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.
Highlights
The development of new molecular methods to identify and delimit species as well as to assess community composition, species richness in biodiversity and ecology studies has greatly accelerated in recent years (Hebert et al, 2003; Amaral-Zettler et al, 2009; Pawlowski et al, 2016; Wangensteen and Turon, 2017; van der Loos and Nijland, 2021)
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)
To assess intra- and inter-specimen genetic variability of the 18S and COI genes in Foraminifera, 200 specimens from the Spermonde Archipelago (Indonesia) and Burger’s Zoo (Netherlands) representing two orders, six families, 13 genera and 22 species were analyzed by single-cell metabarcoding (Figure 1)
Summary
The development of new molecular methods to identify and delimit species as well as to assess community composition, species richness in biodiversity and ecology studies has greatly accelerated in recent years (Hebert et al, 2003; Amaral-Zettler et al, 2009; Pawlowski et al, 2016; Wangensteen and Turon, 2017; van der Loos and Nijland, 2021). Other taxa show too little variability between species, for example only one single nucleotide polymorphism (SNP) was reported between two Amphisorus species in the ITS gene (Macher et al, 2021a). This makes species identification based on 18S rRNA and ITS markers challenging (Morard et al, 2016; Macher et al, 2021a)
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