J. Phycol. 51, 791–807 (2015) © 2015 Phycological Society of America DOI: 10.1111/jpy.12319 SEQUENCING TYPE MATERIAL RESOLVES THE IDENTITY AND DISTRIBUTION OF THE GENERITYPE LITHOPHYLLUM INCRUSTANS, AND RELATED EUROPEAN SPECIES L. HIBERNICUM AND L. BATHYPORUM (CORALLINALES, RHODOPHYTA)1 Jazmin J. Hernandez-Kantun2 Botany Department, National Museum of Natural History, Smithsonian Institution, MRC 166 PO Box 37012, Washington District of Columbia, USA Irish Seaweed Research Group, Ryan Institute, National University of Ireland, University Road, Galway Ireland Fabio Rindi Dipartimento di Scienze della Vita e dell’Ambiente, Universita Politecnica delle Marche, Via Brecce Bianche, Ancona 60131, Italy Walter H. Adey Botany Department, National Museum of Natural History, Smithsonian Institution, MRC 166 PO Box 37012, Washington District of Columbia, USA Svenja Heesch Irish Seaweed Research Group, Ryan Institute, National University of Ireland, University Road, Galway Ireland Viviana Pena~ BIOCOST Research Group, Departamento de Bioloxıa Animal, Bioloxıa Vexetal e Ecoloxıa, Facultade de Ciencias, Universidade da Coruna,~ Campus de A Coruna,~ A Coruna~ 15071, Spain Equipe Exploration, Especes et Evolution, Institut de Systematique, Evolution, Biodiversite, UMR 7205 ISYEB CNRS, MNHN, UPMC, EPHE, Museum National d’Histoire Naturelle (MNHN), Sorbonne Universites, 57 rue Cuvier CP 39, Paris 75005, France Phycology Research Group, Ghent University, Krijgslaan 281, Building S8, Ghent 9000, Belgium Line Le Gall Equipe Exploration, Especes et Evolution, Institut de Systematique, Evolution, Biodiversite, UMR 7205 ISYEB CNRS, MNHN, UPMC, EPHE, Museum National d’Histoire Naturelle (MNHN), Sorbonne Universites, 57 rue Cuvier CP 39, Paris 75005, France and Paul W. Gabrielson Department of Biology and Herbarium, University of North Carolina, Chapel Hill, Coker Hall CB 3280, Chapel Hill, North Carolina 27599-3280, USA DNA sequences from type material in the belonging in Lithophyllum. As well as occurring as a nongeniculate coralline genus Lithophyllum were subtidal rhodolith, L. hibernicum is a common, used to unambiguously link some European species epilithic and epizoic crust in the intertidal zone names to field-collected specimens, thus providing a from Ireland south to Mediterranean France. A set great advance over morpho-anatomical identifi- of four features distinguished L. incrustans from cation. In particular, sequence comparisons of rbcL, L. hibernicum, including epithallial cell diameter, COI and psbA genes from field-collected specimens pore canal shape of sporangial conceptacles and allowed the following conclusion: the generitype sporangium height and diameter. An rbcL sequence species, L. incrustans, occurs mostly as subtidal of the lectotype of Lithophyllum bathyporum, which rhodoliths and crusts on both Atlantic and was recently proposed to accommodate Atlantic Mediterranean coasts, and not as the common, NE intertidal collections of L. incrustans, corresponded Atlantic, epilithic, intertidal crust reported in the to a distinct taxon hitherto known only from literature. The heterotypic type material of Brittany as the subtidal, bisporangial, lectotype, but L. hibernicum was narrowed to one rhodolith also occurs intertidally in Atlantic Spain. Specimens from Ireland and France morpho-anatomically identified as L. fasciculatum and a specimen from 1 Received 3 November 2014. Accepted 7 May 2015. Cornwall likewise identified as L. duckerae were 2Author for correspondence: e-mails [email protected], [email protected] resolved as L. incrustans and L. hibernicum, Editorial Responsibility: C. Lane (Associate Editor) respectively. 791 792 JAZMIN J. HERNANDEZ-KANTUN ET AL. Key index words: anatomy; Lithophyllum bathyporum; Mesophyllum lichenoides ( J. Ellis) Me. Lemoine, while Lithophyllum hibernicum; Lithophyllum incrustans; type material of L. decussatum, which consisted of psbA; rbcL; rhodolith; taxonomy; type specimens two small fragments, could not be assigned with confidence to a species or even a genus. Finally, Abbreviations: BI, Bayesian inference; BP, Bootstrap L. lichenoides is currently recognized morpho-ana- value; GTR, general time reversible; ML, maximum tomically as a heterotypic synonym of L. byssoides likelihood; psbA, Photosystem II D1 protein gene; rbcL, (Lamarck) Foslie (Woelkerling and Lamy 1998: 258- ribulose-1,5-bisphosphate carboxylase/oxygenase large 259). subunit gene In Brittany, Crouan and Crouan (1867) described Lithothamnion depressum P.L. Crouan & H.M. Crou- an; this taxon was transferred by Foslie (1898) to As with many other groups of organisms, one of L. incrustans f. depressum (P.L. Crouan & H.M. Crou- the most important and difficult tasks in current an) Foslie, and later attributed to Atlantic crustose taxonomic studies on algae is to link Linnean sys- forms of L. incrustans (Chamberlain and Irvine tem names with molecular phylogenetic clades 1994). Recently, Ballantine et al. (2011) proposed (Verbruggen 2014). Nowhere is this more evident the novel species name Lithophyllum bathyporum than in coralline red algae (subphylum Coral- Athanasiadis & D.L. Ballantine based on the lecto- linophycidae) and for two main reasons: (i) DNA typification of L. incrustans f. depressum (L. depres- sequencing studies have demonstrated a much sum) described in Chamberlain and Irvine (1994). higher diversity of both non-geniculate (Bittner L. incrustans has been recorded not only from the et al. 2011, Kato et al. 2011, 2013, Mateo-Cid et al. Mediterranean Sea (Hamel and Lemoine 1952, 2014, Pardo et al. 2014, Adey et al. 2015) and genic- Bressan and Babbini 2003) and Atlantic coasts of ulate (Hind and Saunders 2013, Hind et al. 2014b, Europe (Adey and Adey 1973, Ford et al. 1983, Pardo et al. 2015) species compared to previous Edyvean and Ford 1986, Chamberlain and Irvine morpho-anatomical studies, and (ii) morpho-ana- 1994), but also from South Africa (Chamberlain tomical characters have proved inadequate to segre- 1996) and the Caribbean Sea (Ballantine et al. gate species and even genera of coralline taxa 2011). It is reported to occur in a wide range of (Gabrielson et al. 2011, Kato et al. 2011, Martone habitats from the mid-intertidal through the subtid- et al. 2012, Hind and Saunders 2013, Hind et al. al and with diverse habits ranging from epizoic or 2014a,b, Pardo et al. 2014, Adey et al. 2015). The epilithic crusts on bedrock in and outside of pools, most unambiguous method to determine the cor- to rhodoliths (Chamberlain and Irvine 1994, Bres- rect application of a name is to amplify a DNA san and Babbini 2003, Ballantine et al. 2011). Keys sequence from a type specimen to compare with provided by Adey and Adey (1973), Chamberlain complementary sequences from field-collected mate- and Irvine (1994) and Bressan and Babbini (2003) rial (Hughey et al. 2001, Gabrielson 2008, Hind suggested that its habitat and morpho-anatomical et al. 2014a). This method has been applied success- features could be used to identify and separate fully for geniculate (Gabrielson et al. 2011, Hind L. incrustans from similar species. However, in a et al. 2014a,b) and non-geniculate corallines (Pena~ recent molecular study on rhodoliths (Hernandez- et al. 2014, Sissini et al. 2014, Adey et al. 2015, Her- Kantun et al. 2015) the morpho-anatomical identifi- nandez-Kantun et al. 2015). Herein, we sequenced cations of L. dentatum (Kutzing)€ Foslie, L. fascicula- type material to begin resolving the genus Lithophyl- tum (Lamarck) Foslie, and L. incrustans did not lum Phillipi, based on its generitype, L. incrustans match monophyletic clades identified by DNA Philippi (type locality: Sicily) and related species in sequencing, indicating the need for further studies Europe. to resolve the identity of these species. Lithophyllum incrustans, one of four species origi- The identity of the generitype L. incrustans based nally described by Philippi (1837), was designated on reliable molecular and morpho-anatomical fea- as the generitype by Foslie (1898). Philippi’s origi- tures is of crucial importance in defining the genus nal material was lost for over 140 years, until redis- Lithophyllum and for the circumscription of the sub- covered by Woelkerling (1983a,b) in the Nationaal family Lithophylloideae and family Corallinaceae. Herbarium Nederland (L). Along with L. incrustans, To date, molecular systematics studies included type material was also found of L. decussatum (Ellis DNA sequences assigned to L. incrustans without the & Solander) Philippi (based on Millepora decussatum corroboration based on type material (Bailey 1999, Ellis & Solander), L. expansum Philippi, and L. li- Harvey et al. 2003, Bailey et al. 2004, Broom et al. chenoides Philippi. After an exhaustive morpho-ana- 2008, Bittner et al. 2011, Kato et al. 2011, Richards tomical analysis of Philippi’s original material, et al. 2014, Hernandez-Kantun et al. 2015). Woelkerling (1983a) concluded that only L. incru- Taxa ascribed to the genus Lithophyllum (the type stans, the generitype, and L. lichenoides agreed with genus of the subfamily Lithophylloideae with 117 most of the features that define the modern con- specific/infraspecific names currently accepted, cept of Lithophyllum. Type material of L. expansum Guiry and Guiry 2014) represent a major ecological agreed morpho-anatomically with type material of group from tropical to temperate coastal areas, TAXONOMIC IDENTITY OF LITHOPHYLLUM INCRUSTANS 793 demonstrating the importance of this genus in Recent collections were amplified, including