Ann E. Rushing

A Modified, Short Protocol for Preparation of Bryophytes for Scanning Electron Microscopy

Live bryological specimens can be prepared for scanning electron microscopy in under one hour without prior fixation by dehydrating with 2,2-di- methoxypropane followed by critical point drying. Unfixed specimens exhibited little distortion, thus eliminating the requirement for prolonged and/or expensive fixation that may cause collapse of cell walls or other artifacts. Critical point drying of specimens prior to examination by scanning electron micros- copy (SEM) is now a generally accepted procedure and its utility in bryology was dem- onstrated by Magill, Seabury and Mueller in 1974. Muller and Jacks (1975) proposed the use of 2,2-dimethoxypropane (DMP) for the rapid preparation of biological specimens for transmission electron microscopy. This procedure was adapted by Johnson et al. (1976) to specimen preparation for SEM. This adaptation required fixation in glutaraldehyde and osmium, prior to dehydration in 2,2-dimethoxypropane and critical point drying (CPD). The essential element of the procedure is dehydration by DMP, which upon exposure to water instantaneously hydrolyzes to form acetone and methanol. Johnson et al. (1976) suggested that fixation of specimens may be unnecessary. In an effort to simplify and shorten fixation and dehydration schedules, the effects of DMP dehydration on fixed and unfixed material were tested using leafy and thalloid liv- erworts and mosses. The protocol reported here yielded acceptable specimens for SEM

Blepharoplast morphology in Treubia tasmanica (Hepaticae: Treubiales)

Detailed ultrastructural analysis and diagrammatic representation of the blepharoplast in Treubia tasmanica reveal a morphology strongly resembling that reported for Haplomitrium. The major distinguishing features common to both include: 1) A very wide spline (with up to 104 microtubules, Treubias spline is the widest reported for any bryophyte); 2) a spline aperture of the open type; 3) the aperture located on the left side of the spline midline; 4) the anterior basal body located on the left of the spline midline; 5) the left-divergent orientation of the anterior basal body; and 6) nearly total overlap between the anterior and posterior basal bodies. These same six characters exhibit sharply contrasting expressions in the three metzgerialian representatives similarly analyzed. The evidence presentedfor Treubiafurther supports the isolated position of the group and its elevation to ordinal rank. It also reveals an important affinity with the calobryalian line. The Treubiaceae is a small family of liverworts consisting of only two extant genera: Treubia with seven species (Schuster & Scott 1969) and Apotreu- bia with two (Schuster 1984). The initial description of the generic type, Treubia insignis Goebel (1891), and subsequent morphological studies, e.g., those by Campbell (1916), Griin (1914), Schuster and Scott (1969), and Wijk (1928), have shown the members of the Treubiaceae to be anacrogynous while having an intermediate position between the thallose and foliose conditions. Further, this unusual family has long been viewed as having an isolated position in the Metzgeriales; however, the interpretations of its affinities within that order have been largely con- jectural (for examples see the taxonomic mono-

Midstage Spermatid Architecture in Riccia gougetiana (Hepaticae)

Abstract The blepharoplast of Riccia gougetiana displays typical features of the subclass Marchantiidae. The spline reaches a maximum of 24 microtubules and has a closed aperture the equivalent of two microtubule diameters in width. The lamellar strip is longitudinally elongated and asymmetrically spatulate in shape, 1.96 μm long and 0.74 μm wide, the same as the overlying spline. The basal bodies are staggered in insertion, overlapping, and parallel with the spline over most of their lengths. The ABB, situated over the right side of the spline and 0.22 μm from the anterior spline margin, measures 0.71 μm in length including triplet extensions. The PBB is longer, at 1.16 μm, and is situated over the left side of the spline, 0.59 μm from the leading edge of the spline. The uniformity of blepharoplast features in the subclass Marchantiidae, and particularly within the Marchantiales, supports the concept of a monophyletic grouping. The overall similarity of the blepharoplast among those marchantialian genera studied in detail is striking and further supports the use of these features in studying the relationships among bryophytes.

Comparative Studies of Spermatogenesis in the Bryopsida. III. Blepharoplast Morphology in Thuidium delicatulum

Ultrastructural observations of the spermatids of Thuidium delicatulum reveal a blepharoplast comprising a multilayered structure of the typical four-layered type and two super- posed, overlapping basal bodies. The longitudinally elongated lamellar strip is approximately rhom- boidal, 2.6 ,m long and 0.5 Cm wide. Most commonly, the spline comprises 14 microtubules at its widest region. These are separated into two sets (10 at left andfour at right) by a 3 ,m long aperture of the open type. The aperture is three microtubule-diameters wide along its midregion, narrowing to only one at its anterior end. The anterior basal body is 2.1 ,im long and lies directly above and parallel to the spline aperture. The posterior basal body is 7.0 gm long and diverges posteriorly in association with the left marginal microtubule of the spline. The stellate patterns, each measuring 46 nm in length, are the shortest reported for any bryophyte. Although differing substantially in lamellar strip morphology and overall dimensions, the blepharoplast of Thuidium shares more features with that of Funaria than with those of Archidium, Polytrichum or Sphagnum.

THE MATURE SPOROPHYTE-GAMETOPHYTE JUNCTION OF LORENTZIELLA IMBRICATA

The sporophyte-gametophyte junction of Lorentziella imbricata (Mitt.) Broth., in the mature stage, is characterized by transfer cells with wall ingrowths in both the sporophyte foot and the surrounding gametophyte tissue. The foot is globose in shape and does not penetrate extensively into the tip of the gametophyte axis. A cluster of cells, the original hypobasal cell of the embryo and a few cells immediately adjacent to it, do not enlarge with the remainder of the sporophyte but apparently remain intact and are found at the base of the globose foot. Epidermal cells of the sporophyte foot have well developed and extensive wall elaborations, particularly on their outer tangential walls closest to the gametophyte tissue. Cells of the central strand of the seta extend into the foot region and also have wall ingrowths, but typically only on their endwalls. Wall ingrowths in the gametophyte are less extensive than those of the sporophyte and typically are found only on inner tangential walls closest to the sporophyte. The structure of the sporophytegametophyte junction suggests a system of transport from the gametophyte into the sporophyte. A broad based sporophyte foot and an extensive system of wall ingrowths suggests that movement of materials from the gametophyte into the sporophyte is an integral part of the developmental sequence in this minute moss. Movement from the gametophyte into the sporophyte foot and upward through the seta is suggested by the distribution of wall ingrowths in these cells and by the plasmodesmatal connections between the cells of the sporophyte axis. Completing its life cycle in a short period of time likely requires the development and maintenance of an efficient system for rapid movement of materials during favorable times for growth of this ephemeral moss. In bryophytes, because the sporophyte is embedded within and remains permanently attached to the gametophyte, the sporophyte-gametophyte junctional area is critically important to the survival of the developing sporophyte. The area of contact, the placental region, is composed of the sporophyte foot, a placental space, and surrounding gametophyte tissue. Metabolically active cells, many of which have extensive wall ingrowths and can be classified as transfer cells (Gunning & Pate 1974), characterize this placental region and may be found on both sporophyte and the gametophyte sides of the junctional complex (Ligrone et al. 1993). Although the sporophyte in mosses is green during part of its life and presumably develops some degree of autonomous photosynthetic functioning, studies have shown that nutrients are transported across this region, from the gametophyte into the sporophyte (Browning & Gunning 1979a,b; Caussin et al. 1983; Courtice et al. 1978; Proctor 1977; Renault et al. 1989, 1992). The degree of nutritional dependence of the sporophyte on the gametophyte has not been determined, however, and may vary from one moss to another and from one stage of development to another. This study outlines the features of the mature sporophyte-gametophyte junction of the moss Lorentziella imbricata (Gigaspermaceae). Lorentziella is an ephemeral moss, completing its life cycle in late fall to early winter in Texas. It has a minute, highly reduced sporophyte that remains surrounded by leaves of the gametophyte throughout its development, except in the most mature stages in which only the extreme capsule tip may extend slightly above the gametophyte leaves (Rushing & Snider 1980). The cleistocarpous capsule is green and contains large air spaces that open to the surrounding environment through stomata around the base of the truncate capsule. Even though the short seta does not elevate the capsule above the level of the gametophyte axis, presumably the sporophyte becomes photosynthetically active during its development. However, its nutritional dependence on the gametophyte likely extends throughout its lifetime. Other small, ephemeral mosses with cleistocarpous capsules for which the sporophyte-gametophyte junction also has been examined include Acaulon (Rushing & Anderson 1996), Archidium (Brown & Lemmon 1985), Ephemerum (Yip 1994), and Phascum (Chauhan 1992). Observations also exist for the minute, but not cleistocarpous, mosses Physcomitrium (Chauhan & Lal 1987; Lal & Chauhan 1981), Buxbaumia (Ligrone et al. 1982), and Diphyscium (Ligrone et al. 1993). This rather unique collection of mosses is distributed in several different families within the Bryopsida and provide 0007-2745/99/92-98

Taxonomy of Koeberlinia (Koeberliniaceae)

0.85/0 This content downloaded from 157.55.39.102 on Mon, 03 Oct 2016 05:17:10 UTC All use subject to http://about.jstor.org/terms 1999] RUSHING: SPOROPHYTE-GAMETOPHYTE JUNCTION 93 interesting comparisons with these observations of Lorentziella. MATERIALS AND METHODS Specimens of Lorentziella imbricata (Mitt.) Broth. were collected in November and December 1990 and 1991 from populations in Brazos Co. and Burnett Co., Texas. Voucher specimens are deposited in the personal herbarium of the author (Rushing 936, 937, 948, 952). Entire sporophytes (including the foot, seta, and capsule) and the surrounding gametophyte tissues were removed from the remainder of the leafy plant and fixed in a solution of 2.5% glutaraldehyde and 2.0% formaldehyde in 0.1 M PIPES buffer at pH 7.1. The samples were subjected to a vacuum for about 5 minutes. This process was repeated at intervals, particularly in more mature sporophytes with capsules containing visible air spaces, until there were no floating samples. The samples were postfixed in 1.0% osmium tetroxide in the same buffer. After postfixation, samples were rinsed several times in the same buffer, dehydrated in a graded ethanol series, and embedded in Spurrs resin (Spurr 1969). Thick-sections were placed on glass microscope slides and stained with toluidine blue. Thin-sections of samples in suitable stages of development were placed on either nickel thin bar grids or formvar coated single slot grids. Sections were stained with 2.0% methanolic uranyl acetate followed by lead citrate and viewed with a Philips 201 transmission electron microscope operated at 60 kV

The Blepharoplast of the Midstage Spermatid of Andreaeobryum macrosporum (Bryophyta: Andreaeopsida)

Koeberlinia has a natural amphitropical distribution that includes the deserts of central Bolivia, northern Mexico, and the southwestern United States. Despite the long recognition of only one species, K. spinosa, field, herbarium, and SEM studies support the recognition of two species. Koeberlinia spinosa of northern Mexico and adjacent United States is recognized to consist of three varieties: K. spinosa var. spinosa of northeastern Mexico and the adjacent United States, K. spinosa var. tenuispina of the Sonoran Desert of southwestern Arizona, adjacent California, and northwestern Mexico, and K. spinosa var. wivaggii from south central Texas and northern Mexico to Arizona, which is described as new. Koeberlina holacantha, endemic to the deserts of Bolivia, is proposed as new.

Bruchia hallii in Alabama

The blepharoplast of the midstage spermatid of Andreaeobryum macrosporum has a four-layered multilayered structure (MLS) and two superposed, slightly staggered, and overlapping basal bodies. The spline is composed of 30 parallel microtubules at its maximum width. Subtending the spline anteriorly, the lamellar strip (LS) is 1.83 pm long and 0.92 pm wide and has an elongated, wedge-shape with a pointed right anterior tip. Viewing the blepharoplast dorsally from anterior to posterior, the anterior basal body (ABB), approximately 0.95 pm in length, is situated over the right side of the MLS and is inserted at a level 0.42 pCm behind the splines anterior apex. The posterior basal body (PBB), 2.75 pcm long, is positioned over the left side of the MLS and inserted 0.75 pm behind the splines anterior apex. The ABB is essentially parallel with the spline microtubules over its entire length, while the anterior portion of the PBB curves outward gradually and becomes parallel with the spline in its posterior region. The combination of features that distinguish the midstage spermatid of Andreaeobryum from other mosses are its wide spline, wedge-shaped lamellar strip, comparatively short basal bodies, small length of stagger between basal bodies, and the PBB that curves gradually outward from its anterior tip. The data available for Andreaea suggest that its midstage spermatid resembles that of Andreaeobryum, although reconstructions and additional micrographic data are needed for both Andreaea and Takakia before generalizations for the class Andreaeopsida can be made. Andreaeobryum belongs to an unusual line of bryophyte evolution that also includes Andreaea and Takakia (Murray 1988; Renzaglia et al. 1991). While Andreaea is an old and well-established genus, Andreaeobryum is relatively new, having been described in 1976 by Steere and Murray (1976) and further defined by Murray (1988). Andreaea and Andreaeobryum have been grouped together in either the class Andreaeopsida (Crum & Anderson 1981; Murray 1988) or subclass Andreaeidae (Schofield 1985; Vitt 1984). According to Murray (1988), Andreaea and Andreaeobryum belong to separate orders, Andreaeales and Andreaeobryales, within the primitive class Andreaeopsida. The two genera are united by sporeling features and mode SAuthor for correspondence. e-mail: AnnRushing@ baylor.edu FIGUREs 1-6. Blepharoplast anatomy of Andreaeobryum macrosporum. -1-3. Tangential sections. -1. The spline anterior with its acutely curved left margin and closed aperture. Above, the extended triplet is part of the posterior basal body (cf. Fig. 18). -2. Slightly oblique section showing the left-marginal curvature of the subtending lamellar strip and the spline aperture (between arrows). -3. The spline aperture extends posteriorly beyond the lamellar strip where it closes by convergence of the microtubules on its right side with those on the left. -4. Near-median longitudinal section through the anterior basal body and the distal transition zone with its stellate pattern. The anterior mitochondrion is just becoming positioned beneath the lamellar strip. -5. Oblique-tangential section showing the full longitudinal extent of the anterior stellate pattern. -6. Oblique-tangential section section through the posterior basal body and the subtending spline and nucleus. Key to labeling: ABB, anterior basal body; AF, anterior flagellum; AM, anterior mitochondrion; ASP, anterior stellate pattern; EH, extended hub of basal body; ET, extended triplet; LS, lamellar strip; N, nucleus; PBB, posterior basal body; PSP, posterior stellate pattern; S, spline; S1-S4, specific strata of the multilayered structure; SA, spline aperture; bracket ([), lamellar strip. All micrographs are printed at a magnification of X67,500 except Fig. 6 where the magnification is X47,500. Except as otherwise noted for Figure 18, each scale bar represents

An Ultrastructural and Developmental Study of the Sporophyte-Gametophyte Junction in Ephemerum cohaerens

Bruchia hallii Aust. is reported from Alabama. The known range of the species and its distinguishing features are discussed. Although known from numerous sites in Texas, Bruchia hallii Aust. has been reported previously from only two other sites in the United States (Fig. 1), a single locality in Arkansas and one in North Carolina (Rushing 1986). This report of B. hallii represents the first from Alabama. This specimen was collected from a granite outcrop, a previously unreported habitat type for the species. Based on its other typical habitats, such as disturbed fields and road banks, it should be found throughout the southeastern United States. Recent collections have shown B. brevifolia Sull. also to be more widely distributed than previously reported (Rushing 1989). Plants of B. hallii (Fig. 2), including sporophyte, rangefrom about 3.0 to 6.5 mm in height (Rushing 1986). This species is distinguished from other Bru- chia species by its leaves which are ovate to lan- ceolate, acute to short-acuminate, and imbricate (Fig. 2; fig. 79-89 in Rushing 1986). Most species of Bru- chia have leaves which are distinctly subulate and do not so closely surround the stem (Crum & An- derson 1981; Reese 1984; Rushing 1986). Plants of B. hallii most closely resemble those of B. flexuosa (Sw. ex Schwaegr.) C. Miill. and B. texana Aust. Bruchia hallii also is distinguished by its rather large