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

Sten Anslan,Ryan Kerney,Lois Rancilhac,Anja Schwarz,Maria Sachs,Henner Brinkmann,Hartmut Arndt,Jörn Petersen,Miguel Vences,Sven Künzel

doi:10.1007/s00114-021-01734-0

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.

Highlights

Amphibians are characterized by a striking diversity of reproductive modes (Haddad and Prado 2005; Salthe and Duellman 1973) that is reflected in the diversity of eggs and clutches they deposit (Altig and McDiarmid 2007)
In addition to green algae assigned to Chlorophyta (89% of reads), rbcL operational taxonomic units (OTUs) included taxa from diatoms (Bacillariophyceae; 7.8%) and other Ochrophyta (3.1%) in the clutch samples (Fig. 3a)
DNA metabarcoding revealed that R. dalmatina clutches in the Elm region (Germany) provide a distinct ecosystem for an array of algae and other micro-eukaryotes

Summary

Introduction

Amphibians are characterized by a striking diversity of reproductive modes (Haddad and Prado 2005; Salthe and Duellman 1973) that is reflected in the diversity of eggs and clutches they deposit (Altig and McDiarmid 2007). One of the most prominent organisms associated with amphibian eggs is the unicellular green alga, Oophila amblystomatis. The singular interactions of Oophila with its amphibian hosts, in particular with North American salamanders of the genus Ambystoma, were noted as early as in the late nineteenth century (Orr 1888). In these salamanders, the embryo exits the vitelline membrane during neurulation (see Altig and McDiarmid 2007; Salthe 1963 for details of egg capsule structure), and a bloom of O. amblystomatis proliferates outside the embryonic blastopore. The close symbiotic association of Oophila with its amphibian hosts even leads to algal cells invading host embryonic tissues and cells in the spotted salamander (Ambystoma maculatum), and constitutes a unique example of endosymbiosis in vertebrates (Kerney et al 2011, 2019)

Methods

Results

Conclusion