Inheritance of mitochondrial and chloroplast genomes in the isogamous brown alga Scytosiphon lomentaria (Phaeophyceae)

Patterns of inheritance of chloroplasts and mitochondria were examined by fluorescence microscopy and haplotype genome markers in the isogamous brown alga Scytosiphon lomentaria (Lyngbye) Link. Germination of the zygote in this species was unilateral, the growing thallus developed entirely from the germ tube, and the original zygote cell did not develop except for the formation of a hair. Inheritance of chloroplasts was biparental, and partitioning of the two parental chloroplasts into the first sporophytic cells was accidental: either the maternal or the paternal chloroplast was migrated from the zygote into the germ tube cell, whereas the other chloroplast remained in the original cell. In contrast, the mitochondrial genome in all cells of the sporophyte came only from the female gamete (maternal inheritance). These inheritance patterns are similar to those of the isogamous brown alga Ectocarpus siliculosus (Dillwyn) Lyngbye. Maternal inheritance of mitochondria might be universal in brown algae.     

Taxonomic revision of the genus Cutleria proposing a new genus Mutimo to accommodate M. cylindricus (Cutleriaceae,Phaeophyceae)

Molecular phylogenetic analyses of representative Cutleria species using mitochondrial cox3, chloroplast psaA, psbA and rbcL gene sequences showed that C. cylindrica Okamura was not included in the clade composed of other Cutleria species including the generitype C. multifida (Turner) Greville and the related taxon Zanardinia typus (Nardo) P.C. Silva. Instead, C. cylindrica was sister to the clade composed of the two genera excluding C. cylindrica. Cutleria spp. have heteromophic life histories and their gametophytes are rather diverse in gross morphology, from compressed or cylindrical‐branched to fan‐shaped, whereas the sporophytes are rather similar. In contrast, the monotypic species Z. typus has an isomorphic life history and resembles fan‐shaped Cutleria in morphology. Morphological comparisons of these taxa revealed that C. cylindrica is morphologically distinct from other Cutleria spp. and Z. typus in having cylindrical gametophytes with multiseriate trichothallic filaments instead of uniseriate filaments (hairs) characteristic of Cutleriales (or Cutleriaceae, Tilopteridales), and in lacking rhizoidal filaments in the crustose sporophytes. Therefore, based on the molecular and morphological data, the establishment of a new genus Mutimo to accommodate C. cylindrica, and the new combination of M. cylindricus, is proposed.     

Distinct regulation of two flagella by calcium during chemotaxis of male gametes in the brown alga Mutimo cylindricus (Cutleriaceae,Tilopteridales)

Brown algal male gametes show chemotaxis to the sex pheromone that is released from female gametes. The chemotactic behavior of the male gametes is controlled by the changes in the beating of two flagella known as the anterior and posterior flagellum. Our previous study using Mutimo cylindricus showed that the sex pheromone induced an increment in both the deflection angle of the anterior flagellum and sustained unilateral bend of the posterior flagellum, but the mechanisms regulating these two flagellar waveforms were not fully revealed. In this study, we analyzed the changes in swimming path and flagellar waveforms with a high-speed recording system under different calcium conditions. The extracellular Ca²⁺ concentration at 10⁻³ M caused an increment in the deflection angle of the anterior flagellum only when ionomycin was absent. No sustained unilateral bend of the posterior flagellum was induced either in the absence or presence of ionomycin in extracellular Ca²⁺ concentrations below 10⁻² M. Real-time Ca²⁺ imaging revealed that there is a spot near the basal part of anterior flagellum showing higher Ca²⁺ than in the other parts of the cell. The intensity of the spot slightly decreased when male gametes were treated with the sex pheromone. These results suggest that Ca²⁺-dependent changes in the anterior and posterior flagellum are regulated by distinct mechanisms and that the increase in the anterior flagellar deflection angle and sustained unilateral bend of the posterior flagellum may not be primarily induced by the Ca²⁺ concentration.     

Tracing the introduction history of the brown seaweed Dictyota cyanoloma (Phaeophyceae,Dictyotales) in Europe

Dictyota cyanoloma has recently been described from the Mediterranean Sea and Macaronesia but doubt had arisen as to whether this species was truly native in Europe. The species is mainly found on non-natural substrata (harbour walls, marinas, boat hulls, etc.), strongly suggesting that it is an introduction. Molecular sequence information from historical herbarium samples proves the presence of D. cyanoloma in the Adriatic Sea as early as 1935. Since approximately the year 2000, however, the number of records as well as the geographic range of the species has expanded significantly. The present-day distribution of D. cyanoloma occupies most of the Mediterranean Sea, Macaronesia, NW Africa and southern Portugal, but recent records from Galicia and SW England (Falmouth, Cornwall) indicate that the species is rapidly expanding northward. Collections from Australia demonstrated that the species is also present from Perth in Western Australia, over much of the southern Australian coastline up to Minnie Water in New South Wales. Phylogenetic analyses resolve D. cyanoloma in a sister clade to a previously unreported Australian Dictyota species. Analysis of genetic diversity of the mitochondrial markers (nad6–nad11 and atp9–orf11) reveals that even though Australian populations contain a much higher haplotype richness, European populations are also fairly diverse. Furthermore, only two out of 25 haplotypes are shared between both regions. These somewhat counterintuitive results could be indicative of a more complicated introduction history.     

Historical biogeography of the widespread macroalga Sargassum (Fucales,Phaeophyceae)

Sargassum is a cosmopolitan brown algal genus spanning the three ocean basins of the Atlantic, Pacific and Indian Oceans, inhabiting temperate, subtropical and tropical habitats. Sargassum has been postulated to have originated in the Oligocene epoch approximately 30 mya according to a broad phylogenetic analysis of brown macroalgae, but its diversification to become one of the most widespread and speciose macroalgal genera remains unclear. Here, we present a Bayesian molecular clock study, which analyzed data from the order Fucales of the brown algal crown radiation (BACR) group to reconstruct a time-calibrated phylogeny of the Sargassum clade. Our phylogeny included a total of 120 taxa with 99 Sargassum species sampled for three molecular markers – ITS-2, cox3 and rbcLS – calibrated with an unambiguous Sargassaceae fossil from between the lower and middle Miocene. The analysis revealed a much later origin of Sargassum than expected at about 6.7 mya, with the genus diversifying since approximately 4.3 mya. Current geographic distributions of Sargassum species were then analyzed in conjunction with the time-calibrated phylogeny using the dispersal-extinction-cladogenesis (DEC) model to estimate ancestral ranges of clades in the genus. Results strongly support origination of Sargassum in the Central Indo-Pacific (CIP) region with subsequent independent dispersal events into other marine realms. The longer history of diversification in the ancestral CIP range could explain the much greater diversity there relative to other marine areas today. Analyses of these dynamic processes, when fine-tuned to a higher spatial resolution, enable the identification of evolutionary hotspots and provide insights into long-term dispersal patterns.     

Selective disappearance of maternal centrioles after fertilization in the anisogamous brown alga Cutleria cylindrica (Cutleriales, Phaeophyceae): Paternal inheritance of centrioles is universal in the brown algae

The behavior of centrioles in zygotes and female gametes developing parthenogenetically in the anisogamous brown alga Cutieria cyiindrica Okamura was studied using electron and immunofluorescence microscopy. Two pairs of centrioles, detected using anti-centrin antibody, were observed in the vicinity of the male and female nuclei, respectively, just after plasmogamy. The fluorescence intensity of one of the two centrin foci became weak 6 h after plasmogamy and finally disappeared. It was impossible to determine whether the male- or female-derived centrioles disappeared in zygotes, because there was nothing to detect morphological differences between the two centrioles. However, a prominent anti-centrin staining focus was located at the condensed male nucleus in zygotes in which karyogamy had not occurred yet. As a result, it was considered that the maternally inherited centrioles had selectively disappeared during development in C. cylindrica. The paternal inheritance of centrioles in zygotes was also confirmed by electron microscopy. Considering previous observations from oogamous and isogamous species of brown algae, we concluded that the paternal inheriance of centrioles could be universal in the brown algae.     

Electron tomographic analysis of cytokinesis in the brown alga Silvetia babingtonii (Fucales,Phaeophyceae)

In brown algae, membrane resources for the new cell partition during cytokinesis are mainly flat cisternae (FCs) and Golgi-derived vesicles. We used electron tomography coupled with rapid freezing/freeze substitution of zygotes to clarify the structure of transient membrane compartments during cytokinesis in Silvetia zygotes. After mitosis, an amorphous membranous structure, considered to be an FC intermediate was observed near endoplasmic reticulum clusters, lying between two daughter nuclei. FCs were arrayed at the cytokinetic plane, and a tubular membranous network was formed around them. This network might be formed by the consecutive fusion of spherical vesicles that are linked to the edges of FCs to form a membranous network (MN). At the initial stage of the formation of a membranous sac (MS) from the MN, the MS had flat and swollen parts, with the latter showing membranous tunnels. Coated pits were detected with high frequency at the swollen parts of the MS. This observation indicated that membranous tunnels disappeared by recycling of excess membrane via endocytosis, and the swollen part became flat. The MN appeared at the edges of the growing MS. MN and the MN-MS complex were observed along the cytokinetic plane in several spaces. The MS expanded by the incorporation of MN or other MS in its neighborhood. With the maturation of the new cell partition membrane, the thickness of the MS became constant and the membrane cavity disappeared. The changes in the surface area and volume of the transient membrane compartment during cytokinesis were analyzed from the tomographic data.     

Lobophora (Dictyotales) Species Richness,Ecology and Biogeography Across the North-Eastern Atlantic Archipelagos and Description of Two New Species1

The brown alga Lobophora (Dictyotales, Phaeophyceae) is an important macroalga in the North-eastern Atlantic archipelagos (i.e., Macaronesia). Notably in the Canaries it can dominate benthic assemblages. While the genus has been the subject of several ecological studies in the Canaries, no study has yet been conducted to assess species-level diversity of Lobophora in Macaronesia. We reassessed the diversity of Lobophora in Macaronesia, reporting the presence of seven species (L. caboverdeana sp. nov., L. canariensis, L. dagamae sp. nov., L. delicata, L. dispersa, L. littlerorum, and L. schneideri). Lobophora spp. from Macaronesia are morphologically and ecologically distinguishable. In the Canaries, L. schneideri dominates the photophilic assemblages from the intertidal to 20-30 m depth. Lobophora dagamae sp. nov. grows in less illuminated shallow habitats, and replaces L. schneideri from 30 to ~80 m. Lobophora canariensis also has a wide vertical distribution, from the intertidal to deep waters, while L. delicata, L. dispersa and L. littlerorum grow in shallow waters. The dominance of species with an upright habit versus prostrate or crustose species may be mediated by the pressure of herbivores. Four species have an amphi-Atlantic distribution: L. littlerorum, L. canariensis, L. delicata, and L. schneideri. Lobophora schneideri and L. delicata are furthermore distributed in the Mediterranean Sea. By sampling a pivotal region in the Atlantic, this study significantly improves our knowledge of Lobophora biogeography in the Atlantic Ocean. Macaronesia constitutes a species–poor region for Lobophora where no diversification events occurred, and a region of overlap between the Greater Caribbean and the Indo-Pacific.     

Delimitation of cryptic species of the Scytosiphon lomentaria complex (Scytosiphonaceae,Phaeophyceae) in Japan,based on mitochondrial and nuclear molecular markers

Scytosiphon lomentaria (Scytosiphonaceae, Ectocarpales) is believed to include some cryptic species, particularly in the Pacific. We attempted to delimit these species in Japan using mitochondrial cox1 and cox3 and nuclear ITS2 and the second intron of the centrin gene (cetn‐int2). Fifty‐three cox1+cox3 mitotypes, 26 ITS2 ribotypes and 45 cetn‐int2 haplotypes were found in 107 samples collected from 33 localities in Japan. Based on phylogenetic analyses, similar sequence types were grouped into ten mitogroups, eight ribogroups and six cetn‐int2 haplogroups (sequence‐type groups). From the molecular trees and combinations of the mito‐, ribo‐ and haplogroups, three cryptic species were apparent (Groups I–III). Group I, widely distributed on Pacific coasts, was highly supported by all molecular trees, whereas Groups II (North Pacific) and III (Northwestern Pacific and Australasia) were more closely related to each other. However, sequence‐type‐group combinations that would be characteristic of hybrids between Groups II and III were not detected, suggesting no gene flow between the two Groups. Further investigations of an additional 127 sympatrically growing plants supported the absence of gene flow between Groups II and III. Four samples did not belong to any of the Groups I–III and possibly represent additional species.     

Phylogeography of the freshwater red alga Sirodotia (Batrachospermales,Rhodophyta) in Brazil

Considering the lack of knowledge on genetic variation on members of the freshwater red algal of the order Batrachospermales in tropical regions, phylogeographic patterns in Sirodotia populations were investigated using two mitochondrial regions: the cox2‐3 spacer and partial cox1 gene (barcode). Individuals identified as Sirodotia delicatula were analyzed from 14 stream segments across its distribution in Brazil. Phylogenetic analyses based on the ribulose‐1,5‐bisphosphate carboxylase/oxygenase large sub‐unit gene showed three clades, one representing S. delicatula, from all locations in southeastern Brazil and other regions from Brazil. The remaining samples formed two clades, which were highly divergent and distantly positioned from those of S. delicatula: 2.5–2.7% and 3.4–3.7%. This level of variation would warrant the species split of these taxa from mid‐western Brazil. A total of eight cox2‐3 spacer and nine cox1 haplotypes were observed among the 122 individuals studied. One location had two cox2‐3 haplotypes and three locations had two cox1 haplotypes; all others had a single dominant haplotype each. The existence of high intraspecific genetic variation among individuals of distinct locations (several haplotypes), but little variation within a location seems to be a pattern for the Batrachospermales. Haplotype networks showed low variation among the haplotypes from southeastern Brazil (10 locations with divergence of 0.3–1.1% for cox2‐3, 0.1–0.3% for cox1) and high variation among the haplotypes from the mid‐west region (four locations, 4.0–9.3% for cox2‐3, 6.2–8.4% for cox1). Thus, the present data clearly suggest the existence of cryptic species in Sirodotia in Brazil.     

PHYLOGEOGRAPHY OF CUTLERIA CYLINDRICA (CUTLERIALES,PHAEOPHYCEAE) IN NORTHEASTERN ASIA,AND THE IDENTITY OF AN INTRODUCED POPULATION IN CALIFORNIA1

Cutleria cylindrica Okamura was described from Japan in 1902 and has been reported only from northwestern Asia until its relatively recent discovery in California, USA, and Baja California, Mexico. To clarify the genetic relationships within and among the disjunct populations, we carried out a molecular phylogenetic study, as well as the examination of sex ratio and the life‐history patterns, of populations in Japan, Korea, and California. Based on the DNA sequences of mitochondrial genes cox2, cox3, the open reading frame (ORF) region, and the spacer between cox3 and ORF, a total of 23 haplotypes were detected in the 85 individuals from 20 localities in Japan, Korea, and California. All localities in Japan and Korea included multiple haplotypes, but only a single haplotype was found in California. There was a positive relationship between distance and genetic divergence in Japan and Korea. The single haplotype found in California was the same as one occurring in Japan (Aomori Pref. and Fukuoka Pref.) and Korea (Daedaepo, Pusan). Both male and female gametophytes were distributed in most northeastern Asian populations. Only female gametophytes, developing parthenogenetically from female gametes, were found in California and Aomori Pref., Japan. On the basis of these results, we conclude that the disjunct population of C. cylindrica in California originated from a relatively recent introduction from Japan and shares its origin with the parthenogenetic population in the Tsugaru Strait.     

Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae)

Plasmodesmata are intercellular bridges that directly connect the cytoplasm of neighboring cells and play a crucial role in cell-to-cell communication and cell development in multicellular plants. Although brown algae (Phaeophyceae, Heterokontophyta) are phylogenetically distant to land plants, they nevertheless possess a complex multicellular organization that includes plasmodesmata. In this study, the ultrastructure and formation of plasmodesmata in the brown alga Dictyota dichotoma were studied using transmission electron microscopy and electron tomography with rapid freezing and freeze substitution. D. dichotoma possesses plasma membrane-lined, simple plasmodesmata without internal endoplasmic reticulum (desmotubule). This structure differs from those in land plants. Plasmodesmata were clustered in regions with thin cell walls and formed pit fields. Fine proteinaceous "internal bridges" were observed in the cavity. Ultrastructural observations of cytokinesis in D. dichotoma showed that plasmodesmata formation began at an early stage of cell division with the formation of tubular pre-plasmodesmata within membranous sacs of the cytokinetic diaphragm. Clusters of pre-plasmodesmata formed the future pit field. As cytokinesis proceeded, electron-dense material extended from the outer surface of the mid region of the pre-plasmodesmata and finally formed the nascent cell wall. From these results, we suggest that pre-plasmodesmata are associated with cell wall development during cytokinesis in D. dichotoma.     

Ultrastructural observations of mitochondrial morphology through the life cycle of the brown alga,Mutiomo cylindricus (Cutleriaceae,Tilopteridales)

Protoplasma - Mitochondrial morphology varies according to development and the physiological conditions of the cell. Here, we performed electron tomography using serial sections to analyze the...     

Phylogeographic history of the genus Tropheus,a lineage of rock-dwelling cichlid fishes endemic to Lake Tanganyika

Lake Tanganyika contains the oldest and most complex flock of cichlid fishes counting about 200 endemic species. It is comprised of 16 ecologically, morphologically and genetically highly distinct tribes. Many species are further subdivided into arrays of geographic morphs, each colonizing particular sections of the shore line. The genus Tropheus represents the most spectacular and best studied example for this phenomenon, counting more than 100 distinctly colored populations and sister species, some living in sympatry. Their present distribution and genetic structure was shaped by a series of lake level fluctuations which caused cycles of isolation and secondary admixis. The present study extends previous work on the phylogeography of Tropheus and aims at the fine-scale reconstruction of the origin and spread of lineages in the central and southern basin of the lake. The previously defined mtDNA lineages were evaluated on the basis of statistical parsimony networks. Haplotype networks were created for each lineage and related to their centers of diversity in terms of present distribution. A linearized tree analysis and a mismatch distribution analysis corroborate two of the three radiation waves suggested in earlier works, but the new data suggest a different primary colonization scenario for the southern basin.     

Phylogeography of Padina boryana (Dictyotales,Phaeophyceae) around the Thai-Malay Peninsula

The Indo-Pacific Ocean is a biodiversity hotspot for marine organisms. In this area, most of the research has focused on marine animals, such as reef fish, molluscs and other associated coral fauna, but very little has been done on macroalgae. The Thai-Malay Peninsula is an important north–south barrier in this area, which faces two different oceans – the Indian Ocean and the Pacific Ocean. This study aims to investigate genetic distribution patterns of Padina boryana Thivy around the Thai-Malay Peninsula, where it is common. Three DNA marker regions, the mitochondrion-encoded cytochrome c oxidase subunit 3 gene (cox3); the plastid rbcL, and the nuclear internal transcribed spacer 2 (ITS2) were used to evaluate genetic diversity and the relationships within and between populations. Samples were collected from both the Andaman Sea and Gulf of Thailand sides of the peninsula. Parsimony networks and maximum likelihood and Bayesian analyses showed clearly that there are two separated P. boryana lineages, one restricted to the Gulf of Thailand and the other to the Andaman Sea and other areas of the Indo-Pacific. The effect of different ocean currents along the Andaman Sea and Gulf of Thailand may have shaped these populations of P. boryana. This phylogeographic separation, based on persistent currents in the area, may affect other marine organisms along the Thai peninsula.     

New record of Sporochnus dotyi (Sporochnales,Phaeophyceae) from Kii Peninsula,Japan

The subtidal brown algal species Sporochnus dotyi Brostoff (Sporochnales, Phaeophyceae), which has been regarded as a Hawaiian endemic, is reported from Kushimoto, Kii Peninsula, Pacific coast of central Honshu, Japan, for the first time outside Hawai'i. The species grew on subtidal rocks ca. 5–20 m deep attached by a small conical holdfast. The erect thalli were 5–30 cm high, terete, robust and alternately branched in 1–2 orders. When mature, pedicellate receptacles developed on the branches, and formed elliptical sori 1 mm long with a pedicel 3–5 mm long. The apical parts of the thalli and the receptacles were terminated with a tuft of simple assimilatory filaments of up to 4 mm long and showed prominent green to yellow underwater iridescence. Reproductive filaments (paraphyses) were densely packed, simple, up to 200 μm long and bore 4–6 mostly unilateral unilocular zoidangia 20–22 μm long and 5–6 m in diameter. In the genetic analyses, the Sporochnus alga from Kushimoto had partial rbcL sequence identical to S. dotyi from Hawai'i. The cox3 phylogeny revealed that this alga formed a fully supported clade with S. dotyi. Therefore, we identified the alga from Kushimoto as S. dotyi. This finding of S. dotyi from Japan, together with the recent reports of the mesophotic macroalgae Ryuguphycus kuaweuweu (=Umbraulva kuaweuweu), Ulva iliohaha and Newhousia imbricata from various localities in the Pacific Ocean including Japan, suggest closer biogeographical connections of subtidal/mesophotic macroalgae in the Pacific than previously recognized.     

Destruction of maternal centrioles during fertilization of the brown alga, Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae)

In brown algal fertilization, a pair of centrioles is derived from the male gamete, irrespective of the sexual reproduction pattern, i.e., isogamy, anisogamy, or oogamy. In this study, the manner in which the maternal centriole structure is destroyed in early zygotes of the isogamous brown alga Scytosiphon lomentaria was examined by electron microscopy. At fertilization, the zygote had two pairs of centrioles (flagellar basal bodies) derived from motile male and female gametes, and there was no morphological difference between the two pairs. The flagellar basal plate and the axonemal microtubules were still connected with the distal end of centrioles. Ultrastructural observations showed that the integrity of maternal-derived centrioles began to degenerate even in the 1-h-old zygote. At that time, the cylinder of triplet microtubules of the maternal centrioles became shorter from the distal end, and a section passing through the centrioles indicated that a part of the nine triplets of microtubules changed into doublet or singlet microtubules by degeneration of B and/or C tubules. In 2-h-old zygote, there was no trace of maternal centrioles ultrastructurally, and only the paternal centrioles remained. Further, reduction of centrin accompanying destruction of the maternal centrioles was examined in immunofluorescence microscopy. Centrin localized at the paternal and the maternal centrioles had the same fluorescence intensity in the early zygotes. At 4-6 h after fertilization, two spots indicating centrin localization showed different fluorescence intensity. Later, the weaker spot disappeared completely. These results showed that there is a difference in time between the destruction of the centriolar cylinders and the reduction of centrin molecules around them.     

Phylogeny and morphology of the freshwater red alga Nemalionopsis shawii (Rhodophyta,Thoreales) from Nepal

Phylogenetic relationships and morphological characters are presented for a population of Nemalionopsis shawii Skuja collected from Nepal. Molecular data (sequences of rbcL and cox1) were generated and morphological characters were described in detail. The rbcL sequence analyses showed that specimens from Nepal are most similar to N. shawii from Indonesia and Japan and that these entities form a clade with high support (>95% bootstrap and 0.95 posterior probability). The cox1 barcode sequence, however, only had 90.9–91.9% identity with specimens of N. shawii from Hawaii. The rbcL sequence of the specimen from Nepal was positioned in a clade having sequence identity of 99.3–99.7% with three samples: N. shawii from Indonesia and two from Japan identified as N. tortuosa. The comparison of morphological characters of Nemalionopsis from Nepal allowed unequivocal identification with N. shawii. Identifications from previous studies using molecular data were mistaken since most reports of N. shawii are actually of N. tortuosa or vice‐versa. This confusion of names presumably occurred because most specimens previously sequenced were from culture collections or from ‘Chantransia’ stages. Small tufts of ‘Chantransia’ stage were observed growing epiphytically on gametophytes and on the basal system. Carpogonia and spermatangia were fully described in specimens from Nepal. Monosporangia were not observed, whereas carposporangia were unequivocally described for the first time in the genus. An unusual flat strap‐like basal system was observed, interpreted as an additional mode of maintenance in nature under unfavorable environmental conditions.