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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: [email protected].
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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