Taxonomic study of the polyphyletic Dudresnaya (Dumontiaceae, Florideophyceae) with descriptions of Dudresnaya ryukyuensis sp. nov. and two new genera, Himehibirhodia and Nudresdaya

The red algal genus Dudresnaya (Dumontiaceae, Gigartinales) has traditionally been a morphologically well‐defined taxon, but its molecular phylogeny has rarely been studied. To examine the phylogenetic relationships among Dudresnaya species, we generated new partial sequences of mitochondrial cox1, chloroplast rbcL and nuclear 28S rRNA genes from an undescribed Dudresnaya species from Okinawa Island, Japan, alongside five additional described species. Our phylogenetic analyses show that Dudresnaya is genetically diverse and polyphyletic. Based on molecular phylogeny and morphological data, we describe the Okinawan Dudresnaya as a new species, Dudresnaya ryukyuensis, and transferred Dudresnaya minima and Dudresnaya littleri, which were phylogenetically and morphologically distinct from the genuine Dudresnaya, to the new genera Himehibirhodia and Nudresdaya, respectively. Our phylogenetic analyses also showed that the Dumontiaceae is not a monophyletic group including the Gainiaceae and Rhizophyllidaceae (DGR complex). Considering that the DGR complex exhibits female reproductive structures and their post‐fertilization development that are similar to each other, the DGR complex appears to be recognized as the Dumontiaceae sensu lato.


INTRODUCTION
The family Dumontiaceae (Gigartinales, Rhodophyta) is characterized by (i) separated carpogonial branches and auxiliary cell branches that are distinctly differentiated from vegetative branches; (ii) fusion of the carpogonium with one or more cells of the carpogonial branch prior to formation of secondary connecting filaments to auxiliary cells; and (iii) the location of the auxiliary cell, which is mostly intercalary but rarely terminal in the auxiliary cell branch (Mitchell 1966;Shepley & Womersley 1983;Tai et al. 2001).Dudresnaya P.Crouan & H.Crouan nom.et typ.cons. is a dumontiacean genus, having erect thalli that are irregularly branched, lubricous, gelatinous, solitary or gregarious, which are attached by a small discoid crust.Anatomically, the erect thalli have a uniaxial construction, with axial cells producing a whorl of cortical fascicles and the inner cortical cells producing rhizoidal filaments that descend around the axial filaments.Currently, 20 species are recognized in Dudresnaya, although relatively large morphological variation has been reported.For example, (i) spermatangial mother cells can densely cover the outermost 3-6 cells of the spermatangial branches, forming corncob-like spermatangial structures, or can be arranged at the terminus of the outermost 1-2 cortical cells; (ii) the division of tetrasporangia can be zonate, cruciate or irregularly cruciate; and (iii) the life cycle can be either isomorphic or heteromorphic (Robins & Kraft 1985;Notoya 1988;Notoya & Aruga 1989;Tabares et al. 1997).
Although both the Dumontiaceae and Dudresnaya are morphologically well-defined groups, recent molecular phylogenetic studies do not support their monophyly.Dixon et al. (2015) showed that two dumontiacean algae, Kraftia dichotoma  Sherwood et al. (2010) showed that Dudresnaya hawaiiensis R.K.S. Lee and Dudresnaya littleri Abbott are distantly related in the Dumontiaceae, indicating that Dudresnaya is a complex of distinct lineages.
In the present study, we examined the phylogenetic relationships among Dudresnaya species with newly generated partial sequences of mitochondrial cox1, chloroplast rbcL and 28S ribosomal RNA genes from six Dudresnaya species: Dudresnaya babbittiana Abbott & K.J. McDermid, Dudresnaya japonica Okamura, Dudresnaya kuroshioensis Kajimura, D. littleri, Dudresnaya minima Okamura and one undescribed species from Okinawa Island, Japan.Our multigene phylogenetic analyses show that Dudresnaya is currently composed of three non-monophyletic lineages: D. littleri, D. minima and other species examined.To deal with this taxonomic problem, we propose taxonomic revisions for Dudresnaya and discuss taxonomic relationships between the Dumontiaceae and the related families, Gainiaceae and Rhizophyllidaceae.

Specimen collection
In total, nine Dudresnaya plants were collected by skin diving at three localities in Okinawa Island (Fig. 1; see Supporting information, Table S1).Fragments of plants were preserved in 70% ethanol or salt for morphological observations, and in silica gel for DNA extraction.Pressed specimens were made and deposited in the Herbarium of Faculty of Science, Hokkaido University, Sapporo, Japan (SAP) or the National Museum of Nature and Science, Tsukuba, Japan (TNS).In addition to these specimens, pressed specimens of D. babbittiana, D. japonica, D. kuroshioensis, D. minima and Dudresnaya okiensis Kajimura housed in the herbarium SAP and TNS were morphologically examined and included in the molecular phylogenetic analyses (see Supporting information, Table S1) (herbarium acronyms follow Index Herbariorum; http:// sweetgum.nybg.org/science/ih).The specimens of D. japonica and D. minima included those collected near the type localities (Fig. 1).The specimens of D. kuroshioensis and D. okiensis were the holotype specimens, and the specimens of D. babbittiana comprised the paratype specimens that were collected from the type locality, Midway Atoll (Fig. 1).The DNA samples of D. littleri that were used in Sherwood et al. (2010; specimen code: ARS00292 and ARS00359) were also used for molecular phylogenetic analyses; these were both collected from the island of O'ahu (Fig. 1), although not at the exact type locality.In addition to the Dudresnaya specimens, two specimens of the dumontiacean alga Masudaphycus irregularis (Masuda) Lindstrom were collected in Akkeshi, Hokkaido, Japan, for inclusion in the phylogenetic analyses.

Morphological observation and culture experiment
Morphological observations were conducted using a stereo, an inverted and a compound light microscope, and images were taken with Basler Aca2440-75uc (Basler, Ahrensburg, Germany), DFK 37AUX250 (The Imaging Source, Bremen, Germany) and Digital Sight DS-Fi1 (Nikon, Tokyo, Japan) digital cameras.Imaging software, comprising Basler Video Recording (Basler) and IC Capture (The Imaging Source), were used for the first two cameras, respectively.To observe internal anatomy, fragments of material were stained with cotton blue (lactic acid/phenol/glycerol/water 1:1:1:1), mounted in seawater or 50% glycerol/seawater on glass slides and squashed under a cover glass.
© 2024 The Authors.Phycological Research published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Phycology.
light:dark = 16:8 h) and subsequently transferred to a 25 C short day condition (SD; light:dark = 10:14 h) to encourage tetrasporophyte maturation.Tetraspores released from the tetrasporophytes were isolated with a Pasteur pipette and each spore was transferred to a separate Petri dish.Gametophytes developed from the tetraspores were cultured in a 25 C LD (light:dark = 16:8 h).Fluorescent lighting was 30-50 μmol m À2 s À1 photon flux density for all culture conditions.The morphology of the cultured thalli was observed as above, but without cotton blue staining.

Molecular phylogenetic analyses
Partial sequences of the mitochondrial cox1 (up to 664 bp), the chloroplast rbcL (up to 1310 bp) and nuclear 28S ribosomal RNA (up to 684 bp) genes were newly generated for inclusion in the phylogenetic analyses.Total genomic DNA was extracted using GenCheck DNA Extraction Reagent (FASMAC, Atsugi, Japan) in accordance with Hoshino et al.
The newly generated sequences were aligned with sequences downloaded from GenBank and two datasets were made for phylogenetic analysis.The first dataset was constructed to infer the phylogeny of dumontiacean species and consisted of 63 operational taxonomic units (OTUs) of the Dumontiaceae with its five phylogenetically close families (22 OTUs from the Gainiaceae, Rhizophyllidaceae, Kallymeniaceae, Etheliaceae, Ptilocladiopsidaceae) and Polyides rotunda (Polyidaceae) as the outgroup (see Supporting information, Table S3).The second dataset was used to infer the phylogenetic position of the Dumontiaceae in the Gigartinales and consisted of 32 families of Gigartinales and the Acrosymphytales as the outgroup (total 58 OTUs: up to four species per family; see Supporting information, Table S4).The cox1 and rbcL sequences were aligned using CLUSTAL W (Thompson et al. 1994) in MEGA version 7 (Kumar et al. 2016).The 28S sequences were aligned using MAFFT in the GUIDANCE2 Server (Landen & Graur 2008;Penn et al. 2010;Sela et al. 2015) and the positions in the alignment with a score below 0.93 (i.e.poorly aligned positions) were excluded.The alignments of cox1, rbcL and 28S were concatenated using Kakusan4 (Tanabe 2011).The final length of the alignments was 4676 bp for the first dataset (664 bp of cox1, 1363 bp of rbcL, 2649 bp of 28S) and 4804 bp for the second dataset (664 bp of cox1, 1358 bp of rbcL, 2782 bp of 28S).
For the phylogenetic analyses of the concatenated datasets, IQ-TREE, version 2.2.0.3 (Chernomor et al. 2016;Kalyaanamoorthy et al. 2017;Minh et al. 2020) were used for maximum likelihood (ML) analysis, and MrBayes version 3.2.7 (Huelsenbeck & Ronquist 2001;Ronquist et al. 2012) for Bayesian inference (BI) analysis.The ML analysis by IQ-tree was performed with 1000 of standard non-parametric bootstrap replicates and with the best partition and nucleotide models inferred by the flag 'Àm MFP + MERGE'.The BI analysis was performed with the partition and nucleotide models inferred by Kakusan4 based on the Bayesian Information Criterion (BIC; Schwarz 1978) and Monte Carlo Markov chains (MCMC) were run with the following parameters: two runs, four chains, sampling frequency of 100 and burn-in fraction of 0.25.Stationarity of the MCMC run was monitored using Tracer, version 1.7.1 (Rambaut et al. 2018), and the analyses were terminated after 7 253 000 generations for the first dataset and 30 000 000 generations for the second dataset.The ML and the Bayesian trees were visualized using FigTree, version 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree).For the first dataset, ML analyses were also conducted for each gene, separately, following the same procedure as for the concatenated analyses.Uncorrected pairwise genetic distances (p-distances) for interand intra-species comparisons based on the cox1, rbcL and 28S datasets were calculated in MEGA, version 7.

Taxonomic observations
Here, we describe the Dudresnaya specimens from Okinawa Island as a new species, Dudresnaya ryukyuensis.
Dudresnaya ryukyuensis M. Hoshino, Wakeman, Kitayama & Kogame sp.nov.(Figures 2-8) Description: Gametophytes are erect, yellow to wine-red in color, gelatinous and lubricous in texture, and up to 12 cm in height.The stipe is terete.The main branch is terete to slightly compressed, up to 14 mm in width, irregularly radially branched, sometimes gradually densely covered with branchlets towards terminal.Annulations absent even in terminal branchlets.The axial filament of the branchlet produces two to four cortical fascicles in a whorl, sometimes produces secondary axial filaments, terminates not with an apical cell but with cortical fascicles, whereas the position of the apical cell is unclear.The primary axial filaments and secondary axial filaments are not distinguishable except that secondary axial filaments usually have thinner axial cells.The apical cell is recognized only at the unbranched indeterminate axis primordia.Hexagonal crystals are absent in all cells.The cortical fascicles are dichotomously branched, while their distal cells are cylindrical and lack hairs.The rhizoids arise from the basal cell of cortical fascicles, carpogonial branches and auxiliary cell branches, and develop downward along the axes, frequently branching but without producing cortical fascicles.The gametophytes are dioicous.The spermatangial branches arise laterally on cortical fascicles.The spermatangial mother cells are densely arranged to the outermost 2-7 cells of the spermatangial branches, forming corncob-like structures.The carpogonial branches and the auxiliary cell branches lack a mucilage coat; they are morphologically and spatially distinct (non-procarpic).The carpogonial branches are slightly reflexed, usually composed of six to nine cells including the basal cells, and are terminated by a carpogonium with a long trichogyne.The auxiliary cell branches are usually composed of 15-18 cells including the basal cell.The middle 7-10 cells of the branch are rounded, and the auxiliary cell is located at the center of these modified cells and is usually flattened and smaller than its neighboring cells.The mature carposporophytes are spherical to slightly ellipsoidal and reached 140 μm in diameter, and encircle the auxiliary cell branch with a narrow slit.The tetrasporophytes observed in culture condition are discoid, up to 5 mm in diameter, crimson in color, and mature with cruciately divided tetrasporangia.Cox1, rbcL and 28S sequences of the holotype specimen: GenBank accession LC577547, LC577557, LC632012, respectively.
Etymology: This species is named after the Ryukyu Islands which includes its type locality, Okinawa I.
Morphological observation: In total, nine specimens from three localities of Okinawa I. (Fig. 1) were examined.Plants were irregularly and radially branched and reached 12 cm in height (Fig. 2a,b).Annulations were absent even in the young branches (Fig. 2c).Both primary and secondary axial filaments terminated with cortical fascicles and the position of their apical cells were unclear (Fig. 3a).Apical cells were recognized only at the unbranched indeterminate axis primordia (Fig. 3b).The axial cells produced whorls of two to four cortical fascicles, some of which seemed to develop later into secondary axial filaments (Fig. 6a).Hexagonal crystals reported in axial cells in some Dudresnaya species were absent (Fig. 3c).The cortical fascicles lacked hair cells at their tips.Rhizoids arose from the basal cell of cortical fascicles, carpogonial branches and auxiliary cell branches (Figs 3c,5b,6b).They were usually 4-6 μm (up to 10 μm) in diameter, developed downward along the axes, and frequently branched, but did not produce cortical fascicles.Only gametophytes were collected from the field.These gametophytes were dioicous, two being male and seven being female.Spermatangial branches arose laterally on cortical fascicles (Fig. 4a), whereas the spermatangial mother cells were densely arranged to the distal 2-7 axial cells of the spermatangial branches, forming corncob-like structures (Fig. 4b).The carpogonial and auxiliary cell branches arose from the axial cells or basal part of cortical fascicles, replacing cortical fascicles/filaments (Figs 5a and 6a).A mucilage coat was not observed around the carpogonial and auxiliary cell branches.The mature carpogonial branches were slightly reflexed, usually composed of six to nine cells including the basal cells, and were terminated by a carpogonium with a long trichogyne (Fig. 5a,b).Rhizoids or sterile laterals often arose from the basal cell of the carpogonial branch (Fig. 5a,b).The mature auxiliary cell branches were usually composed of 15-18 cells including the basal cell (Fig. 6a-c), but sometimes had a long, terminal cortical filament, and composed of more than 25 cells.The middle 7-10 cells of the branch were modified (i.e.rounded and often darkly (c) Rhizoid from basal cell of cortical lateral (SAP115582).aux, auxiliary cell; auxb, auxiliary cell branch; ax, axial cell; b, basal cell of cortical lateral; cf., connecting filament; cp, carpogonium; cpb, carpogonial branch; fc, fusion complex; gi, gonimoblast initial; lb., lateral branch; nut, nutritive cell; pcf, primary connecting filament; rh, rhizoid; s, spermatia; scf, swollen part of secondary connecting filament; smc, spermatangial mother cell; tr, trichogyne; 2cf, secondary connecting filament.stained) and the auxiliary cell was located at the center of these modified cells.The auxiliary cells are usually flattened and smaller than its neighboring cells (Fig. 6b).The auxiliary cells often had a slightly greater diameter at their distal end (Fig. 6b).Rhizoids sometimes arose from the basal cell of the auxiliary cell branch (Fig. 6b).No sterile laterals were observed on the basal cell of the auxiliary cell branch.
After fertilization, the trichogyne became plugged at its base (Fig. 5b) and the carpogonium extended a primary connecting filament that fused with the fourth and fifth cells (nutritive cells; Fig. 5c-f) of the carpogonial branch.From the resulting fusion complex, secondary connecting filaments, which fuse with the auxiliary cells, arose (Fig. 5f).Disappearance of the trichogyne during the post-fertilization development of the carpogonium, which was reported in D. minima and D. littleri (Kawashima 1959;Littler 1974), was not observed (Fig. 5b-f).After secondary connecting filaments laterally fused with the auxiliary cells (Fig. 6c), the fused portion of the connecting filament swelled and formed gonimoblast initials (Fig. 6d) and additional connecting filaments to other auxiliary cells.The mature carposporophyte was spherical to slightly ellipsoidal and reached 140 μm in diameter, which encircled the auxiliary cell branch with a narrow slit (Fig. 6e).
Life cycle in culture: Released carpospores were spherical and 10-12 μm in diameter (n = 21) (Fig. 7a).The first cell division divided the carpospores into two equal halves (Fig. 7b) and germinated unipolarly (Fig. 7c) or bipolarly.The germlings repeatedly branched and formed discoid plants (Fig. 7d).The plants often had fine hairs on the surface.The discoid thalli repeatedly underwent horizontal cell divisions and became thicker (Fig. 7e,f).In a 20 C LD, they did not produce any spore even after 8 months of cultivation, but some formed erect thalli from their edge (Fig. 7e).The erect thalli that developed in the tetrasporophyte generation did not form tetrasporangia but instead formed spermatangial branches.We did not examine whether the spermatia from the spermatangia were functional.Then, the discoid and erect thalli were transferred to a 25 C SD to encourage tetrasporophyte maturation.The discoid thalli transferred started to release tetraspores after 1 week of cultivation.The tetrasporangia formed on the surface of the discoid thalli without forming nemathecia and were larger than the vegetative cortical cells (Fig. 7g,h).The tetrasporangia were spherical, 9.6-18.9μm in diameter (14.5 μm on average; n = 16) and cruciately-divided (Fig. 7g).The released tetraspores were spherical, 8.1-9.6 μm in diameter (9.1 μm on avg.; n = 13), and were connected by fine and sticky threads (Fig. 7i).
In the 25 C SD, the released tetraspores first divided into two equal halves and subsequently germinated in a unipolar or bipolar direction (Fig. 8a,b).The germlings repeatedly branched and formed compact discs with fine hairs (Fig. 8c).These discs started to form erect thalli in 2 weeks.The erect thalli were separately isolated into new Petri dishes and cultured in the 25 C LD (Fig. 8d).After 7 weeks of cultivation, erect thalli formed either female reproductive structures (carpogonial and auxiliary cell branches) (Fig. 8e) or spermatangial branches (Fig. 8f).The male and female gametophyte strains were deposited in the Kobe University Macro-Algal Culture Collection (strain codes: KU-3451-3453).In summary, our culture experiments indicated that D. ryukyuensis has a heteromorphic life cycle with erect dioicous gametophytes and discoid tetrasporophytes.

Molecular phylogenetic analyses
In total, 32 sequences were generated (14 for cox1, 11 for rbcL and seven for 28S) (see Supporting information, Table S3).Although we could not generate any sequences from D. okiensis, the phylogenetic position of five Dudresnaya species (D. babbittiana, D. japonica, D. kuroshioensis, D. minima and D. ryukyuensis) and M. irregularis were examined for the first time in this study.
Our phylogenetic analyses demonstrate that the family Dumontiaceae is not monophyletic, nesting the families Gainiaceae and Rhizophyllidaceae in it (DGR complex) (Fig. 9; see also Supporting information, Fig. S1).The monophyly of the DGR complex was supported by our concatenated dataset [bootstrap proportion (BP) = 85, posterior probability (PP) = 1.0] (Fig. 9).The DGR complex can be further divided PP = 1.0), although the phylogenetic relationship between the two species was not well resolved (Fig. 9).In clade A, at least three lineages were recognized (lineages i-iii); wellsupported lineage i (BP = 97, PP = 1.0) and weakly supported lineage ii (BP = 76, PP = 1.0) were sister (BP = 100, PP = 1.0), with lineage iii sister to them (Fig. 9).Our phylogenetic analyses showed that D. hawaiiensis, D. japonica and Dudresnaya verticillata (Withering) Le Jolis were not monophyletic.This indicates that some sequences deposited in GenBank may have an incorrect taxonomic name or suggests the existence of cryptic species.We considered the D. hawaiiensis lineage including the topotypes (Sherwood et al. 2022) to be the true D. hawaiiensis and that our D. japonica specimens, collected near the type locality, were the true D. japonica (Fig. 9).For the generitype D. verticillata, there were at least two lineages in lineage ii and one in lineage iii (Fig. 9).Considering that its type locality is in the U.K. (exact type locality is unknown; Withering 1796; Irvine 1983), true D. verticillata would be represented by either the specimens from the UK and Spain belonging to lineage ii, or from Ireland belonging to lineage iii, and probably not from Taiwan belonging to lineage ii (Fig. 9).Dudresnaya cf.babbittiana, which was first reported in the Hawaiian Islands outside the type locality Midway Atoll by Sherwood et al. (2022), was shown to be clustered with the paratype from the type locality (Fig. 9), indicating that this species is widely distributed in the Hawaiian Island chain (Figs 1 and 9).The new species D. ryukyuensis belonged to lineage i with D. hawaiiensis and D. japonica.The intraspecific genetic distance (p-distance) of D. ryukyuensis was up to $1.96% in cox1, $ 0.76% in rbcL and 0.0% (no polymorphism) in 28S.The genetic distance between D. ryukyuensis and its genetically closest described species, D. hawaiiensis, was $8.6% in cox1, $4.2% in rbcL and $ 0.17% in 28S, and the intra-specific genetic distance of D. hawaiiensis was $1.79% in cox1, $1.72% in rbcL and 0.0% in 28S.

DISCUSSION
New Dudresnaya species from Japan Based on morphological and molecular phylogenetic analyses, we describe a new species, D. ryukyuensis.This is the fifth Dudresnaya species from Japan and morphologically distinguishable from other four species, namely, D. minima, D. okiensis, D. japonica and D. kuroshioensis.The first two species differ from D. ryukyuensis in having annulations on their branches (Okamura 1932;Kajimura 1993).Dudresnaya japonica tends to exhibit more or less di/trichotomous and sparse branching (Okamura 1908), whereas D. ryukyuensis exhibits radial and dense branching.Dudresnaya kuroshioensis may exhibit a similar external morphology to D. ryukyuensis (i.e.radial branching, branches without annulations, and wide branches more than 0.5 cm), but it differs in having hexagonal crystals in the axial cells (Kajimura 1994).
Outside of Japan, several species may resemble D. Dudresnaya hawaiiensis is morphologically identical to D. ryukyuensis, except that D. hawaiiensis is monoicous (Lee 1963) and D. ryukyuensis is dioicous.Because these sexual states co-exist in some red algal species, dioicy may not be a stable diagnostic character, whereas the genetic distance between D. ryukyuensis and D. hawaiiensis supports their independence.Phylogenetically, D. ryukyuensis was closest to D. 'japonica' from Taiwan.The label 'D.japonica' has been used in Taiwan since Shen and Fan (1950).Although the detailed morphology of D. 'japonica' from Taiwan has likely not been reported so far, an image of a D. 'japonica' specimen from Taiwan (catalog number: NCU-A-0010791; https://macroalgae.org/portal)resembles D. hawaiiensis and D. ryukyuensis, rather than D. japonica.Detailed observation of the Taiwanese specimens is necessary to discuss their taxonomic relationships with D. ryukyuensis.1).The trichogyne of Himehibirhodia and Nudresdaya disappears during post-fertilization development (Kawashima 1959;Littler 1974, as Dudresnaya lubrica Littler), whereas that of Dudresnaya does not (Okamura 1908;Robins & Kraft 1985;Kajimura 1994; present study), as shown in the generitype D. verticillata (Bornet & Thuret 1876;Kylin 1928;Robins & Kraft 1985).Furthermore, the distal end of the auxiliary cell branches of Himehibirhodia is terminated with rounded modified cells (Hasegawa 1949;Kawashima 1959;Kitayama 1989), whereas the auxiliary cell branches of Dudresnaya and Nudresdaya are usually terminated with cortical filaments (Littler 1974;Robins & Kraft 1985;present study).Furthermore, in H. minima, the tetrasporangia exhibit an irregularly cruciate division (Notoya 1988), whereas, in Dudresnaya and Nudresdaya, they exhibit a zonate division (Robins & Kraft 1985;Kajimura 1994;Abbott 1999) and, in some Dudresnaya, a cruciate pattern is observed (Notoya & Aruga 1989;present study).
As indicated by the fact that the heterotypic synonym of H. minima was established in Thuretellopsis Kylin (T.japonica Segawa & Ichiki;Segawa & Ichiki 1958), H. minima resembles Thuretellopsis peggiana Kylin, which is the sole species recognized in Thuretellopsis, in external morphology (i.e.soft and gelatinous thalli, crimson in color, and branches with annulations) (Lindstrom & Scagel 1987).However, T. peggiana differs from H. minima by possessing monosporangia in both gametophytes and sporophytes (Richardson & Dixon 1970), a feature that has never been reported in Himehibirhodia, Nudresdaya and Dudresnaya (Table 1).Our molecular phylogenetic analyses also support Thuretellopsis as distinct from the three other genera.
Himehibirhodia and Nudresdaya exhibit similar post-fertilization development of the carpogonial branches yet differ in life cycle (Table 1).Notoya (1988) cultured the carpospores of H. minima and reported a heteromorphic life cycle with a crustose tetrasporophyte and tetrasporangia that are irregularly cruciately divided.By contrast, N. littleri has an isomorphic life cycle with the isomorphic tetrasporophyte having zonately divided tetrasporangia (Abbott 1999;Abbott & McDermid 2001).Although the phylogenetic relationship between Himehibirhodia and Nudresdaya is unclear, the differences in life cycle strongly support the distinction of the two genera.

Genetic and morphological variation in Dudresnaya
After the exclusion of H. minima and N. littleri, the genus Dudresnaya can be defined by the persistence of trichogynes even after post-fertilization development, in addition to the traditional characters of Dudresnaya (Robins & Kraft 1985).However, this newly defined Dudresnaya still exhibits significant morphological diversity (Table 1).The most significant trait is the variation in life cycle: Dudresnaya includes species with an isomorphic life cycle and species with a heteromorphic life cycle (Table 1).We observed that the immature discoid tetrasporophyte of D. ryukyuensis developed erect thalli bearing spermatangia.The erect thalli probably did not originate from tetraspores that had formed unnoticed because, if tetraspores had formed, both male and female gametophytes would have developed, but no erect thalli bearing carpogonial branches were observed.Occurrence of both sexual organs and tetrasporangia on a single plant has occasionally been reported in the Rhodophyceae (West & Norris 1966;Edelstein & McLachlan 1967;Rueness & Rueness 1985).However, we speculate that this phenomenon in D. ryukyuensis was an abnormal occurrence as a result of laboratory conditions, because these erect thalli formed after the long cultivation in the culture condition that is unsuitable for tetrasporophyte maturation (i.e.20 C LD). Thus, we consider D. ryukyuensis to have a heteromorphic life cycle with erect gametophytes and discoid tetrasporophytes.Besides D. ryukyuensis, D. japonica is known to have discoid tetrasporophytes (Notoya & Aruga 1989).In the molecular phylogenetic tree, D. japonica and D. ryukyuensis belong to lineage i with D. hawaiiensis and D. 'hawaiiensis' from Australia and New Caledonia.In lineage i, D. ryukyuensis, D. hawaiiensis and D. 'hawaiiensis' form a clade, and D. japonica is sister to the clade.Considering this topology, lineage i is expected to have discoid tetrasporophytes; however, Abbott (1999) and Abbott and McDermid (2001) reported erect tetrasporophytes in D. hawaiiensis.These reports should be reexamined because, except for these, tetrasporophytes have never been found in D. hawaiiensis (Lee 1963) and D. hawaiiensis from Australia (Robins & Kraft 1985;Huisman 2018).It is noteworthy that Robins and Kraft (1985) examined over 100 specimens of D. hawaiiensis from Australia, which is probably the same species as D. 'hawaiiensis' from Australia in Fig. 9, but could never find tetrasporophytes.Furthermore, none of the D. hawaiiensis specimens, from which the sequence data that we used were generated, were morphologically tetrasporophytes.These facts raise the possibility that D. hawaiiensis has discoid tetrasporophytes, and that Abbott misidentified tetrasporophytes of other species as those of D. hawaiiensis.Indeed, a species showing an isomorphic life cycle, D. babbittiana, described by Abbott and McDermid (2001) as an endemic to Midway Atoll, was shown to be distributed around the type locality of D. hawaiiensis (Sherwood et al. 2022;present study).
By contrast to lineage i, lineage ii and iii include species with an isomorphic life cycle (Robins & Kraft 1985;Kajimura 1994;Abbott & McDermid 2001).Their sporophytes exhibit zonately divided tetrasporangia, whereas those of lineage i (D. japonica and D. ryukyuensis) exhibit cruciately divided tetrasporangia.Furthermore, hexagonal crystals are commonly observed in axial cells in lineages ii and iii (Robins & Kraft 1985;Kajimura 1994;Abbott & McDermid 2001), but never in lineage i.
Considering the genetic and morphological variations, Dudresnaya could arguably be separated into multiple genera.Although the phylogenetic position of the generitype D. verticillata was unclear because of its paraphyly, true D. verticillata probably belongs to lineage ii or iii because the lectotype of D. verticillata is an erect tetrasporophyte with zonately divided tetrasporangia (Lindstrom 1985).Therefore, at minimum, lineage i could be recognized as a new genus.Nevertheless, in the present study, we do not make further taxonomic revisions on Dudresnaya because of insufficient molecular data and a lack of information on the life cycle of Dudresnaya species.Out of 19 described Dudresnaya species, reliable molecular data (i.e.data from type specimens or specimens collected close to the type locality) have been obtained for only six species: D. ryukyuensis, D. hawaiiensis, D. japonica, D. babbittiana, D. kuroshioensis and Dudresnaya australis.Tetrasporophyte morphology is not known in some species, probably because some of them have small discoid tetrasporophytes that require culture experiments to observe.For some species, a different morphology and/or habitat information have been reported in the original description and in subsequent reports (e.g.D. verticillata, D. australis, D. capricornica, D. hawaiiensis and Dudresnaya crassa M.A.Howe).Molecular phylogenetic studies on more species/specimens with detailed descriptions of morphology and life cycle are necessary to solve the taxonomic problem of Dudresnaya.

Non-monophyletic dumontiaceae
Our molecular phylogenetic analyses showed that the Dumontiaceae is not monophyletic and includes the Gainiaceae and Rhizophyllidaceae (DGR complex), as shown in other studies (Tai et al. 2001;Dixon et al. 2015).The Gainiaceae is a monotypic family for the Antarctic alga Gainia mollis Moe and was established based on its noncalcareous crustose habit, isomorphic life cycle and nemathecial development of reproductive structures (Moe 1985).The Rhizophyllidaceae consists of four genera: Contarinia Zanardini, Ochtodes J. Agardh, Portieria Zanardini and Nesophila Nelson & Adams.Although these genera are mostly different from each other in vegetative morphology, they share large prominent gland cells and nemathecial development of reproductive structures as the synapomorphy (Wiseman 1975;Nelson & Adams 1996;Payo et al. 2011).In our opinion, the distinction among the Dumontiaceae, Gainiaceae and Rhizophyllidaceae is ambiguous.Most of the diagnostic traits of the Gainiaceae and Rhizophyllidaceae are also seen in dumontiacean taxa.Non-calcareous crustose habit and isomorphic life cycle have been reported in Wearnia (Wilce et al. 2003), and nemathecial development of reproductive structures has been reported in Wearnia and Rhodopeltis (Nozawa 1970;Itono & Yoshizaki 1992;Wilce et al. 2003).
The DGR complex shares (i) separate carpogonial branches and auxiliary cell branches (non-procarpic) that are more than two-celled and distinct from vegetative branches and (ii) fusion of the carpogonium with other cells of the carpogonial branch prior to formation of the secondary connecting filaments (Wiseman 1977;Shepley & Womersley 1983;Moe 1985;Payo et al. 2011).In the Florideophyceae, these features are also observed in the Peyssonneliales and Acrosymphytales (Kylin 1925;Lindstrom 1987;Kato & Masuda 2000), but, within the Gigartinales, they are unique to the DGR complex.Among the Gigartinales, the Ptilocladiopsidaceae, Kallymeniaceae and Etheliaceae were phylogenetically close to the DGR complex.Although the reproductive structure of the Etheliaceae is unknown, the female reproductive structures and their post-fertilization development of the former two families are generally similar to those of the DGR complex.However, the Ptilocladiopsidaceae differs from the DGR complex in that its auxiliary cell branches are two-celled (Rodríguez-Prieto et al. 2014).In the non-procarpic taxa of the Kallymeniaceae, © 2024 The Authors.Phycological Research published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Phycology.
the carpogonia are located in the carpogonial branch system and the auxiliary cells in the auxiliary cell system (Rodríguez-Prieto & Hommersand 2009;D'Archino et al. 2016), which are distinct from those of the DGR complex.Thus, the female reproductive structures and their post-fertilization development mentioned above might be the synapomorphy of the DGR complex in the Gigartinales.
To solve the taxonomic problem of the DGR complex, two proposals can be considered: (i) integrating the Gainiaceae and Rhizophyllidaceae into the Dumontiaceae based on the potential synapomorphy mentioned above or (ii) splitting the DGR complex into multiple families in a manner consistent with the molecular phylogeny.For the second proposal, assuming the current topology of the phylogenetic tree is correct, one possible approach would be to either divide clade E and the others into two families or, in an extreme case, to split each of clade A to E into separate families.However, either case is challenging because there are currently no apparent diagnostic characters for these potential families, except the monogeneric ones.Thus, at present, the first proposal appears to be reasonable.We advocate for the Gainiaceae and Rhizophyllidaceae to no longer be recognized and consider the DGR complex as the Dumontiaceae sensu lato.

Dumontiaceae Bory emend. M.Hoshino & Kogame
Description: The thalli are erect or crustose, uniaxial or multiaxial, non-calcareous or calcareous.The reproductive structures are sometimes in nemathecia.The female reproductive structure is non-procarpic.The carpogonial branches are at least four-celled, usually unbranched, distinctly differentiated from the vegetative branches; the carpogonium terminal on the carpogonium branch.The auxiliary cell branches are at least three-celled, usually unbranched, somewhat to distinctly differentiated from the vegetative branches; the auxiliary cell mostly intercalary in the auxiliary cell branch, but rarely terminal.The fertilized carpogonium generally fuses with one or more cells of the carpogonial branch itself prior to production of secondary connecting filaments that fuse with the auxiliary cells.The carposporophyte develops from the fusion complex of the auxiliary cell and the connecting filament; the gonimoblast filaments radiate, almost all the gonimoblast cells convert to carposporangia, resulting in a globular cluster of carposporangia.The spermatangia are cut off from outer cortical cells.The tetrasporangia are zonately or cruciately or irregularly divided.Life history, where known, is triphasic with isomorphic or heteromorphic gametophytes and tetrasporophytes.
Type genus: Dumontia J.V.Lamouroux.The outer cortical cells are cylindrical.The gametophytes dioicous.The spermatangia are produced at the distal end of the terminal cells of the cortical fascicles.The carpogonial branches are usually 6-7 celled including the basal cell and strongly crooked near the terminus.The auxiliary cell branches are typically 6-9 celled, all cells are modified and rounded, including the terminal cells; the auxiliary cell is intercalary, usually the third cell from the base, the same in size and shape as neighboring cells of the branch, but more transparent and showing a large nucleus.After fertilization, the carpogonium directly fuses with the fourth cell (counting from the terminal carpogonium) of the carpogonium branch that is in contact with it, and then cuts off a primary connecting filament fusing with the fifth cell.The resulting two fusion complexes cut off secondary connecting filaments to the auxiliary cells.The trichogyne degenerates and disappears by the time the primary connecting filament fuses with the fifth cell.Carposporophytes up to 150 μm in diameter.
The released carpospores are 28-35 μm in diameter.The tetrasporophytes are crustose, and the tetrasporangia are irregularly cruciate.The released tetraspores are 20-25 μm in diameter.

Diagnosis:
This genus resembles Dudresnaya, Thuretellopsis and Nudresdaya in being uniaxial, gelatinous and lubricous.However, it differs from Dudresnaya with respect to the disappearance of the trichogyne during the post-fertilization development of the carpogonial branch; from Thuretellopsis with respect to lacking production of monospores; and from Nudresdaya with respect to having a heteromorphic life cycle.
Etymology: The genus is named after the Japanese name of the generitype H. minima, Hime-hibirodo (Okamura 1932).The genus name is feminine in gender.
Remark: The description and the diagnosis of this genus are based on the descriptions of D. minima (Okamura 1932;Hasegawa 1949;Segawa & Ichiki 1958, as Thuretellopsis japonica;Kawashima 1959;Notoya 1988).The carpogonial branches are 6-10 celled including the basal cell, and strongly crooked near its terminal end.The auxiliary cell branches are 10-16 celled of which the 4-6 cells are enlarged and rounded, and the auxiliary cell is the second of the enlarged cells from the proximal end.After fertilization, the carpogonium fuses with the fourth cell (counting from the terminal carpogonium) of the carpogonial branch by an extremely short primary connecting filament, and resulting fusion complex cuts off a second primary connecting filament fusing with the fifth cell.The resulting two fusion complexes cut off secondary connecting filaments to the auxiliary cells.Holotype: BISH 518022 collected offshore of M akua, O'ahu, Hawai'i, deposited in BISH (Abbott 1999).

Fig. 1 .
Fig. 1.Sampling sites in Japan and the Hawaiian Island chain in the present study.Species names are given for the type localities.Species collected from two or more sites are assigned symbols.For the sampling sites, from which specimens were examined in the present study, Arabic numerals (1-12) are given.
Fig. 6.Dudresnaya ryukyuensis sp.nov.Auxiliary cell branch and its post-fertilization development, observed using a compound light microscope.(a) Auxiliary cell branches developed on axial cells (SAP115582).(b) Mature auxiliary cell branch showing slightly flattened auxiliary cell (SAP115582).(c) Auxiliary cell branch showing lateral fusion of connecting filament with auxiliary cell (SAP115582).(d) Gonimoblast initials on swollen part of connecting filament developed on auxiliary cell (SAP115582).(e) Mature carposporophytes with a narrow slit (arrowhead) (TNS-AL152111).

Fig. 7 .
Fig. 7. Development of the carpospores of Dudresnaya ryukyuensis sp.nov.under culture conditions.(a) Released carpospores.Observed using an inverted light microscope.(b) Carpospores after the first cell division.Observed using an inverted light microscope.(c) Germling of the carpospore.Observed using an inverted light microscope.(d) Three-week-old discoid germling with several layers of cells in the central region.Observed using an inverted light microscope.(e) Mature discoid tetrasporophyte.Erect thalli (arrowheads) developed from the margin of the disc.Observed using a stereo light microscope.(f) Cross-section of immature part of a discoid tetrasporophyte.Observed using a compound light microscope.(g) Cross-section of mature part of a discoid tetrasporophyte, showing cruciately divided tetrasporangia.Observed using a compound light microscope.(h) Surface view of a discoid tetrasporophyte, showing tetrasporangia (asterisks).Observed using a compound light microscope.(i) Tetraspores released from a discoid tetrasporophyte.The spores are connected by fine threads.Observed using a compound light microscope.

Fig. 8 .
Fig. 8. Development of the tetraspores of Dudresnaya ryukyuensis sp.nov.under culture condition.(a) Tetraspore after the first cell division.Observed using an inverted light microscope.(b) Tetraspore germinated bipolarly.Observed using an inverted light microscope.(c) Six-day-old germlings of a tetraspore showing a hair (arrowhead).Observed by an inverted light microscope.(d) Young erect thalli (gametophyte) growing on a disc originated from a tetraspore.Observed using a stereo light microscope.(e) Carpogonial and auxiliary cell branches on a mature female gametophyte.Observed using an inverted light microscope.(f) Spermatangial branches on a mature male gametophyte.Observed using an inverted light microscope.

Fig. 9 .
Fig. 9. Maximum likelihood tree based on the first dataset showing the phylogeny of the species of the Dumontiaceae and its related families: 86 OTUs, concatenated DNA sequences of mitochondrial cox1 and chloroplast rbcL and nuclear 28S ribosomal RNA genes (total 4676 bp).Numbers on branches indicate bootstrap values from ML analysis (left) and posterior probabilities from BI analysis (right).The black circles indicate branches with full support (100/1.0).Only bootstrap values >80 and posterior probabilities >0.95 are shown.Possible species misidentifications are indicated by single quoted species names (for details, see the Results section).The taxa synonymized in the present study are indicated by white text.Newly sequenced samples are indicated by bold text.Scale bar = the number of nucleotide substitutions per nucleotide site.
Himehibirhodia minima (Okamura) M.Hoshino, Kitayama & Kogame comb.nov.Nudresdaya M. Hoshino, A. R.Sherwood & Kogame gen.nov.Description: The gametophytes are erect, pale red in color uniaxial, cylindrical, narrow and irregularly branched.Texture is very soft, gelatinous and lubricous.The cortical fascicles are branched dichotomously.The distal cells of the cortical fascicles are oval to obpyriform and the proximal cells are cylindrical.The gametophytes are dioicous.The spermatangia are produced at the distal end of the terminal cells of the cortical fascicles, or form corncob-like spermatangial structures.
The trichogyne degenerates and disappears during the postfertilization development.The carposporophytes are up to 430 μm in diameter.The released carpospores are 24 μm in diameter.The tetrasporophytes are isomorphic to the gametophytes.Tetrasporangia are zonately divided.and Himehibirhodia in that plants are uniaxial, gelatinous and lubricous.However, it differs from Dudresnaya in the disappearance of the trichogyne during the postfertilization of the carpogonial branch, and differs from Thuretellopsis and Himehibirhodia in having an isomorphic life cycle.Generitype: Nudresdaya littleri (I.A.Abbott) M.Hoshino, A.R.Sherwood & Kogame comb.nov.Etymology: The name is an anagram of Dudresnaya.The genus name is feminine in gender.Remark: The description and the diagnosis of this genus are based on the descriptions of Dudresnaya littleri (Littler 1974, as Dudresnaya lubrica; Abbott 1999; Abbott and McDermid 2001).Nudresdaya littleri (I.A.Abbott) M. Hoshino, A. R. Sherwood & Kogame comb.nov.Basionym: Dudresnaya littleri I.A.Abbott, in Abbott, I. A., Abbott 1996, Pacific Science 50: 151.Heterotypic synonym: Dudresnaya lubrica Littler, nom.illeg.
Sherwood et al. (2010)and D. littleri to new genera Our molecular phylogenetic analyses demonstrated that the genus Dudresnaya is polyphyletic as previously shown inSherwood et al. (2010).Here, we propose the transfer of D. minima and D. littleri to the new genera Himehibirhodia and Nudresdaya, respectively (see taxonomic summary below).Himehibirhodia (H.minima) and Nudresdaya (N.littleri) are phylogenetically and morphologically distinct from Dudresnaya (Table

Table 1 .
Morphological comparison of Dudresnaya, Thuretellopsis and the newly established genera, Himehibirhodia and Nudresdaya © 2024 The Authors.Phycological Research published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Phycology.
Color is crimson to light purple.Texture very soft, gelatinous and lubricous.The branches are annulated.The cortical fascicles are branched di-or tri-chotomously, with a colorless hair at the terminus.