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.

两栖动物的离合器被多样化但研究不足的微生物群落定殖。最著名的一种是单细胞绿藻Oophila amblystomatis，但其他微生物在两栖动物离合器周围的囊腔中的发生和作用在很大程度上仍未得到研究。在这里，我们进行了一项多标记 DNA 元条形码研究，以表征与敏捷青蛙 ( Rana dalmatina ) 离合器相关的藻类和其他微真核生物群落。在德国的三个小池塘进行采样，从四种基质：水、沉积物、池塘底部的树叶和R. dalmatina离合器。采样底物强烈决定了藻类和其他微型真核生物的群落组成。因此，正如预期的那样，与青蛙离合器相关的群落形成了明显不同的集群。在我们的研究中，与离合器相关的群落不仅由大量绿藻构成，还由硅藻和其他赭植物构成。离合器样品中最丰富的操作分类单元 (OTU) 是来自衣藻、鱼尾藻的分类群，也来自Nitzschia和其他赭色植物的分类群。Oophila的序列“Clade B”仅在离合器中被发现。基于对 18S rDNA 和源自转录组的 18 个核基因矩阵的额外系统发育分析，我们在我们的样本中证实了在过去的研究中分配给Oophila的两个不同的绿藻进化枝的存在。我们假设“进化枝 B”藻类对应于真正的Oophila，而“进化枝 A”藻类是一系列绿球藻这些物种与其他绿藻、赭藻和原生生物一起，机会主义地在两栖动物的巢穴中定殖，并且由于其强劲的生长性能而经常从离合器样本中培养出来。与离合器相关的群落会通过采样位置进行过滤，这表明在两栖动物离合器中定居的分类群可能因环境条件而有很大差异。