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A description of <scp><i>Aliichlorella ignota</i></scp> gen. et sp. nov. and a comparison of the efficiency of species delimitation methods in the <scp><i>Chlorella</i></scp>‐clade (Trebouxiophyceae, Chlorophyta)

AbstractThe present study reports a new representative of Chlorella‐clade that was newly isolated from the Lake Vos'merka (Samara region, Russian Federation). 18S–ITS1–5.8S–ITS2 sequence analyses indicated that the studied strain ACSSI 368 and related Chlorella chlorelloides, Chlorella pulchelloides, formed an separate cluster in the Chlorella‐clade, unrelated to the holotype of the genus Chlorella – Chlorella vulgaris. The independent place of the genus was also confirmed by genetic distances. The individual species status of the studied strain ACSSI 368, compared with the sister C. chlorelloides, was confirmed by morphological differences (cell number per colony, type of chloroplast, number of autospores), genetic distances of the interspecific level by 18S–ITS1–5.8S–ITS2 fragment, alone by internal transcribed spacer 1 (ITS1) and internal transcribed spacer 2 (ITS2) sequences, one complementary base change in ITS1, and the results of species delimitation using five methods. A description of studied strain ACSSI 368 was provided as an authentic strain of the type species Aliichlorella ignota. Currently, the new genus Aliichlorella also includes Aliichlorella chlorelloides comb. nov. and Aliichlorella pulchelloides comb. nov. In general, six different delimitation algorithms were used in the present study. The ASAP (i.e. assemble species by automatic partitioning) and LocMin (i.e. ‘local minimum’ function) algorithms showed synchronous results, but their accuracy with respect to closely related species was somewhat lower. The mlPTP algorithm simultaneously aimed to combine closely related species in one and at the same time divide slightly distinguishing strains within a species into several molecular operational taxonomic units. The bPTP algorithm separated excessively Chlorella‐clade representatives into individual species. The generalized mixed Yule coalescent (GMYC) algorithm results were consistent the most with the modern understanding of the Chlorella‐clade taxonomy. However, the same method is one of the most time‐consuming because, for its implementation, it is necessary to carry out long preparatory work. The KoT results were less accurate than the GMYC results, although this is less time‐consuming because it does not require the preliminary construction of ultrametric trees.

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<scp><i>Lithophyllum nagaokaense</i></scp> sp. nov. (Corallinales, Corallinophycidae, Rhodophyta): A new rhodolith‐forming non‐geniculate coralline alga from Japan

SUMMARYLithophyllum is a cosmopolitan coralline algal genus with 12 species currently recognized in Japan based on modern morpho‐anatomical taxonomic concepts primarily characterized by having uniporate tetrasporangial and bisporangial conceptacles, the presence of secondary pit‐connections between cells of adjacent filaments and a dimerous thallus construction with a single basal layer of predominantly non‐palisade cells. In the present study, we describe Lithophyllum nagaokaense sp. nov. based on a combination of molecular and morpho‐anatomical data of specimens from the temperate waters of Japan. The new species forms both attached epilithic thalli and free‐living rhodoliths. Phylogenetic analyses of psbA, rbcL and COI markers resolved L. nagaokaense as an independent species. Morpho‐anatomically, L. nagaokaense resembles Lithophyllum johansenii, originally described from Australia, but distinguished from the latter by the absence of enlarged angular cells occluding pore canals of tetrasporangial conceptacle chambers. Lithophyllum nagaokaense shared a fruticose morphology with Lithophyllum kaiseri, Lithophyllum kuroshioense, Lithophyllum neo‐okamurae, Lithophyllum okamurae, and Lithophyllum pygmaeum from Japan. Of these, L. nagaokaense is the most similar to L. neo‐okamurae in having attached and free‐living thalli with encrusting and warty to lumpy growth forms bearing knobby protuberances that are dichotomously branched or anastomosing. The new species is distinct from other Japanese fruticose species collectively by five tetrasporangial conceptacle chamber characters and the absence of trichocytes. A taxonomic key for identifying Japanese fruticose species is provided. This is the fifth Lithophyllum species and the third rhodolith‐forming species verified by DNA sequencing to be found in Japan.

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Sterols of the aberrant plastid, <scp>K</scp>areniacean dinoflagellate <scp><i>Asterodinium gracile</i></scp>: Chemotaxonomic overlap with amphisterol‐producing <scp><i>Amphidinium carterae</i></scp>

SUMMARYDinoflagellates have a rich history of characterization of their membrane‐reinforcing sterols because of a structural diversity and chemotaxonomic utility uncommon to other classes of algae. The Kareniaceae are no exception in that they produce sterols, often containing a Δ8(14) nuclear unsaturation, which are rare in most other dinoflagellate groups. A continuing goal of our laboratory is to examine the sterols of previously uncharacterized dinoflagellates to assess their sterol‐based chemotaxonomy compared to other members of the Dinophyceae. Asterodinium gracile, a member of the Kareniaceae, has not been commercially available for study until recently. To this end, our objective was to characterize the sterols of A. gracile to determine whether they match the 4α‐methyl‐substituted, Δ8(14)‐nuclear‐unsaturated sterols, such as (24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (gymnodinosterol; C29:2) and 27‐nor‐(24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (brevesterol; C28:2), of most species within the canonical genera Karenia, Karlodinium and Takayama, or instead the 4‐desmethyl sterols, such as 27‐nor‐(24R)‐23‐methyl‐ergosta‐8(14),22‐dien‐3β‐ol (C28:2), of its chemotaxonomically atypical, yet closest phylogenetic relative, Karenia papilionacea. We have observed A. gracile to produce two 4α‐methyl‐substituted sterols with the suggested structures of 4α‐methyl‐5α‐ergosta‐8(14)‐en‐3β‐ol (C29:1) and 4α‐methyl‐5α‐ergosta‐8(14),24(28)‐dien‐3β‐ol (amphisterol, C29:2), as major sterols amongst five other minor sterols, which included the common dinoflagellate sterol cholest‐5‐en‐3β‐ol (cholesterol, C27:1), but did not include gymnodinosterol or brevesterol (as in most canonical Kareniaceae) or 27‐nor‐(24R)‐23‐methyl‐ergosta‐8(14),22‐dien‐3β‐ol (as found in K. papilionacea). Detection of amphisterol and 4α‐methyl‐5α‐ergosta‐8(14)‐en‐3β‐ol is notable because they are sterols associated with some species of Amphidinium, such as Amphidinium carterae, and, to our knowledge, have not been observed in a member of the Kareniaceae before. Discovery of these major sterols in A. gracile expands our knowledge of the range of sterols produced by the Kareniaceae and indicates a shared chemotaxonomy with some species of Amphidinium.

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

SUMMARYThe 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.

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Field survey of the phase and sex ratios of the brown alga <scp><i>Dictyota dichotoma</i></scp> (Dictyotales, Phaeophyceae) using sex‐specific molecular markers

SUMMARYDominance of one phase (i.e. diploid or haploid) has been reported in various isomorphic macroalgae; however, studies of the relative frequencies of the two phases are limited to a few taxa owing to the difficulty in identifying phases of sterile thalli. In Dictyota dichotoma, we have previously documented the dominance of fertile sporophytes. In the present study, the phase and sex ratios of sterile thalli of D. dichotoma were determined using newly developed sex‐specific markers. Differentially expressed genes were detected by transcriptome analyses of female and male gametophytic strains and a sporophytic strain aiming to identify candidate sex‐specific genes. These candidates were aligned with genome sequences on the sex chromosomes of Ectocarpus siliculosus and were identified as putative sex‐specific genes of D. dichotoma. Two primer sets were designed to discriminate phase and sex by the presence or absence of PCR products, and these markers were applied to examine the phase and sex ratios of D. dichotoma in various habitats and seasons. The proportion of sporophytes against 33–50 thalli examined in each of the five habitats at Ebisu Island, Shizuoka Prefecture was 88–100%, and the proportion of fertile sporophytes against total sporophytes (fertility proportion) varied among the habitats (0–54%). The fertile sporophytes were remarkably larger than the sterile sporophytes. At Tateyama, Chiba Prefecture, the dominance of sporophytes occurred in all seasons. The fertility proportion of sporophyte was relatively high from February (62.4%) to May (91.9%) and lower than 23% in other seasons. When artificial substrates were anchored in the middle of the D. dichotoma population, newly recruited thalli were all sporophytes, suggesting infrequent reproduction via tetraspores in nature. Based on these findings, we discuss potential fitness differences between the phases of D. dichotoma.

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Differential cell size reduction of two sympatric <i>Epithemia</i> (Bacillariophyta) taxa in Nakaikemi Wetland, Japan

SUMMARYWe observed contrasting temporal changes in cell size between two Epithemia taxa. The life cycle of diatoms is closely linked to their cell size, with a gradual decrease during the vegetative stage and a recovery through sexual reproduction. Sexual reproduction is triggered when cells become smaller than a species‐specific size threshold and receive species‐specific environmental cues. Few studies have documented the life cycles and frequency of sexual reproduction in diatoms, combining field observations and laboratory culture. We collected two Epithemia taxa, Epithemia gibba var. ventricosa and Epithemia sp. from a pond in Nakaikemi Wetland, and measured their valve lengths monthly for almost 3 years. Additionally, we established cultures of both taxa to examine the rates of cell size reduction, which affect the duration of the vegetative stage. In the field, E. gibba var. ventricosa exhibited a wide range of cell sizes and signs of size recovery. Furthermore, the cultures of this taxon showed a clear decrease in cell size through division, suggesting that sexual reproduction is required for size recovery. On the other hand, Epithemia sp. showed no obvious change in cell size both in the field and in the laboratory. This suggests that either sexual reproduction is not necessary for this taxon to maintain its population or their life cycle extends over several years to several decades. Thus, our findings reveal congeneric taxa sharing a common habitat and substratum, yet exhibiting distinct sexual and asexual strategies. Furthermore, we question the validity of taxonomic classifications previously assigned to these diatoms based on the range of morphological characteristics observed in the present study.

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