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

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.

Mitochondrial Cytochrome Oxidase Subunit 1: A Promising Molecular Marker for Species Identification in Foraminifera

“…Sequences for each marker were aligned with the MAFFT v7.450 algorithm using default settings in Geneious (Katoh and Standley, 2013). Sequences that did not align properly [i.e., created important gap regions (>10 bp)] and did not give any BLAST 2 results somewhat similar to the phylum Insecta, which is a common blast result for Foraminifera COI due to a lack of foraminiferal COI sequences in the reference database (Macher et al, 2021b), were removed from the analysis. Here, such sequences are referred to as "flagged" sequences.…”

“…Additional nuclear markers have been studied, such as the ITS (Macher et al, 2021a), which is also often used for molecular analyses of protists in general (Thornhill et al, 2007;Pawlowski et al, 2016), the ribosomal large subunit (Pawlowski et al, 1994), actin (Flakowski, 2005), tubulin (Takishita et al, 2005) and multiple genes based on expressed sequence tags data (Sierra et al, 2013). Recently, Macher et al (2021b) established a barcoding protocol for foraminiferal mitochondrial cytochrome c oxidase I (COI), based on Rotaliida and Miliolida sequences. Here, we use high-throughput sequencing to assess intra-and inter-specimen genetic variability of two foraminiferal molecular markers, in order to test the potential of the newly discovered COI compared to the widely used 18S.…”

“…Amplifications were performed by polymerase-chain reaction (PCR) using a T100 Thermal Cycler (Bio-Rad, Hercules, CA, United States). For amplification of the 18S marker, two different reactions (i.e., TaqMan Environmental Master Mix 2.0 and KAPA HiFi HotStart For amplification of the COI marker, the protocol was improved from Macher et al (2021b). 2.5 µL of diluted 10x DNA template was mixed to 11.7 µL Milli-Q water, 2 µL PCR buffer CL 10X (Qiagen), 0.4 µL MgCl2 25 mM, 0.8 µL BSA 10 mg/mL, 0.4 µL dNTP 25 mM, 0.2 µL Taq-polymerase (Qiagen) 5 U/µL, 1 µL forward primer 10 µM and 1 µL reverse primer 10 µM for a total volume of 20 µL.…”

“…Metabarcoding methods based on high-throughput sequencing enable the identification of a wide range for organisms, especially using mitochondrial markers (Wangensteen and Turon, 2017). Most phyla in different Kingdoms have well defined markers such as the mitochondrial cytochrome oxidase subunit 1 (COI), but this gene has only recently been identified in Foraminifera (Macher et al, 2021b).…”

“…Many studies also show the importance of combining different markers to get a robust species identification and to better understand evolutionary traits and relationships within and between species (Purty and Chatterjee, 2016;Zhao et al, 2016). Macher et al (2021b) recently discovered the first mitochondrial COI gene in Foraminifera. COI is a molecular marker commonly used in molecular research on animals (Hebert et al, 2003) and has been shown to be informative on other protists (Nassonova et al, 2010;Pawlowski et al, 2012;Burki et al, 2021).…”