Botryllus schlosseri, is a model marine invertebrate for studying immunity, regeneration, and stress-induced evolution. Conditions for validating its predicted proteome were optimized using nanoElute® 2 deep-coverage LCMS, revealing up to 4,930 protein groups and 20,984 unique peptides per sample. Spectral libraries were generated and filtered to remove interferences, low-quality transitions, and only retain proteins with >3 unique peptides. The resulting DIA assay library enabled label-free quantitation of 3,426 protein groups represented by 22,593 unique peptides. Quantitative comparisons of a laboratory-raised with two field-collected populations revealed (1) a more unique proteome in the laboratory-raised population, and (2) proteins with high/low individual variabilities in each population. DNA repair/replication, ion transport, and intracellular signaling processes were unique in laboratory-cultured colonies. Spliceosome and Wnt signaling proteins were the least variable (highly functionally constrained) in all populations. In conclusion, we present the first colonial tunicate’s deep quantitative proteome analysis, identifying functional protein clusters associated with laboratory conditions, different habitats, and strong versus relaxed abundance constraints. These results empower research on B. schlosseri with proteomics resources and enable quantitative molecular phenotyping of changes associated with transfer from in situ to ex situ and from in vivo to in vitro culture conditions.

Invasive species are of increasing concern to the local biodiversity and ecology as the magnitude of biological invasions is increasing globally. The genetic structures of newly established invasive populations may reveal insights towards the invasion processes, making population genetics an important tool for understanding current invasions. Here we study newly established populations (<10-20 years before first sampling) of the cosmopolitan alien ascidian Botryllus schlosseri in four Puget Sound (Washington, USA) marinas, using eight polymorphic microsatellites. Up to seven sampling sessions over a period of 19 years revealed populations with fluctuating allelic richness (AR=2.693-4.417) and gene diversity (He=0.362-0.589). The populations were well differentiated on spatial and temporal scales and subjected to moderate genetic drift (Fs’=0.027-0.071). The obtained significant heterozygote deficiencies, positive inbreeding coefficients (Fis) and population structure measures (Fst) revealed that no population was under the Hardy-Weinberg equilibrium. Comparing these parameters with those from two Californian sites (Moss Landing and Santa Cruz, 1200 km southerly; invaded by Botryllus during 1940’s) revealed a connection between Moss Landing and Puget Sound, while Santa Cruz remained isolated. On the US west coast scale, this study revealed no major difference in invasive population dynamics between recently and decade long established populations, except for fewer alleles and lower He. When comparing ten worldwide sites, only few microsatellite loci displayed strong regional differences. With globally the lowest numbers of alleles and lowest genetic indices, the Puget Sound Botryllus populations exhibit genetic characteristics of recently established populations, further emerging as one of the youngest B. schlosseri populations, worldwide.