First report of mitochondrial COI in foraminifera and implications for DNA barcoding

Jan-Niklas Macher,Aleksey Sadekov,Elza Duijm,Elsa B Girard,Richard Wissels,Anouk Langerak,Willem Renema,Jamaluddin Jompa,Jeremy G Wideman,Rutger Vos

doi:10.1038/s41598-021-01589-5

Abstract

Foraminifera are a species-rich phylum of rhizarian protists that are highly abundant in many marine environments and play a major role in global carbon cycling. Species recognition in Foraminifera is mainly based on morphological characters and nuclear 18S ribosomal RNA barcoding. The 18S rRNA contains variable sequence regions that allow for the identification of most foraminiferal species. Still, some species show limited variability, while others contain high levels of intragenomic polymorphisms, thereby complicating species identification. The use of additional, easily obtainable molecular markers other than 18S rRNA will enable more detailed investigation of evolutionary history, population genetics and speciation in Foraminifera. Here we present the first mitochondrial cytochrome c oxidase subunit 1 (COI) gene sequences (“barcodes”) of Foraminifera. We applied shotgun sequencing to single foraminiferal specimens, assembled COI, and developed primers that allow amplification of COI in a wide range of foraminiferal species. We obtained COI sequences of 49 specimens from 17 species from the orders Rotaliida and Miliolida. Phylogenetic analysis showed that the COI tree is largely congruent with previously published 18S rRNA phylogenies. Furthermore, species delimitation with ASAP and ABGD algorithms showed that foraminiferal species can be identified based on COI barcodes.

Highlights

Foraminifera are a species-rich phylum of rhizarian protists[1]
Single-cell shotgun sequencing and assembly resulted in complete mitochondrial c oxidase subunit I (COI) sequences of 21 Foraminifera specimens from 8 species
Transmembrane prediction and comparison with genes deposited in Pfam, UniProt, SwissProt and Ensembl databases confirmed that the closest match of the identified gene sequences with existing references is mitochondrial COI, and blast searches against NCBI Genbank revealed that the closest matches were COI sequences stemming from various eukaryotes

Summary

Introduction

Foraminifera are a species-rich phylum of rhizarian protists[1]. They are highly abundant in a wide range of primarily marine environments and play a major role in global carbon cycling[2,3]. Other species have hypervariable 18S regions[15,23] and show high levels of intragenomic polymorphisms, i.e., highly different variants of the 18S rRNA within single specimens, which is potentially due to the presence of multiple nuclei within the single foraminiferal cell or hybridization of closely related s pecies[21,27] These challenges have been tackled by the advent of molecular species delimitation approaches for Foraminifera based on molecular taxonomic units (MOTUs)[15]. The availability of foraminiferal COI genes allows the deposition of Foraminifera in commonly used repositories for mitochondrial reference sequences like the Barcode of Life database (BOLD40), which are widely used for molecular community a nalyses[41,42] and will help improve species identification in this genetically understudied, but diverse and globally important group of protists

Methods

Results

Discussion

Conclusion