J. Phycol. 53, 1044–1059 (2017) © 2017 Phycological Society of America DOI: 10.1111/jpy.12562 THE CORALLINE GENERA SPOROLITHON AND HEYDRICHIA (SPOROLITHALES, RHODOPHYTA) CLARIFIED BY SEQUENCING TYPE MATERIAL OF THEIR GENERITYPES AND OTHER SPECIES1 Joseph L. Richards 2 Department of Biology, University of Louisiana at Lafayette, Lafayette, Louisiana 70504-3602, USA Thomas Sauvage Smithsonian Marine Station at Fort Pierce, Fort Pierce, Florida 34949-3140, USA Department of Biology, University of Louisiana at Lafayette, Lafayette, Louisiana 70504-3602, USA William E. Schmidt, Suzanne Fredericq Department of Biology, University of Louisiana at Lafayette, Lafayette, Louisiana 70504-3602, USA Jeffery R. Hughey Division of Mathematics, Science, and Engineering, Hartnell College, Salinas, California 93901, USA and Paul W. Gabrielson Biology Department and Herbarium, University of North Carolina at Chapel Hill, Coker Hall CB 3280, Chapel Hill, North Carolina 27599-3280, USA Interspecific systematics in the red algal order reported to be H. woelkerlingii from New Zealand is Sporolithales remains problematic. To re-evaluate likely an undescribed species. Type specimens of all its species, DNA analyses were performed on other Sporolithon and Heydrichia species need to be historical type material and recently collected sequenced to confirm that they are distinct species; specimens assigned to the two genera Sporolithon morpho-anatomical studies have proved inadequate and Heydrichia. Partial rbcL sequences from the for this task. lectotype specimens of Sporolithon ptychoides (the Key index words: COI; Egypt; Gulf of Suez; LSU; generitype species) and Sporolithon molle, both from new species; phylogenetics; psbA; rbcL; Red Sea; El Tor, Egypt, are exact matches to field-collected UPA topotype specimens. Sporolithon crassum and Sporolithon erythraeum also have the same type Abbreviations: bp, base pairs; BS, bootstrap value; locality; material of the former appears to no longer ML, maximum likelihood; NWGMx, Northwest Gulf exist, and we were unable to PCR amplify DNA of Mexico; SEGMx, Southeast Gulf of Mexico from the latter. A new species, Sporolithon eltorensis, is described from the same type locality. We have not found any morpho-anatomical characters that To determine if any genus is monophyletic and distinguish these three species. No sequenced which species belong in the genus, one must begin specimens reported as S. ptychoides from other parts by characterizing the generitype species. Histori- of the world represent this species, and likely cally, this characterization was done for coralline reports of S. ptychoides and S. molle based on algae by morpho-anatomical comparisons using morpho-anatomy are incorrect. A partial rbcL gross morphology at first, but soon thereafter aug- sequence from the holotype of Sporolithon dimotum mented by anatomical characters through decalcifi- indicates it is not a synonym of S. ptychoides, and cation, sectioning and light microscopy, and finally data from the holotype of S. episporum confirm its in the late 20th century aided by scanning electron specific recognition. DNA sequences from topotype microscopy (Johansen 1981, Woelkerling 1988). material of Heydrichia woelkerlingii, the generitype Recently, this approach has been further augmented species, and isotype material of Heydrichia cerasina by DNA sequence data that has, thus far, supported confirm that these are distinct species; the taxon the monophyly of only one genus, Bossiella (Hind et al. 2014a, 2015), but revealed that many others 1Received 6 March 2017. Accepted 15 June 2017. First Published were polyphyletic, e.g., Calliarthron (Gabrielson et al. Online 6 July 2017. Published Online 7 August 2017, Wiley Online 2011), Clathromorphum (Adey et al. 2015), Corallina Library (wileyonlinelibrary.com). 2Author for correspondence: e-mail [email protected]. (Hind and Saunders 2013), Hydrolithon, Porolithon, Editorial Responsibility: M. Vis (Associate Editor) and Spongites (Rӧsler et al. 2016). Furthermore, the 1044 THE CORALLINE GENERA SPOROLITHON AND HEYDRICHIA 1045 generitype species of three additional genera, Pach- had been considered a later heterotypic synonym of yarthron, Serraticardia and Yamadaia were all shown S. ptychoides since the early 20th century, was more to belong in Corallina, and these genera were placed similar to S. erythraeum, leaving only two equivocal in synonymy by Hind et al. (2014b), Hind and characters to distinguish the three species: (i) size Saunders (2013) and Martone et al. (2012), respec- of tetrasporangia (partially overlapping in length tively. Many other coralline genera have yet to be and width), and (ii) whether or not old, buried assessed using this methodology. terasporangia were sometimes infilled (Verheij DNA sequencing of the type specimen of generi- 1993, table 2). To date, no original material of type species has been critical to correctly applying S. crassum has been located in other herbaria. Thus, these names. For example, Bossiella plumosa, the at present, all four species are recognized, and generitype species and B. frondifera both share the S. erythraeum, S. molle, and S. ptychoides are reported same type locality, and sequencing recently collected to be widely distributed based on morpho-anatomy topotype material did not allow names to be correctly (Guiry and Guiry 2017, accessed January 20, 2017). applied until the holotype specimen of each was The generitype species of Heydrichia, H. woelker- sequenced (Hind et al. 2015). In the case of the lingii, is based on specimens collected from Oudek- generitype species of Lithophyllum, L. incrustans, that raal, Western Cape Province, South Africa name had been misapplied to specimens for 60 years (Townsend et al. 1994). Sequencing of the holo- (Hernandez-Kantun et al. 2015). Western European type, isotype, and other specimens examined in the field-collected specimens with rbcL sequences identi- protologue is likely not possible due to formalin cal to the lectotype specimen were subtidal rhodo- and glutaraldehyde preservation. The first published liths, or less commonly, subtidal, epilithic, epizoic, or SSU sequence of H. woelkerlingii was from a speci- epiphytic crusts, not a common, epilithic intertidal men collected from Betty’s Bay near the type locality crust as widely reported (Cabioch and Boudouresque (Bailey and Chapman 1998). Subsequently, Farr 1992, Chamberlain and Irvine 1994). et al. (2009) provided psbA sequences of specimens Four currently recognized Sporolithon species, identified as H. woelkerlingii from New Zealand. S. ptychoides, the generitype, S. erythraeum, S. crassum, More recently, Mateo-Cid et al. (2014) published a and S. molle, all have their type locality at El Tor, psbA sequence of a specimen collected at the type Egypt in the Gulf of Suez, Red Sea, and all have locality in South Africa. Bahia et al. (2015a) showed intermingled taxonomic histories. Sporolithon ery- that the psbA sequence of a specimen identified as thraeum was originally described as Lithothamnion ery- H. woelkerlingii from New Zealand did not form a thraeum (Rothpletz 1893) based on material found clade with the psbA sequence of the specimen from on the beach. In the protologue of S. ptychoides, two South Africa. forms were described, S. ptychoides f. dura (Heydrich Herein, we apply DNA sequencing methodology 1897a, pl. III, figs. 20–23) and S. ptychoides f. mollis to the generitype species, Sporolithon ptychoides and (Heydrich 1897a, pl. III, figs. 15–19). Heydrich Heydrichia woelkerlingii. For the former, we obtained (1897b) subsequently split the S. ptychoides f. mollis an rbcL sequence from the lectotype specimen; for material and recognized two species, S. molle (Hey- the latter, an rbcL sequence from topotype material. drich 1897a, pl. III, figs. 16, 18, 19) and S. crassum We also sequenced the lectotype of S. molle and (Heydrich 1897a, pl. III, fig. 15), leaving S. pty- the holotypes of Sporolithon dimotum (type local- choides, based on S. ptychoides f. dura as the generi- ity: Lemon Bay, near Guanica, Puerto Rico), cur- type species. Woelkerling and Townsend (in rently considered a synonym of S. ptychoides, and Woelkerling 1988: 207) noted that Heydrich’s origi- of S. episporum (type locality: Point Toro, near nal material of S. ptychoides, S. molle, and S. crassum Colon, Panama). Based on the results of the DNA was destroyed, but that syntype material of the first sequence analyses and SEM observations of mor- two was present in the Foslie herbarium (TRH). pho-anatomical characters, we assess the identity of They designated this syntype material of S. ptychoides species of Sporolithon and Heydrichia and describe a in TRH as the lectotype specimen. Much earlier, new coralline alga, S. eltorensis sp. nov. Foslie (1900;) (as Archaeoliththamnium erythraeum) had considered S. ptychoides to be a later, heteroty- pic synonym of S. erythraeum, and this was confirmed MATERIAL AND METHODS by Woelkerling and Townsend (in Woelkerling Specimen collection. Collections of Sporolithon spp. specimens 1988: 207) based on a morpho-anatomical compar- were made in the shallow subtidal zone (1–1.5 m deep) near ison of the two type specimens, both in TRH. How- El Tor, Egypt as well as Dahab, Egypt, along fringing reefs ever, Verheij (1993) examined the arrangement of and reef flats during a field expedition focused on assessing tetrasporangia in the type specimens of both S. pty- algal biodiversity in the northern Red Sea (Table 1). Speci- choides and S. molle and, based on the presence