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European Journal of Phycology

ISSN: 0967-0262 (Print) 1469-4433 (Online) Journal homepage: https://www.tandfonline.com/loi/tejp20

Phaeocystis rex sp. nov. (Phaeocystales,Prymnesiophyceae): a new solitary species thatproduces a multilayered scale cell covering

Robert A. Andersen, J. Craig Bailey, Johan Decelle & Ian Probert

To cite this article: Robert A. Andersen, J. Craig Bailey, Johan Decelle & Ian Probert (2015)Phaeocystis�rex sp. nov. (Phaeocystales, Prymnesiophyceae): a new solitary species thatproduces a multilayered scale cell covering, European Journal of Phycology, 50:2, 207-222, DOI:10.1080/09670262.2015.1024287

To link to this article: https://doi.org/10.1080/09670262.2015.1024287

Published online: 24 Apr 2015.

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Phaeocystis rex sp. nov. (Phaeocystales, Prymnesiophyceae): anew solitary species that produces a multilayered scale cellcovering

ROBERTA. ANDERSEN1, J. CRAIG BAILEY2, JOHAN DECELLE3,4 AND IAN PROBERT5,6

1Friday Harbor Laboratories, University of Washington, 620 University Road, Friday Harbor, WA 98250 USA2Center for Marine Science and Department of Biology and Marine Biology, UNC-Wilmington, 5600 MK Moss Lane,Wilmington, NC 28409 USA3EPEP, Sorbonne Universités, UPMC Univ Paris 06, UMR 7144, Station Biologique de Roscoff, 29680 Roscoff, France4CNRS, UMR 7144, Station Biologique de Roscoff, 29680 Roscoff, France5Roscoff Culture Collection, Sorbonne Universités, UPMC Univ Paris 06, FR2424, Station Biologique de Roscoff, 29680Roscoff, France6CNRS, FR2424, Station Biologique de Roscoff, 29680 Roscoff, France

(Received 22 September 2014; revised 24 November 2014; accepted 16 December 2014)

A morphologically distinct marine species, Phaeocystis rex sp. nov., was described on the basis of light microscopy, transmissionelectron microscopy and DNA sequence comparisons. Non-motile cells were solitary (non-colonial), 6–10 µm in diameter and8–15 µm long, and possessed chloroplasts with distinctive finger-like lobes. TEM observations demonstrated the presence of twoshort flagella and a very short haptonema that arose from an invagination of the protoplast. Non-motile cells were surrounded byone to several dense layers composed of scales, presumably unmineralized, and an amorphous material. Phylogenetic analysesbased upon combined partial nucleotide sequences for five nuclear- or plastid-encoded genes (18S rRNA, 28S rRNA, 16S rRNA,psbA and rbcL) from cultured strains and from uncharacterized acantharian symbionts confirmed that P. rexwas a distinct species.These analyses implied that P. rex occupies an intermediate evolutionary position between solitary and colonial Phaeocystisspecies.

Key words: algae, Phaeocystis rex, Phaeocystales, Prymnesiophyceae, organic scales, systematics, ultrastructure

INTRODUCTION

The haptophyte microalga Phaeocystis Lagerheim isone of the most extensively studied genera of marinephytoplankton. Free-living Phaeocystis are ubiqui-tous from poles to tropics and from coastal to openocean waters (Schoemann et al., 2005). Colonial spe-cies of Phaeocystis rank among a handful of keystoneeukaryotic phytoplankton taxa that shape the structureand functioning of marine ecosystems (Verity &Smetacek, 1996). Phaeocystis is not only a majorcontributor to global carbon cycling and export(Arrigo et al., 1999; DiTullio et al., 2000), but alsoimpacts sulphur cycling by producing substantialamounts of dimethylsulphoniopropionate (DMSP)and its volatile catabolite dimethylsulphide (DMS), aclimatically active trace gas emitted from the ocean(Stefels et al., 2007). In coastal areas, blooms ofPhaeocystis, which can contribute > 90% of totalphytoplankton abundance, may be detrimental to thegrowth and reproduction of marine life, and strongly

impact human activities such as fisheries, aquacultureand tourism (He et al., 1999; Chen et al., 2002;Schoemann et al., 2005; Blauw et al., 2010; Doanet al., 2010). In oceanic regions from poles to tropics,several Phaeocystis species have recently beenreported to form endosymbiotic associations with cer-tain species of Acantharia, a widespread and abundantlineage of radiolarians (Decelle et al., 2012).

The first report of Phaeocystis was by Pouchet(1892) who found large round gelatinous colonies inNorwegian marine waters (Lofoten Islands, Varanger)during summer 1882. Eight years later, he found thesame alga in the North Atlantic Ocean (near Torshavn,Faroe Islands), and formally described it as TetrasporapouchetiHariot (Pouchet, 1892) due to perceived simi-larity to another colony-forming alga, Tetraspora gir-audyiDerbès & Solier (1851). Lagerheim (1893) founda similar alga in 1884 from the Koster Islands, Sweden,and argued that these two marine brownish-colouredalgae should not be placed in the freshwater green algalgenus Tetraspora Link ex Desvaux. Therefore, he pro-posed the new generic name Phaeocystis Lagerheim

Correspondence to: Robert A. Andersen. E-mail: raa48@uw.edu.

Eur. J. Phycol. (2015), 50: 207–222

ISSN 0967-0262 (print)/ISSN 1469-4433 (online)/15/020207-222 © 2015 British Phycological Societyhttp://dx.doi.org/10.1080/09670262.2015.1024287

Published online 24 Apr 2015

and designated Phaeocystis pouchetii (Hariot)Lagerheim as the type species. Hariot (1893) listedPhaeocystis pouchetii but did not describe it; hereferred to Lagerheim’s 1893 paper. Lagerheim(1893) also suggested that Tetraspora fuscescensBraun exKützing, a freshwater colonial alga, belongedto this small group of colonial brown-coloured algae. In1895, DeToni recombined T. giraudyi and T. fuscescensas Phaeocystis giraudyi (Derbès & Solier) DeToni andPhaeocystis fuscescens (A. Braun ex Kützing) DeToni,respectively. Lagerheim (1896) made a more thoroughstudy of Phaeocystis pouchetii and argued that P. gir-audyi and P. fuscescens should be doubtful members ofPhaeocystis until further detailed investigations werecompleted. In confirmation of Lagerheim’s doubt,those two taxa are now known to be heterokont algae,not haptophytes. Chrysoreinardia giraudyi Billard(in Hoffmann et al., 2000) is a member of thePelagophyceae and Tetrasporopsis fuscescens (A.Braun ex Kützing) Lemmermann ex Schmidle belongsin Clade I of the heterokont algae (Entwisle &Andersen, 1990; Bailey et al., 1998; Yang et al., 2012).

A few new species of Phaeocystis were describedduring the next 25 years, beginning with Phaeocystisglobosa Scherffel, which was collected in the NorthSea, from Helgoland, Germany (Scherffel, 1899).Scherffel gave an exceptionally detailed descriptionthat included an illustration of the haptonema (‘thirdflagellum’) (Scherffel, 1900, plate 1, fig. 69). Soonafter, Karsten (1905) described P. antarctica Karstenfrom the Southern Ocean. Although the alga wascommon and abundant, Karsten’s description of indi-vidual cells and colonies was brief, and he was uncer-tain whether there was one deeply lobed chloroplast ortwo separate chloroplasts. He was convinced, how-ever, that the cells were embedded in mucilage andspecifically mentioned the resemblance of P. antarc-tica and P. globosa.

Phaeocystis sphaeroides Büttner and P. amoeboi-dea Büttner were then described from cultures estab-lished from the harbour of Kiel, Germany (Büttner,1911). The two species were distinguished from eachother based on the sizes of their swimming cells aswell as the amoeboid nature of aflagellate cells.Büttner’s descriptions were not as thorough as thoseof Scherffel, but he described brown gelatinous colo-nies visible to the naked eye, a single yellow-brownchloroplast per cell, and swimming cells with twoequal flagella. Although Büttner did not report a‘third flagellum’ (i.e. haptonema), he clearly statedthat there was only a single chloroplast per cell exceptimmediately before cell division when the chloroplastdivides prior to cytokinesis. The gelatinous colony,the yellow-brown colour and the two equal flagella areconsistent with Phaeocytis, but, as Medlin & Zingone(2007) point out, the single chloroplast per cell andmetabolic nature of the cells casts some doubt on thesealgae belonging in Phaeocystis. Unfortunately,

Büttner (1911) made no attempt to compare or distin-guish among the colonial stages of his two new taxa orbetween them and earlier described Phaeocystisspecies.

Mangin (1922) described Phaeocystis bruceiMangin from Antarctica. The cells are 5–6 µm indiameter, each cell possesses two golden chloroplasts,and the colonies are 1–2 mm in diameter. Manginsuggested his new alga was different from P. globosaand P. antarctica because of the distinct form of thecolonies: P. brucei colonies are characterized by thepresence of separate clusters of cells that are intercon-nected by mucilaginous ‘trails’.

Moestrup (1979) described a sixth species, P. scro-biculataMoestrup, from New Zealand coastal waters.Colonies are not known for this species and cells are c.8 µm in size and are covered by a periplast consistingof two scale types. Cells also produce star-like arraysof filaments. Scales and the enigmatic star-like fila-ments were first discovered in Phaeocystis by Parkeet al. (1971), and these scale and star-like features ledMoestrup to conclude that P. scrobiculata belonged toPhaeocystis although it was a solitary (non-colonial)organism. Zingone et al. (1999) described two moresolitary species, P. cordata Zingone & Chrétiennot-Dinet and P. jahnii Zingone from the MediterraneanSea. Both species have small cells (3–4 µm and 3–5µm, respectively) and bear flagella that are slightlyunequal in length. In culture, P. jahnii also sometimesforms loose and small aggregations of immobile (non-flagellate) cells embedded in a gelatinous matrix, butthese are different from classical Phaeocystis coloniesin lacking a definite shape and a regular arrangementof cells as well as a visible external envelope (Zingoneet al., 1999).

Six species have been examined using electronmicroscopy and/or DNA sequence analysis:Phaeocystis pouchetii, P. antarctica, P. cordata, P.globosa, P. jahnii and P. scrobiculata; the remainingthree species (P. amoeboidea, P. brucei, P. sphaer-oides) have not received modern study because theyare unavailable in culture.

In this paper, we describe a new solitary species ofPhaeocystis collected from the Arabian Sea. Unlikeany previously described Phaeocystis species, theArabian Sea isolate has chloroplasts with two elongatelobes and an unusual extracellular cell covering com-prised of scales and other material. Electron micro-scope observations and five-gene phylogeneticanalyses further demonstrate that this alga is a newPhaeocystis species.

MATERIALS AND METHODS

Culture origin and culture conditions

The alga was isolated from a sample collected byW. Balch on12 November 1995 from the Arabian Sea (14.4490N,

R.A. Andersen et al. 208

64.9997E) using a Niskin bottle at 75 m depth (2% of surfacelight level). The first attempt produced a unialgal culture but alabyrinthulid-like parasite was present. A single-cell isolation(culture A9125) was established on 25 September 1998 byselecting a single swimming cell using a micropipette. Thestrain was deposited as CCMP2000 in the Provasoli-GuillardNational Center for Culture of Marine Phytoplankton (nowthe NCMA). Cells were grown in L1 or f/2 seawater culturemedium enriched with soil extract (Guillard, 1975; Guillard& Hargraves, 1993). Cultures were maintained at c. 24ºCunder a 13:11 h light:dark cycle with approximately 60–100µM s−1 m−2 of cool white fluorescent illumination.

Brightfield and transmission electron microscopy

Light microscope (LM) observations were made using aZeiss Axio Imager (Z1) equipped with an Axio Cam HRmdigital camera and Axio Vision 4.5 software (Carl Zeiss,Göttingen, Germany). For transmission electron microscopy(TEM), cells were gently pelleted, the supernatant wasremoved, and the cells were resuspended in 3% glutaralde-hyde in 0.1 M phosphate buffer (pH 6.8) containing 0.6 Msucrose. After 30 s, an equal volume of 2% osmium tetroxidein 0.01 M phosphate buffer with sucrose was added. After 50min, cells were centrifuged, the fixative was discarded andthe pellet was resuspended in 0.1M phosphate buffer withoutsucrose. Cells were centrifuged again, the supernatant wasdiscarded and the pellet was resuspended in 0.1 M phosphatebuffer, and the solution was filtered onto a 13 mm Milliporefilter (Merck-Millipore Corp., Billerica, Massachusetts,USA). The filter and cells were placed in a small plasticdish and enrobed with 1% low gelling point agar at 35ºC(Sigma-Aldrich, St. Louis, Missouri, USA). When the agargelled, 0.5% uranyl acetate in de-ionized water was added,the dish was covered and then refrigerated at 4ºC overnight.The uranyl acetate was decanted and the agar-enrobed filterwas dehydrated in an ethanol series (10%, 30%, 50%, 70%,95%, 100%). Following two changes of 100% ethanol, thefilter and cells were further dehydrated with two changes of100% propylene oxide. Cells were infiltrated and embeddedin Spurr’s epoxy resin. Thin sections were examined on aZeiss 902A transmission microscope (Carl Zeiss).

The culture strains for which new sequences wereobtained in this study are listed in Table 1. Cultures wereharvested in exponential growth phase and concentrated bycentrifugation. Total nucleic acids were extracted using theNucleospin RNA II kit (Macherey-Nagel GmbH, Düren,Germany) and quantified using a Nanodrop ND-1000 spec-trophotometer (Labtech International Ltd, Uckfield, UK). Inthis study, we targeted the nuclear 18S and 28S rRNA, theplastid-encoded 16S rRNA, photosystem II D1 protein(psbA) and form-I ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) genes using different primersets (Decelle et al., 2012). Amplifications were performedwith Phusion high-fidelity DNA polymerase (Finnzymes;Thermo Fisher Scientific/Finnzymes Oy, Vantaa, Finland)in a 25 μL reaction volume, using the following PCR para-meters: 30 s at 98°C; followed by 35 cycles of 10 s denatura-tion at 98°C, 30 s annealing at 50°C for the 18S, 28S andpsbA genes and at 55°C for 16S and rbcL genes, and 30 sextension at 72°C; with a final elongation step of 10 min at72°C. PCR products were then purified by EXOSAP-IT (GE Tab

le1.

Pha

eocystisstrains,geog

raph

icorigin,and

GenBankaccessionnu

mbersforfive

nuclearandchloroplastg

enes.

Strain/ID

number

Pha

eocystisspecies

Isolation

source

Geographicorigin

plastid

16SrRNA

18SrRNA

28SrRNA

psbA

rbcL

P36

0Pha

eocystispo

uchetii

Culture

NorwegianSea

_AF18

2114

__

_P36

1Pha

eocystispo

uchetii

Culture

NorwegianSea

_AJ278

036

__

_SK34

Pha

eocystispo

uchetii

Culture

Greenland

Sea

_X77

475

__

_CCMP13

74(RCC40

23)

Pha

eocystisan

tarctica

Culture

Antarctica(M

cMurdo

Sou

nd)

KP14

4217

KP14

4246

KP14

4256

KP14

4270

KP14

4261

CCMP18

71(RCC40

24)

Pha

eocystisan

tarctica

Culture

Antarctica(A

rthu

rHarbo

ur)

KP14

4233

KP14

4253

KP14

4258

KP14

4272

KP14

4262

RCC13

83(CCMP31

04)

Pha

eocystiscordata

Culture

MediterraneanSea

EF05

1764

JX66

0992

JX66

0941

JX66

0950

JX66

0967

RCC13

84(CCMP24

96)

Pha

eocystisjahn

iiCulture

MediterraneanSea

KP14

4232

AF16

3148

KP14

4257

KP14

4271

KP14

4263

CCMP20

00(RCC40

25)

Phaeocystis

rex

Culture

ArabianSea

(IndianOcean

)KP14

4218

KP14

4247

KP14

4259

KP14

4273

KP14

4264

K13

21Pha

eocystisglob

osa

Culture

WestA

tlanticOcean

_JX

6609

86JX

6609

35JX

6609

42JX

6609

82K13

98Pha

eocystisglob

osa

Culture

EastC

hina

Sea

_JX

6609

87JX

6609

36JX

6609

43JX

6609

83PCC54

0(RCC35

39)

Pha

eocystisglob

osa

Culture

North

EastA

tlanticOcean

KP14

4229

AF18

2115

JX66

0930

JX66

0944

JX66

0978

PCC57

5Pha

eocystisglob

osa

Culture

North

EastA

tlanticOcean

KP14

4231

KP14

4252

JX66

0933

JX66

0946

JX66

0981

PCC64

(RCC35

38)

Pha

eocystisglob

osa

Culture

Eng

lishChann

el_

JX66

0994

JX66

0934

JX66

0948

JX66

0977

RCC17

19Pha

eocystisglob

osa

Culture

Eng

lishChann

el_

_JX

6609

21JX

6609

51JX

6609

68RCC73

9Pha

eocystisglob

osa

Culture

PacificOcean

KP14

4234

_JX

6609

24JX

6609

56JX

6609

71RCC67

8Pha

eocystissp.(un

described)

Culture

North

Sea

_KP14

4254

JX66

0939

JX66

0955

KP14

4266

(con

tinued)

Phaeocystis rex sp. nov. 209

Tab

le1.

Con

tinued.

Strain/ID

number

Pha

eocystisspecies

Isolation

source

Geographicorigin

plastid

16SrRNA

18SrRNA

28SrRNA

psbA

rbcL

PCC55

9(RCC35

41)

Pha

eocystissp.(un

described)

Culture

North

EastA

tlanticOcean

KP14

4230

JX66

0995

JX66

0931

JX66

0945

JX66

0979

RCC87

6Pha

eocystissp.(un

described)

Culture

Sou

thEastP

acificOcean

KP14

4235

_JX

6609

28JX

6609

57JX

6609

75RCC90

8Pha

eocystissp.(un

described)

Culture

Sou

thEastP

acificOcean

KP14

4236

EU10

6761

/JX66

0990

JX66

0919

JX66

0959

JX66

0964

RCC93

5Pha

eocystissp.(un

described)

Culture

Sou

thEastP

acificOcean

KP14

4237

EU10

6782

JX66

0920

JX66

0960

JX66

0965

RCC94

2Pha

eocystissp.(un

described)

Culture

Sou

thEastP

acificOcean

KP14

4238

__

KP14

4278

_RCC99

3Pha

eocystissp.(un

described)

Culture

Sou

thEastP

acificOcean

_EU10

6820

JX66

0922

JX66

0961

JX66

0969

RCC99

4Pha

eocystissp.(un

described)

Culture

Sou

thEastP

acificOcean

KP14

4239

KP14

4255

KP14

4260

KP14

4279

KP14

4265

Oki29

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

KP14

4219

KP14

4248

JX66

0792

JX66

0832

KP14

4267

Oki32

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

_KP14

4249

JX66

0793

KP14

4274

JX66

0892

Oki35

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

_KP14

4250

JX66

0794

JX66

0833

_Oki36

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

KP14

4220

KP14

4251

_JX

6608

34JX

6608

93Oki43

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

KP14

4221

JX66

0740

_JX

6608

35JX

6608

94Oki44

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

KP14

4222

JX66

0741

JX66

0795

JX66

0836

JX66

0895

Oki46

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

KP14

4223

JX66

0742

JX66

0796

JX66

0837

JX66

0896

Oki57

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

KP14

4224

JX66

0747

JX66

0797

KP14

4275

JX66

0898

Oki65

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

KP14

4225

JX66

0749

_KP14

4276

JX66

0900

Oki66

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

KP14

4226

_JX

6607

98KP14

4277

_Oki74

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

KP14

4227

JX66

0752

JX66

0800

JX66

0842

_Oki85

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

KP14

4228

JX66

0754

JX66

0801

JX66

0843

JX66

0903

Oki87

Pha

eocystissp.(un

described)

Sym

bion

tEastP

acificOcean

_JX

6607

55JX

6608

02JX

6608

44JX

6609

04Ros1

Pha

eocystissp.(un

described)

Sym

bion

tEng

lishChann

elKP14

4240

JX66

0757

JX66

0804

JX66

0846

KP14

4268

Ros2

Pha

eocystissp.(un

described)

Sym

bion

tEng

lishChann

elKP14

4241

JX66

0758

JX66

0805

JX66

0847

KP14

4269

Vil4

6Pha

eocystissp.(un

described)

Sym

bion

tMediterraneanSea

KP14

4242

JX66

0766

JX66

0807

JX66

0852

JX66

0910

Vil8

4Pha

eocystissp.(un

described)

Sym

bion

tMediterraneanSea

KP14

4243

JX66

0771

JX66

0809

JX66

0859

JX66

0914

Vil8

6Pha

eocystissp.(un

described)

Sym

bion

tMediterraneanSea

KP14

4244

JX66

0772

JX66

0811

JX66

0861

JX66

0916

Vil9

6Pha

eocystissp.(un

described)

Sym

bion

tMediterraneanSea

KP14

4245

JX66

0774

JX66

0813

JX66

0863

JX66

0918

Outgroup

RCC13

43Im

antoniarotund

aCulture

KP14

4214

AJ246

267

EU72

9457

EU85

1963

AB04

3696

RCC13

48Isochrysisga

lban

aCulture

JF48

9944

AJ246

266

EU72

9474

AJ575

574

AB04

3693

RCC24

76Pleurochrysiscarterae

Culture

KP14

4215

AJ246

263

EU81

9084

AY1197

57D1114

0RCC14

34Prymnesium

parvum

Culture

KP14

4216

AJ246

269

EU72

9443

AY1197

58AB04

3698

R.A. Andersen et al. 210

Healthcare, Little Chalfont, UK) and bidirectionallysequenced on an ABI3130xl automated DNA sequencer(Life Technologies Corporation, Carlsbad, California, USA)using the ABI-PRISM Big Dye Terminator CycleSequencing Kit (Life Technologies Corporation) accordingto the manufacturer’s specifications. Raw sequences wereedited and assembled with Chromas Pro v.1.5(Technelysium Pty Ltd, South Brisbane, Australia).GenBank accession numbers for the sequences are shownin Table 1.

Phylogenetic analyses

To determine the phylogenetic affiliation of CCMP2000, adataset for each of the five loci (16S rRNA, 18S rRNA, 28SrRNA, psbA and rbcL) was constructed including partialgene sequences from culture strains and from unculturedPhaeocystis living in symbiotic association with Acantharia(Decelle et al., 2012). Four taxa from other haptophyte orderswere included in each dataset as outgroups: Isochrysis gal-bana M. Parke, Pleurochrysis carterae (Braarud &Fagerland) Christensen [= Chrysotila carterae (Braarud &Fagerland) Andersen, Kim, Tittley & Yoon], PrymnesiumparvumN. Carter and Imantonia rotundaReynolds (Table 1).

For each locus, sequences were independently alignedwith MUSCLE implemented in Seaview v4 (Gouy et al.,2010). The length of aligned sequences was 734 bp for 16SrRNA, 465 bp for 18S rRNA, 836 bp for 28S rRNA, 572 bpfor psbA and 479 bp for rbcL. Sequence divergence betweenthe five genes was estimated by the p-distance (uniform ratesamong sites) with MEGA version 5 (Tamura et al., 2011).The single-locus datasets were then separately subjected tomaximum likelihood (ML) analyses with 100 bootstrap repli-cates using RAxML (Stamatakis et al., 2008). Visual check-ing of the topology of each tree showed congruence for thephylogenetic placement of highly supported clades. A con-catenation of the five datasets into a single partition (48 taxa,3087 bp length, including gaps) was therefore performedwith the FASconCAT program (Kück & Meusemann,2010). Because the sequences of symbiotic Phaeocystiswere short, a separate dataset (28 taxa, 4824 bp length,including gaps) was constructed without these sequences toincrease the number of nucleotide positions.

For both concatenated datasets, the general-time-reversi-ble model with gamma distributed rates (GTR+G) wasselected by jModelTest v2 (Darriba et al., 2012) as thefavoured model of sequence evolution under the Akaikeand Bayesian Information Criteria (AIC and BIC, respec-tively). Maximum likelihood topologies for the two concate-nated datasets were inferred using RAxML withGTRGAMMA (GTR+Γ) model, and support for nodes wasassessed by performing 2000 bootstrap replicates. TheBayesian inference was performed using MrBayes v3.2.1(Ronquist & Huelsenbeck, 2003). Markov chain MonteCarlo (MCMC) analyses were run for two independent setsof four chains with a chain length of 5 million generations forboth datasets, starting from a random tree with a samplefrequency for trees of 1 in every 1000 generations. Theaverage standard deviation of the split frequencies was< 0.01 and the effective sample size for all parameters washigher than 200, as recommended by Drummond & Rambaut(2007). Twenty-five per cent of the total trees were discarded

as burn-in, and the remaining trees were used to build aconsensus tree and to calculate posterior probabilities (PP)for each node. The final tree was visualized with FigTreev1.3.1.

RESULTS

Taxonomic description

Phaeocystis rex Andersen, Bailey, Decelle & Probert sp.nov. (Figs 1–17)

DIAGNOSIS: Vegetative cells 6–10 µm in diameter and8–15 µm long; cells surrounded by a wall-like cover-ing comprised of multiple layers of organic scales andamorphous material; posterior end of the cell coveringoften, but not always, thickened forming a conspicu-ous knob; two chloroplasts per cell; each chloroplastwith two anteriorly projecting finger-like lobes andone pyrenoid; non-motile cells with two short flagellaand a short haptonema; free-swimming motile cellswith two flagella and a haptonema; cell division insidethe cell covering, with subsequent escape of onenaked daughter cell; multiple wall-like layers accu-mulating with repeated cell divisions; nucleotidesequences distinct (GenBank accession numbersKP144218, KP144247, KP144259, KP144,273,KP144264).

HOLOTYPE HERE DESIGNATED: NY02026433, plasticTEM block RAA-665, deposited in the New YorkBotanical Garden, New York, USA.

ISOTYPE HERE DESIGNATED: Cryopreserved culture strainCCMP2000 deposited in the Provasoli-GuillardNational Center for Marine Algae and Microbiota,Bigelow Laboratory for Ocean Sciences, EastBoothbay, Maine, USA.

TYPE LOCALITY: Arabian Sea (14.4490N, 64.9997E) ata depth of 75 m.

ETYMOLOGY: Latin, rex = king; refers to the crown-likeappearance imparted by the four lobes of the twoplastids.

Description: light microscopy

Cells were typically ellipsoid, 6–10 µm in diameterand 8–15 µm long, although round and oval cells werealso observed immediately after cell division(Figs 1–6). A conspicuous thin, translucent cell cover-ing surrounded the cell (arrowheads, Figs 1, 2, 6). Twochloroplasts occurred in each mature cell and werepressed together forming a cup-like base (Figs 2, 3).Each plastid typically had two long, tapering finger-like lobes that extended towards the (apparent) ante-rior end of the cell, although only two lobes werevisible in most LM images taken at highmagnification(Figs 2–4). When viewed from the tip of the cell, thefour lobes were evident (Fig. 5). Each chloroplast had

Phaeocystis rex sp. nov. 211

an immersed, bulging pyrenoid (Figs 2, 3). The cellswere sometimes attached to older walls or debris(Fig. 6), and the attached region was often thickened(arrows, Figs 2, 3, 6). Plastid-bearing motile cellswere spheroid, 5–7 µm in diameter, and had twoemergent flagella as well as a short, variable-lengthhaptonema (Figs 7–10).

Electron microscopy

Non-motile cells had a single nucleus with darkly stain-ing nucleolar RNA and mitochondrial profiles showed

the presence of tubular cristae (Fig. 11). A single Golgibody was adjacent to the nucleus and contained 15 ormore individual cisternae. Each chloroplast had lamel-lae consisting of three adpressed thylakoids and anembedded pyrenoid was located near the nucleus(Figs 11–13). The pyrenoid was traversed by a singlethylakoid (Fig. 12). Cells sectioned longitudinallyshowed the extended chloroplast lobes (Fig. 13). Thewall-like structure observed by LMwas more complexwhen observed with TEM (Figs 13, 14). There werealternating electron-dense and transparent layers. Theelectron-dense layers included scales (Figs 11, 12, 15,16) that are presumably organic (unmineralized), butthe majority of the dense layer was composed of anamorphous material. The precise pattern on the scaleswas not determined, but the scales do lack a foldedmargin (Figs 11, 12, 15, 16). An enlarged image of thedense layer showed that the amorphous layer was dis-tinct from the scales (Fig. 15). There was always a layerof scales outside the thicker amorphous layer, suggest-ing that after each cell division, daughter cells firstdeposited scales and then deposited the amorphous

Figs 7–10. Differential interference contrast light micrographsof Phaeocystis rex. Motile cells showing one or two flagellaand a haptonema (arrowhead). Scale bars = 5 μm.

Figs 1–6. Differential interference contrast light micrographs of Phaeocystis rex non-motile cells. Fig. 1. Newly formed non-motilecell with rounded protoplasm and oval wall-like covering (arrowhead). Figs 2–4. Mature non-motile cells, more-or-less elliptical inshape; cells with a central nucleus (N), two lobed chloroplasts (P) and a conspicuous pyrenoid (py) in each chloroplast. Arrowindicates thickening at one end of the wall-like covering. Fig. 5. Cell viewed from the tip showing the four chloroplast lobes. Fig. 6.Living and dead cells weakly attached. The wall-like covering (arrrowheads) and thickening at one end of the wall-like covering(arrow) are indicated. Scale bars = 5 μm.

R.A. Andersen et al. 212

Figs 11–12. TEM micrographs of Phaeocystis rex non-motile cells. Fig. 11. Section depicting typical cellular features including thenucleus (N), Golgi body (G), mitochondrial profiles (M), one chloroplast (P) and its pyrenoid (py). Note the densely stainingchromatin in the nucleus and the presence of the scales (arrowheads). Fig. 12. Section showing the pyrenoid (py) transversed by asingle thylakoid and a lipid body (L). Note the scales (arrowheads). Scale bars = 500 nm.

Phaeocystis rex sp. nov. 213

Figs 13–15. TEM micrographs of Phaeocystis rex non-motile cells. Fig. 13. Longitudinal section showing the elongated chloroplastlobe (P) and the nucleus (N). Note the thickened wall-like layers at one end (arrow). Fig. 14. Two daughter cells shortly after celldivision. Note the thickened wall-like layers at one end (arrows). Fig. 15. Enlarged view of a wall-like layer showing the scales(arrowheads) and the electron dense amorphous layer (arrow). Scale bars: Figs 13, 14 = 1 µm; Fig. 15 = 200 nm.

R.A. Andersen et al. 214

Figs 16–19. TEM micrographs of Phaeocystis rex non-motile cells. Fig. 16. Alternating wall-like layers with electron-denseamorphous materials and electron-transparent zones with scales. Fig. 17. Vegetative cell with wall-like layers. Note the basal bodiesin the cell centre (arrow). Fig. 18. Enlarged view of the basal bodies (b), mitochondrion (M) and a microtubular root (arrow). Fig. 19.Section of a flagellum emerging in a flagellar depression. Note the transitional plate (arrowhead), the proximal transitional helix (longarrow) and the microtubular root (short arrow). Scale bars: Figs 16, 17 = 1 µm; Figs 18, 19 = 300 nm.

Phaeocystis rex sp. nov. 215

layer (Figs 15, 16). Furthermore, adjacent to thenucleus-Golgi-chloroplasts complex, the electron-dense layers were thicker and swollen (Figs 13–14).The swollen region was also visible by LM (Figs 2, 3,5). The ornamentation of scales was not studied indetail.

Two rudimentary flagella (c. 500–1000 nm long), ashort haptonema, and at least one nascent microtubu-lar root were present in non-motile cells with the wall-like layers (Figs 17–22). The flagella appeared to havea transitional helix located on the proximal side ofthe transitional plate (Figs 19, 20). The basal bodieswere anchored in the cell near the Golgi body, mito-chondrion and nucleus (Figs 17–20). The flagella andhaptonema emerged from a shallow depression and amicrotubular root ran along the margin of the invagi-nation (Figs 19, 21, 22). Despite numerous effortsduring the past 15 years, we were unable to success-fully fix swimming cells for TEM.

A morphological comparison of Phaeocystis rex tothe type descriptions for other currently recognizedPhaeocystis species is presented in Table 2. The pre-sence of the wall-like layers and the morphology ofthe chloroplasts distinguished P. rex from all pre-viously described Phaeocystis spp.

Phylogenetic analyses

Phylogenetic analyses based on concatenated datasetsof partial DNA sequences from five nuclear and plas-tidial genes were carried out to determine the relation-ships of Phaeocystis rex to other Phaeocystis species.Two phylogenetic trees with and without sequences ofthe symbiotic Phaeocystis of Acantharia were con-structed (Figs 23, 24, respectively). The phylogenetictree involving only the described species (Fig. 24,dataset containing fewer taxa but longer sequences)displayed stronger overall statistical supports for deepnodes. In both trees, P. rex held the same phylogeneticposition with strong support in both Bayesian and MLanalyses, branching between the solitary Phaeocystisspecies (P. jahnii and P. cordata) and the colony-forming species (P. globosa, P. antarctica and P. pou-chetii). Among these Phaeocystis species, sequencedivergence estimates (p-distance) varied according tothe gene, but on average, P. rex was genetically closerto P. antarctica (0.018% of dissimilarity) (Table 3).Phylogenetic analyses also demonstrated that theundescribed Phaeocystis clades represented by sym-bionts of Acantharia were the earliest diverginglineages, followed by P. jahnii, P. cordata, P. rex, P.globosa, P. antarctica and P. pouchetii. Significantly,we confirm here that PLY559 is closely related to P.jahnii, and that P. globosa includes several crypticspecies that require definition. One taxon identifiedas P. pouchetii (sequence AB280613) clearly groupedin the P. globosa clade, indicating that this is probablya mis-identification.

Figs 20–22. TEM micrographs of Phaeocystis rex non-motilecells. Fig. 20. Rudimentary flagellum for a vegetative wall-likecell showing the two basal bodies (b). Figs 21-22.Cross-sectionsof two emergent rudimentary flagella and the haptonema (arrow-head). Note the microtubular root (arrow). Scale bars = 300 nm.

R.A. Andersen et al. 216

Tab

le2.

Morph

olog

icalfeatures

forPha

eocystisrexandotherPha

eocystisspeciesas

determ

ined

from

theirtype

descriptions.

Species

Dom

inategrossmorph

olog

yColon

ysize

Cellsize

Flagellate

cellsize

Flagellarleng

thPlastid

no.

Pyrenoids

Scales

P.am

oebo

idea

1sphaeroidalcolon

ies

??6×10

µm

6×10

µm

equal,10

µm

one

unkn

own

unkn

own

P.an

tarctica2

sphaeroidalcolon

ies

????

??un

know

ntwo

unkn

own

unkn

own

P.brucei3

irregu

larcolonies,g

elatinou

strails

1–2mm

5–6µm

unkn

own

unkn

own

two

unkn

own

unkn

own

P.cordata4

sing

lecellflagellate

??3–

4µm

3–4µm

unequal

two

immersed

present,dimorph

icP.glob

osa5

sphaeroidalcolon

ies

2–3mm

7–15

µm

4–6µm

equal,subequ

altwo

none

unkn

own9

P.jahn

ii4

sing

lecellflagellate

??3–

5µm

3–5µm

unequal

two

immersed

present,dimorph

icP.po

uchetii

6irregu

larcolonies

1–2mm

6–8µm

6–8µm

unequal,~10

–20µm

two

unkn

own

unkn

own

P.scrobiculata

7sing

lecellflagellate

??8µm

8µm

subequ

al,2

3–30

µm

two

unkn

own

present,dimorph

ic;star-lik

ethread

P.rex8

single-celledwithwall-lik

ecovering

??6–

10×8–

15µm

6–8µm

unequal

two

immersed

present

P.spha

eroides1

sphaeroidalcolon

ies

??5–

6µm

5–6µm

equal,5–

6µm

one

unkn

own

unkn

own

1Büttner

(1911),2

Karsten

(190

5),3

. Mangin(192

2),4

. Zingo

neetal.(19

99),

5. Scherffel(190

0),6

. Pou

chet(189

2),7

. Moestrup(197

9),8

. Present

stud

y.

Tab

le3.

Geneticdistances(p-distances)betweenPha

eocystisspeciescalculated

usingpartialsequences

foreach

ofthefive

genesexam

ined

inthisstud

y.

P.jahn

ii(CCMP13

84)

P.cordata

(CCMP13

83)

P.po

uchetii

P.an

tarctica

(CCMP13

74)

P.glob

osa

(RCC17

19)

P.glob

osa

(PCC54

0)

Phaeocystis

rexsp.n

ov.(CCMP20

00)

16SrR

NA(734

bp)

0.03

00.00

9_

0.00

80.00

90.00

918

SrR

NA(106

9bp)

0.05

30.00

40.01

50.00

40.01

50.01

528

SrR

NA(820

bp)

0.113

0.03

2_

0.01

20.06

90.02

0rbcL

(106

1bp)

0.10

30.07

10.05

10.04

30.04

40.04

3psbA

(569

bp)

0.02

70.02

7_

0.02

30.02

10.02

1p-distance

mean

0.06

50.02

90.03

30.01

80.03

20.02

2

Phaeocystis rex sp. nov. 217

Fig.2

3.Phy

logenetic

tree

ofPha

eocystisinferred

from

aconcatenated

alignm

ento

ffive

genes(the

ribo

somal16

SrRNA,1

8SrRNAand28

SrRNA,and

theplastid

ialp

sbAandrbcL

genes)using

RAxM

L(48taxa

with

3087

alignedpo

sitio

ns).Sequences

comefrom

differentcultu

restrainsandun

cultu

redPha

eocystisthatliv

ein

symbioticassociationwith

acantharianradiolarians.R

AxM

Lbo

otstrappercentagesbasedon

2000

pseudo

replicates

andBayesianpo

steriorprob

abilities(PP)areindicatedateach

node

whensupp

ortv

aluesarehigh

erthan

60%

and0.8,respectiv

ely.The

tree

isrooted

with

sequ

encesof

four

haptop

hytespeciesthatdo

notb

elon

gto

theorderPhaeocystales.

R.A. Andersen et al. 218

Fig.24

.Ph

ylogenetictree

ofPhaeocystisinferred

from

aconcatenated

alignm

entof

five

genes(the

ribosomal

16SrRNA,18SrRNA

and28SrRNA,andtheplastid

ialpsbA

andrbcL

genes)using

RAxM

L(28taxa

with

4824

alignedpositio

ns).Onlysequencesfrom

Phaeocystiscultu

reswereincluded

inthisanalysis.R

AxM

Lbootstrappercentagesbasedon

2000

pseudoreplicates

andbayesian

posteriorp

robabilities(PP)areindicatedateach

node

whensupportvaluesarehigherthan

60%

and0.8,respectiv

ely.The

tree

isrooted

with

sequencesof

four

haptophytespeciesthatdo

notbelongtothe

Phaeocystales.

Phaeocystis rex sp. nov. 219

DISCUSSION

Systematics of Phaeocystis rex

Phaeocystis rex is morphologically distinct from allcurrently recognized Phaeocystis species. When thealga was first isolated and examined by TEM it wasinitially presumed to be a new prymnesiophyte genus.However, our DNA sequence analyses clearly demon-strate that this alga is a close relative of otherPhaeocystis species for which comparable moleculardata are available. Like most other Phaeocystis spe-cies, P. rex cells possess two plastids with immersedpyrenoids, motile cells with two flagella and a hapto-nema, and the characteristic arrangement of subcellu-lar organelles (particularly the nucleus, Golgi bodyand flagellar apparatus) common to haptophytes ingeneral (Parke et al., 1971; Chang, 1984; Inouye &Pienaar, 1985). However, P. rex differs from all otherdescribed Phaeocystis species in two principalrespects (Table 2). First, the external cell covering, inmature non-motile cells, is comprised of alternatinglayers of presumably inorganic scales and an under-lying layer of amorphous wall-like material. Second,the finger-like chloroplast lobes are unlike thosedescribed for any other Phaeocystis species.Additionally, the sizes of P. rex cells, the ellipsoidaloutline of mature cells, and the uneven posterior thick-ening of the extracellular covering are useful for iden-tifying the alga, but their possible taxonomic and/orphylogenetic importance is unclear at this time.Importantly, we demonstrated for the P. rex non-motile stage the presence of (1) scales, and also (2)two rudimentary flagella and a haptonema inside(beneath) the extracellular cell covering. These obser-vations for P. rex are not completely in agreement withthe emended generic description for Phaeocystis pre-sented in Medlin & Zingone (2007). We failed inattempts to determine the exact pattern on the scalesof P. rex. A comparative study of the composition andarchitecture of body scales found on cells of P. rex andother Phaeocystis species would be helpful (Parkeet al., 1971; Moestrup, 1979; Lange et al., 1996;Zingone et al., 1999).

In this study we specifically compared morpholo-gical data for P. rex to information only found in theoriginal descriptions for other Phaeocystis species,and for P. rex we designated a cryopreserved culture(CCMP2000) as the isotype that provides a depend-able source for future research on the species.Authentic cultures of P. cordata and P. jahnii arealso available for study (Zingone et al., 1999).However, such biological resources (cultured strainsdesignated as epi- or isotypes), which are associatedwith publicly archived DNA sequences of importancefor systematics and other investigations, are not avail-able for any other species of Phaeocystis. Thus, con-nections between their original descriptions andpublicly available DNA sequence data are much

more tenuous for the many culture stains identifiedas P. antarctica, P. globosa and P. pouchetii (the typespecies). We recommend designating a single cryo-preserved culture strain collected from the type local-ity as a nomenclatural epitype to anchor the namesP. pouchetii, P. globosa, P. antarctica, P. brucei andP. scrobiculata. Until epitypic strains are designatedfor all Phaeocystis species, morphological studies willbe hampered and nomenclatural uncertainty will pre-vail. For example, early studies of Phaeocystis speciesdid not include ultrastructural data or information onscales because electron microscopes had not beeninvented. Thus, for half of all Phaeocystis speciesthis information is lacking and is, obviously, notfound in the original species description. Therefore,when extant or newly derived cultures are examinedby TEM there is uncertainty regarding their identity.For example, Parke et al. (1971) studied two strainsthat they implicitly, if not explicitly, identified asPhaeocystis pouchetii, largely following the reason-ing provided by Kornmann (1955) that there was onlyone highly variable species and that the name shouldbe based upon priority (i.e. P. pouchetii). Twenty-fiveyears later, we find that one of the cultures (Clone 64 =PLY64) used by Parke et al. (1971) was identifiedusing DNA sequence analysis as P. globosa (Langeet al., 1996), and this same strain appears in our treesas P. globosa PCC64. Even now, can we accuratelyinterpret the TEM observations of Parke et al. (e.g.scale morphologies, flagellar transitional region,embedded pyrenoid) as belonging to P. pouchetii orP. globosa – or some new species in a cryptic speciescomplex? The situation becomes even more tenuousfor ecological studies where identifications have oftenbeen based upon LM observations. In summary, mor-phological comparisons (e.g. Medlin & Zingone,2007) are not entirely stable without nomenclaturalanchoring of names (e.g. by epitype). While an epi-type may not actually represent the original taxon, thefirmly anchored names will at least allow for a wayforward in a meaningful manner.

Phylogenetics, evolution and ecology

Phylogenetic analyses of combined DNA sequencedata from the five nuclear- or plastid-encoded genesprovided solid support for the conclusion, based onour microscope observations, that Phaeocystis rex is anew Phaeocystis species. Additionally, the consistentand highly supported topologies obtained provideinsights into the evolutionary history of not only thespecies P. rex, but also more broadly on the genusPhaeocystis. Phaeocystis rex occupies an intermediateposition between the solitary species (P. jahnii and P.cordata) and the colony-forming species (P. globosa,P. antarctica and P. pouchetii). Phaeocystis jahnii hasbeen described as forming small, loose, occasionallypalmelloid aggregations of cells (Zingone et al.,

R.A. Andersen et al. 220

1999), but these do not seem to be colonies in the samesense as those produced by P. globosa, P. antarcticaand P. pouchetii. Phaeocystis rex has not beenobserved to form colonies in culture, and therefore ispresumed not to form colonies in the natural environ-ment. To date, P. cordata and P. jahnii have only beenreported from the Mediterranean Sea, whereas P. glo-bosa has been collected from both temperate andtropical waters worldwide (Schoemann et al., 2005).The polar species P. pouchetii and P. antartica arebetter adapted to cold temperatures and are mainlyrestricted to Arctic and Antarctic waters, respectively(Schoemann et al., 2005). The phylogenetic positionof P. rex therefore suggests that it may represent anevolutionary transition between the solitary and colo-nial lifestyles, and between distinct ecological prefer-ences. Our phylogenetic analyses provide rigoroussupport for the hypothesis that Phaeocystis likelyoriginated in warm waters and that the colonial life-style is a derived character (Medlin & Zingone, 2007).A wider diversity of Phaeocystis need to be morpho-logically characterized, particularly from the P.globosa clade, as well as from the clades that formsymbiotic relationships with the Acantharia. Thesesymbiont sequences that form the basal clades in thePhaeocystis phylogeny were retrieved from the sub-tropical Pacific ocean (Okinawa Islands, Japan), pro-viding additional evidence for an evolutionary originin warm waters.

From a biodiversity perspective it is interesting torecall that the first five species assigned to Phaeocystisare in nature predominately colonial organisms(Pouchet, 1892; Lagerheim, 1896; Scherffel, 1899,1900; Karsten, 1905; Büttner, 1911; Mangin, 1922).These organisms exhibit other life forms in the courseof their life histories (e.g. motile flagellate and/orbenthic palmelloid life forms) but most often takethe form of colonies of cells in mucilage (e.g.Rousseau et al., 2007; Gaebler-Schwarz et al.,2010). The last four species of Phaeocystis described,including P. rex, are predominately solitary organisms(Moestrup, 1979; Zingone et al., 1999). Followingthis trend, the majority of undiscovered Phaeocystisspecies in nature are liable to be predominately soli-tary organisms. Environmental metabarcoding mayhelp to constrain the extent of undiscovered biodiver-sity in this cosmopolitan genus and improve ourunderstanding of the ecological and biogeochemicalsignificance of different Phaeocystis species in coastaland open ocean regions.

FUNDING

R.A.A. was supported by a USA National ScienceFoundation [grant number 0951733]. J.D. was supportedby the project OCEANOMICS, that has received fund-ing from the French government, managed by theAgence Nationale de la Recherche, under the grant

agreement ‘Investissement d’Avenir’ [ANR-11-BTBR-0008].

AUTHOR CONTRIBUTIONSR. A. Andersen: strain isolation, USA algal culture, TEM andwriting; J. C. Bailey: LM, DNA sequencing and writing; J.Decelle: DNA sequencing, phylogenetic analysis and writing;I. Probert: France algal culture and writing.

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