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.