may have considerable consequences for the interpretation of results from ecological, behavioural, physiological and evolutionary studies of the host, as parasitism can significantly influence these traits. Because the host G. duebeni has a complex phylogeography and evolutionary history, the studied host-parasite system may have potential as a model system for investigating processes of co-evolution.

Glacial-driven vicariance in the amphipod Gammarus duebeni.

We have examined the genetic diversity using mitochondrial COI and ND2 sequence data from 306 specimens of the amphi-Atlantic-distributed amphipod Gammarus duebeni. Marine populations from the Atlantic Ocean, the Baltic and North Sea, as well as freshwater populations from Ireland, Cornwall and Brittany were analysed. G. duebeni is a complex of five allopatric lineages. Freshwater populations result from multiple invasions of marine ancestors, represented by distinct lineages. We interpret the recent distribution of lineages as the outcome of a series of spatio-temporal vicariant events caused by Pleistocene glaciations and sea level changes. The freshwater lineages are therefore regarded as 'glacial relicts'. Furthermore, inter-specific competition with, for example, Gammarus pulex (which is absent in Ireland and western Brittany) may be another important determinant in the distribution of freshwater G. duebeni. In Ireland and Brittany, three freshwater refugia are suggested. The significantly limited gene flow detected among marine populations is more likely due to inter-specific competition than to salinity. The G. duebeni-complex represents a model system for the study of allopatric speciation accompanied by major habitat shifts. The pattern of spatio-temporal origins of the freshwater entities we describe here provides an excellent system for investigating evolutionary adaptations to the freshwater environment. Our data did not confirm the presently used subspecies classification but are only preliminary in the absence of nuclear genetic analyses.