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

  • Patrícia Assunção1Email author,

    Affiliated with

    • Ruth Jaén-Molina2,

      Affiliated with

      • Juli Caujapé-Castells2,

        Affiliated with

        • Adelina de la Jara1,

          Affiliated with

          • Laura Carmona1,

            Affiliated with

            • Karen Freijanes1 and

              Affiliated with

              • Héctor Mendoza1Email author

                Affiliated with

                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.
                http://static-content.springer.com/image/art%3A10.1186%2F2046-9063-8-2/MediaObjects/12999_2011_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

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                © Assunção et al; licensee BioMed Central Ltd. 2012

                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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