1. The phylogenetic hypotheses confirmed the monophyly of the Laurencia complex and of the Osmundea genus, but the relationships between the Laurencia and Palisada genera were not totally solved (Fig. 22). 2. The trees topology found in separate analyses of rbcL/rbcL-S (Fig. 20) and in combined analyses (Fig. 21) was similar, forming two monophyletic groups represented by Palisada and Laurencia. However, the combination of the three markers improved the support for most of the established relationships and presented better topology definition. In the cox1 tree (Fig. 19), three groups were formed: the Palisada clade, the Laurencia clade and a third group formed by two unidentified species of Laurencia (Laurencia sp. 2 and L. sp.3). The relationships within Laurencia sensu stricto clade were best determined with the rbcL rather than with cox1, however the later proved to be efficient to identify and delimitate the specific taxonomic categories, corroborating its use for barcoding. 3. The phylogenetic analyses showed that: a. Chondrophycus furcatus belongs to the Palisada genus; b. The conspecificity of Palisada papillosa and P. perforata was confirmed by cox1, as previously suggested by Cassano et al. (2009) based on rbcL sequences; CONCLUSIONS Extraction, amplification of DNA and sequencing followed the usual protocols. A total of 710 pb were amplified for the cox1 gene, 1467 bp for rbcL gene, and 118 pb for rbcL-S spacer. Phylogenetic relationships were inferred with PAUP* 4.0b10 (Swofford, 2002) and MrBayes v.3.0 beta 4 (Huelsenbeck & Ronquist, 2001). Maximum-parsimony trees (MP) were constructed using the heuristic search option, tree-bisection-reconnection branch swapping, unordered and unweighted characters, and gaps treated as missing data. The model used in the Bayesian analysis was the GTR+I+G. One tree was sampled every 10 generations for 1,000.000 generations after generation 100,000 ("burn in") from four MCMC chains. Most species of the Laurencia complex presents great morphologic plasticity making its taxonomic delimitation difficult. In this case, the use of molecular markers, mainly the plastidial rbcL gene, has proven useful to delimit the taxa of the complex. Taxonomic and phylogenetic studies of the Laurencia complex based on morphological and molecular characters were carried out on specimens collected in Brazil, Mexico, Cuba and Canary Islands, Spain. The first sequences for species of the Laurencia complex using the 5’ region of mitochondrial cytochrome c oxidase subunit I gene (cox1) were obtained from 46 taxa of Laurencia and Palisada. The results were compared with rbcL/rbcL-S data and with the combination of the three markers (rbcL, rbcL-S and cox1) from the same set of species. A major analyses was performed using 86 rbcL sequences obtained from this study, added to those available at the GenBank. MATERIAL AND METHODS INTRODUCTION PHYLOGENY OF THE LAURENCIA COMPLEX (RHODOPHYTA) FROM THE ATLANTIC BASED ON rbcL, rbcL-S AND cox1 Valéria Cassano 1 , Mª Candelaria Gil-Rodríguez 2 , Abel Sentíes G. 3 , Jhoana Díaz Larrea 3 , Mariana C. de Oliveira 4 & Mutue T. Fujii 5 Instituto Botânica São Paulo 1. Dept. Biologia Vegetal, Universidade do Estado do Rio de Janeiro, Brazil (vcassano@uerj.br ); 2. Dept. Biología Vegetal (Botánica), Universidad de La Laguna, Canary Islands, Spain; 3. Dept. Hidrobiología, Universidad Autónoma Metropolitana – Iztapalapa, Mexico; 4. Dept. de Botânica, Universidade de São Paulo, Brazil; 5. Seção de Ficologia, Instituto de Botânica, São Paulo, Brazil). cox1 → 146 sites parsimoniously informative in 665 positions, rbcL/rbcL-S → 305 sites parsimoniously informative in 1564 positions; Combined analyses → 451 were parsimoniously informative in 2229 positions. cox1 → interspecific divergence: 5.5 to 13.6% (36 to 90 bp) → intraspecific divergence: 0 to 5.1% (0 to 29 bp) rbcL → interspecific divergence: 1.9 to 6.2% (27 to 89 bp) → intraspecific divergence: 0 to 1% (0 to 14 bp) Range of variation in the percentage of divergence among the markers studied 1 . 0 - 5,1 0 - 1 0 - 1 Intraspecific 1,6 - 13,6 1,5 – 15,6 1,9 – 6,2 Interspecific 3,2 - 11,5 15,7 - 47 5,6 - 11,4 Intergeneric 12,8 - 25,5 35,2 - 59,3 11 - 11,2 Ingroup x outgroup cox1 rbcL-S rbcL Markers / divergence % 1. Excludoed from the comparion Laurencia sp.2 and Laurencia sp.3. c. Laurencia intricata from the type locality (Antilles) is distinct from that reported for Brazil. Part of the citations of this species from Brazil corresponds to L. catarinensis; d. The taxa previously cited to the Brazilian coastline as Laurencia arbuscula, L. filiformis, L. majuscula and L. obtusa are the same taxonomic entity. Examination of materials-type allowed their identification as L. dendroidea, whose type locality is Brazil. Laurencia obtusa from Northern Atlantic is a distinct species, confirmed by the molecular data. Therefore, L. dendroidea must be considered as an independent species and not as a variety of L. obtusa, as currently treated; e. The confirmation of the taxonomic position of the L. aldingensis and Laurencia sp. 1 found in Brazil depends on comparison, at molecular level, with material (not yet available), of L. aldingensis and L. filiformis from Australia, their type locality; f. Laurencia sp.2 and Laurencia sp.3 can be new taxa for science. Financial support Fig. 19. Consensus tree derived from maximum parsimony (MP) analyses (2,000 replicates) of cox1 sequences. Bootstrap supports are given above, MP (left) and neighbor-joining (NJ, 2,000 replicates) (right); below, maximum-likelihood (ML, 100 replicates); * indicates bootstrap supports = 100%. Thick branches indicate Bayesian posterior probabilities ≥ 0.95. Fig. 20. Consensus tree derived from ML analyses (100 replicates) of rbcL/rbcL-S sequences. Bootstrap supports (2,000 replicates) are given above, MP (left) and NJ (right); below, ML. * indicates bootstrap supports = 100%. Thick branches indicate Bayesian posterior probabilities ≥ 0.95. Palisada Laurencia Chondria collinsiana RJ, Brazil Palisada papillosa Mexico (Pacific Ocean) P. perforata Canary Islands (type locality) P. papillosa Canary Islands P. perforata Brazil P. perforata Brazil P. papillosa Brazil P. papillosa Brazil P. perfotata Brazil P. flagellifera Brazil P. flagellifera Brazil Chondrophycus furcatus Brazil Laurencia filiformis Brazil L. filiformis Brazil L. Filiformis Brazil L. filiformis Brazil L. filiformis Brazil L. majuscula Brazil L. majuscula Brazil L. filiformis Brazil L. filiformis Brazil L. arbuscula Brazil L. obtusa Brazil L. obtusa Brazil L. arbuscula Brazil L. arbuscula Brazil Laurencia sp.1 Brazil Laurencia sp.1 Brazil L. intricata Cuba (type locality) L. aldingensis Brazil L. intricata Brazil L. intricata Brazil L. intricata /L. catarinensis Brazil L .intricata Brazil L. intricata Brazil L. intricata Brazil L. intricata Canary Islands L. intricata Canary Islands L. caduciramulosa Brazil L. caduciramulosa Brazil L. caduciramulosa Canary Islands Laurencia sp.2 Brazil Laurencia sp.3 Brazil 0.01 100/100 78 100/100 94 100/90 92 * 98/100 88 86/88 85 * 74/92 78 - / 79 60 * 95/- 97 * 100/100 98 89 69/84 * * 64/96 91 85/- 85 * 79/85 55 73/80 55 L. oliveirana Brazil (type locality) L. aldingensis Brazil L. filiformis Brazil Chondria collinsiana RJ, Brazil P. papillosa Mexico (Pacific Ocean) P. perforata Canary Islands (type locality) P. papillosa Canary Islands P. perforata Brazil P. perforata Brazil P. papillosa Brazil P. papillosa Brazil P. perforata Brazil Chondrophycus furcatus Brazil Laurencia filiformis Brazil L. filiformis Brazil L. filiformis Brazil L. filiformis Brazil L. arbuscula Brazil L. obtusa Brazil L. filiformis Brazil L. majuscula Brazil L. majuscula Brazil L. filiformis Brazil L. obtusa Brazil L. arbuscula Brazil L. arbuscula Brazil Laurencia sp.1 Brazil Laurencia sp.1 Brazil L. caduciramulosa Brazil L.caduciramulosa Brazil L. caduciramulosa Canary Islands L. oliveirana Brazil (type locality) L. aldingensis Brazil L. aldingensis Brazil L. Intricata Brazil L. intricata Brazil L. intricata / L. catarinensis Brazil L. intricata Brazil L. intricata Brazil L. intricata Brazil L. Intricata Canary Islands L. intricata Canary Islands Laurencia sp.2 Brazil Laurencia sp.3 Brazil 0.01 100/100 89 86 100/90 100/100 89 * * 90 93 96/100 100/100 * 97 95/98 100/100 89 61 - / - * 99 95/100 - 62/68 * 92/100 64 88/95 96 * 99 100/100 - / - 97 63 67/97 * 69 72/ - 62/92 56 100/100 98 78 57/69 Laurencia Palisada P. flagellifera Brazil P. flagellifera Brazil L. intricata Cuba (type locality) Chondria collinsiana RJ, Brazil P. flagellifera Brazil P. flagellifera Brazil Chondrophycus furcatus Brazil Laurencia sp.2 Brazil L. majuscula Brazil L. majuscula Brazil L. filiformis Brazil L. filiformis Brazil L. filiformis Brazil L. filiformis Brazil L. filiformis Brazil L. filiformis Brazil L. filiformis Brazil L. arbuscula Brazil L. obtusa Brazil L. arbuscula Brazil L. obtusa Brazil L. arbuscula Brazil L. filiformis Brazil Laurencia sp.1 Brazil Laurencia sp.1 Brazil L. aldingensis Brazil L. aldingensis Brazil L. intricata Brazil L. intricata Canary Islands L. intricata Canary Islands L. intricata Brazil L. intricata Brazil L. intricata Brazil L. intricata Brazil L. intricata Brazil L. caduciramulosa Brazil L. caduciramulosa Brazil L. caduciramulosa Canary Islands L. intricata Cuba (type locality) L. oliveirana Brazil (type locality) 5 100/100 100/100 84 92 100/100 94 78 89/91 91/98 99I/98 94 100/100 90 79 100/100 81 * 74 100/98 85/- - 100/100 84 88 100/100 90/94 56 63/57 80 - 88/79 92/87 - 56/- - L. catarinensis Brasil Laurencia sp.3 Brazil Palisada Laurencia Palisada papillosa Mexico (Pacific Ocean) P. papillosa Canary Islands P. perforata Canary Islands (type locality) P. perforata Brazil P. papillosa Brazil P. perforata Brazil P. papillosa Brazil P. perforata Brazil 0.1 Bostrychia r adicansUSA (AF259497) Centroceras sp. (as C. clavulatum) USA (A F259490) Ceramium brevizonatumMexico (AF259491) 97/98 Chondria dasyphylla USA (U04021) Chondria collinsianaBrazil 76/94 Laurencia c omplanataSouth Africa (AF465813) L. c araibica Mexico (Caribbean Sea) (EF658642) Laurencia sp.1 Brazil L. o btusa Ireland (AF281881) L. v iridis Canary Islands (EF685999) Laurencia sp.4 Canary islands (EF686004) L. i ntricataCuba L. i ntricata Gulf of Mexico (AF465809) L. i ntricataUSA (AY588410) L. i ntricataMexico (Caribbean Sea) L. i ntricata Mexico (Caribbean Sea) L. i ntricataMexico (Caribbean Sea) L. i ntricataMexico (Caribbean Sea) L. a ldingensisBrazil L. i ntricata/ L. catarinensis Brazill L. i ntricataBrazil L. i ntricata Brazil L. i ntricataCanary Islands L. f lexuosaSouth Africa(AF465815) L. c araibica Brazil L. o liveirana Brazil L. r igidaAustralia (AY920852) L. n atalensis South Africa (AF465816) L. v enustaMexico (Caribbean Sea) (EF061655) L. c aduciramulosaBrazil, RJ L. c aduciramulosaCanary Islands L. c f. brongniartii A ustralia (EF061654) L. t ranslucida Brazil, ES (AY588408) L. c f. brongniartii Taiwan (AF465814) L. f iliformis Brazill L. m ajusculaCanary Islands (EF686000) L. f iliformis Brazil L. f iliformis Brazil L. f iliformis Brazil (AF465808) L. o btusa Brazil L. f iliformis Brazil (AF465810) L. m ajuscula Brazil L. cf. filiformis Mexico (Caribbean Sea) L. cf. filiformis Mexico (Caribbean Sea) L. cf. filiformis Mexico (Caribbean Sea) Palisada p oiteaui var. gemmiferaMexico (Caribbean Sea) (EF061648) P. poiteaui var. g emmiferaCuba (EF061650) P. poiteaui var. g emmiferaMexico EF061649) P. poiteaui var. p oiteaui Mexico (Caribbean Sea) (EF061653) P. poiteaui var. p oiteaui USA(EF061652) L. sp.5 Mexico (Caribbean Sea) L. sp.5 Mexico (Caribbean Sea) L. m arilzaeCanary Islands (EF686001) L. m arilzaeCanary islands (EF686002) L. m arilzaeCanary Islands (EF686003) Laurencia sp.3 Brazil Laurencia sp.2 Brazil L. f lexilis Philippines (AF489860) Laurencia III P. papillosaMexico (Pacific Ocean) (AY588409) P. perforata Canary Islands (EU256329) P. perforata B razil P. perforata Brazil (EU256330) P. papillosaBrazil (AF465806) P. papillosaUSA (AF465807) P. perforata Mexico (Caribbean Sea) (EF658641) P. flagellifera Brazil (AF465804) P. flagellifera Brazil Chondrophycuscf. thuyoides Canary Islands (EF685998) P. papillosaPhilippines (AF489861) P. thuyoides PhilippinesFilipinas (AF489863) P. patentiramea Philippines (AF489862) P. corallopsis Mexico (Caribbean Sea) (EF061646) C. f urcatusBrazil Osmundea sinicola USA (Pacific Ocean) (AY588407) O. spectabilisvar. s pectabilis Mexico (Pacific Ocean) (AY172574) O. blinksii USA (Pacific Ocean) (AY172575) O. splendensMexico (Pacific Ocean) (AY172576) O. osmundaIreland (AF281877) O. pinnatifidaCanary Islands (EF686005) O. pinnatifidaIreland (AF281875) O. pinnatifidaFrance (AF259495) O. oederi ( as O . r amosissima) Ireland (AF281880) O. truncata I reland (AF281879) O. truncata Canary Islands O. cf. p innatifida Canary Islands Osmundeasp. Canary Islands 61/ - * 93/100 * * 59/- 55/58 61/97 77/84 75/77 - /89 69/- * 95/100 54/ - 99/100 * 99/100 98/100 * 85/100 * * * 63/67 98/99 73/80 90/90 76/97 * 97/100 63/- 91/93 * 95/99 99/100 * 68/81 77/83 84/86 97/100 * 99/81 * 96/- 99/- 93/94 63/- Laurencia I Palisada I Osmundea Palisada II Laurencia II Fig. 21. Consensus tree derived from ML analyses (100 replicates) of rbcL/rbcL-S and cox1 sequences. Bootstrap supports are given above, MP (left) and NJ (right) (2,000 replicates); below, ML. * indicates bootstrap supports = 100%. Thick branches indicate Bayesian posterior probabilities ≥ 0.95. Fig. 22. Consensus tree derived from Bayesian analyses of rbcL sequences. Bootstrap supports (2,000 replicates) are given above, MP (left) and NJ (right); * indicates bootstrap supports = 100%. Thick branches indicate Bayesian posterior probabilities ≥ 0.95. Phylogenetic analyses RESULTS AND DISCUSSION Taxa of the Laurencia complex included in this study 1 cm Fig. 1. Palisada perforata 1 cm P. papillosa Fig. 7. Laurencia filiformis Fig. 8. L. majuscula Fig. 10. L. arbuscula Fig. 6. Chondrophycus furcatus 2 cm Fig. 2. P. perforata Fig. 5. P. flagellifera Fig. 12. L. aldingensis Fig. 13. L. oliveirana Fig. 15. L. translucida Fig. 14. L. caduciramulosa Fig. 18. Laurencia sp. 3 Fig. 17. Laurencia sp. 2 Fig. 16. Laurencia sp. 1 1 2 3 5 6 7 8 Fig. 9. L. filiformis 9 10 Fig. 11. L. catarinensis 11 12 13 14 15 16 18 17