Morphological and Molecular Studies on Topotype Material

Botanica Marina 2014; aop

Daniel Le ó n- Á lvarez * , Maria L. N ú ñ ez-Resendiz and Michael J. Wynne Morphological and molecular studies on topotype material of Neoralfsia expansa (Phaeophyceae) reveal that Asian specimens assigned to this taxon are genetically distinct

Abstract: The genus Neoralfsia was newly described by specimens, collected by Liebmann from an unspecified P.-E. Lim and Kawai on the basis of rbc L sequence data area on the coast of Veracruz, Mexico, in February 1841 from specimens from Japan and Malaysia identified ( God í nez 2008 ). B ø rgesen (1912) reported on specimens as Neoralfsia expansa , and N. expansa [Myrionema ( ? ) from the Danish West Indies (now the U.S. Virgin Islands) expansum] from Mexico was designated as the type spe- that were very similar to the type of N . expansa (as Ralfsia cies. Our maximum likelihood and Bayesian analyses expansa ), and he described his material in detail includ- show that specimens of N. expansa from the type locality ing reproductive characteristics. His study was directly are grouped into a new, not previously described, clade or indirectly the basis for the recognition of the species in Neoralfsiaceae, distant from that of the putative speci- by other authors ( Weber-van Bosse 1913 , B ø rgesen 1914 , mens attributed to N . expansa from Japan and Malaysia. Taylor 1960 , Joly 1965 , Earle 1969 , Schnetter 1976 , Lawson We show that genuine “ expansa ” does not include Asian and John 1982 ). However, Tanaka and Chihara (1980a,b,c, material. The distribution of the species is not as wide as 1981a,b), in their review of the order Ralfsiales Nakamura has been previously recorded. ex Lim et Kawai, described the reproductive characteristics of specimens of what they called “R. expansa ” from Keywords: brown crustose algae; distribution; morphology; various Japanese islands and generated a different inter- Neoralfsia ; Ralfsia ; rbc L. pretation of the species than that of Bø rgesen. Based on a morphological study of specimens from various localities in Veracruz and the Mexican Tropical Pacific, Le ó n- Á lva- DOI 10.1515/bot-2014-0029 Received May 15 , 2014 ; accepted July 24 , 2014 rez and Gonz á lez-Gonz á lez (2003) were of the opinion that specimens with mainly unilateral symmetry, unangial specimens with sessile unangia, or with unangia with a single stalk cell correspond to N. expansa sensu B ø rgesen Introduction (as R. expansa sensu B ø rgesen), whereas specimens with unilateral to bilateral symmetry and unangia with multi- Neoralfsia expansa (J. Agardh) P.-E. Lim et H. Kawai ex cellular stalks correspond to N. expansa sensu Tanaka and Cormaci et G. Furnari in Cormaci et al. (2012) is a commonly Chihara (as R. expansa sensu Tanaka and Chihara). Lim recorded species [as Ralfsia expansa (J. Agardh) J. Agardh] et al. (2007), however, considered the circumscription of around the world. Poong et al. (2013) described its distri- N. expansa (as R. expansa) based on these characteristics bution as “cosmopolitan”. It was originally described by J. to be insufficient, and they used molecular data from the Agardh (1847) as Myrionema ( ? ) expansum and transferred rbcL region of chloroplast DNA to show that specimens to Ralfsia the following year by J. Agardh (1848) . The origi- identified as R. expansa from Japan and Malaysia were nal description was very brief and based on vegetative located in a distinct clade from other Ralfsia species and described the new genus Neoralfsia in the new family *Corresponding author: Daniel Leó n- Á lvarez, Laboratorio de Neoralfsiaceae of the order Ralfsiales. Their proposed new Ficolog í a and Secci ón de Algas del Herbario de la Facultad de combination N. expansa as the type species of the genus, Ciencias, Universidad Nacional Aut ó noma de M é xico, UNAM, with Myrionema ( ? ) expansum J. Agardh as the basionym, Mexico 04510 D.F., e-mail: [email protected] was later validated by Cormaci and Furnari (in Cormaci Maria L. Nú ñ ez-Resendiz: Laboratorio de Ficologí a and Secció n de Algas del Herbario de la Facultad de Ciencias, UNAM, Mexico et al. 2012). Recent molecular studies on rbc L showed Michael J. Wynne: University of Michigan Herbarium, 3600 Varsity Neoralfsia hancockii (E.Y. Dawson) D. Le ó n- Á lvarez and Drive, Ann Arbor, Michigan 48108, USA M.L. Nú ñ ez-Res é ndiz, another common species in the

Brought to you by | UNAM Authenticated | [email protected] author's copy Download Date | 8/15/14 11:49 AM 2 D. Le ó n- Á lvarez et al.: Topotype material of Neoralfsia expansa eastern Tropical Pacific ( Le ó n- Á lvarez et al. 2014 ), to be Faculty of Sciences, National Autonomous University of distinct from N. expansa (as Ralfsia expansa sensu Tanaka Mexico, FCME (GM649, GM650, GM651) ( Table 1 ). These and Chihara 1980b , as Neoralfsia expansa sensu Lim et al. collections were also compared with three specimens of 2007). Given the geographic distance between Japan N. expansa (FCME: GM289 and GM635), from the Morro de or Malaysia and Veracruz, Mexico, and the differences la Mancha, Veracruz, one of them in unangial reproduc- in morphological characteristics, we have conducted tive condition, and other one identified as Ralfsia expansa molecular studies on topotype material to determine the in plurangial reproductive condition from Punta Lim ó n, relationship between the Mexican (topotype) and Asian Veracruz, from the Herbarium of the National School specimens. of Biological Sciences, National Polytechnic Institute, ENCB4257.

Materials and methods Morphology For this study, we collected samples of Neoralfsia expa nsa from Veracruz, Mexico, on multiple occasions ( Table 1 ). Under an optical microscope, preparations were observed Specimens were collected using a chisel and hammer and starting with vertical-radial sections of tissue made using a knife to remove scrapings from rock substratum. For the a manual rotation microtome, which were stained with molecular studies, samples collected on the dates Decem- safranin and fast green (slides in FCME, Table 1 ). Obser- ber 4, 2009, May 18, 2010, and April 29, 2010 were used, vations were made on diagnostic characteristics of this and these samples were each subdivided into three por- species, such as thickness of the thallus, crust medul- tions. One portion of each was preserved with silica gel lary differentiation, chloroplast number, presence, posi- (for molecular analysis), a second portion with a dilute tion and number of cells in the plurangia, presence and formalin solution in seawater, and the third portion on position of unangia and number of stalk cells, presence/ blotting paper (these latter two portions being used for absence of paraphyses, according to the characteristics morphological analysis and herbarium reference). All described by Tanaka and Chihara (1980a,b), Le ó n- Á lvarez samples were incorporated into the collection of the Gulf and Gonzá lez-Gonz á lez (1995, 2003), Le ó n- Á lvarez (2005), of Mexico of the Algae Section of the Herbarium of the and Lim et al. (2007) for Ralfsia species and with the

Table 1 Herbarium references and data collection sites for the Mexican samples of Neoralfsia expansa and Neoralfsia hancockii included in this study.

Species Locality Collection date Collector Habitat Catalog no. Slides nos. in FCME in FCME

N. expansa Mexico: Morro de la Mancha, 27/09/2010 Mar í a L. Karst rock platform GM635 1174 – 1177 Veracruz. N ú ñ ez and exposed to waves at sea 19 ° 35 ′ 22 ″ N; 96o 22 ′ 43 ″ W Daniel Le ó n ” 29/04/2010 ” ” GM651 1170 – 1174 ” 18/05/2010 ” Vertical walls of rocky GM649 1153 – 1156 barriers or crags ” 04/12/2009 ” Boulders exposed to light GM650 1165 – 1169 and waves ” 14/02/2003 Daniel Le ó n Karst rock platform GM289 244 exposed to waves at sea a Mexico: Punta Lim ón, Veracruz 09/11/1976 – Unspecified ENCB4257 1758 N. hancockii Mexico: San Jos é del Cabo (La 30/09/2009 ” Granite boulders directly PTM9165 1139 – 1142 Palmilla), B.C.S. exposed to waves and 23 ° 03 ′ 31.8 ″ N; 109° 41 ′ 23.3 ″ W light San Jos é del Cabo (Club 30/09/2009 ” Rocky irregular platform PTM9167 1132 – 1138 Campestre), B.C.S. exposed to light, protected 23 ° 01 ′ 04.0 ″ N; 109° 42 ′ 56.9 ″ W from direct waves

a As Ralfsia. GM and PTM, Gulf of Mexico and Mexican Tropical Pacific catalog numbers in FCME; ENCB, Escuela Nacional de Ciencias Biol ó gicas, Herbarium.

Brought to you by | UNAM Authenticated | [email protected] author's copy Download Date | 8/15/14 11:49 AM D. Le ó n- Á lvarez et al.: Topotype material of Neoralfsia expansa 3 terminology proposed by Le ó n- Á lvarez and Norris (2005) and PRB-R3 (Kogame et al. 1999). The PCR product was for crustose brown algae. Because the exact nature of the sequenced at the Molecular Biology Laboratory of the reproductive cells (i.e., gametes or spores) cannot be estab- Institute of Biology, UNAM. lished, we use the morphological terms unangium and plu- rangium instead of unilocular or plurilocular sporangia. Phylogenetic analysis

Molecular analysis Thirty-four rbcL sequences of the order Ralfsiales from GenBank (NCBI) and three sequences from this study were DNA extraction and amplification followed the methods included in the molecular analyses, together with Tilop- described in Le ó n- Á lvarez et al. (2014) , with the addition teris mertensii (Turner) Kü tzing and Sargassum muticum of the following primers: rbc-F4 (Kawai and Sasaki 2004) (Yendo) Fensholt as outgroups ( Table 2 ).

Table 2 Molecular sequence data (NCBI, 2014 and this study) and herbarium references.

Species Accession no. Locality Author

Neoralfsia hancockii KF977828 La Palmilla, San Jos é del Cabo, B.C., M é xico (PTM9165 FCME, 09/30/2009) Le ó n- Á lvarez et al. 2014 N. hancockii KF977827 Country club, San Jos é del Cabo, B.C., M é xico (PTM9167 FCME, 09/30/2009) Le ó n- Á lvarez et al. 2014 Neoralfsia expansa AB250077.1 Okinawa, Ishigaki Island, Hamasaki, Japan Lim et al. 2007 N. expansa AB250078.1 Johor, Desaru, Malaysia Lim et al. 2007 N. expansa AB250079.1 Hyogo, Igumi, Japan Lim et al. 2007 N. expansa A KM032758 Morro de la Mancha, Veracruz, M é xico (FCME-GM649) This study N. expansa B KM032759 Morro de la Mancha, Veracruz, M é xico (FCME-GM650) This study N. expansa C KM032760 Morro de la Mancha, Veracruz, M é xico (FCME-GM651) This study Ralfsia verrucosa AB250072.1 Roscoff, Brittany, France Lim et al. 2007 Ralfsia fungiformis AB250071 Hokkaido, Akkeshi, Japan Lim et al. 2007 R. fungiformis EU579936.1 Cap du Bon Dé sir, Quebec, Canada Bittner et al. 2008 Ralfsia spA AB250073.1 Hyogo, Ako, Japan Lim et al. 2007 Ralfsia spB AB250074.1 Bergen, Norway Lim et al. 2007 Ralfsia spC AB250075.1 Katiki Beach, New Zealand Lim et al. 2007 Ralfsia spD AB250076.1 Durban, South Africa Lim et al. 2007 Ralfsia spH AB250080.1 Shimoda, Nabeta, Japan Unpublished Ralfsia spI AB250081.1 Shimoda, Nabeta, Japan Unpublished Ralfsia spJ AB250082.1 Hyogo, Awaji Island, Maruyama, Japan Unpublished Ralfsia spK AB250083.1 Hyogo, Takeno, Oura, Japan Unpublished Mesospora elongata JQ620004.1 Lombok island, Gili Genting, Indonesia Poong et al. 2013 M. elongata JQ620002 Okinawa Prefecture, Ishigaki Island, Japan Poong et al. 2013 M. elongata B JQ620005.1 Lombok Island, Nipah, Indonesia Poong et al. 2013 M. elongata C JQ620003.1 Okinawa Prefecture, Ishigaki Island, Japan Poong et al. 2013 Mesospora schmidtii JQ620000 Port Dickson, Malaysia Poong et al. 2013 M. schmidtii JQ619998 Malaysia: Johor, Pulau Che Kamat Poong et al. 2013 M. schmidtiiBAT43 JQ620001 Indonesia: Lombok Island, Batukijok Poong et al. 2013 M. schmidtiiBAT7A JQ619999 Indonesia: Lombok Island, Batukijok Poong et al. 2013 Mesospora spA AB250063.1 Okinawa, Ishigaki Island, Fusaki, Japan Unpublished Mesospora spB AB250064.1 Johor, Desaru, Malaysia Unpublished Mesospora spC AB250065.1 Johor: Tg. Gemoh, Malaysia Lim et al. 2007 Mesospora spD AB250066 Okinawa, Ishigaki Island, Nakura, Japan Lim et al. 2007 Mesospora spE AB250067 Johor, Tg Lompat, Malaysia Lim et al. 2007 Mesospora spF AB250068 Johor, Tg. Balau, Malaysia Lim et al. 2007 Mesospora spG AB250069 Shizuoka, Shimoda, Japan Lim et al. 2007 Analipus japonicus AB264042 Hokkaido, Japan Lim et al. 2007 Heteroralfsia saxicola AB250070.1 Ohma, Aomori Pref., Japan Lim et al. 2007 Endoplura aurea AB264039 Inubouzaki, Chiba Pref., Japan Lim et al. 2007 Tilopteris mertensii AB045260 Helgoland, Germany Sasaki et al. 2001 Sargassum muticum AJ287854 Zeeland, Oosterschelde, Zeelandbrug, Netherlands Draisma et al. 2001

Brought to you by | UNAM Authenticated | [email protected] author's copy Download Date | 8/15/14 11:49 AM 4 D. Le ó n- Á lvarez et al.: Topotype material of Neoralfsia expansa – – – – – – – – – 7,1.4 × 10 Basal cell of Ø 2.5 – 3, l/d 4 – 7 diameter, l/d ratio l/d diameter, paraphyses: length, paraphyses:

– – – – – – m), μ 12 – 16 Present Present Present 155, 13 – 15 Paraphyses: Paraphyses: length ( number of cells of number 100 – 190 (-220),

Unangia Terminal on 3-celled stalks Terminal on 3-celled stalks Terminal on 6-celled stalks on Terminal 3- to 6-celled stalks Absent Absent Sessile lateral to paraphyses Absent Absent Absent Absent

Plurangia Subterminal, 1 terminal with sterile cell Subterminal, 1 terminal with sterile cell Subterminal, 1 terminal with sterile cell Subterminal, 1 terminal with sterile cell Absent Subterminal, 1 sterile under cell Absent Absent Subterminal, 1 sterile under cell Subterminal, 1 sterile under cell Subterminal, 1 sterile under cell

– – – – – 2.5 – 3 2.5 – 3 1.5 – 2.6 2.4 – 2.6 2.3 – 2.6 2.5 – 2.6 cortical cell cell cortical Sub- cortical/ diameter ratio

m) μ 5 – 6 3 – 5 4 – 5 5 – 8 4 – 6 5 – 6 × × × × 4 – 5.5 × Present Present Present Present Cortex: cell Cortex: cell 5, 4 – 6 5, 5 – 6.8 3, 5 – 6.5 diameter ( 5, 4.5 – 6 5 – 6, 8 – 13 number, length number,

– – – – – – m) μ 12 – 13 11 – 13 12 – 15 12 – 14 13 – 15 × × × × × 32 32 34 32 examined in this study. in this study. examined Subcortical Subcortical cells: lengthcells: diameter ( 17 – 30

Neoralfsia Symmetry of of Symmetry vegetative filaments curving Bilateral; and upwardly from a downwardly layer central curving Bilateral; and upwardly from a downwardly layer central curving Bilateral; and upwardly from a downwardly layer central curving Bilateral; and upwardly from a downwardly layer central curving Bilateral; and upwardly from a downwardly layer central or Unilateral in partsbilateral Bilateral arching Unilateral layer from a basal Bilateral to edges Unilateral bilateral central and Bilateral

m) μ ( (-1100) 138 – 390 110 – 280 141 – 160 200 – 800 449 – 649 315 – 414 180 – 450 200 – 295 190 – 300 195 – 300 160 – 260 Thickness Thickness

649) GM

(Japan) (Hyogo) Holotype (GM 650) (GM635) GM289 (GM 651) ( . Int. label: Myrionema Morphological comparison of the specimens of of the specimens comparison of Morphological expansa expansa expansa . . . . expansa . expansa . expansa N. expansa N Neoralfsia expansa Neoralfsia N. expansa N. expansa N. expansa ENCB4357

N N N N N (Okinawa) Table 3 Table Taxa a a (Malaysia) a b c d

A681 (as expansum Museum Botanical Copenhagen L78/2002, No.7)

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The sequences were edited and aligned in the program Bioedit ( Hall 1999 ). A matrix of 39 sequences × 1349 bp 1.6 – 2.1 2.2 – 3.7 was subjected to maximum-likelihood (ML) analyses Basal cell of using PAUP* 4.0b 10 ( Swofford 2002 ). To find the model Ø 1.6 – 5.2, l/d Ø 3.1 – 7.3, l/d of nucleotide substitution that best fits the data set, the diameter, l/d ratio l/d diameter, Ø 2 – 6.5, l/d 2 – 2.4

paraphyses: length, paraphyses: program ModelTest v.3.7 (Posada and Crandall 1998) was used with the Akaike Information Criterion (AIC). The AIC m), μ indicated that the least-rejected model for the present 11 – 13

100 – 110, study data set was the Hasegawa-Kishino-Yano model of 95 – 10, 25 10 – 13 (18) Paraphyses: Paraphyses: length ( nucleotide substitutions with invariant sites and γ distrib- number of cells of number

100 – 150 (-175), uted rates for the variable sites (HKY + I + G). The ML was performed using TBR swapping with 10 random sequence additions in PAUP using parameters estimated from the ModelTest. Bootstrap percentage (BP) was computed with 100 replications. Unangia Terminal on 3-celled stalks on Terminal 3- to 6-celled stalks on Terminal 4- to 5-celled stalks

The range for partial sequences of rbc L divergence values within and among species was calculated using uncorrected “ p ” distances using PAUP. The Bayesian analysis was performed using the

Plurangia Absent Absent Absent program MrBayes 3.1.1 ( Huelsenbeck and Ronquist 2001 ),

– using HKY model with a distribution. Five chains of HAHF9 in LAM500460, now in UC, Le ó n- Á lvarez and á lez á lez-Gonz Gonz 2003 . e Markov chain Monte Carlo were used starting with a . 2 – 2.4 1.7 – 2 random tree and sampling the data every 100 generations

cortical cell cell cortical 6 Ralfsia for 5 × 10 generations. The likelihood scores stabilized Sub- cortical/ diameter ratio as d after 250,000 generations, but we used a “ burn-in ” of 25%

m) (1,250,000 generations). μ 5 – 7 5 – 7 × × The uncorrected distances (“ p ” ) for partial sequences Present of rbcL were calculated in PAUP* 4.0b 10. Cortex: cell Cortex: cell diameter ( 6 – 8, 6 – 9 5 – 7, 7 – 9 number, length number, Le ó n- Á lvarez Le ó n- Á lvarez 2005 .

c ×

– m)

μ Results 10 – 12 × Subcortical Subcortical cells: lengthcells:

diameter ( Morphology of the specimens from Mexico: 20 – 25, 10 – 12 22 – 25

Neoralfsia expansa

The specimens consist of orbicular crustose thalli, 4– 12 cm diameter, growing distinctly from each other, sometimes overlapping at the margins, dark brown when wet, and reddish brown when dry, epilithic, adher- Symmetry of of Symmetry vegetative filaments Mainly unilateral; unilateral; Mainly upwardly curving downwardly and layer from a central unilateral; Mainly upwardly curving downwardly and layer from a central unilateral, Mainly also curving in part downwardly

ent to the substrate by rhizoids, with a rough, leathery m)

μ texture, and with conspicuous radial growth lines, warty ( surface, smooth margins discolored to light brown, easily 130 – 210 160 – 300 108 – 306 198 – 360 Thickness Thickness , Tanaka and Chihara 1980b: 231 – 232, Figure 1 B. 1 Figure 232, – 1980b: 231 Chihara and Tanaka ,

(vegetative) (vegetative) detached from the substrate; pseudoparenchymatous, in

(reproductive) radial-vertical section; connate filaments growing from

Ralfsia

. one or two basal layers bending parallel to the substrate as R b and curving upward as a result of repeated ramification (as (PTM and downward, showing an evident bilateral symmetry Holotype)

Continued) with a central axis in main parts of the crust; the delimita- tion between cortex and medulla was evident where there Table 3 Table Lim et al. 2007 . N. hancockii N. hancockii 9167) ( hancockii Ø , diameter; – , not available. Taxa e a Neoralfsia hancockii Neoralfsia 9165) (PTM was an abrupt reduction in cell diameter (GM289, 635,

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649, 650, 651, ENCB4257). Cells with a parietal laminate Phylogenetic analysis chloroplast without a pyrenoid. The specimens from the type locality had sessile unangia that were lateral to para- Molecular sequences were obtained for the rbcL gene of physes (GM289) or subterminal plurangia under a sterile the topotype of Neoralfsia expansa . Overall topologies cell (ENCB4257, GM649, GM650, GM651) in discrete sori and branching order of the main clades were identical in and in distinct thalli. One of them was vegetative (GM635). the Bayesian and ML trees. The bootstrap values result- Diagnostic characteristics of the specimens are included ing from ML and BPP (Bayesian posterior probability) are in Table 3 . incorporated in the Bayesian tree of Figure 1 (as BPP/ML).

Figure 1 Bayesian tree deduced from BPP and ML analyses of rbcL, showing the position of Neoralfsia expansa from Veracruz in Neoralfsi- aceae. The topology of the Bayesian tree was identical to ML. Support values (BPP/ML) are shown at the nodes of the branches. Bootstrap values below 50% are indicated as “ – ” . Substitutions per site (S.P.S.) are indicated by the scale bar.

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The family Neoralfsiaceae formed a strongly supported species from Japan and Malaysia; and second, the latter clade (Clade III, 1.00/100, Figure 1 ), with two subclades specimens represent an undescribed species of a new (Subclade A, 1.00/98, and Subclade B, 1.00/59, Figure 1 ). genus for which new names must be given. Because Lim Among the Asian specimens of so-called “ Neoralfsia et al. (2007) cited N. expansa (basionym of Myrionema ? expansa , ” the values of uncorrected distance ( “ p ” ) ranged expansum J. Agardh) as the generitype of their new genus from 2.3% to 4.4%. In turn, these specimens diverged Neoralfsia , according to Article 10.1 of the ICN ( McNeill from Neoralfsia hancockii specimens by 6.6 – 8.1%, and et al. 2012), that generic name is based on the species they diverged from topotype specimens of N. expansa by name with a type locality of Veracruz, Mexico. The present 6.6 – 9.8%. results show that Lim et al. (2007) mis-identified their specimens from Malaysia and Japan as “ expansa, ” based on previous records of the “ expansa ” species in the region (Tanaka and Chihara 1980b, Yoshida et al. 2000) and Discussion because they considered the morphological diagnostic characters to be insufficient. However, at present, we are Because the holotype of Neoralfsia expansa is not repro- unable to propose a new name of the genus and species ductive, diagnostic features of its reproductive characteris- for the Asian material because we do not have the appro- tics are not available. For this reason, Le ó n- Á lvarez (2005) priate specimens to use as the basis for the delineation of described the holotype specimen and also the reproduc- a new taxon to accommodate them. By this description of tive structures based on the topotype material confirm- Neoralfsia expansa , employing gene-sequence studies on ing the presence of sessile or unicell-stalked unangia in topotype material, we have been able to demonstrate that agreement with the authors, who described or illustrated it is distinct from specimens that have been assigned to the species (as Ralfsia expansa : B ø rgesen 1912 , Schnet- this species from Asian collections. ter 1976, Balakrishnan and Kinkar 1981, Sz é chy-Menezes 1986 , Sartoni and Boddi 1989 ). The topotype has paraphy- Acknowledgments: The authors are indebted to Drs. ses with thicker and shorter basal cells (length/diameter Michele Gold and Den í Rodr í guez for their valuable com- ratio of basal cells 1.4, Table 3 ) than Japanese specimens. ments along this project. Thanks also to Dr. Edith Ponce The contrasting characteristics between the topotype and who advised us on DNA extraction and amplification. Asian specimens, as recorded by Tanaka and Chihara Financial support was partially provided by DGAPA, (1980b, as R . expansa ) and Lim et al. (2007), suggest UNAM, PAPIIT IN215108 and IN229711 projects. that these traits (unangial stalks 3 – 6 cells, length/diameter ratio of basal cells of paraphyses 4– 7, and plurangia sometimes in the same thallus) are specific to the Asian material and correspond to a different species. The Asian References specimens also share considerable morphological simi- larities with Neoralfsia hancockii from the type locality, Agardh, J.G. 1847. Nya alger fr å n Mexico. Ö fversigt af Kongl. but their paraphyses have thinner and larger basal cells. Vetenskaps-Acad. F ö rhand., Stockholm 4 : 5 – 17. Additionally, rbc L data of Le ó n- Á lvarez et al. (2014) and Agardh, J.G. 1848. Species genera et ordines algarum. Volumen primum. Algas fucoideas complectens. C.W.K. Gleerup, Lundae this study show that N. expansa is very distant from the [Lund]. pp. viii+ 363. Asian specimens. Neoralfsia hancockii and the remaining Balakrishnan, M.S. and V.N. Kinkar. 1981. A taxonomic account of Mexican specimens form a monophyletic group distinct Indian Ectocarpales and Ralfsiales. Seaweed Research and from any other previously described. This group appears Utilization 4: 1 – 57. as a sister clade (Subclade A, 1.00/98, Figure 1) to Asian Bittner, L., C.E. Payri, A. Couloux, C. Cruaud, B. de Reviers and F. Rousseau. 2008. Molecular phylogeny of the Dictyotales and Neoralfsia (Subclade B, 1.00/69, Figure 1) in the Neoralf- their position within the Phaeophyceae, based on nuclear, siaceae. It is also distant from Ralfsiaceae, even though it plastid and mitochondrial DNA sequence data. Mol. Phylogen. develops similar crustose forms. Evol. 49 : 211 – 226. The wide nucleotide divergence between the speci- B ø rgesen, F. 1912. Two crustaceous brown algae from the Danish mens of N . expansa from the type locality and those from West Indies. Nuova Notarisia, Padova 23: 123 – 129. Japanese and Malaysian islands (6.6– 9.8%), and their B ø rgesen, F. 1914. The marine algae of the Danish West Indies. Phaeophyceae. Dansk Bot. Arkiv 1: 157 – 226. morphological differences lead us to conclude the follow- Cormaci, M., G. Furnari, M. Catra, G. Alongi and G. Giaccone. 2012. ing: first, authentic N. expansa from the type locality is Erratum. Flora marina bentonica del Mediterraneo: Phaeophy- different from the entities that have been identified as that ceae. Boll. Acad. Gioenia Sci. Nat. Catania. 45: 509 – 510.

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