A SCAR MOLECULAR MARKER SPECIFICALLY RELATED TO THE FEMALE GAMETOPHYTES OF SACCHARINA (LAMINARIA) JAPONICA (PHAEOPHYTA)1

PCR amplification was employed to identify female or male gametophyte associated markers in Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders (=Laminaria japonica Aresch.). One pair of the primers, P5, was screened from five pairs designed based on a specific sequence (GenBank accession no. AB069714 ) of Marchantia polymorpha Y chromosome, resulting in a differential band ～500 bp in size between female and male gametophytes of Rongfu strain of S. japonica. According to the SCAR (sequence‐characterized amplified regions) strategies, one pair of primers, P51, was designed on the basis of the sequence of this band that was only present in female gametophytes. A SCAR marker, designated FRML‐494 (494‐bp Female‐Related Marker of S. japonica, GenBank accession no. EU931619 ), was developed successfully by PCR amplification using the designed P51 primer pair. The SCAR marker was verified to be present only in female gametophytes of another variety 901 of this kelp that was a hybrid between S. japonica as paternal and S. longissima (Miyabe) C. E. Lane, C. Mayes, Druehl et G. W. Saunders (=Laminaria longissima Miyabe) as maternal, suggesting that the FRML‐494 marker was specifically related to female gametophytes of the genus. This marker is the first molecular tool reported for sex identification in kelps. This study was beneficial for identifying gametophyte gender during vegetative growth and for judging whether the monogenetic sporophytes came from exclusive male or female gametophytes, as well as for further research on sex determination at the molecular level in kelps.     

CONSTRUCTION AND CHARACTERIZATION OF A TENTATIVE AMPLIFIED FRAGMENT LENGTH POLYMORPHISM–SIMPLE SEQUENCE REPEAT LINKAGE MAP OF LAMINARIA (LAMINARIALES,PHAEOPHYTA)1

A tentative amplified fragment length polymorphism–simple sequence repeat (AFLP–SSR) linkage map of Laminaria was constructed using a haploid population of 40 gametophyte clones isolated from an individual of Dongfang No. 2, the first commercially cultured hybrid of a female gametophyte clone of L. japonica Aresch. [=Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders] and a male one of L. longissima Miyabe [=Saccharina longissima (Miyabe) C. E. Lane, C. Mayes et G. W. Saunders]. To the map, 263 markers (255 AFLP, seven SSR, and the gametophyte sex) were assigned. The map consisted of 25 linkage groups (LGs) with ≥ four markers, five triplets, and 15 doublets, which is 1,629.0 centiMorgans (cM) in length, covering 66% of Laminaria genome. The maximum space between loci is 24.63 cM. A putative sex‐determining region was identified in LG₂, which was characterized by a dense marker distribution around the gametophyte sex locus. The linkage map itself and the methodology associated with its construction will facilitate the genetic study and further trials of the linkage map construction of Laminaria.     

Nuclear DNA level and life cycle of kelps: Evidence for sex‐specific polyteny in Macrocystis (Laminariales,Phaeophyceae)

Giant kelp, Macrocystis pyrifera (Linnaeus) C. Agardh, is the subject of intense breeding studies for marine biomass production and conservation of natural resources. In this context, six gametophyte pairs and a sporophyte offspring of Macrocystis from South America were analyzed by flow cytometry. Minimum relative DNA content per cell (1C) was found in five males. Unexpectedly, nuclei of all female gametophytes contained approximately double the DNA content (2C) of males; the male gametophyte from one locality also contained 2C, likely a spontaneous natural diploid variant. The results illustrate a sex‐specific difference in nuclear DNA content among Macrocystis gametophytes, with the chromosomes of the females in a polytenic condition. This correlates with significantly larger cell sizes in female gametophytes compared to males and resource allocation in oogamous reproduction. The results provide key information for the interpretation of DNA measurements in kelp life cycle stages and prompt further research on the regulation of the cell cycle, metabolic activity, sex determination, and sporophyte development.     

Temperature requirements for growth and maturation of the warm temperate kelp Eckloniopsis radicosa (Laminariales,Phaeophyta)

The annual kelp Eckloniopsis radicosa is distributed along Japanese coasts and occurs within the area with a February isotherm ranging 15–18°C and August isotherm ranging 25–28°C. In this study, the effects of temperature on the gametophyte growth and maturation, and the young sporophyte growth of E. radicosa were examined and the results are discussed in relation to the distribution of other warm‐adapted kelp species and the potential effects of climate change. The optimal temperature ranges for growth of male and female gametophytes were 23–27°C and 20–26°C, respectively. The upper survival temperature for gametophyte growth was 31°C for males and 30°C for females, respectively. The optimal temperature range for maturation of female gametophytes was ≤23°C. The optimal temperature range for growth of young sporophytes was 14–22°C. It was clarified that E. radicosa has the most warm‐adapted characteristics for growth and maturation of gametophytes among members of the Laminariales studied so far. The natural seawater temperature ranges during the growth and maturation seasons for gametophytes of E. radicosa, as well as the growth season for young sporophytes near to the northern and southern distribution limits (Izu‐Oshima: 14.9–24.5°C, Ichiki‐kushikino: 17.1–29.6°C), agreed with the experimentally determined temperature requirements. The warm‐adapted gametophyte stage and annual lifecycle are major factors enabling survival of E. radicosa in warm waters near tropical regions along the Japanese coast.     

Identification of a genomic region linked with sex determination of Undaria pinnatifida (Alariaceae) through genomic resequencing and genetic linkage analyses of a segregating gametophyte family

Different from the traditional knowledge about kelp, three sexual phenotypes (female, male, and monoecious) exist in the haploid gametophytes of Undaria pinnatifida. However, the sex-determining mechanisms remain unknown. Genetic linkage mapping is an efficient tool to identify sex-linked regions. In the present study, we resequenced a segregating gametophyte family based on the male genome of U. pinnatifida. A high-density genetic linkage map was constructed using 9887 SNPs, with an average distance of 0.41 cM between adjacent SNPs. On the basis of this genetic map and using the composite interval mapping method, we identified 62 SNPs significantly linked with the sexual phenotype. They were located at a position of 67.67 cM on the linkage group 23, corresponding to a physical range of 14.67 Mbp on the HiC_Scaffold_23 of the genome. Reanalysis of the previous specific length amplified fragment sequencing data according to the reference genome led to the identification of a sex-linked genomic region that encompassed the above-mentioned 14.67 Mbp region. Hence, this overlapped genomic range was likely the sex-determining region. Within this region, 129 genes were retrieved and 39 of them were annotated with explicit function, including the potential male sex-determining gene-encoding high mobility group (HMG) domain protein. Relative expression analysis of the HMG gene showed that its expression was higher in male gametophytes during the vegetative phase and monoecious gametophytes during both the vegetative and gametogenesis phases, but significantly lower in male gametophytes during the gametogenesis phase. These results provide a foundation for deciphering the sex-determining mechanism of U. pinnatifida.     

UV‐radiation and elevated temperatures induce formation of reactive oxygen species in gametophytes of cold‐temperate/Arctic kelps (Laminariales,Phaeophyceae)

Enhanced UV‐radiation (UVR) through stratospheric ozone depletion and global warming are crucial stressors to marine macroalgae. Damages may arise through formation of reactive oxygen species (ROS) in gametophytes of ecologically important kelps, brown algae of the order Laminariales, Such stress‐induced damages may have a negative impact on their fitness and further impact their following life stages. In our study, gametophytes of three kelp species Alaria esculenta (L.) Grev., Laminaria digitata (Huds.) Lamour., Saccharina latissima (L.) Lane, Mayes, Druehl, Saunders from the Arctic, and of L. hyperborea (Gunnerus) Foslie from the North Sea were exposed to photosynthetically active radiation, UV‐A, and UV‐B radiation and four temperatures (2–18°C). ROS are formed predominantly in the peripheral cytoplasm and in chloroplasts especially after exposure to UVR. Superoxide (O₂*^‐) is additionally formed in small, globular cytoplasmic structures, possibly mitochondria. In the surrounding medium O₂*^‐‐concentration increased markedly at elevated temperatures and under UV stress in some cases. Ultrastructural damage was negligible pointing to a high stress tolerance of this developmental stage. Our data indicate that stress tolerant gametophytes of three Arctic kelp species should sustain their crucial function as seed bank for kelp populations even under prospective rising environmental perturbations.     

Sex‐linked microsatellite marker detected in the female gametophytes of Undaria pinnatifida (Phaeophyta)

In our microsatellite analysis of three male and three female gametophytes of Undaria pinnatifida (Harv.) Suringar, a microsatellite marker (part of the locus Up‐AC‐2A8, GenBank accession no. AY738602.1) was only polymerase chain reaction‐amplified in three female gametophytes. This putative female‐specific marker was further tested by the use of 32 male and 21 female gametophytes maintained in the Marine Biological Culture Collection Centre, China. In addition, three sporophytes were included for confirmation. Results showed that the marker was present in all of the female gametophytes and sporophyte cultures, but absent in all of the male gametophytes. To our knowledge, this is the first sex‐related marker ever reported in U. pinnatifida. The discovery of this marker will accelerate gender identification and shed light on our understanding of the mechanisms of sex determination at a molecular level in this commercially important seaweed.     

Ocean acidification and seaweed reproduction: increased CO2 ameliorates the negative effect of lowered pH on meiospore germination in the giant kelp Macrocystis pyrifera (Laminariales,Phaeophyceae)

The worldwide effects of ocean acidification (OA) on marine species are a growing concern. In temperate coastal seas, seaweeds are dominant primary producers that create complex habitats and supply energy to higher trophic levels. Studies on OA and macroalgae have focused on calcifying species and adult stages, but critically, they have overlooked the microscopic stages of the reproductive life cycle, which, for other anthropogenic stressors, e.g., UV‐B radiation, are the most susceptible life‐history phase. Also, environmental cues and stressors can cause changes in the sex ratio, which has implications for the mating system and recruitment success. Here, we report the effects of pH (7.59–8.50) on meiospore germination and sex determination for the giant kelp, Macrocystis pyrifera (Laminariales), in the presence and absence of additional dissolved inorganic carbon (DIC). Lowered pH (7.59–7.60, using HCl‐only) caused a significant reduction in germination, whereas added DIC had the opposite effect, indicating that increased CO₂ at lower pH ameliorates physiological stress. This finding also highlights the importance of appropriate manipulation of seawater carbonate chemistry when testing the effects of OA on photosynthetic organisms. The proportion of male to female gametophytes did not vary significantly between treatments, suggesting that pH was not a primary environmental modulator of sex. Relative to the baseline (pH 8.19), gametophytes were 32% larger under moderate OA (pH 7.86) and 10% larger under extreme OA (pH 7.61). We suggest that metabolically active cells can compensate for the acidification of seawater. This homeostatic function minimizes the negative effects of lower pH (high H⁺ ions) on cellular activity. The 6–9% reduction in germination success under extreme OA suggests that meiospores of M. pyrifera may be resistant to future OA.     

Gametogenesis and chromosome number in Postelsia palmaeformis (Laminariales, Phaeophyceae)

Gametophytes of the ‘sea palm’, the kelp Postelsia palmaeformis Ruprecht, produced gametes whether or not chelated iron was supplied in the culture medium, in contrast to the inhibition of gametogenesis seen with the absence of iron in many other kelps. As gametogenesis proceeded, every cell of the gametophytes was converted into a gamete so that the gametophytes did not continue to grow vegetatively. The portion of the life history from spore release through germination, gametophyte growth, gametogenesis, fertilization and growth of the young sporophyte was completed in 9–10 days under laboratory conditions. Chromosome counts showed that sporophytes had a diploid number of 26–34 chromosomes while sporangia and gametophytes had a haploid number of 14–17 chromosomes, indicating a typical haplodiplophasic life history as seen in other Laminariales.     

Occurrence of the gametophyte of Agarum clathratum (Laminariales, Phaeophyceae) as an endophyte in Orculifilum denticulatum (Gigartinales, Rhodophyceae)

An endophytic filamentous brown alga, growing in the red alga Orculifilum denticulatum Lindstrom, was collected from the north‐east Pacific, near Juneau, Alaska. Within the host tissue, its branched filaments formed a network in the space between the filaments of the host tissues embedded in the host intercellular substances. Cells of the filaments contained many discoid chloroplasts without pyrenoids. Neither microscopic morphological observation nor culturing was sufficient to reveal the specific identity or even the familial affinity of the alga; in contrast, molecular phylogenetic analysis of its rbcL gene and rDNA ITS sequences showed that it was the gametophyte of Agarum clathratum Dumortier (Laminariales). There are few reports of laminarialean gametophytes in nature; this is the first report actually identifying the species of laminarialean gametophyte in a red alga.     

Telomeric localization of the Arabidopsis‐type heptamer repeat, (TTTAGGG)n,at the chromosome ends in Saccharina japonica (Phaeophyta)

Telomeres generally consist of short repeats of minisatellite DNA sequences and are useful in chromosome identification and karyotype analysis. To date, telomeres have not been characterized in the economically important brown seaweed Saccharina japonica, thus its full cytogenetic research and genetic breeding potential has not been realized. Herein, the tentative sequence of telomeres in S. japonica was identified by PCR amplification with primers designed based on the Arabidopsis‐type telomere sequence (TTTAGGG)_n, which was chosen out of three possible telomeric repeat DNA sequences typically present in plants and algae. After PCR optimization and cloning, sequence analysis of the amplified products from S. japonica genomic DNA showed that they were composed of repeat units, (TTTAGGG)_n, in which the repeat number ranged from 15 to 63 (n = 46). This type of repeat sequence was verified by a Southern blot assay with the Arabidopsis‐type telomere sequence as a probe. The digestion of S. japonica genomic DNA with the exonuclease Bal31 illustrated that the target sequence corresponding to the Arabidopsis‐type telomere sequence was susceptible to Bal31 digestion, suggesting that the repeat sequence was likely located at the outermost ends of the kelp chromosomes. Fluorescence in situ hybridizations with the aforementioned probe provided the initial cytogenetic evidence that the hybridization signals were principally localized at both ends of S. japonica chromosomes. This study indicates that the telomeric repeat of the kelp chromosomes is (TTTAGGG)_n which differs from the previously reported (TTAGGG)_n sequence in Ectocarpus siliculosus through genome sequencing, thereby suggesting distinct telomeres in brown seaweeds.     

Plasticity of female reproductive resource allocation depends on the presence or absence of prior environmental sex determination in Ceratopteris richardii

Resource allocation plasticity enables individuals to alter patterns of nutrient use between reproductive and vegetative output to better fit their current environment. In sexually labile plant species, abiotic environmental factors can influence expression of dimorphic gender, resulting in environmental sex determination (ESD), which potentially reduces the need for plasticity of resource allocation by preemptively matching an individual’s future nutrient demands to resource availability in its location. Ceratopteris richardii gametophytes exhibit gender‐dependent differences in relative carbon and nitrogen content, and ESD in certain nutrient environments. This study examined whether prior ESD in C. richardii gametophyte populations reduced subsequent plasticity of reproductive allocation compared to instances where no ESD occurred, by quantifying phenotypic responses to reduced P, N, or CO₂ availabilities. All three nutrient‐limited environments resulted in decreased size of egg‐bearing (meristic) gametophytes compared to nonlimited environments, but gametophytes failed to respond to N and CO₂ limitation at the time of sex determination, resulting in no ESD. N limitation resulted in a predictable allometric re‐allocation of resources based on small gametophyte size, whereas CO₂ limitation caused a change in reproductive output consistent with true plasticity. Withholding exogenous P caused ESD and had no effect on relative reproductive output of resultant meristic gametophytes because the size decrease was minor. Under P limitation, ESD matched the resource demands of gender phenotypes to their environment before the onset of developmental dimorphism, reducing the need for large allocation adjustments after sex determination.     

Family‐level variation in early life‐cycle traits of kelp

Temperate kelp forests (Laminarians) are threatened by temperature stress due to ocean warming and photoinhibition due to increased light associated with canopy loss. However, the potential for evolutionary adaptation in kelp to rapid climate change is not well known. This study examined family‐level variation in physiological and photosynthetic traits in the early life‐cycle stages of the ecologically important Australasian kelp Ecklonia radiata and the response of E. radiata families to different temperature and light environments using a family × environment design. There was strong family‐level variation in traits relating to morphology (surface area measures, branch length, branch count) and photosynthetic performance (F_v/F_m) in both haploid (gametophyte) and diploid (sporophyte) stages of the life‐cycle. Additionally, the presence of family × environment interactions showed that offspring from different families respond differently to temperature and light in the branch length of male gametophytes and oogonia surface area of female gametophytes. Negative responses to high temperatures were stronger for females vs. males. Our findings suggest E. radiata may be able to respond adaptively to climate change but studies partitioning the narrow vs. broad sense components of heritable variation are needed to establish the evolutionary potential of E. radiata to adapt under climate change.     

How light and biomass density influence the reproduction of delayed Saccharina latissima gametophytes (Phaeophyceae)

Kelp life-cycle transitions are complex and susceptible to various (a)biotic controls. Understanding the microscopic part of the kelp's lifecycle is of key importance, as gametophytes form a critical phase influencing, among others, the distributional limits of the species. Many environmental controls have been identified that affect kelp gametogenesis, whose interactive effects can be subtle and counterintuitive. Here we performed a fully factorial experiment on the (interactive) influences of light intensity, light quality, and the Initial Gametophyte Density (IGD) on Saccharina latissima reproduction and vegetative growth of delayed gametophytes. A total of 144 cultures were followed over a period of 21 d. The IGD was a key determinant for reproductive success, with increased IGDs (≥0.04 mg DW · mL⁻¹) practically halting reproduction. Interestingly, the effects of IGDs were not affected by nutrient availability, suggesting a resource-independent effect of density on reproduction. The Photosynthetically Usable Radiation (PUR), overarching the quantitative contribution of both light intensity and light quality, correlated with both reproduction and vegetative growth. The PUR furthermore specifies that the contribution of light quality, as a lifecycle control, is a matter of absorbed photon flux instead of color signaling. We hypothesize that (i) the number of photons absorbed, independent of their specific wavelength, and (ii) IGD interactions, independent of nutrient availability, are major determinants of reproduction in S. latissima gametophytes. These insights help understand kelp gametophyte development and dispersal under natural conditions, while also aiding the control of in vitro gametophyte cultures.