Journ. Hattori Bot. Lab. No. 61 : 487-497 (Dec. 1986)

SPORE GERMINATION AND PROTONEMAL DEVELOP­ MENT OF FONTINALIS SQUAMOSA

JANICE M. GLIMEl AND BERND C. KNOOp2

ABSTRACT The germination and sporeling development of Fontinalis squamosa Hedw. was investigated at 3, 14, and 20°C and five light intensities. The sporeling type most closely resembles that of several acrocarpous mosses in that it produces both chloronema and caulonema. The sporeling forms suggest that the protonema form is adaptive rather than genetic, and the ecological implications of the con­ ditions affecting protonemal development are discussed.

INTRODUCTION A study of sporeling development may provide phylogenetic insight, and several researchers have classified germination types (Bopp 1968, Sood 1975, Nishida 1978, Nishida & Iwatsuki 1981, Nehira 1983). On the other hand, we may find that the pattern of development is dependent upon the ecological conditions and is therefore plastic and adaptable (Nishida & Iwatsuki 1982). The most morphologically plastic species are the aquatic ones, as exemplified by such species as Fontinalis antipyretica. Elssmann (1923- 25) has germinated the spores of this species, but germination of spores in Fontinalis has not been observed in the field, and little is known of the development. Fontinalis is known to have two types of spores in a capsule, and these seem to be comprised of functional and non-func­ tional spores; thus the condition has been termed pseudoanisosporous (see Mogensen 1978). The role of these aborted spores is not understood, and we do not know at what point they become non-functional. During its annual cycle. Fontinalis is subjected to submersion and exposure, with warm temperatures corresponding to its time of exposure. If its spores germinate under water, they could easily be washed away and the protonema would have little chance to establish itself on a substrate. On the other hand, if they germinate on an emergent rock, they are likely to be damp, at least early in the growing season, and the protonema would have a chance to become established and attached to a substrate. However, either of these conditions presents unique problems, and the present study is an attempt to determine some possible conditions under which the spores can germinate and the protonema can develop, so that we may postulate the adaptations and suggest where and when to look for germinating spores in the field.

1 Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, U.S.A. 2 Botanisches Institut, Universitat Heidelberg, Heidelberg, Germany. 488 Journ. Hattori Bot. Lab. No. 61 1 986

METHODS Fontinalis squamosa Hedw. was collected at Lydford Gorge, Devon, England (500 38'N, 4°?'W, sec. 502835) on 18 April 1982. Numerous capsules were present on these submersed mosses, and the collections were kept in plastic bags until 23 April, then washed in distilled water and stored in the dark at 11 °C. Germination Conditions: Young Spores Spores from six capsules were inoculated on Knop's medium plus micronutrients on 6 May by cutting the operculum from green capsules and removing spores with a fine glass needle. Four capsules were brown with medium-sized (l5- 18/-lm) brown spores and two were green with small (10 /-lm) greenish spores. Two plates of agar plus cellophane (Bopp 1980) were inoculated with brown spores and wrapped with aluminum foil, two plates with cellophane were inoculated with brown spores and not wrapped, and two plates with no cellophane were inoculated with the smaller greenish spores and not wrapped. One of each type of culture was placed at 20°C, 20 hr Jight/4 hr dark, 2100 lux (28.1 ,uE m-2 S-I, cool white fluorescent lamps) and at 14°C, 12 hr light/12 hr dark, 3000 lux (500 watt Osram HWLM lamp). Uncontaminated portions of plates were transferred to fresh plates when necessary. Spores were examined with light, fluorescence, and scanning electron microscopy to determine chlorophyll fluorescence, morphology, and apparent abortion. Germination Conditions: Mature(?) Spores Once germination occurred, additional spores were inoculated from older olive-green capsules on 28 May. Capsules were soaked in 10 % hypochlorite for 5 minutes, 10 seconds, or not washed to determine degree of sterilization needed. Hypochlorite was rinsed off in distilled water. No color was lost due to hypochlorite and there was no reduction in germina­ tion, but initial contamination was eliminated, so capsules for subsequent Fontinalis spore cultures were immersed for several minutes in 10 % hypochlorite. Spores were again of two sizes (green 13-22,um; brown 10- 13 ,urn). Spores from one capsule were divided among the six treatments, with two light intensities created on each plate by layers of white paper over half the plate, thus reducing genetic variability among treatments. Each plate was inoculated with three capsules and each treatment had five plates, so 15 capsules were used. The following culture conditions were chosen, based on those avail­ able at the Heidelberg laboratory: 3°C 120 lux variable day length, < 12 hr 20 lux variable day length, < 12 hr 14°C 1200 lux 12 hr light/12 hr dark submersed and not submersed 260 lux 12 hr light/12 hr dark submersed and not submersed 20°C 3200 lux 20 hr light/4 hr dark 1300 lux 20 hr light/4 hr dark 20°C 270 lux 20 hr Jight/4 hr dark (6 capsules, 1 per plate) Spore Maturation Capsules were stored in water at 14°C, 12 hr light/12 hr dark, 1200 lux for eight days (28 May to 5 June). Spores from several capsules were examined for stage of maturation and germination. Protonemata Cultures of germinated spores were maintained under the same conditions as for germina- J. M. GLIME & B. C. KNOOP: Spore germination and protonemal development 489 tion and the development of the protonemata was observed. Examination was with light microscope and fluorescence microscopy.

RESULTS Germination No differences could be noted between rate of germination and growth in the darker and lighter halves of plates, and no differences in germination were apparent between 14 and 20°C. Only the spores at 3°C with a short photoperiod failed to ger­ minate until much later (one culture took 15 days and the others did not germinate). Plates covered with water seemed to germinate at about the same rate as those on non-submersed plates, but extensive contamination made the submersed cultures impossible to evaluate. Germination Conditions: Young Spores Observations of capsules in early May revealed two kinds of spores: small (10 ,urn) greenish ones and medium-sized (15-18 ,urn) brown ones. When these spores were cultured at 20°C, 3000 lux on Knop agar, the brown ones required 18 days for germina­ tion, and the small spores did not germinate. The medium-sized brown spores became swollen and green about two days prior to distention (germination). No spores ger­ minated in the dark. Germination Conditions: Mature(?) Spores Observations of capsules in late May revealed three kinds of spores, with no more than two kinds in a single capsule. Again, the smaller spores were present, but these were now brown, and they were accompanied by medium-sized brown spores. In other capsules, there were large swollen green spores (25 ,urn) (Fig. I) and medium­ sized brown spores. No small brown spores could be identified among the germinating spores in either set of cultures, and the small spores seemed to have disintegrated. However, collapsed medium-sized brown spores swelled in water. The medium-sized spores cultured in early May (Young Spores) germinated, but those cultured in late May (Mature Spores) failed to germinate after 25 days. However a few of these became swollen and green. Spores that were swollen and green in the capsule germinated on agar in as few as 5 days at 14 and 20°C. Spores at 3°C required 15 days for the first discernible distention. Spore Maturation Spores that were retained in submersed capsules at 1200 lux for eight days were almost all swollen and green in the capsule. Some were oblong and a few had pro­ trusions, indicating germination in the capsule. A few green spores were 2-3 times the volume of the majority of the spores. The brown spores were nearly all collapsed and exhibited conspicuous trilete markings. A few capsules that were old, thick-walled, and dark in color had only disinte­ grated spores. Protonemata Following distention, the first septum usually formed at the base of the germ 490 Journ. Hattori Bot. Lab. No. 61 1 9 8 6 tube, but in some cases it formed across the center of the spore body. The first fila­ ments had perpendicular cell walls and were full of chloroplasts and very green, as in chloronemata. Only in contaminated cultures did these initial cells form brown walls, frequently with oblique crosswalls (Fig. 2). The apical cell of each filament always had less dense chloroplasts than other cells and its cell apex was colorless. These apical cells exhibited less fluorescence than other filament cells, whereas the spore body retained the greatest fluorescence (Fig. 3). Spores at 14°e usually produced one or two protonemal filaments, whereas those at 20 0 e produced two or three (Fig. 4). A fourth filament appeared on many 20 0 e sporelings, but this filament arose secondarily from the basal cell of one of the germ filaments. Once filaments reached 5- 8 cells in length, subsequent cells often developed oblique walls as they matured, but the apical cell always had a perpendicular wall. These oblique-walled cells had fewer and elongated chloroplasts, as in caulonemata (Fig. 5). The older cells became brown like germ rhizoids, and the nucleus could be seen as a lighter spot in the center of the cell. In some cases, these older primary filaments branched near the spore and the daughter filaments developed in the same way as the primary filaments. In addition to these identical basal branches, other side branches from more distal cells had a totally different character with thick, short, nearly iso­ diametric cells, full of bright green chloroplasts, and never turning brown (Fig. 5). These new filaments were positively phototropic and apparently negatively gravitropic, so that they grew to the opposite direction from the "germ rhizoids." These green "chloronema" branches grew not only from "germ rhizoids" but also from daughter rhizoids. The branching pattern varied widely even in the same culture plate. Some pro­ tonemata branched only near the spore whereas others branched only far away from it. At 20 0 e some protonemata formed several branches, obtaining in rare cases up to ten by 27 days (Fig. 3). Even the position of branching from the mother cell varied, coming usually from the middle, but also arising from either end.

FIG. 1. Electron micrograph of a large, swollen green spore from a capsule of Fontinalis squamosa. FIG. 2. Protonema from a culture of Fontinalis squamosa that has been contaminated with bacteria. Note oblique crosswalls. FIG. 3. Protonema from a culture of Fontinalis squamosa showing a) apical cell with less dense chloroplasts and colorless tip, and b) fluorescence in the same cells. FIG. 4. Protonemal filaments of Fontinalis squamosa from spores cultured at 20De, showing one, two, and three filaments arising from the spore. FIG. 5. Protonema cells of Fontinalis squamosa showing chloronema (ch) cells with perpendicular walls, dense chloroplasts, and short cells, primary caulonema (pc) cells arising from the germ rhizoid, and secondary caulonema (sc) cells arising from the primary caulonema. Note the oblique cross walls and sparse chloroplasts of the caulonema. FIG. 6. Orientation of all protonemata on one plate cultured at 14De showing growth away from the light source (left). FIG. 7. Protonemata cultured at 20De showing a) irregular growth of globose cells, b) droplets appearing on the surfaces of some cells. J. M. GUME & B. C. KNOOP: Spore germination and protonemal development 491 492 Journ. Hattori Bot. Lab. No. 61 1 9 8 6

Orientation and direction of later development of the sporelings was random in many cases, resulting in protonemata that had a tumbled appearance (Fig. 3). How­ ever, at 14°e in two cultures the first filament arose from the shaded side of the spore in all cases, and this resulted in all filaments being oriented in one direction (Fig. 6). Subsequent filaments could not occupy the same position on the spore, and thus they arose from other sides of the spore and then grew negatively phototropically. At places on the plate where there was no cellophane, these filaments grew at an angle of 55° into the agar, whereas the light source made an angle of 35° with the surface of the agar. On the other hand, cultures at 20 0 e grew aerially toward the light (at 90° to the agar). Although no differences in germination due to culture conditions were evident, protonemal development differed. There was no development beyond distention at 3°e. The best growth occurred at 14°e, with more extensive growth on the unshaded side of the plate. At 20 0 e the protonemata formed irregular balls of irregular fila­ ments with irregular cells (Fig. 7). Many cultures at 14°e produced buds, whereas no buds were produced at 20°e. When protonemata cultured at 20 0 e were transferred to 14°e, some the protonemata produced buds, but no buds were produced on plates transferred from 14° to 20 0 e, and growth all but ceased in the latter transfer. Nevertheless, one culture in a later experiment maintained at 22°e produced buds (Glime in prep.). Protonemata maintained at 20 0 e formed brown walls nearly to the tip, presuma­ bly correlated with their slow growth, even though these were not contaminated as were those mentioned earlier. These brown filaments produced external bubbles of liquid (Fig. 7) from cells of various positions. It would be worthwhile to investigate whether these bubbles function in cementing the rhizoids to the substrate. Bud development first appeared after three months and occurred in a manner similar to protonematal branching. It appeared to originate from the basal cell of the secondary caulonema. The young, few-celled bud was nearly colorless due to the few, small chloroplasts. Moreover, these buds formed rhizoids when they reached about eight cells, long before leaf primordia were visible. By the time the leaves were formed, rhizoids were well-developed into a star of filaments. These rhizoids at first grew in all directions, but later they grew negatively phototropically, as do rhizoids on adult gametophores (Glime in prep.). One spore seems to produce only one or few gametophores. Yet in a single plate in a later experiment (Glime in prep) at least ten gametophores arose in the same plate simultaneously, but only that one plate out of 113 had buds.

DISCUSSION Developmental Type Several authors have attempted to classify protonemata on the basis of their development patterns, but Fontinalis has never been classified or described in detail. The developmental patterns exhibited in our study suggest that the growth form of J. M. GUME & B. C. KNOOP: Spore germination and protonemal development 493 the protonema is plastic and that patterns exhibited in the laboratory may not occur or succeed in nature. Nevertheless, certain observations under similar culture conditions are important. Fontinalis squamosa is heterotrichous, producing both chloronema and caulonema. This character is present in all known protonemata of acrocarpous mosses, but is not so common among the pleurocarpous taxa already investigated. Kanda and Nehira (1976) showed that representative members of the pleurocarpous Hypnobryales are not heterotrichous. The aquatic Leptodictyum riparium (Hedw.) Warnst., for example, has no caulonema and produces only one protonema type. Nishida and Iwatsuki (1982), on the other hand, felt that the protonema type was adaptive and reflected habitat more than taxonomic affinity, as evidenced in Entodon. Of the illustrations we have examined, the pleurocarpous isobryalean Fontinalis squamosa protonema most closely resembles that of the acrocarpous Leucobryum bowringii Mitt. (Nehira 1964). In both species, the protonema increases in cell number and develops into a caulonema with brown walls and oblique septa. The chloronema has globose chloroplasts, whereas the caulonema has elongate plastids. The apical part of the protonema lacks completed chloroplasts. The bud arises on the caulonema and several long brown rhizoids develop at the base of the bud before the leafy shoot arises. Some protonemal branches have ovoid or globose cells. In some cases, the chloronema can be recognized as upright green protonemata, as in L. bowringii. Another similar species is Trichosteleum aculeatum Broth. & Par. (Nehira 1965). This species, like Fontinalis, has two spore types, including smaller spores with fewer chloroplasts, and these spores do not germinate. However, in the protonema, chloro­ plast shape does not differ between caulonema and chloronema, but the main fila­ ment has oblique septa. Claopodium assurgens (Sull. & Lesq.) Card. has a similar developmental pattern to that of L. bowringii and F. squamosa, with curved branches like the latter (see Fig. 5), but differs in having bud rhizoids delayed until after a leafy shoot develops (Neh ira 1965). The pattern of cell division between the spore body and the germ tube in Fontinalis squamosa is likewise dependent upon cultural conditions. In some cases the division occurs within the spore body and resembles the pattern found in the acrocarpous Pogonatum aloides (Sood 1975), but more often division occurs at the spore wall, being more typical of such pleurocarpous mosses as Forsstroemia trichomitria or Macromitrium gymnostomum (Nehira 1983). The pattern of development of the caulonema differs somewhat from that of other bryophytes thus far examined. We have pointed out that the green chloronema branches grew not only from germ rhizoids but also from daughter rhizoids. This means that the secondary branches and the primary filaments are physiologically identical. Therefore we suggest the neutral designations of "primary caulonema" for those arising directly from the spore, "secondary caulonema" for the daughter branches, and "chloronema" only for the bright green branches with nearly iso­ diametric cells. Nishida (1978) has described thirteen protonema types based on patterns of 494 Journ. Rattori Bot. Lab. No. 61 1 986 spore germination, presence of thallose or massive protonema, special funnel or vesiculate protonema, cell shape, presence of chloronema, and presence of primary rhizoid. If one uses the Nishida key to types, one must choose the Macromitrium type for Fontinalis squamosa because the chloronema cells are short cylindrical or semiglobose. The caulonema cells are elongate. The unusual developmental patterns observed at 20 0 e can be compared to the somewhat irregular behavior of Funaria at higher temperatures. Kofler (1971) found that higher temperatures augmented the gravitropic response in protonemata, and in our cultures the protonemata grew upward at 20 0 e instead of away from the light as they did at 14°C. Likewise, the nearly round cells we found at 20 0 e have been observed in Funaria cultures at 22°C (Raeymaekers unpub. data). Ecological Implications It appears that Fontinalis squamosa is an opportunist by providing viable spores over a relatively long period of time. One way to accomplish this is by having fertiliza­ tion occur over an extended time period. We already know (Glime 1984) that Fontinalis dalecarlica Br. & Schimp. is an opportunist that produces its archegonia over a span of several months. The presence of archegonia and antheridia in several stages of de­ velopment on F. squamosa at the time of collection suggests that it likewise produces gametangia over a long time span. This observation correlates with the fact that the capsules from Lydford Gorge were not of equal maturity, as evidenced by some with swollen green spores and others with only smaller brown ones. The fact that the fruit­ ing F. squamosa was found at the edge of the stream where the plant would be partly in and partly out of the water during some seasons suggests that it could have an extended period of fertilization similar to that which has been suggested for Fontinalis dalecarlica (Glime 1984). Such a position could likewise provide the proper condi­ tions to hydrate different capsules on the moss clump at successive points in time as the water level drops. Having a series of archegonial maturation and fertilization dates, subsequently causing capsule maturation throughout an extended period of time, could provide the opportunity for spores to be ready for germination at several points in time. The germination of swollen brown spores after 18 days and swollen green spores after 5 days in our study further suggests that both capsule ripening and after ripening of spores are possible in the same population, thus permitting dispersal to occur when­ ever conditions permit it. Elssmann (1923- 1925) found that spores of Fontinalis antipyretica from submersed capsules had germinated and developed up to six cells within 18 days. In his experiments, spores in capsules exposed to air ripened several weeks earlier than those under water, again providing variation among individuals occupying a range of water depths. The ability of spores to germinate at temperatures of 3, 14, and 200 e suggests that this species is ready and able to germinate at nearly any season that its spores get dispersed. This is further supported by the light levels in our experiments, ranging from 49 lux at 3°C to 3000 lux at 20oe, and correlating with seasonal light condi- J. M. GUME & B. C. KNOOP: Spore germination and protonemal development 495

tions in nature. Germinability on both humid and submersed cellophane suggests that the moss could take advantage of almost any moisture condition. Nevertheless, the rate of subsequent development seems to be highly temperature dependent. These germination capabiiities raise questions regarding the necessity for such flexibility. Fontinalis has a well-developed double peristome with teeth that respond to drying. Should we not expect its dispersal to be timed to some meteorological condition that is suitable for dispersal and germination? But if one tries to remove the operculum from a mature capsule, one finds that its abscission ring is not very weak, and the capsule in fact is more likely to break than to dehisce its operculum. Elssmann (1923-1925) likewise found that the operculum did not dehisce after many months in culture. Our observations of field populations of Fontinalis have never revealed capsules with the operculum missing and the peristome intact. In our samples, no capsule that was missing an operculum appeared to have lost it by dehiscence. All open capsules were obliquely opened, apparently due to abrasion or other damage, and none of these retained any part of the peristome. This observation for Fontinalis is further complicated by the disappearance of virtually all the winter capsules of F. novae-angliae Sull. during spring runoff in New Hampshire, USA (Glime, pers. obs.). It appears that abrasion is the major mechanism of capsule opening and spore dispersal. Since major events of abrasion are as un­ predictable as the weather, having capsules simultaneously at several stages of ma­ turity and spores with wide-ranging capabilities for germination and protonema de­ velopment would be advantageous. The fact that growth of the protonema is best at 14°C implies that spring or autumn, with mean temperatures near 14°C, would favor development of proto­ nemata and buds in nature. This is supported by several observations: 1) Tempera­ tures of 20°C are detrimental to mature gametophores of Fontinalis species (Glime 1982, 1984); 2) At 14°C desiccation is less likely than at warmer temperatures, and submersion is more likely to occur, due to spring or autumn rains; 3) At 3°C, the spore remains swollen and healthy but protonemal development does not occur; 4) Capsules are mature during late spring; 5) Abrasion of the capsule, as an aid to spore dispersal, is most likely to occur as winter ends and snow melts or spring spates create rapid, turbid waters; 6) In spring, stream temperatures rise rapidly to 100l2°C following snow melt, particularly in mountain streams. If the gametophore stage developed at temperatures of 15°C or greater, but not at lower temperatures, it could soon be debilitated by the warm summer water before it was sufficiently established to survive. On the other hand, if dispersal were to occur in late winter or early spring, the sporeling could take advantage of the entire spring growing season before being subjected to the heat and desiccation of summer. The fact that so few protonemata produced buds under any condition suggests that we have not determined the complete needs of this species. Since the one plate in 113 at 22°C that produced buds produced them on many filaments (Glime in prep.), it is possible that some hormonal induction factor is necessary. Such has been noted in Funaria (Bopp 1976). This induction factor could easily be exogeneous in the natural 496 Journ. Hattori Bot. Lab. No. 61 1 9 8 6

habitat. On submersed rocks and wood where the moss grows, growth of bacteria (see Spiess et a1. 1982) or algae might provide stimulating hormones or vitamins. The ability of the spore to remain on its substrate in moving water remains un­ explained. Evidence that the spore can germinate in a thin film of water on a Petri plate and continue protonemal development and produce buds in humid air without submersion suggests that we should look for spores and protonemata on emergent, but damp, surfaces. This, however, does not fit with our evidence that the best develop­ ment occurs at 14°C, a temperature at which we might expect maximum water depth in most of its geographic range. If, on the other hand, it germinates and develops during lower temperatures of autumn, the capsules must retain the spores throughout the summer. Dates on the limited herbarium records of capsules suggest that this is not the case. One possibility remains to explain a mechanism permitting springtime germina­ tion above water. Certainly fast-moving water will splash to emergent rocks. If spores do indeed germinate there and become established, the protonema must finally grow to reach the water where the mature plant must spend most of its life cycle. This necessity could explain the variable growth patterns of the protonema, permitting at least some of the buds to be opportunists. Further adaptive behavior could be attributed to the tropic behavior of the pro­ tonemata. The ability of the young protonema to grow negatively phototropically on a horizontal surface, then positively gravitropically (or perhaps negatively photo­ tropically), and finally positively phototropically when chloronema branches arise might permit the protonema to establish itself on a rock surface, then grow around the rock and toward the water (away from the light). One would predict the greatest success of establishment on the downstream side of a rock where it is most protected from abrasion. The limited protonemal branching, especially at lower temperatures, relative to many other mosses such as Funaria, would make a more stream-lined surface and reduce drag caused by flowing water. However, since different growth forms were apparent under different conditions, we cannot suppose what the moss does in nature. Growth by mature gametophores is at first favored and then inhibited at 20°C in several species of Fontinalis (Glime 1982). Since Fontinalis squamosa was producing capsules in April and spores were not ripe, one would predict that the gametophore bud stage in nature might not be reached until late July or August, perhaps even later. Our cultures produced gametophores near the end of August. A delay in de­ velopment due to warm temperatures would increase the chances that the young gametophore would not be present to suffer desiccation during the reduced stream flow of summer. We found that dry protonemata of Fontinalis squamosa will rehy­ drate and become turgid and green. Perhaps the protonemal stage is more resistant than the very thin-walled young bud. One would expect this to be particularly true of the brown-walled caulonemata from which the buds develop. These results can only serve as a model for the potentials of the young gameto­ phyte of Fontinals squamosa. In order to assess the adaptive significance, further ex- J. M. GLIME & B. C. KNOOP : Spore germination and protonemal developmen 497 periments and field observations will be necessary. In particular, the effects of mois­ ture, flow, and associated organisms such as bacteria and algae should be assessed.

LITERA TURE CITED

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