1 | Introduction
Ctenophora is a phylum of gelatinous marine zooplankton with nearly 200
named species (Mills 2017). Their unique body plan separates them from
other gelatinous plankton and from Cnidarians with which they are
sometimes associated (Dunn et al. 2015). They are found
throughout the world ocean, from both poles to the equator and from the
surface to the deep-sea (Harbison et al. 1978), with the deepest
ctenophore observed at over 7,000 meters depth (Lindsay & Miyake 2007).
Many species are common and well-studied, particularly coastal species
like Mnemiopsis leidyi , which is noted for having been introduced
to habitats around the world. However, most deep-living ctenophores
remain undescribed because specimens are delicate, difficult to access,
and often damaged during collection (Haddock 2004). The use of remotely
operated vehicles (ROVs) and specialized sampling equipment have
expanded our ability to observe and collect ctenophores in the deep-sea
during the last 30 years of research (Haddock et al. 2017).
Morphological investigations of specimens collected from the deep sea
suggest that we have only begun to reveal the remarkable diversity
within this phylum. However, there are few taxonomic experts who work on
ctenophores, and morphological identifications often are stymied by
damaged specimens, cryptic morphology, and poor preservation in all
fixatives. Molecular identifications can provide relatively quick
identifications, especially for taxa like ctenophores that have few
taxonomic experts, although polymorphic loci and good reference
libraries are critical to achieve this goal.
Sequence data from the nuclear 18S ribosomal gene
provide a molecular phylogenetic framework for the broad relationships
within ctenophores (Podar et al. 2001; Simion et al.2015), and transcriptomes from a handful of species allow for more
in-depth studies of some representative diversity (Simion et al.2017). However, the 18S rDNA gene fragment is highly conserved,
and the phylogenies often do not effectively discriminate between many
species and closely related genera particularly in groups such as
Lobata. For example, some genera, such as Bathocyroe,
Eurhamphaea , Deiopea and Kiyohimea , have nearly identical18S rDNA sequences, showing the limitations of the utility of the18S fragment with respect to species delineation (Haddock et al.
in prep). The “barcoding” mitochondrial cytochrome-c-oxidase subunit-I
(COI ) sequence fragment is typically useful for species
identification and delimitation (DeSalle & Goldstein 2019). Many
degenerate PCR primer sets are available to amplify a broad swath of
taxa, from bacteria to humans (Folmer et al. 1994; Gelleret al. 2013; Leray et al. 2013; Siddall et al.2009). While the barcode locus is successfully amplified for many taxa,
amplification and/or its utility is problematic for many others, often
for non-model organisms (Vrijenhoek & Waples 2012). One such example is
Ctenophora, since many species have extremely high rates of
mitochondrial evolution, are rich in adenine (A) and thymine (T)
residues, and have variable gene order within the mitochondrial genome,
even within a genus (Arafat et al. 2018; Kohn et al. 2012;
Pett et al. 2011; Schultz et al. 2020). A consequence of
high mitochondrial variability is that common primers are often poorly
suited to amplify the COI fragment. Unsuccessful amplification of
ctenophores by commonly used barcoding primers has a number of important
ramifications including: a lack of quick and easy molecular
identifications that results in difficulties for revealing diversity,
few ctenophore sequences in public databases, and a deficiency of easily
amplified phylogenetic and population-genetic markers.
The lack of robust COI primers and the resulting paucity of
ctenophore sequences available in public repositories also hampers our
understanding of the role of ctenophores in ocean ecology. Although they
are delicate animals, ctenophores are carnivorous and play a critical
role as predators in food webs. Despite their seemingly low nutrient
content, they are also prey for a large range of animals (Choy et
al. 2017; Thiebot & McInnes 2020; Yeh et al.
2020). Metabarcoding and eDNA studies are powerful tools to assess
community diversity, ecosystem monitoring, and function (Eble et
al. 2020). Many manuscripts have highlighted the presence and abundance
of ctenophores in zooplankton metabarcoding analyses based on 18SrDNA fragments (Günther et al. 2018; López-Escardó et al.2018; Preston et al. 2020; Schroeder et al. 2020; Yehet al. 2020); However, since the fragment of 18S is often
used in metabarcoding studies but lacks resolution to discriminate
between most species of ctenophores, often all members of the entire
phylum are lumped together (Günther et al. 2018; López-Escardóet al. 2018; Preston et al. 2020; Schroeder et al.2020; Yeh et al. 2020). In manuscripts that used multiple loci in
a metabarcoding framework, a significant proportion of 18Ssequences were from ctenophores, yet COI often failed to detect
any (Djurhuus et al. 2018; Günther et al. 2018), or the
ctenophore sequences had poor taxonomic assignments so the results were
not addressed (Pitz et al. 2020). For other environmental studies
of metabarcoding and eDNA, the entire phylum of Ctenophora is often
missing from diversity estimates (Lacoursière-Roussel et al.2018) or they are lumped in an ‘unassigned taxa’ category (Leray &
Knowlton 2015).
For this study, we designed multiple primers to amplify COI from
all major clades of ctenophores, including many deep-living, undescribed
species. We applied those primers across the phylum and tested taxonomic
assignments for various groups. Finally, as a case study, we used our
newly generated sequences as a library for an eDNA study along the
eastern Pacific coast, and provided species-level resolution for
taxonomic assignments of ctenophores.