Phycologia Volume 57 (3), 318–330 Published 15 March 2018

A re-evaluation of subtidal species (Corallinales, Rhodophyta) from North Carolina, USA, and the proposal of L. searlesii sp. nov.

1 2 3 4 JOSEPH L. RICHARDS ,PAUL W. GABRIELSON ,JEFFERY R. HUGHEY AND D. WILSON FRESHWATER * 1Biology Department, University of Louisiana at Lafayette, Lafayette, LA 70504-3602, USA 2Department of Biology and Herbarium, Coker Hall CB 3280, University of North Carolina, Chapel Hill, Chapel Hill, NC 27599- 3280, USA 3Division of Mathematics, Science, and Engineering, Hartnell College, 411 Central Ave., Salinas, CA 93901, USA 4Center for Marine Science, University of North Carolina at Wilmington, Wilmington, NC 28409, USA

ABSTRACT: The current identification of crustose coralline from North Carolina is based on a few morphoanatomical studies from the last century. We reassessed the type specimens of the two Lithophyllum species historically reported from offshore communities in North Carolina, L. intermedium with a Caribbean Sea type locality and L. subtenellum with an Atlantic southern France type locality, using scanning electron microscopy images and diagnostic rbcL sequences. Neither of the sequences generated from the type specimens matched rbcL sequences from contemporary specimens collected from subtidal North Carolina epibenthic communities. On the basis of analyses of rbcL and other loci (psbA, UPA, and COI), we instead found L. atlanticum, recently described from Brazil, and L. searlesii sp. nov. from Onslow Bay, North Carolina. These sequence data show that L. atlanticum is related to northeast Pacific species, whereas L. searlesii is related to Mediterranean species.

KEY WORDS: Hard bottom, Lithophyllum atlanticum, Lithophyllum intermedium, Lithophyllum subtenellum, psbA, rbcL, Subtidal, Systematics, UPA

INTRODUCTION communities (Peckol & Searles 1983, 1984; Peckol & Ramus 1988; Freshwater et al. 2016). These studies found that Warm-temperate, subtidal, hard-bottom communities are although CCA do not have the large area coverage seen in poorly studied with respect to a molecular assessment of other hard-bottom communities, they still are important, crustose coralline algal diversity compared with cold- albeit taxonomically unknown, components of warm-tem- temperate to boreal/arctic regions. However, even in these perate epibenthic communities. CCA and Peyssonnelia-like latter, better-studied communities, the crustose coralline species as a group were the largest contributors to epibenthic algae (CCA) are woefully understudied (O’Leary et al. 2017). community structure on North Carolina hard bottoms at The one exception is the subtidal Arctic, subarctic and depths between 18.0 and 31.0 m, and they also made the subarctic/boreal transition zone in the North Atlantic where second greatest contribution among macroalgae at sites studies by Adey and colleagues over the last 5 decades have 32.5–42.0 m deep (Freshwater et al. 2016). resolved the systematics (e.g. Adey 1966; Adey et al. 2005, Hoyt (1920) first reported on from North 2015b), biogeography (Adey & Steneck 2001), and ecology Carolina, listing six genera, including Lithophyllum Phillipi, (Adey et al. 2015a) of a suite of coralline species recognized with one species, L. intermedium Foslie (type locality: Cruz as the ecosystem engineers of this biome. CCA remain Bay, St. John, US Virgin Islands). A second species of poorly studied in the warm temperate and subtropical waters Lithophyllum, L. subtenellum (Foslie) Foslie (type locality of the northwest Atlantic Ocean despite their importance Gue´thary, France), was reported in an unpublished thesis within these hard-bottom communities (Freshwater et al. (Suyemoto 1980), and these two species were listed and 2016). described by Schneider & Searles (1991) in their Seaweeds of North Carolina marine waters are biogeographically the southeastern United States. Unfortunately we could not important as the transition zone between the cold-temperate locate any original material cited by Hoyt (1920) or and warm-temperate/tropical regions of the east coast of Suyemoto (1980). Herein, we narrow the lectotypes of both North America (Spalding et al. 2007). The North Carolina L. intermedium and L. subtenellum, document their mor- continental shelf is characterized by many hard-bottom rock outcrops separated by expanses of sand, especially the warm- phoanatomy with scanning electron microscopy (SEM), and temperate shelf waters of Raleigh, Onslow, and Long Bays, provide diagnostic rbcL sequences for each. We also show where the greatest concentration of subtidal hard bottom that the recently described L. atlanticum Vieira-Pinto, occurs (Riggs et al. 1996). Only a few studies have focused on M.C.Oliveira & P.A.Horta, previously known only from the ecology of North Carolina hard-bottom epibenthic Brazil (Vieira-Pinto et al. 2014), is a common component of North Carolina offshore hard-bottom sites in a depth range of 16.5–33 m, and we describe L. searlesii sp. nov., which was * Corresponding author ([email protected]). DOI: 10.2216/17-110.1 collected at a depth of 28 m from Onslow Bay, North Ó 2018 International Phycological Society Carolina.

318 Richards et al.: Northwest Atlantic subtidal Lithophyllum species 319

products were cleaned and sequenced as described in Taylor et al. (2017). Amplification, sequencing, and editing of sequences of historical type specimens was completed by JRH and followed the protocol of Hernandez-Kantun et al.(2016) pairing the F1150cor primer with the R1460cor to generate the 263-base pair (bp) sequence of Lithophyllum intermedium, and the F1150cor with the RrbcS primer (Freshwater & Rueness 1994) to generate the 293-bp sequence of L. subtenellum. Data sets were compiled and aligned using MUSCLE (Edgar 2004) as implemented in Geneious (v. R9, Biomatters, Auckland, New Zealand). Phylogenetic analyses of DNA sequences were performed on two data sets. The first data set included partial (rbcL-5P or rbcL-3P, 11 species) or nearly full-length rbcL sequences (18 species) for 23 Lithophyllum species from GenBank, two newly sequenced Italian specimens, the two North Carolina species, and the L. intermedium and L. subtenellum types. Three Spongites Ku¨tzing species were included as outgroups and the alignment length was 1436 bp. The longest GenBank Fig. 1. Sites in Onslow Bay, North Carolina where Lithophyllum sequences with identifications verified by comparison with specimens were collected. (1) Southwest ledge, 28-m depth; (2) 200/ type specimen sequences were chosen to represent species 200 ledge, 27-m depth; (3) 5-mile ledge, 16.5-m depth (three sites whenever possible. The second data set included partial psbA along ledge up to 0.6 km apart); (4) U-352 wreck, 33-m depth. sequences (851 bp) for 36 Lithophyllum specimens in GenBank, a Brazilian L. atlanticum specimen verified by MATERIAL AND METHODS type sequencing, a newly sequenced specimen from Italy, the two North Carolina species, and three Spongites species as Type specimens were borrowed from TRH; herbarium outgroups (43 species total). Phylogenetic reconstructions acronyms follow Thiers (2017). These specimens were with maximum likelihood (ML) and Bayesian inference for examined, compared with the relevant, original protologues both data sets were carried out using the RAxML by Foslie, with the photographs in Printz (1929), and with (Stamatakis 2006) and Mr. Bayes (Huelsenbeck & Ronquist later writings about the Foslie collections by Adey & 2001) Geneious plug-ins, respectively. ML analyses were Lebednik (1967), Adey (1970), and especially the thorough performed using the general time-reversible (GTR) CAT I curation done by Woelkerling (1993) and Woelkerling et al. model with the data partitioned by codon position and rapid (2005). Only after a complete understanding of each type hill-climbing algorithm for 50 random starting trees. Node collection was material removed from the same individual confidence was assessed by 500 bootstrap replications using crust for both DNA extraction and SEM. GTR CAT I model with the data partitioned by codon All field samples were collected by hand or using a position and the rapid bootstrapping algorithm. Bayesian hammer while scuba diving on hard-bottom ledges or wrecks analyses were performed using the GTR þ gamma þ in Onslow Bay, North Carolina (Fig. 1, Table S1), and invariable sites model and two simultaneous runs with four placed in silica gel desiccant. Material for DNA extraction (three heated and one cold) Monte-Carlo Markov chains for and SEM was selected from the same individual crust. either 2,500,000 generations, sampling every 2250 genera- Specimens were deposited in NCU or WNC (Table S1). tions and with a burn-in value of 250,000 generations (rbcL DNA extraction of field-collected specimens was done by data set), or 5,000,000 generations, sampling every 4500 PWG in his lab following the protocol of Gabrielson et al. generations and with a burn-in value of 500,000 generations (2011); the extraction of historical type material was done by (psbA data set). JRH in his lab following Hernandez-Kantun et al.(2016), Specimen preparation and SEM procedure followed both following the precautionary guidelines of Hughey & Richards et al. (2017). Cell dimensions were measured from Gabrielson (2012). Amplifications of psbAfromfield-collected SEM micrographs following the protocols of Irvine & specimens were performed by PWG following Adey et al. Chamberlain (1994) and Adey et al. (2005). Ten cells of (2015b); of COI-5P and UPA by DWF using primers M13- each vegetative cell type (hypothallial, perithallial, epithallial LF3/M13-Ri (Saunders & Moore 2013) and p23SrV-fl/ and meristematic cells) were measured for each specimen p23SrV-r1 (Sherwood & Presting 2007) and following Taylor except where otherwise noted, and the number of reproduc- et al. (2017); and of rbcL by PWG following Gabrielson et al. tive structures measured was noted for each specimen. (2011), except that the 50 end was amplified with primer pair Terminology follows Adey et al. (2015b) and Hernandez- F57 (Freshwater & Rueness 1994)/R1150cor (sequence of the Kantun et al. (2016). latter: GCATTTGTCCACAATGTATACC) to overlap F753/RrbcS (Freshwater & Rueness 1994) or /R1460cor (Hernandez-Kantun et al. 2016). Cleaning of amplified RESULTS fragments, sequencing, and editing of rbcLandpsbA followed ML and Bayesian analyses of rbcL sequences for the North Gabrielson et al. (2011). COI-5P and UPA amplification Carolina specimens and other Lithophyllum species (Fig. 2) 320 Phycologia, Vol. 57 (3)

Fig. 2. ML phylogeny based on rbcL sequences for Lithophyllum spp. Species without sequenced type material are indicated by single quotes. GenBank accession numbers and country/island of collection provided. ML bootstrap and Bayesian posterior probability values are shown when . 70% and . 0.80 respectively. demonstrated that they were neither L. intermedium nor L. sequencing type specimens. It is described here as L. searlesii subtenellum, and represented two distinct species. The 13 sp. nov. North Carolina specimens identified as L. atlanticum shared Sequences for COI-5P and UPA were generated for nearly or completely identical sequences (100–99.9% similar- specimens from North Carolina of L. atlanticum [WNC ity). North Carolina L. atlanticum was the sister species to L. 34266 (GenBank# MG515131; MG515132), NCU 651909 grumosum (Foslie) Foslie [ML bootstrap (MLb) ¼ 91%; (GenBank# MG515133) and WNC 34271 (GenBank# Bayesian posterior probabilities (PP)¼ 1.00]. The other North MG515134)]. These were compared with the sequences of Carolina species was represented by only one specimen. It was distinct from all other Lithophyllum species, and was resolved those loci generated from the Brazilian types. The WNC 34266 in a well-supported clade (MLb ¼ 94%;PP¼ 0.98) with L. COI-5P sequence was 99.4% and 99.2% similar to those from bathyporum, L. hibernicum, L. incrustans, and other Litho- the holotype and an isotype specimen, respectively (Table S2). phyllum species whose identities were based only on mor- The WNC 34266, WNC 34271, and NCU 651909 UPA phoanatomical similarities with type material or descriptions sequences were a 100% match with the Brazilian isotype of L. in the literature, but that have not been confirmed by atlanticum. Richards et al.: Northwest Atlantic subtidal Lithophyllum species 321

Fig. 3. ML phylogeny based on psbA sequences for Lithophyllum spp. Species without sequenced type material are indicated by single quotes. GenBank accession numbers and country/island of collection provided. ML bootstrap and Bayesian posterior probability values are shown when . 70% and . 0.80 respectively.

Sequences of psbA were generated for a subset of the North specimens identified as L. stictaeforme (MLb¼ 96; PP ¼ 1.00). Carolina Lithophyllum specimens, and included in ML and Sequence divergences among the five specimens in this clade Bayesian analyses (Fig. 3). Five of the North Carolina ranged from 2.0% to 4.5%. Lithophyllum searlesii had greatest specimens had identical psbA sequences and were 99.9% sequence homology with GenBank accession KX020444 similar to the sequence of a Brazilian L. atlanticum specimen (97.2%), but was resolved with mixed support sister to that had been identified using multiple loci as representative of GenBank accession KX020443 (MLb ¼ 75; PP ¼ 0.99). the type of L. atlanticum.ThepsbA analysis resolved L. searlesii within a strongly supported clade (MLb ¼ 97%;PP¼ 1.00) that included L. bathyporum, L. hibernicum, L. incru- Lithophyllum intermedium Foslie, 1906: 23 stans, species whose names have been linked by DNA sequence to their type specimens, as well as several distinct LECTOTYPE: TRH A6-275, Cruz Bay, St. John Island, US Virgin lineages named either L.‘dentatum’orL.‘stictaeforme’. Islands, 24.iii.1906, no habitat data, leg. Børgesen; designated by Adey & Lebednik (1967), narrowed herein (see Discussion). Multiple distinct species are passing under each of these names whose type specimens have not been assessed by DNA ETYMOLOGY: Foslie (1906) likely named this species for being sequencing. Within this clade, L. searlesii was part of a ‘intermediate’ between L. incrustans and L. discoideum [currently strongly supported subclade that included four Mediterranean known as Spongites discoideus (Foslie) Penrose & Woelkerling]. 322 Phycologia, Vol. 57 (3)

Figs 4–9. Habit and morphoanatomy of lectotype specimen (narrowed herein) of Lithophyllum intermedium, TRH A6-275. Fig. 4. Lectotype collection, TRH A6-275: lectotype specimen (narrowed herein, right specimen), fragment that was not narrowed as lectotype herein (left specimen), and box label. Fig. 5. Crust surface view showing uniporate conceptacles (brackets), radular scrapes (black arrow), and location (white arrows) where portion of crust was removed for DNA extraction. Scale bar ¼ 5.0 mm. Fig. 6. Vertical fracture showing dimerous, unistratose hypothallium (h), and perithallium (brackets). Scale bar ¼ 40 lm. Fig. 7. Perithallium with secondary pit connections (arrows). Scale bar ¼ 10 lm. Fig. 8. Uniporate conceptacle slightly raised above thallus surface and detail of pore canal (arrow). Scale bar ¼ 30 lm. Fig. 9. Surface view of uniporate conceptacle lacking roof showing putative zonately arranged tetrasporangia (arrows, roman numerals). Scale bar ¼ 50 lm.

DNA SEQUENCE: rbcL-3P 263 bp (nucleotide positions 1172–1434 of VEGETATIVE ANATOMY: Hypothallium dimerous, unistratose, with the rbcL gene, GenBank MG515127), about 18% of the rbcL gene in cells that were approximately isodiametric or slightly palisade, 5.0–9.5 , was diagnostic for L. intermedium. lm long 3 6.0–15.0 lm wide (Fig. 6). Perithallium with secondary pit connections and no cell fusions (Fig. 7), with cells 7.2–13.0 lm long 3 HABITAT AND HABIT: Epilithic, smooth to somewhat irregularly 5.3–9.3 lm wide. Epithallium not well preserved, but appeared to be lumpy crust with uniporate conceptacles that were flush with crust two layers of epithallial cells, 3–4 lm long 3 6.0–8.8 lm wide (n ¼ 5). surface to slightly convex (Figs 4, 5). No habitat data were provided Meristematic cells 8.5–17.5 lm long 3 5.0–8.5 lm wide (n ¼ 6). for the lectotype. Trichocytes absent. Richards et al.: Northwest Atlantic subtidal Lithophyllum species 323

REPRODUCTIVE ANATOMY: Uniporate conceptacles that occupied five HABITAT AND HABIT: Nongeniculate coralline, with a smooth to to seven cell layers from conceptacle surface to base were observed in somewhat irregularly lumpy surface and uniporate conceptacles that section view with chambers (29.0–30.0 lm high 3 95.0 lm wide, n ¼ 2) were slightly convex and raised above the crust surface; encrusting a and thin roofs, two to three cell layers thick (Fig. 8). Other gastropod shell in a subtidal, hard-bottom community (Fig. 16). conceptacles lacking roofs with wider chambers were observed from surface view (144–300 lm, n ¼ 4) (Fig. 9). Conceptacles were VEGETATIVE ANATOMY: Hypothallium dimerous, unistratose with interpreted as being tetrasporangial; structures that appeared to be cells that were approximately isodiametric or subisodiametric, 5.4– zonately arranged tetrasporangia were observed from surface view of 10.6 lm long 3 3.0–8.6 lm wide (Fig. 17). Perithallium with secondary a roof-lacking conceptacle (Fig. 9). pit connections and no cell fusions, cells 6.0–10.0 lm long 3 5.4–8.2 DISTRIBUTION: Confirmed only from the type locality by rbcL-3P 263- lm wide (Fig. 18). Epithallium one to two cell layers, cells 2.4–2.6 lm bp sequence. long 3 5.0–6.6 lm wide (n ¼ 3). Trichocytes absent.

REPRODUCTIVE ANATOMY: Conceptacles uniporate (Fig. 19), occu- Lithophyllum subtenellum (Foslie) Foslie, 1909: 11 pying five to seven cell layers from conceptacle surface to base, with round to elliptical chambers (34–48 lm in height 3 57–78 lm wide, n ¼ 4) and thin roofs (three to four cells) (Fig. 20). Conceptacles possibly BASIONYM: Goniolithon subtenellum Foslie, 1899: 11–13. male or aborted females, but not determined with certainty.

LECTOTYPE: TRH A2-124, Gue´thary, France, iii–v.1898, no habitat DISTRIBUTION: Known only from the type locality. data, leg. C. Sauvageau; designated by Adey (1970), narrowed herein (see Discussion). COMMENT: No suite of morphoanatomical characters was diagnostic for L. searlesii. Conceptacles became overgrown by new vegetative ETYMOLOGY: sub (almost, approaching), tenellus (delicate). Foslie growth and were observed infilled with secondary mineral formations (1899) likely used this combination of terms to describe the thin (Fig. 21). thallus that falls apart when scraped.

DNA SEQUENCE: rbcL-3P 293 bp (rbcL 1172-1464, GenBank Lithophyllum atlanticum Vieira-Pinto, M.C.Oliveira & MG515128), about 20% of the rbcL gene in red algae, was diagnostic for L. subtenellum. P.A.Horta, 2014: 361

TYPE LOCALITY: Arvoredo Island, Santa Catarina, Brazil. HABITAT AND HABIT: On cobble (Figs 10, 11), likely collected in the intertidal zone from the extensive rocky reef at the type locality. Epilithic, smooth to somewhat irregularly lumpy crust with uniportate DNA SEQUENCES: Newly generated sequences from the North conceptacles that were flush with the surface crust to slightly convex. Carolina material included 13 rbcL sequences (ranging in length from 336 to 1360 bp), 5 psbA sequences (855 bp), 3 UPA sequences (370 bp) and 1 COI sequence (661 bp) (Table S1). VEGETATIVE ANATOMY: Hypothallium dimerous, unistratose with cells that were approximately isodiametric or subisodiametric, 3.5–8.0 lm long 3 5.4–7.0 lm wide (Fig. 12). Perithallium with secondary pit DISTRIBUTION: North Carolina and Brazil. connections and no cell fusions, with cells 6.0–8.2 lm long 3 3.5–6.0 lm wide (Fig. 13). Occasionally elongated perithallial cells were COMMENTS: Specimens from North Carolina had only a crustose observed (i.e. columnar cells), 12.5–15.0 lm long 3 5.9–7.0 lm wide (n habit (Fig. 22). Hypothallium was primarily dimerous (Fig. 23), with ¼ 2). Epithallium not well preserved, consists of one to two cell layers. cells 5.0–8.7 lm long 3 7.5–12.9 lm wide, and secondarily monomer- Meristematic cells 8.8–12.0 lm long 3 5.6–8.0 lm wide (n ¼ 4). ous where the thallus had grown over uneven substratum, whereas Trichocytes absent. Brazilian specimens were reported as only monomerous (Table S3). Perithallial cells linked by two secondary pit connections on the same lateral sides were occasionally observed (Fig. 24), which was not REPRODUCTIVE ANATOMY: The one conceptacle observed appeared reported for Brazilian specimens. Uniporate conceptacles (Fig. 25) to be a male gametangial conceptacle that occupied five to six cell occupied fewer cell layers (six to nine) from conceptacle surface to layers from conceptacle surface to base, with a shallow flattened base, with smaller chambers (34–47 lm high 3 105–142 lm wide, n ¼ chamber (107.8 lm wide 3 18.6 lm tall, n ¼ 1) and a thin roof of two 2), although the number of cell layers in conceptacle roofs was within to three cell layers (Figs 14, 15). the range reported for Brazilian specimens. It was not determined with certainty if the conceptacles of the North Carolina material were DISTRIBUTION: Confirmed only from the type locality by rbcL-3P 293 tetrasporangial. No suite of morphoanatomical characters was sequence. diagnostic for L. atlanticum.

Lithophyllum searlesii P.W.Gabrielson, Freshwater, DISCUSSION J.L.Richards & Hughey sp. nov. (Figs 1621) Lithophyllum intermedium DIAGNOSIS: DNA sequences of the holotype, including rbcL (GenBank accession MG515129) and psbA (GenBank accession Lithophyllum intermedium was described by Foslie (1906) as MG515130). a crust up to 3 mm thick, first circular, then becoming irregular to slightly crimped in outline, partly smooth, HOLOTYPE: NCU 593934, Southwest Ledge, Onslow Bay, North sometimes slightly glossy and with scattered irregular 0 0 Carolina (34823.576 N, 76853.924 W), 08.ix.2010, epizoic, 27–28 m protuberances. In section the hypothallium was described deep, leg. D.W. Freshwater & R. Mu˜oz. n as partly weak, partly developed with cells 11–25 lm long

ETYMOLOGY: The epithet searlesii honours Dr Richard B. Searles for and 7–11 lm wide; perithallial cells were 9–18–(22) lm long his contributions to marine algal systematics and ecology, particularly and 7–11 lm wide. Conceptacles thought to be sporangial in the subtidal offshore waters of the Carolinas. were slightly convex, 150–200–(300) lm in diameter. Foslie 324 Phycologia, Vol. 57 (3)

Figs 10–15. Habit and morphoanatomy of lectotype of Lithophyllum subtenellum, TRH A2-124. Fig. 10. Habit of lectotype specimen (narrowed herein to crust indicated by arrow) encrusting a rock and collection notes. Fig. 11. Surface view of thallus showing uniporate conceptacles and location where portion of crust was removed for DNA extraction (arrow indicates black dot marked on specimen). Scale bar ¼ 2.5 cm. Fig. 12. Vertical fracture of thallus showing dimerous, unistratose hypothallium (h), and perithallium (bracket). Scale bar ¼ 15 lm. Fig. 13. Perithallium with secondary pit connections (arrows). Scale bar ¼ 10 lm. Fig. 14. Vertical fracture of male conceptacle showing pore canal in part (arrow). Scale bar ¼ 50 lm. Fig. 15. Fracture and partial surface view of male conceptacle with pore (arrow). Scale bar ¼ 30 lm.

(1906) compared his new species to L. incrustans Phillipi and specimens from Florida, Barbados, Jamaica, and Puerto L. discoideum Foslie (currently known as Spongites dis- Rico as well as Børgesen’s specimen from St Jan (St John coideus), but noted that when he obtained conceptacle- Island). bearing specimens collected by Børgesen from the West Adey & Lebednik (1967) selected the ‘Børgesen, West Indies, he considered the species to be closest to L. Indies, St. John, Cruxbay, 24.3.1906, LM57 (7, 8)’ as the type discoideum. He further wrote that L. intermedium differed (lectotype) collection (Fig. 4), LM57 (7, 8) referring to Printz from L. discoideum by having cells that are generally slightly (1929, pl. 57, figs 7, 8), who 20 years after Foslie’s death wider and thicker and conceptacles that are fewer. He also photographed the specimens in his herbarium. As seen in Fig. thought the species would be more variable, as most of the 4, this collection contains two specimens. The specimen shown specimens that he examined were younger. Foslie cited on the left in Fig. 4 was illustrated by Printz (1929, pl. 57, fig. Richards et al.: Northwest Atlantic subtidal Lithophyllum species 325

7) and clearly bears multiple crusts likely of different taxa; the Islands (Taylor 1945). All of these records require confirma- specimen shown on the right in Fig. 4 also was illustrated by tion based on DNA-sequenced specimens. The record from Printz (1929, pl. 57, fig. 8). Moreover, upon close examination the Galapagos Islands is particularly suspect. of the left specimen in Fig. 4, many of the crusts are overgrowing other crusts, so obtaining a fragment of a single Lithophyllum subtenellum taxon for DNA sequencing was highly problematic. The right specimen in Fig. 4 also bears at least two crusts, which likely Foslie (1899) originally described this species as Goniolithon could belong to different taxa, but one of which overlays the subtenellum. He provided an extensive morphological descrip- rock substratum and bears flush to slightly convex concepta- tion, including describing male, female, and tetra/bisporangial cles as described by Foslie (1906) in the protologue of conceptacles and listed the collection localities as several Lithophyllum intermedium. It is from this crust (Fig. 5) that places along the coast of Algeria, Saint Vincent de la we removed material for DNA sequencing and SEM. Here we Barquera, Spain, and Gue´thary, Basses Pyre´ne´es, France. narrow the lectotype as allowed in Article 9.14 (McNeill et al. Foslie (1909), without comment, transferred this species to 2012) to the specimen illustrated by Printz (1929, pl. 57, fig 8) Lithophyllum and, for the first time, provided details about its (Fig. 4, right) and specifically to the crust with radular marks anatomy, including that it had a weakly developed hypothal- and bearing uniporate conceptacles (Fig. 5). All other lus and a thick perithallus, as well as providing cell sizes for materials are excluded from this narrowed lectotype, including each thallus region. Printz (1929, pl. LIII, figs 1–3) photo- anatomical microscope slides made by Foslie or subsequent graphed three pieces of cobble under the name L. subtenellum, investigators, as it could not be ascertained from which crust two from Gue´thary (Atlantic), France and one from Banyuls- the material for the slides was obtained. sur-Mer (Mediterranean), France. Adey (1970) designated the SEM observation of secondary pit connections in Lith- material from Gue´thary collected by Camille Sauvageau as the ophyllum intermedium confirms that this species belongs in lectotype, noting that this was ‘...the only set of material Lithophyllum. Observed sizes of hypothallial cells overlap with originally mentioned by Foslie remaining in the collection and those described by Foslie with the following caveat: Foslie sectioned as well as appearing in the Monograph’, the last likely referred to length as the vertical height of the cells and referring to Printz (1929). Adey (1970) further noted that width as the horizontal breadth of the cells, relative to the Foslie’s slides showed cells linked by secondary pit connec- substratum. This is opposite of the modern definitions of tions and that the hypothallus was single layered (dimerous). length and width in hypothallial cells for members of the He also noted that asexual (tetra/bisporangial), single-pored (sensu Campbell & Woelkerling 1990; Irvine conceptacles were present, and he did not see the ‘lateral pores & Chamberlain 1994), where length is the distance between in the roof’ referred to by Foslie (1899) in his original primary pit connections (oriented horizontally in hypothallial description. cells) and width is the distance between the cell walls at right The first application of Lithophyllum subtenellum,aside angles to the primary pit connections (oriented vertically in from localities cited in the original description, was by hypothallial cells). Observed sizes of perithallial cells overlap Suyemoto (1980) to specimens from offshore North Carolina, with those described by Foslie, although some cells were and this name was adopted by Schneider & Searles (1991). smaller than Foslie’s description. Observed conceptacle sizes Chamberlain (1993) noted that she examined the lectotype of correspond to Foslie’s original description of L. intermedium. L. subtenellum and determined that it was the same as L. In this study, smaller conceptacles of L. intermedium were 95 orbiculatum Foslie. This synonymy has not been accepted by lm in diameter, whereas larger conceptacles were 144–300 lm some workers, and more recently L. subtenellum has been in diameter. These values are similar to conceptacle diameters applied in West Africa (John et al. 2003; 2004) and has (150–250 lm or up to 300 lm) described by Foslie (1906). continued to be recognized in the western Atlantic (Wynne Foslie reported these conceptacles to be ‘sporangia (?)- 2017). conceptacles’ (translated from Norwegian), possibly indicat- The lectotype collection currently comprises three crust- ing that Foslie thought these were tetrasporangial concepta- covered rocks, only one of which (Fig. 10) matches one of the cles. In this study, putative zonately arranged tetrasporangia crust-covered cobbles illustrated by Printz (1929, pl. LIII, fig. were observed (Fig. 9), which corresponded with Foslie’s 1) and is from the lectotype locality. A portion of the most observation. isolated of the crusts in the middle of the rock that matched Both the morphoanatomy and the rbcL 293-bp sequence best the original protologue by bearing uniporate conceptacles that we obtained from the herein narrowed lectotype specimen that are likely male (Figs 14, 15) and is dimerous (Fig. 12) was confirmed that Lithophyllum intermedium is correctly placed in sampled for DNA and SEM. Herein we narrow the lectotype Lithophyllum.TherbcL sequence, however, did not match any as allowed in Article 9.14 (McNeill et al. 2012) to the crust on specimen that we have sequenced or any sequence in GenBank the cobble illustrated by Printz (1929, pl. LIII, fig. 1) and in (Fig. 2). We have few sequences of relatively smooth, crustose, Fig. 11 indicated by the black dot. All other materials are Lithophyllum spp. from the Caribbean Sea region, and we excluded from this narrowed lectotype, including anatomical have no information on the habitat of the lectotype specimen microscope slides made by Foslie or subsequent investigators, to guide us where to collect. However, we are confident that a as it could not be ascertained from which crust the material for concerted collecting effort in the Caribbean Sea will produce the slides was obtained. It may be that the other crusts on the specimens whose rbcL sequences match that of L. interme- rock are the same species, but from these we did not attempt to dium. On the basis only of morphology, L. intermedium was obtain DNA sequences. reported from Bermuda (Howe 1918), Florida, Barbados, SEM observations of secondary pit connections in Lith- Jamaica, and Puerto Rico (Foslie 1906), and the Galapagos ophyllum subtenellum verify that this species belongs in 326 Phycologia, Vol. 57 (3)

L- it- h- o- p- h- y- ll- u-

Figs 1621. Lithophyllum searlesii, NCU 593934, habit and morphoanatomy. Fig. 16. Thallus habit encrusting gastropod shell. Scale bar ¼ 8.0 cm. Fig. 17. Vertical fracture showing dimerous, unistratose hypothallium (h), and perithallium (bracket). Scale bar ¼ 25 lm. Fig. 18. Perithallium with secondary pit connections (arrows). Scale bar ¼ 10 lm. Fig. 19. Surface view of uniporate conceptacles (arrows). Scale bar ¼ 200 lm. Fig. 20. Vertical fracture and partial surface view of uniporate conceptacle with pore (arrow). Scale bar ¼ 40 lm. Fig. 21. Vertical fracture showing overgrown conceptacles infilled with secondary minerals (circle pointers). Scale bar ¼ 60 lm. m. Foslie (1909) described the hypothallium as being ‘weakly (1909); however, the width of perithallial cells observed in this developed with short arches upward’ (translated from study overlapped with the smallest width reported by Foslie or Norwegian). The phrase ‘weakly developed’ may be referring were smaller than Foslie’s values. Foslie (1909) also provided to the dimerous thallus construction; however, the hypothal- measurements for ‘upper cells’, which may be referring to lium observed in this study did not appear to be arched meristematic cells. However, meristematic cells observed in upward. Foslie described the hypothallial cells as being ‘sub- this study were smaller than the upper cells reported by Foslie quadrate’, which corresponds to the shapes of hypothallial (1909). Observations of the conceptacle corresponded with cells observed in this study. However, the hypothallial cells those reported by Foslie (1899), who described male observed in this study were smaller than the sub-quadrate cells conceptacles 100 lm in diameter. reported by Foslie. The length of perithallial cells observed in Both the morphoanatomy and the 293-bp rbcLsequence this study overlapped with the values described by Foslie that we obtained from the herein-narrowed lectotype confirm Richards et al.: Northwest Atlantic subtidal Lithophyllum species 327

Figs 2225. Lithophyllum atlanticum, NCU 651909, habit and morphoanatomy. Fig. 22. Thallus habit encrusting rock (brackets). Scale bar ¼ 2.0 cm. Fig. 23. Vertical fracture showing dimerous, unistratose hypothallium (h), and perithallium (bracket). Scale bar ¼ 20 lm. Fig. 24. Perithallium with secondary pit connections, sometimes two per lateral cell wall (arrows). Scale bar ¼ 20 lm. Fig. 25. Vertical fracture showing detail of conceptacle and pore canal (arrow). Scale bar ¼ 60 lm. that Lithophyllum subtenellum is a species of Lithophyllum. field et al. 2014; Freshwater et al. 2016), and may prevent the The rbcL sequence did not match any specimen that we have growth of stenothermal tropical species. We thought it equally sequenced from North Carolina or elsewhere, nor any likely that L. subtenellum from the east Atlantic would not sequence in GenBank, but was sister to a sequence from an apply. We have no habitat data for either type specimen, but unidentified species of Lithophyllum from Calafuria, Italy L. intermedium, collected by F. Børgesen from Cruz Bay, (Fig. 2). On the basis of the offshore North Carolina Danish West Indies (now US Virgin Islands), could have been specimens that we have sequenced and its NE Atlantic type collected from the subtidal by dredge, whereas L. subtenellum, locality, we do not believe that L. subtenellum occurs in the collected by C. Savageau from Gue´thary, France, likely was NW Atlantic. Reports of this species from West Africa require collected in the extensive rocky intertidal at that locality. Both confirmation from sequenced specimens. At present, L. names were applied by morphoanatomical comparisons with subtenellum is known only from its type locality. specimens from offshore North Carolina, the former by Hoyt (1920) and the latter by Suyemoto (1980), but we have been North Carolina Lithophyllum species unable to locate material studied by either to compare with We demonstrated that the two names discussed above, L. our collections. Because of this, it could not be determined intermedium, with a Caribbean type locality, and L. sub- with certainty if specimens identified as L. intermedium and L. tenellum, with a northeast Atlantic (southern France) type subtenellum by Suyemoto are the same species as reported locality, are incorrectly assigned to specimens from North herein. Suyemoto used sizes of tetrasporangial conceptacles to Carolina. At the outset of this project, we thought it likely that distinguish between the two putative species in her study. Only L. intermedium might apply to North Carolina deepwater L. atlanticum possessed conceptacles that might be tetraspor- Lithophyllum species, as the type locality is in the tropical angial, and these are smaller than the tetrasporangial western Atlantic and the deeper North Carolina continental conceptacles of both species reported by Suyemoto. The shelf waters are warm temperate to subtropical because of the specimens identified as L. intermedium by Suyemoto included Gulf Stream current. However, winter bottom-water temper- a male individual that had overgrown conceptacles similar to atures at the depths where the North Carolina Lithophyllum what we observed in L. searlesii (Fig. 21), but this character is specimens were collected range from c.108Cto188C (Whit- not known to be diagnostic at the species rank. Male 328 Phycologia, Vol. 57 (3) conceptacles of the putative L. intermedium shown by the world on the basis of using comparative DNA sequence Suyemoto are larger than those of L. searlesii. data as opposed to comparative morphoanatomy. This is true Of interest, one of our species was Lithophyllum atlanticum, for both geniculate (Hind & Saunders 2013; Hind et al. 2014, previously described from intertidal and subtidal sites in the 2015) and nongeniculate corallines (Sissini et al. 2014; Adey et state of Santa Catarina, southern Brazil (Vieira-Pinto et al. al. 2015b; Basso et al. 2015; van der Merwe et al. 2015; 2014). Schneider (1976) reported that 50% of the morpholog- Hernandez-Kantun et al. 2016; Hind et al. 2016; Richards et ically identified marine algal species he collected on the North al. 2016, 2017; O’Leary et al. 2017). We estimate that the true Carolina continental shelf were also distributed in Brazil. species diversity of extant corallines is, at minimum, two to However, we know of only two inshore species, Gelidium four times greater than that currently recognized. There just crinale (Hare ex Turner) Gallion and Hypnea stellulifera are not enough morphoanatomical characters that can be used (J.Agardh) Yamagishi & Masuda, that have been verified to distinguish species, and this is exacerbated by low sample through DNA sequencing to be present in both North sizes of collected individuals of each species, such that Carolina and Brazil (Kim & Boo 2012; Freshwater et al. infraspecific variation is difficult to assess, making meaningful 2014; Iha et al. 2015; Jesus et al. 2015). Lithophyllum interspecific comparisons even less reliable. Even when sample atlanticum is the first DNA sequence-verified offshore species sizes are larger, typically for geniculate species, few discrim- with this distribution, and only one other coralline alga has inating characters among species are found (Hind et al. 2016). been confirmed by DNA sequences from both northwest and This makes applying names to corallines on the basis of southwest Atlantic temperate waters, the rhodolith Meso- morphoanatomical comparisons with type specimens nearly phyllum erubescens (Foslie) Me.Lemoine (Sissini et al. 2014). impossible and has caused numerous species to be placed in Further comparisons need to be made between these floras as synonymy incorrectly (Hernandez-Kantun et al. 2016; Hind et additional geniculate and nongeniculate coralline algae are al. 2016). Only rarely can morphoanatomical characters alone sequenced. be used to distinguish coralline species and only in locally Lithophyllum atlanticum occurs in Brazil both as a subtidal known floras where all of the species, have first been identified warty rhodolith and as an intertidal and subtidal crust; in and their morphoanatomical variation documented using North Carolina we have found this only as a smooth epilithic DNA-sequenced specimens. At present, such information or epizoic crust at offshore subtidal hard-bottom and does not exist for any local coralline flora. shipwreck sites (Table S1, Fig. 1) and deeper than the 15-m depth reported from Brazil. Photoacclimation enables some CCA species to grow in variable light environments (Payri et ACKNOWLEDGEMENTS al. 2001; Martin et al.2013),andL. atlanticum exhibits a broad tolerance of light environments ranging from intertidal We are very grateful to Dr Kristian Hassel, curator at TRH, to 33-m depth. Specimens from North Carolina are morphoa- for the loan of type material that is critical to the application natomically similar to specimens from Brazil (Table S3), of coralline names. The authors thank Dr Talita Vieira-Pinto although we found specimens with both monomerous and for providing DNA sequences of Brazilian specimens, and dimerous construction, with fewer cell layers (six to nine) from Dr Gavin Maneveldt and two anonymous reviewers for conceptacle surface to base and with smaller conceptacle helpful comments on the manuscript. JLR acknowledges Dr chambers (34–47 lminheight3 105–142 lmwide,n ¼ 2). Tom Pesacreta and Mike Purpera at the University of Although the number of cell layers in conceptacle roofs were Louisiana Lafayette Microscopy Center for help and advice the same as Brazilian specimens, considering the difference in while using the SEM. PWG thanks Dr Todd Vision, conceptacle sizes and lack of observable tetrasporangia in the University of North Carolina Chapel Hill for use of lab North Carolina specimens, the suite of conceptacle characters space and equipment. DNA extraction and sequencing reported to be diagnostic for Brazilian specimens (Vieira- performed by JRH and PWG were funded by a private Pinto et al. 2014) could not be used to identify this species in family trust (PWG). North Carolina field collections were North Carolina. made possible by support from the National Oceanic and Lithophyllum searlesii is known only from the holotype Atmospheric Adminstration, the University of North collection, but it clearly belongs in Lithophyllum and is distinct Carolina Wilmington’s Coastal Oceans Research & Moni- from all other species in GenBank and in our own databases. toring Program, a Center for Marine Science (CMS) Pilot Interestingly L. searlesii is in a clade of European Lithophyl- Project, and the CMS DNA-Algal Trust. lum spp., whereas L. atlanticum is most closely related to northeast Pacific species (Figs 2, 3). Any meaningful morphoanatomical comparison with other species of Lith- SUPPLEMENTARY DATA ophyllum is precluded by having only one specimen of L. searlesii. It is likely that with extensive collecting in offshore North Carolina waters we will find additional specimens of Supplementary data associated with this article can be found this and other Lithophyllum spp. online at http://dx.doi.org/10.2216/17-110.1.s1.

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