Open Access

Molecular taxonomy of Dunaliella (Chlorophyceae), with a special focus on D. salina: ITS2 sequences revisited with an extensive geographical sampling

  • Patrícia Assunção1Email author,
  • Ruth Jaén-Molina2,
  • Juli Caujapé-Castells2,
  • Adelina de la Jara1,
  • Laura Carmona1,
  • Karen Freijanes1 and
  • Héctor Mendoza1Email author
Aquatic Biosystems20128:2

DOI: 10.1186/2046-9063-8-2

Received: 20 October 2011

Accepted: 30 January 2012

Published: 30 January 2012

Abstract

We used an ITS2 primary and secondary structure and Compensatory Base Changes (CBCs) analyses on new French and Spanish Dunallela salina strains to investigate their phylogenetic position and taxonomic status within the genus Dunaliella. Our analyses show a great diversity within D. salina (with only some clades not statistically supported) and reveal considerable genetic diversity and structure within Dunaliella, although the CBC analysis did not bolster the existence of different biological groups within this taxon. The ITS2 sequences of the new Spanish and French D. salina strains were very similar except for two of them: ITC5105 "Janubio" from Spain and ITC5119 from France. Although the Spanish one had a unique ITS2 sequence profile and the phylogenetic tree indicates that this strain can represent a new species, this hypothesis was not confirmed by CBCs, and clarification of its taxonomic status requires further investigation with new data. Overall, the use of CBCs to define species boundaries within Dunaliella was not conclusive in some cases, and the ITS2 region does not contain a geographical signal overall.

Keywords

Canary Islands Compensatory Base Changes Dunaliella salina Internal Transcribed Spacer Saltworks Taxonomy

Background

The Internal Transcribed Spacer 2 (ITS2) of the nuclear rDNA cistron is one of the most frequently used regions for phylogenetic analysis in algae [13]. Although its application in deep taxonomic levels was initially limited to comparisons of genera within the same family owing to uncertainties in alignment at higher taxonomic levels, the analysis of its secondary structure has provided key solutions to this problem [4]. Thus, the use of an ITS2 secondary structure improves sequence alignments, resulting in a higher robustness and accuracy of phylogenetic reconstructions [5] and providing help to distinguish species [6]. Furthermore, an automatic approach to analysis is possible [7], as a pipeline consisting of the ITS2 Database (annotation/structure prediction), 4SALE (alignment), ProfDistS (inferring phylogenies) and the CBCAnalyzer (distinguishing species) have recently become available (http://​its2.​bioapps.​biozentrum.​uni-wuerzburg.​de/​?​about).

In Dunaliella (Chlorophyceae), the use of ITS2 secondary structure for phylogenetic analysis has a long tradition [813]. The genus Dunaliella comprises twenty-eight recognized species separated in two subgenera, Pascheria (which contains the freshwater species), and Dunaliella (grouping the marine species); the latter is further subdivided into four sections: Tertiolecta, Dunaliella, Viridis and Peirceinae [11]. The species ascribed to these four sections occur in a wide range of marine habitats such as oceans, brine lakes, salt marshes, salt lagoons and salt water dishes near the sea [14], being Dunaliella salina Teodoresco (section Dunaliella) the most studied one. Dunaliella salina, is the most halotolerant eukaryotic photosynthetic organism known to date [14, 15] since it shows a remarkable degree of adaptation to a variety of salt concentrations and it accumulates large amounts of carotenes under extremely stressful conditions such as high salinity, low nitrogen levels, and high solar radiation [14]. Nowadays, it is the best commercial source of natural β-carotene [14, 16], and it also stands out as a source of glycerol [17].

One of the aspects of D. salina that have most intrigued researchers is the enormous variability within strains regarding its geographic, physiological, and morphological characteristics [1824]. Recent phylogenetic analyses of ITS1+ITS2 combined with the analysis of the ITS2 secondary structure of D. salina strains have also revealed a high intraspecific variation [811, 25].

The high genetic diversity detected in D. salina, its morphological plasticity, and the restricted geographical sampling used in all scientific publications to date have hindered taxonomic elucidation in this taxon. Our objectives are (1) to use the ITS2 sequences and secondary structure analysis in a thorough geographic and taxonomic representation of the genus Dunaliella and particularly D. salina (including new Spanish and French strains) to improve understanding of the complex phylogenetic structure in this taxon, (2) to study the relationship of the new strains with the Dunaliella sequences available at the ITS2 Database and/or at GenBank [26]; (3) to investigate if the Compensatory Base Changes (CBCs) analyses could elucidate the species concept in Dunaliella, and reveal potentially new species; and (4) to test if D. salina ITC5105 "Janubio" could be considered a new species.

Results

The ITS2 primary and secondary structure phylogenetic analysis of all the Dunaliella sequences available in the ITS2 Database plus the new sequences revealed great heterogeneity, although some of the clades were not statistically supported (Figure 1). No phylogenetic relationship is supported between the two Dunaliella freshwater species, since D. lateralis was positioned outside the Dunaliella subgenus, while D. acidophila was positioned within this subgenus.
https://static-content.springer.com/image/art%3A10.1186%2F2046-9063-8-2/MediaObjects/12999_2011_Article_2_Fig1_HTML.jpg
Figure 1

Sequence-structure tree and consensus structure of ITS2. (A) Sequence-structure Neighbor-Joining tree obtained by ProfDistS and supporting bootstrap values (100 replicates). Strains on the right written within squares have identical ITS2 sequences. Strains written in bold were sequenced in this study. All strains information can be found in Supplementary Table 1 (B) Consensus structure (51%) for all ITS2 sequences obtained from the complete multiple sequence-structure alignment without gaps. Helices are numbered I-IV. Sequence conservation is indicated from red (not conserved) to green (conserved). Nucleotides which are 100% conserved in all sequences are written as A, U, G or C. Nucleotide bonds which are 100% conserved throughout the alignment are marked in yellow. The figure was generated with 4SALE [41].

The strains positioned in the tertiolecta-clade represent species that belong to different traditionally accepted sections: section Tertiolecta (D. tertiolecta, D. primolecta, D. quartolecta, D. polymorpha), section Viridis (D. bioculata, D. minuta), section Dunaliella (D. salina, D. parva), and section Peircei (D. percei). The majority of these strains had an exact ITS2 sequence (Figure 1). Most D. viridis strains sampled were positioned in a single clade; however, some strains (UTEX200, CCAP19/3) were positioned in a different clade together with D. pseudosalina CONC010 (pseudosalina-clade), but without statistical support.

The majority of the D. salina strains were distributed in two different clades (salina-clade-I and salina-clade II), positioned between the tertiolecta sub-clades, whereas two D. salina strains (CCAP19/30, CCAP19/18) were positioned together in a different clade. Only two of the new ITS2 D. salina sequences, ITC5119 ("salina-clade-I") and D. salina ITC5105 "Janubio" (salina-clade II), had a unique ITS2 sequence profile (Figure 1). The CBC analysis of the ITS2 secondary structure showed that there is at least one CBC between "Janubio" and the other D. salina strains analyzed (see Additional file 1), except D. salina CCAP19/30 and CCAP19/18; however these two strains were phylogenetically distant to Janubio.

The taxonomic identification of the Dunaliella sp. strains (below the D. acidophila strain in Figure 1) was not possible because they did not match with any known Dunaliella species. Despite the phylogenetic tree indicates that this group may indeed represent a new species; the analysis of the CBC did not confirm these results (Additional file 1).

We observed a lack of CBCs in some strains when we compared species in different clades: viridis vs salina, viridis vs tertiolecta, salinas vs tertiolecta, salina vs salina, etc (Additional File 1). Also, the CBC analysis of the different species within the subgenus Dunaliella for the confirmation of the species boundaries was not conclusive in some cases (Additional file 1).

The phylogenetic results of the ITS2 sequences in this study, and previous confirmed identification of some Dunaliella strains (see Table 1), allow us to suggest the re-identification of some strains (Table 1).
Table 1

Taxonomic classification, Culture Collection, Geographic Origin and GenBank acession numbers of the strains included in this study.

Former classification

New suggested classification [Reference] and comments

Culture Collection

Geographic Origin

Isolator (Date)

GenBank acession number

GenBank identifier

Subgenus Pascheria (Freshwater species)

 

Dunaliella lateralis Pascher &Jahoda

  

Nepal

 

AF313445

16596847

   

Nepal

 

DQ377089

87047580

Dunaliella acidophila (Kalina) Massyuk

 

CCAP19/35

Freshwater; acidic sulphurous pool, Pisciarelli, Naples, Italy

Albertano (1981)

HM060646

 

Subgenus Dunaliella

 

Section Tertiolecta (Marine species. Optimum salinity < 6% NaCl)

 

Dunaliella tertiolecta Butcher

 

CCAP19/6B

Brackish; Oslo Fjord, Norway

Foyn (1928 or earlier)

HM243579*

 
  

CCAP19/6B

Brackish; Oslo Fjord, Norway

Foyn (1928 or earlier)

AY572957

47933783

  

CCAP19/27

  

EF473748

145587830

  

CCAP19/27

  

AY654300

55979209

  

Dtsi

Italy: Venezia

 

EF473730

145587823

  

UTEX999

Norway: Oslofjord

 

AF313435

16596842

  

CCMP1320

Salt flat. USA?

 

AF313433

16596841

  

CCMP1302

Salt flat. USA?

 

DQ377096

87047587

  

CCMP364

Salt flat. USA?

 

DQ377097

87047588

  

FHL

  

DSU66956

2627284

  

DCCBC5

  

AY686684

56578596

  

SAG13.86

Norway: Oslofjord

 

EF473738

145587825

  

ATCC30929

United Kingdom: Plymouth

 

EF473742

145587827

  

DCCBC26

  

DQ224338

77955899

Dunaliella quartolecta Butcher

Dunaliella tertiolecta [this study]

CCAP19/8

Marine; Southampton, Hampshire, England

Butcher (1953)

DQ157054

77539932

Dunaliella primolecta Butcher

Dunaliella tertiolecta [this study]

UTEX1000

English Channel, Plymouth, Devon, England

Gross (1936)

AY582942

50952902

  

UTEX1000

English Channel, Plymouth, Devon, England

Gross (1936)

DQ377092

87047583

 

Dunaliella tertiolecta [this study]

hd8

China?

 

DQ116745

71482600

Dunaliella polymorpha Butcher

Dunaliella tertiolecta [this study]

CCAP19/7C

Brackish; River Crouch, Essex, England

Butcher (1954)

DQ157053

77539931

Dunaliella marítima Butcher

Dunaliella tertiolecta [this study]

SAG42.89

  

AY582086

51035303

Section Dunaliella (Halophilic species. Optimum salinity > 6% NaCl. Accumulates carotenes)

Dunaliella salina Teodoresco

 

CCAP19/18

Hypersaline; Hypersaline brines, Hutt Lagoon, Western Australia

Kaethner (1982)

AF546098

33333776

  

CCAP19/18

Hypersaline; Hypersaline brines, Hutt Lagoon, Western Australia

Kaethner (1982)

EF473746

145587829

  

CCAP19/25

  

HM140783

 
  

UTEX1644

Point Colorado Salinas; La Paz, Baja California, Mexico

Loeblich (1967)

AF313429

16596839

  

CONC006

Salar de Atacama, Chile

(1990)

AF313425

16596837

  

CONC001

Laguna La Rinconada, Chile

 

AF546092

33333770

  

CONC007

Salar de Atacama, Chile

(1990)

AF313427

16596838

  

DCCBC1

Lake Tyrell, Victoria, Australia

Polle

AY549442

47499297

  

DCCBC2

South Korea

 

AY512973

46250926

  

hd6

Israel

 

DQ116743

71482598

   

Yucatan, Mexico

 

AF546094

33333772

   

Tanggu, China

 

AF546096

33333774

  

AC144

Tunisia, North Africa

 

AY549441

47499296

 

Dunaliella viridis [this study]

184.80

  

AY577766

49073091

  

OUC66 "hd4"

China

(2005) ?

DQ116741

71482596

  

OUC38 "hd3"

China

(2005) ?

DQ116740

71482595

  

OUC36 "hd2"

China

(2005) ?

DQ116739

71482594

  

OUC21 "hd1"

China

(2005) ?

DQ116738

71482593

  

9802

China ?

(2007) ?

EF695405

151573027

  

"hd5"

Inner-Mongolia

(2005)

DQ116742

71482597

 

Dunaliella tertiolecta [this study]

DS18S1

Mexico?

 

FJ360756

213958821

 

Dunaliella tertiolecta [this study]

DS18S2

Mexico?

 

FJ360757

213958822

 

Dunaliella tertiolecta [this study]

DS18S3

Mexico?

 

FJ360758

213958823

 

Dunaliella tertiolecta [this study]

Dsge

Belgium: Gent

 

EF473732

145587824

 

Dunaliella viridis [ 9, 10, 27]

CCAP 19/3

Brackish; dirty salt lake, Soviet Union

Mainx

EF473744

145587828

 

Dunaliella viridis [ 9, 10, 27]

UTEX200

Brackish; dirty salt lake, Soviet Union

Mainx

AF313423

16596836

  

MSI-1

  

GQ337903

254838316

  

ITC5100

Vargas, Gran Canaria, Spain

de la Jara & Mendoza (2005)

HM035353*

 
  

ITC5101

Punta, Gran Canaria, Spain

de la Jara & Mendoza (2005)

HM035354*

 
  

ITC5102

Tenefé, Gran Canaria, Spain

de la Jara & Mendoza (2005)

HM035355*

 
  

ITC5103

Rio, Lanzarote, Spain

de la Jara & Mendoza (2005)

HM035356*

 
  

ITC5104

Guatiza, Lanzarote, Spain

de la Jara & Mendoza (2005)

HM035357*

 
  

ITC5105

Janubio, Lanzarote, Spain

de la Jara & Mendoza (2005)

HM035346*

 
  

ITC5106

Carmen (Majo), Fuerteventura, Spain

Mendoza & Trujillano (2003)

HM035358*

 
  

ITC5107

Añana, Álava, Spain

de la Jara & Mendoza (2005)

HM035359*

 
  

ITC5118

île de Ré (01), France

Carmona & Mendoza (2006)

HM035348*

 
  

ITC5122

île de Ré (05), France

Carmona & Mendoza (2006)

HM035347*

 
  

ITC5114

La Tapa, Cádiz, Spain

de la Jara & Mendoza (2007)

HM035350*

 
  

ITC5119

île de Ré (02), France

Carmona & Mendoza (2006)

HM035349*

 
 

Aliquot of Dunaliella salina BCA421

ITC5003

Tenefe, Gran Canaria, Spain

Mendoza (1992)

HM035352*

 

Dunaliella bardawil nomen nudum Ben-Amotz & Avron

Dunaliella salina CCAP 19/30 [27], obtained from Dr. Joao Varela (Faro, Portugal)

ITC5000

Marine; salt pond, near Bardawil lagoon, North Sinai, Israel

Ben-Amotz & Avron (1976).

HM035351*

 
 

Dunaliella salina [27]. Reinstated from SAG on April 1996

CCAP19/30

Marine; salt pond near Bardawil lagoon, North Sinai, Israel

Ben-Amotz & Avron (1976).

EU932917

205361369

 

Dunaliella salina [27]

ATCC30861

Marine; salt pond near Bardawil lagoon, North Sinai, Israel

Ben-Amotz & Avron (1976)

AF313431

16596840

 

Dunaliella salina [27]

UTEX2538

Marine; salt pond near Bardawil lagoon, North Sinai, Israel

Ben-Amotz & Avron (1978)

DQ377085

87047576

 

Dunaliella salina [27], Dunaliella tertiolecta [this study]

SAG42.88

Marine; salt pond, near. Bardawil lagoon, North Sinai, Israel

Ben-Amotz & Avron (1976)

EF473741

 
 

Dunaliella tertiolecta [this study]

hd7

China?

 

DQ116744

71482599

Dunaliella parva Lerche

Dunaliella viridis [911]

UTEX1983

Dead Sea

(1973)

AF313441

16596845

 

Dunaliella tertiolecta [10, 11]Dunaliella quartolecta [27]

CCAP19/9

Brackish; salt marsh, Northey Island, Essex, England

Butcher (1956)

AF313439

16596844

 

Dunaliella tertiolecta [10, 11]

CCMP362

 

Gold

AF313437

16596843

 

Dunaliella marítima [27], Dunaliella viridis [this study]

SAG19-1

Marine: Lacul Sarat, Romania

Lerche (Before 1938)

DQ377091

87047582

 

Dunaliella tertiolecta [this study]

hd9

China?

 

DQ116746

71482601

Dunaliella pseudosalina Massyuk & Radchenko

 

CONC010

Salar de Atacama, Chile

 

AF313421

16596835

Section Viridis (Halophilic species. Optimum salinity > 6% NaCl. Cells always green. Do not accumulate carotenes. Cells radially symmetrical)

Dunaliella minuta Lerche

Dunaliella tertiolecta [this study]

CCAP19/5

Marine; sand and sea water, Roscoff, France

Jowett (1967)

HM035345*

 
 

Dunaliella tertiolecta [this study]

SAG23.86

  

AY582085

51035302

Dunaliella bioculata Butcher

Dunaliella tertiolecta [this study]

CCAP19/4

Brackish; salt lake, Soviet Union

Mainx

HM035344*

 
 

Dunaliella tertiolecta [this study]

UTEX199

Brackish; salt lake, Soviet Union

Mainx

DQ157433

76097092

  

UTEX199

Brackish; salt lake, Soviet Union

Mainx

DQ377086

87047577

Dunaliella viridis Massyuk

 

CONC002

Salar de Atacama, Chile

(1990)

AF313419

16596834

  

CONC002

Salar de Atacama, Chile

(1990)

DQ377098

87047589

  

SAG44.89

  

87047600

61200914

  

SHU

China?

 

AY878700

58339343

  

DCCBC4

Great Salt Lake, Utah, USA

 

AY686685

56578597

  

DCCBC3

Great Salt Lake, Utah, USA

 

AY828227

61200913

Section Peirceinae ((Halophilic species. Optimum salinity > 6% NaCl. Cells always green. Do not accumulate carotenes. Cells bilaterally symmetrical)

Dunaliella percei Nicolai & Baas-Becking

Dunaliella tertiolecta [10, 11]

CCAP19/2

Brackish; California, USA

Nicolai (1931)

HM035343*

 
 

Dunaliella tertiolecta [10, 11]

UTEX2192

Brackish; California, USA

Nicolai (1931)

AF313443

16596846

Unknown Dunaliella Species

Dunaliella sp.

Dunaliella tertiolecta [27]

CCAP19/23

Marine;

Pennick

HM035341*

 
 

Dunaliella salina [27]

CCAP19/12

Brackish; North Sinai, Israel

Ginzburg (1976)

HM035342*

 
  

CCMP367

Salt flat

 

DQ377087

87047578

  

CCMP220

Salt flat

 

DQ377095

87047586

 

Dunaliella tertiolecta [this study]

CCMP1923

Salt flat

 

DQ377094

87047585

 

Dunaliella tertiolecta [this study]

CCMP1641

Salt flat

 

DQ377093

87047584

 

Dunaliella tertiolecta [this study]

SAG19.6

  

AY582086

51035303

  

FL1

Salt flat

 

DQ377099

87047590

 

Dunaliella viridis [this study]

BSF1

USA: Utah, Bonneville Salt Flats

William Henley

DQ377081

87047572

 

Dunaliella viridis [this study]

BSF2

USA: Utah, Bonneville Salt Flats

William Henley

DQ377082

87047573

 

Dunaliella viridis [this study]

BSF3

USA: Utah, Bonneville Salt Flats

William Henley

DQ377083

87047574

 

Dunaliella salina [this study]

006

 

A.W. Coleman, U. Brown

AF033278

2645739

 

Dunaliella tertiolecta [this study]

hd10

  

DQ116747

71482602

 

Dunaliella viridis [this study]

ABRIINW M1/1

  

EU927374

197290927

  

ABRIINW M1/2

Iran?

 

EU927373

197290646

 

Dunaliella salina [this study]

ABRIINW U1/1

Iran?

 

FJ164063

205371718

 

Dunaliella viridis [this study]

ABRIINW U2/1

Iran?

 

FJ164064

205371719

  

SPMO112-3

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377103

87047594

  

SPMO201-3

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377110

87047601

  

SPMO128-2

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377105

87047596

  

SPMO109-1

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377105

87047596

  

SPMO112-4

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377104

87047595

  

SPMO207-3

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377115

87047606

  

SPMO200-3

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377107

87047598

  

SPMO201-4

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377111

87047602

  

SPMO201-5

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377112

87047603

  

SPMO201-6

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377113

87047604

  

SPMO112-1

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377101

87047592

  

SPMO112-2

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377102

87047593

  

SPMO300-4

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377118

87047609

  

SPMO210-3

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377116

87047607

 

Dunaliella viridis [this study]

SPMO200-8

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377108

87047599

 

Dunaliella viridis [this study]

SPMO601-1

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377121

87047612

 

Dunaliella viridis [this study]

SPMO200-2

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377106

87047597

 

Dunaliella viridis [this study]

SPMO201-2

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377109

87047600

 

Dunaliella viridis [this study]

SPMO202-4

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377114

87047605

 

Dunaliella viridis [this study]

SPMO300-5

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377119

87047610

 

Dunaliella viridis [this study]

SPMO600-1

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377120

87047611

 

Dunaliella viridis [this study]

SPMO BP3

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377122

87047613

 

Dunaliella viridis [this study]

SPMO 980625-IE

Salt flat, USA: Oklahoma, Salt Plains National Wildlife Refuge

 

DQ377123

87047614

Other groups

Chlamydomonas reinhardtii

 

SAG11-31

  

AJ749628

111073405

Chlamydomonas allensworthii

 

isolate 7

  

AF326855

13274452

Oedogonium nodulosum

    

DQ078301

71482662

Bulbochaete rectangularis var. hiloensis

 

UTEX954

Catawmont, Massachusetts, USA

Cook 1962

AY962677

62183531

Astrephomene gubernaculifera

 

UTEX2479

  

AGU66932

2982755

Pandorina morum

 

EP3

  

AF378359

14165185

Eudorina elegans

 

UTEX 1192

  

AF098173

4007503

Yamagishiella unicocca

 

UTEX2431

  

AF375785

14091689

Phacotus lenticularis

 

970805-20

  

AY009933

18025134

Lobochlamys culleus

 

UTEX1057

Maxville, Florida, USA

Smith

CCU66946

2627275

Asterisks indicate the strains sequenced in this study. Underlined strains correspond to identical isolates stored in different culture collections.

Acronyms: Culture Collection of Algae and Protozoa, UK (CCAP); Sammlug von Algenkulturen, Germany (SAG); University of Texas Culture Collection of Algae, USA (UTEX); American Type Culture Collection, USA (ATCC); Dunaliella Culture Collection at Brooklyn College, USA (DCCBC); Provasoli-Guillard National Centre for the culture of Marine Phytoplankton, USA (CCMP); Universidad de Concepción, Chile (CONC); Banco Canario de Algas, Spain (BCA) ; Instituto Tecnológico de Canarias, Spain (ITC).

Finally, the ITS2 data was not informative regarding the geographic origin of the D. salina strains.

Discusion

The ITS2 Database allows automatic large scale simultaneous analyses of both ITS2 sequences and their secondary structures. Potential pitfalls are in structures obtained by different algorithms; however the main difficulty of performing a phylogenetic analysis of the genus Dunaliella is the misinformation available at Culture Collections and GenBank regarding the identification of strains and sequences. The tracking of the true identification of each strain was only possible after consulting several publications where the authors concluded that they were misidentified and suggested their re-identification [9, 10, 27]. In this study, we have also suggested the re-identification of some strains based on our ITS2 data. To overcome all these unnecessary problems (and given that it is unlikely that all the Dunaliella strains could be openly available), we suggest to establish a "type strain" for each Dunaliella taxon (including subspecies, forms or varieties). These basic data should be easily obtained from any official culture collection, thereby greatly facilitating comparison with new field isolates and avoiding misleading information and/or false conclusions.

Our ITS2 phylogenetic analysis of Dunaliella reveals several major groups, and positions the freshwater D. lateralis clearly outside Dunaliella, confirming that it no longer should be considered a member of this genus [10]. Nevertheless, the other freshwater species analyzed in this study (D. acidophila, CCAP19/35), maintained its position within the subgenus Dunaliella, and was not phylogenetically related to D. lateralis, as recently proposed [28]. Furthermore, the observation that different species belonging to several Sections (Tertiolecta, Viridis, Dunaliella and Peircei) share the exact same ITS2 sequence, make us believe that they correspond to a single species. These data agree with other authors [10, 11, 27, 29], who suggested that the number of Dunaliella species may be much lower than it has been claimed till now. The possibility that the ITS2 gene is not able to discriminate between these species is highly unlikely; therefore, our observations support the suggestion that the morphological and physiological criteria available to discriminate Dunaliella species are either not very reliable [11], or are difficult to interpret..

In an attempt to clarify the species concept within Dunaliella, we searched for compensatory base changes (CBCs). Several case studies have revealed that the detection of a CBC in the ITS2 secondary structure between two organisms is correlated with sexual incompatibility [6, 30, 31], and these changes have been proposed as markers for distinguishing species [6, 7, 30, 31]. In summary, these investigations conclude that while a CBC in a pair of sequences is positively correlated with species distinctness at a confidence level of 93%, the lack of a CBC in the ITS2 secondary structure does not necessarily indicate that two organisms belong to the same species [6]. The overall analysis of the CBC was not able to elucidate completely the species boundaries within the different groups of Dunaliella, since in some cases it was observed that there was a lack of CBCs between known distinct species.

In the special case of D. salina, high variation levels have been reported for decades [1924]. However, only Massjuk [18] translated geographical, physiological, and morphological variables into the recognition of two subspecies (D. salina sp. salina and D. salina sp. sibirica Massjuk and Radch.) and three forms (D. salina sp. salina f. salina, D. salina sp. salina f. oblonga Lerche, and D. salina sp. salina f. magna Lerche). Later on, ITS2 phylogenetic analyses suggested the existence of two distinct phylogenetic species within the taxonomic entity currently known as D. salina [10, 11, 32], indicating the possibility of cryptic speciation [10]. Our ITS2 phylogenetic analysis does confirm the existence of three different groups within D. salina; however, the CBC results did not resolve if these groups may correspond to distinct species, although several strains of each D. salina group shared one CBC with D. salina strains in other groups. On the other hand, the high morphological and physiological variability found within the Spanish D. salina strains under the same lab conditions [24] was not correlated with the phylogenetic observations of this study. This finding indicates that the phylogenetic closeness found with ITS sequences does not reflect common physiological or morphological attributes. Moreover, our data unequivocally suggest that D. salina is not monophyletic, at odds with previous hypotheses [10, 11, 32].

The other objective of this work was to elucidate if the physiological uniqueness found in "Janubio" [such as its unique fatty acid profile and accumulation of high levels of carotenes under low light flux density conditions; Mendoza et al.: A new strategy for carotenogenesis under conditions of cellular stress in Dunaliella (a Potential New Species), submitted] could be confirmed by the ITS2 data and the CBC analysis. However, although we observed that this strain has a unique ITS2 sequence profile, and had more than one CBC with the other phylogenetically related D. salina, our data do not allow us to conclude that this is a new species, and further studies must be performed to find out if the differences observed are just reflecting a high intra-specific variability. Finally, in agreement with previous studies [8, 11, 25], our ITS2 data failed to furnish evidence for isolation by distance among D. salina strains.

Conclusion

This work demonstrates that the taxonomy of Dunaliella should be revised. The great diversity observed within the ITS2 sequences of D. salina suggests that different biological groups may exist within this taxon; however, this was not confirmed with the CBC analysis. Likewise, although the Spanish D. salina strain ITC5105 "Janubio" was characterized by a unique ITS2 sequence, the hypothesis that it may be a new species could not be confirmed by the CBCs analysis, requiring further morpho-physiological and genetic investigation. Overall, the use of CBCs to define species boundaries within Dunaliella was not conclusive in some of the cases assessed.

Methods

Strains, DNA extraction and ITS amplification

We sequenced the ITS2 region of 13 D. salina strains from Spanish and French saltworks, one strain obtained from the Culture Collection of Algae and Protozoa UK (CCAP), and one D. salina strain that has been maintained in the Instituto Tecnológico de Canarias (ITC henceforth) for several years (purchased from CCAP as Dunaliella salina 19/30). We also sequenced other Dunaliella species (D. minuta CCAP19/5, D. tertiolecta CCAP19/23 and CCAP19/6B, D. bioculata CCAP19/4) [Table 1]. The sequences of the other strains analyzed in this study were obtained from the ITS2 Database (http://​its2.​bioapps.​biozentrum.​uni-wuerzburg.​de/​?​about). Detailed information about the strains used in this study can be found in Table 1.

DNA extraction was performed with a chelex-100 (Biorad, CA, USA) resin-based protocol [33]. For the DNA amplification of the ITS region, primers AB28 and TW81 in Goff et al. (1994) [34] were used. DNA amplification was carried out in a total volume of 25 μl with 1X iQ SYBR Green Supermix (Biorad, CA, USA) and 10 pM of each primer in a Smart Cycler thermocyler (Cepheid,CA, USA) as follows: 5 min at 94°C; 5 cycles of 1 min at 94°C, 2 min at 50°C and 1 min at 72°C; 30 cycles of 1 min at 94°C, 1 min at 62°C and 1 min at 72°C, with a final extension of 5 min at 72°C.

PCR products were first electrophoresed in a 1.5% agarose gel to assure that a single band of 500-600 bp was present, then purified using the Real Clean Spin kit (REAL, Durviz S.L.U., Valencia, Spain), and finally bi-directionally sequenced on an ABI PRISM 3730xl automatic sequencer (Applied Biosystems, CA, USA) at the DNA sequencing services of Macrogen (Korea).

Phylogenetic analyses

Sequences and their individual secondary structures were obtained from the ITS2 Database [3537]. Newly obtained ITS2 sequences were annotated according to Keller et al. [38], and their secondary structures predicted by homology modeling [39]. The phylogenetic analysis followed the procedure outlined in Schultz and Wolf [7] in accordance with Keller et al. [5]. The software used for the ITS2 sequence-structure analysis can be obtained from http://​its2.​bioapps.​biozentrum.​uni-wuerzburg.​de/​?​about. A global, multiple sequence-structure alignment was generated in 4SALE v1.5 [40, 41]. The sequences and their individual secondary structures were synchronously aligned making use of an ITS2 sequence-structure specific scoring matrix [40], and the start and end of the alignment was manually adjusted. Based on primary and secondary structure information, phylogenetic relationships were reconstructed by ProfDistS, through the use of an ITS2 specific, general time reversible substitution model [42, 43]. Bootstrap support [44] was estimated on 100 pseudo-replicates. The resulting tree was visualized with TreeView [45].

To study the species boundaries within Dunaliella we followed the "distinguishing species" instructions [6] based on compensatory base changes (CBCs) in the ITS2 secondary structure, and we used the CBCAnalyzer option implemented in 4SALE.

Declarations

Acknowledgements

This research was supported by BANGEN-"Banco Genético de la Macaronesia", MAC/1/C070 (INTERREG-IIIB). We thank the Cabildo de Gran Canaria for allowing us to collaborate with the Departamento de Biodiversidad Molecular at the Jardín Botánico Canario "Viera y Clavijo"- Unidad Asociada CSIC, and for continuous support to all its research lines. We would like to thank Matthias Wolf (University of Würzburg) for helping with the ITS2 sequence-structure analysis and to Frank Förster (University of Würzburg) for alignment adjustments.

Authors’ Affiliations

(1)
Departamento de Biotecnología. División de Investigación y Desarrollo Tecnológico, Instituto Tecnológico de Canarias (ITC)
(2)
Departamento de Biodiversidad Molecular y Banco de ADN, Jardín Botánico Canario "Viera y Clavijo"-Unidad Asociada CSIC

References

  1. Coleman AW, Suarez A, Goff LJ: Molecular delineation of species and syngens in volvocacean green algae (Chlorophyta). Journal of phycology. 1994, 30: 80-90. 10.1111/j.0022-3646.1994.00080.x.View Article
  2. Coleman AW, Mai JC: Ribosomal DNA ITS-1 and ITS-2 sequence comparisons as a tool for predicting genetic relatedness. Journal of Molecular Evolution. 1997, 45: 168-177. 10.1007/PL00006217.View Article
  3. Coleman AW: Pan-eukaryote ITS2 homologies revealed by RNA secondary structure. Nucleic Acids Research. 2007, 35: 3322-3329. 10.1093/nar/gkm233.View Article
  4. Coleman AW: ITS2 is a double-edged tool for eukaryote evolutionary comparisons. Trends in Genetics. 2003, 19: 370-375. 10.1016/S0168-9525(03)00118-5.View Article
  5. Keller A, Förster F, Müller T, Dandekar T, Schultz J, Wolf M: Including RNA Secondary Structures improves Accuracy and Robustness in Reconstruction of Phylogenetic Trees. Biology Direct. 2010, 5: 4-10.1186/1745-6150-5-4.View Article
  6. Müller T, Philippi N, Dandekar T, Schultz J, Wolf M: Distinguishing species. RNA. 2007, 13: 1469-1472. 10.1261/rna.617107.View Article
  7. Schultz J, Wolf M: ITS2 Sequence-Structure Analysis in Phylogenetics: A How-to Manual for Molecular Systematics. Molecular Phylogenetics and Evolution. 2009, 52: 520-523. 10.1016/j.ympev.2009.01.008.View Article
  8. Gomez PI, Gonzalez MA: Genetic variation among seven strains of Dunaliella salina (Chlorophyta) with industrial potential, based on RAPD banding patterns and on nuclear ITS rDNA sequences. Aquaculture. 2004, 233: 149-162. 10.1016/j.aquaculture.2003.11.005.View Article
  9. González MA, Gómez PI, Montoya R: Comparison of PCR-RFLP analysis of the ITS region with morphological criteria of various strains of Dunaliella. Journal of Applied Phycology. 1999, 10: 573-580.View Article
  10. González MA, Coleman AW, Gómez PI, Montoya R: Phylogenetic relashionship among strains of Dunaliella (Chlorophyceae) based on nuclear ITS rDNA sequences. Journal of Phycology. 2001, 37: 604-611. 10.1046/j.1529-8817.2001.037004604.x.View Article
  11. González MA, Gómez PI, Polle JEW: Taxonomy and Phylogeny of the genus Dunaliella. The Alga Dunaliella. Biodiversity, Physiology, Genomics and Biotechnology. Edited by: Ben-Amotz A, Polle JEW, Subba Rao DV. 2009, Science Publishers, Enfield, NH, USA, 15-44.View Article
  12. Buchheim MA, Kirkwood AE, Buchheim JA, Verghese B, Henley WJ: Hypersaline soil supports a diverse community of Dunaliella (Chlorophyceae). Journal of Phycology. 2010, 46: 1038-1047. 10.1111/j.1529-8817.2010.00886.x.View Article
  13. Buchheim MA, Keller A, Koetschan C, Förster F, Merget B, Wolf M: Internal Transcribed Spacer 2 (nu ITS2 rRNA) sequence-structure phylogenetics: towards and automated reconstruction of the green algal tree of life. PLoS One. 2011, 6: 1-10.View Article
  14. Ben-Amotz A: Industrial production of microalgal cell-mass and secondary products - major industrial species. Handbook of Microalgal Cultures, Biotechnology and Applied Phycology. Edited by: Richmond A. 2004, Blackwell, UK, 273-280.
  15. Ben-Amotz A, Avron M: The biotechnology of cultivating the halotolerant alga Dunaliella. Trends in Biotechnology. 1990, 8: 121-126.View Article
  16. Borowitzka MA, Borowitzka LJ: Dunaliella. Microbial Biotechnology. Edited by: Borowitzka MA, Borowitzka LJ. 1988, Cambridge: Cambridge University Press, 27-88.
  17. Ben-Amotz A: Glycerol production in the alga Dunaliella. Biochemical and Photosynthetic aspects of energy production. Edited by: san Pietro A. 1980, Academic Press, New York, 191-208.
  18. Massjuk NP: Morphology, Taxonomy, Ecology and Geographic Distribution of the Genus Dunaliella Teod. and Prospects for Its Potential Utilization. Naukova Dumka, Kiev [original in Russian]. 1973, 242-
  19. Cifuentes AS, González MA, Conejeros M, Dellarossa V, Parra OO: Growth and carotenogenesis in eight strains of Dunaliella salina Teodoresco from Chile. Journal of Applied Phycology. 1992, 4: 111-118. 10.1007/BF02442459.View Article
  20. Cifuentes AS, González M, Parra O: The effect of salinity on the growth and carotenogenesis in two Chilean strains of Dunaliella salina Teodoresco. Biological Research. 1996, 29: 227-236.
  21. Cifuentes AS, González M, Parra O, Zúñiga M: Cultivo de cepas de Dunaliella salina (Teodoresco 1905) en diferentes medios bajo condiciones de laboratorio. Revista Chilena de Historia Natural. 1996, 69: 105-112.
  22. Markovits A, Gianelli MP, Conejeros R, Erazo S: Strain selection for β-carotene production by Dunaliella. World Journal of Microbiology & Biotechnology. 1993, 9: 534-537. 10.1007/BF00386289.View Article
  23. Gómez P, González M, Becerra J: Quantity and quality of β-carotene produced by two strains of Dunaliella salina (Teodoresco 1905) from the North of Chile. Boletín de la Sociedad Chilena Química. 1999, 44: 463-468.View Article
  24. Mendoza H, de la Jara A, Freijanes K, Carmona L, Ramos AA, de Sousa Duarte V, Serafim Varela JC: Characterization of Dunaliella salina strains by flow cytometry: a new approach to select carotenoid hyperproducing strains. Electronic Journal of Biotechnology. 2008, 11 (4): DOI: 10.2225/vol11-issue4-fulltext-2 http://​www.​ejbiotechnology.​info/​content/​vol11/​issue4/​full/​2/​2.​pdf
  25. Gomez PI, Gonzalez MA: Genetic polymorphism in eight Chilean strains of the carotenogenic microalga Dunaliella salina Teodoresco (Chlorophyta). Biological Research. 2001, 34: 23-30.View Article
  26. Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW: GenBank. Nucleic Acids Research. 2011, 39: D32-37. 10.1093/nar/gkq1079.View Article
  27. Borowitzka MA, Siva CJ: The taxonomy of the genus Dunaliella (Chlorophyta, Dunaliellales) with emphasis on the marine and halophilic species. Journal of Applied Phycology. 2007, 19: 567-590. 10.1007/s10811-007-9171-x.View Article
  28. Assunção P, Jaen-Molina R, Caujapé-Castells J, de la Jara A, Carmona L, Freijanes K, Mendoza H: Phylogenetic position Dunaliella acidophila (Chlorophyceae) based on ITS and rbcLsequences. Journal of Applied Phycology.
  29. Oren A: A hundred years of Dunaliella research: 1905-2005. Saline Systems. 2005, 1 (2): doi: 10.1186/1746-1448-1-2, [http://​www.​salinesystems.​org/​content/​1/​1/​2]
  30. Coleman AW: The significance of a coincidence between evolutionary landmarks found in mating affinity and a DNA sequence. Protist. 2000, 151: 1-9. 10.1078/1434-4610-00002.View Article
  31. Coleman AW: In there a molecular key to the level of "Biological species" in eukaryotes? A DNA guide. Molecular Phylogenetics and Evolution. 2009, 50: 197-203. 10.1016/j.ympev.2008.10.008.View Article
  32. Polle JEW, Struwe L, Jin E: Identification and characterization of a new strain of the unicellular green alga Dunaliella salina (Teod.) from Korea. Journal of Microbiology and Biotechnology. 2008, 18: 821-827.
  33. Richlen ML, Barber PH: A technique for the rapid extraction of microalgal DNA from single live and preserved cells. Molecular Ecology Notes. 2005, 5: 688-691. 10.1111/j.1471-8286.2005.01032.x.View Article
  34. Goff LJ, Moon DA, Coleman AW: Molecular delineation of species and species relationships in the red algal agarophytes Gracilariopsis and Gracilaria (Gracilariales). Journal of Phycology. 1994, 30: 521-537. 10.1111/j.0022-3646.1994.00521.x.View Article
  35. Schultz J, Müller T, Achtziger M, Seibel PN, Dandekar T, Wolf M: The internal transcribed spacer 2 database-a web server for (not only) low level phylogenetic analyses. Nucleic Acids Research. 2006, W704-707. 34 Web Server
  36. Selig C, Wolf M, Müller T, Dandekar T, Schultz J: The ITS2 Database II: homology modeling RNA structure for molecular systematics. Nucleic Acids Research. 2008, 36: D377-380. 10.1093/nar/gkn325.View Article
  37. Koetschan C, Förster F, Keller A, Schleicher T, Ruderisch B, Schwarz R, Müller T, Wolf M, Schultz J: The ITS2 Database III - sequences and structures for phylogeny. Nucleic Acids Research. 2010, 38: 275-279.View Article
  38. Keller A, Schleicher T, Schultz J, Müller T, Dandekar T, Wolf M: 5.8S-28S rRNA interaction and HMM-based ITS2 annotation. Gene. 2009, 430: 50-57. 10.1016/j.gene.2008.10.012.View Article
  39. Wolf M, Achtziger M, Schultz J, Dandekar T, Müller T: Homology modeling revealed more than 20,000 rRNA internal transcribed spacer 2 (ITS2) secondary structures. RNA. 2005, 11: 1616-1623. 10.1261/rna.2144205.View Article
  40. Seibel PN, Müller T, Dandekar T, Schultz J, Wolf M: 4SALE - A tool for synchronous RNA sequence and secondary structure alignment and editing. BMC Bioinformatics. 2006, 7: 498-10.1186/1471-2105-7-498.View Article
  41. Seibel PN, Müller T, Dandekar T, Wolf M: Synchronous visual analysis and editing of RNA sequence and secondary structure alignments using 4SALE. BMC Research Notes. 2008, 1: 91-10.1186/1756-0500-1-91.View Article
  42. Friedrich J, Dandekar T, Wolf M, Müller T: ProfDist: A tool for the construction of large phylogenetic trees based on profile distances. Bioinformatics. 2005, 21: 2108-2109. 10.1093/bioinformatics/bti289.View Article
  43. Wolf M, Ruderisch B, Dandekar T, Müller T: ProfdistS: (Profile-) Distance based phylogeny on sequence-structure alignments. Bioinformatics. 2008, 24: 2401-2402. 10.1093/bioinformatics/btn453.View Article
  44. Felsenstein J: Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985, 39: 783-791. 10.2307/2408678.View Article
  45. Page RDM: TreeView: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences. 1996, 12: 357-358.

Copyright

© Assunção et al; licensee BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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