EVIDENCE FOR INHIBITORS OF SPORANGIUM FORMATION IN LAMINARIA DIGITATA (PHAEOPHYCEAE) DURING THE SEASON OF RAPID GROWTH

The pathway from the intercalary frond meristem to distal frond portions was blocked in experimental sporophytes of Laminaria digitata (Huds.) Lamour. in January by cutting 2 or 3 holes (15–25 mm diameter) 5 cm from the base of the frond. This procedure resulted in the formation of sori adjacent to the distal edge of the holes within 5–10 weeks. Frond portions that were cut at least 20 cm away from the stipe–frond transition readily formed sori within 5–7 weeks after isolation from the rest of the thallus. These findings suggest that the basal actively dividing and expanding part of the laminarian frond is the source of inhibitors of sporangium formation that move in a distal direction and keep the young frond free of sori during the season of rapid growth (i.e. during the first part of the year). The natural occurrence of sori during the season of slow growth (i.e. the second part of the year in the Northern Hemisphere) may be due to reduced synthesis and export of inhibitors of sporangium formation from the more or less resting basal meristem.     

AXENIC TISSUE CULTURES FROM THE SPOROPHYTES OF LAMINARIA DIGITATA AND LAMINARIA HYPERBOREA (PHAEOPHYTA)

Starting material for the tissue cultures was the meristematic basal zone of the blade. Pieces treated 30–60 sec in hypochlorite solution were rinsed and placed on agar plates made from the artificial seawater ASP6 F2 solidified with 6 g agar l^?1. After 6 weeks colorless callus-like tissue grew out from some pieces. Treatment with activated charcoal removed some inhibiting substances from the agar medium as numbers of callus developing pieces increased on such plates. A combination of 10^?5 M NAA and 5 · 10^?7 M kinetin gave a yellow-brown tissue. A differentiation in the tissue from L. hyperborea was observed as well as the formation of meiospores, which grew out into male and female plants. Thalli of sporophytes were observed but they never reached a length of more than one mm before they died or changed to an irregular pattern of growth.     

A SMALLER AND IMPAIRED XANTHOPHYLL CYCLE MAKES THE DEEP SEA MACROALGAE LAMINARIA ABYSSALIS (PHAEOPHYCEAE) HIGHLY SENSITIVE TO DAYLIGHT WHEN COMPARED WITH SHALLOW WATER LAMINARIA DIGITATA1

Pigment composition, fluorescence parameters, and oxygen evolution of the deep water Laminaria abyssalis Oliveira and of the shallow water L. digitata Lamoroux were determined in response to high irradiances. This was performed in the presence and absence of an inhibitor of violaxanthin de‐epoxidase (dithiothreitol) or an inhibitor of the chloroplast‐encoded protein synthesis (chloramphenicol). Photochemical quenching in L. digitata was almost 3‐fold that seen in L. abyssalis, whereas both nonphotochemical quenching and PSII photochemical yield were doubled. Laminaria digitata possessed a xanthophyll‐cycle pool nearly double that of L. abyssalis. After photoinhibitory treatment, L. digitata displayed substantial violaxanthin de‐epoxidation, whereas in L. abyssalis de‐epoxidation only took place in limited amounts. Both species were able to fully recover their epoxidation status after transfer back to dim light. Overnight incubation with dithiothreitol fully blocked de‐epoxidation in both species, and both displayed similar fluorescence properties. Chloramphenicol caused no change in their fluorescence parameters. With high light treatment, L. abyssalis was completely and irreversibly inhibited both in the presence and absence of inhibitors, whereas L. digitata showed 60% inhibition of its photosynthetic activity and full recovery in the absence of inhibitors. In the presence of dithiothreitol, L. digitata did not recover to the preillumination conditions and chloramphenicol delayed the recovery of the oxygen evolution activity. We suggest that the xanthophyll cycle is the main mechanism of photoprotection of these Laminaria species and that the higher susceptibility of L. abyssalis to photoinhibition may be due to its limited de‐epoxidation capacity and reduced xanthophyll‐cycle pool size.     

HAPLOID PARTHENOGENETIC SPOROPHYTES OF LAMINARIA JAPONICA (PHAEOPHYCEAE)1

Parthenogenetic sporophytes were obtained from three strains of Laminaria japonica Areschoug. These sporophytes grew to maturity in the sea, producine spores that all grew into female gametophytes. These female gametophytes gave rise to another generation of parthenogenetic sporophytes during the next year, so that by the year 1990 parthenogenetic sporophytes had been cultivated for 12, 9, and 7 generations, respectively, for the three strains. When female gametophytes from parthenogenetic sporophytes were combined with normal male gametophytes, normal sporophytes that reproduced and gave rise to both female and male gametophytes were obtained. The parthenogenetic sporophytes were shorter and narrower than the normal sporophytes of the same strain. Chromosome counts on mature sporophytes showed that normal sporophytes (from fertilized eggs) were diploid (2n = approximately 40) and that the spores they produced were haploid (n = approximately 20), while nuclei from both somatic and sporangial cells in parthenogenetic sporophytes were haploid. All gametophytes were haploid. Young sporophytes derived from cultures with both female and male gametophytes were diploid, while young, sporophytes obtained from female gametophytes from parthenogenetic sporophytes had haploid, diploid, or polyploidy chromosome numbers. Polyploidy was associated with abnormal cell shapes. The presence of haploid parthenogenetic sporophytes should be use in breeding kelp strains with useful characteristics, since the sporophyte phenotype is expressed from a haploid genotype which can be more readily selected.     

CLONAL PROPAGATION OF LAMINARIA DIGITATA (PHAEOPHYCEAE) SPOROPHYTES THROUGH A DIPLOID CELL-FILAMENT SUSPENSION

In vitro cultures of Laminaria digitata (Hudson) Lamouroux stipe medullary explants (Laminariales, Phaeophyceae) gave rise to friable morula-like masses of pigmented cells. Upon subculture under continuous white light, they grew out as filaments, which tended to dissociate into spherical isolated cells, leading to the establishment of a suspended cell-filament culture. Depending on culture conditions, this cell-filament suspension underwent various forms of growth, including regeneration of morphologically normal sporophytes, and this developmental pathway was stimulated by blue light. Genotyping with nine polymorphic microsatellite markers indicated that the regenerated sporophytes and the cell-filament suspension shared the same genotype. As assessed by flow cytometric analysis of isolated nuclei, the cell-filament suspension exhibited a 2C nuclear DNA content, whereas the regenerated sporophytes displayed a 4C level. Chromosome counting, however, showed that both the mother suspension and the regenerated sporophytes were diploid, suggesting the involvement of polyteny in the regeneration process.     

SPOROGENESIS UNDER ULTRAVIOLET RADIATION IN LAMINARIA DIGITATA (PHAEOPHYCEAE) REVEALS PROTECTION OF PHOTOSENSITIVE MEIOSPORES WITHIN SORAL TISSUE: PHYSIOLOGICAL AND ANATOMICAL EVIDENCE1

To study the effect of different radiation conditions on sporogenesis of Laminaria digitata (Huds.) J. V. Lamour., excised disks were induced to form sporangia under PAR (P), PAR + ultraviolet‐A (UVA) (PA), and PAR + UVA + ultraviolet‐B (UVB) (PAB) conditions in the laboratory. Vitality of meiospores, released from sori induced under different radiation conditions in the laboratory and from sori of wild sporophytes acclimated to in situ solar radiation in the presence and absence of ultraviolet radiation (UVR), was measured in terms of their germination capacity. Sorus induction in disks of laboratory‐grown sporophytes was not hampered under light supplemented with UVR, and sorus area was not significantly different among P, PA, and PAB. Vitality and germination rate of meiospores released from sori induced under different radiation treatments was comparable. Likewise, screening of UVR of the natural solar radiation did not promote higher germination rates of meiospores released from wild sporophytes. Germination rates were, however, higher in meiospores released from laboratory‐induced sori compared to sori of wild sporophytes. Higher DNA damage (formation of cyclobutane pyrimidine dimers, CPDs) was observed in laboratory‐grown nonsorus compared to sorus tissue, while CPDs were nondetectable in both sorus and nonsorus tissue of wild sporophytes. To explain the apparent protection of developing meiospores and the unexpected UV resistance of soral tissue, concurrent anatomical investigations of sporogenic tissue were performed. We observed the previously unreported existence of two types of sterile paraphysis cells. One type of paraphysis cells, the most frequent type, contained several red‐fluorescing plastids. The other type, less frequently occurring, was completely filled with substances emitting blue fluorescence under violet excitation, presumably brown algal phenolic compounds (phlorotannins). Cells of this type were irregularly scattered within the sorus and did not contain red‐fluorescing plastids. Meiospore‐containing sporangia were positioned embedded between both types of paraphysis cells. In vegetative tissue, blue autofluorescence was observed only in injured parts of the blade. Results of our study suggest that the sorus structure with phlorotannins localized in the specialized paraphysis cells may be able to screen harmful UVR and protect UV‐sensitive meiospores inside the sporangia.     

DECREASE IN DYNAMIC VISCOSITY AND AVERAGE MOLECULAR WEIGHT OF ALGINATE FROM LAMINARIA DIGITATA DURING ALKALINE EXTRACTION1

Alginates are natural polysaccharides that are extracted from brown seaweeds and widely used for their rheological properties. The central step in the extraction protocol used in the alginate industry is the alkaline extraction, which requires several hours. In this study, a significant decrease in alginate dynamic viscosity was observed after 2 h of alkaline treatment. Intrinsic viscosity and average molecular weight of alginates from alkaline extractions 1–4 h in duration were determined, indicating depolymerization of alginates: average molecular weight decreased significantly during the extraction, falling by a factor of 5 between 1 and 4 h of extraction. These results suggested that reducing extraction time could enable preserving the rheological properties of the extracted alginates.     

EFFECTS OF EXTERNAL INORGANIC NITROGEN CONCENTRATION ON METABOLISM,GROWTH AND ACTIVITIES OF KEY CARBON AND NITROGEN ASSIMILATORY ENZYMES OF LAMINARIA SACCHARINA (PHAEOPHYCEAE) IN CULTURE1

The growth rate of Laminaria saccharina (L.) Lamour. is dependent on inorganic nitrogen in culture. Growth rates were saturated between 5 and 10 μmol · L^?1 nitrate. The activities of ribulose-1,5 bisphosphate carboxylase, phosphoenolpyruvate carboxykinase, mannitol-1-phosphate dehydrogenase, nitrate reductase and glutamine synthetase also varied with the concentration of inorganic nitrogen in the medium. All enzyme activities were lowest at 2.5 μmol · L^?1 nitrate (the lowest concentration used) increasing to a maximum activity between 10 and 30 μmol · L^?1 nitrate. Most enzyme activities followed a hyperbolic curve resembling those described by the Michaelis-Menten equation, with different half-saturation constants.     

CRITICAL LEVELS OF LIGHT AND TEMPERATURE REGULATING THE GAMETOGENESIS OF THREE LAMINARIA SPECIES (PHAEOPHYCEAE)1

Gametophytes of three Laminaria species occurring near Helgoland, North Sea, were cultivated 4 wk in a 12:12 LD regime at different temperatures in artificial light fields, and in the sea at different water depths. In the artificial light fields underwater spectral distribution was simulated according to Jerlov water Types 5, 7, 9. Blue light in the simulated light fields amounted to 17, 12 or 4% of total quanta. The rate of vegetative growth did not depend on spectral distribution, was light-saturated at 4–6 W · m^?2, and increased with temperature up to 15 C. L. saccharina (L.) Lamour. exhibited the highest tolerance towards temperature, light and UV. Gametophytes survived 1 wk at 21 C ± 0.1, but not 22 C ± 0.1. Gametophytes of L. hyperborea (Gunn.) Fosl. and L. digitata (Huds.) Lamour. survived 1 wk at 20 C ± 0.1, but not at 21 C ± 0.1. In sunlight, and in the light field of a xenon lamp, 50% of L. saccharina gametophytes were killed by a quantum dose of 50 μEin · cm^?2, and 100% of the plants by 90 μEin · cm^?2. Approximately half of these quantum doses killed the corresponding percent of the other species gametophytes. Appreciably higher quantum doses were survived in visible light, with red being the most detrimental. Fertility depended on a critical quantum dose of blue light which decreased almost exponentially with decreasing temperature. The quantum dose (400–512 nm) required for induction of fertilization of 50% of the female gametophytes (males react similarly) was 90 μEin · cm^?2 at 5 C, 110 μEin · cm^?2 at 10 C, 230 (560 in L. digitata)μEin · cm^?2 at 15 C, and 560 (L. hyperborea) or about 850 (other 2 species) μEin · cm^?2 at 18 C. In the sea the gametophytes survived the dark winter months in the unicellular stage, with almost no vegetative growth of the primary cell, due to lack of light. In early spring the female gametophytes matured in the unicellular, and the males in a few-celled stage at the depth of 2 m, as did the laboratory cultures under conditions inducing maximal fertility.     

GRAZING IMPACT OF,AND INDIRECT INTERACTIONS BETWEEN MESOGRAZERS ASSOCIATED WITH KELP (LAMINARIA DIGITATA)1

Little is known about the indirect effects of nonlethal grazing impacts in mesograzer–seaweed interactions. Using laboratory experiments, the effect of grazing by the seasonally abundant kelp‐associated gastropod Lacuna vincta on subsequent kelp consumption by one kelp‐associated (Idotea granulosa) and one nonassociated species of isopod (I. emarginata) was determined. Measurements of the toughness and elemental composition of different parts of the sporophyte of Laminaria digitata (Huds.) J. V. Lamour., as well as grazer‐induced changes in the palatability of the blade, were conducted to explore possible mechanisms of indirect effects. In situ grazing pressure was the highest between July and September, with the blade being the preferred part of the kelp sporophyte, despite missing differences in the elemental composition among kelp parts. The laboratory experiments supported our hypotheses in that kelp consumption by both species of isopods was lower on intact than on L. vincta–damaged areas of the blade. This pattern was not caused by grazing‐induced changes in blade palatability. Instead, the observed increase in isopod consumption following grazing by L. vincta resulted more likely from the combined effects of a reduction in the toughness of L. vincta–damaged kelp blades and some unknown gastropod cue(s). These results suggest that kelp‐associated and nonassociated mesograzers may benefit from the nonlethal grazing impact of L. vincta due to changes in physical traits of the seaweed. Thus, the nonlethal grazing impact by one species of mesograzer can positively modify the trophic interactions between kelp and other potential competitors, suggesting that the interactions among mesograzers might be more complex than previously assumed.     

ISOLATION AND CHARACTERIZATION OF A DNA VIRUS INFECTING FELDMANNIA SIMPLEX (PHAEOPHYCEAE)1

We report on the isolation and characterization of a virus that is formed in modified zoidangia of the marine brown alga Feldmannia simplex (Crouan) Hamel (Ectocarpales, Phaeophyceae). Isolated virus particles had a buoyant density of about 1.35 g·mL^?1 in CsCl equilibrium gradients. They contained one major polypeptide (MW = 55,000) and at least six additional polypeptides (MW = 15,000–120,000). Four of these proteins were glycosylated. The viral genome consisted of double-stranded DNA and formed two freely migrating fractions in pulsed-field-gel electrophoresis, namely linear DNA with a size of 220 kilobase pairs, and fragments of 10–60 kilobase pairs. However, electron microscopic examination revealed that a substantial fraction of the viral DNA occurred as closed circles. We suggest that the viral DNA in native particles is circular but tends to break at random sites during the preparation.     

ISOLATION AND CHARACTERIZATION OF PHASE-SPECIFIC COMPLEMENTARY DNAs FROM SPOROPHYTES AND GAMETOPHYTES OF PORPHYRA PURPUREA (RHODOPHYTA) USING SUBTRACTED COMPLEMENTARY DNA LIBRARIES1

The red alga Porphyra purpurea (Roth) C. Agardh has a life cycle that alternates between shell-boring, filamentous sporophytes and free-living, foliose gametophytes. The significant morphological differences between these two phases suggest that many genes should be developmentally regulated and expressed in a phase-specific manner. In this study, we prepared and screened subtracted complementary DNA (cDNA) libraries specific for the sporophyte and gametophyte of P. purpurea. This involved the construction of cDNA libraries from each phase, followed by the removal of common clones through subtractive hybridization. Sampling of the subtracted libraries indicated that 8–10% of the recombinant colonies in each library were specific for the appropriate phase. Of 20 putative phase-specific cDNAs selected from each subtracted library, eight unique clones were obtained for the sporophyte and seven for the gametophyte. After confirming their phase-specificities by hybridization to gametophyte and sporophyte messenger RNA, these 15 phase-specific cDNAs were sequenced, and the deduced amino acid sequences were used to search protein databanks. Two proteins encoded by the sporophyte-specific cDNAs and two by the gametophyte-specific cDNAs were identified by their similarity to databank entries.     

ROLE OF SETTLEMENT DENSITY ON GAMETOPHYTE GROWTH AND REPRODUCTION IN THE KELPS PTERYGOPHORA CALIFORNICA AND MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Laboratory studies were used to examine how variation in the density of spore settlement influences gametophyte growth, reproduction, and subsequent sporophyte production in the kelps Pterygophora californica Ruprecht and Macrocystis pyrifera (L.) C. Ag. In still (non-aerated) cultures, egg maturation in both species was delayed when spores were seeded at densities 300 · mm^?2. Although the density at which this inhibition was first observed was similar for both species, the age at which their eggs matured was not. P. californica females reached sexual maturity an average of 4 days (or ～ 30%) sooner than did M, pyrifera. As observed previously in field experiments, per capita sporophyte production was negatively density dependent for both species when seeded at spore densities of 10 · mm^?2. Total sporophyte production (i.e. number · cm^?2) for both species, however, was greatest at intermediate densities of spore settlement (～ 50 spores · mm^?2). In contrast, total sporophyte production by P. californica steadily increased with increasing spore density in aerated cultures; highest sporophyte density was observed on slides seeded at a density of 1000 spores · mm^?2. Preliminary experiments with P. californica involving manipulation of aeration and nutrients indicate that inhibition of gametophyte growth and reproduction at higher densities of spore settlement in non-aerated cultures was probably caused by nutrient limitation.     

COORDINATNE NUCLEAR AND CHLOROPLAST DIVISION IN UNILOCULAR SPORANGIA OF LAMINARIA ANGUSTATA (LAMINARIALES,PHAEOPHYCEAE)1

Changes in the number of nuclei and chloroplasts were examined during the process of unispore formation in unilocular sporangia of Laminaria angustata. Just before meiosis, eight chloroplasts were always present in unilocular sporangial mother cells. The number of chloroplasts remained constant through meiosis. After the resulting four nuclei divided again (third nuclear division), a close association between a nucleus and a chloroplast developed among each of the eight nuclei and eight chloroplasts. The eight chloroplasts divided ahost synchronously before the synchronous division of the eight nuclei. Following the 16 nucleate stage with 16 chloroplasts and the final 32 nucleate stage with 32 chloroplasts, 32 unispores, each with a nucleus and a chloroplast, were fomd in unilocular sporangza of L. angustata. Immunofluorescence microscopy using an anti-centrin antibody showed that two anticentrin-stained structures (as future mitotic poles) occurred adjacent to each of the premitotic four nuclei, and each spot was located near a chloroplast. Therefore, after the third division, each of the eight nuclei established close contact with a chloroplast presumably mediated by the centrosomes.     

BIOLOGICAL WEIGHTING FUNCTIONS FOR UV INHIBITION OF PHOTOSYNTHESIS IN THE KELP LAMINARIA HYPERBOREA (PHAEOPHYCEAE)1

Different wavelengths of sunlight either drive or inhibit macroalgal production. Ultraviolet radiation (UVR) effectively disrupts photosynthesis, but since UVR is rapidly absorbed in coastal waters, macroalgal photoinhibition and tolerance to UVR depend on the depth of attachment and acclimation state of the individual. The inhibition response to UVR is quantified with a biological weighting function (BWF), a spectrum of empirically derived weights that link irradiance at a specific wavelength to overall biological effect. We determined BWFs for shallow (0 m, mean low water [MLW]) and deep (10 m) Laminaria hyperborea (Gunnerus) Foslie collected off the island of Finnøy, Norway. For each replicate sporophyte, we concurrently measured both O₂ evolution and ¹³C uptake in 48 different light treatments, which varied in UV spectral composition and irradiance. The relative shape of the kelp BWF was most similar to that of a land plant, and the absolute spectral weightings and sensitivity were typically less than phytoplankton, particularly in the ultraviolet radiation A (UVA) region. Differences in BWFs between O₂ and ¹³C photosynthesis and between shallow (high light) and deep (low light) kelp were also most significant in the UVA. Because of its greater contribution to total incident irradiance, UVA was more important to daily loss of production in kelp than ultraviolet radiation B (UVB). Photosynthetic quotient (PQ) also decreased with increased UVR stress, and the magnitude of PQ decline was greater in deepwater kelp. Significantly, BWFs assist in the comparison of biological responses to experimental light sources versus in situ sunlight and are critical to quantifying kelp production in a changing irradiance environment.     

SEASONAL PATTERNS OF PHOTOSYNTHESIS AND LIGHT-INDEPENDENT CARBON FIXATION IN MARINE MACROPHYTES1

The contribution of light-independent carbon fixation (LICF) to the overall carbon gain and the seasonal patterns of maximum photosynthesis (P_max and LICF were characterized in a broad taxonomic range of macrophytes from Monterey Bay, California. P_max and LICF rates (nmol C.g filtered seawater^?1.min^?1) varied among species and taxonomic groups examined, and as a function of tissue type in the phaeophyte Laminaria setchellii Silva (Phaeophyceae). On average, P_max values were higher in the Rhodophyta, whereas LICF rates were greater in the Phaeophyceae. LICF rates were generally less than 5% of P_max in the marine macrophytes studied and, as a consequence, cannot fully compensate for respiratory carbon losses, which usually are greater than 10% of P_max. All species studied possessed the highest P_max and LICF rates when irradiance levels were highest and decreased during periods of low incident irradiance. Seasonal patterns of P_max and LICF in most of the macrophytes from the stenothermal environment of Monterey Bay were strongly correlated with photosynthetic photon flux rather than seawater temperature. The concomitant decrease of LICF and P_max rates in all species examined argues against LICF playing a major role in carbon acquisition under light-limiting conditions as suggested previously. Rather, the strong positive correlation of P_max and LICF indicates the direct coupling of photosynthate (e.g. 3-phosphoglyceric acid) generation with production of substrates for LICF reactions. Our results also suggest that LICF might be a useful indicator of photosynthetic metabolism in marine macrophytes.     

ISOLATION AND CHARACTERIZATION OF GOLGI APPARATUS FROM EGGS OF FUCUS (PHAEOPHYCEAE)1

A golgi-rich cell-free fraction has been obtained from eggs of Fucus serratus L. and characterized by enzyme markers (IDP-ase and TPP-ase) and electron microscopy. The results are correlated with cytochemical localization of IDP-ase and TPP-ase in situ.     

A SEASONAL COMPARISON OF CARBON,NITROGEN, AND PIGMENT CONTENT IN LAMINARIA SOLIDUNGULA AND L. SACCHARINA (PHAEOPHYTA) IN THE ALASKAN ARCTIC1

Laminaria solidungula and L. saccharina inhabit the Beaufort Sea in the Alaskan High Arctic. Laminaria solidungula is an Arctic endemic, whereas L. saccharina extends from north temperate Pacific and Atlantic waters to the Arctic. Previous studies have shown that the two species have different seasonal timing of growth, but little comparative physiological information exists. As a first step in characterizing these two species from a mixed Arctic population, we measured variations in carbon, nitrogen, and photosynthetic pigment content in blade tissue from plants collected under the fast ice in April and during the open water Period in late July, Both species exhibited seasonal differences in many measured variables; seasonal differences in L. solidungula were most pronounced in growing basal blades. For example, the molar CIN ratio of basal blades averaged 11 in April and 21 in July for L. solidungula and 11.5 in April and 28 in July for L. saccharina. Basal and mature second blades differed in pigment content in April but not in July: chlorophyll a + c in L. solidungula basal and mature second blades averaged 19 and 27 nmol.cm^?2 in April and 30 and 29 nmol. cm^?2 in July, respectively. The corresponding values for L. saccharina were 17 and 29 nmol.cm^?2 in April and 16 and 16 nmol.cm^?2 in July (95% confidence intervals approximately 1–3 nmol. cm ^?2). Carotenoids exhibited similar patterns. Species differences in pigments, carbon, and nitrogen were minor and were probably effects rather than causes of the different seasonal patterns of growth and development. The primary difference between the two species may be the ability of L. solidungula to retain multiple metabolically active blades and to fuel areal growth with stored carbohydrates during winter near-darkness, whereas L. saccharina growth is more closely tied to active photosynthesis in the growing basal blade. The cause of old blade retention in L. solidungula and the possibility of other physiological differences between the two species, including gametophytes, remain to be determined.