Svensk Botanisk Tidskrift: Volym 62: Häfte 3, 1968

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Svensk Botanisk Tidskrift. Bd 62, H. 3. 1968.

CAROTENOIDS EXTRACTED FROM MYCOBIONTS

OF COLLEMA TENAX, BAEOMYCES ROSEUS,

AND SOME OTHER LICHENS.

BY

ELISABET HENRIKSSON and L. C. PEARSON.

(Institute of Physiological Botany, University of Uppsala, and Ricks College, Rexburg, Idaho.)

The first investigations of lichen carotenoids were performed on intact lichens. As early as 1902, Kohl reported the presence of

carotene in Baeomyces roseus. Later, Seshadry & Subramanian (1949)

and Murty & Subramanian (1959) reported that the lichen Roccella montagnei contained a relatively large amount of carotenoids.

The carotenoids of lichens can be present either in the phycobiont or the mycobiont. In an earlier investigation, 19 samples of intact lichens representing the genera Collema and Leptogium were examined and carotenoids demonstrated to be present (Henriksson 1963). In the same study, an isolated culture of the mycobiont of Collema tenax was also shown to contain carotenoids.

The research reported in the present paper was undertaken for the purpose of characterizing the carotenoids in some lichen fungi in order that eventual identification of them might be possible.

Materials and methods.

Besides the Collema tenax mycobiont used in the preliminary investiga­ tion, mycobionts of Baeomyces roseus (two strains, 19 and 20). Lecidea coarctata (54), Lecidea enieromorpha (69), and Cladonia cristatella (80) were analyzed. The cultures were obtained from Prof. V. Ahmadjian, Clark University, Worcester, Massachusetts; strain numbers are those of the collec­ tion at Clark University. These mycobionts were chosen because they are all pigmented and also, according to traditional classification, taxonomically diverse. The cultures were all maintained on maltextract-glucose-casein agar (Henriksson 1958), and they were grown in a light and temperature control chamber with continuous illumination at 3000 lux light intensity and 17°C.

442

Pigments were extracted from the material after grinding in either acetone, methanol, or methanol-hexane (2:1) mixture using approximately 10 to 30 ml solvent. If necessary, the mixture was centrifuged. After transferring to a small separatory funnel, about 5 ml hexane was added if it was not already present in the mixture. About 20 ml distilled water or 10% NaCl solution were added in small aliquots during gentle rotation. As the carote­ noid pigments separated from the water-acetone or water-methanol layer, the latter was drawn off. The colored hexane layer was washed three or four times with distilled water to remove all traces of acetone or methanol. The hexane was dried either with anhydrous Na2S04 or by deep-freezing.

Three chromatographic techniques were used in studying the pigments: paper, column, and thin sheet chromatography. Dry concentrated pigments were spotted on Whatman No. 1 “special” filter paper in reduced light and developed in the dark by an ascending front of | % proponol in hexane. Development took an average of approximately one hour during which time the front had advanced about 25 cm. Powdered sugar or A1203 was the sorbent used in column chromatography. For the sugar column, the develop­ ing fluid was the same as for the paper chromatograms; and for the A1203 columns, petroleum ether was used. In the third method, a thin sheet of Kieselgel G was applied to the surface of a rectangular piece of plate glass 5 x 20 cm in size. To ensure applying an even coat of gel to the glass, a brass applicator of the type described by Stahl(1962) was used. I he pigments were spotted in subdued light and allowed to develop in the dark until the front had ascended exactly 10 cm; this took about 45 minutes. Developing fluid was a 50-50 mixture of petroleum ether (90-110°) and benzol. The pigmented zones were scraped from the thin sheets of gel and dissolved in methanol. After elution, the pigments were transferred to hexane, and examined with the spectrophotometer.

Absorption spectra of the carotenoids in hexane solutions were ascer­ tained with Zeiss spectrophotometers M4Q III and PMQ II in the wave length range of 250 to 525 nm.

Results.

In a preliminary report on the physiology of the mycobiont of Collema tcnax (Henriksson, 1958), it was reported that the fungus changed color when growing in the dark. In the control chamber, the cultures had a pink color when grown in light but were yellowish or olivish-grey when grown in the dark under otherwise similar condi­ tions. Experiments with both strains of Baeomyces roseus in the present study have given the same results. It is questionable, however, if any such change occurs in Lecidea spp. and Cladonia cristatella, since even in light the pink color observed in the investigated strains is not especially pronounced.

Columns of sucrose and A1203 and thin sheet chromatograms

CAROTENOIDS EXTRACTED FROM MYCOBIONTS

443

suu nm

Fig. 1. Absorption spectra in hexane of carotenoids extracted from the mycobiont of Collema tenax. Optical density of the solutions has been corrected to equal that of the extract from 0.1 g (dry weight) of plant material dissolved in 1 ml hexane. Total extract

---, pink pigment---, and yellow pigment--- .

showed that extracts of Collema tenax and Baeomyces roseus separated into at least two colored zones and into two fluorescent zones. In order of increasing adsorptive power the zones are listed as follows:

fluorescent zone, pink zone, yellow zone, fluorescent zone.

444

In extracts of Collema tenax the pink color dominated over the yellow one. Fig. 1 shows spectrograms over the pink and yellow pigments eluted from a thin sheet chromatogram. The figure shows also the unseparated extract of Collema tenax. The spectrum of the pink pigment was shown to be essentially identical to that of the total extract. According to Goodwin (1955) the absorption spectrum of

lycopene gives maxima in hexane at 446, 474, and 506 nm, and so it seems possible that the pink pigment consists chiefly of lycopene. Comparisons between the pink pigment in extracts of Collema tenax and in that of Baeomyces roseus have been made on paper chromato­ grams. These chromatograms gave an Rf value of 0.90 for the pink color of Collema tenax and 0.85 for that of Baeomyces roseus (20) showing that these pigments can be considered to be of the same type.

The yellow zone in the Ihin sheet chromatograms of Collema tenax was so faint that a good spectrograph of it was not possible (Fig. 1). In extracts of the Baeomyces strains, the yellow pigment seemed to be more abundant but we did not obtain enough to examine in the spectrophotometer. However, when some of this pigment was mixed with pure /3-carotene, and the mixture chromatogrammed on paper, there was distinct separation of two pigments, one of them identical to the /3-carotene, indicating that in spite of some similarities to ß- carotene, the yellow pigment investigated was not identical to ß- carotene. In addition, according lo Ihe list of Goodwin (1955) on the

adsorption power of different plant carotenoids, the carotenes are lesser sorbed than lycopene. The Rf values in sucrose columns with a developing fluid of 1:1 mixture of petroleum ether (90—110 ) and benzol were 0.06 for the Collema tenax extract and 0.1 7 for Baeomyces roseus (20), whereas in the same column, the pink zone of the Collema tenax extract had an Rf value of 0.85. Such a strongly sorbed pigment could hardly be a carotene. On the other hand, the yellow pigments of C. tenax and B. roseus were probably identical. A similar yellow carotenoid was also extracted from Lecidea coarctala (54), L. entero- morpha (69), and Cladonia cristatella (80).

Spectrographs of hexane extracts of Baeomyces- and Lecidea- species (Fig. 2) gave peaks in the range of 250-300 nm and 320-380 nm, and the maxima indicated the occurrence of phytoene and phyto- fluene. Phytoene has maxima in hexane at 275, 285, and 296 nm and phytofluene at 332, 348, and 367.5 nm (Goodwin 1955).

Phytoene and phytofluene have very low adsorptive power; there­ fore, the fluorescent zones which had the lowest adsorption in the

CAROTENOIDS EXTRACTED FROM MYCOBIONTS

445

Fig. 2. Absorption spectra in hexane showing the occurrence of phytoene and phyto-fluene. Extracts from the mycobionts of Baeomyces roseus (19) ---, Baeomyces roseus (20)---, Lecidea enteromorpha (69)--- , and Lecidea coarctala (54)--- .

The extracts are the same in the two diagrams.

446

chromatograms consisted, probably, of a complex of phytoene and phytofluene.

There were also water-soluble pigments present in all five species, but they were not examined spectrophotometrically. It was observed that the odors distinctive of each mycobiont culture were associated with the water-soluble pigments.

Discussion.

Photoinduced carotenoid synthesis which has been demonstrated for Collema and Baeomyces is rather common in fungi and has been reported in several cases (Carlile 1965).

It is probable that the biosynthesis of the carotenoid pigments in the lichen fungi investigated here follows the common primary pathway: phytoene -> phytofluene -»• f-carotene -*• neurosporene lycopen (Goodwin 1965). It is also likely that the pink color of Baeomyces and

Collema is lycopene (Fig. 1), although this pigment has not been

found to be the major light absorbing pigment in other fungi.

Besides the presence of the pink pigment in both genera, there are other similarities between Collema and Baeomyces: they have similar temperature requirements when grown in the laboratory (Thomas

1939, Henriksson 1964), cell shape is similar, both require relatively

long acid hydrolysis before they can be satisfactorily stained with azure A, both are multinucleate with very small nuclei, and the water-soluble substances from pure cultures of both have a distinct naphthalene odor. It is likely that the two fungi are genetically more closely related than the traditional taxonomic treatments indicate. I he superficial differences of the two intact lichens, caused largely by the genetic differences of the host algae, likely obfuscate the true genetic relationships which studies of the mycobionts grown under essentially identical conditions, on the other hand, can reveal.

Summary.

Carotenoids were found in all the lichen fungi investigated: Baeo­ myces roseus, Collema tenax, Lecidea coarctata, L. enteromorpha, and Cladonia cristatella. From them a relatively strongly sorbed, uniden­ tified, yellow carotenoid was extracted. In extracts from Baeomyces and Collema, a pink pigment which might be lycopene was also separated on paper, sugar columns, and kieselgel G. Presence of

phyto ene and phytofluene was demonstrated. I he study indicated that the mycobionts of Baeomyces and Collema may be more closely related to each other than has been indicated by traditional taxonomic studies.

CAROTENOIDS EXTRACTED FROM MYCOBIONTS

447

Acknowledgments.

We wish to express our appreciation to Professor Nils Fries, Head of the Institute of Physiological Botany, for the use of facilities, to Dr. Sven-Olof Norbergfor help with the thin sheet chromatography technique, and to the National Science Foundation (U.S.) and the Administration of Ricks College for financial aid as well as time to carry out the study. Financial aid was also received through a scholarship by Selma Andersson from Uppsala Uni­ versity.

LITERATURE CITED.

Carlile, M. J., 1965: The photobiology of fungi. — Ann. Rev. Plant Phys. 16: 175.

Goodwin, T. W., 1955: Carotenoids. — Modern Methods of Plant Analyses 3: 272.

__»__; 1965: The biosynthesis of carotenoids. — Proc. Plant Phenolics Group Symposium (Leeds, April 1964) 37.

Henriksson, E., 1958: Studies in the physiology of the lichen Collema. II. A preliminary report on the isolated fungal partner with special regard to its behaviour when growing together with the symbiotic alga. — Svensk Bot. Tidskr. 52: 391.

__»_t 1963: The occurrence of carotenoids in some lichen species belonging to the Collemataceae. — Physiol. Plant. 16: 867.

__»_, 1964: Studies in the physiology of the lichen Collema. V. Effect of medium, temperature, and pH on growth of the mycobiont. — Svensk Bot. Tidskr. 58: 361.

Kohl, F. G., 1902: Untersuchungen über das Carotin und seine physiolo­ gische Bedeutung in der Pflanze. — Verlag Borntraeger, Leipzig.

Murty, T. K. & Subramanian, S. S., 1959: Isolation of Carotene from Roccella montagnei. — J. Scientific & Industrial Research 18 B: 4.

Seshadry, T. R. & Subramanian, S. S., 1949: Chemical investigation of Indian lichens. VIII. Some lichens growing on sandal trees (Ramalina

layloriana and Roccella montagnei). — Proc. Indian Acad. Sei. 30 A:

15.

Stahl, E., 1962: Geräte zur Dünnschicht-Chromatographie und ihre Hand­ habung. — In: Dünnschicht-Chromatographie, ein Laboratoriums­ handbuch. Egon Stahl, Ed. Springer-Verlag, Berlin, Göttingen, Heidelberg.

Thomas, E. A., 1939: Über die Biologie von Flechtenbildnern. - Beiträge zur Kryptogamenflora der Schweiz 9: 1.

Svensk Botanisk Tidskrift. Bd 62, H. 3. 1968.

THE INFLUENCE OF CALCIUM ON DEVELOPMENT,

PHOSPHATE, ASSIMILATION AND ATP LEVEL IN

SYNCHRONIZED CULTURES OF SCENEDESMUS.

BY

GAURANGAKUMAR DAS.

(Institute of Botany, University of Stockholm, Sweden.) Abstract.

Synchronization of our strain of Scenedesmus is reported. The strain seems identical to Sc. obtusiusculus Chod. Deficiency of calcium will lead to dis­ turbances of growth, which can be observed in the order: (1) decrease in the number of autospores produced per mother cell; (2) rounding off of the cells, combined with a slowing down of the rate of increase in cell size; (3) loss of synchrony under the conditions of the experiments, presumably followed by (4) a slow increase in cell size to the proportions noted in unsynchronized cultures. On a dry weight basis, the amount of phosphate assimilated decreased but the level of ATP increased under calcium deficiency. For a given cycle, no variation with time was observed in the amount of phosphate assimilated per unit dry weight, but the level of ATP was lower during the light than during the dark period. On a per cell basis, cyclical variations were observed for phosphate and ATP until the loss of synchrony had occurred.

Introduction.

It has been shown that calcium is necessary as a trace element for Scenedesmus, not only to maintain cell division but also to give the spindle-like form characteristic for cells of this genus (Kylin & Das

1967). At the same time there were indications that cell multiplication and morphogenesis showed different sensitivities towards calcium. In order to analyze these effects further, we have synchronized our cultures and followed their development with and without calcium in the medium. Since it is known that calcium may interfere with energy-dependent conversion of phosphate (Brierley et al. 1963, Saris 1963), we have also collected data on the assimilation of

phosphate and on the levels of ATP in these cultures. It is our scope to present these data, as well as some notes on the synchronization procedure.

INFLUENCE OF CALCIUM ON SCENEDESMUS

₄₄₉

Analytical methods.

Cell numbers and relative cell sizes were determined conductometrically, in a Celloscope 202 automatic particle counter, as described earlier (Kylin & Das 1967). Also the methods to obtain the total cell volume and the dry weight of a sample were as stated there. Phosphate was analyzed colori- metrically and ATP with the luciferin-luciferase method according to the standard procedures of this laboratory (Kylin & Tillberg 1967 a).

Synchronization of the algae.

The medium used was the same as before (Kylin& Das 1967), rein­ forced with 0.1 mill Ca(N03)2 according to the experiences of that investiga­ tion. A preculture was inoculated from an agar slant and grown in continuous light as specified there for about 48 hours, when the cell density reached around 5 millions per ml. The culture was then poured into a high cylinder and left to sediment in the dark at 2-4°C. All operations were made with sterilized utensils and as cleanly as possible.

After several nights, 10 ml were pipetted out from the cylinder below the surface and were used to inoculate 200 ml medium in a 500 ml Erlenmeyer flask. The flask was placed at 30°C in a thermoregulated shaking water bath. Air enriched with 2.5 per cent C02 was bubbled through with a speed of approximately 2 1/h, and a light:dark cycle of 15:9 hours was given. The light was obtained from a ramp of 40 W warm white fluorescent tubes, giving an intensity of about 22,000 erg • cm-2 • sec-1.

After two cycles, the sedimentation procedure was repeated. The syn­ chronized cultures then obtained in the complete medium showed complete division ol' the cells within two hours (Fig. 1). More than 80 per cent of the newly divided cells fell within the size classes represented by diameters between 3.0 and 4.8 n in the calibration particles for the cell counter (Fig. 2 a). Using the method with standard dilution (Lorenzen 1964), we found no difficulties to maintain the cultures for a week or more, using 1-1.1 million cells per ml as the diluted number at the start of a new cycle.

Photographs from different stages of a cycle are shown in PI. 1:1 for comparison with data from the literature (see for instance Tamiya 1966 for a recent review). It may be noted that, contrary to the condi­ tions in Chlorella ellipsoidea as reported by Hase et al. (1957), we find it possible to discern between sporulated and aggregated cells. This may be correlated to the fact that in nature Scenedesnms is normally forming colonies whereas Chlorella is not. Furthermore, our normal cultures show a fairly homogeneous distribution of the cell sizes throughout a light period (Fig. 2 a), whereas Shibata et al. (1964)

found a considerable broadening of the band width of cell size distribution during the middle of the illumination time.

450

Table I. Formation ot autospores in relation to the density of the inoculate. Experiment Inoculate cells/ml Average division number 1 1.08 xlO6 8.1 2 1.30 x 10« 7.4 3 1.45 x 106 6.8 4 3.06 x 10" 6.2

From the taxonomic point of view, the data of PI. 1:1 places our algae as a strain of Scenedesmus obtiisiusculus Chod. (cf. Uherkovich

1966).

Results.

The conditions stated above were arrived at by systematic searching for the optimal results obtainable with our present equipment. As shown by Table I, the average number of cell multiplication per cycle is then somewhat dependent upon the initial cell number. An initial cell number of about 3 millions per ml was chosen for the main investigation, a choice which was done with regard to the demands of the analytical procedures for enough material also in the early stages of a cycle. To obtain a reasonably quick depletion of calcium in the deficient series, the cells were separated from the old culture medium by centrifugation and resuspended in the new medium instead of using simple dilution.

Already during the first cycle in a medium without calcium, the average number of new cells decreased from a little more than 6 in the complete medium to about 5 (Fig. 1). The time necessary to go through the divisions increased from about two to about three hours, and the aggregates formed were somewhat irregular (PI. 1:2A). During the second and third period in the Ca deficient medium, the multiplication number was further decreased, and the synchroniza­ tion was no longer maintained in the present regime of light: darkness. As for size and shape of the cells, only slight or no changes from the normal could be observed during the first cycle with calcium defi­ ciency. This refers to cell volume (Fig. 2 b) as well as to cell weight (Fig. 3) and general appearance of an individual cell (PI. 1:2A). After 10-15 hours of the second day without Ca, the increase in cell volume became slower than in the earlier cycles (Fig. 2 c), and a

INFLUENCE OF CALCIUM ON SCENEDESMUS ₄₅₁

Fig. 1. Number of cells per ml of synchron­ ized cultures of Scenedesmus. (1) o — O

in complete medium; (2) • — • in initial — Ca2+; (3) A— A in intermediate — Ca2+;

(4) x — x in final — Ca2+.

L D

12 16 20 24

HOUR

changed morphology could be noted (PI. 1:2 B). As a symptom of the loss of synchronization, the cell sizes at 24 hours showed a two- peaked distribution instead of the earlier one-peaked curves (Fig. 2 c). The disturbed cell division was reflected also in the weight of the

Fig. 2. Partition of cell sizes in different stages of synchronous cultures of Sce- nedesmus. (a) in complete medium; (b) in initial — Ca2+; (c) in intermediate

- Ca2+. 24 H OCDOtOOCDOOOOOOOO yaoncDOJconONiDinmN ' oj N co co h t ^ in to co n co bi o

diameter of calibration particles in u

,OH

5 H 10 H

15 H

452

L D

;—k.4

HOUR

Fig. 3. Dry weight per average cell during syn­ chronous growth ot Scenedesmus. (1) O — O in complete medium; (2) • — • in initial — Ca2+; (3) A—A in intermediate -Ca2+;(4) x—x in

final - Ca2+.

average cell and microscopically (Fig. 3 and PI. 1:2 C); but not until the third day without calcium did the algae obtain the completely sphaerical appearance of Scenedesmus with advanced deficiency of Ca (PI. 1:2D), which was reported by Kylin & Das (1967).

Taken on a dry matter basis, the assimilation of phosphate showed no significant changes during a cycle. The assimilation decreased with increasing deficiency of calcium (Table II). Contrary to this, the level of ATP changed during a cycle (Table III). During the first hours of illumination, the amount of ATP per g dry weight decreased and then remained low until the dark set in after 15 hours. During the following period of cell division the ATP level increased, and then remained high until dilution and renewed illumination. Super­ imposed upon the cyclic pattern was a steady increase in the level of ATP with increasing Ca deficiency.

In order to facilitate the coordination of these data with the para­ meters of growth, phosphate assimilation and ATP level are also given on a per cell basis (Figs. 4, 5). In both cases, cyclical variations can be observed until loss of cell synchrony has occurred.

Discussion.

First of all, we wish to draw the attention to the parallel investiga­ tion on Chlorella fusca (Soeder & Thiele 1967). They found that the

liberation of the autospores in synchronized cultures is more sensitive towards deficiency of Ca2+ than the growth of Ihe individual cell. This comes close to our own results with unsynchronized Scenedesmus

(Kylin & Das 1967) referred to in the introduction.

INFLUENCE OF CALCIUM ON SCENEDESMUS

453

Table II. Assimilation of phosphate expressed as mg P per g dry weight.

Hours from start

Medium 0 4.30 9.30 14.30 17.30 20.30 23.30

Complete 0.0 8.1 8.0 8.1 8.1 8.2 8.2

Initial -Ca2+ 0.0 6.2 6.1 6.3 6.2 6.1 6.1

Inter. — Ca2+ 0.0 5.1 4.9 5.0 5.0 5.0 5.2

Final -Ca2+ 0.0 4.6 4.5 4.6 4.6 4.6 4.6

One should expect a separation in time during experiments of the present type, so that the parameters of growth that require more Ca2+ are affected earlier than those with less need for Ca2+. As shown in the experimental section, the events when calcium deficiency develops, follow in the order: (1) decrease in the number of autospores pro­ duced per mother cell; (2) rounding off of the cells, combined with a slowing down of the rate of increase in cell size; and (3) loss of synchrony under the conditions of the experiments. This is presum­ ably followed by (4) a slow, continuous increase in cell size, since at extreme deficiencies of Ca (Kylin & Das 1967), average cell dry weights were obtained that were more than 2 times the final value reached here.

In the above sequence of growth, the change in the cell form occurs at a rather early stage. The formation of the giant cells is a late link of the chain, and can thus at most in part be a stereometric consequence of the change from needle-shaped to spherical cells.

As for ATP, the increased level during the dark hours when com­ pared with the light period (Table III), may be due to the differences

Table III. ATP level, ^g/g dry weight.

Hours from start

Medium 0 4.30 9.30 14.30 17.30 20.30 23.30 Complete 95 42 40 40 70 98 97 Initial — Ca2+ 102 45 42 44 78 104 106 Inter. — Ca2+ 105 50 48 50 79 114 113 Final — Ca2+ 112 60 58 68 94 120 152 Sv.Bot. Tidskr., 62 (1968): 3

454

Fig. 4 (left). Phosphate assimilation per average cell during synchronous growth of Scenedesmus. (1) O — O in complete medium; (2) • — • in initial — Ca2+; (3) A— A in

intermediate — Ca2+; (4) x — x in final — Ca2+.

Fig. 5 (right). Amount of ATP per average cell during synchronous growth of Scenedes­ mus. (1) O— O in complete medium; (2) •—• • in initial — Ca2+; (3) A— A in inter­

mediate - Ca2+; (4) x — x in final - Ca2+.

between oxidative and light induced phosphorylation rather than to the growth processes themselves. This caution is made since un­ synchronized cultures of our strain of Scenedesmus contained less ATP in photophosphorylation than in oxidative phosphorylation

(Kylin & Tillberg1967 b). Furthermore, it was reported byCtiRNUTT & Schmidt (1964) that the amount of ATP increases logarithmically with the same rate as the dry weight of synchronized cultures of Chlorella in continuous light; this also seems to indicate that our fluctuation is due to the light-dark change rather than to the cell development.

The increased level of ATP under conditions of Ca deficiency (Table III) is compatible with the situation known from mitochondria

(Brierley et al. 1963, Saris 1963) and chloroplasts (Nobel & Packer1964), where uptake of calcium is an alternative to the forma­

tion of ATP. It should be pointed out, however, that the causal relationship is unclear in the present case, since the rise could also occur as a consequence of a diminished utilization of ATP when the growth disturbances set in.

In our material, we have not observed the cyclical variation in the amount of phosphate taken up per g dry weight (Table II), which has been reported for different types of Chlorella (Hase et al. 1957, Sv. Bot. Tidskr., 62 (1968): 3

INFLUENCE OF CALCIUM ON SCENEDESMUS

455

basis when Ca is lacking (Table II) was observed before (Kylin & Das 1967). It may be noted, however, that calcium deficiency will

lead to increased amounts of phosphate per cell when the late stages of the different cycles are compared with each other (Fig. 4, 20-24 hours).

The investigation was suggested by Dr. Anders Kylin and performed within the group supervised by him. Support from the Natural Science Research Council of Sweden is gratefully acknowledged.

REFERENCES.

Brierley, G., Murer, E., Bachman, E. & Green, D. E., 1963: Studies on ion transport. II. —J. Biol. Chem. 238: 3482-3489.

Curnutt, S. G. & Schmidt, R. R., 1964: Possible mechanisms controlling the intracellular level of inorganic polyphosphate during synchronous growth of Chlorella pyrenoidosa. — Biochim. Biophys. Acta 86: 201- 203.

Hase, E., Morimura, Y. & Tamiya, H., 1957: Some data on the growth physiology of Chlorella studied by the technique of synchronous culture. —- Arch. Biochem. Biophys. 69: 149-165.

Kylin, A. & Das, G., 1967: Calcium and strontium as micronutrients and morphogenetic factors for Scenedesmus. — Phycologia 6: 201-210.

Kylin, A. & Tillberg, J.-E., 1967 a: Action sites of the inhibitor-/? complex from potato and of phloridzin in light-induced energy transfer in Scenedesmus. — Z. Pflanzenphysiol. 57: 72-78.

-—»—, 1967 b: The relation between total phosphorylation, level of ATP, and oxygen evolution in Scenedesmus as studied with DCMU and anti- mycin A. — Z. Pflanzenphysiol. 58: 165-174.

Lorenzen, H., 1964: Synchronization of Chlorella with light-dark changes and periodical dilution of the populations to a standard number. — E. Zeuthen (ed.): Synchrony in cell division and growth, pp. 571— 577. Interscience.

Nobel, P. S. & Packer, L., 1964: Energy-dependent ion uptake in spinach chloroplasts. — Biochim. Biophys. Acta 88: 453-455.

Saris, N. E., 1963: The calcium pump in mitochondria. — Soc. Scient. Fenn. Comment. Phys.-Math. 28 (11): 1-59.

Schmidt, R. R., 1961: Nitrogen and phosphorus metabolism during syn­ chronous growth of Chlorella pyrenoidosa. — Exptl. Cell Res. 23: 209 -217.

Shibata, K., Morimura, Y. & Tamiya, H., 1964: Precise measurement of the change of statistical distribution of cell size occurring during the synchronous culture of Chlorella. — PI. & Cell Physiol. 5: 315-320.

Soeder, C. J. & Thiele, D., 1967: Wirkungen des Calcium-Mangels auf Chlorella fusca Shihira et Krauss. — Z. Pflanzenphysiol. 57: 339-351.

456

Tamiya, H., 1966: Synchronous cultures of algae. — Ann. Rev. PI. Physiol. 17: 1-26.

Uherkovich, G., 1966: Die Scenedesmusarten Ungarns, 1-173.—Akadémiai Kiad6, Budapest.

Explanation of the Plate.

1. Photographic evidence from the life cycle of synchronized cells of Scenedesmus obtusiusculus Chod. (A) Aggregated cells; (B) dark cells; (C) transient stage dark-and- light cells; (D) immature light cells; (E) half mature light cells; (F) mature light cells; (G) sporulated cells.

2. Gradual change of cell morphology due to calcium deficiency during synchronous growth of Scenedesmus. (A) Dividing cells after one day with Ca2+ deficiency (initial deficiency), cf. 1 A; (B) mature light cells in the second day with Ca2+ deficiency (inter­ mediate deficiency) cf. 1 F; (C) dividing cells in the second day with Ca2+ deficiency (intermediate deficiency), cf. 1 G & A; (D) cells from third day with Ca2+ deficiency (final deficiency), cf. 1 B.

G. das: influence of calcium on scenedesmus _{PI. I}

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Svensk Botanisk Tidskrift. Bd 62, H. 3. 1968.

GROWTH AND APPEARANCE OF SCENEDESMUS

AS INFLUENCED RY DEFICIENT

INORGANIC NUTRITION.

BY

GAUBANGAKUMAR DAS.

(Institute of Botany, University of Stockholm, Sweden.) Abstract.

Morphology, growth, formation of chlorophyll and assimilation of phos­ phate were investigated in the unicellular Scenedesmus obtusiusculus (Chod.)

in relation to deficiences of K, Mg, S, P, N, and Fe. Completely different pictures were obtained in the different cases. The potential possibilities of a unicellular system for studies on morphogenesis are pointed out, and stress is placed on the importance of running standard cultures before samples of unicellular algae are taxonomically analyzed.

Introduction.

It was observed that the presence of trace amounts of calcium is necessary to give cells of Scenedesmus their characteristic, needle- shaped appearance (Kylin & Das 1967), and that the absence of

calcium causes disturbances in synchronized cultures of this algae

(Das 1968). A survey of the literature gave at hand that although the

physiology of the mineral nutrition of unicellular algae is compar­ atively well known (Krauss 1953, 1958) and although it is felt that the mineral nutrition will affect the morphological appearance of the cells (Uherkovich 1966), little or no systematic attention has been

given to the connection between deficient mineral nutrition and the appearance of the cells—perhaps since the most intense studies, for instance the interesting paper by Hase et al. (1957), have been con­

cerned with Chlorella, in which the round cells cannot be expected to change their form very much. With this background, experiments were undertaken to investigate the effect of defficiencies of some of the ions necessary for Scenedesmus, where our results with calcium made

Sv. Bot. Tidskr., 62 (1968): 3 30-683873

458

it likely that we had found a suitable material. The nutrients in­ vestigated so far have been K+, Mg2+, sulphate, phosphate, nitrate, and iron.

Materials and methods.

The experiments were performed with a strain of Scenedesmus, which was obtained from Professor E. C. Wassink of the Agricultural University at Wageningen, The Netherlands (Kylin1964), and which has been much used also in studies from the present laboratory (Kylin & Das 1967, Das 1968). The strain appears to belong to Scenedesmus obtusiusculus Chod. as depicted by Uherkovich (1966).

In the main, the culture methods were the same as used before (Kylin & Das 1967). In accordance with the experiences from that paper, the com­ plete medium was reinforced with 0.1 mM Ca(N03)2- The standard medium is given in Table I, as well as the modifications used to obtain deficiencies of the elements investigated. No precautions were taken to avoid the potassium, sulphate and nitrate present in the trace element solution (about 2.5 x 10-5, 7 x 10-3 and 7 x 10“6 mM respectively), an approach which is justified by the results.

The cultures were run in a shaking water bath at 25°C under continuous illumination with warm white fluorescent tubes giving about 22,000 erg -cm-2 sec-1 at the surface level of the algal suspensions.

To obtain the deficient algae, 20 ml were taken from a normal culture and used to inoculate 180 ml of the deficient medium. If necessary, the procedure was repeated as stated in the presentation of the results.

The number, volume and dry weight of the cells in a sample were deter­ mined as stated before (Kylin & Das 1967), and the same is true for the analysis of phosphate. Chlorophyll was determined according to the method of Arnon (1949).

Results.

The analytical data have been collected in Table II, and photo­ graphic evidence from the final stage of each step is given in PI. I.

Deficiency of potassium led to a considerable increase in the average size of the cells and possibly to a slight increase in the per cent dry matter. The contents of chlorophyll were decreased, but not so much as in the case of the other elements. In the final stage of potassium deficiency, phosphate was given off from the cells instead of being assimilated. The shape of the cells became swollen but not completely rounded.

Magnesium deficiency produced large and completely rounded cells, just as the deficiency of calcium (Kylin & Das 1967, Das

1968). However, diverging from the effects of calcium starvation,

GROWTH AND APPEARANCE OF SCENEDESMUS

₄₅₉

Table I. Salts used to obtain different mineral deficiencies.

The sign (—) denotes the same salt as in complete medium.

mM Standard culture medium Medium deficient in K Mg S P N Fe 1 25 kno3 NaNOj

_

4 0.07 FeS04 — — Fe-citrate — — Omitted

0.11 Na-citrate — — NaH2P04 — — _

0.11 Na2-EDTA — — — — _ _

5 0.1 Ca(N03)2 — — — — CaCI2 —

the lack of Mg led to an increase in dry matter content and to a marked granulation of the cytoplasm. The contents of chlorophyll dropped to a low level. Also the assimilation of phosphate was affected, although less severely than in the case of potassium.

Lack of sulphur gave small cells with the same per cent of dry matter as the normal ones. The size of the cells indicates that the growth in volume must stop soon after the last division, which is in accordance with the findings of Hase et al. (1957) on Chlorella. The general appearance of the cells was not much changed from the normal. Ihe contents of chlorophyll were low, and the decrease occurred already when the cell divisions were only slightly affected (cf. Mandels 1943). Some cell divisions still occurred during the last

period investigated, but the experiments were nevertheless broken off since a release of phosphate occurred.

Phosphate deficiency resulted in large cells with a high per cent dry matter as described earlier by Kylin (1964). The shape of the

deficient cells was ellipsoidal and not sphaerical, which makes them different from the cases of -Ca (Kylin & Das 1967) and -Mg. Small

necrotic areas developed in the P deficient cells, but there was no release of phosphate to the medium. The contents of chlorophyll were low.

Without nitrogen in Ihe medium, only the first deficient culture could be quantitatively analyzed. At the end of this the cells were somewhat larger than the normal cells and appeared a 1 ittie swollen, with necrotic spots developing. The per cent dry matter did not differ much from the normal value and the assimilation of phosphate was

460

Table II. The effect of deficiencies in K, Mg, S, P, N, and Fe on the growth, formation of chlorophyll, and assimilation of P in

Scenedesmus.

Cell Chloro- Phosphate

no. Average cell Dry phyll assimila-Growth Experi- ratio /--- *--- . matter formed tion as

period, ment Final: Volume Dry wt. mg/100 mg/g mg P/g Medium hours number Initial fA x 107 mg x 107 /A cells dry wt. dry wt.

Complete 48 1 83 1.20 0.27 23 21.0 13.5 2 85 1.10 0.24 23 21.0 13.5 Initial -K 48 1 66 1.20 0.27 23 20.5 10.5 2 66 1.20 0.27 23 20.0 10.0 Intermediate -K 48 1 8.3 2.70 0.76 28 10.5 7.5 2 8.3 2.65 0.74 28 10.5 7.5 Final -K 25 1 1.7 3.00 0.84 28 10.0 -3.5 2 1.9 3.00 0.81 28 10.0 -3.5 Initial -Mg 48 1 47 2.00 0.58 29 13.5 7.5 2 45 2.00 0.57 29 14.0 8.0 Final -Mg 25 1 1.1 2.45 0.86 35 4.0 5.0 2 1.2 2.45 0.86 35 3.5 5.0 Initial -S 48 1 63 1.40 0.33 24 8.0 7.0 2 62 1.40 0.33 24 8.0 7.0 Final -S 25 1 5.4 1.10 0.26 24 5.0 -2.0 2 5.0 1.05 0.25 23 5.0 -1.5 Initial -P 48 1 20 2.00 0.67 37 5.0 — 2 19 2.00 0.66 37 5.0 — Final -P 25 1 1.1 2.40 1.13 47 4.0 — 2 1.0 2.40 1.12 47 4.0 — -N 48 1 22 2.00 0.51 28 3.0 9.5 2 21 2.00 0.50 29 3.0 10.5 -Fe 48 1 8.8 1.60 0.55 35 3.1 4.0 2 7.8 1.70 0.60 35 3.1 4.0

comparatively little affected, but the contents of chlorophyll were strongly decreased.

Judging from the number of new cells obtained in the first run on deficient medium, iron is the least abundant in the normal medium of the elements investigated. The average cell size was slightly above that in the complete medium. The per cent dry matter was high, the contents of chlorophyll low and there was only a slight assimilation of phosphate. The cell form was about the normal, and development of necrotic areas could be observed.

GROWTH AND APPEARANCE OF SCENEDESMUS

₄₆₁

Qualitative observations were made during a second generation of deficient cultures, both for nitrogen and for iron deficiency. In the case of nitrogen deficiency, the cells became completely colourless, but otherwise no further data of interest here were obtained.

Discussion.

Although the data collected are only the most basic ones, they illustrate and raise some theoretical questions. First of all, since variations in the composition of the medium can produce cells with vastly different morphologic appearances (PI. I, cf. also Kylin & Das

1967), it seems appropriate to stress the necessity for culture in stand­ ard media and under standard conditions before field collections of unicellular algae are taxonomically evaluated. A discussion of earlier experiences with Scenedesmus is given by Uherkovich (1966).

1 he different deficiencies do not lead to the formation of one and the same type of starved cell. Instead, distinctly different cell types develop when the algae respond to different mineral deficiencies. The comparative simplicity of the algal system may then give it theoretical interest for studies on the causal relationships in development and differentiation. The significance of this point becomes particularly clear in a comparison with higher plants, where the whole complex of organization and development of tissues and organs becomes in­ volved.

fhat the various mineral elements affect growth of the algae in different ways, is reflected not only in the morphology but also in the other parameters investigated. Thus one finds that the amounts of the elements available from the complete medium were sufficient to maintain more cell divisions in the cases of -K and -S than in the case of -Mg, which in turn gave more cells than -P or -N, Fe being in the shortest supply. At the same time the effect of N starvation was not very great when the assimilation of phosphate was concerned, whereas lack of Mg or Fe considerably decreased the assimilation of P, and the absence of S or K even led to losses of P. Furthermore, lack of K did not decrease the contents of chlorophyll to the same extent as the other elements tested; and while S deficiency did not at all change the per cent dry matter from the value of normal cells, lack of K or N may have had a slight effect, and definite increases in the per cent dry matter were caused when Mg, Fe or P were not supplied.

1 he order between the elements is thus different in the different cases.

462

For general reasons this result may in itself be expected and also be known from more complicated organisms, the point again being to illustrate the potential possibilities for further analysis inherent in the unicellular algal system.

The studies have been performed within the group supervised by Dr.

Anders Kylin and were supported by grants from the Natural Science Research Council of Sweden, which are gratefully acknowledged.

REFERENCES.

Arnon, D. J., 1949: Copper enzymes in isolated chloroplasts. Polyphenol- oxidase in Beta vulgaris. — Plant Physiol. 24: 1-15.

Das, G., 1968: The influence of calcium on development, phosphate assimila­ tion and ATP level in synchronized cultures of Scenedesmus. — Sv. Bot. Tidskr. 62: 448-456.

PIase, E., Morimura, Y. & Tamiya, H., 1957: Some data on the growth physiology of Chlorella studied by the technique of synchronous culture. — Arch. Biochem. Biophys. 69: 149-165.

Krauss, R. W., 1953: Inorganic nutrition of algae. — In: J. S. Burlew(ed.), Algal Culture from Laboratory to Pilot Plant, pp. 85-102. Carnegie

Inst, of Washington Publication 600, Washington, D.C.

—»—, 1958: Physiology of the fresh-water algae. Ann. Rev. PI. Physiol. 9: 207-244.

Kylin, A., 1964: The influence of phosphate nutrition on growth and sulphur metabolism of Scenedesmus. — Physiol. Plant. 17: 384-402.

Kylin, A. & Das, G., 1967: Calcium and strontium as micronutrients and morphogenetic factors for Scenedesmus. — Phycologia 6: 201-210.

Mandels, G. R., 1943: A quantitative study of chlorosis in Chlorella under conditions of sulphur deficiency. — Plant Physiol. 18: 449-462.

Uherkovich, G., 1966: Die Scenedesmus-Arten Ungarns. — Akadémiai Kiado, 1-173, Budapest.

Explanation of the plate.

Cell morphology as affected by mineral deficiencies. Photographs taken at a total magnification of x 160; numerical aperture of objective 0.65. (ST) Standard medium; (ID) initial deficiency; (INT.D) intermediate deficiency; (FD) final deficiency. Deficient element as given in each case.

G. das: growth and appearance of scenedesmus PI. I \ Cj _fr •Gt A

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Svensk Botanisk Tidskrift. Bd 62, H. 3. 1968.

ÉTUDES URÉDINOLOGIQUES.

PAR

LENNART HOLM.

8. Gymnosporangium Gaeumannii — une espéce primitive?

En 1949 Zoggdécrivit une espéce nouvelle et bien remarquable de

Gymnosporangium qu’il nomma G. Gaeumannii. On la trouvait å quelques stations peu nombreuses en Suisse, dans 1’Engadine, å une altitude de 2000 metres et au-dessus, oü eile parasitait les aiguilles de Juniperus communis var. nana. Aucun stade écidien en avait été vu et on n’en connait encore aucun. D’apres Zogg ce

Gymnosporangium était caractérisé par 1’existence abondante d’uré- dospores; les téliospores, par contre, semblaient relativement rares et « nur mit 1 Promille bis höchstens einigen Prozent vertreten » et on les comptait panni les urédospores.

Cette communication de Zogg était extrémement interessante

puisque jusqu’ä ce jour-lä une espéce seulement de Gymnosporangium était connue ayant un stade urédo, å savoir G. nootkatense de 1’Amé- rique du Nord Pacifique. A ce que nous sachions G. Gaeumannii n’a pas été étudié depuis lors; Pexposé de Gäumann (1959) se base

entiérement, semble-t-il, sur le travail de Zogg. Au corn s d’une étude

sur les Gymnosporangium et surtout leur stade écidien, nous avoirs eu lieu d’examiner cette espéce et nous tenons å remercier notre ami le Docteur Emil Müller, de Zürich, qui a bien voulu en nrettre du matériel original å notre disposition (SUISSE: Graubünden, Münster­ tal, Alp Campatsch, 24.vn.1948, leg. H. Zogg, ZT). Sur la base de celui-ci nous pouvons compléter ici la description de Zogg avec

quelques observations nouvelles et en vertu de celles-ci nous don- nerons une autre interpretation des prétendues urédospores.

Quant a ces structures nous n’avons guére å ajouter å la descrip­ tion de Zogg. Elles sont en général presque globuleuses, 25-30 g de diam. environ, et pourvues d’une paroi assez épaisse, souvent de 2,5 g environ et 3-3,5 g aux pores germinatifs mais parfois plus, et jusqu’a 5 g aux pores. La paroi est d’un brun roux, assez

464

ment verruqueuse et percée d’une dizaine de pores germinatifs bien distincts (non 6-8, comme le dit Zogg). Les spores sont munies d’un long pédicelle hyalin dont elles se détachent facilement (PI. 1:1).

L’aspect de ces « urédospores » est tel qu’on peut å peine suppri­ mer deux reflexions: D’abord, ces structures, å part le pédicelle, présentent une ressemblance parfaite avec le type écidiosporé normal de Gymnosporangium (cf. PL 1:6). 11 est étonnant que Zoggne signale

pas cela. Puis on se demande si ces spores sont de véritables urédo­ spores. Pour déterminer cette question d’une facon délinitive il faut certainement avoir accés å des spores germantes. Cependant, nous croyons plutot qu’il s’agit de téliospores unicellulaires, pour des raisons que nous démontrerons ci-dessous.

Comme Zoggle fit remarquer il y a dans le matériel original aussi

des spores d’un autre type, qui sont indubitablement des téliospores (PI. 1:3):« Die in der Regel zwei-, seltener einzelligen Teleutosporen sind dick- oder dünnwandig, elliptisch oder spindelförmig, oft aber von unregelmässiger Gestalt, abgerundet, oft zugespitzt, leicht ein­ geschnürt, glatt, braun gefärbt und sitzen wie die Uredosporen auf langen Stielzellcn. Jede Sporenzelle besitzt ein bis zwei Keimporen mit farbloser Papille ... Sie sind denjenigen des Gymnosporangium juniperinum (l.)fr. [=G. cornutum] in Gestalt und Grösse sehr ähn­ lich. » (Op. cit., pp. 424-425.) Nous pouvons en somme confirmer cette description; pourtant nous n’avons pas observé de téliospores ä papille distincte, mais cela peut probablement varier. Mais ce qui esl extremement intéressant est la présence d’une tout autre sorte de téliospores bicellulaires, ce que Zogg ne mentionna pas.

En effet nous trouvons, entremélées avec les « urédospores » et les téliospores « normales » aussi des téliospores bicellulaires ä mem­ brane épaisse et verruqueuse, aux loges munies de 8 pores germinatifs environ. La cloison médiane est distinctement double et percée d’un pore double (PI. 1:2). Les pores possédent un renforcement annu- laire. En réalité chaque cellule est presque pareille aux « urédo­ spores ». On trouve aussi des spores intermédiaires entre ces télio- spores-ci et les téliospores « normales », c’est-å-dire les téliospores décrites par Zogg. Ainsi on peut observer des spores dont la cellule supérieure est pareille å une « urédospore » tandisque la cellule inférieure ressemble å la loge correspondante d’une téliospore « normale », ayant une paroi lisse percée d’un seul pore (PI. 1:4).

Ces téliospores bicellulaires aux pores nombreux présentent une ressemblance évidente et bien intéressante avec les téliospores d’une

LENNART HOLM: ETUDES U R ÉDINOLOGIQUES. 8

_{PI. I}

oV f 4r.

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ÉTUDES URÉDINOLOGIQUES. 8

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autre espéce de Gymnosporangium, å savoir G. multiporum (de 1’Amérique du Nord). Celui-ci a également, comme l’indique l’épi- théte spécifique, les téliospores aux pores nombreux et ceux-ci sont pourvus, notez bien, d’un renforcement annulaire (PI. 1:5). La seule différence importante entre ces téliospores est constituée du fait que la membrane sporée n’est pas verruculeuse chez G. multiporum mais tres finement ponctuée.

Toutes ces ressemblances ne sont sürement pas accidentelles mais les manifestations d’une liaison profonde, et elles possédent certaine- ment un intérét considérable du point de vue phylogénetique. II nous par ait que la théorie la plus simple qui coordonne ces nouveaux faits peut étre résumée en ces points:

1. Les spores unicellulaires de Gymnosporangium Gaeumannii (=les « urédospores » sensu Zogg) représentent un type téliosporé tout primitif.

2. D’un tel type s’est évoluée la téliosporé bicellulaire aux pores nombreux, se trouvant également chez G. Gaeumannii, bien que rare. Celle-ci doit étre le type le plus primitif connu parmi les téliospores bicellulaires de Gymnosporangium. De ce type se sont évoluées et

3 a. La téliosporé de G. multiporum et

3 b. La téliosporé de type normal Gymnosporangium, trouvée aussi chez G. Gaeumannii (2 cellules a paroi lisse etc., cfr Pl. 1:3).

4. La téliosporé unicellulaire primitive, telle que nous la trouvons chez G. Gaeumannii a été une sorte de prototype a 1’origine des pre­ miers écidiospores. Nous supposons done que les écidiospores de Gymnosporangium — on devrait plutöt dire « Proto-Gymnosporan- gium » — sont parues comme des formations nouvelles. L’hypothese la plus simple est de dériver les écidiospores des téliospores, germées avant la fusion nueléaire, c’est-å-dire provenant d’une forme de néoténie. Ce phénoméne est considéré comme ayant joué un role évolutif tres important, cfr. Takhtajan, 1959. En tout cas e’est un fait inconstestable que le type écidiosporé normal de Gymnosporangium est bien ressemblant aux spores unicellulaires de Gymnosporangium Gaeumannii.

La théorie ci-dessus présume ainsi ou plutöt eile améne a la sup­ position que Gymnosporangium est un groupe primitif parmi les Urédinales. Dans un artide suivant nous développerons encore les raisons d’une telle supposition.

466

Summary.

Gymnosporangium Gaeumannii is a parasite on the needles of Juniperus communis and known so far only from a few localities in the Swiss Alps. According to the original description it has abundant uredospores, a condition exceptional in this genus. No aecidial stage is known.

The author has investigated type material and reports the discovery of biccllular teleutospores with a thick verrucose wall, pierced by scattered distinct germ pores. They closely resemble the teleutospores of Gymnosporangium multiporum. Each spore cell is also very similar to the alleged uredospores, and the author suggests that these are, in fact, unicellular teleutospores. Finally, the latter have a remarkable similarity to the aecidiospores, characteristic of Gymnosporangium.

A hypothesis is advanced that these unicellular spores represent a primitive type of teleutospores and that the original aecidiospores of the Uredinales were similar to primitive teleutospores.

BIBLIOGRAPHIE.

Gäumann, E., 1959: Die Rostpilze Mitteleuropas. — Beitr. Krypt.-Flora d. Schweiz, 12.

Takhtajan, A., 1959: Die Evolution der Angiospermen. — Jena.

Zogg, H., 1949: Über ein neues, Uredo-bildendes Gymnosporangium: Gym­ nosporangium Gaeumanni n.sp. •— Ber. Schweiz. Bot. Ges., 59.

Legende de la planche.

1. Gymnosporangium Gaeumannii, deux téliospores (?), encore attachées aux pédicelles. La spore supérieure est immature. -— x 480.

2. G. Gaeumannii; plusieurs téliospores (?) unicellulaires et, ä la Iléche, une téliospore bicellulaire aux pores nombreux. — x 480.

3. G. Gaeumannii; téliospore « normale ». — x 480.

4. G. Gaeumannii; plusieurs téliospores (?) unicellulaires et une téliospore bicellulaire, dont la loge supérieure a des pores nombreux. — x 480.

5. G. multiporum; téliospores. La spore au milieu présente une cloison médiane, nette- ment double. Le pore double perfant la cloison est visible å la spore å gauche. —

x 480.

6. G. confusum; écidiospores. Notez les pores germinatifs munis d’un renforcement annulaire. — x 480.

Svensk Botanisk Tidskrift. Bd 62, H. 3. 1968.

LINDBERGIA BOR: A NEW GENUS OF

GRASSES FROM CYPRUS.

BY

N. L. BOR

Royal Botanic Gardens, Kew.

Lindbergia Bor, genus novum.

Poae L. Eremopoaeque Rozhev. affinis, sed ab illo lemmatibus 3-nerviis ab hoc lemmatibus carinatis inter alia diversum. Gramina annua. Culmi plerumque dense fascieulati, laterales basi breviter decumbentes, graciles, leves glabrique. Foliorum laminae planae demum complicatae vel involutae, lineari-acutae; vaginae culmorum complectentes, inferiores illis breviores scariosae; ligulae membranaceae. Panicula juventute angusta, demum perlaxa, ramis 2-5-nis, late patentibus vel paulo declinatis, glaberrimis; rami ramulique paucispiculati; pedicelli longi. Spiculae primo ellipticae a latere compressae, demum hiantes, 2-5-florae; glumae inaequales, 3-nerviae; lem­ mata oblongo-elliptica, 3-nervia, apiculata, inferne dorso inter nervos et lateribus adpresse sericeo-pilosa, carina nervisque scaberula; palea lemmati aequilonga, anguste elliptico-oblonga, 2-carinata, ad $ carinarum dense ciliata, ad ■£ earum denticulata; stamina 3; styli 2; stigmata plumosa; lodi- culae 2, late ovato-acutae; caryopsis ambitu fusiformis, adaxialiter sulcata; hilum punctiforme basi sulci positum; embryo parvus, circiter ^ caryopseos aequans.

Typus generis: Poa persica y alpina Boiss.

Cyprus. Troödos, 20.6.1880, Paul Sintenis881.

Lindbergia sintenisii (LlNDB.) Bor, comb. nov.

Syn. Poa sintenisii H. Lindb. in Årsbok Soc. Sei. Fenn. 20 B, no. 7: 5 (1942) et in Act. Soc. Sei. Fenn., n.s. B., 2, no. 7: 8 (1946). — Poa persica

Trin. var. y alpina Boiss. Fl. Orient. 5: 610 (1884), pro parte. — P. persica

Trin. ssp. cypria Sam. in Rech. f., Ark. för Bot. ser. 2, 1: 417 (1951). Gramen annuum. Culmi 8-21 cm alti, simplices, dense fascieulati, laterales patentes, basi decumbentes. Foliorum laminae utrinque marginibusque scaberulae, usque 6 cm longae, 1-2 mm latae, lineares, acuminatae; vaginae leves glabraeque; ligulae membranaceae, apice laceratae, 2-3 mm longae. Panicula 2-11 cm longa, 1-9 cm lata, ramis 1-5 cm longis late patentibus vel

468

paulo declinatis, levibus. Spiculae juventute ellipticae, maturitate hiantes, 2-5-florae, 3-4,5 mm longae; rhachilla supra glumas inter flores disarticulans, ultra florem producta, flore vestigiale coronata; gluma inferior 3 mm longa, elliptico-acuta, superior 3,5 mm longa, inferiore latior, ambae 3-nerves, nervis et interdum inter nervos scabridae, carinatae, marginibus hyalinae, ceterum purpureo-tinctae; lemmata 4 mm longa, 3-nervia, carinata, expla- nata oblongo-elliptica, acuta, mucronata vel apiculata, inter nervos et lateribus appresse sericeo-pilosa; palea lemmati aequilonga, 2-carinata, carinis dense ciliata; antherae 0,5 mm longae; caryopsis 2,5-2,75 mm longa.

Cyprus. Mons Troödos, in summo: 20.6.1880, Sintenis 881 (Typus, K); Xerokolymbos: 17.5.1937, 4300', E. W. Kennedy 93; 94 —- near the stream, the water’s edge; Platres, Troödos: 24.5.37, 4600', Idem 96 — roadside; Kyros Potamos: 13.6.37, 5000', Idem 95 — by the stream; ibidem: 23.5.38, 4000', Idem 1100 — igneous mountains, rock above the stream; Khionistra, due south: 20.5.38, 5300', Idem 1101 — igneous mountain range, rock above a wintertime rivulet; Platres: 25.5.38, 4000', Idem 1102 — igneous mountain, in rock under pine trees; ibidem: 20.6.38, 5000', Idem 1103 — igneous moun­ tain, bare dry rock, frequent at this altitude; Kyros Potamos: 22.6.38, 5750',

Idem 1104 — igneous mountain, rock in dry river bed, frequent at this alti­ tude; Troödos: 22.6.39, Harald Lindberg s.n. — in Pineto juxta viam haud procul ab “Olympus Camp Hotel”; Mesopotamos: 16.5.41, 4000',

P. H. Davis 3454-A -— dry igneous slopes under Pinus halepensis; Bolon Pedhoulas: 14.6.51, 3300', L. F. Merton 1240 — among rocks by the stream- side, not uncommon at this altitude; Troödos village: 24.5.55, Idem 2317 — on igneous scree; Troödos: 26.5.60, 5500', M. MacDonald 199 — bare shaley ground under pine tree, sturdy little grass with delicate panicle; Prodhromos (near): 5.6.61, 4800', D. P. Young 7270 — dry open places and tracks in forest, frequent; Troödos: 4.7.63, 5600', Idem 7804 —- scree: gregarious annual, frequent; Between Prodhromes and Agios Nikolaos: 4.5.62, 4000',

R. D. Meikle 2844 — in Pinus nigra woodland at base of trees: erect stem purplish at base, flowers bright green; Agios Nikolaos near Kakopetria:

4.5.62, 3900', R. D. Meikle 2853 — on damp ground by roadside; spreading, leaves, stems and inflorescence dull purple.

There are three sheets of Paul Sintenis’ original collection of this plant in the Kew Herbarium. There are also specimens of the other collections cited by Boissier [FI. Orient. 5: 610 (1884)], namely “In

alpinis, montes supra Elmali Lyciae, Bourg. 271, Taurus Cilicicus, 5000-6000', Kotschy 12, BAL., Libani cacumina, Blanche, Kuh Delu, Persiae australis, Kotschy 477”. All of these were named by BoissierPoa persica y alpina. Actually with exception of the Sintenis

collection, all of them are rather depauperate specimens of Eremopoa persica (Trin.) Roziiev. var. persica, and so different in appearance from Lindbergia that it is difficult to comprehend how they came to be considered congeneric let alone conspecific.

LINDBERGIA BOR: A NEW GENUS OF GRASSES ₄₆₉

Fig. 1-10. Lindbergia sintenisii (Lindb.) Bor. 1, Habit x f; 2, spikelet x 8; 3, lower glume x 9; 4, upper glume x 8; 5, floret from side x 8; 6, lemma x 8; 7, palea x 8; 8,

flower x 8; 9, grain x 8; 10, ligule x 6. Sintenis 881.

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Samuelsson [cf. Recii. i', in Arkiv för Bot. ser. 2, 1 (9): 417 (1951)] examined the gatherings Sintenis 881, Haradjian and Lindberg, and found that they were identical and differed from all specimens of Poa persica that he had seen. He came to the conclusion that they were so different in facies that they deserved the rank of subspecies to which he gave the name subspecies cypria Sam., unaware that Lindberghad already called these specimens, all from Troödos, Poa

sintenisii. This new genus and species seem to be confined to the isolated Troödos mountain range in Cyprus at an altitude of about 4000' and above. It is named in honour of Harald Lindbergwho

quite rightly decided that this species was quite different from Poa persica, of which Boissier had made it a variety.

Svensk Botanisk Tidskrift. Bd 62, H. 3. 1968.

MOUSSES RÉCOLTÉES PAR MR. GILLIS EEN DANS

LES ILES MAURICE ET DE LA RÉUNION.

PAR

M. BIZOT.

Laboratoire de Botanique et Cryptogamie de la Faculté Mixte de Médecine et Pharmacie de Dijon, France.

Ces mousses proviennent des récoltes effectuées par M. Gillis Een

en automne 1962 å Tile Maurice et å lTle de la Réunion. Le collec- teur nous en a confié 1’étude apres la détermination des Hépatiques par M. S. Arnell. Cette note faisant suite au travail de cet auteur (1), nous avons cru nécessaire d’utiliser la méme présentation de facon å donner å ces deux publications une forme analogue, en utilisant l’ordre alphabétique des genres, avecla nomenclature de l’lndex (12).

Les stations citées pour l’lle Maurice ont été traduite en francais et toutes les altitudes ont été converties en metres. Les supports n’étant pas précisés sur les etiquettes, nous ne les avons indiqués que lorsque 1’échantillon permettait de les reconnaitre.

Liste des espéces.

Acroporium megasporum (Duby) Fleisch. — MAURICE. Macabé, forét climacique d’altitude, alt. 550 m. Sterile.

Aerobryidium subpiligerum (Hampe) Card. Stérile. Sur débris végétaux. — REUNION. Reserve biologique de Cilaos, alt. 1200 m. — MAURICE. Réserve naturelle de la lande Petrin, alt. 650 m. — Espéce trés semblable aux espéces centrafricaines A. trachyptera et A. longipendula. Sa différen- tiation semble reposer å notre avis plus sur des considérations géogra- phiques qu’anatomiques.

Anaectangium borbonense Besch. Stérile. — MAURICE. Le Pouce sur les rochers minés pour faire passer la vieille route de Port Louis å St. Pierre, alt. 550 m.

Brachymenium eurychelium Besch. Fructifié. A terre. — MAURICE. Plateau au nord du Mt Cocotte, alt. 700 m. Nouveau pour Pile Maurice.

Callicostella fissidentella (Besch.) Broth. Fructifié. Sur un arbre. — MAU­ RICE. Macabé, forét climacique d’altitude, alt. 550 m; le Pouce, forét å mi-hauteur. Nouveau pour Pile Maurice.