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

Abstract

Traditional morphological methods for species identification are highly time consuming, especially for small organisms, such as Foraminifera, a group of shell-building microbial eukaryotes. To analyze large amounts of samples more efficiently, species identification methods have extended to molecular tools in the last few decades. Although a wide range of phyla have good markers available, for Foraminifera only one hypervariable marker from the ribosomal region (18S) is widely used. Recently a new mitochondrial marker cytochrome oxidase subunit 1 (COI) has been sequenced. Here we investigate whether this marker has a higher potential for species identification compared to the ribosomal marker. We explore the genetic variability of both the 18S and COI markers in 22 benthic foraminiferal morphospecies (orders Miliolida and Rotaliida). Using single-cell DNA, the genetic variability within specimens (intra) and between specimens (inter) of each species was assessed using next-generation sequencing. Amplification success rate was twice as high for COI (151/200 specimens) than for 18S (73/200 specimens). The COI marker showed greatly decreased intra- and inter-specimen variability compared to 18S in six out of seven selected species. The 18S phylogenetic reconstruction fails to adequately cluster multiple species together in contrast to COI. Additionally, the COI marker helped recognize misclassified specimens difficult to morphologically identify to the species level. Integrative taxonomy, combining morphological and molecular characteristics, provides a robust picture of the foraminiferal species diversity. Finally, we suggest the use of a set of sequences (two or more) to describe species showing intra-genomic variability additionally to using multiple markers. Our findings highlight the potential of the newly discovered mitochondrial marker for molecular species identification and metabarcoding purposes.

Mitochondrial cytochrome c oxidase subunit I (COI) metabarcoding of Foraminifera communities using taxon-specific primers

“…We aligned all reference sequences using MAFFT (v. 1.4.0) ( Katoh et al, 2002 ) as implemented in Geneious (v2020.2) and translated the sequences using mitochondrial translation table 4 to check for potential stop codons that might indicate sequencing errors or NUMTs ( Macher et al, 2021b ; Girard et al, 2022 ). This led to the exclusion of the sequences of Psammophaga sp.…”

“…Recently, the first mitochondrial cytochrome c oxidase subunit I (COI) gene sequences of Foraminifera were published, together with primers which amplify a COI fragment ( Macher et al, 2021b ; Girard et al, 2022 ) overlapping the established Leray fragment, which is used in metabarcoding studies on a wide range of Eukaryota ( Leray et al, 2013 ; Wangensteen et al, 2018 ). COI is a promising molecular marker for species identification in Foraminifera, but so far the newly developed COI primers were only applied to single foraminiferal specimens for the purpose of reference barcoding and assessment of marker variability ( Macher et al, 2021b ; Girard et al, 2022 ). Here we test whether the new Foraminifera COI primers can be used for metabarcoding of Foraminifera communities from environmental samples.…”

“…We applied metabarcoding to 48 samples taken along three transects spanning a North Sea beach in the Netherlands from dunes to the low tide level, and analysed both sediment samples and meiofauna samples, which contained taxa between 42 µm and 1 mm in body size obtained by decantation from sand samples. We used single-cell metabarcoding ( Girard et al, 2022 ) to generate a COI reference library containing 32 species of Foraminifera, and used this to taxonomically annotate our community metabarcoding data. Our analyses show that the highly degenerate LerayXT primers do not amplify Foraminifera, while the Foraminifera primers are highly Foraminifera- specific, with about 90% of reads assigned to Foraminifera and amplifying taxa from all major groups, i.e., monothalamids, Globothalamea, and Tubothalamea.…”

Establishment, Genetic Diversity, and Habitat Suitability of Aedes albopictus Populations from Ecuador

“…Generally, molecular markers such as allozymes, nuclear, or mitochondrial genes are used to study diversity or inter and intraspecies genetic differences [ 8 ]. A recently published study compared the use of cytochrome c oxidase subunit 1 (COI) and the 18s gene, exploring the genetic variability of each, showing a higher success rate for COI and a more significant potential for species identification compared to the ribosomal marker [ 9 ]. Among the advantages of using the COI gene are its lower variability in the total length and the metrics based on the variability by codon position or the detection of stop codons [ 10 ].…”

A needle in a haystack: a new metabarcoding approach to survey diversity at the species level of Arcellinida (Amoebozoa: Tubulinea)

“…Despite the proven success of these approaches for (organism) barcoding, protocols have been seldom transferred to environmental studies. The exceptions have been ecological research involving diatom barcodes (Kermarrec et al, 2013) and most recently foraminifera (Girard et al, 2022). Both of these groups are excellent ecological and paleoecological bioindicators in marine environments.…”