Discovery of a novel brown algal genus and species Setoutiphycus delamareoides (Phaeophyceae, Ectocarpales) from the Seto Inland Sea, Japan

We describe a new genus and species of brown algae from the Seto Inland Sea, Japan. This species is similar to Delamarea in gross morphology and anatomy, but distinctive in having longer thalli with rare branching and shorter cortical cells. In culture, pluri-zoids derived from plurilocular zoidangia on the erect thalli developed into filamentous gametophytes bearing ectocarpoid plurilocular zoidangia, but also formed parenchymatous erect thalli of sub-sympodial growth similar to Trachynema often having branches, and formed lateral and terminal plurilocular zoidangia. Molecular phylogenies using concatenated chloroplast and mitochondrial gene sequences showed the new alga nested in the clade composed of ectocarpalean genera with diffuse growth, parenchymatous thalli, and multiple chloroplasts, but this species is distinctive. Therefore, we propose Setoutiphycus delamareoides gen. & sp. nov. for this new alga, and provisionally place it in Chordariaceae, Ectocarpales. The Seto Inland Sea repeatedly dried during sea level regressions during glacial periods, and the present sea level recovered after the last glacial maximums (LGM), ca. 10,000 years ago. Therefore, it is unlikely that the species evolved within this area. Its distribution in the area may be explained as a remnant population that survived in refugia in southern Japan during the LGM.


Introduction
The higher rank taxonomy of brown algae (Phaeophyceae) has been considerably revised in the last few decades, especially in certain orders, by the application of life history studies and molecular phylogenetic analyses 1,2,3,4,5 .Ectocarpales and its related orders, comprising taxonomic groups of relatively small, soft thalli and showing heteromorphic life histories, are an example.Traditionally, members of Ectocarpales sensu lato have been classi ed in independent orders within Ectocarpales sensu stricto, Chordariales, Dictyosiphonales and Scytosiphonales, or Ectocarpales s.l.including Ectocarpales s.s., plus Chordariales, Dictyosiphonales and Scytosiphonales, based on the thallus constructions ( lamentous, or pseudoparenchymatous/haplostichous, or parenchymatous/polystichous) and number of chloroplasts per cell (single or multiple).However, life history studies suggested indistinct boundaries separating those orders, and molecular phylogenetic analyses did not support the monophyly of those orders except for Scytosiphonales.Therefore, it was proposed to classify them in a single order Ectocarpales s.l., including Scytosiphonales as a family (Scytosiphonaceae), and merging many genera that used to be placed in Dictyosiphonales and Chordariales into the family Chordariaceae 6,7 .As a result, currently more than a hundred genera are included in Chordariaceae, which is an exceptionally great number in the brown algae, and their morphology, including the basic thallus architecture, is highly diverse.On the other hand, a new monotypic family was proposed primarily based on molecular phylogenetic data (i.e., Petrospongiaceae 8 ).Also, the taxonomic status of some families is still unclear because their diagnostic characters are not consistent, and phylogenetic resolution of the molecular phylogenetic studies is insu cient.Therefore, family level taxonomic delineation of Ectocarpales is still rather confused and substantial revisions are needed.
Among them, genus and species level taxonomy of the ectocarpalean members with terete, parenchymatous thalli having multiple chloroplasts have been relatively well documented because of their easily recognizable macroscopic thalli, distinctive anatomical features applicable to taxonomic comparisons, and the substantial number of life history studies using unialgal cultures elucidating their life histories and early development.Most of their genera were described by the 1960's, and no additional taxa at genus level have been published for several decades, excluding the proposals of new a genus for known species originally described in a different genus 2,4,9,10,11 .However, recently in the western part of the Seto Inland Sea, Japan, which has a unique geography but has had only limited taxonomic surveys, we collected an undescribed brown alga with unique morphological features.Here we describe, based on morphology and molecular phylogenetic studies, a novel member among the terete parenchymatous species belonging to Ectocarpales s.l., and discuss the family level taxonomy.

Morphological studies
This novel brown alga appeared as a spring annual growing on hard substrates of sandy bottoms of the upper subtidal zone, together with various annuals such as Acrothrix gracilis Kylin, Cutleria multi da (Turner) Greville, Striaria attenuata (Greville) Greville, Tinocladia crassa (Suringar) Kylin, Ulva intestinalis Linnaeus, etc. (Fig. 1a).Erect thalli were epilithic or growing on dead shells, solitary or caespitose, liform, simple or rarely branched, attenuated towards the base, blunt at the tip, surface rough yellowish brown in color, up to about 15 cm in height and up to 2 mm in diameter (Figs.1b, 2a, b).The thalli were parenchymatous, solid when young and later becoming hollow, composed of one to two layers of large colorless inner cells, subcortical cells and barrel-shaped or short clavate cortical cells, and terminal and lateral phaeophycean hairs (Fig. 2c-h).Cortical cells measured up to 100 µm in height and up to 60 µm in diameter.Plurilocular and unilocular zoidangia were formed on the same thallus, developed among the cortical cells at the distal end of subcortical cells (Fig. 2h-k).Plurilocular zoidangia were formed by subdivisions of initial cells (Fig. 2i), conical to lanceolate, often branched at the tip (Fig. 2j), becoming longer than the cortical cells (Fig. 2i), up to 120 µm in height and up to 72 µm in diameter.Unilocular zoidangia were ovate, up to about 60 µm in height and up to 50 µm in diameter (Fig. 2k).Each cell contained many discoidal chloroplasts with projected pyrenoids.In the cortical cells, chloroplasts were parietal at the distal end (Fig. 2d, i).

Molecular phylogeny
Molecular phylogenies based on concatenated mitochondrial cox1, cox3 and chloroplast atpB, psaA, psbA and rbcL gene sequences (7,696 bp) showed identical tree topologies by Maximum Likelihood (ML, Fig. 3) and Bayesian (BI) analyses.The novel alga nested in the clade composed of Trachynema, Delamarea, Cladothele, and Punctaria supported by full bootstrap/posterior priority values, although the supports for the nodes connecting the novel alga with other genera were low (clade 1).This clade was sister to the clade of Striaria and Asperococcus supported by full bootstrap/p.p. values (clade 2).
In the molecular phylogeny based on rbcL gene sequences covering a large portion of the genera in Ectocarpales, the novel alga was also included in the clade composed of Cladothele, Delamarea, Hecatonema, Punctaria and Trachynema supported by medium and high bootstrap/p.p. values (95%/1.00),but the relationships among the genera were not resolved (clade 1; Fig. 4).The novel alga was nested in the clade of Punctaria latifolia Greville, Hecatonema sp. and Punctaria plantaginea, but the statistical supports for the nodes connecting them were low.Hecatonema has lamentous thalli, but the sequenced specimen was likely a gametophytic stage of Punctaria sp. 12 .Clade 1 was sister to clade 2 as in the phylogenetic tree based on six genes, but the statistical support was low.In contrast, some of the ectocarpalean genera such as Coelocladia, Litosiphon, Pogotrichum and Stictyosiphon with parenchymatous terete thalli and many chloroplasts with projected pyrenoids, having similar basic thallus constructions as the novel alga, showed distant phylogenetic relationships with these genera, and were scattered in the rbcL tree (Fig. 4).

Discussion
In morphology, the novel alga was most similar to Delamarea attenuata Hariot.They shared terete rough thalli with an attenuated basal portion and blunt tip, thallus architecture composed of large inner cells and a cortical layer developing large clavate or barrel-shaped cortical cells, and the occurrence on the erect thalli of both unilocular and plurilocular zoidangia among large cortical cells.However, the novel alga was distinctive in having longer thalli with rare branching, shorter cortical cells, and occurrence of branches at the tips of the plurilocular zoidangia.Cladothele, Punctaria and Trachynema, Striaria and Asperococcus, which showed close phylogenetic relationships in our molecular phylogeny based on the concatenated DNA sequences of six genes, shared similar thallus morphological features: terete parenchymatous thalli of diffuse growth bearing unilocular and plurilocular zoidangia among large cortical cells, and terminal and lateral phaeophycean hairs.Although Punctaria spp.have foliose thalli, their juvenile thalli show similar terete stages with apical and opposite lateral hairs, which are common to those genera.Our multigene molecular phylogeny, including most of the related genera, showed that the novel alga is distinct from any of those genera.Therefore, for this novel alga we propose the establishment of a new genus and species, Setoutiphycus delamareoides gen.& sp.nov..In contrast, rbcL of other ectocarpalean genera with similar thallus architecture (e.g., Coelocladia, Litosiphon, Pogotrichum and Stictyosiphon) showed distant phylogenetic relationships with the clade, suggesting convergent evolution of the thallus architecture in Ectocarpales.
Because monophyly of the families with parenchymatous thalli in Dictyosiphonales or Ectocarpales s.l., was not supported in molecular phylogenetic studies 6,22 , expansion of Chordariaceae to include members that used to be classi ed in independent families such as Punctariaceae and Striariaceae was proposed 6 .In contrast, small brown algae having parenchymatous thalli and a single chloroplast with projected pyrenoids have been classi ed in Scytosiphonales 23 , and in spite of this unique cytological feature and highly supported monophyly, they are classi ed as a family nested in Ectocarpales.
However, currently roughly 140 genera are included in Ectocarpales s.l., more than 100 of which are of Chordariaceae, and these numbers are exceptionally great, considering the genetic divergence within each order of the Phaeophyceae 2,5,7,11 (Supplementary Information 4).The DNA sequence divergence of rbcL genes ranges from about 5-15% within each brown algal order.However, divergence was less than 10% in Ectocarpales s.l. and not especially high compared with other orders (Supplementary Information 4).In our molecular phylogeny, monophyly of several genera of former members of Dictyosiphonales (i.e., Delamareaceae [Delamarea, Cladothele], Punctariaceae [Punctaria, Trachynema] and Striariaceae [Striaria, Asperococcus] sharing the following morphological features was supported: Parenchymatous, terete or foliose thalli of diffuse growth, with terminal and lateral (often opposite) phaeophycean hairs; normally forming both unilocular and lanceolate to ovoid plurilocular zoidangia; cells with many discoid chloroplasts with projected pyrenoids.Remarkably, in spite of its close morphological similarity with these genera, Coelocladia was shown to have phylogenetic relationship instead with the clade of Cladothele, Delamarea, Punctaria, Setoutiphycus, and Trachynema.Indeed, an independent family Coelocladiaceae has been proposed for Coelocladia, based on the unique morphology of the plurilocular zoidangia showing a clustered or crown-like appearance and the occasional sympodial branching of the primary lament 21 .In contrast, Dictyosiphon, the type of Dictyosiphonaceae and Dictyosiphonales, does have parenchymatous terete thalli, but the genus is unique in showing apical growth by a single apical cell, and forming only unilocular zoidangia embedded in the subcortical and peripheral layers 9 .
Although the number of taxa we examined were rather limited, our multigene molecular phylogeny based on six genes showed considerable improvement of the phylogenetic resolution of families within Ectocarpales s.l.. Therefore, we expect that the application of multigene molecular phylogeny to additional taxa will give clues for obtaining a better taxonomy of the family.As to the taxonomy of the three families traditionally used for the genera comprising the clade including Setoutiphycus (i.e., Delamareaceae A.D.Zinova 1953, Punctariaceae (Thuret) Kjellman 1880, Striariaceae Kjellman 1890), Punctariaceae has taxonomic priority.Therefore, it is possible to reappraise Punctariaceae for the lineage in reorganizing current Chordariaceae by subdividing it to several monophyletic lineages sharing distinctive morphological features.However, for the moment, we suspend any taxonomic treatment, and provisionally place Setoutiphycus in Chordariaceae, Ectocarpales s.l..As to the biogeography of Setoutiphycus delamareoides, at present it has been only found from the western end of the Seto Inland Sea, Japan, and it is possibly endemic to the region (Supplementary Information 1).Similarly, an endemic red alga Neorhodomela enomotoi Masuda & Kogame was described from the Seto Inland Sea, and has not been reported from any other coasts 24 .Members of Neorhodomela are cool-temperate or cold-water species, and the localities of the species appear to represent the southern limits of their distributional ranges 24 .In spite of the low latitude (34º N) and short distance from the main ow of the Kuroshio Warm Current, water temperatures at the locality are relatively low (monthly average is from 9.5 to 26.5ºC; Wanishi 2004), because of the enclosed geography of the area.The Seto Inland Sea repeatedly dried due to sea level regression during the glacial periods.
Therefore, the history of the ora is rather recent, since the present sea level recovered after the LGM of only ca.10,000 years ago 25 .Therefore, it is unlikely that the evolution of the genus and species occurred within this area.It has been noted that the macroalgal ora of the area is more similar to the cooltemperate Paci c coast of northern Honshu (Tohoku region) than that of the adjacent areas in Paci c Shikoku and Kyushu where the water temperature is higher 26 .This is explained as the result of separation of the populations that survived in the refugia in southern Japan during LGM 27 : during the northward expansion of the populations after the LGM, some of them survived in the Seto Inland Sea 26,27,28 .Therefore, it is possible that S. delamareoides has a broader distributional range, at least in Japan, such as northern Honshu.However, if not, the species can be endangered by the rise of seawater temperature in the area due to global climate change 29,30 , because the Seto Inland Sea is enclosed at its northern end, so the population cannot spread to colder northern coasts.

Description And Diagnosis
Setoutiphycus gen.nov.H. Kawai & T. Hanyuda Typus: Setoutiphycus delamareoides H. Kawai & T. Hanyuda Erect thalli, liform, rarely branched, attenuated towards the base, blunt at the tip, parenchymatous, solid when young and becoming hollow with age, composed of large colorless inner cells and barrel-shaped cortical cells and phaeophycean hairs.Plurilocular and unilocular zoidangia formed on the same thallus at the end of subcortical cells among the large cortical cells.Plurilocular zoidangia conical to lanceolate, often branched at the tip.Unilocular zoidangia ovate.Each cell containing many discoidal chloroplasts with projected pyrenoids.
The new genus resembles Delamarea in gross morphology and anatomy, but differs in the longer, rarely branched thallus and shorter cortical cells.The species differs from Cladothele in the epilithic habit and rare branching, and from Trachynema in having large cortical cells.Nucleotide sequences of mitochondrial cox1 and cox3, chloroplast atpB, psbA and rbcL genes are also distinctive.
Erect thalli, epilithic, solitary or caespitose, liform, simple or rarely branched, attenuated towards the base, blunt at the tip, yellowish brown in color, up to about 15 cm in height, up to about 2 mm in diameter, parenchymatous, solid when young and becoming hollow with age, composed of 1-2 layers of large colorless inner cells, barrel-shaped cortical cells, and phaeophycean hairs.Cortical cells measure up to 100 µm in height and up to 60 µm in diameter.Plurilocular and unilocular zoidangia on the same thallus, at the end of cortical cells, among the cortical cells.Plurilocular zoidangia conical to lanceolate, often branched at the tip, projected from the cortical cells, up to 120 µm in height and up to 72 µm in diameter.Unilocular zoidangia ovate, up to ca. 60 µm in height and up to ca.50 µm in diameter.Each cell containing many discoidal chloroplasts with projected pyrenoids.Nucleotide sequences of mitochondrial cox1 and cox3, chloroplast atpB, psbA and rbcL genes are also distinctive.Holotypus SAP115639, Suo-Oshima (33.9407N 132.4016E), Yamaguchi, Japan, 10, April 2017.
Etymology: The genus name refers to the original locality.The speci c epithet refers to the morphological features of the thallus.

Methods
Specimens were collected at Suo-Oshima, Yamaguchi, Japan by SCUBA diving (Supplementary Information 1).Portions of the specimens were quickly dried in silica-gel and used for molecular analyses.For anatomical observations, cross and longitudinal sections were made by hand using a razor blade.Photomicrographs were taken with a VB-7010 Digital Camera (Keyence, Tokyo, Japan) attached to an Axioplan microscope (Zeiss, Oberkochen, Germany).DNA extractions were made from eld-collected specimens rapidly desiccated in silica-gel (KU-d5871, -d17414, -d17417, -d17418) and unialgal culture strains housed in the Kobe University Macroalgal Culture Collection (KU-MACC, KU-1881) (Supplementary Information 2).Genomic DNA was extracted using a

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