Proteomic fingerprinting using MALDI-TOF mass spectrometry is a well-established tool for identifying microorganisms and has shown promising results for identification of animal species, particularly disease vectors and marine organisms. And thus can be a vital tool for biodiversity assessments in ecological studies. However, few studies have tested species identification across different orders and classes. In this study, we collected data from 1,246 specimens and 198 species to test species identification in a diverse dataset. We also evaluated different specimen preparation and data processing approaches for machine learning and developed a workflow to optimize classification using random forest. Our results showed high success rates of over 90%, but we also found that the size of the reference library affects classification error. Additionally, we demonstrated the ability of the method to differentiate marine cryptic-species complexes and to distinguish sexes within species.

Morphological identification of cnidarian species can be difficult throughout all life stages due to the lack of distinct morphological characters. Moreover, in some cnidarian taxa genetic markers are not fully informative, and in these cases combinations of different markers or additional morphological verifications may be required. Proteomic fingerprinting based on MALDI-TOF mass spectra was previously shown to provide reliable species identification in different metazoans including some cnidarian taxa. For the first time, we tested the method across four cnidarian classes (Staurozoa, Scyphozoa, Anthozoa, Hydrozoa) and included different scyphozoan life-history stages (polyp, ephyra, medusa) into our dataset. Our results revealed reliable species identification based on MALDI-TOF mass spectra across all taxa with species-specific clusters for all 23 analyzed species. In addition, proteomic fingerprinting was successful for distinguishing developmental stages, still by retaining a species specific signal. Furthermore, we identified the impact of different salinities in different regions (North Sea and Baltic Sea) on proteomic fingerprints to be negligible. In conclusion, the effects of environmental factors and developmental stages on proteomic fingerprints seem to be low in cnidarians. This would allow using reference libraries built up entirely of adult or cultured cnidarian specimens for the identification of their juvenile stages or specimens from different geographic regions in future biodiversity assessment studies.

We analyzed robustness of species identification based on proteomic composition to data processing and intraspecific variability, specificity and sensitivity of species-markers as well as discriminatory power of proteomic fingerprinting and its sensitivity to phylogenetic distance. Our analysis is based on MALDI-TOF MS data from 32 marine copepod species coming from 13 regions (North and Central Atlantic and adjacent seas). A random forest (RF) model correctly classified all specimens to species level with only small sensitivity to data processing, demonstrating the strong robustness of the method. Compounds with high specificity showed low sensitivity i.e., identification was rather based on complex pattern-differences than on presence of single markers. Proteomic distance was not consistently related to phylogenetic distance. A species-gap in proteome composition appeared at 0.8 Euclidean distance when using only specimens from the same sample. When other regions or seasons were included, intra-specific variability increased, resulting in overlaps of intra- and inter-specific distance. Highest intra-specific distances (> 0.8) were observed between specimens from brackish and marine habitats i.e., salinity likely affects proteomic patterns. When testing library sensitivity of the RF model to regionality, strong misidentification was only detected between two congener pairs. Still, choice of reference library may have an impact on identification of closely related species and should be tested before routine application. We envision high relevance of this time- and cost-efficient method for future zooplankton monitoring as it provides not only in-depth taxonomic resolution for counted specimens but also add-on information e.g., on developmental stage or environmental conditions.

Interoceanic canals can facilitate biological invasions as they connect the world’s oceans and dissolve dispersal barriers between bioregions. As a consequence, multiple opportunities for biotic exchange arise and the resulting establishment of migrant species often causes adverse ecological and economic impacts. The Panama Canal is a key region for biotic exchange as it connects the Pacific and Atlantic Oceans in Central America. In this study, we used two complementary methods (environmental DNA (eDNA) and gillnetting) to survey fish communities in this unique waterway. Using COI (cytochrome oxidase subunit I) metabarcoding, we detected a total of 142 taxa, including evidence for the presence of sixteen Atlantic and eight Pacific marine fish inside different sections of the Canal. Of these, ten are potentially new records of marine taxa detected in the freshwater segment of the Canal. Molecular data did not capture all species caught with gillnets, but generally provided a more complete image of the fish fauna. Diversity indices based on eDNA surveys revealed significant differences across different sections of the Canal reflecting in part the prevailing environmental conditions. The observed increase in the presence of marine fish species in the Canal indicates a growing potential for interoceanic exchange of fishes across the Isthmus. Monitoring using eDNA is a rapid and efficient way to assess potential changes in the fishes of this important waterway.