The occurrence of Laurencia marilzae (Ceramiales, Rhodophyta) in Brazil based on morphological and...

Botanica Marina 53 (2010): 143–152 � 2010 by Walter de Gruyter • Berlin • New York. DOI 10.1515/BOT.2010.018

2010/87

Article in press - uncorrected proof

The occurrence of Laurencia marilzae (Ceramiales,

Rhodophyta) in Brazil based on morphological and molecular

data

Renato Rocha-Jorge1,*, Valeria Cassano2, MarianaCabral Oliveira3 and Mutue Toyota Fujii4

1 Post-Graduate Program ‘‘Biodiversidade Vegetal e MeioAmbiente’’, Instituto de Botanica, Av. Miguel Estefano,3687, 04301-012 Sao Paulo, Brazil,e-mail: [email protected] Departamento de Biologia Vegetal, Instituto de BiologiaRoberto Alcantara Gomes, Universidade do Estado do Riode Janeiro, Maracana, 20550-013 Rio de Janeiro, Brazil3 Departamento de Botanica, Universidade de Sao Paulo,Rua do Matao 277, Sao Paulo 05508-900, Brazil4 Secao de Ficologia, Instituto de Botanica, Av. MiguelEstefano, 3687, 04301-012 Sao Paulo, Brazil

* Corresponding author

Abstract

Laurencia marilzae is recorded for the first time from thewestern Atlantic Ocean; it was found in Laje de SantosMarine State Park, Sao Paulo, southeastern Brazil. The spec-imens were collected in the rocky subtidal zone from 7 to15 m depth. The most distinctive characteristic of this speciesis the presence of corps en cerise in all cells of the thallus,including cortex, medulla, and trichoblasts. The phylogeneticposition of the species was inferred by analysis of the chlo-roplast-encoded rbcL gene sequences from 43 taxa, usingtwo other rhodomelacean taxa and two members of the Cera-miaceae as outgroups. Within the Laurencia assemblage, L.marilzae from Brazil and from the Canary Islands (typelocality) formed a distinctive lineage sister to all other Lau-rencia species analyzed. Male plants are described for thefirst time. This study expands the geographical distributionof L. marilzae to the western Atlantic Ocean.

Keywords: Atlantic Ocean; geographical distribution;Laurencia marilzae; rbcL; taxonomy.

Introduction

Laurencia marilzae Gil-Rodrıguez, Sentıes et M.T. Fujii wasdescribed recently from Tenerife, Canary Islands, Spain(Gil-Rodrıguez et al. 2009). The species is characterized bya distinctive yellow-orange color in its natural habitat, fourpericentral cells per vegetative axial segment, the presenceof secondary pit-connections between adjacent cortical cells,markedly projecting cortical cells, presence of one or twocorps en cerise in all cells of the thallus wcortex, medulla

(including pericentral and axial cells) and trichoblastsx,procarp-bearing segment with five pericentral cells, andtetrasporangia produced from the third and fourth pericentralcells, which are arranged in a parallel manner in relation tofertile branchlets.

During a phycological survey of Laje de Santos MarineState Park (LSMP), Sao Paulo, southeastern Brazil, Lauren-cia marilzae was found growing in the subtidal zone(7–15 m depth). The LSMP is an area of great importancefor ‘‘marine plants’’ in the broadest sense (Oliveira et al.2002) and, in the summer, it receives a strong input of SouthAtlantic Central Waters (SACW), a cold water mass thatbrings nutrients to the park area, reduces water temperatureand forms thermoclines. Because of this, it is possible to findin the park some genera that occur mainly in cool waters(i.e., Plocamium and Osmundea). Amado-Filho et al. (2006)identified 129 taxa of macroalgae collected from only the‘‘continental side’’ of the main rock (an area of ca. 7700 m2);within this small area, 45% of all species listed for the SaoPaulo State were found. The area is classified as a ‘‘hotspot’’for benthic marine algae on the Brazilian coast (Oliveira etal. 2002, Amado-Filho et al. 2006).

This study reports for the first time the presence of Lau-rencia marilzae in the western Atlantic Ocean; it was foundin LSMP, Sao Paulo, Brazil, and identified by morphologicalcharacters and chloroplast-encoded rbcL gene sequences.Male plants are described for the first time for this species.

Materials and methods

Study area

The LSMP is located in the State of Sao Paulo, southeasternBrazil, about 32 km from Santos city, which is the largestport in Latin America. The 5000 ha area comprises a largerock (Laje de Santos), which is 33 m high, 550 m long and185 m wide, a pair of cliffs called ‘‘Calhaus’’ and four reefs(Brilhante, Bandolim, Sul and Novo) (Figure 1). This groupof rocky substrata supports a major stand of high density anddiverse marine life (Neves 1997). Sample collections weremade by SCUBA diving on the Sul reef subtidal zone,between 7 and 15 m depth.

Morphological study

Voucher specimens and material for morphological studywere fixed in 4% formalin/seawater or pressed on herbariumsheets. Living specimens were examined to check for thepresence of corps en cerise. Transverse and longitudinal

144 R. Rocha-Jorge et al.: Laurencia marilzae in Brazil


Figure 1 Laurencia marilzae: southern coast of the State of Sao Paulo, showing the Laje de Santos Marine State Park and sampling sitesfrom which plants were collected; Sul Reef and Laje de Santos (S 24.19.629; W 46.11.109, using datum WGS 84).

hand-sections were made with a stainless steel razor bladeand stained with 0.5% aqueous aniline blue solution acidifiedwith 1 N HCl (Tsuda and Abbott 1985). Measurements oncharacters of taxonomic value were made with an ocularmicrometer. Photomicrographs were captured with a Sony W5digital camera (Tokyo, Japan) coupled to a Nikon EclipseE200 microscope (Tokyo, Japan). Vouchers are deposited inthe Herbarium of the Instituto de Botanica (SP), Sao Paulo,Brazil. Herbarium abbreviations follow Holmgren and Holm-gren (1998).

Molecular study

The sample used for molecular analysis was dried in silicagel. Total DNA was extracted, after grinding in liquid nitro-gen, using the Dneasy Plant Mini Kit (Qiagen, Valencia, CA,USA) following the manufacturer’s instructions. A total of1467 base pairs of the rbcL gene were amplified in threeoverlapping parts with the primer pairs FrbcLstart-R753,F492-R1150 and F993-RrbcS (Freshwater and Rueness1994), using the master mix of the Promega Corp. (Madison,WI, USA) Premix. All PCR products were analyzed by elec-trophoresis in 1% agarose to check product size. The PCRproducts were purified with the Qiagen QIAquick (Qiagen)purification kit in accordance with the manufacturer’sinstructions.

Sequencing was carried out with the BigDye TerminatorCycle Sequencing Reaction Kit (Applied Biosystems�,Hammonton, NJ, USA) on an ‘‘ABI PRISM 3100 GeneticAnalyzer’’ (Applied Biosystems). The primers used for thesequencing were those used for the PCR amplification. Thefull sequence was obtained from both DNA strands. Analysisof the sequences was performed with BioEdit (Hall 1999)software and aligned with the CLUSTAL algorithm (Thomp-son et al. 1994). The specimens sequenced in this study, plusthe other available sequences used in phylogenetic analysisare shown in Table 1, including GenBank accession numbers(NCBI GenBank 2003).

Phylogenetic relationships were inferred with PAUP*4.0b10 (Swofford 2002) and MrBayes v.3.0 beta 4 (Huelsen-beck and Ronquist 2001). Maximum-parsimony (MP),neighbor-joining (NJ) and maximum likelihood (ML) treeswere constructed using the heuristic search option, tree-bisection-reconnection branch swapping, unordered andunweighted characters, and gaps were treated as missingdata. Branch length was optimized using delayed transfor-mation (DELTRAN), which favors parallelisms over rever-sals. Support values for the relationships discovered in eachanalysis were calculated by performing bootstrap analyses(Felsenstein 1985), as implemented in PAUP*. Ten thousandheuristic search replicates were executed using the TBRbranch-swapping algorithm. Consistency (CI), homoplasy(HI) and retention indexes (RI) were computed to evaluate

R. Rocha-Jorge et al.: Laurencia marilzae in Brazil 145


Tab

le1

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inet

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2001

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F259

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leg.

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and

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2001

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ico,

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eB

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una

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n,le

g.C

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1998

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F281

879



the level of homoplasy in the most parsimonious tree. Themodel used in the Bayesian analysis was the general-time-reversible model of nucleotide substitution with invariantsites and gamma-distributed rates for the variable sites(GTRqIqG). This model was selected based on ML ratiotests implemented by the software Modeltest version 3.06(Posada and Crandall 1998) with a significance level of 0.01.For the Bayesian analysis, we ran four chains of the Markovchain Monte Carlo (one hot and three cold), sampling onetree every 10 generations for 1,000,000 generations startingwith a random tree. The first 100,000 generations were dis-carded as ‘‘burn in’’. A 50% consensus tree (majority ruleas implemented by PAUP*) was computed after the ‘‘burnin’’ point. The range of rbcL divergence values within andamong species was calculated using uncorrected ‘‘p’’ dis-tances with PAUP*.

Results

Morphological analyses

Laurencia marilzae Gil-Rodrıguez, Sentıes et

M.T. Fujii (2009: 45, figs 1–5)

(Figures 2–15)

Holotype Deposited in TFC Phyc 13129.

Type locality Punta del Hidalgo, Tenerife, Canary Islands,Spain.

Plants terete, cartilaginous, forming pink-red tufts up to3 cm high, attached to the substratum by a discoid holdfastand by descending branches (Figure 2). Main axes 640–1015mm in diameter, with branches irregularly alternate andarranged in a spiral, bearing to 2–3 orders of branches (Fig-ure 3). One or two corps en cerise present in each cell ofthe cortical and medullary regions of the thallus (Figures 4and 5), including axial and pericentral cells; one per tricho-blast cell. In surface view, cortical cells secondarily pit con-nected, 39–84 mm long and 22–47 mm wide in mid-portionsof thalli. In transverse section, thalli with one layer of pig-mented cortical cells and four or five layers of colorless med-ullary cells (Figure 6). Cortical cells 22–45 mm long and20–40 mm wide; medullary cells rounded or slightly radiallyelongated, 67–138 mm long and 34–92 mm wide in the mid-portions of the main axes. Medullary cell walls lacking len-ticular thickenings. Each vegetative axial segment cuts offfour pericentral cells (Figure 7). In median longitudinal sec-tion, cortical cells near apices of branchlets project markedly(Figure 8). Tetrasporangial branchlets are cylindrical-clavate,simple or compound, 452–3233 mm long and 418–789 mmin diameter (Figure 9). Tetrasporangia produced from thethird and fourth pericentral cells (Figure 10). The fertile peri-central cells cut-off two pre-sporangial cover cells, the tetra-sporangial initial and one post-sporangial cover cell (Figure11). Mature tetrasporangia tetrahedrally divided, 20–80 mmin diameter, in parallel arrangement (Figure 12). Malebranches characteristically swollen, 375–500 mm in diameter(Figure 13). In longitudinal section through a fertile branch-let, the spermatangial pits are cup-shaped, and an axial cell

row is discernible at the base (Figure 14). Spermatangial tri-choblasts arise from axial cells, consisting of fertile and ster-ile branches (Figure 15); the fertile branches produce manyovoid spermatangia, 6.5–8.5 mm long and 2–5 mm in diam-eter, and terminate in vesicular sterile cells, 25–42.5 mmlong and 17.5–35 mm in diameter; each spermatium pos-sesses an apical nucleus. Female plants were not observed.

Habitat Epilithic specimens were collected in the subtidalzone from 7 to 15 m depth, forming turfs with other macro-algae, mainly Plocamiun brasiliense (Greville) M. Howe etW.R. Taylor.

Material examined Brazil, Sao Paulo, LSMP, Parcel doSul, 25 March 2007, leg. R. Rocha-Jorge (SP399814); Lajede Santos, 18 August 2008, leg. R. Rocha-Jorge (SP399826).

Molecular analyses

Variation in rbcL sequences A total of 43 sequenceswere analyzed including the outgroups Bostrychia radicans,Chondria dasyphylla, Centroceras sp. was C. clavulatum (C.Agardh) Montagnex and Ceramium brevizonatum (Table 1).A data set of 1448 base pairs was produced and the sequenc-es were aligned without ambiguity. Intergeneric divergencevaried from 7 to 11.5% for Palisada and Laurencia, from10 to 13% for Palisada and Osmundea, and from 9 to 13.7%for Laurencia and Osmundea. Interspecific divergence forspecies of Laurencia other than L. marilzae varied from 1.2to 9.7%, whereas for those of Palisada divergence variedfrom 1.1 to 6.4%. The clade formed by L. marilzae divergedfrom the rest of the Laurencia species by 8.6–10.7%. Theintra-specific divergence in L. marilzae was very low (0–0.2%). There was a 0–0.07% genetic divergence between therbcL sequences of Canarian and Brazilian samples. Thesequence of the holotype (EF686002) was 100% identical tothat of Brazilian L. marilzae.

Phylogeny The data set consisted of 892 constant char-acters, 416 parsimony informative sites and 140 parsimonynon-informative sites. MP produced 4 trees of 1763 steps(CIs0.442, RIs0.713, HIs0.557). The topology of Baye-sian tree with corresponding bootstrap values is shown inFigure 16. The analyses indicate a monophyletic Laurenciacomplex with moderate to high bootstrap and posterior prob-ability values in relation to the members of the outgroup.The Laurencia complex was separated into three clades cor-responding to the three genera: Laurencia, Osmundea, andPalisada. In all of the analyses, the earliest diverging cladewas the genus Osmundea, with eight species and high boot-strap support; the genera Laurencia and Palisada were sistergroups, but without significant support. The genus Palisada(with six species) formed a well-supported monophyleticclade. The genus Laurencia included 14 taxa and formed amonophyletic clade with support only in the Bayesian anal-ysis (99% PP). Laurencia marilzae was monophyletic, withhigh support in all analyses and was segregated from otherLaurencia sensu stricto, forming a distinct clade. The rela-



Figures 2–7 Laurencia marilzae: morphology and anatomy.(2) Habit of a plant. (3) Detail of a branch. (4, 5) Details of corps en cerise present in all cells of the thallus: (4) Cortical cells in superficialview showing one or two corps en cerise per cell (arrows). (5) Longitudinal section of a branch showing corps en cerise in cortical andmedullary cells (arrows). (6) Transverse section of a main axis. (7) Transverse section of the upper portion of a branch showing an axialcell (a) with four pericentral cells (p). Scale bars: 5 mm in Figure 2; 2 mm in Figure 3; 40 mm in Figures 4 and 5; 100 mm in Figure 6;25 mm in Figure 7.

tionship between the L. marilzae clade and the other Lau-rencia species was not clearly resolved in MP, NJ or MLphylogenetic analyses.

Discussion

The Brazilian specimens share with Laurencia marilzae fromthe type locality the majority of morphological features

described by Gil-Rodrıguez et al. (2009), especially the pres-ence of corps en cerise in all cells of the thallus, projectedcortical cells in apical and subapical portions of the branchesand two fertile pericentral cells in tetrasporangial segments(the third and the fourth). Our specimens differed mainly incolor (pink-red in Brazil vs. yellow-orange in Canarian spec-imens in their natural habitats, although cultured L. marilzaefrom the Canary Islands has pink-red thalli), in the height ofthalli (smaller in Brazilian specimens), and in the number of



Figures 8–15 Laurencia marilzae: anatomy and reproduction.(8) Longitudinal section of a branch showing projecting cortical cells. Note corps en cerise in cortical and medullary cells (arrows). (9)Tetrasporangial branches. (10) Transverse section near the apex of a branchlet showing tetrasporangial axial segment; axial cell (blackarrow) with two vegetative pericentral cells (p) and two fertile pericentral cells (white arrows). (11) Detail of a fertile pericentral cell (fp)with two presporangial cover cells (pr), the tetrasporangial initial (te) and one postsporangial cover cell (po). (12) Longitudinal sectionthrough a tetrasporangial branchlet showing parallel arrangement of the tetrasporangia (arrows). (13) Male branches (arrows). (14) Longi-tudinal section through a male branchlet showing spermatangial branches in cup-shaped tip (arrow). (15) Detail of trichoblast-type sper-matangial branch. Note trichoblast with two laterals branches: sterile (arrowhead) and spermatangial branches (arrow). Scale bars: 40 mmin Figure 8; 1 mm in Figure 9; 25 mm in Figure 10; 20 mm in Figure 11; 100 mm in Figures 12 and 14; 1 mm in Figure 13; 50 mm inFigure 15.



Figure 16 Consensus tree derived from Bayesian analyses of rbcL sequences.Bootstrap supports (2000 replicates) are given above the branches for maximum parsimony (left) and neighbor joining (right) and belowfor maximum likelihood; * indicates bootstrap supportss100%. Thick branches indicate Bayesian posterior pG0.95. L., Laurencia; P.,Palisada; O., Osmundea.

corps en cerise per cell (one or two in Brazil and only onein Canarian specimens).

Serio et al. (2000), Furnari et al. (2001) and Wynne et al.(2005) reported a species of Laurencia morphologically sim-ilar to L. marilzae and named L. majuscula (Harvey) Lucasfrom the Mediterranean Sea (Italy) and off Oman, respec-tively. The Italian specimens are orange in color (reddish in

shaded habitats), possess one, occasionally two corps encerise per cortical cell, rarely also in medullary cells, withtetrasporagia produced from (2nd) 3rd and 4th pericentralcells (Serio et al. 2000, Furnari et al. 2001). In Omani spec-imens, most of the thallus is red in color, and medullary cellsrarely have corps en cerise (Wynne et al. 2005). All othervegetative and reproductive morphological characters are



shared between L. marilzae and L. majuscula from Italy andOman. The similarities observed among these specimens,including overlaps in color, number and location of corps encerise per cell, suggest that they may belong to the sametaxonomic entity, viz., L. marilzae, and this should bechecked with molecular data.

The intergeneric divergence values obtained in this work(7–13.7%) for rbcL sequences are comparable to thosereported by other authors for the Laurencia complex (11–13%, Nam et al. 2000 and McIvor et al. 2002; 10–12%,Dıaz-Larrea et al. 2007; 6–12%, Cassano et al. 2009). Theinterspecific divergence values reported here (1.3–9.7%) forrbcL sequences are comparable to those reported by otherauthors for species of the genera Osmundea, Laurencia andPalisada (5–9%, Nam et al. 2000; 2–9%, McIvor et al. 2002;6–9%, Dıaz-Larrea et al. 2007; 3–10%, Cassano et al. 2009).The molecular phylogeny indicates that the four specimensof L. marilzae (three from Canary Islands and one fromBrazil) form a well-supported clade with low levels of genet-ic variation between rbcL sequences (0–0.2%). This intra-specific divergence value is comparable to those reported forsamples of P. poiteaui (J.V. Lamouroux) K.W. Nam var. poi-teaui-P. poiteaui (J.V. Lamouroux) K.W. Nam var. gemmifera(Harvey) Sentıes, M.T. Fujii et Dıaz-Larrea (0.01–0.02%) byDıaz-Larrea et al. (2007), for samples of P. perforata (Bory)K.W. Nam (0–0.4%) and for O. pinnatifida (Hudson) Stack-house (0.4%) by Cassano et al. (2009). The high geneticdistance between L. marilzae and Laurencia sensu stricto(8.6–10.7%) and the early divergence of L. marilzae withinthe Laurencia lineage suggest that L. marilzae represents anew genus. The morphological similarities and the moleculardata support the occurrence of L. marilzae in Brazil. Thisreport expands the geographical distribution of this speciesto the western Atlantic Ocean.

Acknowledgements

The authors thank the Instituto Florestal for permission to collectin LSMP. This work was supported by the Fundacao de Amparo a¸Pesquisa do Estado de Sao Paulo (FAPESP) to R.R.J., by Coorde-nacao de Aperfeicoamento de Pessoal de Nıvel Superior (CAPES)¸ ¸to V.C. and by Conselho Nacional de Desenvolvimento Cientıficoe Tecnologico (CNPq) to M.T.F. and M.C.O. for a Research Pro-ductivity Fellowship (Proc. 308426/2006-1, and 301217/2007-6).This research was facilitated by support from FAPESP and CNPq,Proc. 478941/2006-4.

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Received 11 September, 2009; accepted 4 January, 2010

The occurrence of Laurencia marilzae (Ceramiales, Rhodophyta) in Brazil based on morphological and...

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