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

Abstract

Environmental DNA‐based diversity studies have increased in popularity with the development of high throughput sequencing technologies. This permits the potential simultaneous retrieval of vast amounts of molecular data from many different organisms and species, thus contributing to a wide range of biological disciplines. Environmental DNA protocols designed for protists often focused on the highly conserved small subunit of the ribosome gene, that does not permit species‐level assignments. On the other hand, eDNA protocols aiming at species‐level assignments allow a fine level ecological resolution and reproducible results. These protocols are currently applied to organisms living in marine and shallow lotic freshwater ecosystems, often in a bioindication purpose. Therefore, in this study, we present a species‐level eDNA protocol designed to explore diversity of Arcellinida (Amoebozoa: Tubulinea) testate amoebae taxa that is based on mitochondrial cytochrome oxidase subunit I (COI). These organisms are widespread in lentic water bodies and soil ecosystems. We applied this protocol to 42 samples from peatlands, estuaries and soil environments, recovering all the infraorders in Glutinoconcha (with COI data), except for Hyalospheniformes. Our results revealed an unsuspected diversity in morphologically homogeneous groups such as Cylindrothecina, Excentrostoma or Sphaerothecina. With this protocol we expect to revolutionize the design of modern distributional Arcellinida surveys. Our approach involves a rapid and cost‐effective analysis of testate amoeba diversity living in contrasted ecosystems. Therefore, the order Arcellinida has the potential to be established as a model group for a wide range of theoretical and applied studies.

How good are global DNA‐based environmental surveys for detecting all protist diversity? Arcellinida as an example of biased representation

“…A possible solution can come from the use of other non‐ribosomal markers, which are often taxonomically more resolutive and clade‐specific (Pawlowski et al, 2012). Recently, the use of the mitochondrial cytochrome oxidase gene as a biodiversity marker for Arcellinida has revealed considerable environmental diversity in soil and freshwater (González‐Miguéns et al, 2023). Such focused approaches, which aim at providing exhaustive pictures of specific clades at the species level, might well be the future of environmental DNA surveys.…”

“…The latter suborder is divided into at least six infraorders (González‐Miguéns, Todorov, et al, 2022). Within these infraorders, Excentrostoma appears to be highly diverse and abundant in soil and freshwater environments, as revealed by metabarcoding studies based on the mitochondrial marker gene COI (González‐Miguéns et al, 2023). Most Arcellinida species are found in continental habitats, with a few exceptions in coastal systems (Meisterfeld, 2002).…”

When ecological transitions are not so infrequent: independent colonizations of athalassohaline water bodies by Arcellidae (Arcellinida; Amoebozoa), with descriptions of four new species

“…We aligned our sequences with other Arcellidae sequences present in Genbank, as well as some Netzeliidae and Hyalospheniformes as outgroups. We also included Arcellidae environmental sequences from González-Miguéns et al ( 2023 ), altogether resulting in a total of 76 sequences. A first alignment was performed using the MAFFT algorithm (Katoh et al 2002 ), as implemented in GENEIOUS PRIME (v. 2019.0.4).…”

“…Ecological transitions between these ecosystems have been documented in several clades (Lee and Bell 1999 ), but are considered to happen rarely in the evolutionary history of eukaryotes, microbial or not (Hutchinson 1975 , Logares et al 2009 ). Still, along with an increasing knowledge of protist systematics, a growing number of new instances of transitions through the salinity barrier are appearing, as in cryptophytes (Filker et al 2017 ), diatoms (Alverson et al 2007 , Roberts et al 2022 ), dinoflagellates (Annenkova et al 2020 ) or testate amoebae (González-Miguéns, Soler-Zamora, Fernando Useros et al 2022 , González-Miguéns et al 2023 ).…”

“…In order to evaluate the relative importance of salinity and humidity in the evolutionary history of other eukaryotic microorganisms, we took the family Arcellidae (Sphaerothecina; Arcellinida; Elardia; Amoebozoa) as a study group. The order Arcellinida, to which Arcellidae belong, occurs worldwide in different terrestrial, wetland and freshwater habitats (Smith et al 2007 ), with very few species recorded in coastal salt marshes and brackish waters (Golemansky 1998 , Charman et al 2002 , Gehrels et al 2006 , González-Miguéns et al 2023 ). They are often ecological specialists with narrow tolerance to alterations of their environment (Singer et al 2018 ), hence their use as bioindicators for environmental health (Roe and Patterson 2014 , Creevy et al 2018 , Kosakyan and Lara 2019 ).…”