Acquisition of new genes often results in the emergence of novel functions and is a key step in lineage-specific adaptation. As the only group of sessile crustaceans, barnacles establish permanent attachment through initial cement secretion at the larval phase followed by continuous cement secretion in juveniles and adults. However, the origins and evolution of barnacle larval and adult cement proteins remain poorly understood. By performing microdissection of larval cement glands, transcriptome and shotgun proteomics and immunohistochemistry validation, we identified 30 larval and 27 adult cement proteins of the epibiotic turtle barnacle Chelonibia testudinaria, of which the majority are stage- and barnacle-specific. While only two proteins, SIPC and CP100K, were expressed in both larvae and adults, detection of protease inhibitors and the cross-linking enzyme lysyl oxidase paralogs in larvae and adult cement suggested functional convergence. Other barnacle specific cement proteins such as CP100k and CP52k likely share a common origin dating back at least to the divergent of Rhizocephala and Thoracica. Different CP52k paralogs could be detected in larval and adult cement, suggesting stage-specific cement proteins may arise from duplication followed by changes in expression timing of the duplicates. Interestingly, the biochemical properties of larval- and adult-specific CP52k paralogs exhibited remarkable differences, reflecting the composition of cement in different life stages of turtle barnacle might be chemically different. We conclude that de novo gene formation and duplicate neofunctionalization are pivotal to the evolution of lineage-specific cement toolkits in barnacles, which may explain how barnacles can inhabit diverse marine substrata.

The enigmatic “y-larvae” (Pancrustacea: Facetotecta) still have an incompletely understood lifecycle, and their adult forms remain unknown despite their discovery more than 100 years ago and their documented global occurrence from shallow waters to the deep-sea. Only two of the 17 formally described species, all based on larval stages, have been investigated using an integrative taxonomic approach that, besides providing descriptions of the morphology of the naupliar and cyprid stages, also made use of exuvial voucher material and DNA barcodes. To improve our knowledge about the systematics and phylogenetics of y-larvae, we developed a novel protocol that maximizes the amount of morphological, ecological, and molecular data that can be harvested from single individuals of these tiny larvae. This revolves around single larva barcoding, and includes daily imaging of y-nauplii reared in culture dishes, mounting of their last naupliar exuviae on a slide as a reference voucher, live imaging of the y-cyprid instar that follows, and fixation, DNA extraction, amplification, and sequencing of the y-cyprid specimen. By developing and testing a suite of new primers for both nuclear and mitochondrial protein-coding and ribosomal genes, we estimated the most comprehensive phylogeny of Facetotecta to date. We expect that our novel procedure will help to unravel the complex systematics of y-larvae and show how these fascinating larval forms have evolved. Moreover, we posit that our protocols should work on larval specimens of a diverse array of molting marine invertebrate taxa.

Marine drifting animals — zooplankton — play essential ecological roles in the pelagic ecosystem, transferring energy and elements to higher trophic levels, such as fishes, cetaceans, and others. Zooplankton are generally considered passive drifting organisms homogeneously distributed throughout waters, where high dispersal is expected. Although empirical observations have demonstrated that many species possess active swimming mechanisms that generate metacommunities with high beta diversity, the role of animal sizes in the process of marine zooplankton community dynamics remains unexplored. Here, we collected a total of 48 size-fractionated zooplankton samples in the vicinity of a coral reef island with environmental gradients and performed metatranscriptome analyses. The samples were collected in two transects (from nearshore to offshore) twice a day (morning and night). Sample size fraction was the only variable that rendered apparent differences in species composition between the samples. Our results demonstrate differential dispersal through the size fractions — smaller size fraction communities had higher compositional homogeneity than larger ones. Contrary to expectation, distance to shore had no significant influence on the composition or diversity of zooplankton communities. This study offers novel insights on the use of metatranscriptomics for analyzing community structures and the role size plays for the marine zooplankton community assembly processes.

The purplish bifurcate mussel Mytilisepta virgata is widely distributed and represents one of the major components of the intertidal community in the northwestern Pacific (NWP). Here, we characterized population genetic structure of NWP populations throughout their whole distribution range using both mitochondrial (mtDNA cox1) and nuclear (ITS1) markers. Population genetic analyses for mtDNA cox 1 sequences revealed two monophyletic lineages (i.e., southern and northern lineages) geographically distributed according to the two different surface water temperature zones in the NWP. The timing of the lineage split is estimated at the Pliocene- mid-Pleistocene (5.49-1.61 Mya), which is consistent with the timing of the historical isolation of the East Sea/Sea of Japan from the South and East China Seas caused by sea level decline during glacial cycles. Historical sea level fluctuation during the Pliocene-Pleistocene and subsequent adaptation of mussels to different surface water temperature zones may have contributed to shaping the contemporary genetic diversity and deep divergence of the two mitochondrial lineages. Unlike mtDNA sequences, a clear lineage splitting between the two mitochondrial lineages was not found in ITS1 sequences, showing a star-like structure that is composed of a mixture of southern and northern mitochondrial lineages. Possible scenarios are proposed to explain this type of mito-nuclear discordance: stochastic divergence in the coalescent processes of the two molecular markers, or balancing selection under different marine environments. Future work is required to address whether the thermal physiology of these mussels correlates with the deep divergence of their mitochondrial genes.