Diversity and substrate-specificity of green algae and other micro-eukaryotes colonizing amphibian clutches in Germany, revealed by DNA metabarcoding

Abstract

Amphibian clutches are colonized by diverse but poorly studied communities of micro-organisms. One of the most noted ones is the unicellular green alga, Oophila amblystomatis, but the occurrence and role of other micro-organisms in the capsular chamber surrounding amphibian clutches have remained largely unstudied. Here, we undertook a multi-marker DNA metabarcoding study to characterize the community of algae and other micro-eukaryotes associated with agile frog (Rana dalmatina) clutches. Samplings were performed at three small ponds in Germany, from four substrates: water, sediment, tree leaves from the bottom of the pond, and R. dalmatina clutches. Sampling substrate strongly determined the community compositions of algae and other micro-eukaryotes. Therefore, as expected, the frog clutch-associated communities formed clearly distinct clusters. Clutch-associated communities in our study were structured by a plethora of not only green algae, but also diatoms and other ochrophytes. The most abundant operational taxonomic units (OTUs) in clutch samples were taxa from Chlamydomonas, Oophila, but also from Nitzschia and other ochrophytes. Sequences of Oophila “Clade B” were found exclusively in clutches. Based on additional phylogenetic analyses of 18S rDNA and of a matrix of 18 nuclear genes derived from transcriptomes, we confirmed in our samples the existence of two distinct clades of green algae assigned to Oophila in past studies. We hypothesize that “Clade B” algae correspond to the true Oophila, whereas “Clade A” algae are a series of Chlorococcum species that, along with other green algae, ochrophytes and protists, colonize amphibian clutches opportunistically and are often cultured from clutch samples due to their robust growth performance. The clutch-associated communities were subject to filtering by sampling location, suggesting that the taxa colonizing amphibian clutches can drastically differ depending on environmental conditions.

Taxonomy and nomenclature of Oophila amblystomatis (Chlorophyceae, Chlamydomonadales)

“…In our tree, the Nema et al (2019) sequence clearly nests outside the Oophila clade, making the nomenclatural transfer to Chlorococcum unnecessary, as it is based on an incorrect taxonomic interpretation . Another sequence generated by Nema et al (2019), called "Suddent Tract," is a 100% match both to the Kingston sequence and Chlorococcum dalmatina, an alga associated with egg masses of the frog Rana dalmatina (Anslan et al, 2021). Thus, the same argument as made above for the Kingston sequence.…”

“…Lambert ex N. Wille is of interest because of its symbiosis with egg masses and larval tissues of the spotted salamander Ambystoma maculatum (Kerney, 2011; Kerney et al., 2011, 2019) and five other amphibian species (Anslan et al., 2021; Kim et al., 2014; Muto et al., 2017). A variety of green algae can be isolated or detected from these egg masses (Anslan et al., 2021, Jurga et al., 2020, Nema et al., 2019, Schultz, 2016), and O. amblystomatis can be detected from the pond water in which the egg masses occur (Jurga et al., 2020; Lin & Bishop, 2015). These observations raise questions regarding the specificity of the symbiosis and the nomenclatural characterization of O. amblystomatis .…”

Developing Inside a Layer of Germs—A Potential Role for Multiciliated Surface Cells in Vertebrate Embryos

“…The closest sequence match to the chytridomycete extracted from the fluid is Rhyzophidium patellarium which is an algal parasite [80], suggesting a potential antagonistic relationship with the algae inside the egg capsule microbiome. Interestingly, the cercozoan and chytridiomycete variants were not found in association with the European agile frog (Rana dalmatina) despite the presence of Oophila amblystomatis in these eggs [27]. Although the sampling techniques for these two studies differed.…”

“…These include tissue techniques such as fluorescent in situ hybridizations (FISH [22]), or newer hybridization chain reaction (HCR-FISH [23]), to detect clade-specific sequences with fluorescent probes. "Meta-barcoding" requires degenerate PCR primers or primers that target highly conserved regions of fungal (internal transcribed spacer region-ITS rDNA [24]), eukaryotic (18s rDNA [25]), bacterial (16s rDNA [26]), or green algal (rubisco [27]) genomes. The amplified PCR products are either analyzed for sequence heterogeneity through denaturing gradient gel electrophoresis [28], hybridized to a taxa-defined DNA microarray chip [29], or prepared as libraries for next generation sequencing and binned according to sequence similarity with curated reference taxonomy classifiers [30].…”

“…The original evidence that extraembryonic fluid of many amphibians is not sterile is attributable to the common presence of the conspicuous green algae, Oophila amblystomatis [76]. To date, it has been found in the fluid of several ranid frog, and ambystomatid and hynobiid salamander species (reviewed in [27]). The stability and persistence of other microbes contained within amphibian egg capsules across evolution and development has not been thoroughly established.…”