A POLYPLOID SPECIES COMPLEX IN SPIROGYRA MAXIMA (CHLOROPHYTA, ZYGNEMATACEAE), A SPECIES WITH LARGE CHROMOSOMES

LIPID SYNTHESIS BY DIATOMS Ben-Amotz, A,, Tornabene, T. G. & Thomas, W. H . 1985. Chemical profile of selected species of microalgae with ernphasis on lipids. ]. Phjcol. 2 1 :72-8 1. Eppley, R. W., Holmes, R. W. & Strickland,J. D. H. 1967. Sinking rates of marine phytoplankton measured with a fluorometer. J . Exp. Mar. Bid. Ecol. 1:191-208. Feinberg, D. A. 1986. Fuel production options from aquatic species: technical and economic considerations. Solar Energy Research Institute technical report, 61 pp. Holm-Hansen, O., Lorenzen, C. J., Holmes, R. N. & Strickland, J. D. H. 1965. Fluorometric determination of chlorophyll. J . Cons. Perm. Int. Explor. Mer 30:3-15. Laing, I. 1985. Growth response of Chaetoceros calcitrans (Bacillariophyceae) in batch culture to a range of initial silica concentrations. Mar. Biol. (Berl.) 85:37-41. Morris, I., Glover, H. E. & Yentsch, C. S. 1974. Products of photosynthesis by marine phytoplankton: the effect of environmental factors on the relative rates of protein synthesis. Mar. Bid. (Berl.) 27: 1-9. Murphy, J. & Riley,J. P. 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta. 27:31-6. Nelson, D. M., Riedel, G . F., Millan-Nunez, R. & Lara-Lara, J. R. 1984. Silicon uptake by algae with no known Si requirement. I. T r u e cellular uptake and pH-induced precipitation by Phaeodactylum tricornutum (Bacillariophyceae) and Platymonas sp. (Prasinophyceae).]. Phycol. 20:141-7. Paasche, E. 1973. Silicon and the ecology of marine plankton diatoms. 11. Silicate-uptake kinetics in five diatom species. Mar. B i d . (Berl.) 19:262-9. Raymond, L. P. 1978. Initial investigations of a shallow-layer algal production system. Technical Report N o . 7. Hawaii Nat-

267

ural Energy Institute, University of Hawaii, Honolulu, 38 PP. Riemann, B. E. F., Lewin, J. C. & Volcani, B. E. 1965. Studies on the biochemistry and fine structure of silica shell formation in diatoms. I. T h e structure of the cell wall of Cylindrotheca fusijormis Reimann and Lewin J. Cell. Biol. 24:3955. Shifrin, N. S. & Chisholm, S. W. 1981. Phytoplankton lipids: interspecific differences and effects on nitrate, silicate and light-dark cycles.J. Phycol. 17:374-84. Smith-Palmer, T . , d e Freitas, A. S. W., McInnes, A. G., Rogerson, A,, McCulloch, A. W. & McLachlan, J. 1985. A study contradicting the “luxury consumption” of silicon by the marine prasinophycean alga, Platjmonas. Bid. Oceanogr. 3:3 15-26. Spoehr, H . A. & Milner, H . W. 1949. T h e chemical composition of Chlorella. Effect of environmental conditions. Plant Physiol. 24: 120-49. Taguchi, S. & Laws, E. A. 1985. Application of a single-cell isolation technique to studies of carbon assimilation by the subtropical silicoflagellate Dictocha perlaevis. Mar. Ecol.-Prog. Ser. 23:251-5. Technicon Industrial System. 1973. Technicon AutoAnalyzer 11, Industrial Method No. 186-72W, Silicates in water and seawater. Technicon Instruments Corporation, Tarrytown, pp. 1-2. Werner, D. 1977. Silicate metabolism. I n Werner, D. [Ed.] The Bioloa of the Diatoms. University of California Press, Berkeley, California, pp. 110-49. Wood, E. D. F., Armstrong, A. J. & Richards, F. A. 1967. Determination of nitrate in sea water by cadmium-copper reduction in nitrite. J . Mar. Bid. Assoc. U.K. 47:23-31.

J. Phycol. 23, 267-273 (1987)

A POLYPLOID SPECIES COMPLEX IN SPIROGYRA MAXZMA (CHLOROPHYTA, ZYGNEMATACEAE), A SPECIES W I T H LARGE CHROMOSOMES’ Robert W. H o s h a w 2 Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 8572 1

Charles V. Wells Department of Biology, Lenoir-Rhyne College, Hickory, North Carolina 2860 1

and

Richard M . M c C o u r t 2 Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 8572 1 ABSTRACT

A species complex in Spirogyra consists of the series of filament morphotypes of various ploidal levels arisingf r o m a n original morphotype within a clonal culture or i n nature. A clonal culture offilaments identijed as Spirogyra maxima (Hassall) Kutzing produced several morphotypes, i.e. filament types of distinctly different widths and ploidal levels. Banding patterns and satellites were visible on chromosomes stained at mitotic prophase and metaAccepted: 9 December 1986. Address for reprint requests.

phase. The original culture of S. maxima contained3laments averaging 1 2 7 p m wide. Vegetative cells of the original culture contained six large chromosomes (> 4 pm long), identijable as three distinct pairs based on banding patterns and presence of satellites: ( I ) one pair of short chromosomes (ca. 5.0 pm); (2) one pair of long chromosomes (ca. 8.0 pm); and (3)a second pair of long chromosomes (ca. 9.0 pm) including a nucleolar organizing region and satellite. A larger morphotype averaging 175 pm in width contained 1 2 chromosomes, with two pairs of short chromosomes and f o u r pairs of long chromosomes (satellites were usually indistinct). Aneuploid chromosome numbers

268

ROBERT W. HOSHAW ET AL.

ranging from 5 to 13 were obserued i n a few cells. Binucleate and trinucleate cells were also obsenled. A twobanded chromosome fragment was observed i n a few cells with 6 chromosomes and a few cells with 12 chromosomes. The variety of morphotjpes derived i n this study could be identzfied as four dzferent species of Spirogyra by conventional taxonomic criteria. The banding patterns and satellites on chromosomes suggest that three pairs of homologous chromosomes are present infilaments of the original clonal culture and that thesefilaments are themselves autopolyploid (diploid) descendants of a n ancestral form with a base chromosome number of x = 3. Kej index words: algal chromosomes; aneuploidj; banding patterns; clonal culture; polyploidq’; species complex; Spirogyra maxima: Zygnemataceae Spirogyra is the most common genus of Zygnemataceae and exhibits the greatest diversity of the 12 to 13 genera recognized in this family of green algae (Transeau 195 1, Randhawa 1959, Kadlubowska 1972). More than 300 species are presently recognized based on morphological characteristics: filament width, chloroplast number, type of endwall, details of the conjugation process, and size, shape, and ornamentation of zygospores. Recently, however, Hoshaw and co-workers (Hoshaw et al. 1985, Wang et al. 1986) have demonstrated that several of these key taxonomic characteristics are affected by ploidal changes within clonal cultures. Such morphological and ploidal changes result in species complexes, which consist of the series of morphotypes of differing ploidal levels arising from an original morphotype within a clonal culture or in nature. Allen (1958) first used the term “species complex” to refer to such autopolyploid series, and we have used the term in our reports (Hoshaw et al. 1985, McCourt et al. 1986, Wang et al. 1986). However, the concept of a species complex in Spirogjra should not be confused with the concept of a polyploid complex in higher plants (Grant 1981). A polyploid complex usually, though not always, involves a hybridization event and subsequent polyploidy. Species complexes in Spirogjra have only been found to involve autopolyploidy without an initial hybridization event. We will use the term “species complex” to refer to autopolyploid variation within a clonal culture in the laboratory or a clonal population in nature. Euploid changes in chromosome numbers within cultures of S. communis (Hassall) Kutzing yielded filaments of distinctly different widths that when subcultured maintained the morphological and ploidal differences between them and the original culture (Hoshaw et al. 1985). In the species complex of S. communis the different morphotypes were interpreted as representing ploidal levels of 2x = 12 (diploid), 3x = 18 (triploid), and 4x = 24 (tetraploid); a possible ancestral form with x = 6 (haploid) was not found. T h e diploid S . communis morphotype derived in the laboratory from the original clonal culture

was later found to exist in nature along with the tetraploid from which it arose in the laboratory (Wang et al. 1986). Earlier work by Allen (1958) also reported a laboratory species complex in S. pratensis Transeau involving ploidal levels of x = 1115, 2x = 26-30, and 4x = 56-60. These reports of species complexes have involved Spirogjra species with small chromosomes (< 1.5 pm long). Although the chromosomes were visible and countable, few details of chromosome structure were observable, and ploidal increase or decrease was inferred from chromosome number and a corresponding increase or decrease in amount of nuclear DNA measured by relative nuclear fluorescence (Hull et al. 1982). We report here on a species complex in S. maxima, a species with large chromosomes. T h e structural details visible on the large chromosomes provided markers to identify homologous chromosomes and allowed a more detailed description of ploidal change than reported previously for species complexes in Spirogjra with small chromosomes. MATERIALS A N D METHODS

Filaments of Spirogjra maxima were obtained in 1982 from a site near where Florida Highway 54 crosses Cypress Creek, 3.8 miles east of Denham, Florida, just north of Tampa. This collection (#RWH82-68) was part of an extensive series of collections in the continental U.S.A. (McCourt et al. 1986). Filaments of one morphologically distinct strain of Spirogjra were established in unialgal culture in soil-water medium on a 16 : 8 hour light : dark cycle at 20” C & 2” C under fluorescent lights at an illumination of 90 pmol.rn-*.s-’. Cultures were transferred every 2 weeks to fresh soil-water medium during a study period of 6 months. Cultures were occasionally kept under low-light conditions for longterm maintenance between cytological experiments. When filaments of several different widths were observed in a sample from a single culture, filaments differing in width from the parental culture were isolated into separate soil-water bottles and maintained under the conditions described above. For cytological investigation, 10- to 14-day-old cultures were sampled 30 to 60 min after the start of the dark period. Previous observations showed this to be the time when chromosomes were in late prophase or metaphase of mitosis. Filaments were fixed in a 3:l solution of 95% ethanol : glacial acetic acid. After one to two hours fixation, filaments were transferred to 70% ethanol for storage at 4”C. Nuclei were stained with propiocarmine with an iron mordant or a modified version of the Feulgen technique (Wells and Hoshaw 1971). Prior to mounting, filaments were soaked for 10 minutes in distilled water to intensify chromosome staining. Stained filaments were then mounted on slides in either 45% acetic acid or a weak solution of propiocarmine without mordant, then covered with a No. 1 cover glass. To flatten the cylindrical cells, prepared slides were covered with paper towels and a wooden board and hit with a hammer. T h e border of the cover slip was sealed to prevent dehydration. Slides were stored in the dark at room temperature. Photomicrographs were made using a Leitz Ortholux microscope equipped with an Orthomat camera. Scanning electron microscopy (SEM) was performed using the methods described in Hull et al. (1985). Zygospores were prepared for SEM by mechanically removing the outer layer (exospore) to expose the ornamented mesospore. RESULTS

Filaments from the original collection were identified as S. maxima on the basis of filament width (9 =

SPECIES COMPLEX IN SPIROCYRA MAXIMA

269

FIGS.1-6. Spirogyra maxima. FIG. 1. Male gametangium (mgm) and female gametangium (fgm) connected by papillae (p). Scale bar = 100 pm. FIG.2. Male gamete (mg) and female gamete (fg) in gametangia connected by conjugation tube (ct). Scale bar = 100 pm. FIG. 3. Gametangia with lenticular-shaped zygospores (2). Scale bar = 100 pm. FIG. 4.Scanning electron micrograph of reticulate ornamentation of mesospore of a zygospore. Scale bar = 50 pm. FIG. 5. Six chromosomes in cell from original culture. Chromosome pairs are labelled (I, 11, and 111). Note satellites (s). Scale bar = 5 pm. FIG. 6. Six chromosomes plus a fragment (f) in cell from original culture. Note satellites (s). Scale bar = 5 pm.

127 pm, n = 1 13), number of chloroplasts per cell (6-7), and ornamentation of the zygospores. Homothallic scalariform conjugation between paired filaments was observed (Fig. l). Anisogametes were produced in male and female gametangia (Fig. 2). T h e smaller male gamete moved through the conjugation tube to fuse with the female gamete and form a lenticular zygospore (Fig. 3), whose mesospore had reticulate ornamentation (Fig. 4). Interphase nuclei of S. maxima contained one large or two smaller nucleoli, each with a typical nucleolar-organizing (N.O.) track. T h e interphase nucleus stained with the modified Feulgen technique was observed to contain approximately 40 heterochromatin bodies (chromocenters) varying in size from 0.5 to 1.5 pm in diameter. These chromocenters appeared to be located close to the interface between the nucleus and the cell cytoplasm. Very thin Feulgen-positive stained strands called chromonemata were sometimes seen to connect adjacent chromocenters. Such interphase cytological features are typical for Spirogyra and have been illustrated in Godward’s (1966) monograph. During mitosis, a nucleolar substance (Godward 1966, Wells and Hoshaw 197 1, Hoshaw and Wells 1982) covered the chromosomes of S. maxima and made the determination of chromosome number and morphology difficult. T h e Feulgen technique, which is DNA-specific and does not stain nucleolar substance, was therefore the stain of preference in this investigation. Chromosomes formed through the coalescence of five to eight chromocenters. Prophase and metaphase chromosomes had bands of heterochromatin connected by unstained euchro-

matic regions (Fig. 5). Chromosomes displayed parallel separation at anaphase. T h e original culture of Spirogjra maxima was found to have six chromosomes per nucleus (Fig. 5). Approximately 23% (n = 145) of the dividing cells in the original culture of S. rnaxiina contained a twobanded fragment in addition to the normal six chromosomes (Fig. 6). T h e origin of the fragment was not determined because the six chromosomes in these cells were the same size and had the same banding pattern as those in cells having six chromosomes but no fragment. T h e six chromosomes constituted three distinct pairs based on size, banding pattern, and the presence or absence of a satellite (Fig. 7, Table 1). Pair I chromosomes were approximately 5.0 pm long and possessed 3 distinct major bands (1, 4, 6) and 3 very thin minor bands (2, 3, 5) (Fig. 7). Pair I1 chromosomes were approximately 8.0 pm long. These larger chromosomes had 5 to 7 visible bands depending on the degree of chromosome contraction. If the chromosomes of pair I1 were strongly contracted, bands 1 and 2 and bands 5 and 6 appeared as two broad bands. Pair I11 chromosomes had 7 to 8 bands and were approximately 9.0 pm long including an N.O. region and satellite (Fig. 7). Bands 6 and 7 of pair 111often appeared as a single band due to chromosome contraction. A few cells in the original culture contained 12, 13, or 7 7 (= binucleate) chromosomes. Widths of these cells were not determined because they were only identified as atypical in chromosome number after filaments had been deformed by squashing. Observations of vegetative filaments revealed con-

+

270

ROBERT W. HOSHAW E T AL.

:hromosome xlir

T1

LENGTH (,urn) 1

2

3

4

5

6

I

I

I

I

I

I

w 1

2

nLL

I

9 I

7

.I

5

6 7

I 1

I

5 6

rn 4

8

three distinct assemblages of chromosomes (Table 1). Cells with 6 chromosomes (the most abundant type) or 6 chromosomes plus a fragment had similar widths. Cells with 13 chromosomes o r 13 13 (= binucleate) were found and were significantly wider than cells with 6, but only a few cells with these higher chromosome counts were observed and their widths were not precisely measurable due to squashing of the cells. T h e few cells with 13 chromosomes (Fig. 10) possessed 4 short chromosomes of pair I and 9 longer chromosomes of either pair I1 or pair 111. Because of the indistinct appearance of satellites and banding patterns on chromosomes in these cells, the pair type (I1 or 111) of the other 9 long chromosomes was not determined. Strain B contained filaments with three different chromosome counts (Table 1). As in strain A, filaments with 6 chromosomes were the most abundant type. Strain B contained several cells with 7 chromosomes and some cells with 6 plus a fragment. Cells with 7 chromosomes contained 2 short chromosomes of pair I and 5 long chromosomes of either pair I1 o r 111. Despite the observation of one satellite on a long chromosome, satellites and banding patterns were usually not clearly visible and assigning the long chromosomes to either pair I1 or I11 was not possible. Strain C contained filaments with cells wider than the original isolate. Most cells contained 12 chromosomes, but chromosome numbers in strain C were the most variable of any strain (Table 1). Aneuploid numbers of 5 , 8, and 13 were observed, although these aneuploid cells were rare. Cells with aneuploid counts were not appreciably different in width from cells with 12 chromosomes, although the squashed condition of the cells precluded accurate width measurements. In one filament, a single dividing cell was

+

4

3

I

7

I R

0

w.-

"i-------. Satellite

FIG. 7. Diagrammatic representation of three chromosome pairs of Spirogyra maxima showing banding patterns and satellites.

siderable differences in filament width (Fig. 8). One narrow filament was observed to give rise directly to a filament almost twice the original width (Fig. 9). Filaments similar to the derived wide form were later found in the clonal culture and isolated into separate cultures. Four subcultures of wide filaments derived from the original culture were established by isolating four filament sections several cells long into the separate soil-water bottles. These cultures were labelled A-D and used for cytological investigations. Although initially wider than the original culture, filaments in cultures A-D displayed some morphological diversity over time. This diversity in filament width is described below. T h e culture of strain A contained filaments with

TABLE 1 . Fzlament wzdths and chromosome numbers of the origtnal culture of S. maxima and four subcultured strains (A-0) Mean wadth and wzdth ranges based on lzvzngjlaments, these values are not e v e n for filaments with chromosome counts that were rare because of squashed condztzon of the fm cells obserued Strain

Original culture A

B C

D a

Filament width (wm) (mean SDj '

*

127 f 8.3 126 k 12.4 122 f 2.8 179 ? 10.9 136 k 7.8 166 ? 8.0 -

n

Width ranee bm) "

113 32 15 15 15

109-140 106-148 106-148 165- 192 127-150

50

156-191 -

175 f 6.7

Binucleate. Satellites not visible; possibly pair type I1 or 111 Trinucleate.

160- 190

Number of chromosomes of pair type Chromosome number

I

6 6 + fragment 12, 13, 7 + 7' 6 , 6 + fragment 13, 13 + 13" 6 , 6 + fragment 7 12 13 5 8 13 + 13= 13 + 13 + 13< 12, 12 fragment

2 2 2 4 2 2 4 4

+

4

11

Ill

2 2

2 2

-

2 6b 2 5b 8b 9b

4

SPECIES COMPLEX IN SPZROGYRA M A X I M A

27 1

FIGS.8-1 1. Spirogjm maxima. FIG.8. Narrow and wide filaments from a single clonal culture. Scale bar = 100 pm. FIG. 9. Filament showing width change in intercalary cells. Scale bar = 100 fim. FIG.10. Thirteen chromosomes in cell of strain A. Four short chromosomes of pair type I are labelled with arrows. Scale bar = 5 pm. FIG. 11. Twelve chromosomes in cell of strain D. Scale bar = 5 um.

found to possess three nuclei, each with 13 chromosomes. In nuclei with 12 or 13 chromosomes, four were of pair type I. T h e remaining chromosomes were longer and clearly of pair types I1 or 111, but further discrimination of pair type was not possible due to poor banding o r indistinct staining of satellites. Cells in strain D contained 12 chromosomes (Fig. 11) and were significantly wider than cells in the original isolate (Table 1). Two cells in strain D were found with 12 chromosomes plus a fragment. Banding patterns and satellites were distinct in stained material from this strain and pair types I, 11, and I11 were each represented by 4 chromosomes (two pairs) (Table 1). DISCUSSION

Mitosis in Spzrogira maxzinn was basically similar to the process as described for other Spzrogjra species (Godward 1966, Harada and Yamagishi 1984a) and Szrogonzum (Wells and Hoshaw 197 1, Hoshaw and Wells 1982). T h e structure of the nucleolus and the activity of the nucleolar substance in S. maxzma were similar to that described for these other zygnematacean algae. T h e number of chromocenters (ca. 40) was roughly equal to the total number of heterochromatin bands (ca. 32), and we assume that the chromocenters were incorporated as heterochromatin bands in the forming chromosomes. T h e lower number of bands was probably due to adjacent chromocenters forming t w o bands with no visible euchromatin between them. T h e process in which the chromocenters are incorporated into the chromosome as heterochromatin bands has been observed for Szrogoizzuin inrlaizosporuin (Randhawa) Transeau, which also has 6 rod-shaped chromosomes (Hoshaw and Wells 1982). In these cytological respects S. mamma appears similar to other Zygnemataceae examined to date. T h e origin of a two-banded fragment in cells with

6 chromosomes (and occasionally in cells with 12 chromosomes) is not known. In cells with fragments the three chromosome pairs were of normal size and possessed the normal banding pattern. This suggests that the fragment was derived from a disjunction event that left an extra fragment in one of the two daughter cells. T h e two-banded fragment would probably retain the diffuse centromeric activity of the chromosome from which it was derived and therefore could duplicate itself and separate normally at anaphase of mitosis. T h e mechanisms responsible for ploidal variations in S. inaxiina a r e unknown. We suspect that the variety of chromosome numbers observed was probably due to the occurrence of general and specific nondisjunction of chromosomes at mitosis. Whatever the mechanism of ploidal variation, it is obvious that S. maxima displays variability in morphology and chromosome numbers. Although cells with aneuploid o r euploid chromosome counts were observed, the latter were more common. We conclude that once polyploid cells arose, they persisted in culture and grew more readily than aneuploid cells. Euploid differences in chromosome number resulted in distinct width differences, but cells with aneuploid chromosome numbers (5, 7, 8 , 13) were not appreciably different in width from other cells in the cultures in which they occurred. Cell volume usually increases with ploidal increase (Lewis 1980) and DNA content is generally correlated with cell volume in higher plants (Price 1976) and unicellular eukaryotes (Shuter et al. 1983). O u r results fit this general trend, but the lack of correlation of cell width with aneuploid chromosome numbers indicates that the relationship of ploidal level to cell size in Spirogjra is not simple. T h e changes in filament width and ploidy observed during the course of this study were typical of species-complex formation in Spirogjra (Allen 1958, Hoshaw et al. 1985, Miang et al. 1986). T h e

272

R O B E R T W. HOSHAW E T AL.

TABLE 2. Characteristics of four Spirogyra (Transeau 1951) species ulhose descriptions encompass the morphological variation displayed by morphotyfies in the species coinplrx of S. maxima in this study. Sortie\ of S I n r o n r o

S. submaxiinaa S.maximaa S.crassiusculac S.megaspova‘

Filarnrnt a i d t h (rrrn)

Chloroplast number

70-1 10 118-140 145-1 70 170-200

8-9 6-7 6-7 6-7

Z>rosvore shape

lenticular lenticular lenticular lenticular

Mesospore ornamentation

smoothb reticulate reticulate reticulate

z\aorporc

00101

brown golden-brown yellow-brown yellow-brown

* S. submaxima found hybridizing with

S. maxima (Transeau 195 1). SEM examination of material identified as S. submaxima with light microscopy revealed the mesospore wall to be pitted or punctate rather than smooth (Hull et al. 1985). S. crassiuscula and S. inegaspora “are similar in appearance to S.maxima, but are larger in all dimensions.” (Transeau 1951, p. 195).

discrete morphotypes produced in a species complex usually fit several species descriptions (Allen 1958, Hoshaw et al. 1985). T h e observation in this study of width change in a single filament of S. maxima (Fig. 9) is the first direct observation of morphological change associated with ploidal change within a filament in a culture that produced morphotypes of a species complex. This observation corroborates the inference of the previous studies on Spzrogjra species complexes that ploidal change occurs in vegetative cells through some process involving mitosis. Ploidal change during the sexual process (conjugation and zygotic meiosis) has also been inferred from the diversity of filaments arising from zygospores produced in homothallic conjugation in Spirogjra (Hoshaw et al. 1985, Wang et al. 1986) and heterothallic conjugation in Zjgnema (Miller and Hoshaw 1974). Previous studies (Godward 1966, Hoshaw et al. 1985) suggested that polyploidy in Spirogjra has produced a number of morphotypes or species derived from an ancestor with a small base chromosome number. However, the small size of chromosomes obscured details of chromosome morphology and prevented direct comparison of possible homologous pairs. In this study, chromosome numbers, sizes, banding patterns and satellites imply that the originally collected filaments contained three pairs of homologous chromosomes and were thus diploid (2x) derivatives of a haploid ancestor with x = 3 chromosomes irl vegetative cells. T h e morphotypes with wider filaments and 12 chromosomes are thus considered tetraploid (4x). Wang et al. (1986) found two Spirogjra morphotypes in nature that were indistinguishable from morphotypes in a laboratory-derived species complex. They also found that ploidal levels of the laboratory morphotypes were equal to the respective field morphotypes. McCourt et al. (1986) provided evidence that polyploid species complexes in Spirogjra may be widespread and common in nature. Harada and Yamagishi (1984a, b) studied mitosis and meiosis in four species of Spirogjra from Japan. They found pairs of homologous chromosomes in vegetative cells of the four species and concluded that each was diploid in vegetative condition. Their stud-

ies also support the hypothesis that polyploidy is common in Spirogjra. Harada and Yamagishi (1984a, b) studied two strains of Spirogjra from Japan that may be analogous to the two morphotypes in the present studies. They identified two field-collected strains as S. crassa Kutzing and S. crassu X. T h e two strains differed morphologically only in cell dimensions. Vegetative cells of the wider strain, S. crussa, contained 12 large chromosomes; the narrower cells of S. crassa X contained 6 large chromosomes. Both strains contained two N.O. chromosomes. Chromosome size and behavior during mitosis were similar in both strains. T h e relationship between these Japanese strains resembles that between the morphotypes of S. maxima in the present study, except that the 12-chromosome morphotype in our study contained four nucleolar organizing chromosomes, and the Japanese strains apparently contained two instead of three chromosome pair types. Harada and Yamagishi did not report on the characteristics they used to identify their material as S. crassa, whose vegetative characteristics (cell width, numerous chloroplasts) are similar to those of S. maxima. It seems possible that they were dealing with field-derived morphotypes of a species complex very similar to the laboratoryderived species complex in our study. T h e width ranges of morphotypes in the strains observed in this study span the width ranges of four species as reported by Transeau (195 1) (Table 2). A comparison of the distinguishing morphological characters of these four species shows that they differ primarily in the widths of vegetative filaments (Table 2). Transeau (1951) noted that S. maxima differed from S. crassiuscula and S. megaspora mainly in dimensions of vegetative cells. T h e only significant difference other than filament width in Table 2 is the occurrence of a smooth mesospore wall in the zygospore of S. submaxima compared to a reticulate mesospore in the other three species. However, Hull et al. (1985) have recently reported that the mesospore wall of S. submaxima is actually pitted instead of smooth. Transeau (1951) noted in his monograph that hybridization had been observed between S. maxima and S. submaxima and between S. maxima and S. nitida, a narrower species outside

SPECIES COMPLEX I N S P I R O G Y R A 'MAXIMi?

the width range of filaments in the S. maxima species complex. In light of the width variations reported in this study and the lack of other morphological differences, the validity of separating these species is questionable, at least in the context of such morphotypes occurring together in the same site. If the morphotypes observed in this laboratory investigation are found at the original collection site, they would probably represent a naturally occurring species complex. Moreover, if species complexes are common in nature, the species concept in Spirogjra requires revision. This research was supported by National Science Foundation Grants BSR-8215730 and BSR-8516681 to R.W.H. T h e authors wish to thank Jen-Chyong Wang for asistance with cytological techniques and H. R. Hauck for editorial and material aid in the preparation of the manuscript. Allen, M. A. 1958. T h e biology of a species complex in Spirogjra. Ph.D. thesis. Indiana University, Bloomington, 240 pp. Godward, M . B. E. 1966. T h e Chlorophyceae. I n M. B. E. Godward [Ed.] The Chromosomes ofthe .4lgar. Edward Arnold, London, pp. 1-77. Grant, V. 1981. Plant Speciation, 2nd ed. Columbia University Press, New York, 563 pp. Harada, A. & Yamagishi, T . 1984a. Mitosis in Spirogjra (ChloroDhvceae). lab. I. P h d . 32: 1-9. -' 1984b. Meiosis in>pirogjra (Chlorophyceae).Jap. J . PhjC O ~ . 32110-8. Hoshaw, R. W., Wang, J. C., McCourt, R. M. & Hull, H. M. 1985. Ploidal changes in clonal cultures of SpzrogJra coininunzs and implications for species definition. AWLJ . Bot. 72:1005-1 I . I

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J . P ~ J c o23, ~ . 273-283 (1987)

ADAPTA T I 0 N 0F P H 0TOSYN T H ESI S I N LAMILVARIA SA CCHARINA (PHAEOPHYTA) TO CHANGES I N G R O W T H TEMPERATURE' I a n R. Davison Department of Botany and Plant Pathology, University of Maine, Orono, Maine 04469

ABSTRACT

The effect of growth temperature on photosjnthetic metabolism was studied in the kelp Laminaria saccharina (L.)Lamour. Photosynthesis was subject to phenotjpic adaptatzon, with almost constant photosjnthetic rates being achtezled at growth ternperatures between 0 and 20" C This response involved: (1)a n inzferse relationship between growth temperature and photosinthetic capactti, (2) a reduction in the & value f o r photosintheszs of L. saccharinagrown at 0 and 5°C compared with 10, 1 5 and 20" C grown sporophjtes, and (3) a n acquired tolerance ofphotosjnthesis to temperatures betzleen 15-25°C (which znhzbzted photosjntheszs in 0 and 5" C grozLln L. saccharina) zn sporophjtesgrown a t 10, 1 5 and 20°C. The I

Accepted: 10 December 1986.

plijsiological basis of these adaptations is discussed in terms of obserzled changes in actitities and kinetics of the Ca lzlin cjcle enzjme ribulose-l,5-bisphosphatecarboxylase (oxjgenase) and eficiencj of light harvesting-electron transport sjsterns. Key index words: brown algae; Laminaria; phenotypic adaptation; photosjnthesis; ribulose-l,5-bisphosphatecarboxjlase-oxjgenase; temperature adaptation Although the genus Laminaria inhabits polar and temperate seas (environments characterized by low sea water temperatures; Kain 1979), Laminaria spp. are highly productive plants (Hatcher et al. 1977, Johnston et al. 1977), suggesting that this genus may possess specific adaptations to counteract the effect of low temperature on metabolism. This supposition