A DNA barcode approach of the Laurencia complex (Ceramiales, Rodophyta) in the tropical and subtropical Atlantic ocean

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RESULTS

DISCUSSION

1 _{Dpto. Botânica, Universidade de São Paulo, São Paulo, Brazil, vcassano@usp.br,}

2 _{Dpto. Biología Vegetal (Botánica). Univ. de La Laguna, Canary Islands, Spain,}

3 _{CIIMAR, CIRN & Departamento de Biologia, Universidade dos Azores, Ponta Delgada, Azores, Portugal,}

4 _{Dpto. Hidrobiología, Univ. Autónoma Metropolitana,}

Iztapalapa, México, D.F.,

5 _{Departament of Biological Sciences, Florida International University, Miami, Florida, USA,}

6 _{Seção de Ficologia, Instituto de Botânica, São Paulo, Brazil.}

The diversity of the Laurencia complex is being assessed in tropical and

subtropical Atlantic by an international cooperation project involving

Brazil, Mexico, Spain (Canary Islands), Portugal (Azores and Madeira)

and USA (Florida) on the base of molecular data allied to a detailed

morphological study of species. The diversity of the complex was

analyzed for the first time for the Atlantic Ocean, including specimens

from all five localities, using the plastid 23S rRNA gene (UPA), which has

been investigated as potential DNA Barcode marker for photosynthetic

eukaryotes. The COI-5P gene was also used as DNA barcode, and the

rbcL gene was used for phylogenetic inferences.

MATERIAL AND METHODS

INTRODUCTION

A DNA BARCODE APPROACH OF THE LAURENCIA COMPLEX (CERAMIALES,

RHODOPHYTA) IN THE TROPICAL AND SUBTROPICAL ATLANTIC OCEAN

Valéria Cassano

1 _{, Maria Machín-Sánchez}

2 _{, Ana I. Neto}

3 _{, Mariana C. Oliveira}

1 _{, Abel Sentíes}

4 _,

Jhoana Díaz-Larrea

4 _{, Mª Candelaria Gil-Rodríguez}

2 _{, Ligia Collado-Vides}

5 _,

Amanda Medeiros

1 _{, Alain Duran}

5 _{& Mutue T. Fujii}

6

Chondria dangeardii Hawaii (HQ421425)

C. arcuata Hawaii (HQ421424)

L. majuscula Hawaii (HQ421434)

L. dendroidea Rio de Janeiro, Brazil

L. dendroidea Bahia, Brazil

L. dendroidea Espírito Santo, Brazil

L. dendroidea Rio de Janeiro, Brazil I L. dendroidea São Paulo, Brazil

L. nidifica Hawaii (HQ421001)

L. viridis Canary Islands, Spain (MMS0094)

L. cf. pyramidalisAzores, Portugal (PIX-11-26)

L. cf. pyramidalisAzores, Portugal (SMG-11-72)

L. pyramidalis Canary Islands, Spain (MMS0096)

L. pyramidalisAzores, Portugal (PIX-11-11)

L. mcdermidiae Hawaii (HQ4214744)

L. brachyclados Hawaii (HQ421426)

L. galtsoffii Hawaii (HQ421136)

L. decumbens Hawaii (HQ420991)

L. oliveirana Rio de Janeiro, Brazil

L. caraibica Rio Grande do Norte, Brazil

L. catarinensis Santa Catarina, Brazil

L. catarinensis Rio de Janeiro, Brazil

L. catarinensis Espírito Santo, Brazil

L. crustiformans Hawaii (HQ421480

)

P. parvipapillata Hawaii (HQ421473)

P. yamadana Havaí (HQ421478)

P. perforata São Paulo, Brazil (IBC0019)

P. perforata Bahia, Brazil

P. perforata Canary Islands, Spain (MMS0093)

P. perorata Espírito Santo, Brazil

P. flagellifera Canary Islands, Spain (MMS0095)

P. flagellifera Espírito Santo, Brazil

P. flagellifera Rio de Janeiro,Brazil

P. flagelliferaBahia, Brazil

Chondrophycus cartilagineus Hawaii (HQ421430)

P. furcata Bahia, Brazil

L. cervicornis Florida, USA

O. sp. 3 Azores, Portugal (PIX-11-12)

O. truncata Canary Islands, Spain

O. truncata Canary Islands, Spain (MMS0098)

O. sp. 7Azores, Portugal (PIX-11-44)

O. sp. 7Azores, portugal (PIX-11-13)

O.cf. pinnatifidaAzores, Portugal (PIX-11-31)

O. cf. pinnatifidaAzores, Portugal (PIX-11-82)

O. sp. 5 Azores, Portugal (SMG-11-78)

C. succisus Hawaii (HQ421477)

C. dotyi Hawaii (HQ421432)

C. undulatus Hawaii (HQ421208)

L. translucida Bahia, Brazil

L. sp.1 São Paulo, Brazil (IBC003)

L. sp. 1 São Paulo, Brazil (IBC0014)

L. sp. 1 Espírito Santo, Brazil L. sp. 1 Espírito Santo, Brazil

L. cf. marilzaeAzores, Portugal (PIX-11-55)

L. marilzae Canary Islands, Spain (MMS0097)

L. marilzae São Paulo, Brazil

0.005 substitutions/site 100 99 86 100 96 86 91 ₉₇ 73 73 80 90 83 78 76 71

Laurencia s.s.

Palisada

Osmundea

Chondrophycus

Laurenciella

Fig. 1. Neighbor-Joining analysis for UPA sequences for the Laurencia

complex. The bootstrap values for 2000 replicates are shown on the

branches (only values above 70 were considered).

Markers/ divergences (%)

rbcL

COI-5P

UPA

Intergeneric

5.6-11.9

6.9-13.1

3.1-7.8

Interspecific

1.9-6.2

4.0-9.3

0.8-2.9

Intraspecific

0-1

0-2.5

0-0.5

Tab. 1. Range of genetic divergence among DNA sequences of the Laurencia

complex for different markers. Problematic taxa were excluded from the

comparison.

The genus Laurenciella established based on rbcL sequences was also confirmed with the

use of two other markers: UPA and COI-5P, forming independent clades with high

support, represented by the taxa: L. marilzae, L. sp. 1 and L. sp. 2. The intergeneric

divergence between Laurencia and Laurenciella for UPA and COI-5P was in the range of

variation obtained from other genera of the complex, 4.9-5.8% and 10.1-13.1%,

respectively.

Laurencia cervicornis from Florida joined with Palisada by the UPA and rbcL markers and

its taxonomic position will also have to be better investigated.

The ‘problematic’ Laurencia translucida, an endemic species from Brazil, remains an

enigmatic species. Its taxonomic position has always been controversial since it shares a

combination of morphological characters to the genera Chondrophycus and Laurencia.

Unlike the results with the rbcL gene, L. translucida, was positioned within the

Chondrophycus clade by UPA and COI-5P markers. The rbcL sequence available of L.

translucida seems to be chimeric. New rbcL sequences are necessary to confirm its

taxonomic position.

In the analyses with the UPA and COI-5P, C. cartilagineus, type species the genus

Chondrophycus, joined with Palisada, which indicates that these two genera can be

congeneric. Further analyses are necessary to clarify the position of these taxa.

Financial support

v

Chondria dangeardiiUSA (GU223879)

Palisadasp. (as P. papillosa) Mexico

P. perforata Spain

P. perforata(as P. papillosa) Spain

P. perforata Canary Islands, Spain (MMS0093)

P. perforata Parati, Rio de Janeiro, Brazil

P. perforata (asP. papillosa) Rio de Janeiro, Brazil

P. perforata (asP. papillosa) Angra dos Reis, Brazil

P. perforata Parati, Rio de Janeiro, Brazil

P. perforata (asP. papillosa) Rio de Janeiro, Brazil

P. flagelllfera Rio de Janeiro, Brazil

P. furcata Paraíba, Brazil

P. parvipapillataUSA (GU223895)

C.cartilagineus USA (GU223896)

Y. poiteauivar. gemmiferaMexico

L. sp. 1 São Paulo, Brazil

L. sp. 2 Espírito Santo, Brazil

L. marilzae São Paulo, Brazil

L. cf. marilzaeAzores, Portugal (PIX-11-55

L. translucida Espírito Santo Brazil

C. cf. undulatus USA (GU223886)

C. succisus USA (GU223884)

Chondrophycussp. USA (GU223885)

O.cf. pinnatifidaAzores, Portugal (PIX-11-31)

L. dendroidea Bahia, Brazil

L. dendroidea Rio de Janeiro, Brazil LL. dendroidea Rio de Janeiro, Brazil

L. dendroidea São Paulo, Brazil (IBC0113)

Laurencia majusculaUSA (GU223887)

L. aldingensisEspírito Santo, Brazil

L. aldingensis Rio de Janeiro, Brazil

L. catarinensisEspírito Santo, Brazil

L. catarinensis) Rio de Janeiro, Brazil

L. catarinensisRio de Janeiro, Brazil

L. catarinensis(as L. intricata) Rio de Janeiro, Brazil

L. catarinensisRio de Janeiro, Brazil

L. catarinensisSão Paulo, Brazil (IBC0005)

L. catarinensisSanta Catarina, Brazil

L. catarinensisRio Grande do Norte, Brazil

L. catarinensisCanary Islands, Spain

L. mcdermidiaeUSA (GU223877)

L. oliveirana Rio de Janeiro, Brazil

L. nipponica Japan (GU223875)

L. nipponica Russia (GU223874)

L. caduciramulosaRio de Janeiro, Brazil

L. caduciramulosa Canary Islands,Spain

L.sp. Hawaii GU223894

L. nidifica USA (GU223888)

Laurencia sp.1 Espírito Santo, Brazil

L. intricataCuba

L. viridis Canary Islands, Spain (MMS0094)

L. pyramidalis Canary Islands, Spain (MMS0096)

L. cf. pyramidalisAzores, Portugal (PIX-11-11)

0.005 substitutions/site 72 92 100 100 100 100 72 100 100 99 98 100 100 100 80 88 72 87 100 100 100 100 78 100 100 83 100 75

Laurencia s.s.

Chondrophycus

Laurenciella

Palisada

Yuzurua

Osmundea

Fig. 2. Neighbor-Joining analysis for COI-5P sequences for the Laurencia

complex. The bootstrap values for 2000 replicates are shown on the branches

(only values above 70 were considered).

Laurenciella sp. 1

Brazil

Laurenciella sp. 2

Brazil

Laurencia sp. 1

Brazil

Laurenciella cf. marilzae

Azores

Laurencia cf. pyramidalis

Azores

Laurenciella marilzae

Canary Inlands

“Laurencia cervicornis”

Florida

“Laurencia translucida”

Brazil

The UPA gene showed to be more conserved, however, the same genetic groups were

resolved with each of the three markers.

Fig. 3. Consensus tree derived from Bayesian analyses of rbcL sequences. The posterior probabilities

are shown as thicker branches. Bootstrap supports for MP/NJ (2000 replicates) are shown at the nodes

(only values above 70 were considered); *indicates bootstrap support =100%.

Samples of the Laurencia complex, collected in Brazil, Portugal (Azores and

Madeira), Spain (Canary Islands), Mexico (Caribbean Sea) and Florida

(USA), were sequenced using the markers Universal Plastid Amplicon (UPA),

COI-5P and rbcL. For COI-5P and UPA were performed neighbor-joining (NJ)

analyses using PAUP 4.0b10. For rbcL, the p

hylogenetic relationships were

inferred with PAUP 4.0b10 and MrBayes v.3.0 beta 4.

The range of genetic

divergence for the markers used was calculated using "uncorrected 'p'

distance with PAUP.

v

Chondria collinsiana Brazil (GU330225)

C. dasyphylla USA (U04021)

L.sp. 3 São Paulo, Brazil (IBC003)

L.sp. 3 São Paulo, Brazil

L. sp. 2 Espírito Santo, Brazil

L. marilzae Brazil (GU938189)

L. cf. marilzaeAzores, Portugal (PIX-11-55)

Y. poiteuai var. gemmifera Cuba (EF061650)

Y. poiteaui var. poiteaui USA (EF061652) Y. poiteaui var. poiteaui Mexico (EF061653)

Yuzurua poiteuai var. gemmifera Mexico (EF061648) “Laurencia” sp. 2 Canary Islands, Spain (MMS0088)

L. dendroidea Bahia ,Brazil (IBC0092)

L. dendroidea Rio de Janeiro, Brazil (GU330233)

L. dendroidea Bahia ,Brazil (GU330228)

L. dendroideaCanary Islands, Spain (MMS0082)

L.sp.1 Espírito Santo, Brazil

L.sp.1 Rio de Janeiro, Brazil

L. intricataCuba (GU330238)

L. sp. 5 Canary Islands, Spain (MMS0069)

L. obtusaIreland (AF281881)

L. viridisAzores, Portugal (SMA-11-06)

L. viridis Canary Islands, Spain (EF685999) L.cf. pyramidalisAzores, Portugal (SMG-11-72)

L.cf. pyramidalisAzores, Portugal (PIX-11-11)

L. pyramidalis France (FJ785316)

L.cf. pyramidalisAzores, Portugal (PIX-11-50)

L.cf. pyramidalis Canary Islands, Spain (MMS0080)

L. caduciramulosaRio de Janeiro, Brazil

L.caduciramulosa Canary Islands , Spain (JF 781525)

L. venusta Mexico (EF 061655)

L. oliveirana Rio de Janeiro, Brazil (JF810352)

L. natalensis South Africa(AF465816) L. flexuosa South Africa (AF465815) L. brongniartii Australia (EF061654)

L. aldingensisEspírito, Brazil

L. aldingensisRio de Janeiro, Brazil (JF810351)

L. catarinensis Espírtito Santo, Brazil

L. catarinensis São Paulo, Brazil (IBC0005)

L. catarinensis Santa Catarina, Brazil

L. catarinensis Rio de Janeiro, BrazilL

L. catarinensis Rio Grande do Norte, Brazil

L. catarinensis Canary Islands, Spain

L. cf. nidifica New Caledonia (FJ785315)

L. cf. mcdermidiae New Caledonia (FJ785314)

L. cf. mariannensis New Caledonia (FJ785313)

L. cf. kuetzingii New Caledonia (FJ785322)

L. translucida Espírito Santo, Brazil (AY588408)

O. cf. truncataAzores, Portugal (SMG-11-59)

O. cf. pinnatifidaMadeira, Portugal (MAD-11-28)

O. pinnatifica France (AF259495)

O. pinnatifidaCanary Islands, Spain

O. osmunda Ireland (AF281877)

O. sp. 1 Madeira, Portugal (MAD-11-126)

O. oederi Ireland (AF281880)

O. truncata Ireland (AF281879) O. truncata Canary Islands, Spain

O. sinicola USA (AY588407) O. blinksii USA (AY172575) O. splendens Mexico (AY172576 )

O. spectabilis var. spectabilis Mexico (AY172574) O. sanctarum São Paulo, Brazil

Laurencia coelenterata Cuba “P. papillosa” Mexico (AY588409)

P. perforataCanary Islands, Spain (EU256329)

P. perforata Rio de Janeiro, Brazil (EU256330)

P. flagellifera Rio de Janeiro, Brazil (GU330227)

P. furcata Paraíba, Brazil (GU330226 )

Laurencia cervicornis Florida, USA

C. cf. undulatusNew Caledonia (FJ785307)

C. sp. 1 New caledonia (FJ785309) C. sp. 2 New caledonia (FJ785310 ) C.sp. 3 (FJ785311 ) 0.005 substitutions/site

Laurenciella

Yuzurua

Laurencia s.s.

Osmundea

Chondrophycus

Palisada

94/90 * * 100/99 100/99 97/97 100/96 * * * * 100/99 * 99/90 91/92 86/82 100/92 * * * * * 83/73 * 80/90 * * 100/96 100/99 * 100/98 75/79 * * 95/88 0.09 – 1.00

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40

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E. Berrendero1_{, M. Bohunicka}2_{, L. Stenclova}1_{and J. H. Kastovsky}1

1_{University of South Bohemia, Czech Republic;}2_{University of South Bohemia/Academy of Sciences of the}

Czech Republic, Czech Republic

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C. Z. Branco1_{, P. C. Bispo}1_{, C. K. Peres}2_{, A. F. Tonetto}1_{and L. H. Branco}1

1_{São Paulo State University - UNESP, Brazil;}2_{Federal University of Latin American Integration –}

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1_{University of Louisiana at Lafayette, USA;}2_{University of North Carolina Wilmington, USA}

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V. Cassano1_{, M. Machín-Sánchez}2_{, A. I. Neto}3_{, M. C. Oliveira}1_{, A. Sentíes}4_{, J. Díaz-Larrea}4_{, M.}

Gil-Rodríguez2_{, L. Collado-Vides}5_{, A. Medeiros}1_{and M. T. Fujii}6

1_{University of São Paulo, Brazil;}2_{University of La Laguna, Spain;}3_{University of the Azores, Portugal;} 4_{Autonomous Metropolitan University, Mexico;}5_{Florida International University, USA;}6_{Institute of}

Botany, Brazil

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I. S. Chapuis1_,_{M. O. Paiano}2_{, M. Aboal}3_{, P. M. Sánchez Castillo}1_{, O. J. Necchi}2_{, and}_{M. M.}

Elmosallamy4

1_{University of Granada, Spain;}2_{UNESP - Campus de São José do Rio Preto, Brazil;}3_{University of}

Murcia, Spain; 4_{Ain Shams Universit, Egypt}

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W. B. Chiasson, E. D. Salomaki, and M. L. Vis Ohio University, USA

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H. G. Choi1_{, S. M. Kim}1_{, J. H. Kim}1 _{and M. S. Hwang}2

1_{Korea Polar Research Institute, Republic of Korea;}2_{National Fisheries Research and Development}

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68

western Atlantic and Red Sea specimens indicate newly found diversity representing four distinct species, with one unreported species each for Caribbean Colombia, Caribbean Panama, the NW Gulf of Mexico, and Egypt. These taxa in all likelihood correspond to new species. In addition, we propose range extensions for previously unnamed Lobophora spp. reported in Sun et al. 2012. Three recently collected

species from the Red Sea are conspecific with recently characterized taxa from Japan, Palau and Malaysia, and one species from the NW Gulf of Mexico is conspecific with a sample from Curaçao in the Lesser Antilles. The morphological evidence for describing the new species of Lobophora will be

discussed in light of the molecular-based results.

43

A DNA Barcode Approach of the Laurencia Complex (Ceramiales, Rhodophyta) in the Tropical and Subtropical Atlantic Ocean

V. Cassano1_{, M. Machín-Sánchez}2_{, A. I. Neto}3_{, M. C. Oliveira}1_{, A. Sentíes}4_{, J. Díaz-Larrea}4_{, M.}

Gil-Rodríguez2_{, L. Collado-Vides}5_{, A. Medeiros}1_{and M. T. Fujii}6

(vcassano@usp.br)

1_{University of São Paulo, Brazil;}2_{University of La Laguna, Spain;}3_{University of the Azores, Portugal;} 4_{Autonomous Metropolitan University, Mexico;}5_{Florida International University, USA;}6_{Institute of}

Botany, Brazil

The diversity of the Laurencia complex is being assessed in tropical and subtropical Atlantic by an

international cooperation project involving Brazil, Mexico, Spain (Canary Islands), Portugal (Azores and Madeira) and USA (Florida) on the base of molecular data allied to a detailed morphological study of species. The diversity of the complex was analyzed for the first time for the Atlantic Ocean, including specimens from all five localities, using the plastid 23S rRNA gene (UPA) which has been investigated as potential DNA Barcode marker for photosynthetic eukaryotes. The mitochondrial cytochrome c oxidase I gene (COI-5P) was also used as DNA barcode for the same set of species, and the rbcL gene was used for phylogenetic inferences. The range of genetic variation was compared for the three markers. The UPA proved to be more conserved; however, the same genetic groups were resolved with each of the three markers confirming the six genera currently established for the complex: Chondrophycus, Laurencia, Laurenciella, Palisada, Osmundea and Yuzurua.

44

Characterization of Batrachospermum gelatinosum (L.) De Candolle and B. arcuatum Kylin (Batrachospermales, Rhodophyta) from the Iberian Peninsula

I. S. Chapuis1_,_{M. O. Paiano}2_{, M. Aboal}3_{, P. M. Sánchez Castillo}1_{and O. J. Necchi}2

(monica_paiano@hotmail.com)

1_{University of Granada, Spain;}2_{UNESP - Campus de São José do Rio Preto, Brazil;}3_{University of}

Murcia, Spain

Freshwater red algae diversity in the Iberian Peninsula (Spain and Portugal) has been poorly studied. The purpose of this study is to approach the morphological and genetic variation of two most common members of the Batrachospermales in the study area, to better understand their biogeographic and phylogenetic relationships in a more global context. We compared genetically six populations each of B.

gelatinosum and B. arcuatum from eight different river basins, using three molecular markers to evaluate

genetic diversity: RuBisCo large subunit (rbcL) (fully sequenced at the moment), cytochrome oxidase 2-3 spacer (cox2-3) and the barcode region of cytochrome oxidase I (cox1) (preliminary data available). For the morphological comparison nine additional populations were included in the analysis. A wide morphological variation was observed for most vegetative and reproductive characters. rbcL sequences showed a relatively low genetic divergence: 98.8-100% for B. gelatinosum and 99.9-100% for B. arcuatum. We found no correlation between genetic diversity and morphological variation among the

populations of both species. Some taxonomic characters are reevaluated aiming at a more reliable characterization of these species.

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