Protist, Vol. 166, 363–373, July 2015
http://www.elsevier.de/protis
Published online date 28 May 2015

ORIGINAL PAPER

Molecular Phylogeny of the Widely
Distributed Marine Protists, Phaeodaria(Rhizaria, Cercozoa)
Yasuhide Nakamuraa,1 , Ichiro Imaia , Atsushi Yamaguchia , Akihiro Tujib ,Fabrice Notc , and Noritoshi SuzukidaPlankton Laboratory, Graduate School of Fisheries Sciences, Hokkaido University,

Hakodate, Hokkaido 041–8611, JapanbDepartment of Botany, National Museum of Nature and Science, Tsukuba 305–0005,

Japan

cCNRS, UMR 7144 & Université Pierre et Marie Curie, Station Biologique de Roscoff,

Equipe EPPO - Evolution du Plancton et PaléoOcéans, Place Georges Teissier, 29682
Roscoff, France
dInstitute of Geology and Paleontology, Graduate School of Science, Tohoku University,
Sendai 980–8578, Japan
Submitted January 1, 2015; Accepted May 19, 2015
Monitoring Editor: David Moreira

Phaeodarians are a group of widely distributed marine cercozoans. These plankton organisms can
exhibit a large biomass in the environment and are supposed to play an important role in marine
ecosystems and in material cycles in the ocean. Accurate knowledge of phaeodarian classiﬁcation isthus necessary to better understand marine biology, however, phylogenetic information on Phaeodaria
is limited. The present study analyzed 18S rDNA sequences encompassing all existing phaeodarianorders, to clarify their phylogenetic relationships and improve their taxonomic classiﬁcation. The monophyly of Phaeodaria was conﬁrmed and strongly supported by phylogenetic analysis with a larger data
set than in previous studies. The phaeodarian clade contained 11 subclades which generally did notcorrespond to the families and orders of the current classiﬁcation system. Two families (Challengeriidae and Aulosphaeridae) and two orders (Phaeogromida and Phaeocalpida) are possibly polyphyleticor paraphyletic, and consequently the classiﬁcation needs to be revised at both the family and orderlevels by integrative taxonomy approaches. Two morphological criteria, 1) the scleracoma type and 2)
its surface structure, could be useful markers at the family level.
© 2015 Elsevier GmbH. All rights reserved.
Key words: Phaeodaria; Cercozoa; Rhizaria; 18S rDNA; single-cell PCR.

1

Corresponding author; fax +81 138 40 5542
e-mail y.nakamura@ﬁsh.hokudai.ac.jp (Y. Nakamura).

http://dx.doi.org/10.1016/j.protis.2015.05.004
1434-4610/© 2015 Elsevier GmbH. All rights reserved.

364 Y. Nakamura et al.

Introduction
Phaeodarians are marine unicellular protists.
This group is deﬁned by 1) the presence of a“central capsule” containing one or several nuclei,2) a mass of brown aggregated particles named“phaeodium”, and 3) a hollow siliceous skeleton
called “scleracoma”, in most species (Howe et al.
2011; Nakamura and Suzuki 2015; Takahashi andAnderson 2000). The size of the scleracoma varies
with the different families and ranges from several
hundred micrometers (e.g. Challengeriidae) to afew millimeters (e.g. Tuscaroridae). The type of thescleracoma and its surface structure are importanttaxonomic characters to classify these organisms
at the order and family levels. Phaeodarians havelong been considered as a member of Radiolaria
but based on molecular analyses (Nikolaev et al.
2004; Polet et al. 2004; Sierra et al. 2013), they
are now classiﬁed as a subclass in the class
Thecoﬁlosea of the phylum Cercozoa (Adl et al.
2012; Howe et al. 2011).
Phaeodarians are heterotrophic plankton organisms, which are distributed in the world ocean fromthe surface to the deep sea (Nakamura and Suzuki2015). Despite their wide range of distribution, thisplankton group has attracted little attention frommarine biologists (Nakamura et al. 2013). Theirabundance has been very likely underestimated,because their scleracoma is fragile and breaks
easily when samples are collected by plankton
nets. Yet, careful examination of the zooplankton community revealed that these protists can be
numerous and their high abundance has occasionally been reported. For instance, the vertical ﬂux ofphaeodarians was higher than that of polycystineradiolarians in the Panama Basin (Takahashi and
Honjo 1983) and in the California coast (Gowing
and Coale 1989). The combined biomass of the
Aulosphaeridae, Sagosphaeridae, Aulacanthidae
and Coelodendridae was estimated to contribute
2.7–13.7% of the total metazoan biomass in the
150–1000 m layer of the western North Paciﬁc(Steinberg et al. 2008). The proportion of an
Aulacanthidae species, Aulographis japonica, withrespect to the total zooplankton biomass is 22.3%
in 250–3000 m layer in the Sea of Japan, andthis percentage is the second largest, followingthat of copepods (Nakamura et al. 2013). Considering that they possess siliceous skeletons andoccur occasionally at high biomass, this plankton
group can play an important role in ecosystems
locally and have a signiﬁcant impact on the silica cycle of the ocean. Their importance in the
carbon cycle is also reported from the Northeast

Atlantic (Lampitt et al. 2009). Cercozoans, including phaeodarians, have recently been considered
important players in the material cycles and foodwebs of the ocean (Howe et al. 2011). Researchon environmental DNA revealed that Cercozoa
represented a high percentage of the 18S rDNA
sequences obtained from sea-ﬂoor sediments of
the Arctic and the Southern Ocean (Pawlowski
et al. 2011). Consequently, improving our knowledge on phaeodarians will be important for theentire ﬁeld of marine biology. Accurate knowledge of phaeodarian classiﬁcation is critical tobetter assess their genetic diversity and ecology. In
the comprehensive systematics framework established by Haeckel (1887), phaeodarian species
were grouped into families and orders according totheir morphological similarities. Since then, someauthors amended this classiﬁcation, but the relationship between the families remains uncertain(Cachon and Cachon 1985; Campbell 1954; Klingand Boltovskoy 1999; Takahashi and Anderson
2000) and has never been examined from themolecular point of view. Comprehensive molecular studies on phaeodarians are difﬁcult to conduct.
They cannot be cultured, and therefore their DNA
has to be extracted from individual specimens collected and sorted from the environment. Somephaeodarian species, however, live in the deep sea,
and it is hard to obtain specimens. In addition,
efﬁcient phaeodarian-speciﬁc primers do not exist,
increasing the risks of amplifying and sequencing
contaminating organisms attached to or ingestedby the Phaeodaria.
Yet, an integrative taxonomy approach merging both morphological and molecular information
has recently been applied successively to otherunicellular and shell-bearing rhizarian taxa suchas Polycystines, Acantharia and Foraminifera (e.g.
Decelle et al. 2012; Kunitomo et al. 2006;
Pawlowski et al. 2013). Such an approach,highlighting match and mismatch between morphology and phylogenetic analysis, contributed
to an overall better understanding of the classiﬁcation and evolutionary patterns among thesegroups.
The information on phaeodarian 18S ribosomal
DNA currently exists for only 7 out of ca. 200
species, 18 families and 7 orders recognized in
the latest phaeodarian classiﬁcation (Nakamura
and Suzuki 2015; Takahashi and Anderson 2000),
and discrepancies between morphological classiﬁcation and analysis of these partial 18S rDNA
were pointed out (Nakamura et al. 2013). Here,
we designed new phaeodarian-speciﬁc primers
and analyzed the whole 18S rDNA sequenceMolecular Phylogeny of Phaeodaria 365

of extant single-cell specimens of phaeodarianspecies, covering all orders recognized in the current classiﬁcation.

Results
A total of 39 phaeodarian specimens belonging
to 26 distinct taxa (essentially distinct species)
were collected from 0 m to 3 000 m depth, andtheir sequences were obtained by single-cell PCR.
Detailed information and micrographs of analyzed
specimens are shown in Table 1 and Figure 1,
respectively.
The sequences from all phaeodarian specimens
formed a single clade far separated from other
Thecoﬁlosea taxa (Cryomonadida, Ebriacea andPseudodifﬂugia), and the monophyly of this clade
was strongly supported by bootstrap values and
BPP (Fig. 2). The phaeodarian clade consisted ofTable 1. List of phaeodarian sequences used for the phylogenetic analysis in this study. the specimens arecoded according to the sampling station: the wesern North Paciﬁc (WNP); the eastern north Paciﬁc (ENP); theEast China Sea (ECS); the Sea of Japan (SJ); the Mediterranean Sea (MS).
Taxon

Accession no.

SpecimennameSampling
stationDepthrange (m)Sampling
date

Protocystis vicina
Protocystis vicina
Challengeriidae sp. 1
Porospathis holostoma
Challengeriidae sp. 2
Challengeron bethelliProtocystis thomsoni
Challengeron tizardi
Challengeron tizardi
Challengeron tizardi
Entocannula infundibulum
Porospathis holostoma
Protocystis harstoniProtocystis murrayi
Tuscaretta belknapi
Coelodendrum furcatissimum
Sagoscena sp. 1
Tuscaretta belknapi
Tuscaretta sp. 1
Auloceros arborescens
Coelodendrum furcatissimum
Conchariidae sp. 1
Challengeron radians
Conchariidae sp. 2
Challengeron diodon
Challengeron radians
Challengeron willemoesii
Challengeron willemoesii
Protocystis xiphodon
Protocystis xiphodon
Aulographis japonica
Auloscena sp. 1
Protocystis vicina
Aulographis japonica
Auloscena sp. 1
Aulacantha scolymantha
Medusetta parthenopaea
Phaeodina sp. 1
Phaeodina sp. 1

AB998884
AB998885
AB998886
AB998887
AB998888
AB998889
AB998890
AB998891
AB998892
AB998893
AB998894
AB998895
AB998896
AB998897
AB998898
AB998899
AB998900
AB998901
AB998902
AB998903
AB998904
AB998905
AB998906
AB998907
AB998908
AB998909
AB998910
AB998911
AB998912
AB998913
AB998914
AB998915
AB998916
AB998917
AB998918
AB998919
AB998920
AB998921
AB998922

WNP1
WNP2
WNP3
WNP4
WNP5
WNP6
WNP7
WNP8
WNP9
WNP10
WNP11
WNP12
WNP13
WNP14
ENP1
ENP2
ENP3
ENP4
ENP5
ENP6
ENP7
ECS1
ECS2
ECS3
ECS4
ECS5
ECS6
ECS7
ECS8
ECS9
SJ1
SJ2
SJ3
SJ4
SJ5
MS1
MS2
MS3
MS4

WN1
WN1
WN2
WN2
WN2
WN2
WN2
WN2
WN2
WN2
WN2
WN2
WN2
WN2
EN1
EN2
EN2
EN3
EN4
EN5
EN5
E1
E2
E2
E3
E3
E3
E3
E4
E4
S1
S1
S1
S2
S3
M1
M1
M1
M1

250–500
250–500
100–150
150–250
500–750
500–750
500–750
750–1000
1500–2000
1500–2000
1500–2000
1500–2000
1500–2000
1500–2000
0–300
0–10000–10000–10000–10000–10000–1000
0–150
200–500
200–500
650–800
650–800
650–800
650–800
0–150
0–150
250–750
250–750
250–750
250–750
500–750
0–40
0–40
0–40
0–40

Aug. 2013
Aug. 2013
Aug. 2013
Aug. 2013
Aug. 2013
Aug. 2013
Aug. 2013
Aug. 2013
Aug. 2013
Aug. 2013
Aug. 2013
Aug. 2013
Aug. 2013
Aug. 2013
Aug. 2012
Aug. 2012
Aug. 2012
July 2012
July 2012
July 2012
July 2012
May 2013
May 2013
May 2013
May 2012
May 2012
May 2012
May 2012
May 2012
May 2012
Apr. 2013
Apr. 2013
Apr. 2013
Apr. 2012
Mar. 2013
Nov. 2012
Nov. 2012
Nov. 2012
Nov. 2012

366 Y. Nakamura et al.

Figure 1. Light micrographs of phaeodarian specimens analyzed in this study. The information on each specimen is presented in Table 1. cc: central capsule; ph: phaeodium; sc: scleracoma.

Molecular Phylogeny of Phaeodaria 367

Figure 1. (Continued)

11 subclades (A–K) deﬁned based on support values and morphological features (Fig. 3). Subclade
A contains the highest number of individuals (n =18). Subclades A, E, F, G, H and K were composedof sequences from distinct sampling locations. For
instance, subclade E contains the specimens from
the Mediterranean Sea (MS), the Sea of Japan (SJ)
and the eastern North Paciﬁc (ENP).

368 Y. Nakamura et al.

(Fig. 3). The order Phaeogromida was scatteredacross subclades A, B and F, and the order Phaeocystida has representatives in subclades I and
K. The order Phaeosphaerida, composed of two
families Sagosphaeridae and Aulosphaeridae (subclade E), turned out to be nested within the order
Phaeocalpida (subclades C and D).

Discussion
Comparison Between the Phylogenetic
Tree and the Current Classiﬁcation
Figure 2. Phylogenetic tree of phaeodarians andother cercozoans based on 18S rDNA alignments
and maximum-likelihood (ML) method. Note that thephaeodarian clade is presented with 11 subclades(A–K) detected according to the support values andmorphological features. The details of this clade
are shown in Figure 3. Numbers at nodes indicate
Bayesian posterior probabilities (BPP) and bootstrap
support values of neighbor-joining (NJ) and ML methods (BPP/NJ/ML). Only values higher than 0.5 pp and
50% are shown.

The 11 subclades and the families classiﬁed by
Takahashi and Anderson (2000) generally did notmatch well, and disagreement was seen for thefamilies Aulosphaeridae, Sagosphaeridae, Challengeriidae and Aulacanthidae (Fig. 3). Sagoscena
sp. 1 (ENP3) of Sagosphaeridae formed the subclade E along with two Aulosphaeridae species,
being nested one into each other. Challengeriidae and Aulacanthidae were scattered acrossthe phylogenetic tree. Most of the challengeriid
phaeodarians appeared in subclade A, but two
specimens of Challengeron willemoesii Haeckel(ECS6 and ECS7) formed the subclade B clearly
distinct from subclade A with high support values(1.0/100/100, Fig. 3). The sequences of C. diodon(Haeckel) (ECS4 and NA (AB218765)) belonged
to subclade H, distant from subclade A, together
with the Medusetta parthenopaea Borgert (MS2) of
Medusettidae (Fig. 3). Two Aulacanthidae species,
Aulographis japonica Nakamura, Tuji and Suzuki(SJ1, SJ4 and SJ (AB820365)) and Auloceros
arborescens Haeckel (ENP6) formed a single subclade K, whereas Aulacantha scolymantha Haeckel(MS1 and MS (AY266294)), which also belongs to
Aulacanthidae, composed subclade I whose exact
position compared to other subclades is unclear.
The orders of the current classiﬁcation did notcorrespond to the phylogenetic analysis overall

Previous studies analyzed partial 18S rDNA ofphaeodarian species (n=6) belonging to 5 distinct families and orders, and reported that theirsequences formed a monophyletic clade outside ofRadiolaria but within cercozoans (Polet et al. 2004;
Yuasa et al. 2006). In this study, the monophyly of
Phaeodaria within Cercozoa is further conﬁrmed
based on a larger data set, including 29 distinct
species distributed over all 7 orders. Phaeodarians
are now classiﬁed as a member of the class Thecoﬁlosea, together with Cryomonadida, Ebriacea
and Pseudodifﬂugia (Adl et al. 2012; Howe et al.
2011). However, these Thecoﬁlosea taxa did notform a monophyletic group in the 18S rDNA tree
(Fig. 2), and their phylogenetic relationship could
not be conﬁrmed in the present study. For better clarifying the phylogenetic relationship between
phaeodarians and other cercozoans, it would benecessary to add more taxa whose 18S rDNA
sequences are not yet available (e.g. Ventricleftida).
The 11 subclades in the 18S rDNA phylogenetic reconstruction did not well correspond to thefamilies and the orders in the current classiﬁcation system (Fig. 3). The family Challengeriidae
is possibly a polyphyletic group, and the family
Aulosphaeridae would be a paraphyletic group.
As for ordinal level categorization, Phaeogromida
turned out to be polyphyletic. The branching pattern of subclades C, D and E suggests that theorder Phaeocalpida is a paraphyletic group. Consequently, the current classiﬁcation system is notsupported by the 18S rDNA phylogeny and needs
to be reconsidered.

Comparison Between the Phylogenetic
Tree and the Morphological Characters
Previous studies classiﬁed phaeodarian families
according to morphological characters, especiallybased on 1) the scleracoma type and 2) the surface structure of the test (Cachon and Cachon

Molecular Phylogeny of Phaeodaria 369

Figure 3. The phaeodarian clade in the phylogenetic tree inferred by maximum-likelihood (ML) method, basedon 46 sequences of 18S rDNA. The outgroups are shown in Figure 2. Note that the 11 subclades (A–K) aredetected based on the support values and morphological features. Each specimen is coded according to its
sampling station as shown in Table 1, and species retrieved from NCBI database are presented with theiraccession numbers. Numbers at nodes indicate Bayesian posterior probabilities (BPP) and bootstrap support
values of neighbor-joining (NJ) and ML method (BPP/NJ/ML). Only values higher than 0.85 pp and 50% areshown. The families and orders are based on Takahashi and Anderson (2000).

1985; Campbell 1954; Haeckel 1887; Kling and
Boltovskoy 1999; Takahashi and Anderson 2000).
The 11 subclades were generally congruent withthese two morphological criteria in this study(Fig. 3). The scleracoma of subclades A, B, C, D,
F and H formed relatively ﬁrm “test”, but displaying distinction between each other: cape-shaped orovate test possessing smooth or small-pored surface (A and B), spherical test with “tubular spines”
and the surface of “trizonal meshwork” (C), testof smooth surface with short “oral spines” andlong “aboral spines” (D), campanulate test possessing developed “peristome” with “arches” (F),and test composed of two “valves” with largepored surface (H). The scleracoma of subclade E
corresponds to a sphere of geometric meshwork

called “lattice-shell”. The phaeodarians of subclade
G possess scleracoma composed of “inner shell”
and numerous branch-like structure called “styles”.
The specimens included in subclades I and K
have scleracoma called “spherical veil”. Specimens
belonging to subclade J do not possess scleracoma(Figs 1, 3).
Thus, the scleracoma type and the surface structure of the test would be valuable characters forthe family-level classiﬁcation. Future studies should
rearrange phaeodarian families and orders according to these two criteria and the branching patternof the phylogenetic tree. The combination of both
detailed morphological examination and molecularphylogeny helped identifying new characters for thetaxonomic classiﬁcation of Phaeodaria. Such an

370 Y. Nakamura et al.

integrative approach has already been proven valuable for a number of taxa, including some among
Rhizaria (e.g. Acantharia, Decelle et al. 2012).

Taxonomical Consideration on the
Families Challengeriidae andMedusettidae
The subclade A contains 18 specimens belonging
to 3 different challengeriid genera: Challengeron,Protocystis and Entocannula. These 3 genera wereerected according to the difference in their morphological characteristics. Phaeodarians of thesegenera, however, did not make subclades clearly
distinct from each other, and there is a possibility
that the current classiﬁcation within the family Challengeriidae is artiﬁcial. In order to further clarify theirintra-family relationship, it is necessary to analyze
higher resolution genetic maker regions, such as
the D1 and D2 regions of the 28S rDNA or ITS.
The genus Challengeron contains ca. 13
species among which 5, including the type species
C. bethelli (Murray) (WNP6), were analyzed inthis study (Campbell 1954). The scleracoma of
C. willemoesii (ECS6 and ECS7) forms a ﬁrm“test”, similar to other challengeriid phaeodarians
composing subclade A (Fig. 1). This species has atest with the surface made of a regularly arranged
amphora-shaped structure, which is common toother challengeriid species, except C. diodon(ECS4) (Takahashi 1991). Consequently, thesurface structure does not distinguish this species
from other challengeriids examined in this study.
Challengeron willemoesii has a developed “peristome” with four “oral spines” (Fig. 1). This complex
peristome structure is not seen in other challengeriids belonging to subclade A, and this charactermay be a morphological criterion distinguishing
this species from other challengeriids.
The fact that C. diodon (ECS4) appeared in
subclade F together with Medusetta parthenopaea(MS2) of the family Medusettidae suggests that theformer is phylogenetically closer to the medusettids
than other challengeriids. Morphological differences of C. diodon from other challenderiids havepreviously been noticed: 1) a dimpled surface; 2)clusters of small pores associated with individual
amphorae (inner shell surface); and 3) thin anddelicate amphorae (Takahashi 1991). This author
also mentioned that the overall appearance of thisspecies was closer to that of medusettid. The two
species composing subclade F have one “aboral
spine” and the developed “peristome” with “arches”
in common (Fig. 1). These structures could also be
important new characteristic features to distinguish

this subgroup. Considering the morphological features and the position in the phylogenetic tree,
these two species would better to be included in
the family Medusettidae.

Wide Distribution of Phaeodarians
Specimens of phaeodarian were collected fromboth deep sea and surface (e.g. MS1–4, Table 1),
emphasizing their wide vertical distribution as previously reported (Nakamura and Suzuki 2015).
Based on sampling location, C. diodon is likely acosmopolitan species. The two sequences of C.
diodon within subclade F (Fig. 3) have been collected at distinct locations, Sogndalsfjord, western
Norway (NA [AB218765], Yuasa et al. 2006) and theEast China Sea (ECS4, Table 1). This species was
also found in the North Sea, the Labrador Sea, theSouth Atlantic and the Mediterranean Sea (Borgert
1901).
The present study provides a strong morphogenetic framework for phaeodarian classiﬁcation,
and will be a valuable tool for accurate interpretationof ongoing environmental diversity surveys basedon DNA sequencing. This morphogenetic approachand the single-cell PCR method can further elucidate the intra- and inter-group relationships ofphaeodarians and other protists by combining otherbiological information such as distribution or physiology.

Methods
Sampling and identiﬁcation: Plankton samples were collected in 2012 and 2013 from 14 stations located in 5 seas
across the northern hemisphere: the western and eastern North
Paciﬁc Ocean, the Sea of Japan, the East China Sea andthe Mediterranean Sea (Supplementary Material Table 1, Supplementary Material Fig. S1). Phaeodarian individuals wereisolated manually from the samples under a stereomicroscopeor inverted microscope, as soon as possible after the sampling. Isolated specimens were put into wells of cell culture
plates and incubated for several hours to allow self-cleaning.
The specimens were carefully identiﬁed according to their morphological characters under an inverted stereomicroscope andphotographed with a digital camera (DIGITAL SIGHT DS-Ri1,
Nikon, Japan). For identiﬁcation, all the documents concerning phaeodarian taxonomy were examined, and a taxonomic
database of synonyms of Phaeodaria was constructed. The
specimens were then individually preserved in tubes ﬁlled withapproximately 2.0 mL of 99.9% ethanol. The tubes containing
the specimens were stored at 4 ◦ C. Each specimen was labeledaccording to its sampling station (Table 1 and Fig. 1).
Single-cell PCR: After conﬁrming that there were no otherorganisms attached to the cell surface, each specimen was
put into 50 ␮L of guanidine-containing extraction buffer (GITCbuffer, Decelle et al. 2012) and preserved in -80 ◦ C overnight.
Some large specimens (e.g. Aulosphaeridae) were dissectedby a sterilized scalpel, and only their “central capsules”

Molecular Phylogeny of Phaeodaria 371

Table 2. Sequences of the primers for 18S rDNA used in this study.
Primer name

Position*

Sequence 5 -3

Direction

Speciﬁcity

Reference

Medlin A

5 -endForward

Universal

Medlin et al.
(1988)

PhaeoF3a288–319

Forward

Phaeodaria

This study

NS2

546–566

ReverseUniversal

White et al.
(1990)

Phaeo11F

559–583

Forward

Phaeodaria

This study

PhaeoF4-2

872–890

Forward

Phaeodaria

This study

Phaeo880F

872–893

Forward

Phaeodaria

This study

Phaeo R

885–904

ReversePhaeodaria

SR6

1761–1777

AAC CTG GTTGAT CCT GCC
AGTTCA TTC AAA
TTT CTG CCC
TAT CAG CTWGAY GGGGC TGC TGGCAC CAG ACTTGCCAG CAG CCG
CKG TAA TTCCAG CTC C
AKG GAT AGT
TGG GGG TGC T
AKG GAT RGT
TGG GGG TGC
TAG T
GGC CAY TRAATA CTA GCA CCTGT TAC GAC
TTT TAC TT

ReverseUniversal

Medlin B

3 -endReverseUniversal

Nakamura et al.
(2013)
designed in
Vilgalys lab,
Duke University.
Medlin et al.
(1988)CCT TCT GCA
GGT TCA CCT AC* Position within 18S rDNA of Protocystis xiphodon(protoplasmic body with nucleus) were transferred into the GITC
buffer. The specimens were heated at 70 ◦ C for 20 min to break
the wall of their central capsules. The extracted DNA was puriﬁed with a chemagic DNA Plant kit (PerkinElmer chemagen,Germany). Single-cell PCR was conducted to amplify 18S rDNA
with a total reaction volume of 25 L. A total of 9 primers wereused, including 4 phaeodarian-speciﬁc primers designed inthis study: Phaeo F3a, Phaeo11F, PhaeoF4-2 and Phaeo880F(Table 2). PCR reactions were performed using the following
protocol: initial denaturation at 95 ◦ C for 10 min, 40 cycles at
95 ◦ C for 20 sec, 58 ◦ C for 15 sec and 72 ◦ C for 120 sec with aﬁnal extension at 72 ◦ C for 7 min. The ampliﬁed PCR products
were puriﬁed with AMPure XP Kit (Beckman Coulter, USA).
Sequencing reactions were conducted using an ABI PRISM
3130xl Genetic Analyzer (ABI, U.S.A.).
Phylogenetic analysis: The sequences obtained wereassembled using ChromasPro (Technelysium Pty Ltd,
Australia), and the alignments were checked manually.
The sequences of 6 phaeodarian species, 57 cercozoans were also retrieved from the Basic Local Alignment
Search Tool (BLAST) at NCBI (http://blast.ncbi.nlm.nih.gov/
Blast.cgi)(Supplementary Material Table 2), and all thesequences were then aligned using MEGA 5 (Tamura et al.
2011). Phylogenetic trees were inferred by maximum-likelihood(ML), neighbor-joining (NJ) and Bayesian analysis methods.
For the ML and NJ trees, the General Time Reversible(GTR) model plus Gamma with the shape parameter foramong-site rate variation (G) was selected based on thelowest Bayesian Information Criterion (BIC) score, and thesubstitution nucleotide matrix parameters were calculated. The
Bootstrap values (Felsenstein 1985) were estimated basedon 1000 pseudo-replicates. GTR model with invariant sites
and the gamma distributed model (IG) were selected for theBayesian analysis, and the Bayesian posterior probabilities(BPP) were calculated with Mr Bayes version 3.2.2. (Ronquist
and Huelsenbeck 2003). The Markov Chain Monte Carlochains ran for 3.0 x 107 generations, sampling every 1000
generations. The ﬁrst 1.0 x 106 generations were discarded asburn-in, checking by a program Tracer version 1.6 (Rambautand Drummond 2009). The remaining data reaching the steadystate were used for building the consensus tree, and the tree
was visualized by Fig Tree version 1.4.0. (Rambaut 2012).

Acknowledgements
We thank the captains and crews of the T/SOshoro-maru (Hokkaido University), T/S Ushiomaru (Hokkaido University) and T/S Toyoshio-maru(Hiroshima University) for their assistance in sampling. We also thank the members of the oceanicplankton group of Station Biologique de Roscoff
for their kind technical advice. Appreciation is also

372 Y. Nakamura et al.

due to the members of Plankton laboratory of
Hokkaido University for their help in analysis. The
comments of two anonymous reviewers and theeditor also greatly improved the manuscript. This
study was ﬁnancially supported by Grant-in-Aid
for JSPS Fellows (no. 26-2889, Y. Nakamura),by the Cooperative Research Project with Centre National de la Recherche Scientiﬁque (CNRS),“Morpho-molecular Diversity Assessment of Ecologically, Evolutionally, and Geologically Relevant
Marine Plankton (Radiolaria)” by the Strategic International Research Cooperative Program hosted by
the Japan Science and Technology Agency (JST)(N. Suzuki and F. Not), and by “Integrated Research
on Biodiversity of Interspecies Relationships” of the
National Museum of Nature and Science (A. Tuji).

Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.protis.2015.05.004.

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