Collections from the Mesophytic Zone Off Bermuda Reveal Three Species of Kallymeniaceae (Gigartinales, Rhodophyta) in Genera with Transoceanic Distributions1

COLLECTIONS FROM THE MESOPHYTIC ZONE OFF BERMUDA REVEAL THREE SPECIES OF KALLYMENIACEAE (GIGARTINALES, RHODOPHYTA) IN GENERA WITH TRANSOCEANIC DISTRIBUTIONS1

Craig W. Schneider 2 Department of Biology, Trinity College, Hartford, Connecticut 06106,USA Thea R. Popolizio Department of Biology, Salem State University, Salem, Massachusetts 01970, USA and Gary W. Saunders Centre for Environmental & Molecular Algal Research, Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada E3B 5A3

A molecular survey of red algae collected by mostly on sorting out taxa above the species level in technical divers and submersibles from 90 m in the order to present a “contemporary genus-level taxo- mesophotic zone off the coast of Bermuda revealed nomic framework” built on the principle of mono- three species assignable to the Kallymeniaceae. Two phyly for other workers to later fill in species. One of the species are representative of recently described genus previously placed in synonymy with Kallymenia genera centered in the western Pacific in Australia was resurrected (Euhymenia; but see Wynne 2018), and New Zealand, Austrokallymenia and Psaromenia several species were moved to newly described gen- and the third from the Mediterranean Sea and the era removing polyphyletic or paraphyletic group- eastern Atlantic, Nothokallymenia. A phylogenetic ings, and ten new genera were erected to house analysis of concatenated mitochondrial (COI-5P) and them (Saunders et al. 2017). chloroplast (rbcL) genes, as well as morphological Many of the species discovered in the mesophotic characteristics, revealed that two are shown to be new zone off Bermuda in 2016 on the Nekton XL Catlin species with distant closest relatives (N. erosa and cruise of the R/V Baseline Explorer are new to science Psaromenia septentrionalis), while the third represents and a few have already been presented (Richards a deep water western Atlantic species now moved to et al. 2018, Schneider et al. 2018). They were col- an Australasian genus (A. westii). lected in areas of descending, low relief hermatyptic coral reefs or rhodolith covered areas from 60 to Key index words: A. westii; Austrokallymenia; Bermuda; N. erosa 178 m depths by technical divers or mechanical COI-5P; Kallymeniaceae; mesophotic zone; arms from two-man submersibles. In this report, we sp. nov.; Nothokallymenia; P. septentrionalis sp. nov.; Psaromenia rbc recognize an additional two new species and one ; L; western Atlantic new combination, all members of the Kallymeni- Abbreviations: COI-5P, 50 region of the cytochrome aceae with their closest known relative from distant oxidase subunit 1 gene; Mya, million years ago seas and not previously known in the western Atlan- tic Ocean. Interestingly, each of the three species represents a genus that has been introduced to A recent phylogenetic monograph of the diverse science during the present decade (D’Archino et al. red algal family Kallymeniaceae (Gigartinales) pro- 2010, Saunders et al. 2017). vided genetic evidence of a much larger number of Since our collaborative work began more than a genera (Saunders et al. 2017) than previous known decade ago, we have reported on a great number of (Schneider and Wynne 2007, 2013, Wynne and Sch- algal genera from Bermuda with genetic biogeo- neider 2010, 2016). The several new genera eluci- graphic connections to the Indo-Pacific region, in dated by Saunders et al. (2017) were part of an most cases from Australia. These include Asteromenia “explosion of molecularly assisted taxonomic modifi- (Saunders et al. 2006), Crassitegula (Schneider et al. cations” that were revealed in an extensive barcode 2006, 2014a), Halopeltis (Schneider et al. 2012), and phylogenetic study of specimens centered in Meredithia (Schneider et al. 2014b), Ethelia (Dixon Australia and western Europe. The authors focused et al. 2015) and Dasya (Schneider et al. 2017). Some of these studies describe Bermuda taxa whose clos- 1Received 23 August 2018. Accepted 28 November 2018. First est known genetic relative is from the Indo-Pacific Published Online 19 December 2018. rather than any known species from the Atlantic 2Author for correspondence: e-mail [email protected]. (Crassitegula, Halopeltis and Meredithia). The species Editorial Responsibility: M. Vis (Associate Editor)

1 2 CRAIG W. SCHNEIDER ET AL. of Austrokallymenia and Psaromenia reported here can another (Saunders et al. 2018). Support was assessed with be added to this latter group with genetic ties to the 500 bootstrap replicates. Indo-Pacific region. RESULTS AND DISCUSSION MATERIALS AND METHODS Molecular observations. DNA barcoding with COI- Standard methods. Collections from the mesophotic zone 5P uncovered three Bermuda species of Kallymeni- were made on the Nekton 2016 XL Catlin cruise of the R/V aceae from the mesophotic zone in Bermuda Baseline Explorer by technical “rebreather” divers or by (Table S1). Based upon the concatenated COI-5P + mechanical arms from submersibles. The difficulty of secur- rbcL phylogeny, these species aligned with Aus- ing sparsely distributed specimens of red-bladed algae from trokallymenia (n = 2 specimens), Nothokallymenia these deep reefs limited the number of samples of any spe- (n = 2 specimens) and Psaromenia (n = 3 speci- cies brought to the ship’s deck. Collection site locations were marked using a Beier Radio DP1 (dynamic positioning, Beier mens) with solid support in their respective genera. Integrated Systems, Mandeville, LA, USA) to receive ship Each is discussed in detail below. board GPS. Live specimens chosen for DNA analysis were Systematic treatment. Austrokallymenia westii (Gane- photographed using a Canon Powershot s90 digital camera san) C.W. Schneider & G.W. Saunders comb. nov. (Canon Inc., Tokyo, Japan) and dried/pressed onto herbar- (Fig. 2, A–C) ium paper as permanent vouchers. Fragments of these vouch- Basionym: Kallymenia westii Ganesan (1976), Boletın ers were dried on silica gel for DNA extraction, and additional fragments were preserved in 4%–5% Formalin in del Instituto Oceanografico, Universidad de Oriente vol. seawater for anatomical study. Thick sections were cut by 15, pp. 169-170, figs. 1-12, 14, 16-21. hand with a razor blade and thin sections (30–50 lm) were Selected specimens observed: Bermuda – W.G. Farlow made with an American Optical freezing microtome model s.n. as Kallymenia perforata, 1881, Cooper’s I., drift 880 (San Diego, CA, USA). Sections were mounted in 30% [FH; MICH 660484, 660487; Herb. CWS]; A.J. Berna- corn syrup with acidified 1% aniline blue in a ratio of 20:1 towitz 53-512 as K. perforata, July 25, 1953, half mile with a few drops of Formalin as a medium preservative. Her- barium specimens were scanned on an Epson ET-2650 scan- south of Castle Harbour, depth 91.5 m [MICH 660485]; J.J. Frederick (JJF)60–588 as K. perforata, July ner (Seiko Epson Corporation, Suwa, Nagano, Japan) and 0 photomicrographs were taken using Zeiss Axioskop 40 micro- 19, 1960, Challenger Bank Sta. 116, 32°06 21″ N, 0 scope (Oberkochen, Germany) equipped with a model 11.2 65°01 38″ W, depth 50.8 m [MICH 660505]; JJF 60- Spot InSight 2 digital camera (Diagnostic Instruments, Ster- 844 as K. perforata, September 1, 1960, Sta. 173 off ling Heights, MI, USA). The digital images were composed in ° 0 ° 0 TM Gurnet Rock, 32 19 N, 65 39 W, depth 50.2 m Adobe Photoshop CS6 v. 13.0.1 (Adobe Systems, San Jose, [MICH 660501]; C.W. Schneider (CWS)/T.R. Popolizio CA, USA). Voucher specimens are deposited in UNB, MICH, the Bermuda Natural History Museum (BAMZ) and CWS’s (TRP) 16-12-1 [BDA1991], July 29, 2016, deep ledge north northeast of St. George’s I., 32°28058.05″ N, personal herbarium. Unless otherwise noted, specimens cited 0 under the taxa, as well as numbers from the Phycotheca Boreali- 64°35 04.31″ W, depth 90 m; CWS/TRP 16-13-2 Americana (P.B.-A.) exsiccata, are found in the first author’s [BDA1995], July 31, 2016, loc. cit., depth 90 m. herbarium. Herbarium abbreviations follow the online Index North Carolina – R.B. Searles (RBS)/CWS s.n., as Herbariorum http://sweetgum.nybg.org/ih/ and standard K. perforata, R/V Eastward Sta.17683, August 4, 1971, author initials are from Brummitt and Powell (1992). 0 0 33°46.5 N, 76°51.1 W, depth 40 m; RBS 85-31-10, Molecular methods. Specimens were assigned to genetic as K. westii, July 28, 1985, east of Cape Fear, groupings after generating COI-5P sequences. To place these ° 0 ° 0 – species into a wider phylogenetic context, an rbcL sequence 33 32 N, 77 25 W, depth 30 33 m. US Virgin Islands was generated for a representative of each genetic group and – D.L. Ballantine 2966, as K. westii, R/V Oregon II Sta. these data, along with the COI-5P sequences, were analyzed 46048, August 11, 1987, St. Thomas, shelf south with 70 other Kallymeniaceae (Fig. 1, Table S1 in the Sup- Charlotte Amalie, depth 36 m. porting Information). DNA extraction (Saunders and McDe- Distribution: Bermuda, North Carolina, Florida, vit 2012) and marker amplification followed published protocols (Saunders and Moore 2013). Sequence data were south in Caribbean Sea to Venezuela (type locality). edited and contigs produced in Geneious 9.1.3 (http:// Remarks:COI-5PandrbcL sequences of freshly col- www.geneious.com; Kearse et al. 2012). Sequences generated lected plants from the mesophotic off Bermuda pre- here and those produced previously by our laboratory, as well viously known as K. westii show them embedded in a as data from other laboratories downloaded from GenBank genetic grouping with Indo-Pacific species of Aus- (Table S1), were aligned in Geneious. Two individual gene trokallymenia (Fig. 1). The name for this recently alignments were prepared for phylogenetic analyses: COI-5P (69 sequences, 664 bp) and rbcL (73 sequences, 1,358 bp). described new genus alluded to its southern hemi- The gene alignments were analyzed independently with maxi- sphere distribution (Saunders et al. 2017), and here mum likelihood (GTR+I+G) using RAxML (Stamatakis 2014) we add to it a species from the Atlantic Ocean and with partitioning by codon in Geneious. Since no conflicts northern hemisphere. In their work, Saunders et al. were detected between individual gene topologies, a concate- (2017, p. 117) considered K. westii aperforate,poly- nated COI-5P + rbcL alignment was constructed (73 carpogonial species similar in many characteristics sequences, 2,022 bp). This tree (Fig. 1) was rooted along the tooneoftheirnewAustralianspeciesLeiomenia lacu- branch between the Meredithia – Psaromenia clade and the remaining Kallymeniaceae as these two groups are unequivo- nata, but without available DNA from the type local- cally reciprocally monophyletic (Saunders et al. 2017), there- ity it was retained in Kallymenia.Amongthespecies fore representing the best choice of outgroup for one that Saunders et al. (2017) transferred from MESOPHOTIC KALLYMENIACEAE 3

FIG. 1. Combined COI-5P and rbcL RAxML analyses (GTR+I+G) 500 bootstrap replicates (only values >70% shown). Generitypes are marked with asterisks (*). 4 CRAIG W. SCHNEIDER ET AL.

FIG.2. Austrokallymenia westii (Ganesan) comb. nov. (A) Mature specimen from the mesophotic (CWS/TRP 16-12-1), scale bar = 5 cm. (B) Young imperforate specimens washed onshore in Bermuda, 1881 (W.G. Farlow s.n.), scale bar = 2 cm. (C) Net-like, highly perforated specimen from Venezuela (MICH 660509), scale bar = 5 cm.

Kallymenia to Austrokallymenia, they selected the only can be seen in FH and MICH on the Macroalgal perforate species as the generitype, A. cribrogloea, Herbarium Portal site (http://macroalgae.org/por from Australia. Their analyses showed that tal/collections/index.php). The Farlow collections A. cribrogloea was a complex of at least three genetic are small (young) and vary from imperforate groups in need of additional study (Saunders et al. (Fig. 2B) to slightly perforated. Collins and Hervey 2017). Interestingly, Littler and Littler (2000) (1917) also based their report of the species entirely reported A. cf. cribrogloea (as K. cf. cribrogloea)forthe on the Farlow collections noting that the plants they first time in the Atlantic Ocean from deep water in had seen showed “much variability as to the amount the Lesser Antilles, but as yet this material has not of perforation” from quite imperforate to others “lit- had its identity confirmed by gene sequencing. As it tle more than a network.” Frederick (1963) (as turns out, the closest known relative of A. westii,now K. perforata) reported on specimens collected on the the second perforate species in the genus, is the offshore Argus and Challenger Banks in 1960–1961 undescribed imperforate Austrokallymenia sp.1_SAfr at depths of 50–62 m, these also demonstrating a (Fig. 1). These two are 4% divergent in their rbcL range of perforations. sequences; however, A. westii displays a similar level Whether the representatives of Austrokally- of divergence from several Australian species menia westii with few perforations or those highly (Fig. 1). Some perforate Kallymenia species (Abbott networked represent the same genetic species, we and McDermid 2002) have yet to be analyzed geneti- cannot determine without additional genetic cally, and as more are sequenced, it is possible that sequences. Saunders et al. (2017) noted that their relationships could change within Austrokallymenia collections of Leiomenia lacunata had blades that var- and the Kallymeniaceae in general as was the case ied from “few to numerous perforations,” and all with the addition of A. westii. forms were a genetic match to the holotype. Our Austrokallymenia westii was first collected by W.G. recent 90 m large collections (to 22 cm tall) contain Farlow from the drift at Cooper’s I., Bermuda in relatively few perforations (Fig. 2A) and are similar 1881 (Fig. 2B). His specimens were later used by in habit and anatomy to specimens from Venezuela Børgesen (1910) and Collins and Hervey (1917) to selected by Ganesan (1976, figs 1, 3) for the type make the first reports of it (as Kallymenia perforata) collection as well as those found in deep waters off for Bermuda. Several of Farlow’s early collections the coast of North Carolina (Schneider and Searles MESOPHOTIC KALLYMENIACEAE 5

1973, as Kallymenia perforata; Schneider and Searles specimens found throughout its range in the west- 1991, as K. westii). However, looking at archival ern Atlantic including some found in Venezuela material in MICH and digitized specimens of this (Fig. 2C). There are no morphological or anatomical species from throughout its geographic range in the characteristics of Bermuda and North Carolina Macroalgal Herbarium portal, there is a continuum specimens to distinguish them from A. westii. Like of perforation density between the two extremes. the 19th century Farlow collections of A. westii in Until more specimens from throughout the western Bermuda, Ganesan (1976) (fig. 3) demonstrated an Atlantic are sequenced, we can only suggest that what imperforate paratype specimen along with the proto- is considered Austrokallymenia westii could possibly be logue of his new species. Since A. westii has only been a complex of at least two species or a phenotypically collected in deep water throughout its range and all variable species in regards to perforation density. of the specimens we are aware of were preserved in Unfortunately, we did not collect highly perforate or Formalin-seawater prior to drying on herbarium networked specimens on the 2016 Nekton cruise, paper, we were unable to find Venezuelan specimens thus we could not test whether A. westii is one or that could provide DNA sequences. Unfortunately, more perforate species in Bermuda and the western the sequenced specimens from Bermuda were vege- Atlantic. For the present, a morphological compar- tative, disallowing a check as to whether they were, ison of our recent specimens with the type of like the type, polycarpogonial. Despite these issues, K. westii shows that the weakly perforate type mate- we are comfortable in taking the conservative rial (Ganesan 1976, fig. 1) is similar to specimens approach in aligning our mesophotic Bermuda speci- from Bermuda and North Carolina (Schneider and mens with A. westii from the Caribbean until proven Searles 1991), not the highly perforate/networked otherwise by sequence data.

FIG.3. Nothokallymenia erosa sp. nov. (CWS/TRP 16-13-4), holotype. (A) Holotype specimen showing position of the holdfast (arrowhead), scale bar = 1 cm. (B) Close-up of irregularly toothed (erose) margin, scale bar = 250 lm. (C) Cross-section of blade with dark staining ganglionic cell (arrowhead), scale bar = 100 lm. (D) Stellate medullary ganglia, scale bar = 100 lm. (E) Surface view of a loose sorus of tetrasporangia displacing outer cortical layer, scale bar = 50 lm. 6 CRAIG W. SCHNEIDER ET AL.

Nothokallymenia erosa C.W. Schneider, Popolizio & but Nothokallymenia erosa has thinner (to 250 lm) G.W. Saunders sp. nov. (Fig. 3, A–E) ligulate blades with erose margins (Fig. 3B). Description: Rosy-red plants composed of irregular Although both species have irregularly cruciate ligulate blades to 3.5 cm long from small discoidal tetrasporangia, those of N. erosa are smaller (12– holdfasts (Fig. 3A), stipes lacking; blades 135– 17 lm diam.) than those of the generitype (18– 250 lm thick, margins with irregular dentations of 24 lm diam.) and are found in loose patches (sori) varying sizes (Fig. 3B); blade structure multiaxial on blade surfaces (Fig. 3E), while those of with cell layers increasing in size from the outer- N. crouaniorum were scattered over the blades most pigmented cell layer to the innermost nonpig- (Robuchon et al. 2014, Saunders et al. 2017). The mented cell layer connecting to the filamentous rbcL for N. erosa was 4% divergent from an unpub- medulla (Fig. 3C); pigmented cortex composed of a lished species from Brittany, France (KM896874). single layer, the small cells globose to ovoid and Psaromenia septentrionalis C.W. Schneider, Popoli- subrectangular to irregular in surface view, trans- zio & G.W. Saunders sp. nov. (Fig. 4, A–F) versely elongated in section, 7–12 lm in diameter; Description: Rosy-red plants composed of subdi- inner cortex of two layers of elongated cells increas- chotomously branched ligulate blades, arising to ing inwardly in size and stretching and thinning, 13 cm tall from small discoidal holdfasts, stipes lack- some appearing stellate, the innermost 28–67 lmin ing, the blades bearing marginal proliferations that length and connecting to the filamentous medulla; eventually develop into fusiform to lanceolate medulla composed of a spiderweb-like network of blades, 300–500 lm thick (Fig. 4A); structure multi- elongated filaments 2–5 lm in diameter, connected axial, the cortex with cell layers increasing in size to occasional large darkly staining stellate ganglia from the outermost pigmented cell layer to the (Fig. 3D); sporangia in scattered loose sori on blade innermost nonpigmented cell layer connecting to surfaces under and displacing outer cortical cells the filamentous medulla (Fig. 4B); pigmented cor- (Fig. 3E), obovoid to ellipsoidal in shape, irregularly tex composed of one (-two) layers, the outermost cruciately divided, 12–17 lm in diameter and 17– cells polyhedral in surface view, subglobose or elon- 22 lm long; gametangia unknown. gated to irregular in section, 3.5–7.5 lm in diame- Diagnosis: Distinguished from the generitype, ter; inner cortex of three layers of subglobose cells Nothokallymenia crouaniorum, by its COI-5P (GenBank increasing inwardly in size and elongating parallel MH777606) and rbcL (GenBank MH777609) to the blade surface, the innermost 33.5–67.5 lmin sequence data, its ligulate blades with erose margins diameter and connecting to the filamentous and smaller sized tetrasporangia formed in sori. medulla; subsurface cortical cells often with clusters Holotype (designated here): CWS/TRP 16-13-4 of refractive globules; medulla composed of a loose [BDA1997], tetrasporic, July 31, 2016, deep ledge network of elongated filaments 7–12 lm in diame- north northeast of St. George’s I. (NNE02), ter, connected to stellate ganglia morphed from 32°28058.05″ N, 64°35004.31″ W, Bermuda, western the innermost cortical cells (Fig. 4, C and D); Atlantic Ocean, depth 90 m, bottom temp. 19.4°C, monocarpogonial supporting cells bearing a single deposited in UNB (Fig. 3A). 3-celled carpogonial branch and 3–4 sterile sub- Etymology: erosa (L, f.), for its irregularly dentate sidiary cells (Fig. 4E), carposporophytes formed in margins. groups within a cystocarp, in part separated by vege- Distribution: At present, endemic to the mesopho- tative filaments, distally producing carpospores tic zone off Bermuda, western Atlantic Ocean. toward the center of the cystocarp (Fig. 4F); car- Remarks: Nothokallymenia was recently segregated pospores obpyriform to spherical and irregular, from Kallymenia with the generitype, N. crouaniorum, 9.5–17.0 lm in longest dimension; cystocarps from the Northeast Atlantic and three undescribed formed over blades, at times on margins, when fully genetic species from the Mediterranean Sea (Saun- developed raising one surface or the other as pus- ders et al. 2017). The mesophotic N. erosa from Ber- tule-like, irregular cystocarps to 1.3 mm in diame- muda represents the second described species in ter, formed singly or at times in clusters of 3–4, the genus and the first from the western Atlantic. ostioles lacking; spermatangia and tetrasporangia After molecular analysis, Robuchon et al. (2014) (as unknown. Kallymenia crouaniorum) found N. crouaniorum as a Diagnosis: Distinguished from the generitype, consistent understory species in the Laminaria hyper- Psaromenia berggrenii, by its COI-5P (GenBank borea community of Atlantic European waters MH777603) and rbcL (GenBank MH777607) sequence confused with, and historically identified as, K. reni- data, its geographic location, lack of a mottled appear- formis. Nothokallymenia crouaniorum is comprised of ance and monocarpogonial supporting cells. blades that are large (to 20 cm tall, 15 cm broad), Holotype (designated here): CWS/TRP 16-13-1 thick (to 320 lm), and deeply lobed to laciniate [BDA1993], female/cystocarpic, July 31, 2016, deep (Robuchon et al. 2014). The new species has an ledge north northeast of St. George’s I. (NNE02), internal anatomy that is remarkably similar to 32°28058.05″ N, 64°35004.31″ W, Bermuda, western Nothokallymenia crouaniorum (Fig. 3C), including Atlantic Ocean, depth 90 m, bottom temp. 19.4°C, their large darkly staining stellate ganglia (Fig. 3D), deposited in UNB (Fig. 4A); isotypes – BAMZ, Herb. MESOPHOTIC KALLYMENIACEAE 7

FIG.4. Psaromenia septentrionalis sp. nov. (CWS/TRP 16-13-1). (A) Holotype specimen, scale bar = 5 cm. (B) Cross-section at margin of thallus, scale bar = 100 lm. (C) Inner cortical cell stretching with attached medullary filaments, scale bar = 50 lm. (D) Fully developed stellate ganglia and attached medullary filaments, scale bar = 50 lm. (E) Monocarpogonial supporting cell (sc) bearing a 3-celled carpogonial branch (branch cells 1 and 2 [c1, c2], carpogonium [cp]) and 3 sterile subsidiary cells (ss). It appears that the subsidiary cells are at early stage of fusion with carpogonial branch and supporting cell, scale bar = 25 lm. (F) Section through a cystocarp demonstrating carposporophytes with carpospores separated by vegetative filaments, scale bar = 250 lm.

CWS (CWS/TRP 16-13-1 [BDA1994]), MICH (CWS/ the first described from the northern hemisphere TRP 16-13-3 [BDA1996]). (D’Archino et al. 2010). It is also the first species of Etymology: septentrionalis (L, f.), northern, for its this genus reported from the Atlantic Ocean. Its being the first species of Psaromenia formally closest relative in our analysis is Psaromenia sp.1_LH, described from north of the equator. an undescribed Australian species in the genus Distribution: At present, endemic to the mesopho- (Schneider et al. 2014b, Saunders et al. 2017). tic zone off Bermuda, western Atlantic Ocean. D’Archino et al. (2010) named their monotypic Remarks: Psaromenia septentrionalis represents the genus Psaromenia from New Zealand for the distinc- second species formally described in the genus, and tive mottled appearance of the blades. The new 8 CRAIG W. SCHNEIDER ET AL. species from Bermuda does not share this character- limited collections from offshore Bermuda, we like- istic, all the blades showing even coloration wise did not find sporophytes. (Fig. 4A). The internal structure of the new species In the molecular analyses of D’Archino et al. is similar to that of a majority of genera and species (2010), Psaromenia formed a clade with Meredithia,at in the Kallymeniaceae. A complete outer layer or that time geographically restricted to the Atlantic, layers of pigmented smaller cortical cells sits outside while their new genus was considered endemic to an inner layer of enlarged cortical cells (Fig. 4B). New Zealand. Since then, and with this report of The polyhedral outer cortical cells form in groups P. septentrionalis, neither Psaromenia or Meredithia or clusters somewhat reminiscent of a rosette pat- remains restricted to either the Atlantic or Indo- tern. As the enlarged inner cortical cells mature, Pacific Oceans (Schneider et al. 2014b). they become separated (Fig. 4C), often widely, as they appear pulled apart by the medullary filaments they are connected to (Womersley and Norris CONCLUSIONS 1971), thus they represent a transitional layer What has been discovered in the mesophotic zone between the cortex and medulla. At maturity, the of the world’s oceans represents a few narrow snap- transitional cells in Psaromenia septentrionalis are star- shots in time and place from a vast landscape, only shaped (stellate) ganglia and are lightly staining a tiny fraction of which has been visited by scientists (Fig. 4D). Overall, the anatomical arrangement of (Ballantine et al. 2016), Unfortunately, the expense the new species is similar to that found in and technical difficulties necessary to work in meso- Psaromenia berggrenii (D’Archino et al. 2010). photic habitats, as well as the massive footprint of Psaromenia septentrionalis has monocarpogonial such areas in our oceans, preclude a worldwide sys- supporting cells (Fig. 4E), whereas P. berggrenii is tematic survey of the biota of these environments. basically polycarpogonial with some monocarpogo- Nevertheless, the “snapshots” taken have uncovered nial supporting cells (D’Archino et al. 2010). In interesting species living at the limits of light pene- P. septentrionalis, we have not seen connecting fila- tration (McDermid and Abbott 2006, Sherwood ments directly produced by postfertilization fusion et al. 2010, Ballantine et al. 2016). Using genetic cells but have discovered clustered cystocarps lead- sequences from specimens collected at a depth of ing to speculation that both exist. Supporting cells 90 m off Bermuda, we have uncovered three species may act as auxiliary cells in P. septentrionalis as is the assignable to the family Kallymeniaceae. Two were case for several Kallymeniaceae such as Callophyllis, described as new species of Nothokallymenia and Euthora and Pugetia among others (Norris 1957). In Psaromenia and the third required a new combina- the few postfertilization systems that we discovered, tion for a western Atlantic species now recognized young gonimoblast filaments were issued directly by molecular data to be representative of the newly from lobate fusion cells. Two fusion cells that we described Austrokallymenia. These three species focus observed were in proximity to each other in the attention on the great depth at which they were inner cortex-outer medulla, but we did not observe found and highlight the uncovered diversity in the discernable connecting filaments entering or leaving oceans’ mesophotic zone. the two systems. There are interesting biogeographic links for the Carposporophytes of Psaromenia septentrionalis three species reported in this paper, two from the form in the pattern as described for other members Pacific and the other from the Atlantic Ocean and of the Kallymeniaceae by Womersley and Norris Mediterranean Sea. An alliance between Atlantic (1971) as a mass of slender gonimoblast cells with and Pacific species is one that we have observed carposporangia separated into what appear as dis- repeatedly for other species that we have discovered tinct “loculi” separated by vegetative filaments in Bermuda. For example, we demonstrated a trans- (Fig. 4F). The gonimoblast filaments produce distal oceanic biogeographic pattern for the kallymeni- carposporangia toward the center of cystocarps pro- acean Meredithia (Schneider et al. 2014b), and since ducing at maturity a mass of sporangia and inter- then additional species in the genus have been dis- mixed vegetative filaments. Externally, older wart- covered in the western Atlantic and Indo-Pacific like pericarps of P. septentrionalis are reminiscent of regions (Ballantine et al. 2015, Saunders et al. those in the genus Cirrulicarpus as described and 2017). Meredithia crenata from Bermuda and M. pul- illustrated by Hansen (1977a,b); however, they have chella from Puerto Rico are distant to the eastern not been shown to form in ring-like clusters as does Atlantic M. microphylla and join species from Norfolk the latter genus. I., Australia, the Philippines and Cocos (Keeling) I. Males were not seen on cystocarpic plants of The two species of Crassitegula in Bermuda are sister Psaromenia septentrionalis and are thus far unknown; to species from Western Australia and Lord Howe I. therefore, it is possible that this species is dioecious (Schneider et al. 2014a). Asteromenia peltata from the as is Psaromenia berggrenii (D’Archino et al. 2010). western Atlantic genetically joins species from Wes- After observing more than 200 specimens from tern Australia and Cocos I., while A. bermudensis depths of 3–25 m, D’Archino et al. (2010) never groups with a new species from Norfolk I. (Saunders found tetrasporangial plants for P. berggrenii. In our et al. 2006). Genetic sequences of Halopeltis pellucida MESOPHOTIC KALLYMENIACEAE 9 from Bermuda and Halopeltis willisii from North Car- Børgesen, F. 1910. Some new or little known West Indian Flori- – olina show that these species do not group together deae. II. Bot. Tidsskr. 30:177 207. Bringloe, T. T. & Saunders, G. W. 2018. Mitochondrial DNA as sister species from the Atlantic Ocean, rather they sequence data reveal the origins of postglacial marine join two different Indo-Pacific region clades within macroalgal flora in the Northwest Atlantic. Mar. Ecol. Prog. this widespread genus (Schneider et al. 2012). Ser. 589:45–58. Our new Austrokallymenia and Psaromenia species Brummitt, R. K. & Powell, C. E. 1992. Authors of Plant Names. reported herein, as well as the other examples noted Royal Botanic Gardens, Kew, London, UK, 732 pp. – Collins, F. S. & Hervey, A. B. 1917. The algae of Bermuda. Proc. above, display rbcL divergence typically in the 2% 5% Amer. Acad. Arts Sci. 53:1–195. range from their Pacific congeners, which indicates D’Archino, R., Nelson, W. A. & Zuccarello, G. C. 2010. Psaromenia separation ~2–4 Mya based on the molecular clock (Kallymeniaceae, Rhodophyta): a new genus for Kallymenia – used in Bringloe and Saunders (2018). This time- berggrenii. Phycologia 49:73 85. Dixon, K. R., Saunders, G. W., Schneider, C. W. & Lane, C. E. frame would allow for Pacific to Caribbean migra- 2015. Etheliaceae fam. nov. (Gigartinales, Rhodophyta), with tions prior to the closing of open waters between the a clarification of the generitype of Ethelia and the addition Americas by the Isthmus of Panama ~2.8 Mya (O’Dea of six novel species from warm waters. J. Phycol. 51:1158–71. et al. 2016). A detailed molecular clock analysis is Frederick, J. J. 1963. The marine algae of the Bermuda platform. necessary to more precisely date species divergences PhD dissertation, Univ. Michigan, Ann Arbor, Michigan, 92 pp. Ganesan, E. K. 1976. On Kallymenia westii sp. nov. (Rhodophyta, which is not the focus of this paper. However, the tec- Cryptonemiales) from the Caribbean Sea. Bol. Inst. Oceanogr. tonic closure of the Pacific-Caribbean passage created Univ. Oriente 15:169–75. a vicariance event that allowed for speciation in Hansen, G. I. 1977a. Cirrulicarpus carolinensis, a new species in the macroalgal species and other marine biota over geo- Kallymeniaceae (Rhodophyta). Occ. Pap. Farl. Herb. Harvard Univ. 12:1–22. logic time (Rosen 1975, Knowlton and Weigt 1998, Hansen, G. I. 1977b. A comparison of the species of Cirrulicarpus McCartney et al. 2000), providing that there was a (Kallymeniaceae, Rhodophyta). Occ. Pap. Farl. Herb. Harvard pathway of dispersal from the southern Pacific Ocean Univ. 12:23–34. to the Americas. Therefore, with the discovery of Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Psaromenia septentrionalis in the western Atlantic, and Sturrock, S., Buxton, S. et al. 2012. Geneious basic: an integrated and extendable desktop software platform for the the movement of Kallymenia westii to Austrokallymenia, organization and analysis of sequence data. Bioinformatics we have added to the pattern of recurring speciation 28:1647–9. events between Australasia and the warm-temperate/ Knowlton, N. & Weigt, L. A. 1998. New dates and new rates for subtropical western Atlantic Ocean. divergence across the Isthmus of Panama. Proc. Royal Soc. B 265:2257–63. Littler, D. S. & Littler, M. M. 2000. Caribbean Reef Plants. An Identi- The mesophotic collections were made as part of the 2016 fication Guide to the Reef Plants of the Caribbean, Bahamas, Flor- XL Catlin Deep Ocean Survey, Nekton’s mission to the ida and Gulf of Mexico. OffShore Graphics Inc, Washington, Northwest Atlantic and Bermuda. Nekton gratefully acknowl- DC, 542 pp. edges the support of XL Catlin and the Garfield Western McCartney, M. A., Keller, G. & Lessios, H. A. 2000. Dispersal bar- Foundation. We thank Dr. Alex Rogers (Mission Science riers in tropical oceans and speciation in Atlantic and east- Director), Capt. Larry Bennett, the crew of the R/V Baseline ern Pacific sea urchins of the genus Echinometra. Mol. Ecol. Explorer, the submersible pilots and the technical divers led by 9:1391–400. Todd Kincaid and Meredith Tanguay. The work at UNB was McDermid, K. J. & Abbott, I. A. 2006. Deep subtidal marine supported by Discovery and Accelerator grants to GWS from plants from the northwestern Hawaiian Islands: new perspec- the Natural Sciences and Engineering Research Council of tives on biogeography. Atoll Res. Bull. 543:525–32. Canada, as well as funding from the Canada Foundation for Norris, R. E. 1957. Morphological studies on the Kallymeniaceae. – Innovation and the New Brunswick Innovation Foundation. Univ. Calif. Publ. Bot. 28:251 333. The genetic sequencing work of the mesophotic specimens at O’Dea, A., Lessios, H. A., Coates, A. G., Eytan, R. I., Restrepo- UNB was funded by the Nekton Foundation, Oxford, Eng- Moreno, S. A., Cione, A. L., Collins, L. S. et al. 2016. Forma- land. Christopher Flook of the Bermuda Institute of Ocean tion of the Isthmus of Panama. Sci. Adv. 2:e1600883. Richards, J. L., Gabrielson, P. W. & Schneider, C. W. 2018. The Sciences (BIOS) and Roger Simmons of the Bermuda Aquar- deepest growing marine alga identified by DNA sequencing, ium, Natural History Museum and Zoo (BAMZ) provided Sporolithon mesophoticum sp. nov. (Sporolithales, Rhodophyta) logistical support in Bermuda. A loan of K. westii specimens from Bermuda. Phytotaxa 385(2):67–76. from MICH was kindly arranged by Dr. Michael Wynne. This Robuchon, M., Le Gall, L., Gey, D., Valero, M. & Verges, Anow. is contribution no. 269, Bermuda Biodiversity Project (BBP) 2014. Kallymenia crouaniorum (Kallymeniaceae, Rhodophyta), of BAMZ, Department of Environment and Natural a new red algal species from the Laminaria hyperborea under- Resources, and Nekton contribution No. 10. The authors storey community. Eur. J. Phycol. 49:493–507. report no conflicts of interest. Rosen, D. 1975. A vicariance model of Caribbean biogeography. Syst. Biol. 24:431–64. Abbott, I. A. & McDermid, K. J. 2002. On two species of Kally- Saunders, G. W., Huisman, J. M., Verges, A., Kraft, G. T. & Le menia (Rhodophyta: Gigartinales: Kallymeniaceae) from the Gall, L. 2017. Phylogenetic analyses support recognition of Hawaiian Islands, Central Pacific. Pac. Sci. 56:149–62. ten new genera, ten new species and 16 new combinations Ballantine, D. L., Ruız, H. & Norris, J. N. 2015. Notes on the ben- in the family Kallymeniaceae (Gigartinales, Rhodophyta). – thic marine algae of Puerto Rico, XI: new records including Crypt. Algol. 38:79 132. new Meredithia (Kallymeniaceae, Rhodophyta) species. Bot. Saunders, G. W., Jackson, C. & Salomaki, E. D. 2018. Phylogenetic Mar. 58:355–65. analyses of transcriptome data resolve familial assignments Ballantine, D. L., Ruiz Torres, H. & Aponte, N. E. 2016. The for genera of the red algal Acrochaetiales-Palmariales com- – mesophotic, coral reef-associated, marine algal flora of plex (Nemaliophycidae). Molec. Phylo. Evol. 119:151 9. Puerto Rico, Caribbean Sea. Smithsonian Contr. Bot. Saunders, G. W., Lane, C. E., Schneider, C. W. & Kraft, G. T. 105:1–41. 2006. Unraveling the Asteromenia peltata species complex with 10 CRAIG W. SCHNEIDER ET AL.

clarification of the genera Halichrysis and Drouetia (Rhody- Schneider, C. W. & Searles, R. B. 1991. Seaweeds of the Southeastern meniaceae, Rhodophyta). Can. J. Bot. 84:1581–607. United States. Cape Hatteras to Cape Canaveral. Duke University Saunders, G. W. & McDevit, D. C. 2012. Methods for DNA bar- Press, Durham, North Carolina, 554 pp. coding photosynthetic protists emphasizing the macroalgae Schneider, C. W. & Wynne, M. J. 2007. A synoptic review of the and diatoms. Meth. Mol. Biol. 858:207–22. classification of red algal genera a half century after Saunders, G. W. & Moore, T. E. 2013. Refinements for the ampli- Kylin’s “Die Gattungen der Rhodophyceen”. Bot. Mar. fication and sequencing of red algal DNA barcode and Red- 50:197–249. ToL phylogenetic markers: a summary of current primers, Schneider, C. W. & Wynne, M. J. 2013. Second addendum to the profiles and strategies. Algae 28:31–43. synoptic review of red algal genera. Bot. Mar. 56:111–8. Schneider, C. W., Freshwater, D. W. & Saunders, G. W. 2012. First Sherwood, A. R., Kurihara, A., Conklin, K. Y., Sauvage, T. & Prest- report of Halopeltis (Rhodophyta, Rhodymeniaceae) from the ing, G. G. 2010. The Hawaiian Rhodophyta Biodiversity Sur- non-tropical Northern Hemisphere: H. adnata (Okamura) vey (2006-2010): a summary of principal findings. BMC Plant comb. nov. from Korea, and H. pellucida sp. nov. and H. willi- Biol. 10:258. sii sp. nov. from the North Atlantic. Algae 27:95–108. Stamatakis, A. 2014. RAxML version 8: a tool for phylogenetic Schneider, C. W., Lane, C. E. & Saunders, G. W. 2006. Crassitegula analyses and post-analysis of large phylogenies. Bioinformatics walsinghamii (Sebdeniaceae, Halymeniales), a new red algal 30:1312–3. genus and species from Bermuda based upon morphology Womersley, H. B. S. & Norris, R. E. 1971. The morphology and and SSU rDNA sequence analyses. Eur. J. Phycol. 41:115–24. taxonomy of Australian Kallymeniaceae (Rhodophyta). Austr. Schneider, C. W., Lane, C. E. & Saunders, G. W. 2018. A revision J. Bot. Suppl. Ser. 2:1–62. of the genus Cryptonemia (Halymeniales, Rhodophyta) in Ber- Wynne, M. J. 2018. Regarding Kallymenia J. Agardh, 1842, Euhyme- muda, western Atlantic Ocean, including five new species nia Ku¨tzing nom. illeg. 1843, and the proposal of Croisettea and C. bermudensis (Collins et M. Howe) comb. nov. Eur. J. gen. nov. (Kallymeniaceae, Rhodophyta). Not. alg. 76:1–4. Phycol. 53:350–68. Wynne, M. J. & Schneider, C. W. 2010. Addendum to the synoptic Schneider, C. W., Popolizio, T. R. & Lane, C. E. 2014a. Notes on review of red algal genera. Bot. Mar. 53:291–9. the marine algae of the Bermudas. 14. Five additions to the Wynne, M. J. & Schneider, C. W. 2016. Third addendum to the benthic flora, including a distinctive second new species of synoptic review of red algal genera. Bot. Mar. 59:397–404. Crassitegula (Rhodophyta, Sebdeniales) from the western Atlantic Ocean. Phycologia 53:117–26. Schneider, C. W., Quach, P. K. & Lane, C. E. 2017. A case for true morphological crypsis: Pacific Dasya anastomosans and Supporting Information Atlantic D. cryptica sp. nov. (Dasyaceae, Rhodophyta). Phycolo- gia 56:359–68. Additional Supporting Information may be Schneider, C. W., Saunders, G. W. & Lane, C. E. 2014b. The monospecific genus Meredithia (Kallymeniaceae, Gigartinales) found in the online version of this article at the is species rich and geographically widespread with species publisher’s web site: from temperate Atlantic, Pacific and Indian Oceans. J. Phycol. 50:167–86. Table S1. Table of specimens with correspond- Schneider, C. W. & Searles, R. B. 1973. North Carolina marine ing GenBank numbers used in study. algae. II. New records and observations of the benthic offshore flora. Phycologia 12:201–11.