Phycologia (1999) Volume 38 (2), 149-155 Published 16 July 1999

Chrysochromulina fragaria sp. nov. (Prymnesiophyceae), a new haptophyte flagellate from Norwegian waters

WENCHE EIKREM AND BENTE EDV ARDSEN

Section for Marine Botany, Department of Biology, University of Oslo, P. O. Box 1069 Blindem, 0316 Oslo, Norway

W. EIKREM AND B. EDVARDSEN. 1999. Chrysochromulina fragaria sp. nov. (Prymnesiophyceae), a new haptophyte flagellate

from Norwegian waters. Phycologia 38: 149-155.

Chrysochromulina fragaria sp. nov. was one of the dominant species during the 1994 and 1995 Chrysochromulina blooms off the southern coast of Norway. It was isolated by the serial dilution culture method from a surface water sample collected during

the bloom in May-June 1994. The cells are spherical, 4 to 8 f.lm in diameter, and possess a coiling haptonema that is shorter than the two equal to subequal flagella. Each cell contains two chloroplasts, a nucleus, and one mitochondrion, which appears to be

reticulate. The periplast is covered by monomorphic scales (scale faces with identical pattern) of two types arranged in layers. The scale faces have a pattern of radiating ribs arranged in quadrants. One type has inflexed rims, whereas the other lype has erect rims. The first internal transcribed spacer (ITS 1) rONA sequence and scale morphology of C. fragaria are compared with that of Chrysochromulina sp. CCMP 1204. Despite the great similarities in scale morphology, their ITS I rONA regions are very different, indicating that they are different species. Both C. fragaria and Chrysochromulina sp. CCMP 1204 are nontoxic to Artemia franciscana nauplii.

INTRODUCTION

The number of known species in the genus Chrysochromulina

Lackey has increased steadily since the description of the first

marine species by Parke et al. (1955, 1956), and it is now one

of the marine phytoplanktonic genera with the greatest num­

ber of species. More than 50 species have been described, and 38 of them have been observed in Scandinavian waters. In

addition, from Scandinavian waters alone we know of 30

forms not described in the literature (Eikrem et al. 1998; Jen­

sen 1998) and the number of Chrysochromulina species

worldwide is expected to exceed 100 (Thomsen et al. 1994).

Members of the genus Chrysochromulina are unicellular,

nanoplanktonic, photosynthetic flagellates. Most species are

marine; only a few occur in freshwater (Green & Jordan

1994). They possess two smooth flagella and a haptonema that

is readily recognized with the light microscope. The length of

the haptonema varies greatly from many times the cell di­

ameter in some species of Chrysochromulina [e.g. c. 160 fLm

long in C. camella Leadbeater et Manton (Lead beater & Man­

ton 1969)] to less than the cell length in others [e.g. c. 5 fLm

in Chrysochromulina spinifera (Fournier) Pienaar et Norris

(Pienaar & Norris 1979)].

Chrysochromulina cells are covered by one to several kinds

of scales arranged in layers. The scales are organic and com­

posed mainly of proteins and carbohydrates (Leadbeater 1994). The basic scale is a round or oval plate composed of

micro fibrils, but the variation is great, and many highly sculp­

tured and elaborate scale forms are found. The scales of many

species consist of a proximal layer of micro fibrils arranged in

a radial pattern, whereas the pattern of the distal microfibrillar

layer is more variable. Scales may have monomorphic faces (the two scale faces having identical patterns) or dimorphic

faces (the scale faces having different patterns). The cells con­

tain two golden-brown chloroplasts, each with a pyrenoid that

may be bulging. The nucleus is posterior or central and the

outer membrane of the nuclear envelope is confluent with the

endoplasmic reticulum enveloping the chloroplast (Hibberd

1980). Located immediately within the cell membrane is a

layer of peripheral endoplasmic reticulum that also extends

into the haptonema (Pienaar 1994). The mitochondrion prob­

ably forms a network, as was shown for the coccolithophorid

Pleurochrysis carterae (Braarud et Fagerland) Christensen

(Beech & Wetherbee 1984). The flagellar apparatus consists

of two basal bodies, the haptonematal base, microtubular and

fibrous roots, and accessory and connecting fibers. The mi­

crotubular root (R,), which is connected to the mature flagel­

lum, may be either simple (a sheet of microtubules) or com­

pound (a sheet and a crystalline bundle), and its structure

varies between the species within the genus as presently de­

limited. The microtubular root R2 has an origin between the

two basal bodies, is simple, and consists of just a few micro­

tubules in the species examined so far. The roots associated

with the right flagellar base contain only a few microtubules (Green & Hori 1994).

With few exceptions, identification of Chrysochromulina

species relies on the examination by electron microscopy of

the organic scales covering the periplast (Lead beater 1994).

We present some morphological and ultrastructural details of

C. fragaria sp. nov., in addition to its sequence of the first

internal transcribed spacer (ITS1) rDNA, and compare it with

that of Chrysochromulina sp. CCMP 1204, which shows a

scale morphology reminiscent of that of C. fragaria sp. nov.

MATERIAL AND METHODS

Cultures

Cultures of Chrysochromulina fragaria were established from

cells isolated by the serial dilution culture method (Throndsen

1978) during a bloom dominated by species of Chrysochrom-

149

150 Phycologia, Vol. 38 (2), 1999

ulina off the southern coast of Norway in May-June 1994.

Strain S19 originated from a surface water sample collected

on 20 May 1994 off FI\'ldevigen (58°25'N, 8°46'E), and strain

U21 was from a surface water sample collected on 2 June

1994 off Grimstad (58°20'N, 8°36'E).

Chrysochromulina sp. strain CCMP 1204, obtained from

the Provasoli-Guillard National Center for Culture of Marine

Phytoplankton (CCMP), was isolated by R. Selvin from a wa­

ter sample collected at Baffin Bay, Canada (76°25'N,

82°55'W) on 3 June 1986.

Stock cultures of C. fragaria were grown in filtered sea­

water diluted to 30 psu, enriched with nutrients, vitamins, and

trace metals as in IMR 112 medium (Eppley et at. 1967), and

supplemented with 10 nM selenite. The cultures were grown

at 15°C under white fluorescent light with a photon fluence

rate (PFR) of about 50 /-Lmol photons m-2 S-I and a 12:12 h

LD cycle. Stock cultures of strain CCMP 1204 were grown

in K-medium (Keller et at. 1987) at 3°C under similar light

conditions as above. Stock cultures were transferred to new medium every 14 d.

Light microscopy

Living cells of the strains S19 and CCMP 1204 were studied with a Nikon Microphot FX fitted with phase contrast and

differential interference contrast optics and electronic flash.

Strain S19 was photographed using phase contrast and flash.

Electron microscopy

Positively stained whole mounts of strains S19 and CCMP

1204 were prepared according to Moestrup (1984). Some of

the preparations were shadowed with gold-palladium using an

Edward's Speedivac 12 E6 coating unit, c. 30° angle of shad­owing.

Thin sections of S19 were prepared according to the fol­

lowing protocol. The cells were fixed in 2% glutaraldehyde in

medium for 2 h and rinsed 3 times at 30-min intervals in

medium followed by 2 times at lO-min intervals in 0.1 M Na

cacodylate buffer (pH 8). Postfixation was carried out in 1 %

osmium tetroxide and 1 % ferricyanide in 0.1 M Na cacodylate

buffer for 3 h. The cells were then rinsed 3 times at IS-min

intervals in Na cacodylate buffer and 2 times at lO-min in­

tervals in distilled water. The samples were left overnight in

2% aqueous uranyl acetate. Subsequently the cells were rinsed

in distilled water and dehydrated in an ethanol series starting

at 30% and gradually rising to 96%. The dehydration was

concluded with changes in 100% ethanol 4 times at lO-min

intervals and changes in propylene oxide 2 times at lO-min

intervals. The pellets were left overnight in a 1: 1 mixture of

propylene oxide and Epon embedding resin (Burke & Giesel­

man 1971). Finally, the cells were changed in Epon 3 times

at I-h intervals before they were polymerized at 50°C for 12

h. The thin sections were stained with lead citrate. Thin sec­

tions and whole mounts were viewed in a Jeol 1200ex trans­

mission electron microscope.

Artemia bioassay

Toxicity tests were performed using a strain of the crustacean

Artemia franciscana (Creasel, Deinze, Belgium) as previously

described (Edvardsen 1993). Sixty nauplii were exposed to C.

fragaria strain U21 (9 X 108 cells 1-1) and Chrysochromulina

sp. strain CCMP 1204 (3 X 108 cells 1-1), and 30 nauplii were

exposed to C. fragaria strain S 19 for 24 h and 48 h at 25°C

in darkness. Seawater was used as control.

DNA extraction, amplification, and sequencing

DNA was extracted using a 3% CTAB (hexadecyltrimethylam­

monium bromide) procedure (Doyle & Doyle 1990). Biotiny­

lated and nonbiotinylated primers ITS 1 and ITS2 ( White et at.

1990) were used to amplify the internal transcribed spacer 1

(ITS 1) region. Polymerase chain reaction (PCR) amplifications

were performed in a Techne Genius using the following am­

plification profile: 95°C for 5 min, 30 cycles at 50°C for 1 min,

72°C for 1 min and 95°C for 1 min, and a final extension at

noc for 7 min. The PCR reaction mixtures (50 /-Ll) contained

1 U Taq DNA polymerase (Advanced Biotechnologies, UK),

50 ng DNA, 0.5 /-LM of each primer, 200 /-LM dNTp, 2.5 mM

MgCI2, 5 /-Ll lOX reaction buffer, and 5% acetamide. Single­

stranded DNA, obtained from the PCR products by using M-

280 streptavidine-coated magnetic Dynabeads (Dynal AS, Nor­

way), were sequenced directly with the use of the Sequenace

version 2.0 DNA Sequencing Kit (United States Biochemical)

according to the manufacturer's protocol and were run manually

on 6% acrylamide gels (Sambrook et al. 1989). DNA sequenc­

es have been submitted to GenBank: C. fragaria S 19,

AJ238708, Chrysochromulina sp. CCMP 1204, AJ238709.

The alignment was performed automatically using Clustal

W in the GCG package. The distance value was estimated by

Kimura two-parameter analysis (Kimura 1980) using PHYLIP

(Felsenstein 1995).

OBSERVATIONS

Description of the new species

Chrysochromnlina fragaria Eikrem et Edvardsen sp. nov.

Figs 1-16

DIAGNOSIS: Cellulae sphaericae-subsphaericae 4-S JJ.m, appendices in tenuem depressionem apical iter insertae. Haptonema (3-9 JJ.m) ftagellis plerumque aequalibus (10-16 JJ.m) brevius. Periplastus squamis duarum formarum tectus. Squamae aut marginibus inftexis (50-100 nm) ovales vel orbiculares 0.35-0.75 X 0.45-0.S JJ.m, aut marginibus erectis (150-200 nm) orbicularis vel ovales 0.5-0.S X

0.5-0.S JJ.m. Facies proximales et distales utriusque formae squa­marum striis radiantibus in quattuor quadrantes dispositis instructae.

Cells spherical to subspherical measuring 4-8 /-Lm, append­

ages inserted apically in a slight depression. Haptonema (3-9

/-Lm) shorter than the usually equal flagella (10-16 /-Lm). Peri­

plast covered with scales of two types. Scales with inflexed

rims (50-100 nm) oval to round 0.35-0.75 by 0.45-0.8 /-Lm

and scales with erect rims (150-200 nm) round to oval 0.5-

0.8 by 0.5-0.8 /-Lm. Proximal and distal faces of both scale

types with radiating ribs arranged in four quadrants.

HOLOTYPE: Figures 1-9.

ETYMOLOGY: Latin fragaria (strawberry). The organism

gives the impression of the shape of a squat strawberry in the

light microscope.

Eikrem & Edvardsen: A new species of Chrysochromulina 151

".

0.5 Jlm

Figs 1-7. Chrysochromulina fragaria sp. nov. Figs 1, 2. Light micrographs (phase contrast) of living cells. Figs 3-5. Shadow-cast whole mounts of scales. Distal scale faces (arrows). proximal scale faces (arrowheads). Outer layer scales (white arrow, white arrowhead), inner layer scales (black arrows, black arrowheads). Figs 6, 7. Thin sections of scales.

Fig. 6. Cross section showing scales with inflexed rims (arrow) and scales with upright rims (arrowhead). Fig. 7. Tangential section of scales.

Microscopic observations of Chrysochromulina fragaria

sp. nov. strain 819

The spherical to subspherical cells (4-8 J-Lm) bear a hapto­

nema (3-8 J-Lm) that is shorter than the two equal or subequal

flagella (10-14 J-Lm) and is capable of coiling. The appendages

are inserted in a slight apical depression (Figs 1,2). Two types

of scales cover the periplast. We have not been able to produce

sections of cells with intact scale investments, but we assume

that the scales are arranged in two layers, with the scales hav­ing the tall, erect rims forming the outer layer. We also assume that the rims are on the distal face of the scales, which is

usually the case in Chrysochromulina species (Lead beater 1994). The inner layer consists of round to oval plate scales (0.35-0.75 by 0.45-0.8 J-Lm) with an inflexed rim (50-100

nm) with concentric fibers. Both scale faces have radiating ribs (c. 65-90) extending from center to edge (proximal face) or rim (distal face). The round scales of the outer layer (0.5-

152 Phycoiogia, Vol. 38 (2), 1999

8 9

0.5 J.Lm

12

•

Figs 8-16. Thin sections showing ultrastructural features of Chrysochromulina Jragaria sp. nov.; b, basal body; c, chloroplast; f, flagellum; h, haptonema; m. mitochondrion; n, nucleus; p, pyrenoid.

Fig. 8. Longitudinal section of cell with posterior nucleus, two chloroplasts, a pyrenoid with part of a traversing thylakoid (arrowhead), mitochondrial profile, flagellum, and a Golgi apparatus (arrow). Fig. 9. Cross section through cell showing the 'peculiar' dilations of the Golgi apparatus (arrow). Figs 10, 11. Scales are produced in the Golgi apparatus, which is in the anterior part of the cell. The scales are released near the base of the appendages. Figs 12-14. The flagellar transition zone. Proximal band (arrow), distal band (arrowhead). Fig. 15. Section through the flagellar apparatus showing flagellum, part of the haptonema, and the basal body. Fig. 16. Section through the haptonema showing the seven microtubules.

Eikrem & Edvardsen: A new species of Chrysochromulina 153

Figs 17, 18. Whole mounts of Chrysochromulina sp. CCMP 1204. Fig. 17. Whole cell with detached scales. Stained with uranyl ace-tate. Fig. 18. Details of scales. Shadow-cast preparation showing the two scale types (arrow, arrowhead).

0.8 by 0.5-0.8 fLm) have tall erect rims (150-200 nm) with

no obvious pattern, and the radiating ribs (c. 75-90) extend

from the center to the rim on the distal face and from center

to edge on the proximal face (Figs 3-7). The pattern created

by the radiating ribs divides the scales into four distinct quad­

rants (Fig. 7) in both scale types.

The cells contain two golden-brown, parietal chloroplasts,

each with an immersed pyrenoid that may be bulging and may

be traversed by thylakoids, a posterior nucleus, a mitochon­

drion, and a Golgi body where the scales are produced (Figs

8-11).

The flagellar apparatus (Fig. 15) has not been studied in

detail, but some features have been revealed. The flagella have

a proximal band consisting of a tripartite plate and a distal

band with a transitional plate (Figs 12-14). The extended part

of the haptonema contains seven microtubules (Fig. 16).

To allow a comparison between the scales of C. fragaria

sp. nov. and those of Chrysochromulina sp. CCMP 1204, we

have included electron micrographs (Figs 17, 18) of a whole

cell and detached scales. Under the light microscope the cells

appear spherical to pear shaped (8-14 by 6-10 fLm). The cells

may be strongly compressed dorsoventrally. The haptonema

(12-20 fLm) is slightly shorter than the flagella (14-24 fLm).

The scales are of two types: one type has an erect rim (85-

100 nm) and measures 0.7-0.9 by 0.8- l .0 fLm; the rim of the

other type appears inflexed (35-50 nm) and it measures 0.5-

0.8 by 0.6-0.9 fLm. All scale faces have radiating ribs (c. 75-

85).

Artemia bioassay

Strains of C. fragaria (S19 and U21) and Chrysochromulina

sp. CCMP 1204 that were grown in complete medium were

nontoxic to Artemia nauplii (all 60/30 nauplii survived in each

test) when exposed for 24 and 48 h under the conditions used.

ITS! nucleotide sequences

Nucleotide sequences of ITS 1 rDNA and flanking stretches of

18S and 5.8S rDNA regions were determined for C. fragaria

strain S19 and Chrysochromulina sp. strain CCMP 1204 (Fig.

19). Terminals of the 18S rRNA coding region and starting

positions of the 5.8S region were determined by comparison

with other haptophytes (Larsen & Medlin 1997; Edvardsen &

Medlin 1998). The length of the ITS1 region was 326 base

pairs (bp) in C. fragaria and 381 bp in Chrysochromulina sp.

strain CCMP 1204. The G/C content of ITS 1 was much higher

in C. fragaria (71%) than in CCMP 1204 (58%). Pairwise

comparisons, including gaps of strain S19 and CCMP 1204,

revealed a distance value of approximately 0.61 (Kimura two­

parameter) for the ITS 1 region.

DISCUSSION

Of the species of Chrysochromulina already described, there

are several that resemble C. fragaria when examined by light

microscopy. For instance, C. minor Parke et Manton, C. kappa

Parke et Manton, C. brevifilum Parke et Manton, and C. ad­

riatica Leadbeater have the same general appearance as C.

fragaria and are in the same size range. Also, species that are

typically oblong or pear shaped may produce cells that are

round (e.g. C. polylepis Parke et Manton) and may then be

confused with C. fragaria at the light microscope level.

Despite the similarities in cell and scale morphology, ITS 1

data show that Chrysochromulina sp. CCMP 1204 should be

considered a separate species from C. fragaria. The scales of

C. fragaria are in the same size range as the scales of Chrys­

ochromulina sp. CCMP 1204 and the number of radiating ribs

overlap. The height of the upright rims and the width of the inflexed rims in C. fragaria are greater than the corresponding

rims in Chrysochromulina sp. CCMP 1204. Length of hap­

tonema and flagella and cell size are somewhat overlapping

in the two species, but Chrysochromulina sp. CCMP 1204

tend to be larger with longer appendages. Another distinguish­

ing character at the light microscope level is the pronounced

dorsoventral compression of many cells in the Chrys­

ochromulina sp. CCMP 1204 cultures; this has not been ob­

served in cultures of C. fragaria sp. nov.

The scales of both C. fragaria and Chrysochromulina sp.

CCMP 1204 closely resemble the scales of what once was be­

lieved to be a mutant of C. chiton Parke et Manton (Manton

1966, 1967a, 1967b). In the light of recent research on C. pol­

ylepis (Edvardsen & Paasche 1992; Edvardsen & Medlin 1998)

one may speculate that C. chiton has two distinct flagellate

forms. In the original culture, C. chiton (Parke et al. 1958)

possessed two types of scales with dimorphic scale faces. After

some time in the history of the culture a flagellate covered with

two types of scales with monomorphic scale faces turned up

(Manton 1966, 1967a, 1967b). According to a hypothesis based

on the morphology of underlayer scales in coccolithophorids,

scales having dimorphic scale faces represent the haploid gen­

eration and scales with monomorphic scale faces the dipl0id

generation (Billard 1994). Applying this hypothesis to the case

154 Phycologia, Vol. 38 (2), 1999

_____________________________ 18S __________________________ _

C. fragaria

CCMP 1204

????????? ?????????? ?????????? ?????????? ?????????? ???????TGCG

CAAGATATC CATCGACAAG AGTTGTGTGT TTGTTGGTTC TTGAGAGGCT ACGCAAG AAA.

18S __ ITS1

C. fragaria

CCMP 1204

GAAGGATCA -TTACCAGGT CTTTCCACCC GCACCCTTGC GTACCGTTTC CGTCTTCCGGG

......... A .... C-... .......... .... GTA ... .... A .. CAA T-.G.A .. CCT

C. fragaria

CCMP 1204

CGCGCACCGT TTCATTATCG GAGCGCGTCC TCTGCGCGTC -GGCGTC-CG CCTCCGTGCG

G. G. GGT .. G C .. GAG. GG. CC. GT . -. .. . ... T . TC.. T ...... A.. GG ....... T

C. fragaria

CCMP 1204

TG-------G CGTCGTCGC- ------GCAG GCACTGCT-G GCTCGTCCGG CAGCCGTCTG

C.CATTGGC . ........ GT ATCTTTTTT . . T.TC.T.C. T.GT ... GT. AG.G .. G.CT

C. fragaria

CCMP 1204

GCCGCCTCGG C-.-TCCCCGC CGTAGCGTGC --------CC CCCGCTGGAG CGCCG--T-­

.GG .. G .... GCA.A.AG .. AC.G.A.A .. AGAAGGTG.G .T .. T ... TT TT.T.TT.TC

C. fragaria

CCMP 1204

------GCC- GCGCGCGTCC GCGTCGTGCG CGTCGCGTG- G-------CC CTT----GCC

AGAAGC ... T ... T.T .. GG T.T.T ..... . CAGC .... C .TTGAGTT.T ... ATGG.AG

C. fragaria

CCMP 1204

CT----GTCT CCA GGGTGCG GTCCTCGCGT GC-GTCTCGC GCGTCGCGCC CGTCC GGCCT

.. TAGC .. GC .. TT .. A .. T TAG .. G.A.A .GT.C .. TTA A .. GG ... TT TT .. TT .. G.

ITS1 __ S.8S __________________ __

C. fragaria

CCMP 1204

--CCACTCGA GTTGTCACAA -CACACAACT CTTGTCGATG GATATCTTGG CTCTCRCATC

AG .. T ... A . . AACCA .... A ........ . ??????????

Fig. 19. Sequence alignment of ITSI rDNA and flanking stretches of l 8S and 5.8S rDNA of Chrysochromulina fragaria strain SI9 and Chrysochromulina sp. strain CCMP 1204 done in Clustal W.

of C. chiton would imply that the 'mutant' is the diploid gen­

eration and the flagellate originally described as C. chiton rep­

resents the haploid generation. Considering the large number

of species in the genus, it is tempting to speculate that mor­

phologically distinct flagellate stages occur in more species, but

for the 18 species of Chrysochromulina in which the 18S rRNA

gene has been analyzed, it has been shown to be the case only

in Chrysochromulina polylepis. The others appear to be sepa­

rate species (Edvardsen, unpublished observations).

The saddle-shaped C. parva Lackey is the type species of

the genus Chrysochromulina. The fact that C. fragaria sp. nov.

and several other Chrysochromulina species differ from the

saddle-shaped species with respect to morphology, ultrastruc­

ture, and I8S rRNA signatures (e.g. Green & Hori 1994; Green

& Jordan 1994; Birkhead & Pienaar 1995; Medlin et al. 1997;

Eikrem & Moestrup 1998; Edvardsen, Eikrem, Green, Medlin,

and Moestrup, unpublished observations) may eventually lead

to the erection of a new genus that will include C. fragaria sp.

nov. Molecular data have shown that Chrysochromulina is not

a monophyletic group, and according to 18S rRNA data, C.

fragaria is more closely related to, for example, C. polylepis,

C. chiton, C. kappa, Prymnesium parvum (N. Carter) Green et

al., P. patelliferum Green et aI., and P. calathiferum Chang et

Ryan than to the saddle-shaped species of Chrysochromulina

(Medlin et al. 1997; Simon et al. 1997). Further studies should

include detailed examination of the flagellar apparatus of C.

jragaria, which should be compared with that of genetically

related species.

The Chrysochromulina bloom in May 1994 off the south

coast of Norway was dominated by C. fragaria, c. polylepis,

C. acantha, and C. brevifilum. In 1995, C. fragaria and C.

polylepis occurred in high concentrations in the same area

(Dahl et aI. 1998). Thus, C. fragaria can be a quantitatively

important species in Scandinavian waters. Scales that may be

assigned to C. fragaria have also been observed in the Baltic

(Hajdu et at. 1996) and in Danish waters (Jensen 1998).

ACKNOWLEDGMENTS

We thank Sissel Brubak and Lisbeth Haukrough for excellent

technical assistance and Bjl1lrg Tosterud for preparing the Latin diagnosis. Dag Klaveness and E. Paasche kindly read the man­

uscript. Electron microscopy was carried out at the laboratories

for Biosciences, Department of Biology, University of Oslo.

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Accepted 24 March 1999