Biodiversity of symbiotic microalgae
associated with meiofaunal marine acoelsin Southern Japan
Siratee Riewluang1 and Kevin C. Wakeman2,3
1

School of Science, Hokkaido University, Sapporo, Hokkaido, Japan
Institute for the Advancement of Higher Education, Hokkaido University, Sapporo, Hokkaido,
Japan
3
Graduate School of Science, Hokkaido University, Sapporo, Hokkaido, Japan
2

ABSTRACT

Submitted 27 June 2023
Accepted 21 August 2023
Published 5 October 2023
Corresponding author
Kevin C. Wakeman,wakeman.k@oia.hokudai.ac.jp
Academic editor
Anastazia Banaszak
Additional Information and
Declarations can be found on
page 22

Acoels in the family Convolutidae are commonly found with microalgal symbionts.
Convolutids can host green algal Tetraselmis and dinoﬂagellates within the family
Symbiodiniaceae and the genus Amphidinium. The diversity of these microalgae has
not been well surveyed. In this study, we used PCR and culture techniques todemonstrate the biodiversity of Tetraselmis and dinoﬂagellates in symbiosis withmeiofaunal acoels. Here, 66 acoels were collected from seven localities around
Okinawa, Ishigaki, and Kochi, Japan. While convolutids were heavily represented inthis sampling, some acoels formed a clade outside Convolutidae and are potentially a
new family of acoels harboring symbiotic microalgae. From the acoels collected, atotal of 32 Tetraselmis and 26 Symbiodiniaceae cultures were established. Molecularphylogenies were constructed from cultured material (and from total host DNA)
using the 18S rRNA gene (Tetraselmis) and 28S rRNA gene (dinoﬂagellates).
The majority of Tetraselmis sequences grouped within the T. astigmatica clade but
strains closely related to T. convolutae, T. marina, and T. gracilis were also observed.
This is the ﬁrst report of Tetraselmis species, other than T. convolutae, naturally
associating with acoels. For dinoﬂagellates, members of Cladocopium andMiliolidium were observed, but most Symbiodiniaceae sequences formed clusters
within Symbiodinium, grouping with S. natans, or sister to S. tridacnidorum. Several
new Symbiodinium sequences from this study may represent novel species. This is
the ﬁrst molecular record of Miliolidium and Symbiodinium from acoels. Microalgal
strains from this study will provide a necessary framework for future taxonomic
studies and research on symbiotic relationships between acoels and microalgae.
Subjects Biodiversity, Marine Biology, Molecular Biology, Plant Science, Taxonomy
Keywords Phylogenetics, Tetraselmis, Symbiodiniaceae, Photosymbiosis, Biogeography,

Taxonomy, Microscopy, Subtropical

DOI 10.7717/peerj.16078

INTRODUCTION

Copyright
2023 Riewluang and Wakeman

Acoels (Acoela, Acoelomorpha) are soft-bodied invertebrates that primarily live in marine
environments (Achatz et al., 2013). Due to their relatively simple body plan, select lineages(e.g., Symsagittifera roscoffensis) act as important model organisms in the ﬁelds ofregenerative research (Arboleda et al., 2018) and developmental (neural) sciences (Bailly
et al., 2014; Raikova et al., 2004). Acoels are also of interest in the ﬁeld of symbiosis because

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How to cite this article Riewluang S, Wakeman KC. 2023. Biodiversity of symbiotic microalgae associated with meiofaunal marine acoelsin Southern Japan. PeerJ 11:e16078 DOI 10.7717/peerj.16078

select lineages acquire and maintain photosynthetic microalgae. Within Acoela, theConvolutidae is the only family known to possess microalgal symbionts (Jondelius &Jondelius, 2020; Achatz et al., 2010). A high diversity of convolutids can be found in
subtropical/tropical waters associated with corals (Barneah et al., 2012; Kunihiro et al.,2019), as epiphytes on macroalgae (Asai et al., 2022), or living as microscopic meiofauna
between grains of sand (Thomas, Coates & Tang, 2022; Trench & Winsor, 1987).
The Convolutidae (~100 species) includes 24 genera, 19 of which have images depicting
an association with microalgal symbionts (Tyler et al., 2006–2022). Hosts typicallyassociate with one symbiont, but up to two symbionts can be found within a single host.
For example, Symsagittifera and Convolutriloba species harbor only Tetraselmis, a green
algal chlorophyte (Balzer, 1999; Parke & Manton, 1967), while Waminoa are found with
two types of dinoﬂagellates, Symbiodiniaceae and Amphidinium (Barneah et al., 2007;
Ogunlana et al., 2005). The genus Amphiscolops contain acoels (e.g., A. oni and A.
potocani) that harbor Tetraselmis and a dinoﬂagellate (Achatz, 2008; Asai et al., 2022).
Kunihiro & Reimer (2018) is the only study that has surveyed the molecular diversity of
microalgal symbionts in acoels. Although they concluded that Symbiodiniaceae within
Waminoa acoels were different to those found in the acoel’s host coral, only a single
lineage, Cladocopium, was recorded. To our knowledge, no other study has explored thebiodiversity of microalgal symbionts of acoels.
Tetraselmis (Chlorodendrophyceae, Chlorophyta) is globally distributed (John, Whitton& Brook, 2011). They are green unicellular organisms with four equal-length ﬂagella(Butcher, 1959). All known Tetraselmis (~30 species) can be found free-living, mostly inmarine and brackish environments (Arora, 2017). Mass cultivations of Tetraselmis havebeen used in applications such as wastewater treatment (Goswami et al., 2022), biofuel
production (Teo et al., 2014), and pharmacology (Schüler et al., 2020). Few (if any) of thesestudies have examined Tetraselmis from a biodiversity perspective. Therefore, thetaxonomy and systematics of this group remain unresolved. The phylogeny and taxonomy
of Tetraselmis have mainly been based on pyrenoid or ﬂagella ultrastructure, as their outerappearance can vary (Butcher, 1959; Hori, Norris & Chihara, 1982, 1983, 1986; Hori &Chihara, 1974; Marin, Matzke & Melkonian, 1993). Morphological-based phylogeny,
however, has been questioned after molecular work revealed multiple species were
genetically identical (Arora et al., 2013; Hyung et al., 2021). Although Tetraselmis is
widespread among acoels (over half of known convolutids contain Tetraselmis symbionts),
no research has been performed to clarify the diversity of Tetraselmis species naturally
associating with acoels. To date, only T. convolutae from Symsagittifera roscoffensis has
been identiﬁed to the species level (Parke & Manton, 1967).
The family Symbiodiniaceae and the genus Amphidinium are the only known
dinoﬂagellate symbionts of acoels (Taylor, 1971; Kunihiro et al., 2019). It is difﬁcult todistinguish between the two types of symbionts, as line drawings from past literature aresomewhat ambiguous and refer to both as “zooxanthellae” (Dorjes, 1968; Hyman, 1939).
Symbiodiniaceae are well known as symbionts of corals and can associate with a wide
variety of hosts including sponges (Carlos et al., 1999), molluscs (Lee, Jeong & Lajeunesse,2020), protists (Foraminifera) (Pochon et al., 2007; Pochon & LaJeunesse, 2021), and acoels
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(Taylor, 1971; Kunihiro & Reimer, 2018). As for acoel-associated Amphidinium, past
research focused largely on natural product chemistry, looking at cytological effects ofamphidinols and other polyketides extracted from mass cultivations (Satake, Murata &Yasumoto, 1991; Yang et al., 2023). One of many species used as a source of amphidinols
was Amphidinium gibbosum which is also found as a symbiont of the acoel Heterochaerus
langerhansi (Taylor, 1971). Amphidinium gibbosum is also a symbiont of Waminoa litus(Hikosaka-Katayama et al., 2012) and presumably Amphiscolops oni (Asai et al., 2022).
Symbiodiniaceae and Amphidinium have been reported as symbionts within several generaof convolutids (Kunihiro et al., 2019; Lopes & Silveira, 1994). Nevertheless, A. gibbosum isthe only acoel-associated dinoﬂagellate that has been identiﬁed to the species level (Taylor,1971; Trench & Winsor, 1987); Cladocopium is the only acoel-associated Symbiodiniaceae
genus that has been identiﬁed with genetic data (Kunihiro & Reimer, 2018).
The goal of the present study is to investigate the biodiversity of endosymbiotic
microalgae found in meiofaunal marine acoels. Here, acoels containing microalgal
symbionts were collected in subtropical localities in Southern Shikoku (Kochi) and in the
Ryukyu Islands (Okinawa and Ishigaki), Japan. Tetraselmis and dinoﬂagellates (primarily
in the family Symbiodiniaceae) were isolated from these acoels and were investigated using
PCR (18S rRNA and 28S rRNA genes) and culture techniques. Results from this work aim
to better understand acoel-microalgal symbiosis, by providing novel insights into acoel andmicroalgal biodiversity; we also aim to establish several new strains of microalgae that will
improve our current and future understanding of microalgal systematics and taxonomy.

MATERIALS AND METHODS
Sample collection, light microscopy, and establishing microalgal
cultures
Macroalgal and sediment samples were collected from intertidal and subtidal zones atseven sites in Okinawa, Ishigaki, and Kochi (Fig. 1 and Table S1) from April–October
2022. Both macroalgal and sediment samples were collected from all sites. Areas exposed
to sunlight which had seagrass and corals in the vicinity were targeted. Samples werecollected in plastic containers and transported back to the laboratory for further
processing. Acoels were detached from macroalgae and sediment samples by replacing
seawater with an isotonic magnesium chloride solution (Schockaert, 1996). The solution
was stirred and then ﬁltered through a 60–100 mm mesh. The mesh was then placed in apetri dish with ﬁltered seawater and removed after 10–20 minutes. The contents of boththe petri dish, and the material retained on the mesh, were then examined under an
Olympus CKX53 (Olympus, Tokyo, Japan) inverted microscope and Olympus SZ61
(Olympus, Tokyo, Japan) stereomicroscope. Acoels were identiﬁed based on their planula
body-shape, and other characteristics including the statocyst, eyespots, rhabdoid glandcells, and microalgal symbionts (Achatz et al., 2013; Jondelius & Jondelius, 2020). Using
hand-drawn glass capillary tubes, acoels were washed multiple times with ﬁltered seawaterto eliminate any contaminants, and isolated into 24-well tissue culture plates containing
ﬁltered and autoclaved seawater. Acoels were imaged with either an Olympus CKX53

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Figure 1 Map of Japan indicating the sampling sites. Sampling localities in Kochi (green marker),
Okinawa (blue marker), and Ishigaki (orange marker) are indicated on the map and insets. Scale bar isshown in kilometers.
Full-size  DOI: 10.7717/peerj.16078/ﬁg-1(Olympus, Tokyo, Japan), or Zeiss Axioskop 2 Plus (Zeiss, Oberkochen, Germany)
connected to a Canon EOS Kiss X8i digital camera (Canon, Tokyo, Japan). Individual
acoels were either preserved in 99.5% ethanol in 1.5 ml microcentrifuge tubes or broken
apart under a dissection microscope and the internal microalgae were transferred to
24-well tissue culture plates. A small portion of acoels (isolates 22–29) were broken andwashed in multiple wells to check for multiple symbionts. When cultures were visually
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pigmented to the unaided eye, they were transferred to small petri dishes (Tetraselmis) or
full-sized petri dishes (dinoﬂagellates). Microalgal cultures were maintained inhalf-concentration Daigo’s IMK Medium (Wako Pure Chemical Industries, Tokyo, Japan)
and were transferred to new dishes with fresh culture media every month. Where possible,
after host acoels were broken apart, host material was transferred into a 1.5 ml
microcentrifuge tube for DNA extraction. Cultures were maintained at 25  C (12:12, light:
dark hours) in Biotron Incubator LH-350 (NK Systems, Tokyo, Japan). Motile stages of
Tetraselmis and Symbiodiniaceae were mounted on glass slides and imaged under a Zeiss
Axioskop 2 Plus microscope (Zeiss, Oberkochen, Germany) connected to a Canon EOS
Kiss X8i digital camera (Canon, Tokyo, Japan).

DNA extraction, PCR amplification and sequencing
Genomic DNA from cultured microalgae and whole-acoel isolates was extracted according
to manufacturer protocols with one of the following kits: DNeasy Blood & Tissue Kit(Qiagen, Hilden, Germany), QuickExtract FFPE DNA Extraction Kit (Lucigen, Middleton,
WI, USA), or MasterPure Complete DNA and RNA Puriﬁcation Kit (Epicentre, Madison,
WI, USA) (Table S1).
For acoel isolates, nested PCR was performed on extracted DNA using generaleukaryotic primers (Tables S1 and S2), and the products were cloned with an NEB PCR
Cloning Kit (New England Biolabs, Tokyo, Japan) and then sequenced. For cultured
microalgal material, nested PCRs were performed and then directly sequenced, or cloned
and then sequenced. An additional Tetraselmis culture (K19), established from acoels
collected in 2019 from Ikenoura, was also sequenced. All PCR reactions were ﬁrst
ampliﬁed using the SR1–28-1483R primer pair and then nested. From both thewhole-acoel isolates and microalgal cultures, the 18S rRNA gene (henceforth, 18S) was
ampliﬁed for Tetraselmis using SR1–18SRF and SR4–SR12 primer pairs, and a partial 28S
rRNA gene (henceforth, 28S) was ampliﬁed for Symbiodiniaceae using the D1RF1–281483R primer pair. Apart from general eukaryotic primers, extracted host DNA was also
ampliﬁed using a speciﬁc primer pair, Tear F–Tear R, designed from the sequences
obtained from cloning. Polymerases used in the PCRs and thermocycler conditions arelisted in Table S1; primers are listed in Table S2.
Following the nested reaction, PCR products were puriﬁed using Polyethylene Glycol(PEG) and directly sequenced or cloned. Acoel DNA inserts were screened by colony PCR
using the S1512A–S1513A primer pair provided in the NEB cloning kit. Colony-PCR
products were cut using a HAEIII restriction enzyme (Takara, Shiga, Japan) following the
manufacturer’s protocol. Digested products were checked on a 1% agarose gel. A
minimum of four replicates of each banding pattern were selected. Select colony-PCR
products were puriﬁed again with PEG, and then sequenced with BrilliantDye Terminatorv3.1 (NimaGen, Nijmegen, Netherlands), according to manufacturer protocol on a 3130
genetic analyzer (Applied Biosystems, Waltham, MA, USA). Occasionally, additionalsequencing primers were used to join sequence fragments (refer to Table S2).

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Phylogenic analyses of the 18S and 28S rRNA genes
Geneious Prime 2023.1.2 (https://www.geneious.com) was used to process Sanger
sequencing reads. Cloning primers and low-quality regions (HQ% ≤ 50) were manuallytrimmed and assembled with the “De Novo Assemble” tool. Tetraselmis and dinoﬂagellatesequences were identiﬁed with NCBI’s BLAST search tool, sorted into their respective data
sets, and compiled with existing data from Genbank. Multiple Alignment using Fast
Fourier Transform (MAFFT) (Katoh et al., 2002) within Geneious was then used to alignboth data sets. Alignments were manually trimmed, resulting in a ﬁnal alignment of 1,807
bps and 1,391 bps for Tetraselmis 18S and Symbiodiniaceae 28S, respectively.
Iqtree v1.6.12 (Nguyen et al., 2015) was used to select the best-ﬁt model for bothMaximum-Likelihood and Bayesian analyses. Under the Akaike Information Criterionwith correction (AICc), the models TN+F+R2 and TIM+3+F+R3 were selected for
Tetraselmis and Symbiodiniaceae, respectively. Bootstrap analysis was performed on 1,000
pseudo-replicates. Under the Bayesian Information Criterion (BIC), the general time
reversible (GTR) substitution model was selected. MrBayes 3.2.7a (Huelsenbeck &Ronquist, 2001) was used to calculate Bayesian Posterior Probabilities. Four Markov Chain
Monte Carlo (MCMC) chains were run with the general time reversible (GTR)
substitution model for 10,000,000 generations and sampled every 100th generation.
The rate variation across sites were drawn from a gamma distribution with a proportion
being invariable (lset rates = invgamma). Other parameters were set to the default settings.
Chain convergence was checked with Tracer v1.7 and the posterior probability values wereobtained from the consensus tree after the ﬁrst 25% was discarded as burn-in. Molecular
Evolutionary Genetics Analysis version 11 (MEGA11) (Tamura, Stecher & Kumar, 2021)
was used to compute the uncorrected pairwise distance for Symbiodiniaceae sequences.
Default settings were used: no variance estimation method, uniform rates among sites, andcomplete deletion of gaps and missing data.
Sequences generated from this study were deposited to NCBI. Accession numbers arelisted in Table S1.

RESULTS
Sampling and identification of host acoels
A total of 66 acoels were sampled from seven sites (Fig. 1; Table S1). Acoels consisted offour main morphotypes that were differentiated based on symbionts, as well as features
including ocelli, rhabdoid type, and body shape. The ﬁrst (Fig. 2A) and second (Fig. 2B)
morphotype harbored exclusively Tetraselmis. These were distinguishable, based on the
numerous blunt-ended colored rhabdoids seen only in the second morphotype. A third
type of acoel harbored exclusively Symbiodiniaceae and did not have any visible ocelli orcolored rhabdoids (Fig. 2C). The fourth type were acoels collected from Ikenoura that
contained Tetraselmis and Amphidinium. These also possessed three posterior caudal lobes(Fig. 3).
Of the 66 acoels collected, 36 contained exclusively Tetraselmis symbionts (Figs. 4–6);
26 contained exclusively Symbiodiniaceae (dinoﬂagellate) symbionts (Figs. 7 and 8); one

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Figure 2 Representative light micrographs of live meiofaunal marine acoels. (A–C) Dorsal view of live
acoels. Individuals are oriented with the anterior end pointed to the right. (A) Acoel harboring Tetraselmis symbionts (T) with ocelli (Oc) on both sides of the statocyst (St), pigmentation near the mouth(M), a paired bursal nozzle (Bn), developing oocytes (O), cilia (C), and two types of rhabdoid gland cells:
sagittocysts (Sag), and translucent pigmented rhabdoid cells (Rh). (B) Acoel harboring Tetraselmis
symbionts (T) with ocelli (Oc) on both sides of the statocyst (St), cilia (C), and numerous red orange
rhabdoid gland cells (Rh) distributed throughout the parenchyma. (C) Acoel harboring Symbiodiniaceae
symbionts (S), with a statocyst (St), cilia (C), but no ocelli or colored rhabdoid gland cells. Scale bars:
100 µm.
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contained both Symbiodiniaceae and Amphidinium symbionts (Fig. 8B); and threecontained a mix of Tetraselmis and Amphidinium symbionts (Fig. 3).
Maximum-Likelihood analysis of 23 host acoel 18S sequences (Fig. 9) formed three
genetic groups that corresponded with three different morphotypes: the ﬁrst and second
morphotype within Convolutidae and the third morphotype comprising a clade with

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Figure 3 Representative light micrographs of live acoels collected from Ikenoura, Kochi and theircultured Tetraselmis and Amphidinium endosymbionts. (A) Dorsal view of an individual containing
Tetraselmis (T) and Amphidinium (A) endosymbionts. Internally, several features including the malegonopore (Mgp), seminal bursal (Sb), bursal nozzle (Bn), mouth (M), rhabdoid gland cells (Rh), statocyst(St), and ocelli (Oc) were visible. (B) General morphology of Amphidinium gibbosum in an establishedculture (K22). The operculum (Op), pyrenoid (P), and ﬂagella (F) are shown. (C) General morphology of
Tetraselmis closely related to T. convolutae in an established culture. A single pyrenoid (Py), red eyespot(E), and two pairs of ﬂagella (F) are shown. Scale bar: 100 µm (A); 5 µm (B and C).
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Mecynostomidae. Genetic data were not obtained from 43 out of 62 acoel hosts, likely due
to host DNA being lost while breaking apart host tissue to free algal symbionts.
No sequences for the fourth morphotype were obtained. Isolates within Convolutidaewith corresponding genetic data harbored either Tetraselmis or Symbiodiniaceae
symbionts, while isolates sister to Mecynostomidae harbored exclusively Symbiodiniaceae
symbionts. Information on the isolate number, symbiont type, locality, and dates can befound in Table S1.

Cultivation of Tetraselmis and dinoflagellate microalgal strains
In the majority of cases, a single microalgal culture was established from one host acoel.
Exceptions to these included isolates 19–21 collected from Ikenoura, isolate 61 (Fig. 8B)
from Shiraho, and isolates 27 and 28 (Figs. 7H and 7J) from Kabira. Isolates 19–21
contained two types of symbionts, Tetraselmis sp. and Amphidinium gibbosum (Fig. 3).
Cultures were established for both microalgae. Isolate 61 contained Symbiodinium sp. andA. gibbosum; but only Symbiodinium sp. was successfully cultured. Isolate 27 contained
Cladocopium and Symbiodinium (AIV), and isolate 28 contained Symbiodinium, subclades
AIII and AIV; all symbionts were cultured. In total, 32 Tetraselmis cultures, 26
Symbiodiniaceae cultures, and two Amphidinium cultures were established (andsequenced) from acoels (Fig. S2).

Molecular diversity of Tetraselmis
Maximum-likelihood analysis of Tetraselmis 18S sequences (Fig. 10) formed four maingroups that clustered around T. convolutae, T. astigmatica, T. marina, and T. gracilis.
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Figure 4 Light micrographs of live acoels containing Tetraselmis symbionts. (A–I) Acoels harboring
symbiotic microalgae from the Tetraselmis astigmatica clade. Acoels are oriented with the anterior to theright. (A) Isolate 4 collected from Kabira showing Tetraselmis symbionts (T), pigmentation surrounding
the mouth (M), and the statocyst (St). (B) Isolate 1 collected from Shiraho, a juvenile acoel with ocelli(Oc) located on both sides of the statocyst (St). (C) Isolate 17 collected from Mizugama. (D) Isolate 44
collected from Kabira. (E) Isolate 9 collected from Kabira. (F) Isolate 8 collected from Kabira. (I) Isolate 3
collected from Kabira. (J and K) Isolates 58 and 59 collected from Shiraho harboring Tetraselmis closely
related to T. convolutae with rhabdoid gland cells (Rh). Scale bars: 100 µm.
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The most often encountered Tetraselmis was clustered with T. astigmatica (26 cultures,and eight sequences from total-host DNA), followed by T. convolutae (four cultures, andthree sequences from total-host DNA), T. marina (two cultures), and T. gracilis (oneculture). Deeper nodes in the Tetraselmis phylogenetic analyses were largely unresolved.

Molecular diversity of Symbiodiniaceae
Maximum-Likelihood analysis of Symbiodiniaceae 28S sequences (Fig. 11) formed six
clusters across three different clades: Miliolidium (one sequence from total-host DNA),
Cladocopium (three cultures), and Symbiodinium (23 cultures, and eight sequences fromtotal-host DNA). The Miliolidium clade included isolate 16 (Fig. 7E) that grouped with ﬁve

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Figure 5 Light micrographs of live acoels with red rhabdoid gland cells in the parenchyma
containing Tetraselmis symbionts. (A–L) Acoels harboring symbiotic microalgae from the Tetraselmis astigmatica clade. Acoels are oriented with the anterior to the right. (A) Isolate 36 collected from
Kabira. (B) Isolate 35 collected from Kabira. (C) Isolate 50 collected from Kabira. (D) Isolate 49 collectedfrom Kabira. (E) Isolate 54 collected from Kabira. (F) Isolate 34 collected from Kabira. (G) Dorsal view ofisolate 48 collected from Kabira, a juvenile acoel with red rhabdoid cells (Rh), a statocyst (St), and ocelli(Oc). (H) Isolate 18 collected from Odo. (I) Isolate 52 collected from Kabira. (J) Isolate 45 collected from
Kabira. (K) Isolate 46 collected from Kabira. (L) Isolate 37 collected from Kabira, a juvenile acoel withvisible Tetraselmis astigmatica symbionts (T). Scale bars: 100 µm.
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other sequences isolated from Foraminifera and Porifera. The (uncorrected pairwise)
distance between the type species, Miliolidium leei Pochon & LaJeunesse, 2021, and the
Miliolidium sequence obtained in the present study was 0.5%. The Cladocopium clade
included strains 27.1–27.3 from isolate 27 which were sister to C. inﬁstulum, C.
thermophilum, and C. goreaui. The intra-clade distances between our sequences and these
species ranged from 1–4%. These strains had a 1.6% difference to the type species,
Cladocopium goreaui. Many sequences in Symbiodinium grouped together with S. natans(13 cultures, and two from total-host DNA); the genetic distance did not exceed 1%.
The remaining sequences formed a sister group with either S. tridacnidorum (ninecultures), S. microdriaticum (one culture and four sequences from total-host DNA
extractions), or S. pilosum (two sequences from total host DNA extractions); genetic
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Figure 6 Light micrographs of live acoels with red rhabdoid gland cells in the parenchyma
containing Tetraselmis symbionts. (A–J) Acoels harboring symbiotic microalgae from the Tetraselmis
astigmatica clade. Acoels are oriented with the anterior to the right. (A) Isolate 22 collected from Odo, ajuvenile acoel showing the red rhabdoid gland cells (Rh), statocyst (St), ocelli (Oc), and Tetraselmis
astigmatica symbionts (T). (B) Isolate 25 collected from Odo. (C) Isolate 53 collected from Kabira. (D)Isolate 41 collected from Kabira. (E) Isolate 40 collected from Kabira. (F) Isolate 39 collected from Kabira.
(G) Isolate 24 collected from Odo. (H) Isolate 32 collected from Kabira. (I) Isolate 31 collected from
Kabira. (J) Isolate 23 collected from Odo. (K and L) Isolates 57 and 5 collected from Kabira, possessing a
blunt posterior end and a statocyst (St) towards the middle of the body and containing algal symbionts
from the Tetraselmis marina clade. Scale bars: 100 µm.
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distances between our sequences and sequences they grouped with ranged between
0.5–2%, 2–3%, and 4–6%, respectively.

Phylogeny of other dinoflagellates sequenced from acoels
In addition to Symbiodiniaceae, the dinoﬂagellate Amphidinium was also observed(isolates 19–21) and cultured (isolates 61 and K22) from acoels. Amphidinium cultures
were identiﬁed to the genus level based on the presence of an operculum. The 28S
sequences from these Amphidinium isolates, 61 and K22, clustered with A. gibbosum(Fig. S1), including an A. gibbosum from the acoel Waminoa sp. (AB626894). Additionally,a Heterocapsa sequence (isolate 7) was sequenced, and was positioned sister to H.
pseudotriquetra (Fig. S3). It should be noted, however, that Heterocapsa was not cultured
or directly observed within acoels during this study.

DISCUSSION
Meiofaunal marine acoels harbor diverse lineages of Tetraselmis
In this study, we found at least four distinct lineages of Tetraselmis associated with marine
acoels. Several Tetraselmis from Kochi and Ishigaki (isolates 19–21, and cultures K19, K22,
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Figure 7 Light micrographs of live acoels containing dinoﬂagellates from the family
Symbiodiniaceae. (A–L) Acoels harboring dinoﬂagellates from the family Symbiodiniaceae. Acoels areoriented with the anterior to the right. No individuals, juvenile or adult, were observed with ocelli orcolored rhabdoid gland cells. (A) Isolate 10 collected from Blue Cave containing a statocyst (St) at theanterior end and Symbiodinium cf. natans endosymbionts (S). (B) Isolate 13 collected from Manzamo.
(C) Isolate 2 collected from Kabira. (D) Isolate 12 collected from Manzamo. (E) Isolate 16 collected from
Manzamo containing an oocyte (O), and harboring Miliolidium sp. endosymbionts. (F) Isloate 26 collected from Kabira. (G) Isolate 14 collected from Manzamo. (H) Isolate 27 collected from Kabira containing both Cladocopium and Symbiodinium endosymbionts. Rhabdoid gland cells (Rh) are needle
shaped and translucent, observed to be evenly distributed along the parenchyma. (I) Isolate 15 collectedfrom Manzamo, a juvenile with statocyst (St) visible. (J) Isolate 28 collected from Kabira. (K) Isolate 29
collected from Kabira. (L) Isolate 11 collected from Manzamo. Scale bars: 100 µm.
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Figure 8 Light micrographs of live acoels containing dinoﬂagellates from the family
Symbiodiniaceae. (A–N) Acoel harboring dinoﬂagellates from the family Symbiodiniaceae. Acoels areorientated with the anterior to the right. (A) Isolate 63 collected from Shiraho. (B) Isolate 61 collectedfrom Shiraho containing Symbiodinium and Amphidinium. (C) Isolate 42 collected from Kabira. (D)Isolate 62 collected from Shiraho. (E) Isolate 38 collected from Kabira. (F) Isolate 43 collected from
Kabira. (G) Isolate 65 collected from Shiraho. (H) Isolate 30 collected from Kabira. (I) Isolate 64 collectedfrom Shiraho. (J) Isolate 33 collected from Kabira. (K) Isolate 60 collected from Shiraho. (L) Isolate 56
collected from Kabira. (M) Isolate 66 collected from Shiraho showing translucent rhabdoid gland cells(Rh) distributed along the parenchyma. (N) Isolate 47 collected from Kabira showing a statocyst (St) and
its Symbiodiniaceae symbionts (S). Scale bars: 100 µm.
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58, and 59) were closely related to T. convolutae from Symsagittifera roscoffensis (Parke &Manton, 1967). Tetraselmis convolutae strains from S. roscoffensis from Roscoff, France(RCC1563 and RCC1564) are the only Tetraselmis to be formally described from acoels(Parke & Manton, 1967). The novel Japan isolates from this study formed a sister clade to
T. convolutae, but statistical support at shared nodes was weak. Whether Tetraselmis
strains from this study are the same species as T. convolutae (from Roscoff) is difﬁcult to

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Figure 9 Maximum-Likelihood (ML) tree of Acoelomorpha inferred from the 18S rRNA gene. Sequences generated in this study are highlighted
in colors that denote sampling times: April (blue), May (green), July (pink), and October (orange). Maximum-Likelihood bootstrap values <50 andBayesian Posterior Probabilities (BPP) <0.90 were omitted. Black dots indicate fully supported branches (100 ML/1.00 BPP). Long branches areshortened by multiples of the substitutions/site scale bar (indicated on the branch). Representative light micrographs of acoels are shown on theright. Scale bars: 100 µm.
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Figure 10 Maximum-Likelihood (ML) tree of Tetraselmis inferred from the 18S rRNA gene. Sequences generated in this study are highlighted incolors that denote sampling times: April (blue), May (green), July (pink), and October (orange). Maximum-Likelihood bootstrap values <50 andBayesian Posterior Probabilities (BPP) <0.95 were omitted. Black dots indicate fully supported branches (100 ML/1.00 BPP). Long branches areshortened by multiples of the substitutions/site scale bar (indicated on the branch). Representative light micrographs of microalgae from different
cultures are connected to their respective lineages. Scale bars: 5 µm.
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Figure 11 Maximum-Likelihood (ML) tree of Symbiodiniaceae inferred from the 28S rRNA gene. Sequences generated in this study arehighlighted in colors that denote sampling times: April (blue), May (green), July (pink), and October (orange). Maximum-Likelihood bootstrap
values <50 and Bayesian Posterior Probabilities (BPP) <0.95 were omitted. Black dots indicate fully supported branches (100 ML/1.00 BPP). Longbranches are shortened by multiples of the substitutions/site scale bar (indicated on the branch). Representative light micrographs of microalgae
from different cultures are connected to their respective lineages. Note: Clade I and Clade J are based on results from Pochon & Gates (2010), andYorifuji et al. (2021), respectively. Scale bars: 5 µm.
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conclude based on 18S data alone. Additional morphological data are needed to make a
distinction between these closely related strains.
Apart from T. convolutae, we report members of the T. marina, T. gracilis, and T.
astigmatica clades as symbionts of acoels for the ﬁrst time. Speciﬁcally, isolate 5 and culture
57 from Kabira were closely related to T. verrucosa (KX904702), a free-living strain
collected from Messolonghi, Greece (Chantzistrountsiou et al., 2016). Both Japan isolates
were well-nested within the T. marina clade (Fig. 10), with a genetic distance ranging from
0.3–1.1%. The red-orange coloration indicated that culture 57 is T. verrucosa f. rubens,
however, measurements of both the position and distribution of the chloroplast is needed
to identify these isolates (Chantzistrountsiou et al., 2016; Hori, Norris & Chihara, 1983).
Free-living T. marina and T. verrucosa f. rubens have been described (with morphologicaldata) from Japan (Hori, Norris & Chihara, 1983). The present study provides the ﬁrst
molecular data of these Japanese lineages. Isolate 24 from Odo was positioned sister to T.
gracilis (KU352751), a free-living strain collected from the Arabian Sea (unpublished
NCBI data). However, another unpublished NCBI sequence, T. gracilis (KJ756816), waspositioned in a separate clade and represents a strain from the North Sea (Butcher, 1959).
Thus, the identity of isolate 24 from Odo, Japan remains uncertain though it is likely
distinct from the T. gracilis described by Butcher (1959).
The majority of Tetraselmis sequences generated in this study clustered within the T.
astigmatica clade. Isolates from multiple sites around Okinawa and Ishigaki tended toform multiple subclades (Fig. 10, I—IV). Currently, only two sequences that represent T.
astigmatica exist (KSN-2002 and CCMP880). The strain CCMP880 (JN376804) ispresumably the same strain used in the description of T. astigmatica (Hori, Norris &Chihara, 1982, 1983, 1986; Norris, Hori & Chihara, 1980). Both sequences are from
unpublished sources. The topology of the tree shows multiple subclades closely related toT. astigmatica, but molecular phylogenetic analysis based on 18S alone lacks the resolution
to clearly resolve these (Piganeau et al., 2011). Of note are inconsistencies found between
light micrographs taken of Tetraselmis strains (culture 31, Fig. S2D) and the original T.
astigmatica description (Hori, Norris & Chihara, 1982). The most intriguing of these is thepresence of a stigma, a feature absent in the description of T. astigmatica by Hori, Norris &Chihara (1982). A more detailed investigation utilizing additional genetic markers (ITS,
psbA. and rbcL), as well as ultrastructural data, is warranted to resolve what might be a
species complex. Nonetheless, this study is the ﬁrst to report lineages in T. marina, T.
gracilis, and T. astigmatica as symbionts of acoels.

Reconciling novel molecular data into the contemporary taxonomic
framework of Tetraselmis
Assigning species names to molecular clades and reconciling novel (molecular) data within
this genus remains difﬁcult. Classiﬁcation of Tetraselmis species is based on themorphological characteristics of cells including symmetry, presence/position of the stigma,
and the ultrastructure of the pyrenoid and ﬂagella (Butcher, 1959; Hori, Norris & Chihara,
1982, 1983, 1986; Marin, Matzke & Melkonian, 1993; Norris, Hori & Chihara, 1980).
Currently, molecular data (18S) is not available for over half the accepted (~30) Tetraselmis
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species. Only three species (T. jejuensis, T. indica, and T. verrucosa) have type strains forwhich both molecular and morphological data is available (Arora et al., 2013;
Chantzistrountsiou et al., 2016; Hyung et al., 2021). Moreover, different sequences with thesame name (T. convolutae, T. gracilis, and T. chuii) appear in different clades, further
clouding Tetraselmis taxonomy (Arora et al., 2013; Hyung et al., 2021). This study
established 32 cultures of Tetraselmis from acoels providing a vital resource to address
fundamental issues surrounding Tetraselmis taxonomy and systematics.

Marine acoels also contain a diversity of dinoflagellate microalgae
The majority of dinoﬂagellate sequences generated from acoels in this study belonged to
the family Symbiodiniaceae. Identiﬁcation of these symbionts in acoels (except for those in
Waminoa) has been completely based on observational data and lacks corresponding
molecular information (Achatz, 2008; Achatz & Hooge, 2006; Hooge & Smith, 2004;
Yamasu, 1982). Kunihiro & Reimer (2018) found that Waminoa in Okinawa contained
only a single lineage of Cladocopium but speculated that acoels harbor a much morediverse range of Symbiodiniaceae.
Novel (28S) sequences generated in this study formed six clusters across Symbiodinium,
Cladocopium, and Miliolidium: four in Symbiodinium, and one each in Cladocopium andMiliolidium (Fig. 11). This study is the ﬁrst report of four lineages of Symbiodinium andMiliolidium, as symbionts of acoels. Within Symbiodinium, one cluster was positioned in
subclade AIII; the sequences were grouped with S. natans. The presence of S. natans in
cultures established from cnidarian or other metazoan hosts has been attributed to theirabundance in the environment (e.g., contamination) or possible background symbiosis.
They have also not been established as a symbiont under experimental conditions(LaJeunesse et al., 2018). This is intriguing, as acoels may be a possible reservoir, serving asa primary host for S. natans.
The remaining three clusters, one in subclade AII and two in subclade AIV, could
represent novel lineages. These latter three formed separate branches with moderate
support. The presence of S. natans in cultures established from cnidarian or othermetazoan hosts has been attributed to their abundance in the environment (e.g.,contamination) or possible background symbiosis. They have also not been established as a
symbiont under experimental conditions (LaJeunesse et al., 2018). This is intriguing, asacoels may be a possible reservoir, serving as a primary host for S. natans. Other
Symbiodinium sequences from this study did not group with known species in thephylogenetic analyses. The identity (or novelty) of these Symbiodinium strains will have to
be conﬁrmed through sequencing of a more variable genetic region (e.g., ITS), and bymorphological comparisons.
Cladocopium sequences (isolate 27) grouped with other undescribed species, separate
from C. goreaui and other described coral symbionts within that same clade. Our
Cladocopium sequences (cultures 27.1–27.3) could not be compared with symbionts of
Waminoa acoels—because different genes (ITS-2 and psbAncr) were utilized (Kunihiro &Reimer, 2018). It is important to note that a distinct strain of Symbiodinium (culture 27.4)
was also established, along with Cladocopium, from isolate 27. This potentially shows someRiewluang and Wakeman (2023), PeerJ, DOI 10.7717/peerj.16078

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adaptability in acoels, like corals that also host a variety of Symbiodiniaceae at the sametime.
A Miliolidium sequence was obtained from isolate 16. The genus Miliolidium has beenreported as a symbiont of Foraminifera and Porifera, or as free-living (Pochon &LaJeunesse, 2021). It is unclear if our isolate is novel compared to Miliolidium leei, the typespecies. While sequences in this clade are genetically divergent from each other, our data
did not have an associated culture that could aid in differentiating these species(morphologically). Still, the diversity of hosts that acquire Miliolidium make this genus aninteresting target for understanding symbiosis between microalgae, metazoans, andprotists.
Amphidinium and Heterocapsa were also sequenced in this study. Amphidinium fromisolate 61 (Fig. 8B) and those from Ikenoura (from 2019 and 2022) (Fig. 3) were identiﬁed
as A. gibbosum based on morphology and molecular phylogenics (Fig. S1). Amphidinium
gibbosum has been reported from Waminoa litus and Heterochaerus langerhansi(Hikosaka-Katayama et al., 2012; Taylor, 1971). Both Waminoa and Heterochaerus are
relatively large acoels (measuring approximately 2 mm). As Amphidinium are much larger,
in comparison to other acoel symbionts, they may occur more frequently in larger acoels.
Future collections, targeting larger acoels, may uncover a higher diversity of Amphidinium.
Heterocapsa species have never been reported as a symbiont of acoels. The single
Heterocapsa sequence in our data likely represents a food, or other environmental
contaminant, not a symbiont of acoels. Heterocapsa was neither abundant nor cultured inour experiments.

Identity of acoels belonging to Convolutidae collected in this study
Fourty-four Tetraselmis-containing acoels were collected in this study. These are all
believed to be Convolutidae, based on the presence of microalgae (Atherton & Jondelius,
2022; Jondelius & Jondelius, 2020). We observed two morphotypes, distinguished by thepresence of colored rhabdoids (compare Figs. 2A to 2B). Of the 16 Convolutidae isolatessequenced (using 18S), individuals with colored rhabdoids formed a clade consisting of 14
identical sequences. This included isolate 33 which did not possess colored rhabdoids andcontained Symbiodinium instead of Tetraselmis. This clade resembles the genus Convoluta—based on the distribution of colored rhabdoid gland cells, and body size (Hooge & Tyler,2008). Only two individuals without colored rhabdoids (isolates 1 and 17) were sequenced.
Both differed from any known Symsagittifera or Convolutriloba sequences; but they are
believed to belong to one of these families as sagittocysts were visualized in a representative
photo taken under oil-immersion (Fig. 2A). Isolates 1 and 17 (Figs. 1B and 1C) were only
imaged with an inverted microscope, and sagittocysts were not apparent. Three of theisolates from Ikenoura (isolates 19–21), although lacking corresponding sequences, wereidentiﬁed as putative Amphiscolops, based on the three posterior caudal lobes and thepresence of two symbionts (Tetraselmis and Amphidinium). This is similar to otherAmphiscolops species.
Even though a large portion of the acoels collected in this study were not sequenced, it islikely that these represent a variety of lineages in Convolutidae. For example, light
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micrographs of isolates 5 and 57 (Figs. 6K and 6L) have a sparse distribution of rhabdoids,
a blunt posterior end, and a centrally-located statocyst (not anterior) (Achatz, 2008; Achatz
et al., 2010). Similarly, isolates 58 and 59 (Figs. 4J and 4K) have notably rounded anterior
and posterior ends. Molecular identiﬁcation of acoels was challenging because hostindividuals were broken apart to establish microalgal cultures. This likely lowered thesuccess rate (~70% of hosts were not sequenced). Breaking apart the hosts also madehistological observations impossible.

A potential novel family of acoels containing Symbiodiniaceae outside
the Convolutidae
One of the more exciting discoveries from this study was a novel clade of acoels containing
symbionts that fall outside the Convolutidae. The acoels from this clade contained adiversity of Symbiodiniaceae symbionts from Symbiodinium (subclades AII, AIII, and AIV)and Miliolidium. This is potentially the ﬁrst report of non-convolutids containing
microalgal symbionts. A prominent diagnostic characteristic of the family Convolutidae isthe presence of microalgal symbionts—no other acoel family is known to containmicroalgal symbionts (Achatz et al., 2013; Jondelius & Jondelius, 2020). This novel clade(seven individuals; isolates 2, 10, 12–16) was grouped sister to the Mecynostomidae, with
only limited ML/BPP support (69/0.98). The shape and size of copulatory organs, or thepresence of bursal nozzles could not be observed under bright ﬁeld microscopy because themicroalgal symbionts were dense. Most individuals also were not fully mature. Therefore,
it was difﬁcult to compare the morphology of these outstanding acoels to a contemporary
taxonomic key (Jondelius & Jondelius, 2020). Histological slides and sperm axoneme
structure are needed to conﬁrm the identity (family) of this clade (Achatz et al., 2010).

Microalgal symbionts are diverse, but what drives preference?
The acoel-microalgal symbiosis is likely driven by the needs of the host acoel. If juvenile
acoels do not take up symbionts from the environment, they fail to fully mature (Douglas,1983). Vertical transmission of microalgae from parent acoels to offspring is rare and is
only known in Waminoa (Hikosaka-Katayama et al., 2012). Acoels provide nitrogen for
microalgal symbionts, and in return the microalgae provide necessary photosynthates forthe acoel (Meyer, Provasoli & Meyer, 1979). The mechanism behind the recognition andselection of symbionts is still unknown, however, the acoel Symsagittifera roscoffensis isselective towards T. convolutae (Arboleda et al., 2018).
Observations in the present study suggest that meiofaunal acoels prefer either
Tetraselmis or Symbiodiniaceae. Acoels appear to be repeatedly associated (loyal) with oneof these symbionts. Acoels with and without colored rhabdoids all hosted Tetraselmis
symbionts. However, isolate 33, a convolutid hosting Symbiodinium, was also observed.
Isolate 33 was genetically identical to 13 other convolutids that all harbored Tetraselmis.
This means that a single acoel species can contain either Tetraselmis or Symbiodiniaceae;
but this was not observed concurrently within the same individual.
There were subtle patterns that could be observed within our dataset at the species level.
Isolates 5 and 57, which were morphologically different from other acoels, harbored
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Tetraselmis belonging to the T. marina clade. Similar observations were made with isolates
58 and 59, with symbionts that were closely related to T. convolutae. Arboleda et al. (2018)
demonstrated that S. roscoffensis could associate with six other species of Tetraselmis, albeit
with varying levels of abundance. For example, T. rubens and T. subcordiformis appearedless dense within the host acoel, relative to T. convolutae. A larger sample size, based onmolecular data from acoel hosts, would be needed to substantiate further claims regarding
host-symbiont preference. In our study, dinoﬂagellates (Amphidinium) and Tetraselmis
appeared together in only one acoel, the putative Amphiscolops from Ikenoura. Tetraselmis
from these isolates were closely related to S. roscoffensis (Parke & Manton, 1967) andAmphiscolops oni (Asai et al., 2022). Tetraselmis found in these acoels seems to be limited
to a single lineage, but this may have resulted from a sampling bias, as Amphiscolop-likeacoels were not collected from Okinawa or Ishigaki. This association of Amphidinium andTetraselmis might also be attributed to the relative size of the acoel (e.g., Amphiscolops)(Achatz, 2008; Asai et al., 2022; Ogunlana et al., 2005; Trench & Winsor, 1987).
Regarding seasonality, no clear patterns were observed with respect to either Tetraselmis
or Symbiodiniaceae. While seasonality cannot be completely disregarded as a driver ofsymbiotic preference, regular sampling at set locations targeting the same acoel species
would have to be performed, to address such questions.

CONCLUSIONS
Meiofaunal marine acoels in Japan host a variety of Tetraselmis and Symbiodiniaceae
species. The majority of host acoels collected in this study were members of the family
Convolutidae. We also introduce intriguing evidence that there may be a family outside ofConvolutidae that harbors microalgal symbionts. Our survey of symbiotic microalgae
found at least four lineages of Tetraselmis from acoels, including lineages closely related toT. convolutae, T. astigmatica, T. marina, and T. gracilis. This is the ﬁrst report of
Tetraselmis species, other than T. convolutae, as symbionts of acoels. Analysis of thesesequences further highlights the need for taxonomic and systematic revision of
Tetraselmis. Symbiodiniaceae from acoels included Symbiodinium lineages in subclades
AII, AIII, and AIV, as well as Miliolidium and Cladocopium. Lineages from Symbiodinium
and Miliolidium were identiﬁed for the ﬁrst time as symbionts of acoels. We also were able
to culture multiple lineages of Symbiodiniaceae from a single acoel. This shows that acoels
containing multiple types of Symbiodiniaceae do exist, but their prevalence is unknown.
Therefore, the true diversity of these acoel-dinoﬂagellate associations is likely still
underestimated. Further genomic sequencing information would shed light on this issue.
Strains of Tetraselmis and Symbiodiniaceae established in this study will be an important
tool for taxonomic and experimental work. Future studies should focus on theidentiﬁcation of both acoels and symbionts using molecular data. This would be useful inmapping out the selectivity, seasonality, feeding ecology, and the symbiotic relationshipbetween acoels and their microalgae.

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ACKNOWLEDGEMENTS
We would like to express our gratitude to the Takehara Marine Laboratory, Hiroshima
University (Dr. Yusuke Kondo and Dr. Susumu Ohtsuka) for providing laboratory space
during our sampling in Hiroshima/Kochi. We would also like to thank the members of the
Wakeman Lab (Eric Odle and Dr. Samuel Kahng), Biodiversity Division (Dr. Hiroshi
Kajihara) at Hokkaido University, Dr. Yonas Tekle at Spelman College, and Dr. Jonathan
Banks for taking the time to read through drafts of the manuscript and/or provide
constructive feedback throughout the course of this project.

ADDITIONAL INFORMATION AND DECLARATIONS
Funding
This work was supported by the Japanese Society for the Promotion of Science (No.
18K14774). The authors were also supported by the ISP program at Hokkaido University.
There was no additional external funding received for this study. The funders had no rolein study design, data collection and analysis, decision to publish, or preparation of themanuscript.

Grant Disclosures
The following grant information was disclosed by the authors:
Japanese Society for the Promotion of Science: 18K14774.
ISP program at Hokkaido University.

Competing Interests
The authors declare that there are no competing interests.

Author Contributions
 Siratee Riewluang conceived and designed the experiments, performed the experiments,
analyzed the data, prepared ﬁgures and/or tables, authored or reviewed drafts of thearticle, and approved the ﬁnal draft.
 Kevin C. Wakeman conceived and designed the experiments, performed theexperiments, analyzed the data, prepared ﬁgures and/or tables, authored or reviewed
drafts of the article, and approved the ﬁnal draft.

Field Study Permissions
The following information was supplied relating to ﬁeld study approvals (i.e., approving
body and any reference numbers):
Hokkaido University; No ﬁeld permits were required for this study.

DNA Deposition
The following information was supplied regarding the deposition of DNA sequences:
The ribosomal sequences generated in this study are available at NCBI GenBank:
OQ439302; OQ439515–OQ439516; OQ449206–OQ449249; OQ449250–OQ449284;
OQ779018–OQ779040.
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Data Availability
The following information was supplied regarding data availability:
The sequence data, sample data, and the primer data are available in the Supplemental
Files.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.16078#supplemental-information.

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