gomez_adenoides pseudadenoides.pdf

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A new de1047297nition of Adenoides eludens an unusual marinesand-dwelling dino1047298agellate without cingulum and

Pseudadenoides kofoidii gen amp comb nov for the species

formerly known as Adenoides eludens

FERNANDO GOacuteMEZ13 RYO ONUMA2 LUIS F ARTIGAS3 AND TAKEO HORIGUCHI4

1 Laboratory of Plankton Systems sala 100 Oceanographic Institute University of Satildeo Paulo Praccedila do Oceanograacute 1047297co 191

Cidade Universitaacuteria Butantatilde Satildeo Paulo 05508-900 Brazil 2 Department of Natural History Sciences Graduate School of Science Hokkaido University North 10 West 8 060-0810Sapporo Japan3

Laboratoire d rsquo Oceacuteanologie et Geacuteosciences CNRS UMR 8187 Universiteacute du Littoral Cocircte d

rsquo Opale MREN ULCO 32 av

Foch 62930 Wimereux France4 Department of Natural History Sciences Faculty of Science Hokkaido University North 10 West 8 060-0810 Sapporo

Japan

(Received 9 Jul 2014 revised 24 Sep 2014 accepted 8 Nov 2014)

The species Amphidinium eludens as described by Herdman (1922 Proc Trans Liverpool Biol Soc 36 18) based on her drawing in 1047297g 1 has been investigated for the 1047297rst time by scanning electron microscopy and phylogenetic analysis Themorphological and molecular data reveal that this species is distantly related to other known dino1047298agellates Balech [1956 Rev

Algol n ser 2(1 ndash 2) 30] cited Amphidinium eludens Herdman (1922 1047297g 1) as the basionym of the type of Adenoides while hedescribed and illustrated Amphidinium kofoidii Herdman (1922 1047297g 2) as Adenoides eludens As the nomenclatural rules do not allow the change of basionym we have re-de1047297ned the genus Adenoides based on the characteristics of Amphidinium eludens

Herdman (1922 1047297g 1) The thecal plate formula of Adenoides eludens is Po 5prime 6Prime 0c 3+s 5primePrime 3p 1PrimePrime This species lacks acingulum The apical pore complex resembles that of peridinioid dino1047298agellates while the absence of a cingular groove isreminiscent of desmokont prorocentroids We also propose Pseudadenoides kofoidii gen amp comb nov based on Herdmanrsquos 19221047297g 2 of Amphidinium kofoidii which was described by Balech in 1956 and re-named Adenoides eludens

Key words Adenoides kofoidii Dinophyta microphytobenthos molecular phylogeny new genus psammophilic Dinophyceae

INTRODUCTION

Dino1047298agellates are unicellular organisms with twodimorphic 1047298agella Based on the arrangement of the1047298agella the dinokonts (= two 1047298agella in grooves) aredino1047298agellate cells in which two 1047298agella are insertedventrally one1047298agellum is transverse wrapped aroundthe cell and housed in a groove called the cingulum or girdle (equatorial or transverse) and the other one is atrailing longitudinal 1047298agellum and housed in a sulcus(longitudinal) The other type desmokonts (= twoanterior 1047298agella) are cells in which two dissimilar 1047298agella emerge from the anterior part of the cellThey have two leading 1047298agella inserted apicallyrather than ventrally One 1047298agellum extends forwardand the other circles its base and there are no 1047298agellar grooves (cingulum or sulcus) The best known

example of desmokonts is the genus Prorocentrum

CG Ehrenberg However not all dino1047298agellates 1047297t into this division The descriptions of new benthic

species have largely increased in recent years(Goacutemez 2012) The presence of an incomplete cingu-lum is reported in some sand-dwelling dinokonts( Amphidiniopsis J Woloszynska Cabra Sh Murray ampDJ Patterson Herdmania JD Dodge Rhinodinium

Sh Murray Hoppenrath S Yoshimatsu S Toriumi ampJ Larsen) (Murray amp Patterson 2004 Yamaguchiet al 2011) Planktonic dino1047298agellates such as

Podolampas F Stein lack a groove (cingulum) toharbour a transversal 1047298agellum that encircles the cell(Goacutemez et al 2010) However the absence of a cingu-lum has never been reported in benthic dinokonts

In one of the earlier studies of sand-dwelling dino-1047298agellates Herdman (1922) described the species

Amphidinium eludens and A kofoidii In her 1047297g 1she described Amphidinium eludens as oval to ovoidCorrespondence to Fernando Goacutemez (e‑mail

fernandogomez1047297toplanctoncom)

Eur J Phycol (2015) 1 ndash 14

ISSN 0967-0262 (print)ISSN 1469-4433 (online)15000001-14 copy 2015 British Phycological Societyhttpdxdoiorg1010800967026220151009174



in shape has a barely visible episome and a hump inthe sulcal area (Herdman 1922 p 22 1047297g 1) In her 1047297g 2 she described Amphidinium kofoidii as round tosquarish in shape with a more visible button-likeepisome (Herdman 1922 p 26 1047297g 2) Balech(1956) proposed the new thecate genus Adenoides

based on Herdmanrsquos 1047297g 2 of Amphidinium kofoidiiHowever Balech (1956) proposed as basionym

Amphidinium eludens Herdman (1922 1047297g 1) LaterDodge (1982) proposed Adenoides kofoidii for

Amphidinium kofoidii Herdman 1922 1047297g2Thetaxo-nomic and nomenclatural history of Adenoides elu-

dens is reported in Hoppenrath et al (2003) Adenoides eludens based on Balechrsquos description and

as described by Herdmanrsquos 1047297g 2 of Amphidinium

kofoidii was investigated again by Dodge amp Lewis(1986) and Hoppenrath et al (2003) and molecular data have been available since Saldarriaga et al

(2001) To date there are 45 sequences of different molecular markers of Adenoides eludens and another 39 sequences labelled as two uncultured AdenoidesThe sequences of Adenoides eludens available inGenBank are from the cultures CCCM 683 andCCMP 1891 isolated from the Paci1047297c coasts of Canada or 1047297eld material collected from the same loca-tion Another culture CCMP 2081 was isolated fromEurope However there are no molecular data for

Adenoides eludens from the European Atlantic coast where the species was described While Adenoides

eludens (= Amphidinium kofoidii) has been subjected

to morphological and molecular studies little is knownabout the species described as Amphidinium eludens

Herdman (1922 1047297g 1) This organism is one of the1047297rst described sand-dwelling dino1047298agellates andaccording to Herdman (1922) is responsible for discolourations in the sands of the European Atlanticcoasts However the detailed morphology of thisspecies remains unknown

In this study we provide light microscopy picturesthe 1047297rst scanning electron microscopy pictures and the1047297rst molecular data for Amphidinium eludens

Herdman (1922 1047297g 1) and the 1047297rst molecular datafor Amphidinium kofoidii Herdman (1922 1047297g2)fromthe French coasts of the English Channel whereBalech (1956) described the genus Adenoides Themorphological and molecular data support the conclu-sion that the two species described in 1047297gs 1 and 2 byHerdman (1922) belong to independent genera

Amphidinium eludens is an unusual dino1047298agellatecharacterized by the lack of a cingular groove similar to desmokont dino1047298agellates

MATERIALS AND METHODSSource isolation and microscopy observations

This study was undertaken in the soft sandy sediments of theshore of Wimereux France (50ordm46prime 12Prime N 1ordm36prime 42Prime E)

collected during low tide in June 2012 Two sites on the beach in front of the LOG laboratory (MREN ULCO andMarine Station of Wimereux UL1 Goacutemez amp Artigas 2014)were sampled the moist sands around the border of a large

pool (~50 m diameter ~1 m depth) and several smaller poolsand moist sands showing a faint brownish discolourationThe upper centimetre of sand was collected with a spoon

and deposited into a bottle containing seawater collected at the same location Then the sand with seawater was stirredvigorously and the suspension settled in a composite settlingchamber The settled material was examined with an invertedmicroscope (Nikon Eclipse TE2000-S Tokyo) and photo-graphed with a Nikon Digital Sight DS-2M camera The cellsize described as length (apical to antapical axis) and depthie the length along the lateral sides (ventral to dorsaldistance) was measured in 25 specimens The width(transdiameter) was measured in 1047297ve specimens

For scanning electron microscopy the sand samples withseawater were stirred vigorously and the suspension was1047297

xed with glutaraldehyde (5) and 1047297

ltered onto a 08 microm pore size Whatman Nucleporereg membrane 1047297lter washedwith distilled water 1047297xed with osmium tetroxide dehydratedwith a graded series of ethanol and critical point dried withCO2 Filters were mounted on stubs sputter-coated with goldand viewed using a Hitachi S4800 scanning electron micro-scope Images were presented on a black background usingAdobe Photoshop CS3

PCR ampli1047297cation and DNA sequencing

For molecular analysis each specimen of Amphidinium elu-

dens and A kofoidii was micropipetted individually with a1047297ne capillary into a clean chamber and washed several timesin serial drops of 02 μm 1047297ltered and sterilized seawaterFinally 1 ndash 5 specimens of each species were deposited in a02 ml Eppendorf tube 1047297lled with several drops of absoluteethanol The sample was kept at room temperature and indarkness until the molecular analysis could be performed

Prior to DNA extraction the 02 ml Eppendorf tubes werecentrifuged for 10 min at 14462 times g in a TOMY MX-201centrifuge (Tokyo Japan) Ethanol was then evaporated in avacuum desiccator Cells were resuspended in 10 μl of QuickExtract DNA extraction solution (EpicenterMadison) and incubated at 56ordmC for 1 h and 98ordmC for 2 min

in a thermal cycler (GeneAmp PCR System 9700 AppliedBiosystems Foster City) The product was used as DNAtemplate for the following polymerase chain reaction(PCR) In the 1047297rst round of PCR to obtain almost completeSSU rDNA and partial LSU rDNA sequences two sets of

primers (SR1-SR12b for SSU rDNA and D1RF1 28-1483R for LSU rDNA respectively) were simultaneously appliedIn the second round of PCR 05 microl of the 1047297rst PCR productswas used as DNA template and three pairs of primers (SR1-SR5 SR4-SR9p and SR8-SR12b see Yamaguchiet al 2006) were used for SSU rDNA ampli1047297cation andtwo pairs of primers (D1RF-25R1 and D3A-28-1483R seeDaugbjerg et al 2000) were applied for LSU rDNA ampli-

1047297cation PCR conditions for both rounds of ampli1047297cationconsisted of one initial cycle of denaturation at 94ordmC for 5min followed by 35 cycles of denaturation at 94ordmC for 30 sannealing at 55degC for 30 s and extension at 72ordmC for 30 sThe PCR process was completed by a 1047297nal extension cycle

F Goacutemez et al 2



at 72ordmC for 7 min The PCR products were directlysequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and a DNAautosequencer ABI PRISM3100 Genetic Analyzer (Applied Biosystems) Both forward and reverse strandswere sequenced

Phylogenetic analyses

Both SSU rDNA and LSU rDNA sequences were aligned based on the secondary structure of the rDNA molecule(database no longer available) and the alignments werere1047297ned manually We used sequences from GenBank of Amoebophrya spp as outgroups for SSU rDNA and a ciliateTetrahymena pyriformis (CG Ehrenberg) Lwoff and an api-complexa Eimeria tenella (Raillet amp Lucet) Fantham wereused for outgroups of LSU rDNA analyses respectively Theaccession numbers of sequences included in the alignment

are indicated in each tree The aligned sequences were exam-ined using maximum likelihood (ML) analyses with PAUPversion 40b10 (Swofford 2003) and Bayesian analysis withMrBayes 321 (Huelsenbeck amp Ronquist 2001) The pro-gram Modeltest version 304 (Posada amp Crandall 1998)which employs the hierarchical likelihood ratio test (hLRT)was used to explore the best ML sequence evolution modelfor the dataset The hRLT model selected for ML analysis of the dataset was TrN+I+G In the ML analysis a heuristicsearch was performed with a TBR branch-swapping algo-rithm and the starting tree was obtained by the neighbor-

joining (NJ) method The parameters in this analysis wereassumed nucleotide frequencies A = 02756 C = 01898 G =

02392 and T = 02954 substitution rate matrix with Alt-gtC= 10000 Alt-gtG = 35126 Alt-gtT = 10000 Clt-gtG =10000 Clt-gtT = 77026 and Glt-gtT = 10000 proportionof sites assumed to follow a gamma distribution with shape

parameter = 05633 and number of rate categories = 4Bootstrap analysis for ML was calculated for 100 pseudo-replicates For LSU rDNA analysis the hLRT model selectedfor ML analysis of the dataset was TrN+I+G In the MLanalysis a heuristic search was performed with a TBR

branch-swapping algorithm and the starting tree wasobtained by the NJ method The parameters in these analyseswere assumed nucleotide frequencies A = 02865 C =01806 G = 02701 and T = 02628 substitution rate matrix

with Alt-gtC = 10000 Alt-gtG = 28299 Alt-gtT = 10000Clt-gtG = 10000 Clt-gtT = 66145 and Glt-gtT = 10000

proportion of sites assumed to follow a gamma distributionwith shape parameter = 06675 and number of rate categories= 4 Bootstrap analysis for ML was calculated for 100

pseudo-replicates For Bayesian analysis GTR+I+G modelwas selected by MrModeltest 22 (Nylander et al 2004) as asuitable evolutionary model Markov chain Monte Carloiterations were carried out until 3 000 000 generationswere attained for SSU rDNA phylogeny while 5 500000 generations were required for LSU rDNA phylogenywhen the average standard deviations of split frequenciesfell below 001 indicating a convergence of

the iterations Our sequences were deposited in DDBJ EMBLGenBank under accession numbers LC002839-LC002848

RESULTS

Based on the morphological and molecular data (see below) the species described as Amphidinium eludens

Herdman (1922 1047297g 1) and Amphidinium kofoidii

Herdman (1922 1047297g 2) belong to separate genera If we had proposed a new genus name based on

Herdman (1922 1047297g 1) it would have been illegiti-mate as a super 1047298uous later homotypic synonym of

Adenoides [International Code of Nomenclature for algae fungi and plants (ICN) art 521 McNeillet al 2012] As de1047297ned by Balech (1956) the basio-nym of the type of Adenoides Adenoides eludens is

Amphidinium eludens Herdman (1922 1047297g 1)According to article 73 of ICN lsquoA new combina-tion or a name at new rank (Art 610) istypi1047297edbythetype of the basionym even though it may have beenapplied erroneously to a taxon now considered not toinclude the type (but see Art 481)rsquo The characteris-tics of the genus Adenoides are de1047297ned by

Amphidinium eludens Herdman (1922 1047297g 1) inde- pendently of the fact that Balech (1956) provided for Adenoides eludens the description and illustrations of the species described as Amphidinium kofoidii byHerdman (1922 1047297g 2) Hereafter we rede1047297ned thegenus Adenoides based on the morphology of

Amphidinium eludens Herdman (1922 1047297g 1)

Taxonomic descriptions

Adenoides Balech emended F Goacutemez R OnumaArtigas amp Horiguchi

DIAGNOSIS Armoured cell laterally compressedlacking the cingulum and 1047298agella inserted ventrallyThecal plate formula Po 5prime 6Prime 0c 3+s 5primePrime 3p 1PrimePrime An alternative interpretation is Po cp 4prime 7Prime 1c 1+s5primePrime x 2p 1PrimePrime

TYPE SPECIES Adenoides eludens (Herdman) Balech

BASIONYM Amphidinium eludens Herdman 1922 p 22 1047297g 1

SYNONYM Adenoides kofoidii sensu Dodge 1982

Adenoides eludens F Goacutemez R Onuma Artigas ampHoriguchi epitype

Given the lack of type material we have designated1047297g 1 in Herdman (1922) as the lectotype for thisspecies and the cell shown in Fig 23 of this study asthe epitype under Article 97 of the ICN (McNeillet al 2012) A detailed description of the epitype is

presented below

EPITYPE Fig 23 A SEM stub was deposited in theherbarium of the Faculty of Science HokkaidoUniversity as SAP114711

Pseudadenoides gen amp comb nov and Adenoides 3



The slightly laterally 1047298attened cells are ellipsoidalin lateral view The sulcal area is depressed and lies onthe anterior third of the cell neither extending onto theepitheca nor reaching the antapex In lateral view theventral contour of the cell showed a hump (Figs 2 6)Thecellswere30plusmn2(27 ndash 37)micromlong23plusmn2(18 ndash 27)

microm deep and 13 ndash

17 microm wide (Figs 1 ndash

15) There is anintergradation of the size with large and small speci-mens co-existing in the same sample (Fig 6) Theepitheca is directly connected to the hypotheca with-out any cingulum We presume that the vestigial cin-gular groove was placed in the suture between the pre-and postcingular plates that is at the level of the1047298agellar insertion

The height of the epitheca is about one-third thelength of the cell Some specimens showed a large

pusule in the anterior dorsal part of the cell (Figs 1 ndash 4)The transversal 1047298agellum encircled the cell (Fig 5)

The cell possesses yellow-brown plastids The species Amphidinium kofoidii sensu Herdman is more pigmented and brownish when compared with Adenoides eludens (Fig 7) Some specimens of Adenoides were found devoid of pigments and withnumerous granules (Figs 8 ndash 10) The cell showed two

pyrenoids with a starch sheath each pyrenoid placedin the sides of the mid anterior hypotheca (Figs 6 11)The nucleus is oval and situated in the posterior region although hardly visible because it is hidden

by plastids Some specimens experienced ecdysis or exuviation releasing empty thecae (Figs 11 ndash 12)

The empty theca was usually divided into two partswhich are considered the epitheca and hypotheca(Figs 13 ndash 15) One plate that is interpreted as a sulcalanterior plate is attached to the epitheca (Fig 13)

The tabulation is illustrated in detail in Figs 16 ndash 46The thecal plate pattern is an apical pore plate (Po)1047297ve apical plates (5prime ) six precingular plates (6Prime ) nocingular plate (0c) at least three sulcal plates (3+s)1047297ve postcingular plates (5primePrime ) three posterior interca-lary plates (3p) and one antapical plate (1PrimePrime ) The cellsurface is smooth with round pores (approximately015 microm) The pores are evenly distributed in the

plates with a trend to form rows near the sutures(Fig 19) There is an accumulation of pores in the

posterior end of the 1047297rst and second posterior inter-calary plates (1p 2p) and in the dorsal end of theantapical plate (Figs 16 40 ndash 41) The pores are absent in the sulcal plates (Figs 22 27 ndash 30 34) The boundaryof the plates shows a thick (~1 microm wide) smooth anddepressed margin These intercalary bands were moreconspicuous in the hypotheca (Figs 19 21)

The apical pore plate showed a pentagonal to roundshape being bordered by 1047297ve apical plates

(Figs 24 37) The junction of the apical pore plateand the apical series formed a ridge except in the 1047297rst apical plate suture which is shorter than in the other apical plates (Figs 24 25 37) The apical pore platecontains a row of marginal pores (10 ndash 13 pores) and a

round central cover plate (Figs 26 37) From thecentral cover plate emerged a rim that protruded andextended towards the anterior part of the 1047297rst apical

plate (Figs 24 26 37)There are 1047297ve apical plates (Figs 20 23 37) Plate

3prime is the largest and is located in the dorsal side of the

epitheca Plates 2prime and 5

prime are intermediate in sizePlates 4prime and 1prime are the smallest of the apical series

The apical plates 2prime to 5prime showed a curved ridge in thesuture with the apical pore plate Plate 1 prime did not showa ridge in the junction with the apical pore plate Plate1prime is a six-sided irregular smooth-surfaced polygonwith very few pores when compared with the other apical plates (Figs 24 ndash 26) An alternative interpreta-tion of the apical tabulation is to consider plate 1prime as awide canal plate (cp) Plates 2prime and 4prime are pentagonalespecially plate 4prime which is a quasi-regular pentagonPlates 3prime and 5prime are six-sided Plate 5prime shows a distinc-

tive undulating 1047298ange in the sutures except in theanterior end in the suture with plates 5Prime and 6Prime

(Figs 25 ndash 26 37)At the level of the precingular plate series there are

six precingular plates and a large smooth-surfaced andconcave plate This plate is joined to the precingular

plates when the epitheca is separated from thehypotheca (Fig 13) Based on the proximity to the1047298agellar pores the differences in the shape (concave)and the absence of ornamentation (smooth-surfacedlacking pores and intercalary bands) as in the other sulcal plates we have considered it as the right anterior

sulcal plate (Sda) (Figs 21 ndash 22 34) An alternativeinterpretation is to consider this large plate as plate 1Prime Following the interpretation as an anterior sulcal platethe 1047297rst precingular plate (1Prime ) is displaced to the left side in the ventral view Plates 1Prime and 5Prime are the

biggest of the epitheca plates 2Prime 3Prime and 4Prime are inter-mediate in size and plate 6Prime is the smallest of the

precingular series (Figs 16 ndash 20 35 ndash 36) Plate 1Prime is polygonal and curved in the suture with the anterior sulcal plate (Figs 21 ndash 23) Plates 2Prime 4Prime and 5Prime aresix-sided and plates 3Prime and 6Prime are quadrangular (Figs 16 ndash 20) Plate 3Prime is nearly square-shaped(Fig 20) and plate 6Prime is an elongated rectangle orientedalong the antero-posterior axis (Figs 28 34 ndash 35)

The cell does not have a cingulum Howeverwhen the theca is empty it tended to separatealong the suture between the precingular and post-cingular plate series (Figs 13 ndash 15) This suggests that this thick suture could be occupying the space of thecingulum of a hypothetical ancestor The observa-tions of live specimens revealed that the transversal1047298agellum encircles this area (Fig 5) The cell showsa depression in the sulcal area (Figs 21 ndash 22 32 ndash 35)

The left anterior sulcal (Ssa) plate is smoothand located between the right anterior sulcal plate(Sda) and a large plate 1primePrime This Ssa plate iselongated and concave and it could be alternativelyinterpreted as a 1047297rst cingular plate (Figs 21 ndash 23 34)




There are two 1047298agellar pores in the depressed area of the sulcus The longitudinal 1047298agellum is inserted ina pore located in the right side of the sulcus

adjacent to the suture of plates 6Prime

and 5primePrime

(Figs 2227 ndash 30 34) The right half of this pore is surrounded by a prominent ridge (Figs 28 ndash 29) The pore of thetransversal 1047298agellum is located in the middle of thesulcus It is bordered by a rectangular structure

(Figs 28 ndash 30) Below the pores a large sulcal poster-ior plate with a rounded posterior end is subdividedinto three plates connected by wedge-shaped con-

tours (Figs 29 ndash

30) The right anterior sulcal (Sda) plate is in contact with the 1047298agellar pores (Fig 22)This plate shows an anterior-posterior oriented linewhich suggests the presence of a subdivision of the

plate (Fig 30)

Figs 1 ndash 15 Light microscopy pictures of live specimens and empty thecae of the rede1047297ned Adenoides eludens (Fig 7 also includes Amphidinium kofoidii) Figs 1 ndash 4 Different views of the same specimen Fig 5 Note the transversal 1047298agellum that encircles the cellFig 6 Note the different sizes Fig 7 Note the different pigmentation between Adenoides eludens (yellowish on the left) and

Amphidinium kofoidii (brownish on the right) Figs 8 ndash 10 Specimens lacking pigmentation Figs 11 ndash 12 Ecdysis or exuviation fromthe theca Figs 13 ndash 15 Empty theca with separated epitheca and hypotheca Fig 13 Note that the sulcal right (= dexter) anterior plate(Sda) is attached to the epitheca LF = longitudinal 1047298agellum TF = transversal 1047298agellum Scale bar = 10 microm




There are 1047297ve postcingular plates (Figs 16 19)Plate 1primePrime is the longest plate of the cell and extendedfor 23 the height of the hypotheca The anterior part

of this 1047297ve-sided plate is curved and overhangs at the anterior end (Figs 18 22 34) Plates 2primePrime and 4primePrime

are large and broad plate 3primePrime is intermediate in size

Figs 16 ndash 30 Scanning electron micrographs of the re-de1047297ned Adenoides eludens Figs 16 ndash 17 Right lateral view Fig 18 Ventral viewFig 19 Left lateral view Fig 20 Dorsal view Figs 21 ndash 23 Ventral lateral view Figs 24 ndash 25 Detail of the apical plates Fig 26 Detail of the apical pore plate Figs 27 ndash 30 Detail of the sulcal plates Fig 30 The arrowheads pointout tentative sutures in the sulcal plates Sda =Sulcal dexter (= right) anterior plate Ssa = Sulcal sinister (= left) anterior plate Sp = Sulcal posterior plate LFP = longitudinal1047298agellar

pore TFP = transversal 1047298agellar pore Scale bar = 10 microm except Figs 22 24 ndash 30 where scale bar = 1 microm




and plate 5primePrime is the smallest of the postcingular series Plate 2primePrime is four sided plate 3primePrime is a regular

pentagon and plate 4primePrime is 1047297ve-sided and elongated posteriorly Plate 5primePrime is smaller and more anteriorly placed than the other plates of the postcingular ser-ies (Figs 34 ndash 35) An alternative interpretation is that

plate 5primePrime is equivalent to plate x reported in some

sand-dwelling dino1047298

agellates with incompletecingulumFour large plates that do not belong to the sulcal and

postcingular series are considered posterior interca-lary and antapical plates Three plates are considered

posterior intercalary plates and the plate situated at the bottom of the cell is considered the antapical plate 1PrimePrime

(Figs 16 19) The antapical plate is bordered by plate1primePrime and three posterior intercalary plates (1p 2p 3p)The 1047297rst and second posterior intercalary plates(1p 2p) are large and with the shape of an obliqueirregular pentagon (Figs 16 19) Plates 1p and 2p

have an accumulation of pores in the posterior endat the contact with the antapical plate (Figs 39 ndash 41)Plate 3p is an elongated pentagon oriented in thelongitudinal axis (Figs 32 ndash 33) The antapical plate ishexagonal and it showed at the dorsal end the special

Figs 31 ndash 41 Scanning electron micrographs of a single specimen of Adenoides eludens Fig 31 Ventral view Figs 32 ndash 33 Right lateral view Fig 34 Detail of the sulcal area Fig 35 Latero-ventral view Figs 36 ndash 37 Detail of the epitheca Figs 38 ndash 39 Dorsal

view Figs 40 ndash

41 Antapical view Fig 40 The inset shows the pores of the plates 2p (up) and 1PrimePrime

(down) Sda = Sulcal dexter (= right) anterior plate Ssa = Sulcal sinister (= left) anterior plate Sp = Sulcal posterior plate Scale bar = 20 microm except Figs 34and 37 where scale bar = 1 microm




pore 1047297eld found in plates 1p and 2p (Fig 41) Analternative interpretation of the tabulation of thehypotheca may appear when plate 5primePrime is consideredtobeanxplate(Fig 48) then plate 3p is considered to

be 5primePrime plate

A new genus name for Amphidinium kofoidii Herdman(1922 1047297g 2)

We had to rede1047297ne the genus Adenoides due to thedesignation of Amphidinium eludens Herdman (19221047297g 1) as basionym of the type of AdenoidesMorphological and molecular data reveal that thespecies Amphidinium kofoidii Herdman (1922 1047297g 2)does not belong to the new de1047297nition of the genus

Adenoides or to the other known dino1047298agellate genusTherefore a new genus name is proposed here The

Figs 42 ndash 46 Line drawings of the re-de1047297ned Adenoides eludens with the Kofoid system of tabulation Fig 42 Right lateral Fig 43Left lateral Fig 44 Apical Fig 45 Antapical Fig 46 Ventral

Figs 47 ndash 56 Light microscopy pictures of live specimens (47 ndash 50) and empty thecae (51 ndash 56) of Pseudadenoides kofoidii gen ampcomb nov Fig 47 The arrows point the transversal 1047298agella encircling the cingulum Fig 50 Megacytic cell Figs 51 ndash 56 Emptythecae Scale bar = 10 microm




description of the genus and its type species is heresimpli1047297ed because the morphological characteristicshave been already reported from the English Channeland North Sea (Balech 1956 Dodge amp Lewis 1986Hoppenrath et al 2003) For that reason we havesummarized the description which is similar to that

found with more detail in Hoppenrath et al (2003)

Pseudadenoides F Goacutemez R Onuma Artigas ampHoriguchi gen nov

DIAGNOSIS Armoured cell laterally compressed with aminute epitheca The plate formula is Po 4prime 0Prime 6c4s5primePrime 5p 1PrimePrime (or alternatively it can be interpreted as Po4prime 0Prime 6c 5s 5primePrime 3p 2PrimePrime )

SYNONYM Adenoides sensu Balech 1956

ETYMOLOGY Pseudo- pseud - (before vowels)Ancient Greek ψευδής (pseudes) false not genuinefake The type species has been confused with

Adenoides eludens The gender is feminine (art 624of ICN)

TYPE SPECIES Pseudadenoides kofoidii (Herdman) FGoacutemez R Onuma Artigas amp Horiguchi comb nov

BASIONYM Amphidinium kofoidii Herdman (1922 p 26 1047297g 2)

SYNONYM Adenoides eludens sensu Balech 1956

EPITYPE Fig 60

Pseudadenoides kofoidii (Herdman) F Goacutemez ROnuma Artigas amp Horiguchi comb nov

Cells are asymmetrical round to squarish slightly 1047298at-tened laterally 28 ndash 37 microm long and 21 ndash 29 microm deep Theminute epitheca is cup-shaped depressed and scarcelyvisible (Figs 47 ndash 50) The cingulum lies almost at theanterior end of the cell completely encircling theepitheca and meeting without displacement The sulcalarea lies on the anterior third of the cell neither extend-ing onto the epitheca nor reaching the antapex and isslightly depressed The transverse 1047298agellum completely

encircles the cell at the cingulum level (Fig 47) Most of the cells show one large pusule in the anterior hypotheca The cell is full of brown plastids There aretwo conspicuous pyrenoids The nucleus is oval andsituated in the posterior region although hardly visible

because it is hidden by plastids (Fig 47 ndash 50)The thecal plate pattern is an apical pore plate (Po)

four apical plates (4prime ) without precingular plates (0Prime )six cingular plates (6c) four sulcal plates (4s)1047297ve postcingular plates (5primePrime ) 1047297ve posterior intercalary

plates (5p) and one antapical plate (1PrimePrime ) (Figs 51 ndash 62)

The apical pore plate is an angular square-shape bordered by four apical plates which form a ridgerunning around it (Fig 60) The apical pore platecontains few marginal pores (~7 pores) and a round

central cover plate From the central cover plateemerged a narrow rim that protruded and extendedventrally towards the anterior part of the 1047297rst apical

plate (Fig 62) The apical plates 1prime and 4prime are incontact with the sulcus (Fig 62) Plate 2prime is verynarrow and 4prime is relatively large covering nearly the

entire right half of the epitheca There are no precin-gular plates The shallow cingulum consists of six plates and there are four small sulcal plates surround-ing the 1047298agellar pore (sa ss sp sd) (Figs 59 ndash 60)The hypotheca consists of 11 plates comprised of 1047297ve

postcingular 1047297ve posterior intercalary plates and oneantapical plate Plates 1primePrime and 2primePrime lie at the left lateralcell side in the upper fourth of the hypotheca Thesmall pentagonal plate 3primePrime lies dorsally and the pen-tagonal posteriorly pointed plate 4primePrime is relativelylarge lying at the right lateral side The six-sided

plate 5primePrime is in contact with the sulcus All 1047297ve posterior

intercalary plates are large and cover most of thehypotheca (Figs 57 ndash 59) The plates 1p and 5p are incontact with the sulcus and bordered its posterior margin Both are in contact with each other and forma lsquoventral suturersquo (Figs 59 ndash 60)

Molecular phylogeny

We have sequenced specimens of the species Amphidinium eludens Herdman (1922 p 22 1047297g 1now Adenoides eludens emended) and the species

Amphidinium kofoidii Herdman (1922 p 26 1047297g 2now Pseudadenoides kofoidii gen amp comb nov) TheSSU- and LSU rDNA sequences of Amphidinium

kofoidii Herdman from specimens of the Paci1047297cOcean are available in GenBank (retrieved as

Adenoides eludens) We have also provided the 1047297rst SSU- and LSU rDNA sequences from specimens of the European Atlantic coasts where Amphidinium

kofoidii was described The aligned length for SSU-rDNA was 1725 bp and 954 bp for LSU rDNA (D1 ndash

D3 region excluding the variable region) Thesequences of the Atlantic and Paci1047297c specimens of

Amphidinium kofoidii were identical (Figs 63 64)We provided the 1047297rst SSU- and LSU rDNA sequencesof Amphidinium eludens All the sequences obtainedfrom different samples were identical (Figs 63 64)The species Amphidinium eludens and Amphidinium

kofoidii described in Herdman (1922) are distantlyrelated in the SSU rDNA (Fig 63) and LSU rDNAtrees (Fig 64) Our data strongly support the splittingof these two species into two distinct genera based onthe considerable evolutionary distance of their respec-tive SSU- and LSU rDNA sequences Because of the

low resolution of basal positions of the phylogenetictrees it is also dif 1047297cult to establish the accurate rela-tionships between these genera with other dino1047298agel-lates (Figs 63 64)




DISCUSSION

In pioneering studies of sand-dwelling dino1047298agel-lates Herdman (1922) described in her 1047297g 1

Amphidinium eludens as forming discolourations in

a beach of the British Isles Despite the tradition of taxonomic studies in the European Atlantic coastsand the relative abundance of A eludens to date that species has remained under investigation Confusionhas been present since the original descriptionHerdman (1922) illustrated Amphidinium eludens

as slightly larger than A kofoidii in line drawingslacking any reference to the size and reported alength of 70 microm and 50 ndash 80 microm for A eludens and

A kofoidii respectively However in a note at theend of a subsequent publication (Herdman 1923 p63) she explained that the size measurements were

overestimated and should be 30 microm and 25 ndash

40 micromfor Amphidinium eludens and A kofoidii respec-tively Consequently the cells of Amphidinium kofoi-

dii tend to be larger than those of A eludens This

contrasts with the relative sizes of Herdmanrsquos origi-nal 1047297gures In our observations we found an aver-age length of 30 microm for Amphidinium eludenswhich is usually slightly less than for A kofoidiiThe error in the relative size of the original descrip-

tion could have contributed to further confusion inthe identi1047297cation of both species When Balech(1956) proposed Adenoides eludens he consideredthat Herdmanrsquos 1047297gure 1 and 2 corresponded to thesame species Dodge amp Lewis (1986 p 224) main-tained this view when they reported lsquothe cell con-tents of the two species is very similar it may be that they are only variantsrsquo As a consequence the spe-cies Amphidinium eludens has received little atten-tion and has been cited as Adenoides eludens sensu

Balech (Paulmier 1992) a situation that has deprivedus from knowing the unusual morphology of

Amphidinium eludens until nowOur specimens studied from a sandy beach in the

English Channel correspond to Herdmanrsquos 1047297g 1

Figs 57 ndash 62 Scanning electron micrographs of Pseudadenoides kofoidii gen amp comb nov Figs 57 ndash 58 Right lateral view Fig 59Ventral view Fig 60 Apical view Fig 61 Dorsal view Fig 62 Detail of the pore and apical plates Scale bar = 20 microm except Fig 62 where scale bar = 1 microm




Fig 63 Maximum likelihood phylogenetic tree of Adenoides eludens and Pseudadenoides kofoidii with other dino1047298agellatesinferred from SSU rDNA sequences The species newly sequenced in this study are bold The numbers at each node represent

bootstrap value (maximum likelihood ML) and posterior probability (PP) Only values of gt 70 (bootstrap) and gt 09 (PP) areindicated Closed circle denotes 10010 = bootstrapPP




Fig 64 Maximum likelihood phylogenetic tree of Adenoides eludens and Pseudadenoides kofoidii with other dino1047298agellates

inferred from partial LSU rDNA sequences The species newly sequenced in this study are bold The numbers at each node represent bootstrap value (maximum likelihood ML) and posterior probability (PP) Only values of gt 70 (bootstrap) and gt 09 (PP) areindicated Closed circle denotes 10010 = bootstrapPP




reported as Amphidinium eludens The shared charac-teristics with the original description including (1)general cell contour and size (2) lack of apparent raised epitheca viewed from the lateral side (3) the

presence of a hump in the ventral contour of the cellwhere 1047298agella arise and (4) the position and shape of

the nucleus and pyrenoids (Figs 1 ndash

6) The main char-acteristic of the rede1047297ned Adenoides eludens is theabsence of the cingular groove With the exceptionof Podolampas and allied genera the absence of acingular groove is a rare feature in planktonic specieswith a globular shape Although there is no groovethe transversal 1047298agellum nonetheless encircles thecell As a general trend the cell 1047298attening of the

benthic species makes it dif 1047297cult to interpret the tabu-lation This is even more dif 1047297cult in some sand-dwell-ing dino1047298agellates that have an incomplete cingulum

Adenoides is an extreme case where the cingulum is

absent or the sulcal left anterior plate could be interpreted as a vestigial 1047297rst cingular plate We can spec-ulate an evolution towards the loss of the cingular groove as an adaptation to benthic habitats In factdino1047298agellates that lack the cingular groove such as

Prorocentrum are widespread and diverse in benthichabitats Then we can speculate that the loss of thecingular groove may be an advantage in benthic habi-tats and tentatively propose that the rotational move-ment of the transversal 1047298agellum is less useful ininterstitial space between the sand grains than in pela-gic habitats

With the molecular phylogeny we can 1047297nd arelationship between the planktonic and benthicdino1047298agellates that is useful to interpret their tabula-tion For example as suggested by Saldarriaga et al

(2003) the sand-dwelling genus Roscof 1047297a Balech isrelated to Podolampas (Goacutemez et al 2010) The genera

Amphidiniopsis or Herdmania are related to planktonic Protoperidinium-like cells such as Archaeperidinium

minutum (Kofoid) E Joslashrgensen (Goacutemez et al 2011Yamaguchi et al 2011) The morphology of the apical

pore of Adenoides is reminiscent of peridinioid generasuch as Heterocapsa F Stein or Azadinium Elbraumlchter ampTillmann (Tillmann et al 2009) The tabulation of theepitheca of Adenoides is 5prime and 6Prime but it can also beinterpreted as 4prime and 6Prime or 5prime and 7Prime These are commontabulation features of dino1047298agellates from different phy-logenetic origins

Taxonomic sampling in the phylogenetic treesremains incomplete and for the moment there areno dino1047298agellate sequences close to Adenoides or

Pseudadenoides Numerous sand-dwelling dino1047298a-gellates also remain as monotypic genera lackingany known relative with which they can be classi-

1047297ed at least at the family level The lack of thecingular groove in Adenoides or the absence of precingular plates in Pseudadenoides makes thesegenera particularly interesting for the evolution of dino1047298agellates

ACKNOWLEDGEMENTS

We thank Pierre Compegravere and Gerry Moore for their useful advice on nomenclature

FUNDING

FG was supported by a UL1 post-doctoral grant and aCNRS convention of research and an invited lecturer grant from Universiteacute Littoral-Cocircte Opale FG iscurrently supported by the Brazilian Conselho Nacionalde Desenvolvimento Cientiacute1047297co e Tecnoloacutegico (grant num-

ber BJT 3706462013-14)

AUTHOR CONTRIBUTIONS

F Goacutemez collection isolation light and electron micro-scopy drafting and editing manuscript R Onuma mole-cular analysis LF Artigas collection and editingmanuscript T Horiguchi phylogenetic analysis andediting manuscript

REFERENCES

BALECH E (1956) Eacutetude des dino1047298agelleacutes du sable de Roscoff Revue Algologique Nouvelle Serie 2 29 ndash 52

DAUGBJERG N HANSEN G LARSEN J amp MOESTRUP Oslash (2000)Phylogeny of some of the major genera of dino1047298agellates basedon ultrastructure and partial LSU rDNA sequence data includingthe erection of three new genera of unarmoured dino1047298agellates

Phycologia 39 302 ndash 317DODGE JD (1982) Marine Dino 1047298 agellates of the British Isles Her

Majestyrsquos Stationery Of 1047297ce LondonDODGE JD amp LEWIS J (1986) A further SEM study of armoured

sand-dwelling marine dino1047298agellates Protistologica 22221 ndash 230

GOacuteMEZ F (2012) A quantitative review of the lifestyle habitat andtrophic diversity of dino1047298agellates (Dino1047298agellata Alveolata)Systematics and Biodiversity 10 267 ndash 275

GOacuteMEZ F amp ARTIGAS LF (2014) High diversity of dino1047298agellatesin the intertidal sandy sediments of Wimereux (north-east EnglishChannel France) Journal of the Marine Biological Association of

the United Kingdom 94 443 ndash 457GOacuteMEZ F MOREIRA D amp LOacutePEZ-GARCIacuteA P (2010) Molecular

phylogeny of the dino1047298agellates Podolampas and Blepharocysta

(Peridiniales Dinophyceae) Phycologia 49 212 ndash 220GOacuteMEZ F LOacutePEZ-GARCIacuteA P amp MOREIRA D (2011) Molecular

phylogeny of the sand-dwelling dino1047298agellates Amphidiniopsis

hirsuta and A swedmarkii (Peridiniales Dinophyceae) Acta

Protozoologica 50 255 ndash 262HERDMAN EC (1922) Notes on dino1047298agellates and other organisms

causing discolouration of sand at Port Erin II Proceedings and

Transactions of the Liverpool Biological Society 36 15 ndash 30HERDMAN EC (1923) Notes on dino1047298agellates and other organ-

isms causing discolouration of the sand at Port Erin III Proceedings and Transactions of the Liverpool Biological

Society 38 58 ndash 63HOPPENRATH M SCHWEIKERT M amp ELBRAumlCHTER M (2003)

Morphological reinvestigation and characterization of the marinesand-dwelling dino1047298agellate Adenoides eludens (Dinophyceae)

European Journal of Phycology 38 385 ndash 394

HUELSENBECK JP amp R ONQUIST F (2001) MrBayes Bayesian infer-ence of phylogenetic trees Bioinformatics 17 754 ndash 755MC NEILL J BARRIE FR BUCK WR DEMOULIN V GREUTER W

HAWKSWORTH DL HERENDEEN PS K NAPP S MARHOLD KPRADO J PRUDrsquoHOMME VAN R EINE WF SMITH GF WIERSEMAJH amp TURLAND NJ (2012) International Code of Nomenclature




for Algae Fungi and Plants (Melbourne Code) RegnumVegetabile 154 Koeltz Koumlnigstein

MURRAY S amp PATTERSON DJ (2004) Cabra matta gen nov spnov a new benthic heterotrophic dino1047298agellate European

Journal of Phycology 39 229 ndash 234 NYLANDER JAA R ONQUIST F HUELSENBECK JP amp NIEVES-

ALDREY JL (2004) Bayesian phylogenetic analysis of combineddata Systematic Biology 53 47 ndash 67

PAULMIER G (1992) Catalogue illustreacute des microphytes planctoni-ques et benthiques des cocirctes Normandes (Rapport interneRV-92007RH) IFREMER Issy-Les-Moulineaux

POSADA MA amp CRANDALL KA (1998) Modeltest testing themodel of DNA substitution Bioinformatics 14 817 ndash 818

SALDARRIAGA JF TAYLOR FJR K EELING PJ amp CAVALIER -SMITHT (2001) Dino1047298agellate nuclear SSU rRNA phylogeny suggestsmultiple plastid losses and replacements Journal of Molecular

Evolution 53 204 ndash 213SALDARRIAGA JF LEANDER BS TAYLOR FJR amp K EELING

PJ (2003) Lessardia elongata gen et sp nov (Dino1047298agellata

Peridinales) Podolampaceae and the taxonomic position of thegenus Roscof 1047297a Journal of Phycol o gy 39 368 ndash 378

SWOFFORD DL (2003) PAUP Phylogenetic Analysis using

Parsimony (and Other Methods) Version 4 Sinauer AssociatesSunderland MA

TILLMANN U ELBRAumlCHTER M K ROCK B JOHN U amp CEMBELLA A(2009) Azadinium spinosum gen et sp nov (Dinophyceae) iden-ti1047297ed as a primary producer of azaspiracid toxins European

Journal of Phycology 44 63 ndash 79YAMAGUCHI A K AWAMURA H amp HORIGUCHI T (2006) A further

phylogenetic study of the heterotrophic dino1047298agellate genus Protoperidinium (Dinophyceae) based on small and large subunit ribosomal RNA gene sequences Phycological Research 54317 ndash 329

YAMAGUCHI A HOPPENRATH M POSPELOVA V HORIGUCHI T ampLEANDER BS (2011) Molecular phylogeny of the marine sand-dwelling dino1047298agellate Herdmania litoralis andan emended descrip-tion of the closely related planktonic genus Archaeperidinium

Joumlrgensen European Journal of Phycology 46 98 ndash 112




in shape has a barely visible episome and a hump inthe sulcal area (Herdman 1922 p 22 1047297g 1) In her 1047297g 2 she described Amphidinium kofoidii as round tosquarish in shape with a more visible button-likeepisome (Herdman 1922 p 26 1047297g 2) Balech(1956) proposed the new thecate genus Adenoides

based on Herdmanrsquos 1047297g 2 of Amphidinium kofoidiiHowever Balech (1956) proposed as basionym

Amphidinium eludens Herdman (1922 1047297g 1) LaterDodge (1982) proposed Adenoides kofoidii for

Amphidinium kofoidii Herdman 1922 1047297g2Thetaxo-nomic and nomenclatural history of Adenoides elu-

dens is reported in Hoppenrath et al (2003) Adenoides eludens based on Balechrsquos description and

as described by Herdmanrsquos 1047297g 2 of Amphidinium

kofoidii was investigated again by Dodge amp Lewis(1986) and Hoppenrath et al (2003) and molecular data have been available since Saldarriaga et al

(2001) To date there are 45 sequences of different molecular markers of Adenoides eludens and another 39 sequences labelled as two uncultured AdenoidesThe sequences of Adenoides eludens available inGenBank are from the cultures CCCM 683 andCCMP 1891 isolated from the Paci1047297c coasts of Canada or 1047297eld material collected from the same loca-tion Another culture CCMP 2081 was isolated fromEurope However there are no molecular data for

Adenoides eludens from the European Atlantic coast where the species was described While Adenoides

eludens (= Amphidinium kofoidii) has been subjected

to morphological and molecular studies little is knownabout the species described as Amphidinium eludens

Herdman (1922 1047297g 1) This organism is one of the1047297rst described sand-dwelling dino1047298agellates andaccording to Herdman (1922) is responsible for discolourations in the sands of the European Atlanticcoasts However the detailed morphology of thisspecies remains unknown

In this study we provide light microscopy picturesthe 1047297rst scanning electron microscopy pictures and the1047297rst molecular data for Amphidinium eludens

Herdman (1922 1047297g 1) and the 1047297rst molecular datafor Amphidinium kofoidii Herdman (1922 1047297g2)fromthe French coasts of the English Channel whereBalech (1956) described the genus Adenoides Themorphological and molecular data support the conclu-sion that the two species described in 1047297gs 1 and 2 byHerdman (1922) belong to independent genera

Amphidinium eludens is an unusual dino1047298agellatecharacterized by the lack of a cingular groove similar to desmokont dino1047298agellates

MATERIALS AND METHODSSource isolation and microscopy observations

This study was undertaken in the soft sandy sediments of theshore of Wimereux France (50ordm46prime 12Prime N 1ordm36prime 42Prime E)

collected during low tide in June 2012 Two sites on the beach in front of the LOG laboratory (MREN ULCO andMarine Station of Wimereux UL1 Goacutemez amp Artigas 2014)were sampled the moist sands around the border of a large

pool (~50 m diameter ~1 m depth) and several smaller poolsand moist sands showing a faint brownish discolourationThe upper centimetre of sand was collected with a spoon

and deposited into a bottle containing seawater collected at the same location Then the sand with seawater was stirredvigorously and the suspension settled in a composite settlingchamber The settled material was examined with an invertedmicroscope (Nikon Eclipse TE2000-S Tokyo) and photo-graphed with a Nikon Digital Sight DS-2M camera The cellsize described as length (apical to antapical axis) and depthie the length along the lateral sides (ventral to dorsaldistance) was measured in 25 specimens The width(transdiameter) was measured in 1047297ve specimens

For scanning electron microscopy the sand samples withseawater were stirred vigorously and the suspension was1047297

xed with glutaraldehyde (5) and 1047297

ltered onto a 08 microm pore size Whatman Nucleporereg membrane 1047297lter washedwith distilled water 1047297xed with osmium tetroxide dehydratedwith a graded series of ethanol and critical point dried withCO2 Filters were mounted on stubs sputter-coated with goldand viewed using a Hitachi S4800 scanning electron micro-scope Images were presented on a black background usingAdobe Photoshop CS3

PCR ampli1047297cation and DNA sequencing

For molecular analysis each specimen of Amphidinium elu-

dens and A kofoidii was micropipetted individually with a1047297ne capillary into a clean chamber and washed several timesin serial drops of 02 μm 1047297ltered and sterilized seawaterFinally 1 ndash 5 specimens of each species were deposited in a02 ml Eppendorf tube 1047297lled with several drops of absoluteethanol The sample was kept at room temperature and indarkness until the molecular analysis could be performed

Prior to DNA extraction the 02 ml Eppendorf tubes werecentrifuged for 10 min at 14462 times g in a TOMY MX-201centrifuge (Tokyo Japan) Ethanol was then evaporated in avacuum desiccator Cells were resuspended in 10 μl of QuickExtract DNA extraction solution (EpicenterMadison) and incubated at 56ordmC for 1 h and 98ordmC for 2 min

in a thermal cycler (GeneAmp PCR System 9700 AppliedBiosystems Foster City) The product was used as DNAtemplate for the following polymerase chain reaction(PCR) In the 1047297rst round of PCR to obtain almost completeSSU rDNA and partial LSU rDNA sequences two sets of

primers (SR1-SR12b for SSU rDNA and D1RF1 28-1483R for LSU rDNA respectively) were simultaneously appliedIn the second round of PCR 05 microl of the 1047297rst PCR productswas used as DNA template and three pairs of primers (SR1-SR5 SR4-SR9p and SR8-SR12b see Yamaguchiet al 2006) were used for SSU rDNA ampli1047297cation andtwo pairs of primers (D1RF-25R1 and D3A-28-1483R seeDaugbjerg et al 2000) were applied for LSU rDNA ampli-

1047297cation PCR conditions for both rounds of ampli1047297cationconsisted of one initial cycle of denaturation at 94ordmC for 5min followed by 35 cycles of denaturation at 94ordmC for 30 sannealing at 55degC for 30 s and extension at 72ordmC for 30 sThe PCR process was completed by a 1047297nal extension cycle
















RESULTS

















presented below








































and 5primePrime





plate (Fig 30)
























view Figs 40 ndash


























EPITYPE Fig 60














Molecular phylogeny













DISCUSSION












































ACKNOWLEDGEMENTS


FUNDING


ber BJT 3706462013-14)



REFERENCES

























































RESULTS

















presented below








































and 5primePrime





plate (Fig 30)
























view Figs 40 ndash


























EPITYPE Fig 60














Molecular phylogeny













DISCUSSION












































ACKNOWLEDGEMENTS


FUNDING


ber BJT 3706462013-14)



REFERENCES
















































































and 5primePrime





plate (Fig 30)
























view Figs 40 ndash


























EPITYPE Fig 60














Molecular phylogeny













DISCUSSION












































ACKNOWLEDGEMENTS


FUNDING


ber BJT 3706462013-14)



REFERENCES















































and 5primePrime





plate (Fig 30)
























view Figs 40 ndash


























EPITYPE Fig 60














Molecular phylogeny













DISCUSSION












































ACKNOWLEDGEMENTS


FUNDING


ber BJT 3706462013-14)



REFERENCES































































view Figs 40 ndash


























EPITYPE Fig 60














Molecular phylogeny













DISCUSSION












































ACKNOWLEDGEMENTS


FUNDING


ber BJT 3706462013-14)



REFERENCES























































view Figs 40 ndash


























EPITYPE Fig 60














Molecular phylogeny













DISCUSSION












































ACKNOWLEDGEMENTS


FUNDING


ber BJT 3706462013-14)



REFERENCES

































































EPITYPE Fig 60














Molecular phylogeny













DISCUSSION












































ACKNOWLEDGEMENTS


FUNDING


ber BJT 3706462013-14)



REFERENCES























































EPITYPE Fig 60














Molecular phylogeny













DISCUSSION












































ACKNOWLEDGEMENTS


FUNDING


ber BJT 3706462013-14)



REFERENCES













































DISCUSSION












































ACKNOWLEDGEMENTS


FUNDING


ber BJT 3706462013-14)



REFERENCES







































































ACKNOWLEDGEMENTS


FUNDING


ber BJT 3706462013-14)



REFERENCES


































































ACKNOWLEDGEMENTS


FUNDING


ber BJT 3706462013-14)



REFERENCES





























































ACKNOWLEDGEMENTS


FUNDING


ber BJT 3706462013-14)



REFERENCES































































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