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Haraldiophyllum hawaiiense sp. nov. (Delesseriaceae, Rhodophyta): a new mesophotic genus record for the Hawaiian Islands

Copyright © 2020 The Korean Society of Phycology 337 http://e-algae.org pISSN: 1226-2617 eISSN: 2093-0860 Haraldiophyllum hawaiiense sp. nov. (Delesseriaceae, Rhodophyta): a new mesophotic genus record for the Hawaiian Islands Monica O. Paiano*, John M. Huisman, Feresa P. Cabrera, Heather L. Spalding, Randall K. Kosaki and Alison R. Sherwood School of Life Sciences, University of Hawai‘i at Mānoa, 3190 Maile Way, Honolulu, HI 96822, USA Western Australian Herbarium, Biodiversity and Conservation Science, Department of Biodiversity, Conservation and Attractions, Locked Bag 104, Bentley Delivery Centre, Western Australia 6983, Australia Department of Biology, College of Charleston, 66 George St., Charleston, SC 29424, USA NOAA, Papahānaumokuākea Marine National Monument, 1845 Wasp Boulevard, Building 176, Honolulu, HI 96818, USA

eral locations in Europe, as well as the Atlantic Islands and the Middle East (Guiry and Guiry 2020). The remaining species of both genera have more limited distributions, for example H. infossum, which occurs only along the eastern coast of Australia and New Zealand (Guiry and Guiry 2020). There are no records of either genus for the Hawaiian Archipelago to date.
Over the past 15 years, thousands of algal specimens have been collected during expeditions to Papahānaumokuākea Marine National Monument (PMNM) led by the US National Oceanic and Atmospheric Administration (NOAA), as part of an ongoing effort to characterize the algal biodiversity of Hawaiian Mesophotic Coral Ecosystems (MCEs). Here we describe a new species, Haraldiophyllum hawaiiense sp. nov., which is also a new genus record for the Hawaiian Islands, based on morphological and molecular characterization of six specimens collected in 2015 and 2019 from PMNM.

MATERIALS AND METHODS
Six Haraldiophyllum specimens were collected during technical diving operations on cruises to PMNM between 2015 and 2019 (Table 1). Four specimens were collected from Kure Atoll (Hōlanikū), and one specimen each was collected from Pearl & Hermes Atoll (Manawai), and Salmon Bank, at a depth range of 82 to 93 m. Specimens were preserved in 4% formalin for morphological studies and in silica gel desiccant for molecular analyses. Voucher specimens were mounted on herbarium paper and accessioned at the Bernice P. Bishop Museum (BISH) under the accessions BISH 776069-776071 and BISH 776120-776122. Morphological and anatomical investigations of the samples were conducted by gently rehydrating the thalli in a detergent (Modified Pohl's Solution, Clark, unpublished: https://www.eeob.iastate.edu/research/ bamboo/pdf/anatomy_protocols.pdf) for at least 2 h, hand sectioning with a double-edged razor blade, staining with 0.5% aniline blue (Figs 6 & 13 were not stained), and mounting in 30% Karo (ACH Food Companies, Inc., Memphis, TN, USA). Photomicrographs were taken on a Zeiss AxioImager A1 compound light microscope (Pleasanton, CA, USA) with an Infinity2-1RC digital camera (Lumenera Corporation, Ottawa, ON, Canada).
Genomic DNA was extracted using the OMEGA E.Z.N.A. Plant DNA DS Kit (OMEGA Biotek, Norcross, GA, USA) protocol. The genes selected for the molecular analyses were the chloroplast-encoded rbcL and the nuclear-encoded LSU. The rbcL gene was amplified using

INTRODUCTION
The delesseriacean genus Haraldiophyllum was established by Zinova (1981) to include three previously described species: Nitophyllum bonnemaisonii (Kylin) Kylin (= Myriogramme bonnemaisonii Kylin) and N. versicolor Harvey from Atlantic Europe, and N. mirabile Kylin from Washington, USA. In these three species the two pericentral cells that are cut off from the fertile central cell in the procarp are located one on top of the other (i.e., parallel to the longitudinal axis of the thallus), while in the generitype of Nitophyllum, N. punctatum (Stackhouse) Greville (Kylin 1934), the pericentral cells are organized perpendicular to the axis (Wynne 2014, Kang et al. 2017. Furthermore, N. punctatum is characterized by a single sterile-cell group in the development of the procarp, while the other three species have two sterile-cell groups (Kylin 1925(Kylin , 1934 (Nam and Kim 1996), was described as a new species based on vegetative morphological comparisons and phylogenetic analysis of the ribulose-1,5-bisphosphate carboxylase-oxygenase largesubunit (rbcL) gene (Kim and Kang 2011). In 2017, Kang and Kim proposed the genus Neoharaldiophyllum, establishing N. udoense (M. S. Kim & J. C. Kang) J. C. Kang & M. S. Kim as the generitype based on phylogenetic analyses of rbcL and nuclear large-subunit rRNA gene (LSU) markers and the following combination of morphological features: the primary gonimoblast filaments are secondarily fused with many floor cells of the cystocarp cavity, and arrangement of tetrasporangia within the tetrasporangial sori is symmetrical in two layers (Kang et al. 2017 (Miller et al. 2010) with 1,000 bootstrap replicates and the GTRCAT model. Bayesian inference analysis (BA) was performed using the MrBayes plug-in v. 3.2.6 (Huelsenbeck and Ronquist 2001) through Geneious Prime 2019.1.3 (http://www.geneious.com) using four chains of Metropolis-coupled Markov Chain Monte Carlo for 1,000,000 generations and with sampling every 100 generations; 100,000 chains were removed as burn-in prior to determining posterior probabilities.

RESULTS
Comparison of the COI barcode sequences from the Hawaiian specimens ARS 09801, ARS 09803, and ARS 09938 to those in GenBank and BOLD returned the following values: 91.0% and 90.7% similarity with H. bonnemaisonii (BOLD: ABMMC21854-16) from Norway and N. mirable from Canada (BOLD: ABMMC1053-06), respectively, and 90.8% similarity with eight Neoharaldiophyllum udoense specimens (GenBank: KY497914-KY497921) from Korea (Kang et al. 2017). Haraldiophyllum currently includes four species, but only the type, H. bonnemaisonii, has a COI barcode sequence; for this reason, a neighbor-joining analysis was not performed.
Phylogenetic analyses were conducted using the rbcL and rbcL + LSU concatenated alignments of Haraldiophyllum and closely related taxa belonging to the sub-primers for two overlapping fragments: rbcLF7 and rb-cLJNR1 (Gavio andFredericq 2002, Kang andKim 2013) for the first fragment, and rbcLF762 and rbcLR1442 (Kim et al. 2010) for the second fragment. A 1,390-bp fragment of LSU was amplified using the following overlapping primer pairs: T16 and T24, and T25 and T10 (Harper and Saunders 2001). A 630-bp fragment from the DNA barcode region near the 5′ end of the mitochondrial cytochrome oxidase subunit I (COI) gene was generated using the GazF1 and GazR1 primer pair (Saunders 2005). Polymerase chain reaction products that amplified successfully were sent to be sequenced at the University of Hawai'i at Mānoa Advanced Studies in Genomics, Proteomics, and Bioinformatics (ASGPB) core facility. To date, there are no other COI sequences for Haraldiophyllum on GenBank and only a single sequence on Barcode of Life Database (BOLD) (ABMMC218554-16, H. bonnemaisonii); the COI sequence generated in the current study was aligned to this sequence from BOLD for comparison. For phylogenetic analyses of the rbcL and LSU sequences, raw sequences were assembled and aligned using the MUSCLE v. 3.8.425 plug-in (Edgar 2004) in Geneious Prime 2019.1.3 (http://www.geneious.com), and edited manually where necessary. Two alignments were generated: one for rbcL and a concatenated alignment for rbcL + LSU. For each, contiguous sequences were aligned with reference sequences and analyzed with PartitionFinder v. 1.1.1 (Lanfear et al. 2012). Maximum likelihood (ML) analysis was conducted on both alignments using RAx-  epiphyte on an unknown red alga, attached by creeping stolons. When alive, plants were rose-pink, and turned to brownish-red when dry. Thalli are composed of one to many membranous blades (Fig. 3A & B), arising from the holdfast, or creeping stolons when epiphytic, blades frequently cleft with denticulate margins (Fig. 3C), with an obtuse or apiculate apex (Fig. 3D). The blades are thin and mostly monostromatic (Fig. 3E), with no midribs or microscopic veins on the blades except for the lower portion of some blades where occasionally a fan-shaped thickened nerve was observed, becoming polystromatic ( Fig. 3F & G). Marginal growth occurs by transverse or oblique divisions. Tetrasporangial sori are circular to elliptical ( Fig. 3D & H), numerous and randomly scattered on both sides of the blade surface (Fig. 3D), close to the margins and upper parts of the blade. Tetrasporangial sori protrude on either side of the blade and are produced from inner and subcortical cells; in transverse section tetrasporangia are clearly visible in two layers between both surface cells, separated by a central medullary cell layer (Fig. 3J). Mature tetrasporangia are spherical, 43-68 µm in diameter and divided tetrahedrally (Fig. 3I), with numerous tetrasporangia formed within a single sorus (Fig. 3K). No male or female reproductive structures were observed.

DISCUSSION
Haraldiophyllum ( Kang et al. in 2017 with N. udoense as the type, and was differentiated from Haraldiophyllum by the primary gonimoblast being secondarily fused with floor cells of the cystocarp (versus being free, without secondary fusions in Haraldiophyllum) and a symmetrical arrangement of tetrasporangia in two layers (as opposed to the asymmetrical presentation in Haraldiophyllum). The number of layers in the tetrasporangial arrangement does not seen to be a sig- Fig. 3) Description. Plants erect, epilithic, or as epiphytes on other algal species. Thalli irregularly or dichotomously branched, composed of one to numerous ovate to elongate blades, attached by a small rounded holdfast, with occasional polystromatic basal nerves, 425-559 µm thick, with 8-10 cell layers. Thalli rose-pink to brownish-red, 2.2-7.8 cm high and 2.2-13.2 cm broad. Blades with a delicate texture, denticulate margins, monostromatic at the tips and margins. Blades in surface view composed of polygonal cells, 50-94 µm long × 75-106 µm wide, presenting parietal granular chloroplasts; cells are connected by primary and secondary pit connections. Vegetative growth by transverse division of marginal cells. Tetrasporangial sori circular to oval, 300-488 µm long by 238-475 µm wide, scattered throughout both sides of the blade, except along the margins, up to 210 µm thick. Mature tetrasporangia spherical to slightly ovate, 43-67 µm long by 45-68 µm in diameter, tetrahedrally divided. Gametophytes not observed. Habit and vegetative morphology. All specimens were collected from the mesophotic coral ecosystem (81-93 m) in the PMNM. Plants are erect, up to 7.8 cm high and 13.2 cm in breadth, epilithic, attached by a small rounded holdfast or as epiphytes on other algal species. As examples, ARS 09984 was found associated with the holdfast of a brown alga, Sporochnus, while ARS 09985 was an haraldiophyllum, with support, based on the rbcL tree (Fig. 1). The subfamily Myriogrammeae was clearly separated in two robust clades, with the Hawaiian specimens resolved as a basal taxon in the main Haraldiophyllum clade, grouping with H. bonnemaisonii, H. crispatum and Haraldiophyllum sp. 1 with high support, and supporting the conclusion that the Hawaiian specimens represent a distinct taxonomic entity from other congeners and also from Neoharaldiophyllum species. Two possible solutions could resolve the non-monophyly of Haraldiophyllum, given the phylogenetic position of Neoharaldiophyllum: (1) transfer all Neoharaldiophyllum species to Haraldiophyllum, making the genus monophyletic, or (2) recognize the different clades within Haraldiophyllum + Neoharaldiophyllum as a series of genera that include Haraldiophyllum sensu stricto. However, no morphological data are currently available for Haraldiophyllum sp. 1 and sp. 2 (vouchers MK125389, LT969700, LT969717, LT969714, and LT969716) (Supplementary Table S1) to aid in the delineation of these taxa. Additional investigation of morphological features for Haraldiophyllum species from Australia and Europe would be of great value in defining whether these specimens belong to this genus, or whether they represent a new genus to be described. Furthermore, a previous study already reported the necessity of additional morphological investigation to better define the genus: Lin et al. (2007) assessed H. bonnemaisonii, reported from Namibia and South Africa, and showed that it does not belong in Haraldiophyllum. Moreover, they demonstrated that H. sinuosum was established based on the description by Lucas (1913) of Nitophyllum sinuosum A. H. S. Lucas, and that it has not been reinvestigated in detail. To date there are no sequences for H. infossum and H. sinuosum, and phylogenetic analysis of these two species would be a valuable contribution toward understanding the diversity and evolutionary history of the group.

Haraldiophyllum hawaiiense M. O. Paiano, Huisman & A. R. Sherwood sp. nov. (
Many taxonomic investigations employing molecular phylogenies have considered the red algal female anatomy and post-fertilization structures as useful characters for defining generic boundaries in the Gigartinales, Halymeniales and Ceramiales (e.g., Lin et al. 2002, Díaz-Tapia et al. 2013, Gargiulo et al. 2013. Although classification based on the arrangement of carposporangia has been used to describe the Myriogrammeae, comparisons are only valid when all developmental stages are reported (Hommersand and Fredericq 1997). In the present study it was decided to classify our specimens as members of Haraldiophyllum rather than Neoharaldiophyllum, even though the molecular similarity of our nificant character for genera within the Myriogrammeae, varying as one or two layers within Haraldiophyllum and Neoharaldiophyllum, versus two layers in Myriogramme and Platyclinia (Maggs and Hommersand 1993, Lin et al. 2007, Kim and Kang 2011, Kang et al. 2017.
Within the tribe Myriogrammeae, which was described by Hommersand and Fredericq (1997), Haraldiophyllum, Neoharaldiophyllum and Myriogramme are very similar in morphology, and the following features characterize the tribe: procarps associated with anterior cover cells, and consisting of a supporting cell, a one-to two-celled lateral sterile group, a four-celled carpogonial branch, and a one-celled basal sterile group. They also described a character found exclusively in Haraldiophyllum: the nuclei lie beneath the chloroplast (a feature observed for members in the tribe Cryptopleureae) while for other members of the Myriogrammeae the nuclei are positioned along the interior side walls of the cell (Lin et al. 2007). Additionally, Haraldiophyllum and Neoharaldiophyllum have carposporangia that are solitary and terminal, while carposporangia in Myriogramme are borne in terminal chains (Wynne 1983, Lin et al. 2007. The Hawaiian species of Haraldiophyllum described here is clearly a member of the Myriogrammeae in terms of molecular phylogeny. No characteristics of reproductive development have been observed, which are considered diagnostic features for the group and the distinction from their known congeners, because no gametophytic plants were collected. Nonetheless, recognition of the species is supported by molecular phylogenetic data and comparisons of vegetative morphology. The molecular analyses included sequences representing the tribe Myriogrammeae (Figs 1 & 2 The phylogenetic trees based on LSU and rbcL sequences revealed the same topology: within the Myriogrammeae, Myriogramme sequences formed a distinct clade that was sister to the clade containing all Haraldiophyllum and Neoharaldiophyllum species, with strong bootstrap and posterior probability support. These results are similar to those reported by Lin et al. (2007), Kang (2011), andKang et al. (2017), confirming the position of the three genera in the Myriogrammeae and the tribal relationships within the subfamily Phycodryoideae. Interestingly, a third clade of Haraldiophyllum specimens from European waters (collected from private aquaria) identified as Haraldiophyllum sp. 2 was positioned as an early diverging lineage from Haraldiophyllum and Neo-isms from shallow waters after experiencing stress, or are unique environments that merit recognition for their distinct diversity. Additionally, by exploring the endemism levels of species and the distinctiveness of shallow and deep floras, biogeographic comparisons will be possible with other mesophotic regions of the world. specimens is greater with N. udoense than with H. bonnemaisonii and H. crispatum, due to the fact that for both molecular analyses, the Hawaiian specimens grouped with good support with other species of Haraldiophyllum, including the generitype H. bonnemaisonii.
In 2017, when the genus Neoharaldiophyllum was established, species of Haraldiophyllum were re-evaluated and two major changes were suggested: the Korean H. bonnemaisonii, described by Nam and Kang (2012) was found to be in agreement with the Neoharaldiophyllumtype, and Australian specimens of Neoharaldiophyllum nottii, as recorded by Womersley (2003, as Haraldiophyllum nottii) were found to be of the Haraldiophyllumtype and referable to H. crispatum (Kang et al. 2017), although the original material used to describe Neoharaldiophyllum nottii is from Washington, USA, and was recognized as Nitophyllum nottii by Norris and Wynne (1968) due to it presenting a Neoharaldiophyllum-type development of the cystocarp. Thus, the genus Haraldiophyllum has the following distribution: the type species H. bonnemaisonii has records for the Atlantic Ocean and the Mediterranean Sea, and H. crispatum has the broadest reported distribution including Australia and New Zealand, Chile, temperate South America, Antarctica and Subantarctic Islands. The remaining three species have more restricted distributions; H. sinuosum is known from Australia and Brazil, H. infossum has just one record in New South Wales, Australia, and H. hawaiiense is thus far known only from PMNM, Hawai'i. Prior to this study, the depth range recorded for the genus was from shallow waters to 24 m (Kim and Kang 2011), but the specimens in this study were collected at depths between 81-93 m, making H. hawaiiense the only Haraldiophyllum species recorded from mesophotic depths thus far.
Haraldiophyllum hawaiiense is described here as a new species and as a new genus record from mesophotic depths in the Hawaiian Islands. Recent studies have characterized the algal biodiversity of both shallow and mesophotic environments, and based on a combination of molecular and morphological data, new species were described, including four new species of Ulvaceae (Spalding et al. 2016), two new species of Ramicrusta (Sherwood et al. in press), one new species of Sonderophycus (Sherwood et al. 2020), and four new species of Martensia (Sherwood et al. 2019). These are some of the first studies in an ongoing effort to use molecular and morphological data to characterize the mesophotic algal flora of the Hawaiian Islands. The biodiversity characterization of these deep-water environments will be essential to understand whether these MCEs serve as refugia for other organ-