Protist 172 (2021) 125808
http://www.elsevier.de/protis
Published online date 13 May 2021

ProtistORIGINAL PAPER

Ecology, Morphology, Phylogeny andTaxonomic Revision of Giant Radiolarians,
Orodaria ord. nov. (Radiolaria; Rhizaria; SAR)
Yasuhide Nakamura a,b,1, Akihiro Tuji b, Katsunori Kimoto c, Atsushi Yamaguchi d,
Rie S. Hori e, and Noritoshi Suzuki faEstuary Research Center, Shimane University, 1060, Nishikawatsu-cho, Matsue 690-8504, Japan
Department of Botany, National Museum of Nature and Science, 4-1-1, Amakubo, Tsukuba 305–0005, Japanc
Research Institute for Global Change, Japan Agency for Marine-Earth Science and Technology (JAMSTEC),
2-15, Natsushima-cho, Yokosuka 237–0061, Japand
Plankton Laboratory, Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1, Minato-cho,
Hakodate 041–8611, Japane
Department of Earth Sciences, Graduate School of Science and Engineering, Ehime University, 3, Bunkyocho, Matsuyama 790–8577, Japanf
Department of Earth Science, Graduate School of Science, Tohoku University, 6-3, Aramaki Aoba, Aoba-ku,
Sendai 980-8578, Japanb

Submitted September 25, 2020; Accepted May 4, 2021
Monitoring Editor: Alastair Simpson

The phylogenetic affiliation of Oroscenidae, a family of Radiolaria possessing large siliceous skeletons, was thoroughly revised in this study. Plankton sampling was conducted over seven years inseven sea areas of the Northern Hemisphere. Molecular analyses on the obtained specimens revealed
that living oroscenids are phylogenetically different from hitherto-known radiolarian orders. The
detailed examinations by Microfocus X-ray Computed Tomography and Scanning Electron Microscopy clarified that oroscenids and the related family (Thalassothamnidae) have unique skeletal characteristics different from other radiolarian orders. Judging from their phylogenetic distinctiveness andthe difference in the morphology and ecology, Oroscenidae and Thalassothamnidae should be classified in a single order distinct from other radiolarian orders, and consequently, a new order, Orodaria,
is established.
Ó 2021 Elsevier GmbH. All rights reserved.
Key words: MXCT; single-cell PCR; silica; shell-bearing protist; unicellular zooplankton.

1

Corresponding author;e-mail jasnakamura@gmail.com (Y. Nakamura).

https://doi.org/10.1016/j.protis.2021.125808
1434-4610/Ó 2021 Elsevier GmbH. All rights reserved.

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Y. Nakamura et al.

Introduction
Oroscenidae Haeckel, 1887 is a family of Radiolaria,
belonging to the phylum Retaria of the supergroup
Rhizaria (SAR). Oroscenids have large cells wholly
covered with siliceous skeletons. Although biological studies on other large rhizarians have been vigorously conducted (e.g., Ikenoue et al. 2019; Biard
and Ohman 2020), there are only limited numbersof studies on oroscenids as extant plankton (e.g.,
Riecke 1910). Yet, oroscenids are often detected
in Cenozoic strata (Friend and Riedel 1967; Kling
1971; Norris et al. 2014; Palmer 1988, Resig et al.
1970), and these radiolarians have a potential to
be used as environmental indicators connecting
the present and past.
The systematic study of oroscenids involves (1)nomenclatural and (2) phylogenetic problems,
which obstruct the taxonomic arrangement of thisgroup. (1) The family Oroscenidae is also known
as Orosphaeridae Haeckel, 1887, but the validityof this family name is questionable because the typegenus Orosphaera Haeckel, 1882 is a nomen
dubium. (2) The phylogenetic affiliation of oroscenids is also controversial. Oroscenids (the family
Oroscenidae) were initially classified under Phaeodaria (Chediya 1959; Haeckel 1887), but later studies assigned them to different radiolarian orders:
Spumellaria (e.g., Riedel and Sanfilippo 1977), Collodaria (e.g., Haecker 1908; Hollande and Enjumet
1953; Petrushevskaya 1984), or Entactinaria (DeWever et al. 2001; Kiessling 1999; Kozur andMostler 1982). A recent study incorporating
sequence analysis reported that the radiolarian phylogenetic tree contains a large clade composed oforoscenids (monophyletic), Collodaria (monophyletic) and Nassellaria (paraphyletic), and suggested that oroscenids could be classified as anindependent order (Nakamura et al. 2020). This
recent study, however, analyzed only two individuals, and further study with more data is indispensable to solve the controversy concerning thephylogeny of oroscenids. Nakamura et al. (2020)
also suggested that the rare radiolarian family
Thalassothamnidae Haecker, 1906, which is classified in the same order as Oroscenidae (De Weveret al. 2001), is possibly related to Collodaria. Nevertheless, the position of Thalassothamnidae is also
an unresolved issue in the radiolarian taxonomy.
In order to collect specimens of extant oroscenids, plankton surveys targeting on large radiolarians were conducted over seven years in sevensea areas in the Northern Hemisphere. Fossil spec-

imens were also collected from the ocean sediment.
The obtained specimens were examined by Microfocus X-ray Computed Tomography (MXCT) and
Scanning Electron Microscopy (SEM) to understandtheir morphology in detail, and the data were compared with the information extracted from almost
all documents concerning oroscenids. The phylogenetic relationship of this group was thoroughly
examined, and its taxonomic affiliation was rearranged in this study.

Results
Field Sampling and Molecular Analysis (Plankton
Specimens)

The investigations were conducted in a large area in
the North Pacific (Fig. 1), and 154 bulk plankton
samples were collected (Supplementary Material
Table S1). From these samples, only 13 individualsof Oroscenidae and 1 individual of Thalassothamnidae were obtained (Fig. 2, Supplementary Material Fig. S1; Tables S1–S2). Most of these
individuals were found from the mesopelagic(200–1,000 m) or bathypelagic (1,000–4,000 m)
zones (Supplementary Material Table S2).
The specimens Or5, Or6 and Or7 were foundfrom the inside of salps and gelatinous zooplankton(Supplementary Material Fig. S2a–b, d, e–f;
Table S2), and within Or6, there was a foraminiferancell (Supplementary Material Fig. S2b–c). No
symbiont-like algae were observed inside of any
oroscenid specimens by our morphological observations and dissection under the light microscope.
The SSU (18S) rRNA sequences were obtained
from 9 specimens, and long sequences from the
SSU to the LSU (28S) rRNA gene (i. e., SSU ITS1 5.8S - ITS2 – LSU) were successfully readin 8 specimens (Supplementary Material
Table S2). The oroscenids formed an independent
clade distinct from Nassellaria and Collodaria in
the phylogenetic trees inferred from the concatenated alignment of SSU and LSU (D1 and D2
regions) (Fig. 3, Supplementary Material Fig. S3)
and from the alignment of SSU (Supplementary
Material Fig. S4). Approximately 3,000 nucleotides
were obtained in 8 oroscenid specimens, but only
1 or 2 nucleotides were different from each other(the similarities among the 8 specimens were
99.93–99.97%)(Supplementary
Material
Table S3). These different nucleotides were notseen in variable regions, suggesting that they comefrom sequencing errors. Remarkable subcladesOrodaria ord. nov. 3

Figure 1. Plankton sampling stations of the investigations conducted in 2011–2017. The detailed information
on each station is shown in Supplementary Material Table S1.

Figure 2. Images of Oroscena huxleyi and O. gegenbauri (Oroscenidae) analyzed in this study. rs: radial
spine. as: apical spine. ss: side spine. bs: basal spine. rdg: ridge. th: thorn. cc: central capsule. The ridges withradial spines compose the primary spines, and the thorns compose the secondary spines. Three morphologicalcategorizations are also shown for O. gegenbauri. Note that the skeleton of Or1 is covered with the soft part
(protoplasm and gelatinous substance) (b), whereas the soft part and some spines were removed by acidtreatment in Or2 (a). All scale bars are 1 mm.

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Y. Nakamura et al.

Figure 3. Maximum-likelihood (ML) phylogenetic trees of Orodaria ord. nov. and related radiolarian orders.
The tree was derived from a concatenated alignment of SSU and LSU (D1, D2) rRNA. Numbers at nodes
indicate ML bootstrap support values and Bayesian posterior probabilities (BPP). Only values higher than 70%(ML) and 0.7 (BPP) are shown. The uncollapsed tree is shown in Supplementary Material Figure S3.

were not recognized within the large oroscenid
clade both in the SSU + LSU and SSU trees(Fig. 3, Supplementary Material Figs S3–S4). We
were unable to obtain the sequence data for Thalassothamnidae (Cytocladus tricladus, Haecker, 1908:the specimen Coe7) (Supplementary Material
Fig. S1n). The DNA had probably been decomposed before the sampling.
Morphological Examination (Plankton and Fossil
Specimens)

The maximum diameter of Oroscenidae was
6.54 mm (the specimen Ori1) (Fig. 4c–d, Supple-

mentary Material Fig. S1m; Table S2), and that ofThalassothamnidae was 7.53 mm (the specimenCoe7) (Fig. 4e–f, Supplementary Material
Fig. S1n, Table S2).
The morphological examination with SEM and
MXCT revealed the fine skeletal structure of Oroscenidae. The general form of this group is apparently similar to that of the order Nassellaria, which
possesses a conical shell completely covering thecell. However, the skeletal structure of oroscenids
is significantly different from the other radiolarianorders. The whole skeleton is generally composedof irregular polygonal structure, but there is a regu-

Figure 4. MXCT images of Oroscena huxleyi (Oroscenidae) and Cytocladus tricladus (Thalassothamnidae).
The side views (a, c, e) and ventral views (b, d, f) are shown. Scale bars are 1 mm (a–d) and 5 mm (e–f).

Orodaria ord. nov. 5

larity in the detailed structure: radial spines always
extend from the ridges located in a regular meshwork structure (Figs 2, 4). A gelatinous substance(or possible cell membrane) covering whole skeleton was recognized in plankton specimens(Fig. 2b, Supplementary Material Fig. S1a, c, d, k).
No mouth (i.e., large opening for feeding) was
observed in any specimens (Fig. 4b, d).
Detailed morphological examination with SEM
and MXCT revealed that all the oroscenids in theplankton specimens have the characteristics ofOroscena huxleyi Haeckel, 1887 (Supplementary
Material Figs S1a–m, S5; Table S2). It is, nevertheless, noteworthy that the specimens Or1, Or2, Or3
and Or11 possess uncompleted external layers(Fig. 2a–b, 4b; Supplementary Material Fig. S1a,k), which are characteristics of another oroscenid
species, O. diplosphaera Haeckel, 1887. The morphology of the specimen Ori1, which was analyzed
with MXCT, generally corresponds to that of O. huxleyi. On the other hand, the ventral view of Ori1
(Fig. 4d) resembles the illustration of O. arborescens Haeckel, 1887, and their basal spines
have a similar branching pattern.
Various types of spines and surface structures
were observed in oroscenids from the deep-sea
sediment. These oroscenids were identified as O.
gegenbauri Haeckel, 1887 and O. carolae Friend& Riedel, 1967 on the basis of the general morphological characteristics (Fig. 2d–f, Supplementary
Material Fig. S6; Table S4). The surface structuresof the specimens Mc1b and Mc1c were, however,similar to those of O. peruviana Berry, 1929. Furthermore, the specimens Mc1d and Mc3a havecharacteristics that are intermediate between O. gegenbauri and O. carolae (Fig. 2e; Supplementary
Material Fig. S6c, j).
The morphological examination also clarified thatthe skeleton of C. tricladus (Coe7) is composed oftwelve spines, branching from the central junction(Fig. 4e–f). This skeletal structure has a resemblance to that of an extinct genus, Orostaurus
Friend & Riedel, 1967. Before the soft part wasremoved by acid treatment, the whole skeleton of
Coe7 was covered with a gelatinous substance (orpossible cell membrane) (Supplementary Material
Fig. S1n). The central capsule (including nuclei) of
this species is not spherical but tubular, and it
branches from the center along the radial spines(Supplementary Material Fig. S1n).

Discussion
Ecology

Despite our large effort in field sampling (Fig. 1;
Supplementary Material Table S1), only 13 individuals of oroscenids were obtained (Supplementary
Material Tables S1–S2), implying that their abundance is very low in the present ocean (simple
occurrence probability is 0.05, since the number of
total plankton samples is 154, and the number ofsamples in which oroscenids were detected is 8)(Supplementary Material Table S1). Fossils of oroscenids are commonly or abundantly found from the
Oligocene to lower Middle Miocene in the northeastern Atlantic (Norris et al. 2014), but only a small
number of survivors dwell in the deep sea of today’s
ocean.
Oroscenids are distributed in the mesopelagic
and bathypelagic zones (Supplementary Material
Table S1), and no algal symbionts have been
detected so far. By contrast, collodarians are chiefly
distributed in the epipelagic zone (Biard and Ohman
2020), and collodarians and nassellarians generally
have symbiotic algae (Biard et al. 2015) (Fig. 5).
There are, consequently, large differences between
oroscenids and their well-known relatives from theecological viewpoint.
Although the predator–prey relationships ofradiolarians and phaeodarians are largely
wrapped in mystery (Nakamura and Suzuki
2015a, b; Suzuki and Not 2015), a piece of information was clarified by this study. Oroscenids
would be eaten by gelatinous zooplankton suchas salps (Supplementary Material Fig. S2a–b, d,e–f; Table S2). Salps are generally filter-feedingplankton, and they were also reported to feedon phaeodarians (Gowing 1989; Raymont 1983).
Nassellarians, a sister group of oroscenids, generally bear a shell-type skeletal structure with amouth-like opening, inside of which the prey isdigested (e.g., Matsuoka 2007). In contrast, oroscenids have no mouth-like opening (Fig. 4b, d),
implying that they possibly digest the prey outsideof the skeleton. The foraminiferan individual foundfrom the inside of Or5 (Supplementary Material
Fig. S2b–c) was presumably eaten by this oroscenid, but it is also possible that the foraminifer
fed on the oroscenid from the inside or that they
perhaps had a symbiotic relationship.

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Y. Nakamura et al.

Figure 5. General characteristics of all extant radiolarian orders. The information was referred from Suzuki
and Aita (2011), Suzuki and Not (2015) and Nakamura et al. (2019). The topology is based on Nakamura et al.
(2020). * Length or diameter of a colony. ** Wholly-covered shell composed of numerous rods forming
meshwork. +: reported in some species. -: not reported.
Morphology and Phylogeny

Rhizaria are known for gigantism: the members of
xenophyophores (Foraminifera) are the largest unicellular benthos, while the largest unicellular zooplankton are Phaeodaria (e.g., Swanberg et al.
1986). Although C. tricladus (Coe7) examined in thisstudy (Supplementary Material Table S2) is smaller
than the largest Phaeodaria (ca. 8 cm), the present
species has the largest body among Radiolaria.
The morphology of oroscenids is largely different
from those of their well-known relatives, i.e., Collodaria and Nassellaria. Oroscenids have a shelltype skeleton wholly covering the cell, unlike collodarians which bear dispersed shell/spicules or possess no skeleton (Fig. 5). Most of nassellarians also
have a shell-type skeleton, but their shell sizes aremuch smaller than those of oroscenids (Fig. 5).
The colonial form, in which numerous individual
cells are covered with gelatinous substance, isknown in collodarians. Yet, the colonial form has
never been reported for oroscenids.
Nakamura et al. (2020) suggested that oroscenids could be classified as an order different from
Collodaria. The current study analyzed seven moreindividuals of Oroscenidae (Supplementary Material
Table S2), and it was confirmed that living oroscenids are monophyletic and sister to a robust Collodaria clade (Fig. 3; Supplementary Material
Fig. S3–S4). Considering this reciprocal monophyly
and the differences in the ecology and morphology,
oroscenids are highly distinct from collodarians (andfrom nassellarians), and Oroscenidae should betaxonomically separated from the other two orders.
One possibility is that the large clade composed oforoscenids, collodarians and nassellarians is treated as a single highly diverse order, with oroscenids
distinguished from the others at a lower taxonomic
level (e.g., family, superfamily or suborder). However, since Collodaria are currently accepted as anindependent order (e.g., De Wever et al. 2001;
Suzuki et al. (in press)), our view is that oroscenids
should also be classified as an independent order(Supplementary Material Table S4). The analogy
between the family Thalassothamnidae (including
C. tricladus) (Fig. 4e–f) and Orostaurus (Oroscenidae) was suggested by Friend and Riedel (1967).
The morphological similarity of these two groupwere confirmed by our morphological analyses,
and therefore, we proposed that Thalassothamnidae is classified to the same order as
Oroscenidae.
Genus-level Classification

Some specimens of oroscenids apparently havemorphological characteristics of different species(Supplementary Material Table S2). Yet, thesequences of these oroscenids were almost identical (Supplementary Material Table S3), and their
phylogenetic differences could be relatively small.
Judging from this point, all living oroscenid specimens analyzed in this study would be classified into
one single species, whose name should be Oros-Orodaria ord. nov. 7

cena huxleyi Haeckel, 1887 due to the Principle ofPriority and the questionable existence of thename-bearing type of the genus Orosphaera (seealso the taxonomic section below). The morphological diversity of analyzed specimens would be theintra-specific variation of O. huxleyi. This species
may be the only extant species among oroscenids,
since all the other members are reported from thesea sediment, and they have never been collectedby our plankton sampling (Supplementary Material
Table S2).
Morphological characteristics of O. gegenbauri
and O. carolae were observed in oroscenids fromthe sediment. According to Friend and Riedel(1967), O. gegenbauri differs from O. carolae in
the following three points: (1) lower spines tend to
branch, (2) greater prominence of ribs and (3) moreorderly arrangement of rectangular pores. These
differences are, however, obscure, and specimens
having intermediate characteristics of these two
species were recognized in this study (Mc1d andMc3a) (Fig. 2e; Supplementary Material Fig. S6c,j, Table S2). Considering the high morphologicaldiversity in O. huxleyi mentioned above, the morphological variation in the fossil specimens would
be an intra-specific differences, and O. carolaeshould be unified with O. gegenbauri. The observed
morphological diversity of O. gegenbauri possibly
comes from the difference in the ontogenetic stage,
the living environment and the geologic age.
Taxonomy

Considering the results of this study, the order- andfamily-level classification of Oroscenidae and its
related taxa is emended as follows (see also Supplementary Material Table S4):Supergroup Rhizaria Cavalier-Smith, 2002, sensu
emend. Cavalier-Smith, 2003
Phylum Retaria Cavalier-Smith, 1999, stat. CavalierSmith, 2002
Infraphylum Radiolaria Müller, 1859, sensu emend.
Krabberød et al., 2017Class Polycystinea Ehrenberg, 1839, stat. Levine et al.,1980
Order Orodaria Nakamura & Suzuki ord. nov.

Diagnosis: Skeleton composed of solid siliceous
primary spines and secondary spines (thorns). Primary spines form spherical or subspherical shelland meshwork zone in some species. Gelatinous
substance (or possible cell membrane) covers
whole skeleton.

Etymology: “oro-” is from the type family of thisorder, Oroscenidae (“oro-“ comes from Greek
oqo1, hill or mountain) and “-daria” is from the suffixof “Collodaria”, the group phylogenetically related tothis new order.
Type family: Oroscenidae Haeckel, 1887
Included families: Oroscenidae Haeckel, 1887;
Thalassothamnidae Haecker, 1906
Remarks: A total of 42 names at order level in
Polycystinea have been proposed in previous
papers (e.g., Ehrenberg 1839, 1847, 1876;
Haeckel 1887; Müller 1859) but no appropriate
taxon names have been proposed for grouping
Oroscenidae and Thalassothamnidae (excluding
Collodaria). A new order name is, therefore, proposed herein. Note that the following two taxa have
similar composition: superfamily Orosphaericae
Campbell, 1954; suborder Orosphaeridea Van dePaverd, 1995.
Family Oroscenidae Haeckel, 1887 sensu emend.
Nakamura & Suzuki herein(Figs 2, 4a–d, Supplementary Material Fig. S1a–m,
S5a–i, S6)

Synonyms: family Orosphaerida Haeckel, 1887;
family Orosphaeridae Haecker, 1908
Revised diagnosis: Spherical or subsphericalsolid skeleton composed of primary spines (ridges
and radial spines) forming irregular polygonal structures with regular meshwork zones. Radial spines
extend from the ridges located in meshwork zones.
External layer develops in some species. Skeleton
empty (no internal layer) containing central capsule.
Gelatinous substance (or possible cell membrane)
covers whole skeleton. No algal symbionts reported
so far.
Geological range: Eocene to the present
Type genus: Oroscena Haeckel, 1887 (type species: Oroscena gegenbauri Haeckel, 1887, subsequently designated by Campbell (1954))
Included genera: Oroscena Haeckel, 1887;
Orostaurus Friend & Riedel, 1967; Oropelex
Friend & Riedel, 1967
Nomina dubia in this family: subfamily Oronida
Haeckel, 1887; Orosphaera Haeckel, 1882; Orodictyum Haeckel, 1887; Orona Haeckel, 1887; Oronium Haeckel, 1887
Remarks: The family name, Orosphaeridae, isreplaced with Oroscenidae due to the nomen dubium
status for the genus name, Orosphaera, and thequestionable existence of the name-bearing type of

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Y. Nakamura et al.

this genus. Orodapis Friend & Riedel, 1967 was classified to the family Oroscenidae (Orosphaeridae inthe original text), but this genus has a complex internal spicular system, and no polygonal structures
exist on the surface. Considering these points,
Orodapis is excluded from the family Oroscenidae.
Note that the following three taxa have similar
composition: subfamily Oroscenida Haeckel, 1887;
subfamily Orosphaerinae Campbell, 1954; subfamily
Orosceninae Campbell, 1954.
Family Thalassothamnidae Haecker, 1906 sensu
emend. Nakamura & Suzuki herein(Fig. 4e–f; Supplementary Material Fig. S1n, S5j)

Synonym: family Cytocladidae Schröder, 1908
Revised diagnosis: Radiolaria with single skeleton composed of branching primary spines (radial
spines) extended from the central junction. Gelatinous substance (or possible cell membrane) covers
whole skeleton.
Geological range: possibly Holocene to thepresent
Type genus: Thalassothamnus Haecker, 1906
(type species: Thalassothamnus genista Haecker,
1906, subsequently designated by Campbell(1954)).
Included genera: Thalassothamnus Haecker,
1906; Cytocladus Schröder, 1906
Nomen dubium in this family: Conostylus
Popofsky, 1907
Remarks: The genus Triassothamnus Kozur andMostler, 1981 was also included in Thalassothamnidae, and this genus was found only from Triassic
strata (De Wever et al. 2001; Kozur and Mostler
1981). In contrast, Thalassothamnus is reported
from the present. This stratigraphic gap is larger
than the geologic range of Oroscenidae, the sistergroup of Thalassothamnidae. The skeletal structures of Thalassothamnus and Triassothamnus
are relatively simple, and it is difficult to discuss their
phylogenetic relationship. Nevertheless, their large
stratigraphic gap would indicate that the skeletal
structure of Triassothamnus is analogous to that ofThalassothamnus, and therefore, the former should
be excluded from Thalassothamnidae. This family
has branching primary spines extended from thecentral junction, and this structure apparently
resembles that of the family Astracanthidae
Haecker, 1906 belonging to Phaeodaria. However,the spines of Thalassothamnidae are solid (there
are no spaces inside) (Nakamura et al. 2020), unlike
hollow (tubular) skeleton of Phaeodaria.

Methods
Field sampling and treatment: Plankton sampling was conducted
in 2011–2017 at 37 stations located in 7 sea areas of the Northern
Hemisphere (Fig. 1; Supplementary Material Table S1). Radiolarians
were manually isolated from the samples under a stereomicroscopeor inverted microscope (e.g., TMS, Nikon, Japan). The isolated
individuals were photographed with a digital camera (e.g., Nikon
1 V3, Nikon, Japan) attached on the microscopes (Supplementary
Material Fig. S1) and individually preserved in tubes filled withapproximately 2.0 mL of 99.9% ethanol. The tubes containing thespecimens were stored at 4 °C.
Deep-sea sediments were also observed under the light microscope in order to examine the morphology of fossil radiolarians. The
sediment samples were obtained in 1978 at sta. CP1 (5,225 m indepth) located in the eastern North Pacific (Supplementary Material
Table S1). The ages of the sediment samples were estimated as
Lower Miocene (23.03–15.97 million years ago), but they possibly
included sediments of the Eocene (56.0–33.9 million years ago).
Skeletons of oroscenids were picked up from the samples and put on
stubs for the SEM observation.
Molecular analysis: The ethanol-preserved plankton specimens
were dissected by a sterilized scalpel, and the “central capsule”(protoplasmic body including nuclei) of each specimen was put into
100 lL of guanidine-containing extraction buffer (GITC buffer, Decelleet al. 2012). The DNA extraction, amplification and sequencing were
performed following the protocol shown in Nakamura et al. (2015). A
total of 7 primers were used, including 4 oroscenid-specific primers
newly designed in this study (Table S5). Polymerase Chain Reactions (PCR) were performed with a thermal cycler (Veriti, Applied
Biosystems, U.S.A.) using the following protocol: The initial denaturation at 95 °C for 10 min, 45 cycles at 95 °C for 20 sec, 52 °C for
15 sec (SSU) or 53 °C (LSU) for 15 sec, and 72 °C for 70 sec(SSU) or 72 °C for 80 sec (LSU), with a final extension at 72 °C for
2 min. The amplified PCR products were purified using AMPure XP
Kit (Beckman Coulter, U.S.A.). The sequencing was conducted withABI PRISM 3130xl Genetic Analyzer (ABI, U.S.A.).
The obtained sequences were assembled using ChromasPro(Technelysium Pty Ltd, Australia), and the alignments were manuallychecked. The SSU and LSU (D1, D2) rRNA sequences of otherradiolarians were also retrieved from the NCBI database (Supplementary Material Table S6). Some SSU sequences which were too
short (less than 1,500 nucleotides) or low quality were removed fromthe alignments since they may decrease the accuracy of the analysis. The SSU and LSU rRNA sequences were individually aligned
using MEGA 7 (Tamura et al. 2011), and then, the alignments wereconcatenated using SeaView v.4 (Gouy et al. 2010). Although theconcatenated alignment consisted of 2,305 nucleotides and 74
sequences (Supplementary Material Table S6), ambiguously aligned
positions were removed, and the final data set containing 2,129
nucleotides was used for constructing the phylogenetic trees. The
GTR + G (general time reversible + gamma) model was selectedusing Kakusan4 (Tanabe 2011), and Maximum-likelihood (ML) analysis with 1,000 bootstrap replicates (Felsenstein 1985) was carriedout using RAxML (Stamatakis 2014). The GTR + G + I were selected
for Bayesian analysis, and the Bayesian posterior probabilities (BPP)
were calculated with MrBayes v.3.2.2. (Ronquist and Huelsenbeck2003). The Markov chains were run for 1.0  107 generations, andevery 100 generations were sampled. The first 1.0  106 generations
were discarded after checking by a program Tracer v.1.6 (Rambautand Drummond 2009). The remaining data reaching the steady state
were used for building the consensus tree. The obtained phylogenetic trees were visualized using FigTree v.1.4.4 (Rambaut 2009),
and Spumellaria were used as outgroup since this order is monophyletic and closely related to the three orders discussed in this study

Orodaria ord. nov. 9(i.e., Nassellaria, Collodaria and Orodaria) (Nakamura et al. 2020).
Additional phylogenetic trees were also constructed based on thealignments of SSU rRNA. The GTR + G + I and GTR + G models
were selected for ML and neighbor-joining (NJ) methods, respectively. All positions containing gaps and missing data were eliminated. The Bootstrap values were estimated based on 1,000
pseudo-replicates.
Morphological observation and documentary survey: Afterthe morphological observation by an inverted microscope, all thefossil specimens and a part of the plankton specimens were carefully
observed with a SEM (JSM-6390LV with LaB6 gun, JEOL, Japan) to
examine their skeletal morphology in detail (Supplementary Material
Figs S5–S6). The conditions and parameters were the same as
those in Nakamura et al. (2016).
For further understanding the three-dimensional (3D) structure,
three plankton specimens were examined by the MXCT, which is a
non-destructive 3D imaging technique. The specimens were analyzed by ScanXmate-DF160TSS105 (Comscantechno, Japan) witha high-resolution X-ray setting (X-ray focus spot diameter of 0.8 lm,
X-ray tube voltage of 80 kV, detector array size of 1024x1024 pixel,
1800 projections /360°, 4 average/projection. The spatial resolution
was ranged from 5.00–8.96 lm/voxel). By collecting X-ray transmission images, a series of two-dimensional (2D) cross-sectional images were generated. The 2D images of each specimen were thenstacked using an image analysis software (MolcerPlus, White Rabbit,
Japan), and a 3D image was constructed, whose interior structure isobservable from whole angles. The 3D data of MXCT analysis
obtained in this study were stored in a marine biological database,
BISMaL (https://www.godac.jamstec.go.jp/bismal/j/).
Almost all the documents reporting on oroscenid radiolarians
were thoroughly checked, and the information about this radiolariangroup was extracted to comprehend their morphological and ecological characteristics.
Ethical approval: All applicable international, national, and/or
institutional guidelines for the care and use of animals were followedby the authors.

CRediT authorship contribution
statement
Yasuhide Nakamura: Conceptualization, Investigation, Resources, Formal analysis, Methodology,
Data curation, Writing - original draft, Writing review & editing. Akihiro Tuji: Formal analysis,
Supervision. Katsunori Kimoto: Formal analysis,
Methodology, Data curation. Atsushi Yamaguchi:
Investigation, Resources. Rie S. Hori: Investigation,
Resources. Noritoshi Suzuki: Supervision.

Dr. Fabrice Not (CNRS) and Dr. Miguel M. Sandin(Uppsala Univ.) for their kind advice and support.
Appreciations are also due to Dr. David Lazarus(Museum für Naturkunde) for his help in searchingreferences and to Dr. Moira Galbraith (Fisheries
and Oceans Canada) for sending her unpublisheddata. We are grateful to the captains and crew of T/
S Oshoro-maru (Hokkaido Univ.) and T/S Toyoshiomaru (Hiroshima Univ.). The comments from threeanonymous reviewers substantially improved themanuscript. This work was financially supported by
the Sasakawa Scientific Research Grant from theJapan Science Society (2020-4025, Y. Nakamura),
by JSPS KAKENHI Grant Number JP20K15589 (Y.
Nakamura) and 16K07475 (N. Suzuki) and by the
Cooperative Research Project with the Japan
Science and Technology Agency (JST) and Centre
National de la Recherche Scientifique (CNRS,France) “Morpho-molecular Diversity Assessment of
Ecologically, Evolutionally, and Geologically Relevant Marine Plankton (Radiolaria)”.

Appendix A. Supplementary Material
Supplementary Material (Figs S1–S6 and Tables
S1–S6) is available for this study. Supplementary
data to this article can be found online athttps://doi.org/10.1016/j.protis.2021.125808.

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