J. Phycol. 52, 412–431 (2016) © 2016 Phycological Society of America DOI: 10.1111/jpy.12404 PHYLOGENETIC RELATIONSHIPS OF CORALLINACEAE (CORALLINALES, RHODOPHYTA): TAXONOMIC IMPLICATIONS FOR REEF-BUILDING CORALLINES1 Anja Rosler€ 2 Departamento de Estratigrafıa y Paleontologıa, Universidad de Granada, Campus Fuente Nueva, Granada 18002, Spain Francisco Perfectti Departamento de Genetica, Universidad de Granada, Campus Fuente Nueva, Granada 18002, Spain Viviana Pena~ Grupo de investigacion BIOCOST, Facultade de Ciencias, Universidade da Coruna,~ Campus de A Coruna,~ A Coruna~ 15071, Spain Phycology Research Group, Ghent University, Krijgslaan 281, Building S8, Ghent 9000, Belgium 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 F-75005, France and Juan Carlos Braga Departamento de Estratigrafıa y Paleontologıa, Universidad de Granada, Campus Fuente Nueva, Granada 18002, Spain A new, more complete, five-marker (SSU, LSU, son 2009) through their sensitivity to climate psbA, COI, 23S) molecular phylogeny of the family change and ocean acidification (Hall-Spencer et al. Corallinaceae, order Corallinales, shows a paraphyletic 2008, Burdett et al. 2012, Diaz-Pulido et al. 2012, grouping of seven well-supported monophyletic clades. Kato et al. 2014, McCoy and Kamenos 2015), to The taxonomic implications included the amendment their use as paleoenvironmental indicators (Braga of two subfamilies, Neogoniolithoideae and Metago- et al. 2010, Abbey et al. 2011, Aguirre et al. 2012, niolithoideae, and the rejection of Porolithoideae as an Novak et al. 2013). Coralline red algae are impor- independent subfamily. Metagoniolithoideae contai- tant components in many shallow-water marine ned Harveylithon gen. nov., with H. rupestre comb. nov. ecosystems (Adey and Mcintyre 1973, Adey 1986). as the generitype, and H. canariense stat. nov., They are major builders in coral reefs (Littler and H. munitum comb. nov., and H. samoense€ comb. nov. Littler 1984, Mallela 2013) and contribute signifi- Spongites and Pneophyllum belonged to separate clades. cantly to the preservation of reef health and biodi- The subfamily Neogoniolithoideae included the versity (Morse et al. 1996, reviewed in Nelson 2009, generitype of Spongites, S. fruticulosus, for which an Vermeij et al. 2011). The main coralline reef epitype was designated. Pneophyllum requires reass- builders are members of the order Corallinales, fam- esment. The generitype of Hydrolithon, H. reinboldii, ily Corallinaceae, that are predominant in the fossil was a younger heterotypic synonym of H. boergesenii. record (since the Miocene) and on recent coral The evolutionary novelty of the subfamilies Hydro- reefs to a depth of ~50 m (Adey et al. 1982, Braga lithoideae, Metagoniolithoideae, and Lithophylloideae et al. 2010). was the development of tetra/bisporangial conceptacle Despite their significance, the taxonomy of coral- roofs by filaments surrounding and interspersed line algae is not resolved at any rank, although it among the sporangial initials. has been subject to continuous debate since the 19th century (Johansen 1981, Woelkerling 1988, Le Key index words: coral reef; Corallinales; coralline Gall et al. 2010, Bittner et al. 2011, Kato et al. 2011, algae; Hydrolithoideae; Mastophoroideae; Metago- Woelkerling et al. 2012). Recent systematic work niolithoideae; molecular phylogeny; Neogonioli- based on multigene analyses resulted in the separa- thoideae tion of the new order Hapalidiales from Corallinales sensu Silva and Johansen (1986), characterized by multiporate and uniporate tetrasporangial concepta- cles, respectively (Nelson et al. 2015). Coralline The ongoing scientific interest in coralline algae algae show extreme phenotypic plasticity dependent is based on many factors, from their ecological on external factors (Steneck 1986, Woelkerling importance in various shallow marine habitats (Nel- 1988, Braga et al. 1993, Woelkerling et al. 1993a, Farr et al. 2009) and, on the other hand, they 1Received 13 June 2014. Accepted 19 January 2016. possess a relatively simple thallus with limited 2Author for correspondence: e-mail [email protected]. morphoanatomical character variation. Several Editorial Responsibility: P. Gabrielson (Associate Editor) 412 PHYLOGENYOFREEFCORALLINES 413 diagnostic features (e.g., gametangial conceptacle conicum (E.Y.Dawson) Keats, Y.M.Chamberlain & primordia or developing gametangial conceptacles) M.Baba. On the other hand, H. munitum (Foslie & are rarely observed in samples with the added diffi- M.Howe) Penrose, H. cymodoceae (Foslie) Penrose, culty that sample preparation to detect those and H. rupestre (Foslie) Penrose were not included features is arduous (Woelkerling 1988, Farr et al. in the analyses of Kato et al. (2011). 2009). Woelkerling (1988) (pp. 62–73 and table Using four genetic markers (SSU, LSU, psbA and 5.2) listed the characters historically considered COI), Bittner et al. (2011) also separated Porolithon delimiting for subfamilies and genera and proposed Foslie from Hydrolithon. In this work, Porolithon com- a classification scheme for both taxonomic ranks. prised those coralline taxa possessing primarily Since this seminal publication, the diagnostic fea- monomerous thalli and trichocytes in large horizon- tures and circumscription of a number of genera tal pustulose fields without interspersed filaments. and subfamilies have changed (Harvey et al. 2003, Woelkerling et al. (2012), however, pointed out that Le Gall and Saunders 2007, Le Gall et al. 2010, Kato the separation based on horizontal trichocyte fields et al. 2011, Adey et al. 2015), demonstrating that would be untenable because this character is not even at the subfamily rank the taxonomy of coral- present in every specimen, as already stated by Men- line algae is still unresolved (Bittner et al. 2011, doza and Cabioch (1986). In Bittner et al. (2011) Woelkerling et al. 2012). and Kato et al. (2011), Metagoniolithoideae was sis- As stated by Harvey et al. (2003), one of the main ter to Porolithon. impediments to classifying coralline taxa is to deter- In this study, the phylogeny of Corallinaceae was mine which morphological characters result from analyzed with a set of five DNA markers (SSU, LSU, homoplasy and which from common ancestry. The psbA, COI, and 23S). SSU was the first marker used uncertainties in the answers to this question led to in coralline algal phylogeny (Bailey and Chapman the proposal of very different classification schemes 1996) and has been confirmed to be a highly infor- during the last decades (e.g., Adey 1965, Cabioch mative marker in phylogenetic studies for a very 1971, Bailey 1999, Le Gall et al. 2010, Nelson et al. broad group of organisms (White et al. 1990, 2015). However, molecular phylogenetics is now Armando Sanchez et al. 2003, Meyer et al. 2010). providing new insights into biological systematics. In However, SSU is a conservative marker for coralline fact, in the last 10 years the use of molecular tools algae, showing little variation in shallow phylogenies in phylogeny and species identification has seen a (Bailey and Freshwater 1997). Later, Broom et al. sharp increase in genetic sequences of coralline (2008) also used psbA, a plastid gene with substan- algae available in GenBank. In 2003, for example, tial variation between genera and between taxa to the orders Corallinales and Hapalidiales (sensu Nel- improve phylogenetic resolution within the order son et al. 2015) had 38 entries of only the nuclear Corallinales. The combination of the first four marker SSU (18S rDNA) (Bailey et al. 2004), which already showed good resolution of the phylogeny of had risen to 5,845 total sequences by 2015, includ- coralline algae in Bittner et al. (2011). UPA (univer- ing 463 SSU sequences (http://www.ncbi.nlm.nih. sal plastid amplificon, 23S) has been used as a fifth gov/nuccore/, verified 21 October 2015). marker due to its utility in evolutionary studies and Bailey et al. (2004) recognized that the subfamily high amplification success (Sherwood and Presting Mastophoroideae sensu Harvey et al. (2003) was 2007, Clarkston and Saunders 2010). It is able to polyphyletic. Later, Bittner et al. (2011) and Kato distinguish species but is also applicable over a et al. (2011) analyzed with multigen approaches the broad phylogenetic distance. Herein, we provided new placement of the genera once grouped in this new insights in the evolutionary history of Coralli- obsolete subfamily within Corallinaceae. Based on naceae, and clarified some taxonomic uncertainties two genetic markers, Kato et al. (2011) established still remaining in this family. the subfamily Porolithoideae, including Porolithon onkodes (Heydrich) Foslie, the generitype species, MATERIALS AND METHODS P. pachydermum (Foslie) Foslie, and P. gardineri (Foslie) Foslie, characterized by horizontal fields of Fifty-seven intertidal and subtidal samples were collected, trichocytes without interspersed vegetative cells. The snorkeling and scuba diving in different localities in the Paci- subfamily Hydrolithoideae, whose members lack fic, Indian, and Atlantic Oceans and the Caribbean and Mediterranean Seas (see Table 1 for details). Samples col- those “tightly packed” trichocyte fields, was lected by AR and JCB were air-dried for at least one hour restricted to Hydrolithon reinboldii (Weber-van Bosse and then stored in silica gel that was changed several times