| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
2 Department of Biology, Vanderbilt University, Nashville, Tennessee 37235 USA; and 3 School of Biological Sciences, University of Auckland, Private Bag 92109, Auckland, New Zealand
Received for publication July 18, 1999. Accepted for publication October 21, 1999.
ABSTRACT
Mature, gametangia-bearing photosynthetic gametophytes of Phylloglossum can be grown from the nonphotosynthetic, cylindrical, negatively gravitropic immature stages in illuminated axenic culture on a nutrient medium with or without 0.2% glucose. The gametangial-bearing region of these gametophytes, the photosynthetic crown, grows horizontally from the apex of the immature cylindrical stage. At maturity the photosynthetic crown is thickened and bilaterally symmetrical. It is usually narrow and bean-shaped with dorsal and ventral regions. Occasionally, the ventral region becomes thicker and the crown is deltoid in cross section. The dorsal edge or ridge of the crown is the gametangial region with archegonia and antheridia. The gametangia are often hidden because they are interspersed among numerous uni- or bicellular paraphyses. The lateral surfaces below the gametangial region lack outgrowths, and the ventral region is covered with long rhizoids. The apical meristem at the anterior end of the crown is overarched by the developing dorsal tissue of the gametangial ridge. The lower derivatives of the meristem form the ventral region with rhizoids. Phylloglossum gametophytes are unusual because their mode of organic nutrition changes from mycorrhizal to photosynthetic during ontogeny.
Key Words: development • gametophyte • Lycopodiaceae • morphology • Phylloglossum • pteridophyte
The monotypic genus Phylloglossum is from Australia and New Zealand, and little is known about its mature gametophyte (Øllgaard, 1987
). Although Thomas (1902)
, Sampson (1916)
, and Holloway (1935)
found gametophytes, only Thomas (1902)
obtained large numbers of them. Sampson (1916) found a poorly preserved gametophyte with few structural details attached to a young sporophyte. A single well-preserved gametophyte with antheridia was described by Holloway (1935)
. No additional discoveries of mature gametophytes have been made since 1935 and what knowledge there is depends almost completely on the observations of Thomas (1902)
.
Thomas (1902)
described the mature gametophyte as having a primary tubercle, an upright shaft, and an irregularly shaped crown bearing gametangia. The primary tubercle and cylindrical shaft were subterranean. The tubercle and basal part of the shaft were nonphotosynthetic and contained an endophytic fungus. The photosynthetic crown was described as being conical, rounded, or projecting to one side and looking "like the head of a horse." All the described crown shapes and the absence of any illustrations in the Thomas report make understanding the basic shape and morphology of the crown difficult.
Since many details of the gametophyte structure remain unknown, the present study was initiated to provide a more complete description of the mature Phylloglossum gametophyte. The use of axenic culture techniques (Whittier and Braggins, 1992
) has confirmed the observations by Thomas (1902)
on young gametophytes up to the initiation of the photosynthetic crown. Besides demonstrating the early stages of gametophyte development, these techniques can make available numerous gametophytes for study without having to depend on finding them in nature.
MATERIALS AND METHODS
Spores were obtained from strobili of Phylloglossum drummondii Kunze collected at Lake Ohia and Ahipara, New Zealand. Vouchers of sporophytes are on deposit at the Vanderbilt University Herbarium (VDB). The system of classification followed in this report is that of Øllgaard (1987, 1989)
. Under this system the Lycopodiaceae are composed of four genera: Lycopodium, Lycopodiella, Huperzia, and Phylloglossum. The techniques employed to germinate the spores and grow the gametophytes were those of Whittier and Braggins (1992)
. The spores were germinated, and young gametophytes were grown in the dark on the nutrient medium with 0.2% glucose. The young nonphotosynthetic gametophytes were moved into light to turn green. Mature gametophytes were obtained by transferring young photosynthetic gametophytes to fresh nutrient medium with or without glucose and allowing them to grow.
The gametophytes were grown on 15 mL of nutrient medium in 20 x 125 mm culture tubes with screw caps, which were tightened to reduce moisture loss. A litre of nutrient medium contained 100 mg MgSO4·7H20, 40 mg CaCl2, 100 mg NH4NO3, and 100 mg K2HPO4. The medium was completed with 0.5 mL of a minor element solution (Whittier and Steeves, 1960
), and 4 mL of a FeEDTA solution (Sheat, Fletcher, and Street, 1959
), and 2 g or no glucose. The medium was solidified with 1.0% agar and had pH 5.0 after autoclaving. The cultures were maintained at 21° ± 1°C in light for 12 of every 24 h. The irradiance level was 50 µmol·m-2·s-1 from Gro-lux fluorescent lamps. Glucose in the nutrient medium accelerated the growth of the gametophytes but did not alter their morphology. The youngest gametophytes examined in this study were 2 yr old.
The gametophytes were fixed with Randolph's Modified Navashin Fluid (Johansen, 1940
) for light microscopy. After fixation, the gametophytes were embedded in paraffin and sectioned by conventional techniques (Johansen, 1940
). The sections were stained with Heidenhain's hematoxylin, safranin O, and fast green. Twenty antheridia and 20 archegonia were measured to determine their average size.
For scanning electron microscopy, the gametophytes were fixed overnight on ice in a 1:1 solution of 4% glutaraldehyde and 10% acrolein in 0.1 mol/L Hepes buffer (pH 6.8). The gametophytes were postfixed in 1% osmium tetroxide in 0.1 mol/L Hepes buffer (pH 6.8) at room temperature for 1 h. They were then treated with 1% aqueous thiocarbohydrazide for 30 min after the osmium postfixation. The gametophytes were refixed with 2% osmium in water for 1 h and then dehydrated in a graded acetone series. All specimens were critical point dried and coated with gold-palladium before observing with a Hitachi 4500 or 370 scanning electron microscope at 10 or 15 kV.
RESULTS
The mature photosynthetic crown of Phylloglossum is bilaterally symmetrical and tuberous. Most are bean-shaped and narrow, with dorsal and ventral regions (Figs. 1, 2). Some, often the older and larger ones, are more deltoid in cross section. For these gametophytes the ventral region is wider than the dorsal region, which forms a ridge along their length (Figs. 3, 4). The posterior portion of the crown may have two projections (Figs. 2, 3), however, they are not as well developed on some gametophytes (Fig. 1). The dorsal edge or ridge can extend into a narrow, horizontal, pointed projection beyond the main posterior portion of the crown (Figs. 2, 3). A second irregular finger-like projection often is found connected to the posterior ventral region. This projection, which can vary in length, is the cylindrical shaft and basal portion of the young gametophyte and often all of it can be photosynthetic. An absence of chlorophyll in the basal region of long cylindrical shafts may be related to their developmental stage at the time the young gametophytes were moved from the dark into the light. If the cylindrical shaft is short when placed in the light, the ventral projection can appear to be absent on mature gametophytes (Fig. 1).
|
The lateral surfaces of the gametophytes are smooth. There are no outgrowths from the surface cells between the gametangial and ventral regions (Figs. 1–4) either on the vertical surfaces of the bean-shaped gametophytes or the slanted surfaces of the gametophytes with wide ventral regions. The lower surface of the ventral region has numerous long unicellular, nonphotosynthetic rhizoids (Figs. 1, 4, 7).
The apical meristem is at the anterior end of the photosynthetic crown. It is subterminal and overarched by tissue of the dorsal region (Figs. 7, 8). Cells from the upper portion of the meristem contribute to the dorsal gametangial region. Cells from the lower portion give rise to the ventral region with the rhizoids. The width of the apical region is about that of the dorsal gametangial region of the bean-shaped gametophytes. However, it is narrower than the main body of the gametophytes with wide ventral regions. With these gametophytes the anterior end of the crown narrows to the width of the apical meristem.
In regions where the paraphyses have not reached their full size or where they are sparsely distributed, the necks of the archegonia and the opercular cells of the antheridia are evident (Figs. 9, 10). The necks of the archegonia are short with two tiers of neck cells exposed at the surface (Figs. 9, 11). The neck cells are almost colorless because they have fewer and smaller chloroplasts than the other cells of the crown. Mature archegonia have an average length of 190 µm from the base of the egg cell to the tip of the neck, and there are three cells in the neck canal region above the egg (Fig. 11).
The antheridia are completely sunken into the tissue of the gametangial region (Figs. 10, 12). There are no raised areas to indicate their position. However, bulging opercular cells with a few small chloroplasts identify the antheridia (Fig. 13). Each antheridium has one or often two triangular opercular cells (Figs. 10, 13). The antheridium contains a narrowly ellipsoidal mass of gametes at maturity (Fig. 12). The average length of an antheridium is 181 µm from the base of the gamete mass to the tip of the opercular cell. At its widest the mass of gametes has an average diameter of 78 µm.
DISCUSSION
Although earlier studies did not present a clear description of the crown's shape, Thomas (1902)
and Holloway (1935)
did describe other aspects of the crown that agree with what has been found in this study. Some details of the dorsal gametangial region were noted previously by these workers. Younger gametophytes bear only antheridia in the gametangial region as noted by Holloway (1935)
. Mature gametophytes have both antheridia and archegonia intermixed in the gametangial regions as found by Thomas (1902)
.
The sunken antheridia can be tightly packed on the dorsal surface of the crown (Holloway, 1935
). Both Holloway (1935)
and Thomas (1902)
recognized that the outer jacket of antheridia was one cell thick, and Holloway (1935)
noted a cap or opercular cell for each antheridium. The present study extended these observations to show that there are often two opercular cells for each antheridium, and they are almost clear because they contain a few small chloroplasts.
The archegonia, previously seen by Thomas (1902)
, have the same structure on gametophytes from either soil or culture. A short neck of two tiers of clear neck cells was noted previously by Thomas (1902)
. New observations include the three cells in the axial row above the egg. How these cells originate awaits a detailed study on archegonial development.
Thomas (1902)
and Holloway (1935)
both recognized that the crown can grow to one side from the top of the cylindrical shaft. Otherwise, there are few similarities among these studies on the shape of the crown. In culture, the crowns are usually bean-shaped with a dorsal gametangial region and a ventral rhizoid-bearing region. The only variation was in the width of the ventral region. With larger and older gametophytes, the ventral region of the crown can be much wider than the gametangial region. Even with this type of variation, these crowns have a more consistent shape than those from soil. The variations in the gametophyte crown from culture are minor modifications to the basic bean shape.
Besides not understanding the basic shape of the crown, there was uncertainty about how it developed (Thomas, 1902
; Holloway, 1935
; Whittier and Braggins, 1992
). The site of meristematic activity was not clearly identified for the crown in these earlier studies. There is no uncertainty about the site of meristematic activity in the mature gametophytes from culture because they have a well-defined apical meristem. It is subterminal with overarching dorsal tissue at the anterior end of the crown. It is not unlike the meristems of Huperzia lucidula (formerly Lycopodium lucidulum) (Whittier and Webster, 1986
) and Lycopodium digitatum (Bruce, 1979
; Whittier, 1981
) in the sense that young tissue of the gametangial region overarches the meristematic area and the ventral region of the gametophyte forms from the lower portion of the meristem.
Thomas (1902)
and Holloway (1935)
did not find paraphyses in the gametangial region. Similarly, Whittier and Braggins (1992)
did not observe them on gametophytes in early stages of crown development. Although they are not present on the young gametophytes grown in culture, they arise on gametophytes once the early stages of crown development have taken place. Mature gametophytes of Phylloglossum have paraphyses on their dorsal surface intermixed with the gametangia. Prior to this study, paraphyses have been reported for the Lycopodiaceae only on Huperzia gametophytes.
From the descriptions of Thomas (1902)
it would appear that the gametophytic crowns from nature are more variable in shape than those from culture. We speculate on the reasons for these differences. First, Thomas (1902)
reports variations caused by soil irregularities and particle size and hardness that would not occur in culture. Another possibility may relate to the size of the gametophytes. The gametophyte studied by Holloway (1935)
was sexually immature and those studied by Thomas (1902)
may have been young, sexually mature gametophytes. Early formation of archegonia and rapid sexual reproduction could have occurred before the mature form of the crown was established by these soil-grown gametophytes. Also, developing embryos may have altered the shape of small crowns. The gametophytes examined from culture were larger than those observed previously. The growth of gametophytes with larger crowns allowed these Phylloglossum gametophytes to develop a more mature form in culture than observed in earlier studies.
In the past, similarities between gametophytes of Phylloglossum and Lycopodiella cernua (formerly Lycopodium cernuum) have been emphasized (Thomas, 1902
; Holloway, 1935
; Hackney, 1950
; Bruce, 1976
; Øllgaard, 1987
). In a more recent comparison of gametophytes of Phylloglossum and Lycopodiella (formerly Lycopodium subgenus Lepidotis) by Whittier and Braggins (1992)
it was noted that there were few similarities. More comparisons can now be made because the structural details of mature Phylloglossum gametophytes are known. Lycopodiella gametophytes lack the paraphyses that develop on mature Phylloglossum gametophytes. Gametangia from the gametophytes of Phylloglossum and Lycopodiella cernua grown in culture are different. The sex organs of Phylloglossum are larger than those of L. cernua. Phylloglossum archegonia have two tiers of neck cells above the gametophyte surface and those of L. cernua have one. The archegonial length from the base of the egg to the tip of the neck for L. cernua is less than half that of Phylloglossum. The antheridia of L. cernua are some of the smallest in the Lycopodiaceae, and their volume is at most one-third the volume of those of Phylloglossum. In terms of archegonial length and antheridial volume the gametangia of Phylloglossum are more similar to those of Lycopodium and Huperzia than to those of Lycopodiella. Other than mature gametophytes growing above ground and being photosynthetic, the gametophytes of Lycopodiella and Phylloglossum appear to be less similar than previously noted by Whittier and Braggins (1992)
.
The dissimilarities between gametophytes of Lycopodiella and Phylloglossum are many, but they should not be unexpected. Wikström and Kenrick (1997)
using rbcL data have shown that Phylloglossum is more closely related to Huperzia than to Lycopodiella or Lycopodium. As noted earlier, paraphyses on the gametophytes of Phylloglossum, previously known only on Huperzia gametophytes for this family, would support the conclusions of Wikström and Kenrick (1997)
.
The present and past studies present an essentially complete description of the Phylloglossum gametophyte, which is an unusual gametophyte even for the Lycopodiaceae. It begins subterranean and mycorrhizal and then becomes surficial and photosynthetic. Germination of the spores in the dark insures that the young gametophyte (primary tubercle) will be subterranean. Growth at the negatively gravitropic cylindrical stage brings the apex to the soil surface and to light where the photosynthetic crown develops. Gametophyte growth is completed with a bilaterally symmetrical, bean-shaped crown having a dorsal gametangial region and a rhizoid-bearing ventral region. Mycorrhizal gametophytes and photosynthetic gametophytes are not unusual for the Lycopodiaceae. However, under normal circumstances gametophytes of the other genera in the Lycopodiaceae are either photosynthetic or mycorrhizal and they do not switch from mycorrhizal to photosynthetic or vice versa. The change in type of organic nutrition from mycorrhizal to photosynthetic during the normal ontogeny of a pteridophyte gametophyte is only known for Phylloglossum.
FOOTNOTES
1 The authors thank R. L. Peterson for the use of his laboratory facilities in the Department of Botany at the University of Guelph (Canada) where the scanning electron microscopy was done. This study was supported in part by the Vanderbilt University Research Council.
Please note that in a break with the past the A. P. W. Thomas reference is being listed as published in 1902. The Thomas paper was read to the Royal Society of London in December 1901. This appears to be the reason why the Thomas reference has always been listed as 1901. However, the article appeared in Volume 69 of the Proceedings of the Royal Society of London, which was published in 1902. The correct year (1902) is listed for Volume 69 in this paper. 
LITERATURE CITED
Bruce, J. G. 1976 Development and distribution of mucilage canals in Lycopodium. American Journal of Botany 63: 481–491.
———. 1979 Gametophyte of Lycopodium digitatum. American Journal of Botany 66: 1138–1150.
Hackney, F. M. V. 1950 A review of and contribution to knowledge of Phylloglossum drummondii Kunze. Proceedings of the Linnean Society of New South Wales 75: 133–152.
Holloway, J. E. 1935 The gametophyte of Phylloglossum drummondii. Annals of Botany 49: 513–518.
Johansen, D. A. 1940 Plant microtechnique. McGraw-Hill, New York, New York, USA.
Øllgaard, B. 1987 A revised classification of the Lycopodiaceae s. lat. Opera Botanica 92: 153–178.
———. 1989 Index of the Lycopodiaceae. Biologiske Skrifter 34: 1–135.
Sampson, K. 1916 Note on a sporeling of Phylloglossum attached to a prothallus. Annals of Botany 30: 605–607.
Sheat, D. E. G., B. H. Fletcher, and H. E. Street. 1959 Studies on the growth of excised roots. VIII. The growth of excised tomato roots supplied with various inorganic sources of nitrogen. New Phytologist 58: 128–154.[CrossRef]
Thomas, A. P. W. 1902 Preliminary account of the prothallium of Phylloglossum. Proceedings of the Royal Society of London 69: 285–291.
Whittier, D. P. 1981 Gametophytes of Lycopodium digitatum (formerly L. complanatum var. flabelliforme) as grown in axenic culture. Botanical Gazette 142: 519–524.[CrossRef]
———, and J. E. Braggins. 1992 The young gametophyte of Phylloglossum (Lycopodiaceae). Annals of the Missouri Botanical Garden 79: 730–736.[CrossRef][ISI]
———, and T. A. Steeves. 1960 The induction of apogamy in the bracken fern. Canadian Journal of Botany 38: 925–930.
———, and T. R. Webster. 1986 Gametophytes of Lycopodium lucidulum from axenic culture. American Fern Journal 76: 48–55.[CrossRef][ISI]
Wikström, N., and P. Kenrick. 1997 Phylogeny of Lycopodiaceae (Lycopsida) and the relationships of Phylloglossum drummondii Kunze based on rbcL sequences. International Journal of Plant Sciences 158: 862–871.[CrossRef]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
