Arquipelago  -­‐‑  Life  and  Marine  Sciences    ISSN:  0873-­‐‑4704   The  crustose  red  algal  genus  Peyssonnelia  (Peyssonneliales,   Rhodophyta)  in  the  Azores:  from  five  to  one  species     DANIELA  GABRIEL,  W.E.  SCHMIDT,  D.M.  KRAYESKY,  D.J.  HARRIS  &  S.  FREDERICQ   Gabriel, D., W.E. Schmidt, D.M. Krayesky, D.J. Harris & S. Fredericq 2015. The crustose red algal genus Peyssonnelia (Peyssonneliales, Rhodophyta) in the Azores: from five to one species. Arquipelago. Life and Marine Sciences 32: 1-9. The family Peyssonneliaceae comprises a worldwide group of non-calcified to calcified, crust-forming red algae of great ecological significance. Of the genera currently recognized in the family, Peyssonnelia has been widely considered to contain the largest number of species, with five members reported for the Azores. Using rbcL as a molecular marker, we here report on the taxonomic identity of recent collections of Peyssonneliaceae from the Azorean islands of São Miguel, Graciosa and Pico, and compare those specimens in a worldwide context. Only a single Peyssonnelia species, P. squamaria, is confirmed for the Azorean archipelago, with three different haplotypes. Although the populations in the Azores are genetically different from those occurring in the Mediterranean, this separation appears to be relatively recent. Key words: Biodiversity, haplotypes, North Atlantic, phylogeny, rbcL D. Gabriel (e-mail: danielalgabriel@gmail.com) & D.J. Harris, Research Center in Biodiversity and Genetic Resources (CIBIO), University of the Azores, PT-9501-801 Ponta Delgada, Portugal; D.M. Krayesky, Biology Department, Slippery Rock University, Slippery Rock, PA 16057-1326, USA; W. E. Schmidt & S. Fredericq, Department of Biology, University of Louisiana at Lafayette, Lafayette, LA 70504-3602, USA. INTRODUCTION The family Peyssonneliaceae (Denizot, 1968), recently elevated to ordinal rank (Krayesky et al. 2009), comprises a worldwide group of noncalcified or calcified, crust-forming red algae that are of great ecological significance (Peña & Barbara 2013). Of the genera currently recognized in the family, Peyssonnelia (Decaisne, 1841) has been considered the richest in terms of species number (Pueschel & Saunders 2009). A combination of vegetative and reproductive characters are currently used to distinguish species of Peyssonnelia, such as location and degree of calcification, variations in crust adherence, morphology, anatomy and differences in reproductive development (Maggs & Irvine 1983). The identification of Peyssonnelia species is challenging, resulting in a number of species usually underestimated or overestimated (Dixon & Saunders 2013). Comparative morphology and DNA sequence analysis confirm that most species originally reported as belonging in Peyssonnelia in fact belong to other genera (Fredericq et al. 2014) within the Peyssonneliales (Krayesky et al. 2009). Peyssonnelia sensu stricto (following Krayesky et al. 2009) represents species characterized by a hypothallus that cuts off additional cells forming multicellular rhizoids (Krayesky 2007). Recent studies based on worldwide collections indicate that species of Peyssonnelia sensu stricto have a narrow distribution and do not occur in most ocean basins, for example, the Gulf of Mexico (Krayesky et al. 2009; Fredericq et al. 2014). Based on general flora studies, five species of Peyssonnelia have been reported for the Azores (Parente 2010): the generitype P. squamaria ((S.G. Gmelin) Decaisne, 1842) described from Italy; P. rubra ((Greville) J. Agardh, 1851) described from the Ionian Sea, Greece; P. polymor1          Gabriel  et  al.   pha ((Zanardini) F. Schmitz in Falkenberg, 1879) described from the Adriatic Sea; P. coriacea (Feldmann, 1941) described from Tangier, Morocco; and P. rosa-marina (Boudouresque & Denizot, 1973) described from Port-Cros, Mediterranean France (see Guiry & Guiry 2015). Only one species of Peyssonnelia, P. squamaria (Fig. 1), was recognized for the Azores by Krayesky (2007) and Krayesky et al. (2009) after examination of multiple collections. Using rbcL as a molecular marker, we report on the taxonomic identity of recent collections of Peyssonneliaceae from the islands of São Miguel, Graciosa and Pico in the Azores, and discuss the connection between the various Azorean haplotypes. The identity of the Azorean specimens is compared with those of Peyssonnelia sensu stricto in a worldwide context. Fig. 1. Habit of Peyssonnelia squamaria from the Azores. MATERIAL AND METHODS Samples of Peyssonneliaceae were collected in the Azores and the Mediterranean during low tide or by snorkeling and SCUBA diving. Samples were kept in coolers until processed, and then dried in silica gel. Dried samples were ground with mortar and pestle, and total DNA was extracted using DNeasy Plant mini Kits (Qiagen Valencia, CA, USA). All the resulting DNA extracts were deposited in the Seaweed Lab at the University of Louisiana at Lafayette (ULL). Chloroplast-encoded rbcL gene sequences were amplified using PCR primers and protocols described in Lin et al. (2001) and Gabriel et al. (2010). Resulting PCR products were gel-purified 2 and sequenced in both directions using Bigdye terminator v 3.1 (Life Technologies Grand Island NY, USA) on the ABI 3130xl genetic analyzer at ULL and assembled with Sequencher v. 5.2 (Gene Codes Corporation). Newly acquired sequences, in addition to 14 rbcL sequences downloaded from GenBank, were then manually aligned in Mega v 5.2.2 (Tamura et al. 2011). The subsequent alignment was analyzed in Partitionfinder (Lanfear et al. 2012) to determine the best fitting model of evolution and data partition. The analysis resulted in the selection of the General Time Reversible model plus gamma and a proportion of invariable sites applied separately to each codon position on the basis of the three information criteria, i.e. Akaike information criteri-          Peyssonnelia  in  the  Azores   on with correction (AICc), Akaike information criterion (AIC) and Bayesian information criterion (BIC). The alignment was analyzed by Maximum likelihood (ML) as implemented by RAXML v 2.4.4 (Stamatakis 2006) with the above models and partition scheme with 1000 restarts to find the tree with the lowest likelihood score and 1000 Bootstrap (BS) replications. A Bayesian MCMC (Markov Chain Monte Carlo) was also applied to the aligned dataset using MrBayes v. 3.2.5 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003). The Bayesian analysis consisted of two independent runs of 5 million generations with sampling every 1,000 generations for a total of 10,002 trees. Convergence was visualized using Tracer v1.6 (Rambaut & Drummond 2007) and the first 10 percent of the trees of each run was discarded as the burn-in. The resulting Bayesian Posterior Probabilities derived from the consensus tree were mapped on the ML tree. A distance matrix was also resolved from the branch lengths of the ML tree using the function cophenetic.phylo of the APE Package in R (Paradis et al. 2004). The resulting distance matrix was used to find species boundaries in a stand-alone version of Automatic to Barcode Gap Discovery (ABGD). General Mixed Yule Coalescence (GMYC) model, as implemented by the Splits Package in R (Fujisawa & Barraclough 2013) with a single threshold model, was also used to determine species boundaries. The requisite ultrametric tree for the GMYC analysis was generated in Beast v 1.8.1 (Drummond et al. 2012) using a relaxed log-normal clock with a constant population coalescent as a prior and the best fitting model and partition as described above. MCMC Chains were run for 10 million generations with sampling every 1000th generation resulting in 10,000 trees. The quality of the run was assessed in Tracer v1.6 (Rambaut & Drummond 2007) to ensure that effective sample size (ESS) values were >200 with the default burn-in (1,000 trees). Tree annotator v 1.8.1 (Drummond et al. 2012) was used to summarize the resulting 9001 trees after burning, targeting the maximum clade credibility tree with preserved node heights. A statistical parsimony method implemented in the TCS 1.21 software (Clement et al. 2000) was used to infer genealogical relationships among haplotypes. The maximum number of differences resulting from single substitutions among haplotypes was calculated with 95% confidence limits, treating gaps as missing data. RESULTS The final dataset was composed of 34 rbcL sequences of Peyssonneliaceae, 20 of which were newly generated, with 31 sequences representing hitherto confirmed Peyssonnelia species and three sequences of Sonderopelta capensis ((Montagne) Krayesky, 2009) and S. coriacea (Womersley & Sinkora, 1981) (Table 1). Sonderopelta (Womersley & Sinkora, 1981) was selected as an outgroup based on previous studies that established the genus as a sister taxon of Peyssonnelia (Kato et al. 2006; Krayesky et al. 2009; Dixon & Saunders 2013). [Note: Sonderopelta has been viewed to be an illegitimate name by Wynne (2011); however, Sonderophycus (Denizot, 1968) is not a valid name (Womersley & Sinkora 1981) in agreement with Article 41.5 of the International Code of Botanical Nomenclature (2012, Melbourne Code)]. The results of the ABGD and GMYC analyses showed the existence of six species of Peyssonnelia within the dataset: P. replicata (Kützing, 1847), P. bornetii (Boudouresque & Denizot, 1973), P. rubra and three closely related species initially identified as P. squamaria (Fig. 2). All Azorean collections belonged to P. squamaria, along with Mediterranean representatives from Catalonia (Spain), Sicily (Italy), Malta and Greece (not shown). Its two sister clades were only observed in the Mediterranean, and are here referred to as P. coriacea from Malta and P. polymorpha from Sicily. Sequence JX969797, referred to as P. squamaria by Dixon & Saunders (2013), corresponds to material of Sicily that has a unistratose hypothallus layer in contrast to the 2-layered hypothallus of P. squamaria (Boudouresque & Denizot, 1975). Besides the generitype, four species that are also true Peyssonnelia were only collected in the 3 Table 1. Summary of specimens included in the present study. Asterisk marks newly generated sequences. Letters within brackets refer to the haplotypes of Peyssonnelia squamaria in Fig. 3. Extraction Collection number Taxa Locality Depth Collection date Collector Accession number K163 SMG-05-242 Peyssonnelia squamaria (A1) São Miguel, Azores intertidal 09-jul-2005 EU349177 K164 SMG-05-152 Peyssonnelia squamaria (A1) São Miguel, Azores 8m 08-oct-2005 EU349178 LAF4197 MD0002162 Peyssonnelia squamaria (A1) Pico, Azores 26 m 10-aug-2011 D. Gabriel, J. Micael KR732897* LAF6390 MD0002066 Peyssonnelia squamaria (A1) São Miguel, Azores intertidal 10-jul-2011 M.I. Parente, D. Gabriel KR732900* LAF6393 MD0001927 Peyssonnelia squamaria (A1) São Miguel, Azores 15 m 29-oct-2010 A. Botelho KR732899* LAF6395 MD0001933 Peyssonnelia squamaria (A1) São Miguel, Azores 12 m 05-jul-2011 A. Botelho, M. Dionísio, C. Lopes KR732898* K229 SMG-06-88 Peyssonnelia squamaria (A2) São Miguel, Azores 16 m 11-aug-2006 EU349174 K231 GRW-06-100 Peyssonnelia squamaria (A3) Graciosa, Azores 15 m 22-jun-2006 EU349176 K230 CAT-06-10 Peyssonnelia squamaria (B) Catalonia, Spain intertidal 16-jul-2006 EU349175 LAF5459 PG-08-1353 Peyssonnelia squamaria (C) Malta 10 m 06-aug-2008 M.I. Parente, J. Micael KR732910* LAF5357 PG-08-1210 Peyssonnelia squamaria (D) Malta 15 m 02-aug-2008 M.I. Parente KR732905* LAF5360 PG-08-1146 Peyssonnelia squamaria (D) Malta 30 m 02-aug-2008 M.I. Parente, J. Micael KR732909* LAF5362 PG-08-1248 Peyssonnelia squamaria (D) Malta 26 m 05-aug-2008 M.I. Parente, J. Micael KR732901* LAF5363 PG-08-1088 Peyssonnelia squamaria (D) Malta 30 m 02-aug-2008 M.I. Parente, J. Micael KR732902* LAF5455 PG-08-1100 Peyssonnelia squamaria (D) Malta 30 m 02-aug-2008 M.I. Parente, J. Micael KR732907* LAF5458 PG-08-1153 Peyssonnelia squamaria (D) Malta 30 m 02-aug-2008 M.I. Parente, J. Micael KR732904* LAF5463 PG-08-795 Peyssonnelia squamaria (D) Sicily, Italy intertidal 12-mar-2008 M.I. Parente, R. Sousa, J. Matzen KR732903* Extraction Taxa Locality Depth Collection date Collector Accession number LAF5464 PG-08-756 Peyssonnelia squamaria (D) Sicily, Italy intertidal 12-mar-2008 M.I. Parente, R. Sousa, J. Matzen KR732906* LAF5465 PG-08-747 Peyssonnelia squamaria (D) Sicily, Italy intertidal 12-mar-2008 M.I. Parente KR732908* LAF5355 PG-08-1268 Peyssonnelia coriacea Malta 26 m 05-aug-2008 M.I. Parente, J. Micael KR732911* LAF5361 PG-08-1173 Peyssonnelia coriacea Malta 30 m 02-aug-2008 M.I. Parente, J. Micael KR732912* LAF5457 PG-08-1291 Peyssonnelia coriacea Malta 26 m 05-aug-2008 M.I. Parente, J. Micael KR732913* LAF5461 PG-08-804 Peyssonnelia polymorpha Sicily, Italy intertidal 12-mar-2008 M.I. Parente, R. Sousa, J. Matzen KR732915* LAF5462 PG-08-790 Peyssonnelia polymorpha Sicily, Italy intertidal 12-mar-2008 M.I. Parente, R. Sousa, J. Matzen KR732916* GWS018179 Peyssonnelia polymorpha Sicily, Italy - 10-apr-2010 G. Furnari PG-08-551 Peyssonnelia polymorpha Sicily, Italy intertidal 09-mar-2008 M.I. Parente KR732914* LAF4199   Collection number JX969797 K166 LAF-8-2-1-1-12 Peyssonnelia rubra Liguria, Italy 3m 02-aug-2001 B. Gavio EU349179 K217 LAF-7-30-1-1-2 Peyssonnelia bornetii Liguria, Italy 3m 30-jul-2001 B. Gavio EU349180 K218 LAF-7-28-1-1-2 Peyssonnelia bornetii Liguria, Italy 2-20 m 28-jul-2001 B. Gavio EU349181 K241 LAF-2-6-01-2-2 Peyssonnelia replicata KwaZulu-Natal, South Africa intertidal 06-feb-2001 T. Schils EU349182 K243 LAF-7-23-93-1-1 Peyssonnelia replicata KwaZulu-Natal, South Africa Drift (beach) 23-jul-1993 M. Hommersand EU349183 K214 LAF-2-6-01-1-15 Sonderopelta capensis KwaZulu-Natal, South Africa 30 m 06-feb-2001 S. Fredericq & O. De Clerck EU349186 K215 LAF-2-6-01-1-1 Sonderopelta capensis KwaZulu-Natal, South Africa 30 m 06-feb-2001 S. Fredericq & O. De Clerck EU349187 K220 LAF-7-13-95-1-1 Sonderopelta coriacea Victoria, Australia - 13-jul-1995 M. Hommersand EU349190          Gabriel  et  al.   Fig. 2. Consensus phylogram obtained from the Bayesian Inference analysis under the best partition scheme. Numbers besides nodes indicate posterior-probabilities (BI) and bootstrap values (ML), respectively. Vertical bars correspond to the different species found with ABGD (blue) and GMYC (pink) analyses. Mediterranean and corresponded to P. coriacea from Malta, P. polymorpha from Sicily, P. rubra and P. bornetii both from Liguria, Italy. The other true Peyssonnelia species besides the Mediterranean taxa is the Indian Ocean taxon P. replicata from KwaZulu-Natal, South Africa. Of all the Peyssonnelia species recognized in this study, P. squamaria has the widest distribution range, encompassing Sicily, Malta, Mediterranean Spain, Greece (not shown) and three islands of the Azorean archipelago. 6 Six haplotypes were observed within the Peyssonnelia squamaria clade (Fig. 3), three in the Azores (A1, A2, A3) and three in the Mediterranean (B, C, D). P. squamaria is found to be more genetically diverse in the type locality, i.e., in the Mediterranean than in the Azores. In the former, three haplotypes are observed with 2 to 4 mutational steps between them, while in the latter, three separate haplotypes recovered have only 1 to 2 mutational steps.          Peyssonnelia  in  the  Azores   Fig. 3. RbcL haplotype network of Peyssonnelia squamaria and its spatial distribution in the North Atlantic. Haplotype and population sizes are proportional to the number of individual; dach haplotype is represented by a color and a letter (A1 to D). CONCLUSION ACKNOWLEDGMENTS Only a single, true Peyssonnelia species is confirmed for the Azorean Archipelago, in contrast to the five previously reported (Parente 2010). Although the populations in the Azores are genetically different from those occurring in the Mediterranean, this separation might be relatively recent, since the archipelago emerged about 8 My ago (Rumeu et al. 2011). Further studies including more islands and more samples are necessary to assess the variability of the species within the archipelago and the connection between its populations (Gabriel et al. 2014). We greatly acknowledge support from a grant from the National Science Foundation Systematics Program (DEB-1027110 to SF). During this research, DG was supported by FCT grant SFRH/BPD/64963/2009. We are grateful to Manuela I. 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Received 28 May 2015. Accepted 12 Aug 2015. Published online 14 Sept 2015. 9