Direction of illumination controls gametophyte orientation in seedless plants and related algae

Abstract

The environmental influences that determine dorsiventral or axial gametophyte orientation are unknown for most modern seedless plants. To fill this gap, an experimental laboratory system was employed to evaluate the relative effects of light direction and gravity on body orientation of the dorsiventral green alga Coleochaete orbicularis, and gametophytes of liverworts Blasia pusilla and Marchantia polymorpha, early-diverging moss Sphagnum compactum, and fern Ceratopteris richardii, the latter functioning as experimental control. Replicate clonal cultures were experimentally illuminated only from above, only from below, or from multiple directions, with the same near-saturation PAR level for periods brief enough to minimize nutrient limitation effects, and orientation of new growth was evaluated. For all species tested, direction of illumination exerted stronger control over gametophyte body orientation than gravity. When illuminated only from below: 1) axial Sphagnum gametophores that had initially grown into an overlying air space inverted growth by 180°, burrowing into the substrate; 2) new growth of dorsiventral Blasia, Marchantia, and Ceratopteris gametophytes–whose ventral rhizoids initially penetrated agar substrate and dorsal surfaces initially faced overlying airspace–twisted 180° so that ventral surfaces bearing rhizoids faced overlying air space and rhizoids extended into the air; and 3) Coleochaete lost typical dorsiventral organization and diagnostic dorsal hairs. Direction of illumination also exerted stronger control over orientation of liverwort new growth than surface contact did. These results indicate that early land plants likely inherited light-directed gametophyte body orientation from ancestral streptophyte algae and suggest a mechanism for reorientation of gametophyte-dominant land plants after spatial disturbance.

Introduction

The colonization of land by early streptophytes (embryophytes + related algae) and associated microbiota was one of the most important events in Earth history.^1,2 Early gametophyte-dominant streptophytes, thought to have had ecological roles and biogeochemical impacts much like those of modern streptophyte algae and bryophytes, likely contributed to soil formation and stability, provided nutritional resources for early terrestrial heterotrophs, and influenced atmospheric chemistry for tens of millions of years prior to the rise of vascular plants.^3,4 Because the latest-diverging modern streptophyte algae and the earliest-diverging modern embryophytes display prostrate dorsiventral or erect axial bodies, these growth forms are also inferred for earliest terrestrial vegetation.^5,6 Even so, the particular environmental influences that determine dorsiventral or axial gametophytic body orientation in modern seedless plants are largely unknown. Such information could be used to infer the environmental controls most important in determining body orientation of early terrestrial streptophytes, and to interpret genomic data gathered for evolutionary studies that are focused on early plant history.

Understanding factors important in body orientation is also ecologically important. When surface-dwelling gametophytes of seedless plants become spatially disoriented by wind or water disturbance, re-orientation in response to an environmental cue would improve survival. Such ecological considerations were likewise relevant in the early history of land plants. Based on the widespread occurrence of unconsolidated sandy substrata during the Cambrian-Ordovician time period^7,8 that is widely associated with earliest land plants,¹ it can be inferred that early terrestrial streptophytes were susceptible to positional disturbance. Such early vegetation may have adapted by sensing and responding to environmental cues by means of growth changes that restored normal dorsiventral or axial orientation. The direction of incident light, gravity, and contact with a surface present themselves as likely factors that might elicit phototropic, gravitropic, or thigmotropic responses.

Previous studies of light effects on early-diverging streptophytes have focused on sporophyte orientation, spore germination, stages in the development of filamentous protonemata, gametophytic rhizoid growth, or plastid movements. Examples include phototropic and gravitropic responses of liverwort sporophytes^9,10 phototropic or gravitropic aspects of growth by filamentous moss protonemata;^11-15 gravitational responses of characean rhizoids;^16-17 phototropic behavior of liverwort and fern rhizoids;^18,19 phototropic growth of characean branches;²⁰ light influences on chloroplast movement in streptophyte green algae,²¹ and other land plants,²² and the role of phototropin in chloroplast movements in Marchantia.²³ Much less is known about the relative effects of light direction, gravity, or surface contact on the orientation of mature gametophytes of mosses, liverworts, or related streptophyte algae.

Previous studies have indicated that directional white light is the dominant factor influencing prothallus orientation in the fern Adiantum.^24,25 The experiments described here thus tested the hypothesis that direction of white light illumination is not more important than gravity in controlling orientation of mature gametophytes of representative modern bryophytes and streptophyte algae that also stand as reasonable proxies for ancient streptophytes. Responses of erect axial gametophores of the moss Sphagnum compactum, dorsiventral thalloid liverworts Blasia pusilla and Marchantia polymorpha, and the dorsiventral aquatic-grown streptophyte alga Coleochaete orbicularis were studied.^5,26 The thalloid liverwort species were also employed to test a second hypothesis that direction of white light illumination is not more important than surface contact in controlling orientation of mature gametophytes. In addition, the axial gametophores of Sphagnum moss were used to examine the effects of directional illumination under supersaturating (high fluence) levels of photosynthetically active radiation. The fern Ceratopteris richardii, whose gametophytic growth responses to directional white light illumination were expected to be similar to those of previously-studied Adiantum,^24,25 functioned as a control.

Material and Methods

Cultures and growth media

Sphagnum was selected as a representative of mosses and of axial gametophyte body structure because the Sphagnopsida occupy a relatively early-diverging position among embryophytes in general and mosses in particular.²⁷ We note that very young (protonemal-stage) Sphagnum sporelings are monostromatic and dorsiventral with ventral rhizoids and dorsal cuticle-like materials.²⁸ In view of this morphological difference, to avoid confusion, we henceforth refer to axial stages of Sphagnum gametophytes as “gametophores,” even though gametangia were not observed in experimental materials cultivated in white light. Cultures identified as the peat moss Sphagnum compactum, and having no detectable microbial contamination, were kindly supplied by M. Sargent (University of Illinois, Urbana-Champaign). Replicate clonal cultures of S. compactum gametophores were propagated asexually from vegetative branches in 25 mm tall transparent plastic Petri dishes on 1/3 Gamborg's B5 basal medium with minimal organics (Sigma-Aldrich Catalog No. G5893–1L solidified with 2% Bacto agar (Sigma-Aldrich Catalog No. A5306). The relatively short stature of this species facilitates cultivation of axial gametophores in such dishes.²⁹ Clonal cultures were generated and maintained in a walk-in growth room at 20˚C with cool white fluorescent illumination from above and 16 hr day length. The average photosynthetically active radiation (PAR) level was ∼320 µmol photons·m⁻² ·s⁻¹, determined with a Biospherical Instruments Quantum Scalar Laboratory Radiometer QSL-100 with cosine-corrected detector. Gametophores several cm in length were used in experimental manipulations.

Marchantia and Blasia represent thalloid liverworts that produce ventral rhizoids and dorsal asexual gemmae and gametangia. ^30,31 Replicate clonal cultures of M. polymorpha were propagated asexually from meristematic tips of our axenic isolate and maintained in standard transparent Petri dishes on 1/3 Gamborg's B5 basal medium with 1% glucose added, solidified with 2% Bacto agar. The addition of glucose speeded the growth of replicate mature M. polymorpha clonal cultures, which were generated and maintained under the same growth conditions as S. compactum described earlier. Mature gametophytes that had produced gemmae were used for experimental manipulations.

We established Blasia pusilla cultures from spores harvested from fresh sporangia present in a field collection (a roadside bank near Paradox, NY) generously provided by Norton S. Miller. Non-axenic clonal cultures of B. pusilla were generated by asexual propagation of meristematic tips in standard transparent Petri dishes and maintained on the same basal growth medium with minimal organics and under the same growth conditions as S. compactum. Mature gametophytes that had produced gemmae were used for subsequent experimental manipulations.

Coleochaete orbicularis was selected for study because in nature and aquatic cultures this streptophyte green algal species occurs as attached monostromatic disks having dorsiventral orientation indicated by dorsal hairs and cuticle-like materials,²⁸ and is more-or-less closely related to embryophytes depending upon the molecular data set employed for phylogenetic analysis.^32-34 Among streptophyte algae, only certain Coleochaete species such as C. orbicularis display discoid parenchymatous bodies having definitive indicators of dorsiventral orientation; by contrast, other streptophyte algae occur as unicells, colonies, or filaments.³⁵ Although the streptophyte algae known informally as charaleans produce axial filaments that include parenchymatous regions,³⁵ these relatively tall organisms are not cultivated in the same types of culture dishes used for other species studied in this project. Our unialgal isolate of C. orbicularis was propagated asexually from tissue fragments that generate flagellate asexual propagules (zoospores). Such asexual propagules are functionally equivalent to gametophytic tip fragments or gemmae of bryophytes. Replicate clonal cultures were grown in standard plastic Petri dishes containing liquid DY-III medium, an inorganic medium designed for cultivation of algae isolated from oligotrophic aquatic habitats.³⁶ Experiments were conducted in liquid media rather than on agar-solidified media because C. orbicularis has been shown to display different morphology when grown subaerially.²⁶ Prior to experimental manipulations, clonal algal cultures were generated and maintained in the same growth room as the bryophytes studied.

Ceratopteris richardii gametophytes were employed as controls, because directional white light responses of dorsiventral gametophytes (prothalli) of closely related Adiantum have previously been described.^24,25 Sterilized spores of C. richardii obtained from Carolina Scientific (Burlington, North Carolina, USA) were germinated and subsequent gametophyte growth was conducted in standard plastic Petri dishes on Basic C-Fern Medium (Carolina Biological Supply Co., catalog No. Fifteen–6780) (minimal organics) solidified with 2% Bacto agar. Because they originated from sexually produced spores, fern gametophytes had the potential to display greater genetic variability than asexually generated gametophytes of the bryophytes and green alga studied. Replicate culture dishes, each containing multiple mature fern gametophytes of the same heart-shaped developmental stage were generated for directional light experiments. Because this tropical fern has an optimal growth temperature of 30˚C, gametophyte cultures for experimentation were initiated and maintained in a 30°C culture chamber, illuminated from above with cool white fluorescent lamps, with 16 hr day length.

To avoid contamination, in all cases culture dishes were sealed at their edges with a single layer of Parafilm M, which according to supplier information is permeable to carbon dioxide at 1200 mL·m⁻² ·d⁻¹ and oxygen at 150 mL·m⁻² ·d⁻¹ at 23˚C and 0% RH, and water vapor at 1 g·m⁻² at 38˚C and 90% RH (SPI Supplies, catalog No. 01852-AB). Previous experiments with the same isolates of Sphagnum compactum, Marchantia polymorpha and Coleochaete that had been cultivated similarly revealed the occurrence of carbon limitation relieved by exogenous organic carbon.^37-39 In the present study, any such carbon limitation would have affected all experimental materials equally. To facilitate careful evaluation of results, investigations of the 5 selected taxa were conducted serially, with care taken to ensure that temperature and irradiance levels were the same during each experiment conducted with near-saturating levels of photosynthetically active radiation (PAR).^40-44

Experimental design

Consistent with an initial hypothesis that light direction is not more important than gravity in controlling gametophyte orientation, under all experimental direction of illumination treatments, new growth of axial Sphagnum generated during the treatment period would be expected to orient toward the top of the overlying air-filled space, as in nature. Under the same hypothesis, under all experimental direction of illumination treatments, new growth of dorsiventral liverwort and fern gametophytes occurring during the treatment period would be expected to occur as in nature, with rhizoids extending into the underlying agar substratum. Under the same hypothesis, under all experimental direction of illumination treatments, discoid Coleochaete orbicularis produced during the treatment period would be expected to occur attached to the dish surface, display dorsiventral body organization, and produce hair cells that extend into the overlying water-filled space, as in nature. Results consistent with these expectations would support the hypothesis that gravity exerts a stronger control on gametophyte orientation than does direction of illumination. Exceptions to these expectations would be evidence against the hypothesis that direction of illumination is not more important than gravity, indicating instead that direction of illumination or some other factor exerts a stronger control on gametophyte orientation than does gravity.

To test the hypothesis that direction of illumination is not more important than gravity in controlling gametophyte orientation, replicate culture dishes, each containing at least 5 mature individuals, were placed on a 6 mm thick transparent plexiglass shelf, below and above which were mounted cool-white fluorescent lamps of the same type used to generate replicate cultures. Experimental dishes received an average photosynthetically active radiation (PAR) level of ∼320 µmol photons·m⁻² ·s⁻¹, determined with a Biospherical Instruments Quantum Scalar Laboratory Radiometer QSL-100 with cosine-corrected detector. Irradiances were measured by placing the detector beneath a standard plastic petri dish lid or beneath a standard dish bottom 1/3 full of nearly transparent agarized mineral growth medium, as in the case of experimental cultures. Because the same ∼320 µmol photons·m⁻² ·s⁻¹ irradiance level was used to generate all replicate cultures of all species examined, such cultures were pre-adapted to experimental irradiance conditions. Irradiance was provided to all experimental cultures by means of cool-white lamps, with a 16 hour daylength, in a temperature-controlled (20°C) walk-in growth room. Consequently, all species except the fern (for which initial cultures were generated at a higher temperature) were pre-adapted to experimental temperature conditions.

To explore the effect of higher, super-saturating irradiance, 8 replicate clonal Sphagnum compactum cultures of the same age were illuminated from above and below with Sylvania 100W compact fluorescent lamps (daylight 6500K), housed in aluminum reflectors, at irradiance levels ranging over an order of magnitude (250 to 2755 µmol photons·m⁻² ·s⁻¹), for a 2-month growth period in a temperature controlled growth room (20°C). Photoinhibition of at least one Sphagnum species is known to occur at 800 µmol photons·m⁻² ·s⁻¹.⁴³ Because manufacturer instructions indicated that timers should not be used with these high-intensity lamps, plants were illuminated with the higher intensity lamps on a continual basis.

Culture dishes of all species illuminated at a standard, near-saturating PAR level (∼320 µmol photons·m⁻² ·s⁻¹) and only from above were placed onto a black-painted cardboard surface through which light was unable to penetrate from below, determined by use of a PAR meter. Cultures that were illuminated at a standard, near-saturating level and only from below were placed nearby, but under a black-painted cardboard box through which light was unable to penetrate from above, as determined by use of a light meter. In addition, replicate cultures were placed directly and uncovered on the plexiglass shelf and thus received standard, near-saturating illumination from all directions.

Ninety replicate dishes, each containing multiple mature S. compactum gametophores, were divided randomly into 6 experimental groups; 30 dishes were illuminated only from above, and of these, 15 were placed right side up and 15 placed upside down. Such right side up and upside down placement was employed to check for any variation that might result from illumination passing through the translucent agar medium on which bryophyte and fern gametophytes were cultivated, and to provide additional information regarding the relative effects of gravity and direction of illumination. Likewise, 30 S. compactum culture dishes were illuminated only from below; with half the dishes positioned right side up and half upside down. Equal numbers of S. compactum culture dishes were illuminated from multiple directions; half positioned right side up and half upside down.

Eighty replicate dishes, each containing multiple mature gametophytes of M. polymorpha, were divided randomly into 6 experimental groups. Of 27 plates illuminated only from above, 27 plates illuminated only from below, and 26 plates illuminated from multiple directions, approximately half were placed right side up, and the rest upside down. Seventy replicate dishes, each containing multiple gametophytes of B. pusilla, were randomly divided into 6 experimental groups. Of 23 plates illuminated only from above, 23 plates only from below, and 24 plates illuminated from multiple directions, equal numbers of dishes were positioned right side up and upside down.

A separate experiment was conducted with M. polymorpha and B. pusilla to test the hypothesis that surface contact was more important than gravity or direction of illumination (at the same near-saturating PAR level employed in most other experiments). Fifteen culture dishes were illuminated only from above; one-third of these were placed right side up, one-third upside down, and one-third on the dish side, supported by a binder clip. At the same time, 15 culture dishes were illuminated only from below; one-third of these disches were placed right side up, one-third upside down, and one-third on their sides, supported by a binder clip. Side-oriented dishes allowed investigation of surface influence (touch). We expected that if surface contact were more important than direction of illumination in determining the orientation of new growth, dorsiventral gametophytes would continue to lie flat against the substrate surface. We expected that if direction of illumination were more important than surface contact, new growth in dishes positioned on their sides would contact the substrate only at a gametophyte edge and that rhizoids would extend in the direction away from illumination.

Forty-five replicate dishes, each containing attached disks of C. orbicularis large enough to produce zoospores were randomly divided into 3 groups of 15 each. Of these, one group was illuminated only from above, one group only from below, and the third group from all directions. Ninety replicate dishes, each containing multiple C. richardii prothalli, were randomly divided into 6 experimental groups. Thirty culture dishes were illuminated only from above, an equal number illuminated only from below, and an equal number illuminated from multiple directions; in each case half the culture dishes were placed right side up and half upside down.

As a consequence of differences in growth rate, the 5 experimental species were exposed to directional light treatments for different periods. These periods represented the amount of time required to observe responses of new growth but before the onset of chlorosis that would indicate mineral nutrient deficiency. Sphagnum compactum cultures were exposed to directional light treatments for 2 months. Marchantia polymorpha, Blasia pusilla, and Coleochaete orbicularis were exposed to directional light treatments for 4 weeks. Ceratopteris richardii cultures were exposed to directional light treatments for 3 weeks. Given the time periods required for growth responses, it was not possible to investigate the effect of darkness, because, though we had previously established that C. orbicularis, M. polymorpha, and S. compactum are capable of mixotrophic growth, these species are not known to be capable of heterotrophic growth for months-long periods.^31,33,34 Further, the research was designed to focus on effects of illumination direction, rather than presence or absence of illumination, intensity of illumination, or spectral quality of illumination.

At the end of experimental treatment periods, new gametophytic growth that had occurred during the treatment period—namely branch tips of bryophyte gametophytes, new Coleochaete discs arising from zoospores, and edges of fern gametophytes—was immediately imaged using a camera-enabled Leica EZ4D stereoscope or a Zeiss Axioplan compound light microscope equipped with a Nikon D300s Digital Camera and Camera Control Pro software. At least 10 random images were collected of new growth for each of the 5 species, in each of at least 5 replicates in each of the 3 illumination direction treatments, and in upside down versus right side up dishes (>1500 total images). Images are archived in the Wisconsin State Herbarium, Department of Botany, University of Wisconsin, Madison, WI 53706, USA.

In the case of axial gametophytes of Sphagnum, assessment consisted of determining the percentage of main axis capitula which, during the treatment period, extended into the air-filled container space vs. those growing in some other direction (e.g. into the agar substratum). In the cases of dorsiventral gametophytes of Blasia, Marchantia, and Ceratopteris, assessment of light direction effects involved estimating the percentage of new-growth rhizoids extending into the agar substratum versus some other direction (e.g., the air-filled container space). For Coleochaete, assessment of the effects of variation in the direction of illumination involved estimating percentages of young disks (representing new growth from zoospores produced during the treatment period) that were attached and displayed normal dorsiventral morphology with hair cells extending into the liquid medium, vs. young growths having an alternate orientation. Effects of surface contact were assessed in Blasia and Marchantia by comparing the direction of new plant growth (and rhizoid orientation) occurring during the treatment period in dishes lying flat (perpendicular to the gravity vector and light direction) versus new growth in dishes placed on their sides.

Results

All tested species were examined with the use of the same experimental system (Fig. 1), which was designed to discriminate among the effects of variation in direction of illumination, gravity, or surface contact, as described in Table 1. In all cases, the direction of illumination by white light at near-saturation levels of PAR played a more important role in body orientation than did gravity. When illuminated only from below, axial gametophytes of Sphagnum moss burrowed into the substratum (Fig. 2). When illuminated only from below, new growth of dorsiventral gametophytes of Blasia and Marchantia twisted 180° so that ventral surfaces bearing rhizoids faced the overlying air space (Fig. 3), and edges (new growth) of gametophytes of the fern Ceratopteris richardii responded similarly. (Because the fern gametophytes responded in the same way as previously-published fern gametophyte studies,^24,25 images of C. richardii are not shown.) In all cases of gametophytes cultivated on agar surfaces, placement of culture dishes right side up or upside down yielded results expected if direction of illumination were the primary control on gametophyte orientation (Table 1). When illuminated only from below, Coleochaete lost normal attached dorsiventral orientation and did not produce hairs diagnostic for the order Coleochaetales (Fig. 4).

Figure 1.

Experimental system. All tested species were treated with the same level of cool white illumination deployed only from above (left), only from below (center), or from multiple directions (right). Equal numbers of replicate culture dishes of each species were positioned right side up or upside down. In the cases of the thalloid liverworts Blasia and Marchantia, culture dishes were also positioned on their sides, to explore effects of surface contact.

Table 1.

Expected and actual responses of mature dorsiventral gametophytes to variation in direction of illumination under constant gravity. Italicization indicates results critical to distinguishing the effects of illumination direction from those of gravity.

	Initial body orientation	Expected response if gravity is major control on orientation of new growth	Expected response if direction of illumination is major control on orientation of new growth	Observed response
Direction of Illumination Treatments
I. Illumination only from above	A. Dishes upside down; rhizoid tips extending upward	180° inversion of new growth with rhizoid tips extending downward	180° inversion of new growth with rhizoid tips extending downward	180° inversion of new growth with rhizoid tips extending downward
	B. Dishes right side up; rhizoid tips extending downward	No change in body orientation	No change in body orientation	No change in body orientation
II. Illumination only from below	A. Dishes upside down; rhizoid tips extending upward	180° inversion of new growth with rhizoid tips extending downward	No change in body orientation	No change in body orientation
	B. Dishes right side up; rhizoid tips extending downward	No change in body orientation	180° inversion of new growth with rhizoid tips extending upward into the overlying air space	180° inversion of new growth with rhizoid tips extending upward into the overlying air space
III. Illumination from multiple directions	A. Dishes upside down; rhizoid tips extending upward	180° inversion of new growth with rhizoid tips extending downward	Growth in multiple orientations; rhizoid tips extending in multiple directions	Growth in multiple orientations; rhizoid tips extending in multiple directions
	B. Dishes right side up; rhizoid tips extending downward	No change in body orientation	Growth in multiple orientations; rhizoid tips extending in multiple directions	Growth in multiple orientations; rhizoid tips extending in multiple directions

Figure 2.

Responses of Sphagnum compactum gametophores to experimental variation in direction of illumination. (A) Orientation of gametophores experimentally illuminated only from above. Gametophores elongated toward the light source. (B) Orientation of gametophores that had initially been illuminated from above, then experimentally illuminated only from below for a 2-month growth period. All gametophores that had initially extended toward the light source curved 180° and grew downward into the agar medium. Older growth occurring while illuminated from above shown in rectangle; newer growth occurring during illumination from below shown in oval. Direction of new growth indicated by downward-pointing arrow. (C). Orientation of gametophores that had initially been illuminated from above, then experimentally illuminated from all directions for a 2-month growth period. On average, about half of the gametophores continued to extend upward (upward-pointing arrow) and about half curved 180° and grew downward into the agar medium (downward-pointing arrow).

Figure 3.

For figure legend, see page 8.

Figure 4.

Coleochaete orbicularis responses to experimental variation in direction of illumination. (A) During a period of experimental illumination only from above, monostromatic disks grew from single-celled asexual zoospores that attached to the lower surfaces of culture dishes. Such disks produced narrow, colorless, hairs several hundred micrometers long that are diagnostic for the genus. These hairs extended upward into the medium and were thus not visible in this plane of focus. (B) New growth originating from zoospores produced during a period of experimental illumination only from below occurred as irregular masses of cells that were not attached to culture dish surfaces and lacked diagnostic hairs. (C) New growth originating from zoospores produced during a period of experimental illumination from all directions occurred as irregular masses of cells that were not attached to culture dish surfaces and lacked diagnostic hairs.

Sphagnum compactum

In all culture dishes positioned right side up, and illuminated only from above for a 2-month long period, 100% of new growth was oriented with basal portions embedded in the agar substratum and terminal capitula facing the air-filled space (Fig. 2a). Branch fascicles were oriented downward (away from the source of illumination), as normally occurs in nature. By contrast, in all culture dishes placed right side up and illuminated only from below, at the end of the treatment period, in 100% of gametophores new growth had curved 180° downward so that terminal capitula had grown into the agar and fascicles were oriented upward, away from the source of illumination (Fig. 2b). In all culture dishes placed right side up or upside down, and illuminated from multiple directions, at the end of the treatment period, ∼50% of gametophores had curved 180° downward and terminal capitula had grown into the agar substratum, and ∼50% of gametophores continued growth into the air-filled space (Fig. 2c).

In all S. compactum culture dishes placed upside down, and illuminated only from above, at the end of the treatment period 100% of new gametophore growth was oriented into the agar and toward the direction of light, while fascicles extended in the direction of the air-filled space (away from the direction of light). In all culture dishes that were positioned upside down and illuminated only from below, at the end of experimental treatment, 100% of the gametophores had continued to extend into the culture dish air space.

None of the replicate gametophore cultures of S. compactum exposed to PAR irradiance levels of 2505 and 2755 µmol photons·m⁻² ·s⁻¹ survived. Plants occupying culture dishes that were positioned right side up and received PAR illumination from above, at levels of 250, 367, 434, and 1201 µmol photons·m⁻² ·s⁻¹ displayed normal axial orientation, with capitula extending into the air space.

Blasia pusilla, Marchantia polymorpha

Dorsiventral thalloid gametophytes of the 2 liverworts studied responded similarly to experimental treatments; for this reason, results are illustrated only for B. pusilla. In all B. pusilla culture dishes positioned right side up and illuminated from above, 100% of the new growth of all gametophytes displayed normal dorsiventral orientation, with dorsal surfaces of new growth facing an air-filled space and rhizoid tips extending into the agarized medium. In all Marchantia culture dishes placed right side up and illuminated from above, 100% of new growth of all gametophytes likewise displayed normal dorsiventral orientation, with dorsal surfaces of new growth facing an air-filled space and rhizoid tips extending into the agarized medium. In other words, when culture dishes were placed right side up and illuminated only from above, new growth did not undergo change in body orientation during the 4-week treatment period. New growth of both liverworts produced gemmae on the dorsal surface, facing the source of illumination.

In all Blasia and Marchantia dishes positioned upside down and illuminated only from below, no growth reorientation was observed; ventral surfaces of new growth of 100% of gametophytes were oriented toward the culture dish lid. In all culture dishes of Blasia (Fig. 3A) and Marchantia dishes that were positioned upside down and illuminated from above, for 100% of gametophytes, new growth occurring during the 4-week treatment period responded to change in illumination direction by twisting 180°. When illuminated only from below, in all culture dishes of Blasia (Fig. 3B) and all dishes of Marchantia, for 100% of gametophytes, new growth in dishes positioned right side up had likewise twisted 180°, so that the ventral surfaces faced upward and rhizoid tips extended into the air-filled space, away from the direction of illumination.

When illuminated from multiple directions, in all dishes of Blasia and Marchantia that were positioned right side up and in all dishes of Blasia and Marchantia that were positioned upside down, for 100% of gametophytes, new growth produced during the 4-week experimental period was oriented in multiple directions. When dishes were positioned on their sides to evaluate the relative impact of surface contact on body orientation, by the end of the 4-week experimental period, 100% of new growth of both B. pusilla (Fig. 3C) and M. polymorpha had twisted 90° so that only the edges of these plants were in contact with the growth medium surface, and 100% of rhizoids extended in the direction away from light.

Coleochaete orbicularis

Coleochaete orbicularis displayed different morphology depending on the direction of illumination. In all culture dishes illuminated only from above, 100% of individuals displayed the morphology typical of specimens collected from natural aquatic environments, namely monostromatic disks attached to surfaces, with hair cells extending upward into the overlying liquid medium (Fig. 4A). In all culture dishes illuminated only from below (Fig. 4B), and in all culture dishes illuminated from all directions (Fig. 4C), 100% of individuals occurred as floating, irregularly-shaped aggregations of hairless cells.

Ceratopteris richardii

In all culture dishes placed right side up and illuminated only from above, at the end of the treatment period, archegonia and antheridia had developed normally on 100% dorsal surfaces of new growth of C. richardii gametophytes and no rhizoids were observed to extend into the overlying air-filled space. In contrast, in all C. richardii culture dishes placed right side up and illuminated only from below, at the end of the treatment period, in 100% of gametophytes the ventral surfaces of new growth at gametophyte edges faced upward and rhizoids on those surfaces extended into air-filled space. In all dishes positioned right side up and all dishes positioned upside down that were illuminated from multiple directions, dorsal surfaces of new growth were oriented in multiple directions. In all culture dishes positioned upside down, and illuminated only from below, at the end of treatment 100% of the new growth produced dorsal surfaces toward the air-filled space; no rhizoids were observed to extend into the air-filled space. However, in all culture dishes positioned upside down, and illuminated only from above, 100% of the new growth occurred with the dorsal side facing toward the agar surface and rhizoids extending into the air-filled space.

Discussion

Our observations of C. richardii gametophyte responses to directional light treatments, similar to those previously reported for another fern,^24,25 indicate that our experimental design was suitable for exploring environmental effects on gametophyte orientation in other seedless plants. As in the case of ferns, our results for representative bryophytes were consistent with the hypothesis that the direction of illumination exerts primary control over orientation of both upright axial and prostrate thalloid gametophytes. Our observations of C. orbicularis were consistent with the hypothesis that early-diverging embryophytes inherited light-directed orientation responses from ancestral streptophyte algae. This hypothesis is also supported by a report that branching patterns of some (though not all) Chara species seem to be controlled by directional light.²⁰

Bryophytes are generally known to readily reproduce asexually, by the continued growth of body fragments, illustrated by methods used in this study to generate clonal populations of thalloid liverworts and Sphagnum moss for experimentation. Control of gametophyte orientation by light direction would have allowed earliest plants to produce from such fragments multicellular gametophytes having ventral rhizoids that function in attachment to the substratum and dorsal reproductive structures, whether the substrate was horizontal, angled, or vertical. Diverse modern liverworts produce dorsiventral gametophytes that display the same morphology whether they grow on vertical or horizontal surfaces. Were gravity the controlling factor in determining gametophytic orientation, gametophytes occurring on vertical surfaces might be expected to display orientations different from those occurring on horizontal surfaces.

Control of gametophyte orientation by illumination direction also allows gametophytes that become spatially disoriented by wind or water to reorient by means of new growth. Such responses would have been particularly useful during the Cambrian-Ordovician time period when climatic conditions were favorable for plant growth,¹ but unconsolidated, disturbance-prone sandy substrata were common.^7,8 The Cambrian-Ordovician time period is associated with the occurrence of microfossils resembling the terrestrial growth form of the modern streptophyte alga Coleochaete²⁶ and microfossils interpreted as evidence of early, bryophyte-like land plants.⁴⁵ The results presented here indicate that early terrestrial streptophytes, whether classifiable as streptophyte algae, embryophytes, or something in-between, probably possessed the ability to respond to physical disorientation by reorienting new growth. Such reorientation would allow early terrestrial streptophytes to re-establish substratum association of ventral rhizoids and production of dorsal reproductive structures in a position most favorable for wind dispersal of sexual and asexual propagules. Mechanisms for reorientation are also adaptive for modern seedless plants that produce free-living, surface-growing, photosynthetic dorsiventral gametophytes. Likewise, the dorsiventral gametophytes of non-photosynthetic species of Aneura (formerly Cryptothallus), which may occur several cm below the ground surface such as beneath moss,⁴⁶ may receive sufficient unidirectional light to control orientation responses. Similar growth responses to direction of illumination by axial gametophores of Sphagnum compactum suggest that the earliest axial land plants likewise benefitted from directional light control of gametophyte orientation.

Although some experts might regard the growth responses described here to represent photomorphogenesis and reserve the term “phototropism” for sporophytic plant bodies, according to Matsuoka et al.,⁴⁷ phototropism can be defined as “a mechanism to orient a whole plant to light.” This concept suggests that responses observed in the present study could also be interpreted as phototropism. The observations we report indicate that the species investigated possess light-sensing systems and signal transduction processes allowing the translation of variation in the direction of illumination into phototropism-like growth responses, and that responses occur during the generation of new tissues. This conjecture is supported by evidence for production of canonical phototropin by the early-diverging liverwort Marchantia polymorpha²³ since phototropin-based light-sensing is now firmly linked to higher plant phototropism responses.⁴⁸ Evidence for the occurrence of auxin regulation in 4 liverworts and the streptophyte alga Nitella,⁴⁹ polar auxin transport in charalean algae,⁵⁰ and genomic evidence for occurrence of short PIN5-type proteins in streptophyte algae such as Coleochaete orbicularis and long PIN1-type proteins in mosses suggests that part or all of a similar response pathway might have originated in streptophyte algae and have been inherited by earliest land plants.^51,52 These possibilities can be examined as additional genome sequence becomes available.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank S. Friedrich and K. Elliot for assistance with images, and S. Fields for laboratory assistance.