Phytotaxa 224 (2): 140158www.mapress.com/phytotaxa/ Copyright 2015 Magnolia Press Article PHYTOTAXA

ISSN 1179-3155 (print edition)

ISSN 1179-3163 (online edition)

140 Accepted by Rafael Riosmena-Rodriguez: 2 Aug. 2015; published: 28 Aug. 2015

http://dx.doi.org/10.11646/phytotaxa.224.2.2

Sporolithon yoneshigueae sp. nov. (Sporolithales, Corallinophycidae, Rhodophyta), a new rhodolith-forming coralline alga from the southwest Atlantic

RICARDO G. BAHIA1, GILBERTO M. AMADO-FILHO1*, GAVIN W. MANEVELDT2, WALTER H. ADEY3, GABRIEL JOHNSON3, MICHEL B. JESIONEK4 & LEILA L. LONGO11Instituto de Pesquisas Jardim Botnico do Rio de Janeiro, Diretoria de Pesquisa Cientfica, Rua Pacheco Leo 915, 22460-030, Rio de Janeiro, RJ, Brazil2Department of Biodiversity and Conservation Biology, University of the Western Cape, P. Bag X17, Bellville 7535, South Africa3Department of Botany, National Museum of Natural History, Smithsonian Institution, Washington, D.C., 20560, USA 4 Instituto de Biologia, Centro de Cincias da Sade, Universidade Federal do Rio de Janeiro (UFRJ). Ilha do Fundo. Av. Brigadeiro Trompowsky, s.n., 21941-900, Rio de Janeiro, RJ, Brasil.*Corresponding author (gilbertoamadofilho@gmail.com)

Abstract

The aim of this study was to describe the new rhodolith-forming coralline alga species, Sporolithon yoneshigueae sp. nov., based on both morpho-anatomical and molecular data. Specimens were collected in rhodolith beds between 28 and 66 m depths in northeastern and southeastern Brazil. The new species can be distinguished from all other species of the genus Spo-rolithon by its wide tetrasporangial compartment pore diameter (3543 m) and the correspondingly large number (1924) of rosette cells surrounding the tetrasporangial compartment pore. Phylogenies inferred from psbA and SSU markers support it as a new species within Sporolithon with interspecific genetic divergence varying from 8.8610.94 %, and 3.674.63%, respectively. Observations from recent gathering and from herbarium collections show that specimens previously designated as Sporolithon mediterraneum in Brazil correspond to Sporolithon yoneshigueae.

Key words: genetic marker, molecular phylogeny, Sporolithaceae, taxonomy

Introduction

The Sporolithaceae (Sporolithales, Corallinophycidae, Rhodophyta) encompasses those crustose coralline algae (CCA) with cruciately divided tetrasporangia occurring individually in calcified compartments rather than conceptacles (Verheij 1993, Le Gall et al. 2010). Currently comprising the extant genera Sporolithon Heydrich and Heydrichia R.A.Townsend, Y.M.Chamberlain & Keats, the family was previously included in the Corallinales (Verheij 1993) but was recently elevated to ordinal rank as the Sporolithales by Le Gall et al. (2010), because of its unique tetrasporangial development and its closer alliance in molecular phylogenies to the Rhodogorgonales rather than the Corallinales. In Heydrichia tetrasporangia are borne on multiple stalk cells that result from the production of successive sporangia, and tetrasporangial compartments are surrounded by an involucre of narrow elongate cells that differ from the ordinary vegetative cells (Townsend et al. 1994, Maneveldt & van der Merwe 2012). Sporolithon differs from Heydrichia in possessing tetrasporangia that are borne on a single stalk cell that does not produce successive sporangia, and the absence of an involucre of narrow elongate cells surrounding the individual tetrasporangial compartments. Coralline algae have traditionally been described and subsequently identified on the basis of typological species concepts using only morphological/anatomical criteria. However, in the last decade, taxonomy based on DNA analysis has become a complementary tool for delimiting taxa (e.g. Pea et al. 2011, Bahia et al. 2014), determining cryptic speciation (e.g. Broom et al. 2008, Bittner et al. 2011, Sissini et al. 2014), and enabling the identification of unfertile material. The usefulness of molecular markers for phylogenetic inferences and species delimitations depend on the existence of a database containing DNA sequences from other related taxa. The GenBank genetic sequence database hosts some Sporolithon species DNA sequences of different genetic markers (e.g., psbA, rbcl, COI, SSU, LSU). However, due to cryptic speciation and high phenotypic plasticity observed in CCA (e.g., Riosmena-Rodriguez et al. 1999, Bittner et al.

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2011) the correct application of a species name must be complemented by a comparison of DNA sequences of both the type material and specimens collected recently (e.g. Sissini et al. 2014). Unfortunately, most CCA type collections comprise herbarium specimens that are older than 50 years (e.g, Woelkerling et al. 2005), therefore, the extraction and amplification of DNA from type material is challenging and often yield unfruitful results which preclude the DNA comparison. In cases where DNA comparison with type is not possible, a remaining reliable option is to obtain sequences from adequately prepared, recently collected topotypesample (from the type locality) after a rigorous morpho/anatomical identification and positive comparison with the type specimen. In order to construct reliable databases for future CCA taxonomic research and to better understand the evolutionary relationships between CCA, it will be important to obtain DNA sequences (preferably from those commonly used genetic markers) for all newly collected fresh material designated as type specimens for a new species. Among the molecular markers used for CCA, nuclear SSU rDNA and plastidial psbA sequences have been the most commonly used in phylogenetic analyses. These markers have been shown to be the best molecular markers to use to infer phylogenetic relations within the rank of genus and species (Broom et al. 2008, Harvey et al. 2005, Far et al. 2009, Bittner et al. 2011, Pea et al. 2011, Kato et al. 2011, 2013, Bahia et al. 2014). These genes vary at different ratespsbA tends to be more variable while the nSSU is more conservative (Farr et al. 2009). During the course of an investigation of the rhodolith-forming coralline algae from Brazil, a frequently encountered alga from deep waters was collected. The alga possessed the vegetative and reproductive features diagnostic of the genus Sporolithon (Verheij 1993). Some seemingly minor anatomical features appeared to differ from the currently known species of Sporolithon, leading us to further investigations. The aim of this paper is to describe a new Sporolithon species based on both morpho-anatomical and molecular data.

Material and Methods

Morphological/Anatomical Analysis

Specimens were collected by SCUBA and mixed-gas diving in rhodolith beds between 28 and 66 m depths. Collections were made from 20082013 on the Abrolhos Continental Shelf, as well as in the Vitria-Trindade Seamount Chain, northeastern and southeastern Brazil, respectively. In addition, herbarium specimens ascribed in the PhD thesis of Tomita (1976) to Sporolithon mediterraneum Heydrich (1899: 227) were re-examined due to their morphological and anatomical similarity to the recent collections. Samples for light microscopy were prepared using the histological methods described by Maneveldt & van der Merwe (2012). For scanning-electron microscopy (SEM), the procedures followed those of Bahia et al. (2010). Growth-form terminology follows Woelkerling et al. (1993), with anatomical terminology following Adey & Adey (1973). For cell length measurements, the distance between primary pit connections was determined, whereas for cell diameters the maximum widths of cell lumens made at right angles to the length was determined. Typification data follow Woelkerling (1993). Herbarium abbreviations follow Thiers (2015, continuously updated). Identified samples, including permanent slides, were deposited on the Herbarium of Jardim Botnico do Rio de Janeiro (RB).

Molecular Analysis

Four specimens of Sporolithon yoneshigueae (RB 570782, RB 600359, RB 600360, RB 600362) were desiccated in silica gel. The protocols for DNA extraction followed Saunders (2005). A piece of fragmented alga (ca. 1.0 cm diameter) of each specimen was crushed in paper napkins with a hammer and then further pulverized with glass and steel beads in a Qiagen Tissue Lyser. Extraction buffer optimized for red algae (Saunders & Druehl 1993) was added to these tissue powders and DNA extraction was performed with the DNeasy Plant Kit (Qiagen Gmbh, Hilden, Germany). The psbA locus was amplified using primers psbA-F/psbA 600R and psbA 500F/psbA R2 (Yoon et al. 2002), and for SSU the primers SSU-G01F/SSU-G14R and SSU-G04F/SSU-G07R (Harper & Saunders 2001) were used. The PCR reactions were conducted on a final volume of 25 l with 2.5 l of Bioline Ammonium Buffer (10x), 2.0 l of dNTP mix (10mM), 1.25 l of magnesium chloride (50 mM), 1.0 l of each Primer (10 M), 0.5 l of Bovine Serum Albumin (10 mg/mL), 14.05 l of nuclease-free water, 0.2 l of Biolase Taq Polymerase (5U/l) and 2.5 l of DNA template (~20 ng/l). PCR conditions implemented for psbA and SSU are summarized in Table 1. Products were analyzed on 1.5% agarose gel and purified using ExoSapIT enzyme mixture (Affymetrix-USB). Sequencing reactions

BAHIA ET AL.142 Phytotaxa 224 (2) 2015 Magnolia Press

were conducted using 8l of a cycle sequencing reaction mixture (including 0.8 l Big Dye (Terminator 3.1, Applied Biosystems), 1.0 l 1 M primer, 2.0 l 5x buffer, 3.7 l water, and 0.5 l dimethyl sulfoxide) combined with 4l of DNA template. The amplification conditions were 30 cycles of 95C for 30 sec, 55C for 30 sec and 60C for 4 min. The cycle sequencing products were purified using Sephadex G-50 (GE HealthCare)