Li and Endo Progress in Earth and Planetary Science
https://doi.org/10.1186/s40645-020-00384-6(2020) 7:70

Progress in Earth andPlanetary Science

RESEARCH ARTICLE

Open AccessPhylogenetic positions of “pico-sized”
radiolarians from middle layer waters of thetropical Pacific
Luyan Li1 and Kazuyoshi Endo2*

Abstract
In order to determine whether truly pico-sized adult radiolarians exist, we compared spumellarian sequences fromindividual adult samples collected in the central Pacific, with filtered sea water samples of juvenile (5–42 μm) andgamete (0.2–5 μm) sized fractions to see whether the gene sequences are similar or different. Environmental
spumellarian-affiliated sequences we sampled were mostly concentrated in samples from 250 to 400 m depth andonly appeared in the RAD-III clade, which corresponds to the family Astrosphaeridae (including Arachnosphaera,
Astrosphaera, and Cladococcus). None of the same ITS (internal transcribed spacer) sequences were found in both
filter membranes of the same sea water samples. Pairwise distances among these environmental spumellarianaffiliated sequences are within or slightly above the range of intra-morphospecific variations. We propose a model
to explain our observations based on the hypothesis that the “pico-sized radiolarians” represent gametes ofradiolarians of normal size, assuming different sinking speeds of parents and offspring.
Keywords: 18S rDNA, Phylogenetic tree, Genetic distance, Gametes, Pico-sized spumellarians

1 Introductioncommonly observed stages (Kimoto et al. 2011; Yuasa and
Takahashi 2014, 2016).

1.1 What are radiolarians?

Radiolarians are marine protist zooplankton, belonging to
the classes Acantharea, Taxopodia, and Polycystinea (Adlet al. 2019). Extant Polycystinea are further subdivided
into the orders Collodaria, Nassellaria, and Spumellaria(Cavalier-Smith et al. 2018; Adl et al. 2019). Extant Entactinaria of De Wever et al. (2001) are not regarded as anestablished taxon in the currently accepted taxonomy, due
to the lack of a connection with true Devonian Entactinaria (Nakamura et al. 2020). Their sizes range from tens
to hundreds of micrometers (Suzuki and Not 2015). In
general, adult specimens are around 100–300 μm. Lifecycles of radiolarians are largely unknown. Small, flagellated reproductive cells (swarmers) are the most

* Correspondence: endo@eps.s.u-tokyo.ac.jp
2
Department of Earth and Planetary Science, University of Tokyo, 7-3-1
Hongo, Tokyo 113-0033, Japan
Full list of author information is available at the end of the article

1.2 Why we focus on the pico-sized range?

DNA sequences identified with radiolarians have beenfound in the pico-sized fraction of marine environmental
libraries sampled at the surface to thousands of meters
below (< 2 μm) (Not et al. 2007) and from various geographic locales: Sargasso Sea (< 2 μm) (Not et al. 2007),
Arctic Ocean (3–0.22 μm) (Lovejoy et al. 2006), and theAntarctic Ocean (5–0.2 μm) (Lopez-Garcia et al. 2001).
Do living pico-sized radiolarian cells really exist, as implied by some published reports? The smallest polycystines confirmed by molecular phylogenetic analysis are
reproductive swarmers 1.6–3 μm × 2.5–10 μm long
(Kimoto et al. 2011; Yuasa and Takahashi 2014, 2016),
small enough that they can just pass through 2–5 μm
membrane pores. These sequences may also have comefrom unknown “new” species that are genuinely “picosized” as adults. Focusing on “pico-sized plankton,”© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
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(2020) 7:70

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considerable percentages of environmental radiolariansequences have been found in the South China Sea(SCS) and the Atlantic and Pacific Oceans in filteredseawater samples: 15 to 20% at 75 m depth (locality A1)
near the basin of SCS, 5 to 10% at 75 m depth (South
East Asia Time-series Study station) (Wu et al. 2014b),
12 to 25% in clone libraries from depths of 60 m at fivesites along a south–north transect at 155° E (Wu et al.
2014a), and 14 to 15% in full-length 18S rDNA sequence
data from the North Atlantic and Pacific Oceans (Lieet al. 2014). In those studies, large plankton were removed by filtration, but plankton filtered at different
mesh diameters were not compared.

to the secular changes of the paleoenvironment (DeWever et al. 2001).

Li and Endo Progress in Earth and Planetary Science

1.3 What are the environmental radiolarian groups?

By phylogenetic analyses of 18S rRNA gene clone libraries, five environmental radiolarian clades, RAD-I to
RAD-V, have been identified in the Sargasso Sea (Notet al. 2007). However, none of them completely matchessequences of individually isolated specimens. We do notknow whether these environmental sequences represent“unknown” species. RAD-I and RAD-II belong to the
Acantharea. It was suggested by Decelle et al. (2012) thatthe clade, Acanth II, corresponding to RAD II, is represented by the family Acanthoplegmidae. The taxonomic
status of the environmental clade, Acanth I, corresponding to RAD I, remains undetermined. RAD-IV and
RAD-V are composed of Taxopodida-like sequences.
The Taxopodida are very fragile, with easily dissolved
skeletons (Suzuki and Not 2015). A third environmental
clade, RAD III, is neighbor to the spumellarian family
Spongodiscidae and comprises deep Sargasso Sea sequences and sequences from the South China Sea (Yuanet al. 2004; Li et al. 2011). Among these five clades, RAD
III most likely consists of spumellarians with siliceous
skeletons, which can be preserved as fossils. In a reclassification study of Spumellaria, 1165 environmental sequences affiliated with the Spumellaria were included ina phylogenetic analysis (Sandin et al. 2020). Most environmental sequences analyzed by Sandin et al. (2020) are
related to the superfamily Liosphaeroidea (Matsuzaki
et al. 2015), a clade named EnV5 by Sandin et al. (2020).
Considering its phylogenetic position relative to others,
Env5 in Lineage II corresponds to RAD III, named by
Not et al. (2007).
1.4 Why we are interested in spumellarians

Spumellaria and Nassellaria have morphologically complex siliceous architectures, and the fossil record of theformer first appeared in the Cambrian (Ma et al. 2019;
Zhang and Feng 2019), while the record of the latterdates to the late Devonian (Suzuki and Not 2015; Sandin
et al. 2019). These fossil records provide an opportunity
to understand how polycystines evolved, corresponding

1.5 Previous efforts to find tiny adult radiolarians and the
purpose of our study

Fossil radiolarians have been conventionally studied using
63 μm sieves to concentrate larger specimens. Smaller
sieves (45 μm or even 38 μm) are regularly used to collectsmall species and small specimens (Matul et al. 2002; Itaki
et al. 2003). Considerable effort to find tiny adult radiolarians (~ 5–45 μm in diameter) has been expended since thenineteenth century (Haeckel 1887; Frenguelli 1940, 1941;
Swanberg and Bjørklund 1987; Takahashi and Honjo
1991); however, they only illustrated juveniles of Spumellaria and Nassellaria. No papers reported pico-sized radiolarians (< 5 μm). Therefore, we set out to determine
whether pico-sized radiolarians exist, using a molecularbiological approach, by analyzing environmental DNA collected by filtration of seawater.
Radiolarians are indicators of water masses having specific temperatures and salinities (Kamikuri et al. 2008;
Suzuki and Not 2015). RAD-III sequences have been reported from warm, oligotrophic blue waters (Not et al.
2007; Li et al. 2011). Thus, we collected samples in theCentral Equatorial Pacific in this study. Primers specificonly for spumellarians were newly designed in this study.
In the same water column, single-cell samples were obtained to compare their sequences with those of environmental DNA samples. We separated samples into
three different size classes: adult (> 42 μm), juvenile (5–42 μm), and gamete or pico-sized (0.2–5 μm) samples.

2 Materials and methods
Seawater samples were collected during Cruise KH10-4 ofthe R/V Hakuho-maru operated from September to October 2010. Five stations (A, C, D, E, F) were located in thetropical Pacific (Fig. 1). Seawater was collected using a 10
L Niskin bottle, at 4 or 5 depth strata for each station.
In order to avoid contamination with free DNA from disintegrating organisms, a 42 μm mesh screen was used to remove adults, and subsequent filtrations were performed
under low pressure (< 1 kPa). Two different filters were used:
5 μm MF-Millipore SMWP and 0.2 μm Whatman nuclepore
PC, allowing us to fractionate the samples into juvenile andpico-sized subsamples, respectively. Total environmental
DNA was extracted using a Power Water DNA Kit (MOBIO). Primers specific for spumellarians were newly designed(SITSF 5′CAGCGACGTGTCATTCAAATTTC3′ and SITS
R 5′GCAGTCCCAAGCAACACGACTC3′). The 2.5 kb
rDNA cistron (18S-ITS1-5.8S-ITS2-28S) regions of environmental DNA were amplified. PCR products were cloned
using a Mighty TA cloning kit (TaKaRa). All clones were sequenced with the primers M13 RV and M13 M4.

Li and Endo Progress in Earth and Planetary Science(2020) 7:70

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Fig. 1 Location of the 5 sampling sites in the Central Pacific Ocean

Preliminary taxonomic affiliations of the sequenceswere assessed using BLASTN against GenBank database.
Phylogenetic relationships of the 18S rDNA part of sequences were inferred by ML analysis with IQ-Tree 2(Minh et al. 2020) and Bayesian analysis with MrBayes
Version 3.2.7 (Huelsenbeck and Ronquist 2001; Zhang
et al. 2020). Genetic distances between 18S-ITS1-5.8SITS2-28S sequences were calculated with the distancemodule of Mega X (Kumar et al. 2018). Sequences provided in this paper have been submitted to the NCBI nucleotide sequence database under accession numbers:
KP175029-KP175040.

3 Results and discussion
Results of our search for “pico-sized” spumellarians are
summarized in Table 1. The tree in Fig. 2 shows phylogenetic positions of DNA sequences isolated fromsingle-cell samples (Fig. 3) and membrane-filtered seawater samples. Figure 4 illustrates more detailed relationships between the DNA sequences from themembrane-filtered seawater samples and those available
in public databases.
We found environmental spumellarian-affiliated sequences in the filter membranes at only two sites: a

subsample in the size range between 42 and 5 μm (juvenile size) from 400 m at site C, and subsamples of bothjuvenile and gamete sizes from 250 m depth at site D(Table 1).
The 18S rDNA phylogenetic tree is shown in Fig. 2.
Environmental spumellarian-affiliated sequences from
Pacific seawater samples only appeared in the RAD-III
clade named by Not et al. (2007) for environmental sequences from size-fractionated samples (< 2 μm) fromthe Sargasso Sea. Our spumellarian-specific primers
yielded positive results from spumellarians collected atsite D, but those species do not belong to RAD III:
Heliodiscus asteriscus D397, Spongotrochus? aff. Glacialis
D188, Pylonioidea gen. et sp. indet. D401 and D382
(Figs. 2 and 3), in contrast to PCR products amplified
from total environmental DNA, which yielded sequencesthat belong only to RAD III.
Using more RAD III sequences stored in public databases to cover a broader range of different localities, aphylogenetic tree was constructed by focusing on RAD
III (Fig. 4). RAD III sequences can be clearly divided into
several subclades, three of which are represented by anamed taxon (Ar. myriacantha-like clade, As. hexagonalis-like clade, and C. viminalis-like clade). The fourthLi and Endo Progress in Earth and Planetary Science(2020) 7:70

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Table 1 Results of molecular cloning for the environmental DNA from five sites
Site

Depth (m)

A

1000

A

A

C

C

C

C

D

D

D

D

E

E

E

E

F

F

F

CTD information

400

120

Chlorophyll max

750

400

150

100

Chlorophyll max

400

250

120

90

Chlorophyll max

400

150

105

70

Chlorophyll max

400

200

130

Chlorophyll max

Volume of filtered seawater (L)DNA (ng/μl)

PCR

5

A

2.6

−

5

B

183.4

−

5

A

47.0

−

5

B

56.5

−

5

A

77.9

+

5

B

48.3

−

5

A

94.0

−

5

B

197.0

−

5

A

113.8

+

5

B

96.1

−

5

A

79.7

+

5

B

57.0

−

5

A

64.3

+

5

B

100.8

−

5

A

50.8

−

5

B

22

−

Clone number

Spumellarian clones

0

5

3

0

0

5

A

58.6

+

11

2

5

B

15.8

+

18

1

0

5

A

78.4

+

5

B

23.7

−

5

A

39.9

+

7

0

5

B

33

+

8

0

5

A

74.0

−

5

B

31.5

−

5

A

62.8

−

5

B

18.0

−

5

A

47.0

+

5

B

30.4

−

5

A

53.1

−

5

B

39.5

−

5

A

5.6

−

5

B

15.6

−

5

A

89.1

−

5

B

60.6

−

5

A

93.9

−

5

B

23.4

−

0

Site A: Sep 20, 2010, 10:30 PM, N 14′ 00″, W 162′ 05″, depth 5662 m; site C: Sep 22, 2010, 10:30 PM, N 07′ 09″, W 166′ 33″, depth 4933 m; site D: Sep 23, 2010, 9:00
PM, N 2′ 42″, W 165′ 33″, Depth 5346 m; site E: Sep 29, 2010, 8:14 PM, N 05′ 40″, W 164′ 13″, Depth 4874 m; site F: Oct 9, 2010, 9:00 PM, N 16′ 492″, W 172′ 30″;
Depth 5299 m
CTD is an oceanography instrument used to determine the conductivity, temperature, and depth of the ocean; A and B represent the following fractions: 42 μm >A > 5 μm, 5 μm > B > 0.2 μm. The value “+” in PCR column means a positive result with spumellarian-specific primers. Clone column means the number of M13primer, PCR-positive white colonies. The value in spumellarian clones means the number of spumellarian clones that were confirmed by sequencing

major clade includes no identified species (denoted here
as clade A). The phylogenetic position of Cladococcus
viminalis within the C. viminalis-like clade has not been
well resolved, showing a polychotomous relationshiptogether with a cluster of sequences from 100 m depthsof SCS (Fig. 4). The deep-sea sequences seem to be clustered mainly in clade A and in the C. viminalis-likeclade. Five of our environmental sequences (C400A3,

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Fig. 2 Phylogenetic tree of spumellarians based on 18S rDNA sequences. Sequences obtained from the Central Pacific Ocean in this study are in
bold. Numbers at nodes represent posterior probabilities (PP) of Bayesian inferences, bootstrap values (BV) of the ML analysis. The scale bar in theupper left corner is the substitution rate per site for the Bayesian analyses. Sequences obtained in this study are in bold

Fig. 3 Scanning electron microscope photos of a D188 and b D397. The scale bar in the bottom of photo represents 100 μm

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Fig. 4 A phylogenetic tree of RAD III based on 18S rDNA sequences. Sequences obtained from the Central Pacific Ocean in this study are in bold.
Numbers at the nodes represent posterior probabilities (PP) of the Bayesian inferences, bootstrap values (BV) of the ML analysis. The scale bar in
the upper left corner is the substitution rate per site for the Bayesian analyses. Sequences obtained in this study are shown in bold

C400A31, C400A171, D250A6, and D250B1) clusteredin the C. viminalis-like clade and two (C400A27 and
D250A8) somewhat separately in clade A and the Ar.
myriacantha-like clade (Fig. 4).
We did not find the same ITS (internal transcribedspacer) sequences in juveniles and pico-sized subsamples. The largest of the 18S-ITS1-5.8S-ITS2-28S distances in those environmental spumellarian-affiliatedsequences is in the range of 0.070 (between D250A6 and
C400A31), no different than the interspecific distanceobserved within a family (Ando et al. 2009; Krabberød
et al. 2011). The lowest is 0.008, between C400A3 and
C400A31, which might have come from two different“juvenile”-filtered subsamples smaller than 42 μm. The
distance between D250B1 and D250A6 is 0.062, being
close to the distance between D250B1 and D250A8(0.064). This value is close to interspecific distancesamong spumellarians isolated in site D in this study:
0.083, 0.084, and 0.010 in Hexalonchidae; 0.096 in
Pyloniidae.
No identical rDNA sequences were found in both filter
membranes used for the same sea water samples. Radiolarian skeletons are quite fragile; however, for three reasons, we think it is unlikely that the pico-sized
sequences could have come from broken cells. First, a
couple of previous studies (Diez et al. 2001; Not et al.
2007) indicated that the sequences of known multicellular or larger single-celled organisms constitute very small
percentages of the pico-sized environmental samples(2.5% and 1.7%, respectively). Second, astrosphaerid cells
are not as fragile as their skeletons. Radiolarian DNA sequences we obtained from the membranes are mainlyLi and Endo Progress in Earth and Planetary Science(2020) 7:70

concentrated in the RAD-III clade of Not et al. (2007) or
Lineage II of Sandin et al. (2020). Thus, those sequences
could have originated from broken cells of astrosphaerids. However, the central capsule of astrosphaerids is solarge (80–250 μm in diameter), robust, and rigid that it
cannot be broken easily (Hollande and Enjumet 1960;
Suzuki and Sugiyama 2001). Besides, astrosphaerids are
generally tangled with organic matter to form aggregatesor “meat balls” up to 1 cm in diameter when collected(Zhang et al. 2018), so the material inside the capsularmembrane basically cannot trickle out. Therefore, we
consider it unlikely that the sequences we obtained arecontaminants from broken adult astrosphaerid cells.
Third, we carefully treated the samples to avoid damaging the cells, eliminating the possibility, as far as possible, that DNA could be released from radiolarian cells
broken during filtering. As a consequence of doing filtration at low pressure to prevent crushing the radiolarians,
the amount of sample we collected was very limited.
That might be the reason why we obtained only several
clones from the filtered samples, making it difficult tofully assess the genetic diversity of sequences in the
RAD-III clade, which now embraces a considerable
number of environmental sequences stored in public databases (Fig. 4).
Pairwise distances among these environmental
spumellarian-affiliated sequences (0.008–0.060) are withinor slightly above the range of intra-morphospecific variation. For example, Dictyocoryne truncata (Ando et al.
2009; Krabberød et al. 2011) and Actinomma boreale(Krabberød et al. 2011) have been sequenced to estimate
intraspecific distances (0.017 and 0.005, respectively).
These juvenile sequences from site C may have originatedfrom different individuals of an unidentified small (5–42 μm) spumellarian species.
Another possible origin of pico-sized radiolarian sequences could be the pico-sized swarmers or gametes ofradiolarians. But the ITS region sequences of rDNA sequences we found in juveniles and pico-sized subsamples are different. One possibility for this difference
between juvenile and pico-sized subsample sequences isthat spumellarians have intragenomic ITS sequence variation. Intraspecific variations in the ITS region of radiolarians were analyzed and quantified by Ando et al.
(2009), Krabberød et al. (2011), and Ishitani andTakishita (2015), but no intra-individual differences havebeen studied in radiolarians. ITS sequences evolve fasterthan 18S or 28S rRNA gene sequences and are mostcommonly used as markers to discriminate species. Multigenic family members of ITS regions are subject toconcerted evolution, which homogenizes their sequences(Dover 1982). However, exceptions have been observed,
and intragenomic polymorphism of ITS has been detected in many species (Wang et al. 2017; Matthias et al.

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2018; Itskovich 2020), including even the Foraminifera(Pillet et al. 2012), which, together with the Radiolaria,
belongs to the Retaria. If spumellarians produce gametes
showing intragenomic polymorphism, the ITS sequencesfrom each reproductive cell and juvenile could differ.
This would even apply to ITS sequences of the same 18S
rDNA sequences, but in radiolarians, such intracellularvariability is very limited (Decelle et al. 2014). In contrast, even the 18S rDNA sequences in our samples differ between juvenile and pico-sized subsamples. Thus, itappears likely that the different ITS sequences represent
different species. Since no molecular data are available
for any of the 23 known species belonging to the Astrosphaeridae, these different 18S rDNA sequences frommembrane samples could conceivably be pico-sized
swarmers or gametes of different astrosphaerid species.
The observation that no identical sequences were
found among adult, juvenile, and pico-sized subsamples
can be explained by the “gametes hypothesis,” assuming
different sinking speeds of parents and offspring (Fig. 4).
According to the results of our phylogenetic analysis(Figs. 2 and 3), only three identified species fell into the
RAD III clade. They are Astrosphaera hexagonalis, Arachnosphaera myriacantha, and Cladococcus viminalis, inthe size range 80–150 μm. They were recorded by
Haeckel in the nineteenth century from warm surface
waters (Boltovskoy et al. 2010). Living astrosphaerids
have been collected even at depths of 500–1000 m fromFebruary to May in the Mediterranean (Hollande andEnjumet 1960). Obviously, they are not pico-sized radiolarians, but they could be the mother cells of “pico-sizedradiolarians.” Due to differences in size and structure,
the forces that mother cells and swarmer cells experience in seawater would be different. Radiolarians float
passively in convection currents of the mixed layer byhydrodynamic drag, due to their elongated pseudopodia(Ichinohe et al. 2019). Swarmers with celestine crystals
inside quickly sink (Anderson 1983; Yuasa and Takahashi 2014).
In general, plankton exhibit three kinds of movementin the ocean that need to be considered to infer thewhereabouts of radiolarians: (1) movement driven by the
daily migration of nektons between 0 and 500 m, whichswim to deeper waters in the daytime, presumably toseek refuge from large predators, and return to the surface at night to feed (Bianchi et al. 2013). Mother cells
would be affected by this kind of process, but skeletonsof dead cells would not. Swarmers that do not haveenough buoyancy would not be able to return to the sea
surface. We collected all our samples at night. Thus,
mother cells should have been near the surface. (2) Vertical movement of water masses with cold or warm eddies that occur everywhere at bi-weekly to seasonalscales. This is a passive, but very powerful movement.

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Eddy diffusivities for the east Pacific sector range from −0.2 to 0.1 m s−1 (Klocker and Abernathey 2014). Mothercells, skeletons, and swarmers of radiolarians would beequally subject to this kind of movement. This might be
the reason why astrosphaerids can be collected at water
depths from 500 to 1000 m. (3) Pacific circulation, whichforms vertical profiles of equatorial zonal velocity, withthe peak appearing at 100 to 200 m above 0.50 m s−1 at alongitude of 170° W (Johnson et al. 2001). Mother cells
in the surface water (0–500 m) would be greatly affectedby this circulation, while it would have no effect onswarmers and skeletons that have sunk to more than
500 m. Some observations in an experimental study(Yang et al. 2020) concluded that cyclonic eddies enhance the looping path, but anticyclonic eddies decrease
it. If cyclonic eddies occur in a sampling site, the separation of mother cells and swarmers would increase.
Combining the effects of those forces, Fig. 5 shows situations that would occur under our “gametes hypothesis.”
When the research vessel stopped at site A2 and sampled swarmers at B2, the skeletons of the dead mothercells would have arrived at point B1. At this time, immature cells in this population would have floated alongwith the current and have arrived at site A3. Therefore,
in the sample at point B2 of site A2, it would be difficult
to find gametes together with adults or juveniles of thesame species. This model would also explain highpercentages (ca. 20%) of radiolarian DNA recorded insome deep water (500–3000 m) environmental samples(Not et al. 2007) (B3 in Fig. 5).
Still another possibility, especially for those specimensthat did not cluster with known shallow water species(such as C400A27), is that the sequences we found in
the pico-sized samples indeed represent pico-sized adultradiolarians.
A peculiarity of RAD III is that the three identified
species in it belong to the family Astrosphaeridae, theonly family among extant spumellarians with no structure inside the cortical shell (De Wever et al. 2001; Sandin et al. 2020). Other groups of Spumellaria havevarious internal structures. Further studies on unidentified astrosphaerid species may help to reveal evolutionary relationships among radiolarians with and without
internal structures and to understand how the initial
spicular system developed and evolved. The existence ofso many potential astrosphaerid environmental sequences indicates that a number of undescribed astrosphaerid species await morphological studies. It would
be ideal to observe directly the morphogenesis of siliceous skeletons in those radiolarians by capturing themother cells of RAD III in surface waters of deep-sea
sites with rich environmental sequences.
Our results (Table 1) indicate that juvenile (5–42 μm)-, and gamete (0.2–5 μm)-sized spumellarians are

Li and Endo Progress in Earth and Planetary ScienceFig. 5 Diagram showing a model based on the hypothesis that “pico-sized radiolarians” represent radiolarian gametes. Three kinds of movementin the ocean should be considered: ① the daily migration of nektons between 0 and 500 m; ② vertical movement of water masses with cycloniceddies; ③ pacific circulation. We show a case in which cyclonic eddies occurred at a sampling site (night in the Northern Hemisphere), withcyclonic eddies enhancing the looping path, and increasing the distance between mother cells and swarmers

Li and Endo Progress in Earth and Planetary Science(2020) 7:70

not restricted to particular water depths. Still, some discernible patterns exist. They are often found 100 to 300 m
below the highest chlorophyll zone. Although the PCR results for samples with the highest chlorophyll peak werepositive, cloning and sequencing showed that they were
not spumellarian sequences but false positives caused bymismatches to other plankton sequences. The greatest
depth of chlorophyll at site D was shallower than that atsite C, and the spumellarian sequences also appeared at ashallower depth. But the two phenomena are not parallel.
Swarmers continually sink in the sea, so they do not concentrate at a certain depth. For the three reasons discussed
above, juvenile cells can also migrate up or down. The
deep sea below 1000 m is a blank that we have not explored. The presence of RAD III in the deep sea can beseen from clade A (Fig. 4), or from records in the Sargasso
Sea (Not et al. 2007). Their distributions and morphologies are also worthy of further study.
One possible way to clarify the origin of “pico-sized”
sequences would be to do hybridization in situ. Studies
have been done (Gilg et al. 2010) by hybridization to see
whether they can catch unidentified acantharian cells.
Samples collected at 500 m successfully hybridized to
the UC1 (corresponding to RAD I) CARD-FISH probe.
But the difficulty of studying living radiolarians is that
until now, no lab has been able to culture radiolarians
from swarmers to next generation adults. Thus, whether
or how their genomes change during reproduction,
when their skeletons first form, is very hard to know.
Combinations of whole-cell in situ hybridization andflow cytometry may be the solution. For in situ
hybridization, fluorescent oligonucleotides derived fromthe 18S rRNA sequences may be used as probes. For
bacteria, a combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry has been
employed by Fuchs et al. (1998) and Fernández-Lagoet al. (2000). Therefore, we think it is theoretically practical to carry out similar assays with plankton. Our sequences offer some help to design the probes. The
combination of these technologies will not only allow us
to solve the problem of “pico-sized” radiolarians, butalso to recover all “the fish that miss the net” (Biardet al. 2016). Even though use of fine sieves will help collect more living and fossil radiolarians, true radiolarian
diversity would still be underestimated.

4 Conclusions
Considering the phylogenetic positions of the environmental sequences, as well as the genetic distances toknown species, the most plausible explanation for “picosized radiolarians” is that they represent gametes of radiolarians of a normal size. The existence of a large number of environmental sequences in warm deep-sea water
indicates that our understanding of life cycles andPage 9 of 10

morphological diversity of Spumellaria is inadequate.
Most of these unknown sequences can be attributed to
the family Astrosphaeridae, which is of great significancefor understanding evolution of radiolarian skeletons.
Abbreviations
18S rDNA: 18 subunit ribosomal DNA; DNA: Deoxyribonucleic acid;
ITS: Internal transcribed spacer; RAD: Radiolarian clade
Acknowledgements
We are grateful to the captain, crew, and scientists of the cruise KH10-4 of R/
V Hakuno-Maru for their assistance in plankton and seawater sampling. We
thank Drs. Yoshiyuki Ishitani and Yurika Ujiie for their comments on the
manuscript and their support in collecting samples. In addition, we also
thank Dr. Ishitani and the Kochi Core Center (Kochi University/JAMSTEC) for
taking SEM photos.
Authors’ contributions
LL initiated this study and was primarily responsible for the sequence
analysis. KE hosted LL, and they designed the experiments together. Bothauthors contributed to writing the paper. The authors read and approved
the final manuscript.
Funding
This study was funded by the Japanese Society for the Promotion of Science(JSPS) 11P11741 and was carried out at the University of Tokyo.
Availability of data and materials
Datasets supporting this article are included within it. Sequences discussed
in this paper can be found in Genbank (accession numbers: KP175029KP175040).
Competing interests
The authors declare that they have no competing interest.
Author details
1State Key Laboratory of Palaeobiology and Stratigraphy (SKLPS), Nanjing
Institute of Geology and Palaeontology, Chinese Academy of Sciences (CAS),
Nanjing 210008, China. 2Department of Earth and Planetary Science,
University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
Received: 12 August 2020 Accepted: 28 October 2020

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