morphological delineation and distribution patterns of four … · morphological delineation and...

Morphological delineation and distribution patterns of fournewly described species within the Synura petersenii speciescomplex (Chrysophyceae, Stramenopiles)

PAVEL ŠKALOUD, MAGDA ŠKALOUDOVÁ, ANNA PROCHÁZKOVÁ AND YVONNE NĚMCOVÁ

Department of Botany, Charles University in Prague, Benátská 2, CZ-12801, Prague, Czech Republic

(Received 18 October 2013; revised 22 January 2014; accepted 28 January 2014)

The Synura petersenii species complex represents a common, cosmopolitan and highly diverse taxon of autotrophic freshwaterflagellates. In this paper, we describe and characterize four new species (S. borealis, S. heteropora, S. hibernica and S. laticarina)that have been identified during our extensive sampling of freshwater habitats in 15 European countries. Morphometric analysesof siliceous scales led to the significant phenotypic differentiation of all four newly described species, and their separation fromother related species of the S. petersenii complex. Two of these newly described species (S. hibernica and S. borealis) can beclearly distinguished by characteristic large colonies consisting of elongated, lanceolate-shaped cells. Development of stronglyelongated, narrow cells in S. hibernica could be explained by the adaptation of this species to oligotrophic conditions. Thoughmorphologically distinct, S. borealis possesses an exceptionally high degree of genetic diversity, possibly indicating recentspeciation and evolutionary diversification within this taxon. Three of the four newly described species exhibit restrictedbiogeographic distribution. The evolutionarily related S. borealis and S. laticarina occur only in Northern Europe, and seem to beadapted to colder areas. The most remarkable distribution pattern was observed for S. hibernica, which has a geographicdistribution that is restricted to western Ireland.

Key words: biogeography, cox1, cryptic species, ITS rDNA, morphology, phylogeny, rbcL, speciation, Synura, taxonomy

Introduction

The order Synurales (Chrysophyceae, Stramenopiles)contains several genera of scale-bearing flagellates thatare important components of phytoplankton commu-nities of various freshwater bodies (Kristiansen &Preisig, 2007; Škaloud et al., 2013a). The most con-spicuous genus of the order, Synura, is a colonialorganism formed by a variable number of cells joinedtogether at their posterior ends. Each cell is covered byimbricate silica scales (Wee, 1997), consisting of aperforated basal plate with various upturned or bentparts, and a secondary ornamentation, which is used todelimit the different species (Kristiansen, 1986).According to the recently published phylogeny of theSynurales, the genus Synura should be split into fivesections: Echinulatae, Peterseniae, Spinosae,Splendidae and Synura (Škaloud et al., 2013a).Taxabelonging to the section Peterseniae are well character-ized by body scales with a central keel that may end in aspine-like projection. The most widely recognized spe-cies of this section, Synura petersenii sensu lato, isconsidered to be one of the most widely distributedfreshwater chrysophytes (Kristiansen, 1975), even

causing taste and odour problems in water supplies(Nicholls & Gerrath, 1985). This species has beensubjected to various ecophysiological experiments(e.g. Saxby-Rouen et al., 1997; Kim et al., 2008;Pichrtová & Němcová, 2011).

Due to the species-specific morphology of silicascales, Synura could be supposed to have one ofthe best morphological species concepts withinprotists. Synura scales are often well preserved inbenthic sediments and used in palaeoecologicalstudies. Because many taxa are distributed withina narrow range of ecological variables (e.g. pH,temperature and nutrient concentration), investiga-tion of fossilized Synura scales is used as a tool toassess eutrophication, acidification and shifts inclimate (Smol, 1995; Smol & Cumming, 2000).In addition, the records of fossilized Synura scalesdiscovered in Eocene sediments are used to datethe diversification and evolution of the genus (Booet al., 2010; Siver, 2013).

Recent molecular phylogenetic investigationshave revealed a conflict between the traditionalmorphological species concept based on ultrastruc-ture of silica structures and the phylogenetic data.Synura petersenii sensu lato represents the most

Correspondence to: Pavel Škaloud. E-mail: [email protected]

Eur. J. Phycol. (2014), 49(2): 213–229

ISSN 0967-0262 (print)/ISSN 1469-4433 (online)/14/020213-229 © 2014 British Phycological Societyhttp://dx.doi.org/10.1080/09670262.2014.905710

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investigated species in this respect. First, Wee et al.(2001) demonstrated the existence of two well-sup-ported S. petersenii clades with different distributionpatterns. Later, Kynčlová et al. (2010) investigated anumber of European isolates, and revealed the exis-tence of six cryptic species. Almost concurrently,Boo et al. (2010) published a multigene phylogenyof approximately 100 S. petersenii isolates, confirm-ing the high degree of hidden, species-level diversitywithin this species. A taxonomic assessment ofobserved cryptic diversity redefined the species con-cept within the S. petersenii morphotype, and recog-nized six cryptic lineages as separate species(Škaloud et al., 2012).

Phylogenetic analyses performed by Boo et al.(2010) and Škaloud et al. (2012) clearly indicate thatthere are more than six hidden species within theS. petersenii complex. To further investigate the reallevel of species diversity within this complex, weperformed extensive sampling in 15 European coun-tries, including Greenland. According to the analysesof internal transcribed spacer (ITS) rDNA sequences,58 of more than 800 investigated Synura isolatesbelonged to four novel, undescribed species. Theprincipal aim of this study was to expand our knowl-edge and understanding of the diversity in theS. petersenii complex, and to describe and character-ize all these new taxa. In addition, we performed adetailed morphological investigation of all crypticspecies, and investigated whether they could be deli-neated by the morphology of silica scales.

Materials and methods

Origin and cultivation of the investigated strains

The strains used in this study were obtained during aninvestigation of silica-scaled chrysophytes in Europe andGreenland during the period 2008–2013 (Table 1). Thematerial was usually sampled from plankton and meta-phyton of various water bodies, using a plankton net with20-μm mesh. To ensure the highest viability of Synurastrains, individual colonies were isolated on the same daythat they were sampled. The colonies were removed fromthe natural sample by micropipetting, and transferred intoseparate wells of a 96-well plate containing 400 μl ofeither MES- or HEPES-buffered DY IV liquid medium(Andersen et al., 1997). The wells were kept at 15°C(fridge bag TK 51, Ardes SpA, Ponte Nossa, Italy), underconstant illumination of 50–200 μmol m−2 s−1 providedby 6 W LED diodes (LB115A-6W-X, Yuyao LianliangElectric Appliance Co Ltd, Ningbo, China). After return-ing them to the laboratory, the strains were cultivated at15°C (cooling box C5G, Helkama Oy, Helsinki, Finland),under illumination of 40 μmol m−2 s−1 and a 16-h light :8-h dark cycle (TLD 18W/33 fluorescent lamps, Philips,Amsterdam, the Netherlands). In addition, isolated colo-nies were separately transferred to 200 μl PCR tubes andsubsequently frozen at −24°C.

DNA extraction, PCR and DNA sequencing

For DNA isolation, 100 µl of the growing culture was cen-trifuged in PCR tubes (6000 rpm for 3 min), and 30 µl ofInstaGene matrix (Bio-Rad Laboratories, Hercules, CA,USA) was added to the pellet. The solution was vortexedfor 10 s, incubated at 56°C for 30 min, and heated at 99°C for8 min. After vortexing a second time, the tubes were centri-fuged at 12000 rpm for 2 min, and the supernatant wasdirectly used as a PCR template. Three molecular markerswere amplified by PCR: nuclear ITS rDNA, chloroplast rbcLand mitochondrial coxI. The amplification of ITS rDNAwasperformed as described in Kynčlová et al. (2010), using theprimers Kn1.1 (5ʹ-CAAGGT TTC CGTAGG TGAACC-3ʹ;Wee et al., 2001) and ITS4 (5ʹ-TCC TCC GCT TAT TGATAT GC-3ʹ; White et al., 1990). Amplification of the rbcLmarker was performed according to Jo et al. (2011), using theprimers rbcL_2F (5ʹ-AAAAGTGACCGT TATGAATC-3ʹ;Daugbjerg & Andersen, 1997) and rbcL_R3 (5ʹ-GTA ATATCT TTC TTC CATAAATCTAA-3ʹ; Jo et al., 2011). Thecox1 marker was amplified according to Boo et al. (2010),using the primers F692 (5ʹ-TTG TDT GGT CAG TTT TAATTA C-3ʹ) and R1433 (5ʹ-GGC ATA CCT GCWARA CCTAA-3ʹ; Boo et al., 2010). The PCR products were purifiedand sequenced at Macrogen Inc. in Seoul, Korea.

Phylogenetic analyses

A multiple alignment of the newly determined ITS rDNA,rbcL and coxI gene sequences and other sequences selectedfrom the GenBank/EMBL/DDBJ databases was built usingMAFFT, version 6, applying the Q-INS-i strategy (Katohet al., 2002). The sequences were selected to encompass allknown lineages within the Synura petersenii species complex(Table S1). ITS rDNA sequences were then aligned on thebasis of their rRNA secondary structure information(Kynčlová et al., 2010) with MEGA 4 (Kumar et al., 2008).The three loci were concatenated, yielding an alignment of2308 bases. The final matrix contained 51 ITS rDNA,36 rbcL and 33 coxI sequences. A suitable partitioning strat-egy and partition-specific substitution models were selectedin a multistep process (Verbruggen et al., 2010). Initially, aguide tree was obtained by carrying out a second-level max-imum likelihood (ML) search on the unpartitioned datasetwith a HKY + Γ8 model using TreeFinder (Jobb et al., 2004).Then, the dataset was divided by 17 different partitioningstrategies (combining different levels of locus segmentation).Subsequently, Bayesian information criterion (BIC) calcula-tions were performed for all 17 potential partitioning strate-gies, assuming the guide tree and HKY + Γ8 model for eachpartition. The five best-scoring partitioning strategies (lowestBIC scores) were retained for further analysis. In the nextstep, models of sequence evolution were selected for indivi-dual partitions using BIC. For each partition present in thefive retained partitioning strategies, 12 different nucleotidesubstitution models were evaluated (F81, HKY, GTR andtheir combinations with Γ, I and Γ + I). Finally, the partition-ing strategies were re-evaluated using the selected models forthe particular partitions. This BIC-based model selectionprocedure selected the following model with eight partitions:(1) internal transcribed spacers ITS1 and ITS2 –GTR + Γ; (2)5.8 S ribosomal locus – F81; (3), first codon position of therbcL gene – GTR + Γ; (4) second codon position of the rbcL

P. Škaloud et al. 214

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Tab

le1.

Originandsamplingdetails

ofanalysed

strains.

Taxo

nStrain

Locality

Geographiccoordinates

Sam

plingdate

Tem

p.(°C)

pHCon

d.(µScm

−1)

S.am

erican

aS81

.C7

Záplavy

NR,C

zech

Repub

lic50

.146

43°N,1

4.01

389°

E19

.3.201

25

7.2

670

S.am

erican

aS10

4.C4

Schinkelbos,N

etherlands

52.299

27°N,4

.807

42°E

7.12

.201

24

7.9

1257

S.bo

realis

S58

.C7

Lillesjön,

Sweden

56.955

43°N,1

4.71

428°

E22

.4.2011

126.5

81S.

borealis

S62

.D7

unnamed

lake,D

isko

island

,Greenland

69.289

13°N,5

3.49

322°

W28

.7.2011

76.3

81S.

borealis

S90

.F3

Rutajärvi,H

arjulahti,Finland

61.906

36°N,2

6.02

054°

E5.5.20

128

5.8

–S.

borealis

S90

.G4

Harjujärvi,Finland

61.885

47°N,2

6.01

479°

E5.5.20

127

4.9

–S.

borealis

S90

.M34

Jämsänjarvi,F

inland

62.250

60°N,2

5.18

766°

E5.5.20

126

5.2

–S.

borealis

S110.E2/F3

asm

allp

ooln

earKallijõgi,E

ston

ia58

.335

39°N,2

7.26

385°

E10

.5.201

321

6.7

157

S.bo

realis

S110.B9

Apn

ajõgi,E

ston

ia58

.343

05°N,2

7.26

793°

E10

.5.201

319

6.7

110

S.bo

realis

S113.F4/F6

Emajõg

i,Eston

ia58

.396

36°N,2

6.31

094°

E10

.5.201

316

7.5

322

S.bo

realis

S114.B8/B9/C8

unnamed

lake

(A1),S

weden

68.348

05°N,1

9.03

784°

E14

.6.201

313

6.3

134

S.bo

realis

S114.F3

unnamed

lake

near

Paittasjärvi,S

weden

67.862

23°N,1

9.02

746°

E15

.6.201

316

6.5

23S.

borealis

S114.G6

Syv

äjärvi,S

weden

67.756

64°N,2

0.09

297°

E16

.6.201

312

6.6

49S.

borealis

S115.F4

apo

olnear

Arosnjarkajaute,S

weden

68.428

76°N,1

8.35

156°

E19

.6.201

34

6.8

88S.

borealis

S115.G2/G3

apo

olnear

Vassijaure,Sweden

68.433

05°N,1

8.23

966°

E19

.6.201

37

6.9

45S.

borealis

S115.G7

Vassijaure,Sweden

68.430

72°N,1

8.12

755°

E19

.6.201

311

6.9

24S.

borealis

S116.B6

Kjerringd

alsvatnet,Norway

68.660

19°N,1

5.54

052°

E19

.6.201

316

6.2

37S.

borealis

S117.D3

apo

olnear

Paittasjärvi,S

weden

67.858

03°N,1

9.02

454°

E15

.6.201

316

6.0

13S.

borealis

S118.C6

apo

olnear

Torneträsk,

Sweden

68.355

76°N,1

8.82

491°

E17

.6.201

314

7.4

173

S.bo

realis

SCFIN

13A

Ojala,F

inland

61.847

78°N,2

6.29

145°

E5.5.20

1210

6.5

–S.

borealis

SCFIN

22B

Heinäjärvi,Finland

62.88112

°N,2

5.49

724°

E5.5.20

127

5.9

–S.

borealis

SCGRL34

0Sanning

assupTasia,G

reenland

69.269

39°N,5

3.47

820°

W28

.7.2011

156.8

72S.

borealis

SCGRL36

4amorrainelake,D

isko

island

,Greenland

69.297

54°N,5

3.51

961°

W28

.7.2011

5–

–S.

borealis

SCSWE9

Helgassjön,

Sweden

56.956

11°N,1

4.71

630°

E22

.4.2011

116.6

72S.

cono

pea

S10

3.B3

apo

olnear

Smědava,Czech

Repub

lic50

.846

06°N,1

5.23

6750

°E

14.10.20

126

5.0

40S.

glab

raS89

.F5

Saarijärvi,Finland

62.701

73°N,2

5.26

388°

E5.5.20

126

6.5

–S.

heteropo

raS20

.1Lochan

Add

,Scotland

,UK

56.032

98°N,5

.534

510°

W4.6.20

08–

––

S.heteropo

raS20

.45

CrinanCanal,S

cotland

,UK

56.060

51°N,5

.477

146°

W4.6.20

08–

––

S.heteropo

raS40

.F11

Pod

hradskápo

ol,C

zech

Repub

lic50

.46112

°N,1

4.9117

6°E

3.4.20

1116

7.4

660

S.heteropo

raS54

.E11

Klejnarka

river,Czech

Repub

lic49

.969

56°N,1

5.32

123°

E19

.4.2011

––

–S.

heteropo

raS86

.F2

Zbý

šovpo

nd,C

zech

Repub

lic49

.81160

°N,1

5.35

409°

E20

.3.201

2–

––

S.heteropo

raS87

.C6

anephemeralpu

ddlenear

Kufstein,

Austria

47.597

91°N,1

2.15

115°

E22

.4.201

2–

––

S.heteropo

raS10

1.F7

apo

ndin

Nyg

årdsparken,B

ergen,

Norway

60.386

14°N,5

.324

47°E

21.10.20

12–

6.2

117

S.heteropo

raS112.E2

Saardepaisjärv,E

ston

ia58

.143

55°N,2

4.97

033°

E10

.5.201

319

8.7

313

S.heteropo

raS112.F5

Rahum

eri,Eston

ia58

.182

46°N,2

5.05

247°

E10

.5.201

317

7.5

213

S.heteropo

raS113.C8

KarulaJarv,E

ston

ia58

.398

58°N,2

5.59

201°

E10

.5.201

315

6.9

458

S.heteropo

raS117.G6

Torneträsk,

Sweden

68.355

73°N,1

8.82

172°

E17

.6.201

312

7.5

53S.

heteropo

raSCIRL8

GarlanLou

gh,Ireland

55.003

43°N,7

.905

883°

W29

.9.2011

166.1

121

S.hibernica

SIE

E4

The

Lon

gRange,Ireland

51.997

25°N,9

.550

89°W

17.7.200

916

8.0

23S.

hibernica

SIE

E8

unnamed

lake

near

Maam

Cross,Ireland

53.450

49°N,9

.544

07°W

19.7.200

916

6.2

56S.

hibernica

SIE

E11

Glend

ollagh

Lou

gh,Ireland

53.466

58°N,9

.738

63°W

18.7.200

917

7.9

53S.

hibernica

SIE

M38

The

Lon

gRange,Ireland

51.997

25°N,9

.550

89°W

19.9.200

8–

7.6

32S.

hibernica

SIE

103.C8/C9/C11

Gow

laun

Lou

gh,Ireland

51.783

16°N,9

.765

03°W

6.3.20

107

4.4

31

(con

tinued)

Four new species of Synura 215

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gene – F81; (5) third codon position of the rbcL gene – GTR+ Γ; (6) first codon position of the coxI gene – GTR + Γ; (7)second codon position of the coxI gene – F81 + Γ; and (8)third codon position of the coxI gene – GTR + Γ.

The phylogenetic tree was inferred with Bayesian infer-ence (BI) using MrBayes version 3.1.2 (Ronquist &Huelsenbeck, 2003). Two parallel MCMC runs were carriedout for 10 million generations. The dataset was divided intoeight partitions, for which different substitution models wereselected according to the BIC-based model selection. Treesand parameters were sampled every 100 generations.Convergence of the two runs was assessed during the runby calculating the average standard deviation of split frequen-cies (SDSF). The SDSF value between simultaneous runswas 0.003908. The burn-in was determined using the ‘sump’command. Bootstrap analyses were performed with ML andweighted parsimony (wMP) criteria using GARLI version0.951 (Zwickl, 2006) and PAUP* version 4.0b10(Swofford, 2002), respectively. ML analyses consisted ofrapid heuristic searches (100 pseudoreplicates) using auto-matic termination (genthreshfortopoterm command set to100 000). The dataset was divided into eight partitions withdifferent substitution models. The wMP bootstrapping (1000replications) was performed using heuristic searches with100 random sequence addition replicates, tree bisectionreconnection swapping, random addition of sequences (thenumber limited to 10 000 for each replicate), and gap char-acters treated as a fifth character state. Character weightswere assigned using the rescaled consistency index on ascale of 0 to 1000. New weights were based on the mean fitvalues for each character over all trees in the memory.

Morphological investigations and statistical analyses

For morphological observations, the strains were cultivated in50-ml Erlenmeyer flasks for 1–2 months. To avoid depletionof nutrients, the strains were grown under lower light andtemperature regimes. Light microscopy (LM) observationswere performed using an Olympus BX51 microscope. Forscanning electron microscopy (SEM) observations, a drop ofglutaraldehyde (GA)-fixed (2%GA overnight) cell suspensionwas sedimented for 60 min on polyL-lysine-coated glass cov-erslips to ensure appropriate cell adhesion. The coverslipswere washed by repeated transfer into drops of deionizedwater dispensed onto the hydrophobic surface of a Parafilmstrip, and subsequently dehydrated via acetone series. Thecells were dried to a critical point with liquid carbon dioxide(Bal-Tec CPD 030), coated with platinum for 90 s with a Bal-Tec SCD 050 sputter coater, and observed with a JEOL JSM-740 1F FESEM scanning electron microscope. For transmis-sion electronmicroscopy (TEM) investigations of silica scales,a drop from the living cultures was placed onto formvar-coatedcopper grids and dried. After washing in a series of waterdroplets, the grids were examined in a TEM JEOL 1011electron microscope. At least three strains were randomlychosen from each of the clades identified by the molecularstudy, including the cultures deposited in the CultureCollection of Algae of Charles University in Prague (CAUP)and the National Center for Marine Algae and Microbiota inMaine (designated as CCMP). For each clade, the followingcharacters were measured in 30 randomly selected scales: (1)scale length; (2) scale width; (3) area of a base hole; (4)T

able1.

Con

tinued.

Taxo

nStrain

Locality

Geographiccoordinates

Sam

plingdate

Tem

p.(°C)

pHCon

d.(µScm

−1)

S.hibernica

SIE

104.D11

Glanm

oreLake,Ireland

51.739

01°N,9

.771

94°W

6.3.20

109

5.2

40S.

hibernica

SIE

105.F6

CahaLakes,Ireland

51.721

71°N,9

.659

94°W

6.3.20

107

5.4

31S.

hibernica

SCIRL20

aDerrynaherrivaLou

gh,Ireland

54.070

93°N,9

.512

78°W

30.9.2011

145.8

87S.

hibernica

SCIRL41

Easky

Lou

gh,Ireland

54.157

17°N,8

.844

04°W

2.10

.2011

146.1

51S.

hibernica

SCIRL60

Maumwee

Lou

gh,Ireland

53.474

38°N,9

.543

63°W

19.7.200

9–

6.9

39S.

laticarina

S89

.D5

Ojala,F

inland

61.847

78°N,2

6.29

145°

E5.5.20

1210

6.5

–S.

laticarina

S90

.C8

Tehriselkä,Finland

61.756

52°N,2

6.48

581°

E5.5.20

126

5.5

–S.

laticarina

S110.C9

Apn

ajõgi,E

ston

ia58

.343

05°N,2

7.26

793°

E10

.5.201

319

6.7

110

S.laticarina

S113.E5

apo

olnear

Emajõg

i,Eston

ia58

.396

37°N,2

6.31

094°

E10

.5.201

316

7.6

357

S.laticarina

S115.B2

Bardu

elva,N

orway

69.013

95°N,1

8.48

489°

E17

.6.201

310

7.0

80S.

laticarina

S115.D2

apo

olnear

Arosnjarkajaute,S

weden

68.428

76°N,1

8.35

156°

E19

.6.201

34

6.8

88S.

laticarina

S115.E5

apo

olnear

Vassijaure,Sweden

68.433

05°N,1

8.23

966°

E19

.6.201

37

6.9

45S.

macropo

raS71

.B4

Pod

hradskápo

ol,C

zech

Repub

lic50

.46112

°N,1

4.9117

6°E

4.3.20

125.5

6.5

230

S.macropo

raS10

4.D7

acanalinOosteinde,N

etherlands

52.288

16°N,4

.809

22°E

7.12

.201

23.3

7.9

850

S.macropo

raS10

4.F11

apo

olin

Aalsm

eer,Netherlands

52.271

48°N,4

.761

43°E

7.12

.201

24.1

7.9

975

S.petersenii

S89

.D6

Ojala,F

inland

61.847

78°N,2

6.29

145°

E5.5.20

129.5

6.5

–S.

petersenii

S89

.F9

Heinäjärvi,Finland

62.88112

°N,2

5.49

724°

E5.5.20

127.0

5.9

–S.

truttae

S62

.B5

apeatbo

gnear

Přebu

z,Czech

Repub

lic50

.383

08°N,1

2.59

668°

E24

.5.2011

12.6

4.7

107


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average area of a keel pore; (5) average area of a base-platepore; (6) keel width; and (7) number of struts (Fig. 1). Themeasurements were performed using the program ImageJ 1.45s (Schneider et al., 2012). Average values of keel and base-plate pores were obtained by measuring areas of approxi-mately 30 and 60 pores per scale, respectively. Statisticalanalyses of measured data (principal component and canonicaldiscriminant analyses) were performed using Statistica 8.0(StatSoft, Inc., Tulsa, Oklahoma, USA).

Results

Phylogenetic analyses

The Bayesian analysis based on the concatenateddataset (ITS rDNA, rbcL and cox1 sequences)revealed the existence of at least 16 lineages withinthe Synura petersenii species complex (Fig. 2). Inaddition to the six described species (S. americana,S. conopea, S. glabra, S. macropora, S. petersenii andS. truttae) and lineages resolved in previous studies,the phylogenetic analysis detected the existence offour distinct, novel lineages. The first two lineages(here referred to as S. borealis sp. nov. and S. laticar-ina sp. nov.) formed a well-supported clade togetherwith S. americana, S. macropora and S. petersenii.

The latter two newly recognized lineages (herereferred to as S. heteropora sp. nov. and S. hibernicasp. nov.) were inferred to be members of another well-supported clade including S. truttae and the strainsUTEX LB 239 and KNU01. Synura borealis repre-sented the most diverse novel lineage, containing anumber of isolates clustering into several distinctlineages. The most common genotype was repre-sented by eight isolates originating from Estonia,Finland, Norway and Sweden. By contrast, S. laticar-inawas represented by seven sequences clustered intotwo related sub-clades. The third novel lineage, S.heteropora, consisted of 13 isolates originating fromAustria, Czech Republic, Estonia, Great Britain,Ireland, Sweden and Norway. The strain CCMP2898, which was isolated from an Austrian lake, wasinferred as a member of this lineage. The last newlyrecognized lineage, S. hibernica, consisted of 12 iso-lates that all originated from western Ireland.

Morphological observations and taxa descriptions

Molecular phylogenetic analyses show the existence offour novel lineages in the genus Synura, sectionPeterseniae. The detailed TEM investigation of siliceousscales demonstrated their clear distinctness from allspecies without molecular characterization (S. obesa,S. australiensis, ‘S. petersenii’ f. columnata, ‘S. peter-senii’ f. praefracta, and ‘S. petersenii’ f. taymyrensis).Therefore, these novel lineages represent four new spe-cies, which we describe and illustrate below.

Synura borealis Škaloud & Škaloudová, sp. nov.

(Figs 3–10)

DESCRIPTION: Colonies are spherical, up to 86 µm in diameter,consisting of approximately 16–38 cells associated by theirposterior ends. Cells are significantly elongated, lanceolate-shaped, posteriorly elongated into the tail, 31–42 µm long and7–12 µm wide (Fig. 3). Each cell is surrounded by a layer ofimbricate siliceous scales (Fig. 4). Body scales are 4.0–5.8 µmlong and 1.6–2.6 µm wide, consisting of a basal plate with acentrally raised keel protruding into an acute tip (Figs 5 and 6).The keel is often anteriorly widened, ornamented by medium-sized pores (diameter, 54–88 nm). Anteriorly, keel pores areproduced on both sides, so that the keel pore pattern is notablyover-layered (Figs 6, 7). The ratio between scale and keelwidth varies from 1.7 to 2.9. The basal plate is ornamentedby numerous small pores (diameter, 17–26 nm), and anteriorlyperforated by a large, rounded or elongated base hole (dia-meter, 0.27–0.55 µm). Numerous struts (28–38), often inter-connected by transverse folds, extend regularly from the keelto the scale perimeter. Apical scales are 3.2–4.2 µm long and1.7–2.5 µm wide (Fig. 8). The keel of the apical scales ends ina prominent, acute tip (Fig. 9). Rear scales are long andnarrow, 4.3–5.7 µm long and 1.1–1.6 µm wide (Fig. 10).

ETYMOLOGY: The specific epithet ‘borealis’ refers to the north-ern (boreal) occurrence of the species, which is found innorthern Europe and Greenland.

Fig. 1. Morphological features measured in Synura scales;scale length (sl), scale width (sw), base hole area (bha), keelpore area (kpa), base-plate-pore area (bpa), keel width (kw) andnumber of struts (sn). Scale bar represents 1 µm.


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Fig. 2. Phylogeny of the genus Synura, section Peterseniae, obtained by Bayesian inference of the concatenated ITS rDNA, rbcL andcox1 dataset. The analysis was performed under a partitioned model, using different substitution models for each partition. Values atthe nodes indicate statistical support estimated by three methods; MrBayes posterior node probability (left), maximum likelihoodbootstrap (middle), and maximum parsimony bootstrap (right). Only statistical supports higher than 0.90/50/50 are shown. Thickbranches highlight nodes receiving the highest posterior probability (PP) support (1.00). Newly sequenced strains are marked in bold.Strains used for the statistical analyses of morphological features are marked by asterisks. Scale bar represents the expected number ofsubstitutions per site.


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TYPE LOCALITY: Unnamed lake, Disko Island, Greenland(69.28913°N, –53.49322°W).

HOLOTYPE: Synura borealis strain S 62.D7, frozen materialdeposited in the Culture Collection of Algae of CharlesUniversity in Prague (CAUP). Figure 6 presents an illustra-tion of the holotype.

HOLOTYPE DNA BARCODE: GenBank Accession no.HG514176.

DISTRIBUTION (Table 1): Estonia, Finland, Greenland, Norwayand Sweden.

Synura heteropora Škaloud, Škaloudová &Procházková, sp. nov.

(Figs 11–19)

DESCRIPTION: Colonies are spherical, up to 50 µm in diameter,consisting of approximately 16–36 cells associated by theirposterior ends (Fig. 11). Cells are pyriform, posteriorly elon-gated into the tail, 20–25 µm long and 7–11 µm wide. Eachcell is surrounded by a layer of imbricate siliceous scales(Fig. 12). Body scales are 2.5–3.8 µm long and 1.1–1.9 µmwide, consisting of a basal plate with a centrally raised keelprotruding into an acute tip (Fig. 13). The keel is cylindrical,occasionally slightly widened anteriorly, and ornamented by

larger pores (diameter, 49–100 nm) (Fig. 14). The ratiobetween scale and keel width varies from 1.7 to 3.9. Thebasal plate is ornamented by numerous small pores (diameter,20–31 nm), and anteriorly perforated by a rounded base hole(diameter, 0.19–0.42 µm). Numerous struts (22–28), ofteninterconnected by transverse folds, extend regularly from thekeel to the scale perimeter (Fig. 15). Apical scales are 2.6–3.0µm long and 1.3–1.6 µm wide (Fig. 16). The keel of theapical scales ends in a rounded tip (Fig. 17). Rear scales are1.8–3.2 µm long and 0.6–1.0 µm wide (Figs 18, 19).

ETYMOLOGY: Named for the different sizes of the keel- andbase-plate pores.

TYPE LOCALITY: Crinan Canal, Scotland, UK (56.060461° N,5.481813° W).

HOLOTYPE: Synura heteropora strain S 20.45, frozen materialdeposited in the Culture Collection of Algae of CharlesUniversity in Prague (CAUP). Figure 14 presents an illustra-tion of the holotype.


DISTRIBUTION (Table 1): Austria, Czech Republic, Estonia,Ireland, Norway, Sweden and the UK.

Synura hibernica Škaloud & Škaloudová, sp. nov.

Figs. 3–10. Scale morphology of Synura borealis sp. nov. (Fig. 3: LM; Figs 4, 5: SEM; Figs 6–10: TEM). Scale bars represent 10 µm(Fig. 3) and 1 μm (Figs 4–10). Fig. 3. Colony consisting of elongated, lanceolate-shaped cells (strain S 90.G4). Fig. 4. Single cellsurrounded by a layer of siliceous scales (S 90.G4). Fig. 5. Body scale (S 90.G4). Fig. 6. Body scale with transverse foldsinterconnecting the struts. Note the over-layered pore pattern at the scale keel (S 62.D7). Fig. 7. Body scale with obviously anteriorlywidened keel (S 62.D7). Fig. 8.Apical scale (S 62.D7). Fig. 9.Apical scales with prominently protruding keel tips (S 58.C7). Fig. 10.Rear scale (S 62.D7).


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(Figs 20–28)

DESCRIPTION: Colonies are spherical, up to 94 µm in diameter,consisting of approximately 12–52 cells associated by theirposterior ends (Fig. 20). Cells are significantly elongated,anteriorly cylindrical, posteriorly tapering into the tail, 26–47 µm long and 6–12 µm wide. Each cell is surrounded by alayer of imbricate siliceous scales (Fig. 21). Body scales are3.4–5.6 µm long and 1.2–2.0 µm wide, consisting of a basalplate with a centrally raised keel protruding into an acute tip(Fig. 22). The keel is cylindrical, ornamented by medium-sized pores (diameter, 49–89 nm) (Figs 23, 24). The ratiobetween scale and keel width varies from 1.9 to 3.9. The basalplate is ornamented by numerous small pores (diameter, 18–27 nm), and anteriorly perforated by a rounded base hole(diameter, 0.14–0.38 µm). A large number of struts (30–47),often interconnected by transverse folds, extend regularlyfrom the keel to the scale perimeter (Figs 22, 23). Apicalscales are 2.9–3.3 µm long and 1.6–1.9 µm wide (Fig. 25).The keel of the apical scales ends in a prominent, acute tip(Fig. 26), sometimes terminated by two short teeth. Rearscales are long and narrow, 3.0–4.9 µm long and 0.9–1.0µm wide (Figs 27, 28).

ETYMOLOGY: The specific epithet ‘hibernica’ refers to thecommon occurrence of the species in Ireland.

TYPE LOCALITY: Glanmore Lake, Ireland (51.732724° N,9.767121° W).

HOLOTYPE: Synura hibernica strain S IE 104.D11, frozenmaterial deposited in the Culture Collection of Algae ofCharles University in Prague (CAUP). Figure 22 presentsan illustration of the holotype.


DISTRIBUTION (Table 1): Currently known only in Ireland.

Synura laticarina Škaloud & Škaloudová, sp. nov.

(Figs 29–36)

DESCRIPTION: Colonies are spherical, up to 64 µm in diameter,consisting of approximately 12–28 cells associated by theirposterior ends (Fig. 29). Cells are pyriform, posteriorly elon-gated into the tail, 21–32 µm long and 7–13 µmwide. Each cellis surrounded by a layer of imbricate siliceous scales (Fig. 30).Body scales are 3.1–4.3 µm long and 1.6–2.1 µm wide, con-sisting of a basal plate with a centrally raised keel protrudinginto an acute tip (Fig. 31). The keel is often anteriorly widened,ornamented bymedium-sized pores (diameter, 52–76 nm) (Figs32, 33). Anteriorly, keel pores are produced on both sides, sothat the keel pore pattern is notably over-layered (Fig. 33). Theratio between scale and keel width varies from 1.9 to 2.9. Thebasal plate is ornamented by numerous small pores (diameter,19–25 nm), and anteriorly perforated by a rounded base hole(diameter, 0.20–0.33 µm). Numerous struts (24–32), ofteninterconnected by transverse folds, extend regularly from the

Figs. 11–19. Scale morphology of Synura heteropora sp. nov. (Fig. 11: LM; Figs 12, 13, 16: SEM; Figs 14, 15, 17–19: TEM). Scalebars represent 10 μm (Fig. 11) and 1 μm (Figs 12–19). Fig. 11. Colony consisting of densely grouped, pyriform cells (strain S 20.45).Fig. 12. Single cell surrounded by a layer of siliceous scales (S 54.E11). Fig. 13.Body scale (S 54.E11). Fig. 14.Body scale (S 20.45).Fig. 15. Body scale with transverse folds interconnecting the struts (S 87.C6). Fig. 16. Apical scale (S 54.E11). Fig. 17. Apical scalewith a pronounced, rounded keel tip (S 101.F7). Figs 18, 19. Rear scales (S 101.F7).


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keel to the scale perimeter (Fig. 34). Apical scales are 2.5–3.0µm long and 1.7–2.0 µm wide (Fig. 35). The keel of the apicalscales ends in a prominent, sometimes rounded tip. Rear scalesare 2.7–4.3 µm long and 0.7–1.3 µm wide (Fig. 36).

ETYMOLOGY: The specific epithet is derived from the Latin‘latus’ (broad) and ‘carina’ (keel), referring to the remarkableanterior widening of the keel.

TYPE LOCALITY: Ojala Lake, Finland (61.847778° N,26.291447° E).

HOLOTYPE: Synura laticarina strain S 89.D5, frozen materialdeposited in the Culture Collection of Algae of CharlesUniversity in Prague (CAUP). Figure 33 presents an illustra-tion of the holotype.


DISTRIBUTION (Table 1): Estonia, Finland, Norway andSweden.

Morphological analyses

To investigate morphological differences among the10 recognized, closely related Synura species in

detail, we morphologically characterized each speciesby measuring 30 randomly chosen body scales. Thespecies were obviously heterogeneous in the size ofbody scales (Fig. 37). Synura borealis, S. hibernicaand S. petersenii had significantly longer scales thanthose of the remaining species. In addition, twogroups of species could be recognized by the scalewidth: S. conopea, S. heteropora, S. hibernica andS. truttae had much narrower scales than those of theother species. Obvious morphological differences alsowere observed in the number of struts (Fig. 38), whichrepresented the least variable morphologicalcharacter. Therefore, some species could be clearlyrecognized only by counting the number of struts ina few scales. Heterogeneity also was observed in thesize of all three pores measured. The base-plate porearea was the most discriminating character among thespecies (Fig. 38), with exceptionally large pores pre-sented in S. macropora. All four newly recognizedspecies were characterized by rather small base-platepores. Both the keel pore and the base hole areashowed relatively high size variability, whichrestricted their use as good discriminant features

Figs. 20–28. Scale morphology of Synura hibernica sp. nov. (Fig. 20: LM; Figs 21, 22: SEM; Figs 23–28: TEM). Scale barsrepresent 10 μm (Fig. 20) and 1 μm (Figs 21–28). Fig. 20. Colony consisting of significantly elongated cells (strain S IE 104.D11).Fig. 21. Single cell surrounded by a layer of siliceous scales (S IE 104.D11). Fig. 22. Body scale (S IE 104.D11). Fig. 23. Body scale(S IE 103.C9). Fig. 24. Body scale (S IE E11). Fig. 25.Apical scale with prominently protruding keel tip (S IE E11). Fig. 26. Lateralview on apical scales with protruding keel tips (environmental sample, Lough Anillaun, Co. Galway, Ireland). Fig. 27. Rear scale (SIE M38). Fig. 28. Rear scale (S IE E11).


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(Fig. 39). Synura borealis and S. truttae were wellcharacterized by a notably large base hole. Strongheterogeneity was observed in the base-plate area tokeel area ratio (Fig. 40). The keel pores of S. macro-pora and S. glabrawere approximately comparable insize to the base-plate pores; however, those of S.borealis, S. heteropora, S. hibernica and S. laticarinawere usually distinctively larger. Synura americana,S. hibernica and S. laticarina were well characterizedby an anteriorly widened keel (Fig. 40). The centralmorphological characteristics of these 10 lineages,and other members of the genus Synura, sectionPeterseniae, are presented in Table 2. A key to thesespecies is given in Table S2.

The principal component analysis (PCA) of theentire dataset resulted in a relatively well-definedgrouping of scales belonging to the particular species(Fig. 41). Scales of S. macropora and S. borealisformed two distinct clusters with negative values onthe first PCR axis. By contrast, scales of S. conopeawere mostly intermixed with other species. The cano-nical discriminant analysis (CDA) yielded much bet-ter grouping of scales based on their morphologicaldata (Fig. 42). With the exception of S. conopea andS. americana, all species formed distinct, separateclusters. The discriminant analysis (DA) indicated

strongly significant differentiation among the 10 spe-cies (Wilk’s λ = 0.0008; P < 0.00001). The forwardstepwise analysis indicated that all tested morpholo-gical characters were significant for species recogni-tion (P < 0.00001), and selected the base-plate porearea (Partial Wilk’s λ = 0.20), the keel width (PartialWilk’s λ = 0.41) and the number of struts (PartialWilk’s λ = 0.46) as the three best discriminating char-acters. The discrimination of species was highly sig-nificant even when only these three characters wereanalysed (Wilk’s λ = 0.0058; P < 0.00001).Congruently, whereas the first CDA axis was princi-pally correlated with the base-plate pore area and thenumber of struts (correlation coefficients −0.82 and0.63, respectively), the second CDA axis correlatedwith keel and scale widths (correlation coefficients0.68 and 0.64, respectively). The average correct dis-crimination of individual scales on the basis of theirmorphology reached 92% (Table 3). The lowest cor-rect discrimination levels were recovered in S. con-opea (70.0%) and S. laticarina (80.0%).

Biogeography

A 6-year sampling of Synura isolates performed in 15different countries enabled us to determine their

Figs. 29–36. Scale morphology of Synura laticarina sp. nov. (Fig. 29: LM; Figs 30 and 31: SEM; Figs 32–36: TEM). Scale barsrepresent 10 μm (Fig. 29) and 1 μm (Figs 30–36). Fig. 29. Colony consisting of pyriform cells (strain S 90.C8). Fig. 30. Single cellsurrounded by a layer of siliceous scales (S 90.C8). Fig. 31.Body scale (S 90.C8). Fig. 32.Body scale with anteriorly widened keel (S89.D5). Fig. 33. Body scale. Note over-layered pore pattern at the scale keel (S 89.D5). Fig. 34. Body scale with transverse foldsinterconnecting the struts (S 90.C8). Fig. 35. Apical scale (S 89.D5). Fig. 36. Rear scale (S 89.D5).


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biogeographic distribution across the European conti-nent. Different distribution patterns can be recognizedin four newly described Synura species (Figs 43–46).Synura heteropora is distributed across much ofEurope, whereas the remaining three species are morerestricted in their occurrence. Evolutionarily related S.borealis and S. laticarina exhibit similar biogeographicpatterns, occurring in the northern regions (Figs 43,46). Synura laticarina is regionally more restricted,currently reported only from Estonia, Finland,Norway and Sweden (Fig. 46). The most restricteddistribution pattern was observed in S. hibernica. Thisspecies was found only in the blanket peat bogs locatedalong the western coast of Ireland (Fig. 46).

Discussion

Synura petersenii sensu lato is one of the most widelydistributed and common groups of freshwater micro-organisms. It is relatively easily cultivated and inves-tigated with molecular methods (Wee et al., 2001; Booet al., 2010; Kynčlová et al., 2010; Škaloud et al.,2012, 2013a), so it represents an ideal model taxon forinvestigating evolutionary patterns in protists.Particular species are relatively young in evolutionaryterms, diverging on the order of several million yearsago (Jo et al., 2013). Therefore, it allows us to inves-tigate speciation processes, rates of morphologicaland ecological differentiation of species, and overallspecies diversity within this recently diverging group

Figs. 37–42. Morphological comparisons and statistical analyses of siliceous scales. Figs 37–40. Scatterplots of morphologicalfeatures measured in the 10 investigated species; average values and standard deviations are given. Fig. 37. Scatterplot of scale lengthversus scale width. Fig. 38. Scatterplot of base-plate pore area versus number of struts. Fig. 39. Scatterplot of base hole area versuskeel pore area. Fig. 40. Scatterplot of keel pore to base-plate pore area ratio versus keel width. Fig. 41. Principal component analysis(PCA) of the entire measured morphological features dataset. Fig. 42. Canonical discriminant analysis (CDA) of the same dataset.


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of microorganisms. This study was the next stepnecessary for uncovering the real species diversityand to delimit and characterize particular specieswithin this complex.

Overall species diversity

Biological variation within Synura petersenii is verycomplex. Before information was available aboutgenetic differentiation, S. petersenii was generallyconsidered as a single species, with recognition of afew forms or varieties (Kristiansen & Preisig, 2007).In fact, this taxon contains a number of morphologi-cally similar, yet genetically distinct species. In addi-tion to the four newly proposed species, S. peterseniis.l. now includes 10 well-defined, genetically charac-terized species. Phylogenetic analysis clearly recog-nizes six lineages comprised of several strainsdeposited in various culture collections, which veryprobably represent new, yet undescribed species (Fig.2). Several taxa belonging to the section Peterseniaeare currently uncharacterized with molecular markers,and their phylogenetic position is still unknown. Allthese taxa, including S. australiensis (Playfair, 1915),‘S. petersenii’ f. columnata (Siver, 1988), ‘S. peterse-nii’ f. praefracta (Asmund, 1968), ‘S. petersenii’ f.taymyrensis (Kristiansen et al., 1997), and S. obesa(Němcová et al., 2008), could form additionallineages within S. petersenii s.l. Therefore, it is verydifficult to estimate the total number of species in thistaxon. In an extensive molecular investigation of morethan 100 S. petersenii s.l. strains, Boo et al. (2010)identified a large number of genotypes clustered intoseven groups. The strains originated from differentareas located in four continents. Of the four newspecies proposed in the present study, only S. hetero-pora was identified with a previously recognized gen-otype, the strain CCMP 2898 belonging to clade G IBoo et al. (2010). Obviously, there is still a largedegree of hidden diversity that cannot be fullyresolved without additional molecular and morpholo-gical data. However, even if we can predict that sev-eral tens of currently undiscovered taxa exist, it isprobable that the most frequently occurring speciesare already known and characterized.

Species delineation and morphological evolution

The six species delineated in previous studies(Kynčlová et al., 2010; Škaloud et al., 2012) wereshown to be distinguishable by the siliceous scalemorphology. However, the present study includesdescriptions of four additional, morphologicallyhighly similar taxa. Morphometric analyses of silic-eous scales enabled the significant phenotypic differ-entiation of all species of the S. petersenii complex,including the newly described species. The CDA ana-lysis significantly recognized all 10 investigatedT

able2.

Scalecharacteristicsof

selected

Synu

raspecies(sectio

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ithsimilarscalemorph

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ies.New

lydescribedspeciesaregiveninbo

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term

inolog

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measuredcharacteristics,

seeFig.1

.

Taxo

nCelld

imension

s(µm)

Scaledimension

s(µm)

Num

berof

struts

Rearscales

long

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body

scales?

Keelw

idth

Baseho

lediam

eter

(nm)

Base-platepo

rediam

eter

(nm)

Keelp

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diam

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(nm)

Bod

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thto

width

ratio

Con

nection

ofstruts

S.am

erican

a22

–28×8–

122.8–

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0.6–

0.9

167–

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27–43

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S.cono

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22–2

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189–

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S.glab

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17–2

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0.7

144–

327

29–41

44–100

1.3–

1.9

none

S.macropo

ra17

–25×8–

122.2–

3.4×1.5–2.2

15–2

2yes

0.4–

0.8

156–

391

50–78

69–137

1.3–

2.1

none

S.petersenii

20–31×8–

123.4–

4.7×1.7–2.3

26–3

7no

0.6–

0.8

244–

461

18–30

45–8

21.7–

2.5

frequent

S.truttae

22–31×11–13

3.0–

3.9×1.5–1.9

27–3

4no

0.5–

0.8

270–

557

18–25

47–7

01.8–

2.4

frequent

S.b

orealis

31–42×7–

124.0–

5.8×1.6–2.6

28–3

8no

0.8–

1.3

266–

554

17–26

54–8

81.7–

2.9

frequent

S.h

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20–2

5×7–

112.5–

3.8×1.1–1.9

22–2

8no

0.4–

0.7

187–

415

20–31

49–100

1.8–

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frequent

S.h

ibernica

26–47×6–

123.4–

5.6×1.2–2.0

30–4

7no

0.5–

0.8

138–

378

18–27

49–8

92.2–

3.5

frequent

S.laticarina

21–32×7–

133.1–

4.3×1.6–2.1

24–3

2no

0.6–

1.0

200–

334

19–25

52–7

61.7–

2.4

frequent

‘S.p

etersenii’

f.columna

ta15

–23×8–

112.7–

3.5×1.5–2.2

22–2

7–

2.6–

3.1

––

–1.6–

1.8

none

‘S.p

etersenii’f.praefracta

–3.2–

3.4×1.8–2.0

24–2

7–

3.0–

3.5

––

–1.7–

1.8

frequent

‘S.p

etersenii’f.taym

yrensis

–2.7–

2.8×1.2

23–2

5no

1.7

––

–2.3

none


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species (Fig. 42), and identified S. conopea as thespecies with the lowest correct discrimination level(70%). When analysing only the three best discrimi-nating characters (base-plate pore area, keel width andnumber of struts), the correct discrimination leveldecreased to 57% in S. conopea and S. petersenii,

but remained quite high in the majority of species(Table 3). Because the presented percentages repre-sent post-hoc classifications, we can expect a loweraccuracy when new scales will be classified. However,the CDA analyses clearly indicated that simply mea-suring three morphological features in four body

Table 3. Classification matrix of the canonical discriminant analyses of all seven morphological characters/three best discriminatingcharacters (base-plate pore area, keel width, number of struts). Rows: observed classification. Columns: predicted classification.

Species

Predicted classification

% correctly classified(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

(1) S. americana 27/25 0/0 2/2 1/1 0/1 0/0 0/1 0/0 0/0 0/0 90/83(2) S. borealis 0/0 29/27 0/0 0/0 0/0 0/0 1/3 0/0 0/0 0/0 97/90(3) S. conopea 2/3 0/0 21/17 0/0 4/3 0/0 3/6 0/0 0/1 0/0 70/57(4) S. glabra 0/0 0/0 0/1 29/29 1/0 0/0 0/0 0/0 0/0 0/0 97/97(5) S. heteropora 0/1 0/0 0/0 0/1 29/28 0/0 0/0 0/0 0/0 1/0 97/93(6) S. hibernica 0/0 0/0 0/0 0/0 0/0 30/24 0/0 0/0 0/2 0/4 100/80(7) S. laticarina 1/1 1/0 0/0 0/0 0/0 1/0 24/25 0/0 3/4 0/0 80/83(8) S. macropora 1/1 0/0 0/0 0/0 0/0 0/0 0/0 29/29 0/0 0/0 97/97(9) S. petersenii 0/0 0/0 0/0 0/0 0/0 1/2 1/1 0/0 27/17 1/10 90/57(10) S. truttae 0/0 0/1 0/0 0/0 0/1 0/1 0/1 0/0 0/7 30/19 100/63Total 31/31 30/28 23/20 30/31 34/33 32/27 29/37 29/29 30/31 32/33 92/80

Figs. 43–46. Distribution of newly described Synura species. Light grey hexagons show all studied regions where the occurrence of anySynura species has been recorded. Filled hexagons show the known distribution pattern of the new species. Fig. 43. S. borealis. Fig. 44.S. heteropora. Fig. 45. S. hibernica. Fig. 46. S. laticarina. The hexagon edge length corresponds to 80 km (hexagon area ≈ 16 600 km2).


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scales should be sufficient to correctly classify each ofthe species.

We detected a new lineage sister to S. americana,S. glabra and S. petersenii that we interpreted as twonovel species, the genetically diverse S. borealisand the more molecularly homogeneous S. laticarina(Fig. 2). Synura borealis possessed an exceptionallyhigh degree of genetic diversity, particularly in thecox1 gene, which could be interpreted as resultingfrom several independent species. However, therecognition of just these two species was stronglysupported by our morphological data. The silica scalesof all S. borealis strains shared characteristic morpho-logical features, including the anteriorly widened keelwith notably over-layered pore pattern, and large scaledimensions. Further, S. borealis and S. laticarina spe-cies could be undoubtedly recognized under the lightmicroscope. All investigated S. borealis strains werecharacterized by large colonies consisting of signifi-cantly elongated, lanceolate-shaped cells, whereasS. laticarina produced notably smaller colonies con-taining rounded, pyriform cells. Therefore, we inter-pret the high genetic diversity in S. borealis as anindication of recent speciation and ongoing evolution-ary differentiation. Further investigation of S. borealisgenotypes, including their ecophysiological differen-tiation, ecological preferences and distributional pat-terns could provide important insights into the driversof species diversification and mechanisms of popula-tion-level structuring in Synura species.

Particular species of the S. petersenii groupobviously underwent some degree of morphologicaldifferentiation, although they are relatively young inevolutionary terms. Some species were more signifi-cantly differentiated with respect to siliceous scalesthan others, and can be recognized at first glance. Forexample, S. macropora can be unambiguously identi-fied by its extremely large base-plate pores (Fig. 38).We can only speculate about the causes of this rapidmorphological evolution. Large pores could signifi-cantly decrease the density of the cell, and hencereduce the sinking rate. However, cells with largerpores could be more susceptible to viral infection(Losic et al., 2006). Synura macropora seems tooccur only in eutrophic habitats characterized byhigh conductivity values (Table 1; Škaloud et al.,2013b). It was shown that eutrophic conditionsdecreased the availability of silica, which often causeda shift from heavily silicified to less silicified diatomsin freshwater biotopes (Rabalais et al., 1996).Similarly, adaptation of S. macropora to eutrophicconditions could lead to the formation of less silicifiedscales characterized by large base-plate pores.

Synura hibernica represents another morphologi-cally well-defined species characterized by very nar-row, long siliceous scales bearing a large number ofstruts (Figs 37, 38). This species can even be distin-guished under the light microscope by the

characteristically large colonies consisting of stronglyelongated, narrow cells. The significantly differentmorphology of this Irish taxon has been discussedpreviously by Řezáčová & Škaloud (2005), whoemphasized its striking similarity with the tropicalspecies S. australiensis. Obviously, the characteristi-cally elongated shape of the siliceous scales is relatedto the elongated cell shape in both species. Synuraaustraliensis and S. hibernica represent another exam-ple of rapid morphological evolution within theS. petersenii group, leading to the speciation of organ-isms producing big colonies consisting of long andnarrow cells. Unfortunately, in the absence of mole-cular data for S. australiensis, it remains unclearwhether these two taxa are evolutionarily related toeach other. Significant morphological transformationmay have been caused by an ecological niche-shiftduring the speciation of both taxa, similarly to that ofS. macropora. In protists, a negative correlationbetween cell size and temperature has been proposedby Atkinson et al. (2003). However, S. hibernica iscurrently known only from Ireland, whereas the dis-tribution of S. australiensis is restricted to tropical andsubtropical regions (Kristiansen & Preisig, 2007).Therefore, we can rule out temperature-related mor-phological speciation of these two taxa.

The development of elongated cells in S. austra-liensis and S. hibernica could be explained by adapta-tion to oligotrophic conditions. It is known that mostoligotrophs achieve a high surface-to-volume ratio,which increases the organisms’ capacity to scavengeavailable nutrients (Reynolds, 2006). Elongation ofSynura cells would be the most effective way to sig-nificantly increase this ratio. All 12 isolated strains ofS. hibernica originated from nutrient-poor localities,with conductivity values ranging between 23–87 µScm−1 (Table 1). To our knowledge, all records ofS. australiensis have been associated with clear, oli-gotrophic water bodies (Croome & Tyler, 1985;Cronberg, 1989, 1996; Saha & Wujek, 1990; Weeet al., 1993; Hansen, 1996). The measured conductiv-ity of these water samples was 10–123 µS cm−1.

Biogeographic patterns

The biogeography of protists has become a highlycontroversial topic over the last 10 years (Martinyet al., 2006; Caron, 2009). It has been postulated thatthe small size, extremely large populations and highdispersal potential of protists result in the cosmopoli-tan distribution of the vast majority of species (Finlay,2002; Finlay & Fenchel, 2004). Conversely, limitedgeographical distributions have been implied byFoissner (1999), primarily based on the observedrestricted distribution of flagship species with easilyrecognizable morphologies and easily demonstrablepresence/absence (Foissner, 2006, 2008). Synurapetersenii s.l. represents a common group of


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freshwater microorganisms, known to occur in allcontinents except Antarctica (Kristiansen, 2008).Because its molecular diversity has been investigatedin three continents (Wee et al., 2001; Boo et al., 2010;Škaloud et al., 2012), it represents an ideal taxon tostudy the distribution patterns in protists.

The first evaluation of biogeographic patterns in theS. petersenii group was published by Boo et al.(2010), who described the presence of restricted bio-geographic distributions for many species. However,Škaloud et al. (2012) compared the genetic data withpublished morphological observations, and reportedmuch broader distributions for some of the investi-gated taxa. For example, putative North Americanendemic S. americana, although dominantly occur-ring in the USA and Canada, was also reported inSouth America and Europe (Cronberg, 1989;Škaloud et al., 2012). However, the restricted distri-bution of several lineages is evident (e.g. clades D andF sensu Boo et al. 2010). Our results support therestricted distribution of Synura species, includingthe strict regional endemism observed in S. hibernica.To the best of our knowledge, all four newly proposedspecies have been reported only in Europe andGreenland (Figs 43–46). A comparison of the mor-phological characteristics with previously publishedreports reveals that S. borealis and S. hibernica havebeen observed previously in Greenland and Ireland,respectively (Kristiansen, 1992;Řezáčová & Škaloud,2005); however, we found no other reports outsideEurope.

Three of four newly described species exhibitrestricted biogeographic distribution. Evolutionarilyrelated S. borealis and S. laticarina seem to be adaptedto colder areas, and their distributions appear to belimited by high summer temperatures (Figs 43, 46).The occurrence of S. borealis in Greenland indicatesno obvious dispersal limitations, so we expect itscircumboreal distribution. The most remarkable dis-tribution pattern, however, is that of S. hibernica. Thisspecies is restricted in its geographic distribution towestern Ireland (Fig. 45). Althoughwe found S. hiber-nica in nine localities located along the western coastof Ireland (Table 1), it was never detected in any of theother sampled localities. To check this highly unusualdistribution pattern, we undertook two expeditions in2008 and 2012 to investigate the diversity of Synuraspecies in neighbouring Scotland. Ecological and lim-nological conditions are very similar in westernIreland and Scotland: both areas are characterized bythe occurrence of extensive peat deposits restricted toa cool oceanic climate, known as blanket bogs(Moore, 1982). Moreover, the British Isles lie at theintersection of major bird migration corridors, andseveral bird species frequently pass through one partof the Isles to reach another (Newton, 2010).Therefore, the algal flora of the western British lake-lands, including western Ireland and northwestern

Scotland, are highly similar. The significant similarityof western British desmid flora was reported pre-viously (West & West, 1909). Although we sampleda total of 71 localities in northwestern Scotland, wedid not discover a single colony of S. hibernica. Thisdistribution pattern, which is restricted to an extre-mely small biogeographic area, is remarkable andentirely outstanding. Future work should study themechanisms underlying the obvious dispersal limita-tion of S. hibernica, including ecological and habitatrequirements, desiccation tolerance and mechanismsof cyst formation.

Acknowledgements

The authors would like to thank Fabio Rindi andMichael D. Guiry (Ryan Institute, National Universityof Ireland, Galway); Thomas Pröschold and John G. Day(Culture Collection of Algae and Protozoa, ScottishMarine Institute, Oban, UK); David John (NaturalHistory Museum, London, UK); Ole Stecher andFrantz Nielsen (Arctic Station, University ofCopenhagen, Greenland); Eugen Rott (Institute ofBotany, University of Innsbruck, Austria); HelenaBestová, Lucie Jelínková, Katarína Nemjová, PavlaSlámová, Pavel Svoboda and Vojtěch Scharfen(Department of Botany, Charles University in Prague,Czech Republic) for their kind support during sampling.This work was supported by the Czech ScienceFoundation [grant number P506/11/P056].

Supplementary information

The following supplementary material is accessible viathe Supplementary Content tab on the article’s onlinepage at http://dx.doi.org/10.1080/09670262.2014.905710

Table S1. Strain names, geographic origins, and thecorresponding ITS rDNA, rbcL, and cox1 GenBankaccession numbers for the taxa used in the phylogeneticanalyses. Newly obtained sequences are given in bold.

Table S2. Key to species of the genus Synura, sectionPeterseniae.

Alignment S1. The final, partitioned alignment ofconcatenated ITS rDNA, rbcL, and cox1 sequencesused for phylogenetic reconstructions.

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Key to species of the genus Synura, section Peterseniae

1. Base plate covered by struts extending from the keel to the scale perimeter ..................... 2

Struts missing, base plate with fine regular reticulation ............................... S. longisquama

2. Transverse struts connected in a dense net.......................................................................... 3

Transverse struts unconnected, or interconnected by single transverse struts .................... 4

3. Keel narrow, with a large base plate hole .................................................... S. macracantha

Keel extremely broad, with a very small base plate hole ......................................... S. obesa

4. Scales long and narrow (scale length to width ratio higher than 3.6) ........... S. australiensis

Scale length to width ratio less than 3.5 .............................................................................. 5

5. Scales with a very broad rim ............................................................................................... 6

Rim of scales narrower ........................................................................................................ 7

6. Struts of different length, irregularly spacered ................................................ S. asmundiae

Struts of similar length, regularly spacered ........................................................... S. bjoerkii

7. Inner portion of the rim ornamented by row of posts (in SEM) or dots (in TEM) ...............

.................................................................................................. “S. petersenii” f. columnata

Rim not ornamented ............................................................................................................ 8

8. Keel ornamented by a hexagonal pattern ............................... “S. petersenii” f. taymyrensis

Keel not ornamented ........................................................................................................... 9

9. Keel of apical scales terminates into a rounded tip, provided with a number of small teeth10

Keel of apical scales terminates into an acute tip ............................................................. 11

10. Body scales narrower, with 27-34 thickened and conspicuous struts ..................... S. truttae

Body scales broader, with 24-27 inconspicuous struts ............ “S. petersenii” f. praefracta

11. Body scales rounded to oval, struts never or very rarely connected, large base-plate pores

(diameter 25-78 nm) .......................................................................................................... 12

Body scales lanceolate, struts frequently connected by transverse ribs, small base-plate

pores (diameter 17-31 nm) ................................................................................................ 15

12. Rear scales longer than body scales .................................................................................. 13

Rear scales short ................................................................................................................ 14

13. Base-plate pores very large (diameter 50-78 nm) ............................................ S. macropora

Base-plate pores smaller (diameter 27-43 nm) ................................................ S. americana

14. Keel and ribs less developed, scales often almost rounded .................................... S. glabra

Keel and ribs well developed, scales oval ............................................................ S. conopea

15. Keel anteriorly greatly widened, with the over-layered pore pattern (in TEM) ............... 16

Keel more or less cylindrical, ornamented by a single layer of pores .............................. 17

16. Body scales short (length 3.1-4.3 m), cells shorter than 32 m ......................S. laticarina

Body scales long (length 4.0-5.8 m), cells longer than 32 m .......................... S. borealis

17. Keel anteriorly slightly widened, scale width up to 1.8(-2)m........................................ 18

Keel cylindrical, scale width greater than (1.7-)1.9m ..................................... S. petersenii

18. Body scales short (length 2.5-3.8 m), with 22-28 struts ................................ S. heteropora

Body scales long (length 3.4-5.6 m), with 30-47 struts ................................... S. hibernica

ITS

rbc

Lc

ox

1

Syn

ura

am

eric

an

a K

yn

člo

vá

& Š

ka

lou

dC

CM

P 8

62

Win

ter’s

Cre

ek, M

I, US

AG

U3

38

12

4G

U3

25

48

5G

U2

95

52

9

Syn

ura

am

eric

an

a K

yn

člo

vá

& Š

ka

lou

dC

CM

P 8

63

roa

d d

itch

ne

ar W

inte

r’s C

ree

k, M

I, US

AG

U3

38

12

5G

U3

25

48

6G

U2

95

53

0

Syn

ura

am

eric

an

a K

yn

člo

vá

& Š

ka

lou

dC

CM

P 8

66

New

fou

nd

lan

d, C

an

ad

aA

F3

08

84

0-

-

Syn

ura

am

eric

an

a K

yn

člo

vá

& Š

ka

lou

dU

TE

X L

B 2

40

4S

po

rtsm

an

’s L

ake

, WA

, US

AG

U3

38

13

2G

U3

25

49

5G

U2

95

53

8

Syn

ura

am

eric

an

a K

yn

člo

vá

& Š

ka

lou

dS

81

.C7

Zá

pla

vy N

R, C

ze

ch

Re

pu

blic

HG

51

41

66

--

Syn

ura

am

eric

an

a K

yn

člo

vá

& Š

ka

lou

dS

10

4.C

4S

ch

inke

lbo

s, N

eth

erla

nd

sH

G5

14

16

7-

-

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 5

8.C

7L

illesjö

n, S

we

de

nH

G5

14

16

8H

G5

14

23

4H

G5

14

25

5

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 9

0.F

3R

uta

järv

i, Harju

lah

ti, Fin

lan

dH

G5

14

16

9-

-

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 9

0.G4

Harju

järv

i, Fin

lan

dH

G5

14

17

0-

-

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

10

.B9

Ap

na

jõg

i, Esto

nia

HG

51

41

71

--

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

10

.E2

a s

ma

ll po

ol n

ea

r Ka

lli jõg

i, Esto

nia

HG

51

41

72

--

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

16

.B6

Kje

rring

da

lsva

tne

t, No

rwa

yH

G5

14

17

3-

-

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

SC

FIN

13

AO

jala

, Fin

lan

dH

G5

14

17

4-

-

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

SC

SW

E 9

Helg

assjö

n, S

we

de

nH

G5

14

17

5-

-

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 6

2.D

7a

sm

all la

ke

, Dis

ko

isla

nd

, Gre

en

lan

dH

G5

14

17

6H

G5

14

23

5H

G5

14

25

6

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

SC

GR

L 3

64

a m

orra

ine

lake

, Dis

ko

isla

nd

, Gre

en

lan

dH

G5

14

17

7-

-

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 9

0.M

34

Jä

msä

nja

rvi, F

inla

nd

HG

51

41

78

HG

51

42

36

HG

51

42

57

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

10

.F3

a s

ma

ll po

ol n

ea

r Ka

lli jõg

i, Esto

nia

HG

51

41

79

--

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

13

.F6

Em

ajõ

gi riv

er, E

sto

nia

HG

51

41

80

--

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

15

.G2

a p

oo

l ne

ar V

assija

ure

, Sw

ed

en

HG

51

41

81

--

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

18

.C6

a p

oo

l ne

ar T

orn

eträ

sk, S

we

de

nH

G5

14

18

2-

-

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

13

.F4

Em

ajõ

gi riv

er, E

sto

nia

HG

51

41

83

--

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

14

.B8

un

na

me

d la

ke

, Sw

ed

en

HG

51

41

84

HG

51

42

37

HG

51

42

58

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

14

.C8

un

na

me

d la

ke

, Sw

ed

en

HG

51

41

85

HG

51

42

38

HG

51

42

59

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

14

.B9

un

na

me

d la

ke

, Sw

ed

en

HG

51

41

86

--

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

14

.F3

un

na

me

d la

ke

ne

ar P

aitta

sjä

rvi, S

we

de

nH

G5

14

18

7-

-

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

17

.D3

a p

oo

l ne

ar P

aitta

sjä

rvi, S

we

de

nH

G5

14

18

8H

G5

14

23

9H

G5

14

26

0

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

SC

GR

L 3

40

Sa

nn

ing

assu

p T

asia

, Gre

en

lan

dH

G5

14

18

9-

-

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

14

.G6

Syvä

järv

i, Sw

ed

en

HG

51

41

90

HG

51

42

40

HG

51

42

61

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

15

.F4

a p

oo

l ne

ar A

rosn

jark

aja

ute

, Sw

ed

en

HG

51

41

91

HG

51

42

41

HG

51

42

62

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

15

.G3

a p

oo

l ne

ar V

assija

ure

, Sw

ed

en

HG

51

41

92

--

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

S 1

15

.G7

Va

ssija

ure

, Sw

ed

en

HG

51

41

93

HG

51

42

42

HG

51

42

63

Sy

nu

ra a

me

rica

na

Ta

xo

nS

train

nu

mb

er

Orig

inG

en

Ba

nk

ac

ce

ss

ion

Sy

nu

ra b

ore

alis

Syn

ura

bo

rea

lis Š

ka

lou

d &

Ška

lou

do

vá

SC

FIN

22

BH

ein

äjä

rvi, F

inla

nd

HG

51

41

94

--

Syn

ura

co

no

pe

a K

yn

člo

vá

& Š

ka

lou

dC

CM

P 8

59

so

uth

ea

st b

ridg

e p

on

d, A

R, U

SA

GU

33

81

21

GU

32

54

82

GU

29

55

26

Syn

ura

co

no

pe

a K

yn

člo

vá

& Š

ka

lou

dS

10

.2H

uťs

ký p

on

d, C

ze

ch

Re

pu

blic

FM

17

85

07

--

Syn

ura

co

no

pe

a K

yn

člo

vá

& Š

ka

lou

dN

IES

10

07

To

ma

ko

ma

i, Ja

pa

nG

U3

38

11

9G

U3

25

47

9G

U2

95

52

4

Syn

ura

co

no

pe

a K

yn

člo

vá

& Š

ka

lou

dK

NU

13

Dals

eo

ng

gyo

, Ko

rea

GU

33

80

69

GU

32

54

30

GU

29

54

72

Syn

ura

co

no

pe

a K

yn

člo

vá

& Š

ka

lou

dC

AU

P B

70

7 (S

7.1

0)

Ba

bín

po

ol, C

ze

ch

Re

pu

blic

FM

17

85

06

--

Syn

ura

co

no

pe

a K

yn

člo

vá

& Š

ka

lou

dS

10

3.B

3a

po

ol n

ea

r Sm

ěd

ava

, Cze

ch

Re

pu

blic

HG

51

41

95

--

Syn

ura

gla

bra

Ko

rsh

iko

vN

IES

23

3H

iga

sh

iya

ta R

ive

r, Ja

pa

nG

U3

38

11

8G

U3

25

48

0G

U2

95

52

3

Syn

ura

gla

bra

Ko

rsh

iko

vC

NU

09

Jio

kji p

on

d, K

ore

aG

U3

38

07

7G

U3

25

43

8G

U2

95

48

0

Syn

ura

gla

bra

Ko

rsh

iko

vK

NU

10

Bo

mu

n re

se

rvo

ir, Ko

rea

GU

33

80

66

GU

32

54

27

GU

29

54

69

Syn

ura

gla

bra

Ko

rsh

iko

vS

9.2

co

nflu

en

ce

of th

e M

ora

va

an

d D

yje

rive

rs, C

ze

ch

Re

pu

blic

FM

17

85

13

--

Syn

ura

gla

bra

Ko

rsh

iko

vS

14

.1S

wa

mp

NR

, Cze

ch

Re

pu

blic

FM

17

85

14

--

Syn

ura

gla

bra

Ko

rsh

iko

vS

89

.F5

Sa

arijä

rvi, F

inla

nd

HG

51

41

96

--

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

CC

MP

28

98

Wa

llers

ee

, Au

stria

GU

33

81

36

GU

32

54

98

-

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

S 2

0.1

Lo

ch

an

Ad

d, S

co

tlan

d, U

KH

G5

14

19

7-

-

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

S 2

0.4

5C

rina

n C

an

al, S

co

tlan

d, U

KH

G5

14

19

8H

G5

14

24

3-

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

S 4

0.F

11

Po

dh

rad

ská

po

ol, C

ze

ch

Re

pu

blic

HG

51

41

99

--

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

S 5

4.E

11

Kle

jna

rka

rive

r, Cze

ch

Re

pu

blic

H

G5

14

20

0-

-

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

S 8

6.F

2Z

býšo

v p

on

d, C

ze

ch

Re

pu

blic

HG

51

42

01

--

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

S 1

12

.E2

Sa

ard

e p

ais

järv

, Esto

nia

HG

51

42

02

--

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

S 1

12

.F5

Rah

um

eri, E

sto

nia

HG

51

42

03

--

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

S 1

13

.C8

Ka

rula

Ja

rv, E

sto

nia

HG

51

42

04

--

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

S 1

17

.G6

To

rne

träsk, S

we

de

nH

G5

14

20

5-

-

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

SC

IRL

8G

arla

n L

ou

gh

, Irela

nd

HG

51

42

06

--

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

S 8

7.C

6a

n e

ph

em

era

l pu

dd

le n

ea

r Ku

fste

in, A

ustria

HG

51

42

07

HG

51

42

44

HG

51

42

64

Syn

ura

he

tero

po

ra Š

ka

lou

d, Š

ka

lou

do

vá

& K

yn

člo

vá

S 1

01

.F7

a p

on

d in

Nyg

ård

sp

ark

en

, Be

rge

n, N

orw

ay

HG

51

42

08

HG

51

42

45

HG

51

42

65

Syn

ura

hib

ern

ica

Ška

lou

d &

Ška

lou

do

vá

S IE

E4

Th

e L

on

g R

an

ge

, Irela

nd

HG

51

42

09

HG

51

42

46

HG

51

42

66

Syn

ura

hib

ern

ica

Ška

lou

d &

Ška

lou

do

vá

S IE

E1

1G

len

do

llag

h L

ou

gh

, Irela

nd

HG

51

42

10

--

Syn

ura

hib

ern

ica

Ška

lou

d &

Ška

lou

do

vá

S IE

10

3.C

11

Go

wla

un

Lo

ug

h, Ire

lan

dH

G5

14

21

1-

-

Syn

ura

hib

ern

ica

Ška

lou

d &

Ška

lou

do

vá

SC

IRL

41

Ea

sky L

ou

gh

, Irela

nd

HG

51

42

12

--

Syn

ura

hib

ern

ica

Ška

lou

d &

Ška

lou

do

vá

S IE

E8

un

na

me

d la

ke

ne

ar M

aa

m C

ross, Ire

lan

dH

G5

14

21

3H

G5

14

24

7H

G5

14

26

7

Syn

ura

hib

ern

ica

Ška

lou

d &

Ška

lou

do

vá

S IE

10

3.C

8G

ow

lau

n L

ou

gh

, Irela

nd

HG

51

42

14

--

Syn

ura

hib

ern

ica

Ška

lou

d &

Ška

lou

do

vá

S IE

10

3.C

9G

ow

lau

n L

ou

gh

, Irela

nd

HG

51

42

15

--

Syn

ura

hib

ern

ica

Ška

lou

d &

Ška

lou

do

vá

S IE

10

4.D

11

Gla

nm

ore

La

ke

, Irela

nd

HG

51

42

16

HG

51

42

48

HG

51

42

68

Sy

nu

ra g

lab

ra

Sy

nu

ra c

on

op

ea

Sy

nu

ra h

ibe

rnic

a

Sy

nu

ra h

ete

rop

ora

Syn

ura

hib

ern

ica

Ška

lou

d &

Ška

lou

do

vá

S IE

M3

8T

he

Lo

ng

Ra

ng

e, Ire

lan

dH

G5

14

21

7-

-

Syn

ura

hib

ern

ica

Ška

lou

d &

Ška

lou

do

vá

SC

IRL

20

aD

erry

na

he

rriva

Lo

ug

h, Ire

lan

dH

G5

14

21

8-

-

Syn

ura

hib

ern

ica

Ška

lou

d &

Ška

lou

do

vá

S IE

10

5.F

6C

ah

a L

ake

s, Ire

lan

dH

G5

14

21

9H

G5

14

24

9H

G5

14

26

9

Syn

ura

hib

ern

ica

Ška

lou

d &

Ška

lou

do

vá

SC

IRL

60

Ma

um

we

e L

ou

gh

, Irela

nd

HG

51

42

20

--

Syn

ura

latic

arin

a Š

ka

lou

d &

Ška

lou

do

vá

S 8

9.D

5O

jala

, Fin

lan

dH

G5

14

22

1-

-

Syn

ura

latic

arin

a Š

ka

lou

d &

Ška

lou

do

vá

S 9

0.C

8T

eh

rise

lkä

, Fin

lan

dH

G5

14

22

2H

G5

14

25

0H

G5

14

27

0

Syn

ura

latic

arin

a Š

ka

lou

d &

Ška

lou

do

vá

S 1

10

.C9

Ap

na

jõg

i, Esto

nia

HG

51

42

23

--

Syn

ura

latic

arin

a Š

ka

lou

d &

Ška

lou

do

vá

S 1

13

.E5

a p

oo

l ne

ar E

ma

jõg

i, Esto

nia

HG

51

42

24

--

Syn

ura

latic

arin

a Š

ka

lou

d &

Ška

lou

do

vá

S 1

15

.B2

Ba

rdu

elv

a riv

er, N

orw

ay

HG

51

42

25

--

Syn

ura

latic

arin

a Š

ka

lou

d &

Ška

lou

do

vá

S 1

15

.D2

a p

oo

l ne

ar A

rosn

jark

aja

ute

, Sw

ed

en

HG

51

42

26

HG

51

42

51

HG

51

42

71

Syn

ura

latic

arin

a Š

ka

lou

d &

Ška

lou

do

vá

S 1

15

.E5

a p

oo

l ne

ar V

assija

ure

, Sw

ed

en

HG

51

42

27

--

Syn

ura

ma

cro

po

ra Š

ka

lou

d &

Kyn

člo

vá

S 5

.3a

lluvia

l po

ol, P

rag

ue

, Cze

ch

Re

pu

blic

FM

17

84

96

--

Syn

ura

ma

cro

po

ra Š

ka

lou

d &

Kyn

člo

vá

S 1

4.2

Sw

am

p N

R, C

ze

ch

Re

pu

blic

FM

17

84

97

--

Syn

ura

ma

cro

po

ra Š

ka

lou

d &

Kyn

člo

vá

S 7

1.B

4P

od

hra

dská

po

ol, C

ze

ch

Re

pu

blic

HG

51

42

28

HG

51

42

52

HG

51

42

72

Syn

ura

ma

cro

po

ra Š

ka

lou

d &

Kyn

člo

vá

S 1

04

.D7

a c

an

al in

Ooste

ind

e, N

eth

erla

nd

sH

G5

14

22

9-

-

Syn

ura

ma

cro

po

ra Š

ka

lou

d &

Kyn

člo

vá

S 1

04

.F1

1a

po

ol in

Aa

lsm

ee

r, Neth

erla

nd

sH

G5

14

23

0-

-

Syn

ura

pe

ters

en

ii Ko

rsh

iko

vA

CO

I 17

07

Ab

ran

tes, P

ortu

ga

lG

U3

38

15

0G

U3

25

51

2G

U2

95

55

2

Syn

ura

pe

ters

en

ii Ko

rsh

iko

vC

CA

P 9

60

/3P

riest P

ot, E

ng

lan

d, U

KG

U3

38

14

3G

U3

25

50

5G

U2

95

54

5

Syn

ura

pe

ters

en

ii Ko

rsh

iko

vS

6.4

Horn

i Lu

zn

ice

NR

, Cze

ch

Re

pu

blic

FM

17

85

01

--

Syn

ura

pe

ters

en

ii Ko

rsh

iko

vC

CM

P 8

60

Ced

ar L

ake

, IL, U

SA

GU

33

81

22

GU

32

54

83

GU

29

55

27

Syn

ura

pe

ters

en

ii Ko

rsh

iko

vS

AG

12

0.7

9L

ün

eb

urg

er H

eid

e, M

ea

do

w p

oo

l, Ge

rma

ny

GU

33

81

44

GU

32

55

06

GU

29

55

46

Syn

ura

pe

ters

en

ii Ko

rsh

iko

vC

AU

P B

70

3 (S

7.7

)B

ab

ín p

on

d, C

ze

ch

Re

pu

blic

FM

17

85

04

--

Syn

ura

pe

ters

en

ii Ko

rsh

iko

vS

89

.D6

Oja

la, F

inla

nd

HG

51

42

31

--

Syn

ura

pe

ters

en

ii Ko

rsh

iko

vS

89

.F9

Hein

äjä

rvi, F

inla

nd

HG

51

42

32

--

Syn

ura

trutta

e (S

ive

r) Ška

lou

d &

Kyn

člo

vá

Nem

co

va

D5

Ka

ch

ní p

oto

k, C

ze

ch

Re

pu

blic

GU

33

81

40

GU

32

55

02

GU

29

55

42

Syn

ura

trutta

e (S

ive

r) Ška

lou

d &

Kyn

člo

vá

S 1

5.3

Úpské

raše

liniš

tě, C

ze

ch

Re

pu

blic

FM

17

85

08

--

Syn

ura

trutta

e (S

ive

r) Ška

lou

d &

Kyn

člo

vá

S 1

5.9

Úpské

raše

liniš

tě, C

ze

ch

Re

pu

blic

FM

17

85

10

--

Syn

ura

trutta

e (S

ive

r) Ška

lou

d &

Kyn

člo

vá

CA

UP

B7

05

(S 3

4.1

)fo

rest p

oo

l ne

ar M

ezn

í lou

ka

, Cze

ch

Re

pu

blic

FR

81

97

49

HG

51

42

53

-

Syn

ura

trutta

e (S

ive

r) Ška

lou

d &

Kyn

člo

vá

S 6

2.B

5a

pe

at b

og

ne

ar P

řeb

uz, C

ze

ch

Re

pu

blic

HG

51

42

33

HG

51

42

54

-

Syn

ura

sp

.C

NU

01

Sa

mh

wa

po

nd

, Ko

rea

GU

33

80

70

GU

32

54

31

GU

29

54

73

Syn

ura

sp

.C

NU

10

Bih

aksa

n, K

ore

aG

U3

38

07

8G

U3

25

43

9G

U2

95

48

1

Syn

ura

sp

.C

NU

12

Ge

om

uye

okri, K

ore

aG

U3

38

08

0G

U3

25

44

1G

U2

95

48

3

cla

de

D

cla

de

F

Sy

nu

ra p

ete

rse

nii

Sy

nu

ra la

tica

rina

Sy

nu

ra m

ac

rop

ora

Sy

nu

ra tru

ttae

Syn

ura

sp

.C

NU

47

Se

on

gd

on

g p

on

d, K

ore

aG

U3

38

11

4G

U3

25

47

5G

U2

95

51

8

Syn

ura

sp

.K

NU

01

Uksu

rese

rvo

ir, Ko

rea

GU

33

80

57

GU

32

54

18

GU

29

54

60

Syn

ura

sp

.U

TE

X L

B 2

39

un

kn

ow

nG

U3

38

13

5G

U3

25

49

3G

U2

95

53

6

Syn

ura

sp

.C

CA

C 0

05

2C

olo

gn

e, G

erm

an

yG

U3

38

14

6G

U3

25

50

8G

U2

95

54

8

Syn

ura

sp

.C

CM

P 8

69

To

ba

cco

Riv

er, M

I, US

AG

U3

38

12

8G

U3

25

48

9G

U2

95

53

3

oth

ers

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