Description of Hypnea pseudomusciformis sp. nov., a new species based on molecular and morphological analyses, in the context of the H. musciformis complex (Gigartinales, Rhodophyta)

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Journal of Applied Phycology ISSN 0921-8971 J Appl PhycolDOI 10.1007/s10811-014-0488-y

Description of Hypnea pseudomusciformissp. nov., a new species based on molecularand morphological analyses, in thecontext of the H. musciformis complex(Gigartinales, Rhodophyta)Fabio Nauer, Valéria Cassano & MarianaC. Oliveira

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Description of Hypnea pseudomusciformis sp. nov., a new speciesbased on molecular and morphological analyses, in the contextof the H. musciformis complex (Gigartinales, Rhodophyta)

Fabio Nauer & Valéria Cassano & Mariana C. Oliveira

Received: 30 June 2014 /Revised: 1 December 2014 /Accepted: 2 December 2014# Springer Science+Business Media Dordrecht 2014

Abstract Hypnea musciformis, a red macroalga widely dis-tributed in tropical and subtropical coasts around the world,has great economic importance as a source of carrageenan forindustrial production. In this work, the DNA barcode markerCOI-5P and the plastid rbcL gene, in addition to morpholog-ical studies, were used to investigate this species on the coastof Brazil and compare it with specimens from other countries.A total of 128 sequences were obtained in this study for 100specimens fromBrazil and 15 specimens from other countries,including the type locality in Italy. The divergence betweenSouth American sequences and sequences from Italy forH. musciformis was significantly high for both markers, indi-cating that the specimens found on the Brazilian coast belongto a different species. Considering the data gathered frommolecular markers and morphological analysis, the specimenspreviously identified morphologically as “H. musciformis”,“Hypnea nigrescens”, and “Hypnea valentiae” collected inBrazil were considered morphological variations of the newspecies descr ibed in this paper, named Hypneapseudomusciformis Nauer, Cassano & M.C. Oliveira, sp.nov. The identification of specimens based only on morpho-logical characteristics proved to be unsatisfactory for reasonsthat could be attributed to phenotypic plasticity in this species.Thus, the technique of DNA barcoding, especially with re-spect to the COI-5Pmarker, was essential for the identificationand definition of species, revealing scenarios that would oth-erwise be ignored by using only morphological analysis.

Keywords Hypnea . COI-5P . rbcL . DNAbarcode .

Morphology . Phylogeny . Taxonomy

Introduction

The genus Hypnea J.V. Lamouroux (1813) includes approxi-mately 67 species distributed mostly in warm waters aroundthe world (Mshigeni and Chapman 1994; Geraldino et al.2009). The species Hypnea musciformis (Wulfen) J.V.Lamouroux is economically important as a source of carra-geenan, a sulfated polysaccharide extracted from the cellwalls, and widely used in the food, cosmetics, and pharma-ceutical industries (Knutsen et al. 1995). In Brazil,“H. musciformis” is the most common and important speciesof Hypnea, presenting a wide geographical distribution alongthe Brazilian coastline (Nunes 2005). In the intertidal region,this species can be found attached to rocks in the shallowsubtidal zone, as epiphytes, mainly on Sargassum spp., andpresents a large phenotypic plasticity, including color varia-tion (Schenkman 1986; Reis et al. 2003).

“H. musciformis” has been used for the production ofcarrageenan in Brazil. The content of k-carrageenan of“H. musciformis” is between 25 and 48 % of its dry weight,and it is of excellent quality (Reis and Yoneshigue-Valentin1998). In 2004, the consumption of carrageenan was100 T month−1, of which 10 T month−1 was produced usingbiomass obtained from natural beds of “H.musciformis” in thenortheastern region of the country (Furtado 2004).Mariculture studies conducted in Brazil (Oliveira-Filho andBerchez 1987; Reis et al. 2006) showed that “H. musciformis”is the only species of the genus that can be used as rawmaterial for extraction of κ-carrageenan as a result of its largebiomass found on the Brazilian coast.

Schenkman (1986) made the first mention of Hypneanigrescens (Wulfen) J.V. Lamouroux and Hypnea valentiae

Electronic supplementary material The online version of this article(doi:10.1007/s10811-014-0488-y) contains supplementary material,which is available to authorized users.

F. Nauer (*) :V. Cassano :M. C. OliveiraDepartment of Botany, Biosciences Institute, São Paulo University,Rua do Matão 277, São Paulo 05508-090, Brazile-mail: [email protected]

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(Turner) Montagne for the Brazilian coast, but the authorargued that these two species could be easily confused with“H. musciformis” and only the combined analyses of habitat,morphology, anatomy, and growth would permit correctidentification. Guimarães (2011) carried out a study of thegenus based on molecular markers (COI-5P, UPA, and rbcL)and morphology for the coast of São Paulo state and verifiedthat the specimens identified as “H. nigrescens” were, in fact,a morphological variation of “H. musciformis”.

Researchers have increasingly used molecular tools to aidin the taxonomic and biogeographic studies of seaweeds. Theplastid molecular marker rbcL was used for phylogeneticstudies within the genus Hypnea in Asia and South Africa(Yamagishi and Masuda 2000; Fredericq and Hommersand2003; Yamagishi et al. 2003; Geraldino et al. 2006). Themitochondrial marker COI-5P was used as a DNA barcodefor specimens of Hypnea in different locations in Asia(Geraldino et al. 2006, 2009, 2010).

In this paper, we combined molecular (DNA Barcodes andrbcL) and morphological analyses to resolve the taxonomicposition of “H. musciformis”, “H. nigrescens”, and“H. valentiae” from Brazil. Our data indicate the existenceof a H. musciformis species complex worldwide and led us todescribe a new species, Hypnea pseudomusciformis Nauer,Cassano & M.C. Oliveira, to accommodate these taxa previ-ously referred to Brazil.

Materials and methods

Taxon sampling and morphology

In Brazil, 100 specimens morphologically identified as“H. musciformis”, “H. nigrescens”, or “H. valentiae” werecollected along the coast (ranging from 2° 54′ 14.51″ S to27° 08′ 42.44″ S in latitude) in intertidal and subtidalzones. Another 15 specimens were obtained from othercountries, including Uruguay, Barbados, and theDominican Republic from the Caribbean Sea; Floridaand North Carolina from the USA; Namibia in Africa;and Gulf of Trieste, Italy (Table S1 in the SupplementaryMaterial). Voucher specimens were housed at SPF and SPherbaria (abbreviations based on Thiers 2014).

For molecular analyses, apical regions were separated fromthe rest of the thallus, cleaned, and stored in silica gel. Theremainder of the samples was stored in 4 % formalin solutionfor morphological studies. Samples were sectioned by handwith a razor blade and stained with 1 % aniline blue acidifiedwith 1 N HCl. Following Masuda et al. (1997), habit anddiagnostic characters of each specimen were obtained withdigital capture and image analyses, using a compound micro-scope with a Leica DM4000 digital camera.

DNA extraction, PCR, and sequencing

For amplification of molecular markers, the Phire Plant DirectPCR Kit (Thermo Scientific Company, USA) was used, ac-cording to the protocol of the supplier, where the step of DNAextraction is suppressed, and the polymerase chain reaction(PCR) is made with the direct addition of fragments of thespecimen into the PCR tube itself. Specific primer pairs wereused for the amplification and sequencing reaction of eachgene, including FrbcL-R753, F492-R1150, and F993-RrbcSstart for rbcL (Freshwater and Rueness 1994) and GazF1-GazR1 for COI-5P (Saunders 2005). All PCR amplificationswere carried out in a Techne TC-4000 thermal cycler (BibbyScientific Ltd., UK). For rbcL, PCR was performed with aninitial denaturation step at 95 °C for 4 min, followed by 35cycles of 1 min at 94 °C, 2 min at 45 °C, and 3 min at 72 °C,with a final 5-min extension cycle at 72 °C. For COI-5P, PCRwas performed with an initial denaturation step at 95 °C for5 min, followed by 35 cycles of 30 s at 94 °C, 1 min at 52 °C,and 2 min at 72 °C, with a final 1 min extension cycle at72 °C.

The PCR products were purified by column PCR™ GTXDNA and Gel Band Purification Kit (GE Healthcare,Pittsburgh, USA), according to the protocol of the supplier.Sequencing of 10 to 40 ng of purified PCR product was doneusing the same primers of the PCR reactions described aboveand the sequencing kit BigDye Terminator Cycle SequencingReady Reaction (Applied Biosystems, USA), according to theprotocol of the supplier. The samples were sequenced on amodel 3100 ABI PRISM™ automatic sequencer (AppliedBiosystems). Consensus sequences were generated for eachmarker per sample aligning forward and reverse sequencesmanually using the program BioEdit (Hall 1999).Inconsistencies in the sequences were reviewed by checkingthe chromatograms.

A total of 73 Hypnea sequences for rbcL (17 new and 56from GenBank) and 114 sequences for COI-5P (111 new and3 from GenBank) were used in the analyses. For the analysesof the rbcL,Calliblepharis fimbriata (AF385653) was used asoutgroup.

The COI-5P cluster tree was constructed by neighbor-joining (NJ) and by maximum likelihood (ML) using theMEGA5 program (Molecular Evolutionary GeneticsAnalysis, Tamura et al. 2011) with 2,000 bootstrap replicates.For rbcL phylogenetic analyses, the model of evolution wasselected usingMrModeltest 2.2 (Nylander 2004), based on theAkaike information criterion (AIC). Phylogenetic trees wereconstructed in MrBayes v3.1.2. (MB, Huelsenbeck &Ronquist, 2001) based on two runs with four MCMC chainsof 4,000,000 generations each with sampling every 1,000generations, where the first 100,000 generations in both runswere discarded as burn-in to build the consensus tree andPAUP 4.0b8 using NJ (with substitution model Tamura and

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Nei 1993), maximum parsimony (MP), and ML. Maximumparsimony and NJ analyses were performed under heuristicsearch with 2,000 bootstrap replicates, and ML was doneusing stepwise addition for 10 bootstrap replicates(Felsestein, 1985).

Results

In the rbcL phylogeny of Hypnea (Fig. 1), South American(Brazil and Uruguay) specimens obtained in this work formed amonophyletic group with high support in all analyses. Samplesof H. musciformis from the North Atlantic and theMediterranean Sea, including the type locality, Gulf ofTrieste, Italy, formed another monophyletic group with highsupport in all analyses. These two groups formed a monophy-letic clade in all analyses, indicating their close relationship.The divergences of South American and Italian specimens are1.7 to 2.3 % for rbcL (Table 1), and the maximum intraspecificdivergence found for this marker for the South Americanspecimens was 0.7 %. These results indicate that the Braziliansamples, previously identified as “H. musciformis”, are repre-sentatives of a distinct species.

Based on the COI-5P marker (Fig. 2), sequences of spec-imens from Brazil previously identified as “H. musciformis”,“H. nigrescens”, and “H. valentiae” formed a monophyleticcluster, with high bootstrap support, sister to another clustercontaining sequences of specimens from other regions. Thesequence of H. musciformis from Italy formed a clade with aspecimen from Namibia on the Atlantic coast of Africa, andthis clade grouped with a specimen from France (GQ141881).The two sequences from the USA western Atlantic groupedtogether, but the sequence from the Dominican Republic wasnot found to be closely related to them. Finally, the twosequences of Hawaii were grouped with the sequence ofBarbados with 0.9 % of divergence. Divergence betweenSouth American sequences and the sequence ofH. musciformis from Italy is 5.9 to 7.1 % for the COI-5Pmarker (Table 1), indicating that they are not the same species.

The Brazilian specimens were further divided into twogroups, one formed by sequences from specimens collectedin the northeast region of the country (group 1) with highbootstrap support and the other formed by sequences from thesoutheast and southern regions, which also includes a fewsamples collected in the northeast (group 2). The specimensfrom Uruguay also grouped within group 2 (Fig. 2). Thedivergence between Brazilian specimens and Uruguayanspecimens is 0.5 to 1.8 % (Table 1).

The morphology by which each of the specimens waspreviously identified is marked in Fig. 2. Group 1 includesspecimens presenting both the typical morphology of“H. musciformis” (epiphyte with numerous tendrils at the api-ces of the branches; marked by a black circle) as well as

specimens originally identified as “H. nigrescens” (marked bya square). Group 2 includes specimens morphologically identi-fied as “H. musciformis”, “H. nigrescens”, and “H. valentiae”(marked by a gray triangle). This last morphological type seemsto be found only in southeastern and southern Brazil. The COI-5P sequences of these different morphological variants are verysimilar or, in many cases, identical to each other. Based on thesemolecular data, South American specimens identified as“H. musciformis”, “H. valentiae”, and “H. nigrescens” aremorphological variations of the same species.

Intraspecific variation for marker COI-5P (465 bp) was 0–9 bp (0–1.8 %), and for marker rbcL (1,355 bp), it was 0–10 bp (0–0.7 %). Including the sequences fromGenBank usedin the analyses, interspecific variation was 69–130 bp (9.8 to18.2 %) for COI-5P and 48–92 bp (3.6 to 6.8 %) for rbcL.Therefore, no overlap between intraspecific and interspecificvariation was observed. Brazilian specimens showed a signif-icant divergence with respect to specimens of H. musciformisfrom other parts of the world, including the region of the typelocality for the species.

Based on our molecular results, a new species is describedfor the taxon previously named as “H. musciformis”,“H. nigrescens,” and “H. valentiae” from Brazil:

Hypnea pseudomusciformis Nauer, Cassano & M.C.Oliveira, sp. nov. (Figs. 3 and 4)Description: Plants erect, epilithic or epiphytic, terete, carti-laginous in texture, forming tufts up to 16 cm tall, reddish,brownish, greenish when alive, attached to the substrate bydiscoid holdfast and stolon-like branches or often by tendrilswhen epiphytic. Irregular branching in various planes, in acuteor right angles, with up to three orders of branches. Spiny-likebranchlets numerous and irregularly arranged throughout thethalli with 225–1,200 μm in length. Anastomoses absent.Habit highly variable, depending on the habitat (Fig. 3a–h).Epiphytes often growing at the shallow subtidal zone, fixed tothe host plant by abundant tendrils at the ends of branches andbranchlets (Figs. 3a–c, f and 4b, c), with branches entangledby tendrils of the same plant or different plants of the samespecies. Branches and branchlets may also have straight orcurved apices (Figs. 3e and 4a). Thalli have substantiallyuniform diameter throughout their length. Main axis withmany lateral branches 1.5–9.0 cm in length and 300–800 μm in diameter. In some cases, the diameter of the first-order branches can be close to that of the main axis but neverequal. Epilithic plants form tufts at the intertidal zone withnumerous erect axes, 1.0–11 cm in length and 500–1,700 μmin diameter, arising from a discoid holdfast and from stolon-like branches (Fig. 4d). Tendrils are rare, and the main andlateral branches are usually straight or curved. In some popu-lations, all the main axes reach nearly the same height with thelonger axis placed at the central portion of the tufts. Thisarrangement of the thalli resembles the bristles of a brush.

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Fig. 1 Bayesian analysis for rbcL sequences for Hypnea species. On thebranches are the bootstrap values (only values above 70 were considered)for neighbor-joining (NJ), maximum parsimony (MP), and maximumlikelihood (ML). Posterior probabilities are represented as branch

thickness. Sequences generated in this work are in bold. Accessionnumbers and country of origin, when available, are included forsequences obtained from GenBank

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In cross section in subapical region, thalli present one totwo layers of pigmented cortical cells and two to three layersof colorless medullary cells, which decrease in size toward thecortex (Fig. 4h). Small cortical cells, elongated, 7.5–12.5 μmin length and 5.0–12.5 μm in diameter. Large medullary cells,rounded to elliptical, 42.5–125 μm in length and 37.5–137.5 μm in diameter (Fig. 4h–i). Axial cells rounded toelliptical, usually equal to, or less than, periaxial cells, or,rarely, larger. Each axial cell is surrounded by five to sevenperiaxial cells (Fig. 4i), 37.5–175 μm in length and 37.5–137.5 μm in diameter. Medullary cell walls uniformly thick-ened. Lenticular thickening frequent in periaxial cells andother medullary cells (Fig. 4j). Tetrasporangial branchletsdistributed from the basal to the apical region of the branch-lets, 600–2,000 μm in length and 225–625 μm in diameter(Fig. 4f–g). Tetrasporangial sori completely encircling thebasal portions of spiny branchlets or partially encircling themiddle portions of branchlets (Fig. 4k–l). Tetrasporangiazonately divided, 25–50 μm in length and 15–55 μm indiameter. Cystocarps globose, solitary or in clusters, foundin the apical portions of the branches, measuring up to 150 μm

in length and up to 187.5 μm in diameter (Fig. 4e, m). Malegametophytes not found.Remarks: The size and number of branches and spinybranchlets vary in the northeast, southeast, and southern re-gions of the country. In general, specimens of the northeasternregion may reach almost three times the size (~16 cm inlength) of specimens from the rest of the country, with morebranches and numerous and long spiny branchlets that canreach 1.5 cm in length (Jesus 2012), while some northeasternspecimens and specimens of southeastern and southern re-gions are smaller in size and can reach up to 9 cm tall, withbranching that carries fewer spiny branchlets compared to thespecimens of the northeast region but more abundant whencompared with other species of Hypnea; distinct fromH. musciformis by analysis of rbcL (GenBank KM509072and KM509073) and COI-5P (GenBank KM523207) molec-ular sequences.Holotype: Praia de Itaoca, Itapemirim, State of Espírito Santo,Brazil, 06.V.2012, tetrasporic plant, F. Nauer, C. Iha & B.Torrano-Silva (SPF57526). (KM509065) rbcL, (KM523183)COI-5P sequences of holotype, respectively.

Table 1 Divergence values for COI-5P (lower triangle) and rbcL (upper triangle) among specimens of Hypnea musciformis andHypnea pseudomusciformis sp. nov. sequenced in this work

Divergence (%)

H. pseudmusciformis sp.nov.

Specimen 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 rbcL1. Brazil, São Paulo (SPF57441) – 0.0 0.0 0.2 0.4 0.5 0.6 0.2 0.2 0.2 0.1 0.1 2.2 1.9 1.8 1.7

2. Brazil, Espírito Santo(SPF57526)

0.0 – 0.0 0.2 0.4 0.5 0.6 0.2 0.2 0.2 0.1 0.1 2.2 1.9 1.8 1.7

3. Brazil, Santa Catarina(SPF57496)

0.0 0.0 – 0.2 0.4 0.5 0.6 0.2 0.2 0.2 0.1 0.1 2.2 1.9 1.8 1.7

4. Brazil, Bahia (SPF57608) 1.3 1.3 1.3 – 0.2 0.2 0.3 0.3 0.3 0.3 0.3 0.3 2.3 2.0 1.9 1.8

5. Brazil, Ceará (SPF57560) 1.6 1.6 1.6 0.0 – 0.5 0.6 0.6 0.6 0.6 0.5 0.5 2.6 2.2 2.1 2.0

6. Brazil, Ceará (SPF57554) 1.6 1.6 1.6 0.0 0.0 – 0.7 0.6 0.6 0.6 0.6 0.6 2.6 2.3 2.2 2.2

7. Brazil, Paraíba (SPF57577) 1.6 1.6 1.6 0.0 0.0 0.0 – 0.7 0.7 0.7 0.6 0.6 2.7 2.3 2.3 2.3

8. Uruguay, Punta del Este(SPF57824)

0.5 0.5 0.5 1.3 1.3 1.3 1.3 – 0.0 0.0 0.3 0.3 2.3 2.0 1.9 1.9

9. Uruguay, Punta del Este(SPF575825)

0.5 0.5 0.5 1.3 1.3 1.3 1.3 0.0 – 0.0 0.3 0.3 2.3 2.0 1.9 1.9

10. Uruguay, Punta del Este(SPF57826)

0.5 0.5 0.5 1.3 1.3 1.3 1.3 0.0 0.0 – 0.3 0.3 2.3 2.0 1.9 1.9

11. Uruguay, Jose Ignácio(SPF57830)

0.9 0.9 0.9 1.8 1.8 1.8 1.8 0.5 0.5 0.5 – 0.0 2.3 1.9 1.8 1.7

12. Uruguay, Jose Ignácio(SPF57829)

0.9 0.9 0.9 1.8 1.8 1.8 1.8 0.5 0.5 0.5 0.0 – 2.3 1.9 1.8 1.7

H. musciformis 13. Barbados, Caribbean Sea(SPF57447)

4.4 4.4 4.4 4.8 4.8 4.8 4.8 4.4 4.4 4.4 3.9 3.9 – 1.5 1.6 1.7

14. USA, Florida (SPF57598) 5.6 5.6 5.6 6.5 6.5 6.5 6.5 5.2 5.2 5.2 5.6 5.6 3.5 – 0.3 0.3

15. USA, N. Carolina (SPF57598) 5.6 5.6 5.6 6.5 6.5 6.5 6.5 5.2 5.2 5.2 5.6 5.6 3.5 0.0 – 0.4

16. Italy, Triestea (SPF57594) 6.3 6.3 6.3 7.1 7.1 7.1 7.1 5.9 5.9 5.9 6.3 6.3 3.3 1.6 1.6 –

COI-5P

The data in bold represent H. pseudomusciformis of different geographic locations’ intraspecific divergence. Data in bold and italics represent thedivergences between the specimen from Italy (type locality) and from the other geographic regionsa Specimen from type locality: Trieste, Italy (Adriatic Sea)

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Etymology: The specific name refers to the resemblance toH. musciformis—pseudo, false.Habitat: The specimens were collected from the intertidalregion in areas exposed to the impact of waves or in protectedrocky shores and reefs near beaches of the subtidal zone, up to2 m deep, growing on Sargassum C. Agardh.

Previous misapplied names for Brazil are as follows:H. musciformis sensu Joly (1965, pp. 165–166, Figs 371–373, 378), Oliveira-Filho (1977, pp. 57–58), Cordeiro-Marino (1977, pp. 70–72, Figs. 182–184), Yoneshigue(1985, pp. 183–184), Schenkman (1986, pp. 27–28,Figs. 28, 33), Horta (2000, p. 85), Nunes (2005, p. 137,

Fig. 2 Neighbor-joining (NJ) analysis for COI-5P sequences forHypneaspecies. On the branches are the bootstrap values for 2,000 replicates andthe maximum likelihood (ML) values for 2,000 replicates (only valuesabove 70 were considered). Accession numbers and country of origin,when available, are included for sequences obtained from GenBank.Specimens morphologically identified as H. valentiae are marked bygray triangle. Specimens morphologically identified as H. nigrescens

are marked by a square. Specimens morphologically identified asH. musciformis are marked by black circle. The states of Brazil are,from the northeast region, PI = Piauí, CE = Ceará, RN = Rio Grande doNorte, PB = Paraíba, AL = Alagoas, PE = Pernambuco, BA = Bahia;southeast region: ES = Espirito Santo, RJ = Rio de Janeiro, SP = SãoPaulo; south region: SC = Santa Catarina. ① indicates group 1 and ②

group 2 mentioned in the text

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Fig. 141), Guimarães (2006, p. 157), Guimarães (2011, pp.43–44, Figs. 18–23), Jesus (2014, pp. 9–10, Fig. 7a–i);H. nigrescens sensu Schenkman (1986, pp. 29–30, Figs. 28,34), Nunes (2005, p. 139, Figs. 142–145), Guimarães (2006,p. 157, Fig. 9), Jesus (2014, pp. 11–13, Fig. 10a–l),H. valentiae sensu Schenkman (1986, pp. 31–32, Figs. 28,29, 35), Horta (2000, pp. 85–86), Nunes (2005, p. 141,

Fig. 147), Guimarães (2006, p. 158, Fig. 11), and Jesus(2014, pp. 16–18, Fig. 15a–j).Specimens examined: Brazil. Ceará. Praia da Lagoinha,Paraipaba, 02. VI. 2012, F. Nauer (SPF57553). Praia daLagoinha, Paraipaba, 02. VI. 2012, F. Nauer (SPF57555).Praia de Flecheiras, Trairi, 03. VI. 2012, F. Nauer(SPF57556). Praia de Ponta Grossa, Icapuí, 05. VI. 2012, F.

Fig. 3 Habits of Hypneapseudomusciformis. a Habit ofholotype, a tetrasporangial thallusshowing fertile branchlets denselygrouped at the apical portions andbranches with hooks and tendrils.b Habit of a non-fertile thallus. cHabit of a tetrasporangial plantfrom Northeast region. d Habit ofa tetrasporangial plant previouslyidentified as H. nigrescens. e, fHabit of a non-fertile thalluspreviously identified asH. nigrescens. g, hHabit of a non-fertile thallus with few and sparsespiny-like branchlets and withouthooks or tendrils, previouslyidentified as H. valentiae. h Habitof a tetrasporangial thallus fromNortheast region. Scale bars =3 cm (c), 2 cm (a, b, d–f), 1 cm(g–h)

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Nauer (SPF57562). Praia de Ponta Grossa, Icapuí, 05. VI. 2012,F. Nauer (SPF57563). Praia de Morro Branco, Beberibe, 06. VI.2012, F. Nauer (SPF57568). Paraíba. Praia de Carapibus,Conde, 20. VII. 2012, F. Nauer & C. Azevedo (SPF57573).Praia de Tabatinga, Conde, 21. VII. 2012, F. Nauer & C.Azevedo (SPF57578). Praia de Tambaba, Conde, 23. VII.2012, F. Nauer & C. Azevedo (SPF57581, SPF57582,SPF57584). Bahia. Praia de Coroa Vermelha, Santa Cruz deCabrália, 15. IX. 2012, F. Nauer & C. Azevedo (SPF57615,SPF57616). Ponta Grande, Porto Seguro, 16. IX. 2012, F. Nauer& C. Azevedo (SPF57618). Praia de Guaiú, Santa Cruz deCabrália, 17. IX. 2012, F. Nauer & C. Azevedo (SPF57622).Praia de Apuã, Santa Cruz de Cabrália, 17. IX. 2012, F. Nauer &C. Azevedo (SPF57623). Espírito Santo. Praia dos Castelhanos,Anchieta, 05. V. 2012, F. Nauer, C. Iha & B. Torrano-Silva(SPF57521). Ponta das Arraias, Marataízes, 07. V. 2012, F.Nauer, C. Iha & B. Torrano-Silva (SPF57530). Praia de Setiba,Guarapari, 08. V. 2012, F. Nauer, C. Iha & B. Torrano-Silva

(SPF57537). Praia dos Namorados, Guarapari, 08. V. 2012, F.Nauer, C. Iha & B. Torrano-Silva (SPF57538). Rio de Janeiro.Prainha, Arraial do Cabo, 29.IX.2012, F. Nauer, C. Iha & B.Torrano-Silva (SPF57424). Praia do Forno, Armação dosBúzios, 25.IX.2012, F. Nauer, C. Iha & B. Torrano-Silva(SPF57427). Praia da Ferradura, Armação dos Búzios,25.IX.2012, F. Nauer, C. Iha & B. Torrano-Silva (SPF57430).Praia de João Fernandes, Armação dos Búzios, 27.IX.2012, F.Nauer, C. Iha & B. Torrano-Silva (SPF57439). Ponta do Costa,Saco do Mamanguá, Parati, 10.XII.2011, F. Nauer, C. Iha & B.Torrano-Silva (SPF57478). Praia Grande da Cajaíba, Saco doMamamguá, Parati. 10. XII. 2011, F. Nauer, C. Iha & B.Torrano-Silva (SPF57485). São Paulo. Praia do Éden, Guarujá,12. IX. 2011, F. Nauer. C. Iha &A.Medeiros (SPF57459). Praiada Lagoinha, Ubatuba, 22. X. 2011, F. Nauer & F. Kino(SPF57473). Santa Catarina. Praia da Armação, Florianópolis,26. II. 2012, F. Nauer. C. Azevedo & B. Torrano-Silva(SPF57492). Praia Ganchos de Fora, Governador CelsoRamos, 27. II. 2012, F. Nauer. C. Azevedo & B. Torrano-Silva(SPF57502).Maldonado, Uruguay. Playa de los Ingleses, Puntadel Este, 20. III. 2014, F. Nauer (SPF57824). Playa el Emir,Punta del Este, 20. III. 2014, F. Nauer (SPF57825). Playa Brava,José Ignacio, 21. III. 2014, F. Nauer (SPF57829).Additional Specimens Examined: Italy. Gulf of Trieste, F.Rindi (SPF57594)

Observations on H. musciformis from the Gulf of Trieste,Italy (Fig. 5a–f)

Themorphological analysis of the material from the Gulf ofTrieste, Italy, showed that the Italian specimens are somewhatsimilar to the epiphytic specimens of H. pseudomusciformisgrowing on upper subtidal, whose thallus is long, flexible, and

Fig. 5 Hypnea musciformis fromthe type locality, Gulf of Trieste,Italy. a, b Habit of a non-fertilethallus. cDetail of a portion of thethallus. d Upper portion ofbranches showing tendrils. eTransverse section of the thallus. fDetail of medullary regionshowing an axial cell (a) with fiveperiaxial cells (pa). Scale bars =2 cm (a), 1 cm (b), 500 μm (c, d),100 μm (e), 75 μm (f)

�Fig. 4 Holotype of Hypnea pseudomusciformis. a Upper portion of abranch showing straight apex. b Upper portion of a branch showingcurved apex. c Upper portion of a branch showing tendril. d Portion ofthe thallus with discoid holdfast. e Portion of the thallus with cystocarps. fDetail of a portion of the thallus showing tetrasporangial disposed ondorsal side of branchlets (arrow). g Detail of a portion of the thallusshowing tetrasporangial disposed on the middle portion of the branchlets(arrow). h Transverse section of the thallus. i Detail of medullary regionshowing an axial cell (a) with seven periaxial cells (pa). j Detail oflenticular thickening (arrows). k Transverse section of a tetrasporangialbranchlet showing tetrasporangia zonately divided. l Transverse sectionof a tetrasporangial branchlet showing tetrasporangia disposed only ondorsal side of branch. m Transverse section of the thallus with twocystocarps. Scale bars = 500 μm (a–e), 300 μm (f, g), 100 μm (h–j),75 μm (k–m)

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abundantly branchedwith tendrils at the apices of the branchesand branchlets. However, the Italian material has fewer andshorter spiny branchlets scattered throughout the thallus(Fig. 5a–c), and fewer tendrils are present in branches andbranchlets (Fig. 5d); the apices may be curved in hooks orstraight. In cross section, the thallus has one to two layers ofpigmented cortical cells and two to three layers of colorlessmedullary cells (Fig. 5e). The axial cell, smaller than periaxialcells, is surrounded by five to six periaxial cells (Fig. 5f).

Discussion

Hypnea musciformis , originally described for theMediterranean, is widely distributed in tropical and subtropi-cal regions around the world and has been cited for the Northand South Atlantic, as well as the Indian and Pacific Oceans(Guiry and Guiry 2014). Based on molecular data of twodifferent markers, the plastid rbcL, and the mitochondrialDNA barcode COI-5P, the specimens globally known asH. musciformis represent a complex of closely related speciesrather than a single cosmopolitan species.

For the genus Hypnea, the COI-5P marker was first used byGeraldino et al. (2006), who sequenced 21 samples in the studyof Hypnea flexicaulis Y. Yamagishi & M. Masuda from thecoast of South Korea. Subsequently, Geraldino et al. (2009)used the marker in the study of Hypnea charoides J.V.Lamouroux from Asia, which presented an intraspecific diver-gence of 2.06%. In 2010, Geraldino et al. expanded coverage ofthe study to 10Hypnea species, with a total of 27 sequences andinterspecific divergence of 11 to 13%. In this work, intraspecificdivergence for COI-5P (0–1.8%) forH. pseudomusciformis andinterspecific divergence between H. pseudomusciformis andH. musciformis (3.9 to 7.1 %) are within the range found forother red algae (e.g., Saunders 2005; Lane et al. 2007).

The intraspecific divergence of 1.8 % found in this studyrefers to divergence between the samples from northeast andthe samples from the southeastern and southern regions ofBrazil. Despite the relatively high intraspecific divergence forCOI-5P, the variation of the rbcL marker is relatively low.

For the rbcL, the interspecific divergence betweenH. pseudomusciformis and H. musciformis (1.7–2.7 %) iswithin the range found by Geraldino et al. (2010) in theirstudy of the genus Hypnea for the Asian region, where theinterspecific divergence found was 1.3–6.8 %.

H. pseudomusciformis is the most common and abundantHypnea species on the Brazilian coast, occurring fromMaranhão (north coast) to Santa Catarina (south coast) anddown to Uruguay. Specimens with typical morphology areeasily recognizable by their irregular branching in variousplanes, spiny-like branchlets numerous, and irregularly ar-ranged and abundant tendrils at the ends of branches andbranchlets. Nonetheless, this species does present phenotypic

plasticity, depending on the environment. On the rockyshores, morphological variation is typically seen from theupper to mid intertidal zone toward the lower fringe of theintertidal zone, mainly in exposed shores.

H. pseudomusciformis and H. valentiae

I n t h e u p p e r i n t e r t i d a l z o n e , s p e c im e n s o fH. pseudomusciformis forming small greenish to yellowish-brown tufts up to 3-cm long, intertwined, are commonly foundgrowing attached to rocks. The thalli are rigid with littleflexibility, spiny-like branchlets are short in the format ofsmall thorns, and the branches are often straight, withouttendrils (Fig. 3g–h). Specimens with this morphology werepreviously identified as “H. valentiae” (Schenkman 1986;Jesus et al. 2014).

H. valentiae was first proposed as Fucus valentiae byTurner (1809) from the Red Sea. Harvey (1834) reducedF. valentiae to a variety of H. musciformis. Later, Montagne(1841) restored the variety valentiae to specific level, justify-ing the separation ofH. valentiae fromH. musciformismainlyby the presence of star-shaped spines with three, four, or fiverays and with a reddish purple color, whereas the rest of thethallus is greenish, features that were neither described byTurner (1809) nor by Harvey (1834). Despite Montagne’sdescription, based on (1) material from the type locality, whichwas collected at Thor, Red Sea, by M.M. Bovè and Schimper,and (2) Turner’s illustration (plate 78), stellate branchlets havenot been described for H. valentiae from other localities, suchas Brazil (Schenkman 1986; Jesus et al. 2014), Australia(Womersley 1994), Japan (Yamagishi and Masuda 1997),Taiwan (Chiang 1997), and Thailand (Lewmanomont 1997).Furthermore, the absence of these branchlets in H. valentiaehas been used as a characteristic to separate it from Hypneacornuta (J.V. Lamouroux) J. Agardh (Tsiamis and Verlaque2011), a species that is well documented as having stellatebranchlets (Schenkman 1986; Bangmei and Yongqiang 1997;Chiang 1997; Lewmanomont 1997; Jesus et al. 2014). On theother hand, Agardh (1852), analyzing the material collectedby Schimper from the type locality, possibly the same materialanalyzed by Montagne (1841), quoted stellate branchlets forH. valentiae, as well as Børgesen (1943) for Mauritius,Schneider and Searles (1991) for the southeastern USA andMshigeni and Chapman (1994) for Mauritius and the Red Sea.Price et al. (1992) considered H. cornuta as a synonym ofH. valentiae. However, Mshigeni and Chapman (1994), ex-amining authentic specimens of H. cornuta and H. valentiaefrom herbaria C and L, maintained the species as separate taxaand confirmed that both species have stellate branchlets.Mshigeni and Chapman (1994) segregated the species by thebranches and branchlets disposed at angles significantlynarrower in H. cornuta, whereas in H. valentiae, thebranches and branchlets are disposed almost at right angles,

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resulting in a pattern of branching more openly and moreloosely arranged in H. valentiae. Mshigeni and Chapman(1994) also showed differences in the stellate branchlets ofH. cornuta andH. valentiae. InH. cornuta, the stellate branch-lets arise as tiny special branchlets loosely attached to theaxes, whereas in H. valentiae, the branchlets appear to belonger and more firmly attached to the axes.

The presence or absence of stellate branchlets inH. valentiae still seems to be quite controversial, especiallybecause, as pointed out by Schneider and Searles (1991), thesebranchlets may be lacking, which confuses this species withspecimens ofH. musciformiswhen tendrils are absent. Abbott(1999) corroborated the statement made by Schneider andSearles, based on the absence of stellate branchlets in mostspecimens of H. valentiae from Hawaii, suggesting to thisauthor that branchlets are an unreliable diagnostic feature forH. valentiae.

Schenkman (1986) reported “H. valentiae” from SãoPaulo, Brazil, growing on Sargassum and intermixed with“H. musciformis”. It was pointed out that the type materialof H. valentiae was not examined and that stellate branchletswere never found in Brazilian material. For this reason, theauthor considered the identification of “H. valentiae” forBrazil to be provisional. Despite the provisional characterand doubt in the identification of H. valentiae for Brazil, thespecies continued to be cited for other regions of the Braziliancoast (Horta 2000; Pereira et al. 2002; Amado-Filho et al.2006; Guimarães 2006; Figueiredo and Tâmega 2007; Lyraet al. 2007; Jesus et al. 2014). Our results clearly showed thatthe specimens identified as “H. valentiae” for Brazil corre-spond to H. pseudomusciformis. The reports of H. valentiaeon a worldwide basis need a critical review, including DNAsequence data from the type locality to clarify the taxonomicposition of this species.

H. pseudomusciformis and H. nigrescens

The morphological variant, previously identified as“H. nigrescens” (Schenkman 1986; Jesus et al. 2014), is foundexclusively in rocky shores with strong wave impact, growingattached to rocks in the middle intertidal zone. Specimenscollected in this zone present a range of color variation,including vinaceous red, blackish red, light green, and darkgreen; the thalli are more robust and rigid, forming tufts ofmultiple main axes of similar height resembling the bristles ofa brush, with up to 9 cm in height, with numerous thin andlong spiny branchlets, no longer resembling small thorns.Tendrils may be present in the apices of lateral branches, butmore commonly, they are present in the main apex (Fig. 3d, e).

The original description of H. nigrescens (J. Agardh 1851)from India is very short and based only on gross morphology,whose characteristics overlap with other species of Hypnea.

Schenkman (1986) cited “H. nigrescens” for the first timefor Brazil and the Atlantic Ocean, noting that this species wasrestricted to shores strongly exposed to the impact of waves,as also observed in our study. We collected specimens thatcorresponded to the morphology of “H. nigrescens” at thesame location originally collected by Schenkman (1986). Thissample (SPF56891) had COI-5P and rbcL sequences identicalto specimens of H. pseudomusciformis. Furthermore, speci-mens presenting the external morphology of “H. nigrescens”(Fig. 3d–f) cannot be anatomically distinguished from otherH. pseudomusciformis specimens.

H. pseudomusciformis and H. musciformis

In the shallow subtidal zone, epiphytic specimens ofH. pseudomusciformis have the typical musciformismorphol-ogy. It can form large populations, being found both inprotected areas and reefs.

The samples of H. musciformis collected in the type local-ity region (Gulf of Trieste, Italy) were sequenced in this studyand were also morphologically examined and compared withBrazilian material. These analyses do not showmorphologicalcharacteristics with the sufficiency required to distinguishauthentic H. musciformis from the Brazilian specimens de-scribed as H. pseudomusciformis. However, H. musciformisand H. pseudomusciformis can be distinguished based onmolecular analysis. The detailed morphological study of theBrazilian specimens demonstrated a continuum of morpho-logical variation in the habit of plants with intermediate formsbetween typical morphologies of musciformis, valentiae, andnigrescens. H. pseudomusciformis is closely related toH. musciformis, but significant genetic divergence was foundbetween both species.

Acknowledgments We would like to thank Fabio Rindi and M.Hommersand for sending samples of H. musciformis. We also greatlythank Michael Wynne for comments and exchange of relevant informa-tion on the taxonomy of species of Hypnea. We are grateful for thesupport from FAPESP (Biota 2013-11833-3), CNPq (Br BOL-564945-2010-2, 301491/2013-5), CAPES (Proex), and NP-BioMar. We alsothank all members of the Marine Algae Laboratories (LAM), Universityof São Paulo, Brazil.

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