DNA BARCODING OF CHLORARACHNIOPHYTES USING NUCLEOMORPH ITS SEQUENCES1
ABSTRACT Chlorarachniophytes are a small group of marine photosynthetic protists. They are best known as examples of an intermediate stage of secondary endosymbiosis: their plastids are derived from green algae and retain a highly reduced nucleus, called a nucleomorph, between the inner and outer pairs of membranes. Chlorarachniophytes can be challenging to identify to the species level, due to their small size, complex life cycles, and the fact that even genus-level diagnostic morphological characters are observable only by EM. Few species have been formally described, and many available culture collection strains remain unnamed. To alleviate this difficulty, we have developed a barcoding system for rapid and accurate identification of chlorarachniophyte species in culture, based on the internal transcribed spacer (ITS) region of the nucleomorph rRNA cistron. Although this is a multicopy locus, encoded in both subtelomeric regions of each chromosome, interlocus variability is low due to gene conversion by homologous recombination in this region. Here, we present barcode sequences for 39 cultured strains of chlorarachniophytes (>80% of currently available strains). Based on barcode data, other published molecular data, and information from culture records, we were able to recommend names for 21 out of the 24 unidentified, partially identified, or misidentified chlorarachniophyte strains in culture. Most strains could be assigned to previously described species, but at least two to as many as five new species may be present among cultured strains.
- SourceAvailable from: Yoshihisa Hirakawa[Show abstract] [Hide abstract]
ABSTRACT: Chlorarachniophytes are marine unicellular algae that possess secondary plastids of green algal origin. Although chlorarachniophytes are a small group (the phylum of Chlorarachniophyta contains 14 species in 8 genera), they have variable and complex life cycles that include amoeboid, coccoid, and/or flagellate cells. The majority of chlorarachniophytes possess two or more cell types in their life cycles, and which cell types are found is one of the principle morphological criteria used for species descriptions. Here we describe an unidentified chlorarachniophyte that was isolated from an artificial coral reef that calls this criterion into question. The life cycle of the new strain includes all three major cell types, but DNA barcoding based on the established nucleomorph ITS sequences showed it to share 100% sequence identity with Lotharella globosa. The type strain of L. globosa was also isolated from a coral reef, but is defined as completely lacking an amoeboid stage throughout its life cycle. We conclude that L. globosa possesses morphological diversity between culture strains, and that the new strain is a variety of L. globosa, which we describe as Lotharella globosa var. fortis var. nov. to include the amoeboid stage in the formal description of L. globosa. This intraspecies variation suggest that gross morphological stages maybe lost rather rapidly, and specifically that the type strain of L. globosa has lost the ability to form the amoeboid stage, perhaps recently. This in turn suggests that even major morphological characters used for taxonomy of this group may be variable in natural populations, and therefore misleading.PLoS ONE 01/2011; 6(8):e23193. · 3.53 Impact Factor
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ABSTRACT: High intra-individual sequence variation in the nuclear rDNA LSU-5S intergenic spacer in the Sargassaceae (Fucales, Phaeophyceae)Journal of Applied Phycology 12/2012; 24(6):1373-1379. · 2.49 Impact Factor
Article: Algal taxonomy: A road to nowhere?[Show abstract] [Hide abstract]
ABSTRACT: The widespread view of taxonomy as an essentially retrogressive and outmoded science unable to cope with the current biodiversity crisis stimulated us to analyze the current status of cataloguing global algal diversity. Contrary to this largely pessimistic belief, species description rates of algae through time and trends in the number of active taxonomists as revealed by the webresource AlgaeBase show a much more positive picture. More species than ever before are being described by a large community of algal taxonomists. The lack of any decline in the rate at which new species and genera are described, however, is indicative of the large proportion of undiscovered diversity and bears heavily on any prediction of global algal species diversity and the time needed to catalogue it. The saturation of accumulation curves of higher taxa (family, order and classes) on the other hand suggest that at these taxonomic levels most diversity has been discovered. This reasonably positive picture does not imply that algal taxonomy does not face serious challenges in the near future. The observed levels of cryptic diversity in algae, combined with the shift in methods used to characterize them, has resulted in a rampant uncertainty about the status of many older species. As a consequence there is a tendency in phycology to move gradually away from traditional names to a more informal system whereby clade-, specimen- or strain-based identifiers are used to communicate biological information. Whether these informal names for species-level clades represent a temporary situation stimulated by the lag between species discovery and formal description, or an incipient alternative or parallel taxonomy, will be largely determined by how well we manage to integrate historical collections into modern taxonomic research. Additionally, there is a pressing need for a consensus about the organizational framework to manage the information about algal species names. An eventual strategy should preferably come out of an international working group that includes the various databases as well as the various phycological societies. In this phycologists should link up to major international initiatives that are currently being developed, such as the compulsory registration of taxonomic and nomenclatural acts and the introduction of Life Science Identifiers. © 2012 Phycological Society of AmericaJournal of Phycology 01/2013; 49(2):215-225. · 2.53 Impact Factor
DNA BARCODING OF CHLORARACHNIOPHYTES USING NUCLEOMORPH
Gillian H. Gile,2Rowena F. Stern,2Erick R. James, and Patrick J. Keeling3
Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, British Columbia,
Canada V6T 1Z4
Chlorarachniophytes are a small group of marine
photosynthetic protists. They are best known as
examples of an intermediate stage of secondary
endosymbiosis: their plastids are derived from green
algae and retain a highly reduced nucleus, called a
nucleomorph, between the inner and outer pairs of
membranes. Chlorarachniophytes can be challenging
to identify to the species level, due to their small
size, complex life cycles, and the fact that even
genus-level diagnostic morphological characters are
observable only by EM. Few species have been for-
mally described, and many available culture collec-
tion strains remain unnamed. To alleviate this
difficulty, we have developed a barcoding system
for rapid and accurate identification of chlorarach-
niophyte species in culture, based on the internal
transcribed spacer (ITS) region of the nucleomorph
rRNA cistron. Although this is a multicopy locus,
encoded in both subtelomeric regions of each chro-
mosome, interlocus variability is low due to gene
conversion by homologous recombination in this
region. Here, we present barcode sequences for
39 cultured strains of chlorarachniophytes (>80%
of currently available strains). Based on barcode
data, other published molecular data, and informa-
tion from culture records, we were able to recom-
mend names for 21 out of the 24 unidentified,
partially identified, or misidentified chlorarachnio-
phyte strains in culture. Most strains could be
assigned to previously described species, but at least
two to as many as five new species may be present
among cultured strains.
Key index words: Bigelowiella; Chlorarachnion; cul-
ture collections; Gymnochlora; internal transcribed
spacer; Lotharella; Norrisiella; Partenskyella
bp,base pairs;ITS, internal
Chlorarachniophytes are a small group of marine
photosynthetic protists belonging to the phylum
Cercozoa in the supergroup Rhizaria (Bhattacharya
et al. 1995, Cavalier-Smith and Chao 1996, Keeling
2001, Nikolaev et al. 2004). They can take the form
of amebae, coccoid cells, or flagellates, and many of
them alternate among all of these forms and varia-
tions thereof during their complex life cycles
(Fig. 1). A major distinguishing feature of the chlor-
arachniophytes is their retention of a nucleomorph.
Chlorarachniophytes acquired their plastid by the
secondary endosymbiotic uptake of a green alga,
and the nucleomorph is a relict nucleus of that
endosymbiont that persists between the inner and
outer pairs of their four membrane-bound plastids
(Ludwig and Gibbs 1989, Ishida et al. 1997, Rogers
et al.2007). Chlorarachniophyte
genomes are invariably composed of three chromo-
somes ranging in size from roughly 100 to 200 kbp
and bearing rRNA cistrons at each of the chromo-
some ends (Silver et al. 2007). The nucleomorph
has attracted the attention of molecular evolutionary
biologists as a window into processes of endosymbi-
otic organelle integration, such as endosymbiotic
gene transfer, genome compaction, and development
of protein-targeting systems (Gilson and McFadden
1996, 2002, Archibald et al. 2003, Rogers et al. 2004,
Gilson et al. 2006, Gile and Keeling 2008). As a result,
knowledge about chlorarachniophytes is heavily
biased toward their molecular evolution: the entire
chloroplast and nucleomorph genomes of Bigelowiella
natans, the model chlorarachniophyte for molecular
studies, have been sequenced (Gilson et al. 2006,
Rogers et al. 2007).
The type species, Chlorarachnion reptans Geitler,
was first described in 1930 by Lothar Geitler, but
there were no further studies of chlorarachnio-
phytes until it was rediscovered, described more
fully with EM, and brought into culture by Hibberd
and Norris in 1981 (Geitler 1930, Hibberd and
Norris 1984). Today, seven genera and 12 species
have been described, of which nine species from
five genera are represented among the 48 strains in
public culture collections worldwide. Chlorarachnio-
phyte genera are defined mainly by the morphology
of the bulbous pyrenoid that protrudes from the
1Received 15 May 2009. Accepted 28 April 2010.
2These authors contributed equally to this work.
3Author for correspondence: e-mail firstname.lastname@example.org.
J. Phycol. 46, 743–750 (2010)
? 2010 Phycological Society of America
plastid toward the center of the cell and is capped
on the cytoplasmic side by a carbohydrate storage
vesicle. The pyrenoid can have a shallow cleft
caused by an invagination of the inner pair of plas-
tid membranes, as in Norrisiella S. Ota et Ishida (Ota
et al. 2007a); a deep cleft, as in Lotharella Ishida et
Y. Hara (Ishida et al. 1996, 2000, Ota et al. 2005,
2009a); or a deep cleft that encloses the nucleo-
morph, as in Chlorarachnion (Hibberd and Norris
1984). Otherwise, the pyrenoid can be invaded with
small tubular invaginations of the innermost plastid
membrane, as in Gymnochlora Ishida et Y. Hara
(Ishida et al. 1996), or it may be lacking entirely,
as in Partenskyella S. Ota, Vaulot et Ishida (Ota et al.
scheme: there are no EM data concerning pyrenoid
morphology in Cryptochlora E. Caldero ´n et Schnetter
(Calderon-Saenz and Schnetter 1987, 1989), and
the genus Bigelowiella Moestrup (Moestrup and
Sengco 2001) was defined by its dominant life-cycle
stage being flagellate cells (pyrenoids of Bigelowiella
are invaded by a shallow cleft, as in Norrisiella).
The principle behind DNA barcoding is to enable
accurate species identification through a short sig-
nature sequence of DNA (Hebert et al. 2003a).
Where feasible, barcoding has the advantage of
allowing nonexperts to accurately and rapidly iden-
tify species even when diagnostic morphological fea-
tures are missing (as for immature or damaged
specimens), misleading (as for common convergent
morphologies, such as coccoid green algae), or
not easily observable (as for simple microscopic
Fig. 1. Differential interference contrast light micrographs illustrating a diversity of chlorarachniophyte cell types. (A) Chlorarachnion
reptans CCCM 449 ameboid cells, (B) Bigelowiella longifila characteristic ameboid cell with single extended filopodium, (C) Gymnochlora
stellata CCMP 2057 ameboid cells, (D and E) Lotharella oceanica CCMP 622 flagellate cells, (F) C. reptans CCCM 449 coccoid cell, (G)
Lotharella amoeboformis CCMP 2058 ameboid cell, (H) C. reptans CCMP 238 ameboid cell, (I) Bigelowiella natans CCMP 621 flagellate cell,
(J) Lotharella vacuolata CCMP 240 thick-walled resting cysts (a sealed pore is visible in the wall of one cell). All scale bars represent
GILLIAN H. GILE ET AL.
organisms). Chlorarachniophytes exemplify the lat-
ter two points in that in their coccoid or flagellate
forms they can easily be mistaken for other green-
pigmented algae [CCMP 621 B. natans was initially
misidentified as Pedinomonas minutissima, a chloro-
phyte (Moestrup and Sengco 2001)], and their diag-
nostic features can be observed only by EM.
Moreover, a great many chlorarachniophyte cultures
in available collections have not been identified to
the species level and for many years were automati-
cally assigned to the type genus Chlorarachnion.
A barcoding system for chlorarachniophytes would
therefore be a useful tool for clarifying the existing
diversity of cultures as well as accurately distinguish-
ing chlorarachniophyte species from each other and
from other groups of algae.
The most commonly used DNA sequence marker
for barcoding is the 5¢ end of the cytochrome c oxi-
dase I (cox1) gene. This locus can effectively dis-
criminate species in many animal groups (Hebert
et al. 2003b), as well as red algae (Saunders 2005),
brown algae (Lane et al. 2007, Kucera and Saun-
ders 2008), diatoms (Evans et al. 2007), and the cil-
iate genus Tetrahymena (Chantangsi et al. 2007).
The cox1 locus is not universally applicable, how-
ever, either because it does not display sufficient
variation to distinguish species, as is the case with
land plants (Chase et al. 2005, Fazekas et al. 2008),
or because it is missing, as is the case with many
anaerobic protists. A commonly used alternative is
the ITS of the rRNA cistron. Although this locus
can be problematic due to length variation among
the multiple copies typically present per nuclear
genome, the ITS locus has been used successfully
for groups including sponges (Park et al. 2007),
anthozoans (Sinnigeret al.
(Li et al. 2008, Nguyen and Seifert 2008, Ortega
et al. 2008).
Here, we present a method for barcoding chlor-
arachniophytes using nucleomorph ITS sequences,
in which intragenomic variation is limited by gene
conversion in nucleomorph genomes. We have
determined barcode sequences for 39 out of 48
chlorarachniophyte strains in culture collections
plus Norrisiella sphaerica S. Ota et Ishida, repre-
senting nine described species from seven genera
(Cryptochlora perforans E. Caldero ´n et Schnetter and
Lotharella polymorpha C. Dietz, K. Ehlers, C. Wilh.,
Gil-Rodriguez et Schnetter are not available from
culture collections). This marker clearly resolved
described species, assigned most unidentified or
provisionally named chlorarachniophyte strains to a
previously described species, and revealed that one
‘‘chlorarachniophyte’’ strain, UTEX 2631, is a mem-
ber of the Eustigmatophyceae. Up to 25 strains are
redundant, being either directly derived from other
strains or isolated at the same time and place and
found to have identical barcode sequences, indicat-
ing a lower diversity of chlorarachniophytes in
culture than the number of strains would suggest.
MATERIALS AND METHODS
DNA extraction, PCR, and sequencing. Chlorarachniophyte
cultures (Table S1 in the supplementary material) were either
purchased or donated from the Provasoli-Guillard National
Center for Culture of Marine Phytoplankton (CCMP, West
Boothbay Harbor, ME, USA); the Roscoff Culture Collection
(RCC, Roscoff, France); the Plymouth Culture Collection of
Marine Algae (PCC, Plymouth, UK); the Culture Collection of
Algae at the University of Texas (UTEX, Austin, TX, USA); the
Culture Collection of Algae, Philipps-University Marburg
(Marburg, Germany); the Canadian Centre for the Culture of
Microorganisms (CCCM, Vancouver, Canada); and the Micro-
bial Culture Collection at the National Institute for Environ-
mental Studies (NIES, Tsukuba, Japan). Chlorarachniophyte
cells from ?10 mL of culture were harvested by centrifugation
at 1,150g in an Eppendorf 5415 D benchtop centrifuge
(Eppendorf, Hamburg, Germany) and subjected to two rounds
of snap-freeze treatment with liquid nitrogen. DNA was
extracted using the DNeasy Plant mini kit (Qiagen, Mississauga,
ON, Canada) according to the manufacturer’s instructions.
Previously published primers (Inagaki et al. 1998) failed to
amplify the most commonly employed locus for barcoding, the
5¢ region of cytochrome c oxidase subunit 1 (cox1) under
various reaction conditions. Because cox1 sequences are pub-
licly available for only two chlorarachniophytes and only two
other Cercozoa, primer optimization for this locus in the
chlorarachniophytes is impracticable at this time, and we
shifted our focus to the ITS between the LSU and SSU rRNAs,
which includes the 5.8S rRNA. Previously published universal
ITS primers, ITS1 (Gottschling et al. 2005) and ITS4 (White
et al. 1990), amplified either the nuclear or nucleomorph ITS
region, so we designed new primers based on published
nuclear and nucleomorph SSU sequences (Silver et al. 2007) to
selectively amplify either nuclear or nucleomorph ITS loci
along with a short stretch of SSU sequence to aid in positive
identification and orientation of this variable region. Nuclear
ITS primers also include a short stretch of the LSU sequence.
Nucleomorph ITS sequences were amplified using nmITSF, 5¢-
AACGAGGAATGCCTAGTAAGC-3¢ and ITS4, 5¢-TCCTCCGCT-
TATTGATATGC-3¢ primers. Nuclear ITS sequences required a
heminested PCR using nucITSF 5¢-AACGAGGAATTTCTAGT-
AAAC-3¢ as a forward primer in both reactions, and nucITSR-
outer 5¢-CAATCCCAAACAACACGACTCTTCG-3¢ and nucITSR-
inner 5¢-TCTGTACGGGGTTCTCACCCT-3¢ as reverse primers.
Some of the sequences presented here were initially obtained
using ITS1 and ITS4 primers; for each of these sequences, the
nucleomorph-specific ITS primers were subsequently con-
firmed to amplify products of the expected size. PCR reactions
were carried out using Econotaq DNA polymerase (Lucigen,
Middleton, WI, USA) or PuReTaq Ready-to-Go PCR beads (GE
Life Sciences, Piscataway, NJ, USA) with an initial denaturation
at 94?C for 3 min followed by 35 cycles of 94?C for 30 s, 50?C
for 30 s, and 72?C for 1 min for nucleomorph or 2 min for
nuclear ITS amplification, and a final extension step at 72?C
for 10 min. PCR products were purified using QIAquick PCR
purification kit (Qiagen) and sequenced directly on both
strands at Macrogen (Seoul, Korea). Barcode sequences
determined in this study were deposited in GenBank under
accession numbers FJ821375–FJ821382, FJ821384, FJ821385,
FJ821387–FJ821428, FJ937328–FJ937333, and FJ937351.
sequences were aligned separately using ClustalX (Thompson
et al. 1997) with the gap opening penalty reduced to five and
gap extension penalty reduced to two. Sequences from strains
belonging to the same species were nearly identical, but each
species was quite divergent relative to the others. To improve
the resolution of among-species relationships, ambiguously
aligned regions were removed by GBlocks 0.91b (Castresana
and nucleomorph ITS
2000) with all gap positions allowed and all other parameters
set to default, which reduced the alignment sizes from 903 to
610 and 1,459 to 679 characters for nucleomorph and nuclear
data sets, respectively. Modeltest v3.7 (Posada and Crandall
1998, Posada and Buckley 2004) was used to determine the
best-fit model of nucleotide substitution using the Akaike
information criterion (AIC) from log likelihoods computed
were inferred using distance, maximum-likelihood (ML), and
Bayesian methods using the HKY+I model for the nuclear data
set and the TVMef+I+G model for the nucleomorph data set
and specifying transition⁄transversion ratios, equilibrium base
frequencies, substitution category rates, proportion of invari-
able sites, and gamma shape parameter alpha as specified by
Modeltest. Distance trees were computed using the neighbor-
joining (NJ) method from 1,000 bootstrap replicate data sets,
and a consensus tree was generated in PAUP*. ML trees with
1,000 bootstrap replicates were computed in PhyML v3.0
(Guindon and Gascuel 2003) using a BIONJ starting tree and
NNI search procedure. Bayesian analyses were performed with
MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001) using six
substitution categories and a proportion of invariable sites for
the nucleomorph data set and two substitution categories and a
proportion of invariable sites for the nuclear data set. For each
data set, two independent chains, sampled each 100 genera-
tions, were run until they converged (the average standard
deviation of partition frequency values between the chains
dropped below 0.01) with 25% of the trees discarded as burn-
in. The nuclear data set runs continued for 230,000 genera-
tions, and the nucleomorph data set runs continued for
650,000 generations. Consensus trees were computed from the
final 75% of saved trees of both runs, for a total of 3,450
nuclear trees and 9,750 nucleomorph ITS trees. Uncorrected
‘‘p’’ pair-wise distances were computed in PAUP* 4.0b10
RESULTS AND DISCUSSION
Assessment of nucleomorph ITS for barcoding. Here,
we present barcodes for chlorarachniophyte strains
in culture worldwide using the nucleomorph ITS
locus. Nucleomorph ITS was chosen over nuclear
ITS and the more commonly used cox1 locus due to
its ability to clearly differentiate described chlor-
arachniophyte species and the efficiency with which
it can be amplified and directly sequenced. Nucleo-
morph ITS PCR products are ?700 base pairs long,
an ideal length for directly sequencing both strands,
and are expected to display little or no interlocus
variability on the basis of the B. natans nucleo-
morph genome.In chlorarachniophyte
morphs, the rRNA cistrons are typically located in
both subtelomeric regions of all three chromo-
somes, and their sequence homogeneity is main-
tainedby gene conversion
recombination (Gilson et al. 2006). In the nucleo-
morph genome of B. natans, five out of six ITS
regions are identical in sequence, and the sixth,
located on one end of chromosome 1 (1a), differs
only by four nucleotides (Fig. 2). In contrast, the
nuclear ITS-amplified product length is roughly
1,300 base pairs, and for many chlorarachniophytes,
it could not be sequenced directly on both strands
due to a variable region near the 3¢ end of the
amplified fragment. We searched the incomplete
nuclear genome of B. natans by BLASTn and found
four distinct loci with an indel in the ITS2 region,
which accounts for difficulties sequencing the anti-
sense strand and the 3¢ end of the sense strand.
More rRNA cistrons may exist in the genome, as the
genome is incomplete and the nuclear ITS sequence
in GenBank (accession AF289035) is not identical to
any of the four loci we identified. Nuclear ITS
sequences were also more easily sequenced from
B. natans strains than others, so interlocus length poly-
morphism may be more pronounced in other taxa.
The relationships between both nuclear and nu-
cleomorph ITS regions (Figs. 2 and 3) were roughly
as expected based on phylogenies of SSU rRNA
(Silver et al. 2007), and both molecules effectively
Because the majority of chlorarachniophyte species
in culture are represented by only one strain (see
below), we do not yet know whether either locus will
effectively discriminate between chlorarachniophyte
species in nature. There is cause to be optimistic that
the nucleomorph ITS will remain a useful barcode
marker, however, because the minimum interspecies
distance is more than five times greater than the
maximum intraspecies distance (Table 1). This gap
is more pronounced in the nucleomorph than it is
in the nucleus, mainly due to the lower intraspecies
maximum distance in the nucleomorph.
Assignment of cultured strains to described species. In
this study, we have determined barcode sequences
for 39 out of 48 chlorarachniophyte strains in cul-
ture collections, plus N. sphaerica, representing
>80% of available strains. From this sample, we were
able to assign all but two strains to previously
described species (Table S1). Our analyses suggest
that CCMP 2014 could be a new species. Based on
the barcode, it is most likely a member of the genus
Gymnochlora, but this assignment requires EM to
confirm that its pyrenoid is invaded by tubular
invaginations of the innermost plastid membrane.
Furthermore, confirmation that CCMP 2014 is mor-
phologically distinct from Gymnochlora stellata would
help to support our inference that the nucleo-
morph ITS locus can assign species to genera in
addition to distinguishing species from one another.
The other unassignable strain, UTEX 2631, yielded
an anomalous ITS sequence with 90% sequence
identity to Nannochloropsis limnetica, a freshwater
eustigmatophyte. Because this strain was collected
from the sporangiophores of the lichen-like fungus
Multiclavula corynoides, and the plastids visible in
light micrographs on the UTEX Web site (http://
5115) do not show the bulbous pyrenoid character-
istic of chlorarachniophytes, we conclude that this
culture is not a chlorarachniophyte and should be
reassigned to the Eustigmatophyceae.
We were unable to barcode 10 chlorarachnio-
phyte strains due to inefficient DNA extraction from
GILLIAN H. GILE ET AL.
sparse culture samples. For six of these strains
(CCMP 242, CCMP 3166, Marburg CCMP 621,
CCMP 1259, CCMP 1408, and CCMP 1487), we
have, nonetheless, recommended updated names.
One (CCMP 242) has been formally named as
B. longifila (Ota et al. 2007b), and two (CCMP 3166
and Marburg CCMP 621) are synonyms of the
type strain for their species (P. glossopodia and
B. natans)—that is, their collection records indicate
that they are each directly derived from another
strain. We have also recommended updated names
for CCMP 1259, CCMP 1408, and CCMP 1487
mainly on the basis of previously published molecu-
lar data (Silver et al. 2007, Ota et al. 2009b), but
supported in the case of CCMP 1259 and CCMP
1487 by collection information indicating they are
not independent isolates from other strains we have
barcoded here. For each of these six strains,
therefore, we expect the nucleomorph ITS barcodes
to be identical with those of their related strains.
However, one or more new species may be repre-
sented among the remaining four strains that we
were unable to barcode (RCC 375, RCC 376, CCMP
2285, and RCC 531). Previous analyses have indi-
cated that RCC 375 is likely a new species of Lotha-
rella, and a formal description is underway (Ota
et al. 2009c). A potentially related strain, RCC 376,
from the same collection date and location, has no
published molecular data. Similarly, there is no
molecular data for CCMP 2285, but it was collected
at the same time and place as CCMP 2314, which
we have here assigned to Lotharella globosa. Without
molecular data, we cannot determine whether RCC
376 and CCMP 2285 are identical to the barcoded
strains bearing the same isolation information, but
in all other cases so far, strains sharing the same
B. natans nucleomorph 3a
B. natans nucleomorph 2a
B. natans nucleomorph 1a
Marburg Chlorarachnion sp.1
Fig. 2. Maximum-likelihood (ML) phylogenetic tree of nucleomorph-encoded internal transcribed spacer (ITS) sequences. Filled cir-
cles at nodes indicate 100% bootstrap support for both ML and distance analyses and a Bayesian posterior probability of 1.0. An asterisk
indicates the type species; (1) indicates strain synonyms of the type species; (2) indicates strains from the same isolation date and location
as the type species. MB, MrBayes; NJ, neighbor joining.
isolation information have yielded identical bar-
codes. Finally, RCC 531 represents an independent
isolation for which there are no molecular or micro-
graphical data yet available.
On the basis of barcode sequences, other pub-
lished molecular data, and collection records, we
here recommend names for all but three of the 48
strains in culture (Table S1), the vast majority of
which belong to previously described species. Half
of these (24 strains) have been previously identified
to species and are correctly labeled, but 16 are cur-
rently unidentified, five are identified only to genus,
and three are incorrectly labeled. We consider the
13 strains currently labeled either ‘‘Chlorarachnion
sp.’’ or ‘‘Chlorarachnion cf. sp.’’ to belong to the
unidentified category, rather than identified to
genus, as these names were equivalent to ‘‘unidenti-
fied chlorarachniophyte’’ until 2001 when a second
genus was described, and each of these cultures was
deposited before 2000. Only three strains were
determined to be incorrectly named according to
our data. The barcode for CCMP 2314, currently
labeled ‘‘Chlorarachnion reptans,’’ is identical to that
of L. globosa, and nuclear and nucleomorph SSU
sequences support this designation (Silver et al.
renamed. Another misidentification, UTEX 2631,
is actually a eustigmatophyte. Finally, the Culture
Collection of Algae at Philipps University in Mar-
burg holds a strain derived from CCMP 621 labeled
C. reptans that should be updated to B. natans. Alto-
gether, the number of potentially new chlorarach-
niophyte species present among the unidentified
strains in culture collections is between two (CCMP
2014 and RCC 375, descriptions underway) and five
(if all of the strains lacking molecular data turn out
to be new species), bringing the total number of
cultured species to between 14 and 17, a surpris-
ingly low range given the total of 48 strains.
Aside from the number of species, how much
diversity is represented among the chlorarachnio-
phytes in culture? Many strains are synonymous,
meaning that they were directly derived from
another strain, either to be shared with another cul-
ture collection or to establish a clonal strain. In
addition, many strains share the same collection
date and location, so although they are not strictly
synonyms, they were likely derived from the same
population of chlorarachniophytes. Taken together,
only 23 out of the 48 strains can be considered truly
independent isolates. This is reflected in the unex-
pectedly high sequence similarity among nucleo-
morph ITS barcodes from each species (Fig. 2). For
the most extreme example, C. reptans, there are 11
strains in culture, of which nine were collected at
Puerto Pen ˜asco, Mexico, by Richard Norris, on June
20, 1966. Four of these are labeled as strain syn-
onyms of the type culture, CCAP 815⁄1, now
deceased, although it is possible that all 10 derive
from the same collection vessel. One, NIES 1408,
was collected independently at Amami Island, Japan
(Fig. 2, Table S1), and another was inferred to
have been collected in Tunisia, as it was deposited
Table 1. Uncorrected p distances.
B. natans genome locus 1
B. natans genome locus 4
B. natans genome locus 2
B. natans genome locus 3
B. natans AF289035
Fig. 3. Maximum-likelihood (ML) phylogenetic tree of nucleus-encoded internal transcribed spacer (ITS) sequences. Filled circles at
nodes indicate 100% bootstrap support for both ML and distance analyses and a Bayesian posterior probability of 1.0. An asterisk indicates
the type species; (1) indicates strain synonyms of the type species. MB, MrBayes; NJ, neighbor joining.
GILLIAN H. GILE ET AL.
by K. Grell, who published observations of C. reptans
collected at the Gulf of Hammamet (Grell 1990).
Although only three independent isolations cannot
provide insight into within-species genetic diversity,
the nucleomorph ITS sequence similarity among
the Japan, Tunisia, and Mexico isolations shows that
this species is widespread. Likewise, there are 13
strains of B. natans in culture, derived from only
four independent isolations. Six of these strains are
synonymous with the type strain CCMP 621 accord-
ing to the collection records. Two separate isola-
tions from the Mediterranean Sea, RCC 337 and
RCC 435, show some genetic differentiation relative
to the remaining strains, all of which were isolated
from the Sargasso Sea (Fig. 2). The eight strains of
B. longifila are derived from five independent isola-
tions, although four of these came from the Sar-
collection, RCC 530, shows no sequence divergence.
L. globosa has been isolated from the coasts of both
France and Guam. The remaining seven chlorarach-
niophyte species in culture (seven out of 11, includ-
ing the two strains
underway) are represented by one isolation each,
thereby providing no information as to their diver-
sity or distribution.
homogeneous within a culture to be efficiently
amplified and sequenced, and they contain suffi-
cient variability to distinguish chlorarachniophyte
species. This will be a useful tool for future species
identifications, as it removes the need for EM.
Based on these data, other published molecular
data, and inferences from strain information, we
have recommended updated names for 21 strains
that are currently unidentified, identified only to
genus, or, in only three cases, misidentified. There
are fewer distinct chlorarachniophytes in culture
than the number of strains would suggest, due
mainly to the same strain being held at multiple cul-
ture collections, but also due to strains derived from
the same collection. At least some chlorarachnio-
phyte species are widely distributed, but an under-
future environmental sequencing studies.
We thank Ken Ishida for providing a culture of N. sphaerica
from his private collection; Tia Silver and John Archibald for
sharing the nucleomorph ribosomal cistron sequence from
Lotharella oceanica CCMP 622; the Joint Genome Institute’s
Community Sequencing Program (http://www.jgi.doe.gov/
sequencing/why/50026.html) for their ongoing efforts to
sequence the nuclear genomes of Guillardia theta and B. na-
tans; K. Barry and E. Lindquist of the JGI for project manage-
ment and data availability; and M. W. Gray, J. Archibald, G. I.
McFadden, and C. E. Lane for their contributions to the pro-
ject. We also thank the Provasoli-Guillard National Center for
Culture of Marine Phytoplankton, USA; the Roscoff Culture
Collection, France; the Plymouth Culture Collection of
Marine Algae, UK; the Culture Collection of Algae at the
University of Texas, USA; the Culture Collection of Algae,
Philipps-University Marburg, Germany; the Canadian Centre
for the Culture of Microorganisms, Canada; and the Micro-
bial Culture Collection at the National Institute for Environ-
mental Studies, Japan for their kind donation of cultures for
this project. This work was supported by a grant from Gen-
ome Canada to P. J. K. as part of the Canadian Barcode of
Life Network. G. H. G. is supported by a postgraduate doc-
toral fellowship from the Natural Sciences and Engineering
Research Council of Canada. P. J. K. is a Fellow of the Cana-
dian Institute for Advanced Research and a Senior Investigator
of the Michael Smith Foundation for Health Research.
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Table S1. Chlorarachniophyte strain informa-
tion and recommended names.
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