ABSENCE OF CAP MEMBRANES AS A CHARACTERISTIC OF PIT PLUGS OF SOME RED ALGAL ORDERS1

The current general model of the organization of a fully developed rhodophycean pit plug includes a cap membrane associated with each end of the plug. Continuity of the plasmalemma and cap membrane results in the plug core being extracellular. However, routine ultrastructural methods have not convincingly demonstrated cap membranes in the Bangiales or the Corallinales. In this study, a variety of fixation regimes that enhance membrane contrast were used in an attempt to detect cap membranes in representatives of these two orders. Although cap membranes were readily and clearly demonstrable by these methods in a representative of an order previously known to have cap membranes, they could not be detected in members of the Bangiales or Corallinales. Should the absence of cap membranes prove to be an invariant feature of these two orders, presence or absence of cap membranes will constitute a new character for phylogenetic analysis. It is proposed that the ancestral character state of red algal pit plugs was a homogeneous plug without caps and without cap membranes.     

AN EXPANDED SURVEY OF THE ULTRASTRUCTURE OF RED ALGAL PIT PLUGS1

The fine structure of pit plugs in 90 species of red algae was examined, bringing the total number of species in the continuing survey to 153. The organization of plug caps was confirmed to be a stable, predictable trait within thalli, between generations in heteromorphic life histories, and within the presently recognized orders, with one exception—the Acrochaetiales. Two forms of the outer cap were found in this group, a thin plate, as in the Nemaliales and Palmariales, and a dome, as in Batrachospermales and Corallinales. Variation of pit plug structure indicates that the Acrochaetiales are a heterogeneous assemblage and that pit plugs will be useful in reappraising their systematics. The systematic affinities of several species of uncertain affinities are clarified. Schmitziella endophloea Bornet et Batters is excluded from both orders, Corallinales and Acrochaetiales, with which it previously was allied. Although other ordinal attributions are not precluded by pit plug structure alone, pit plug structure is consistent with placement of Apophlaea sinclairii Harvey and Hildenbrandia rivularis (Liebman) J. Agardh in the Hildenbrandiales, Plagiospora gracilis Kuckuck, Schmitziella endophloea, and Wurdemannia miniata (Duby) J. Feldmann et Hamel in the Gigartinales, and Pseudorhododiscus nipponicus Masuda in the Palmariales.     

The nature of the ancestral red alga: inferences from a cladistic analysis

A cladistic analysis of the orders of Rhodophyta is presented. Sixteen taxa and 34 characters comprise the data matrix. Included in the analysis are biochemical and ultrastructural features of pigments, cell walls, cell organelles, mitosis and pit connections as well as vegetative and reproductive characters. The traditional recognition of two classes or subclasses, Bangiophycidae and Florideophycidae, is not supported regardless of whether Porphyridiales, Rhodochaetales or Bangiales is designated the outgroup. Florideophycidae, however, appears to be monophyletic with Bangiales as its sister group. Relationships among taxa with one or two plug cap layers, i.e. Acrochaetiales, Palmariales, Corallinales, Nemaliales, Batrachospermales, Gelidiales and Hildenbrandiales are unresolved. Rhodochaetales, Bangiales and possibly Erythropeltidales are monophyletic, but Porphyridiales is polyphyletic. The class Cyanidiophyceae is not recognized and the included genera are considered to be unicellular red algae belonging to Porphyridiales. Taxa that have been proposed as sister groups for red algae, including Cyanobacteria, Cryptophyta, Glaucophyta and Chlorophyta, and Ascomycetes and Basidiomycetes are discussed in relation to the proposed phylogeny of Rhodophyta.     

SYSTEMATIC SIGNIFICANCE OF THE ABSENCE OF PIT-PLUG CAP MEMBRANES IN THE BATRACHOSPERMALES (RHODOPHYTA)1

Four species of the Batrachospermales were examined by transmission electron microscopy to determine whether or not cap membranes, a typical structural component of pit plugs in several orders of red algae, were present. Routine specimen preservation methods used in past studies led to contradictory reports, so Batrachospermum keratophytum Bory, B. sirodotii Skuja ex Reis, Sirodotia suecica Kylin, and S. tenuissima (Collins) Skuja ex Flint were prepared by secondary fixation in potassium permanganate or a combination of potassium ferrocyanide-osmium tetroxide to enhance membrane contrast. These fixation procedures produced clear, well-contrasted images in which cap membranes were absent. The absence of cap membranes in S. suecica and S. tenuissima and the presence of cap membranes in two members of the Nemaliales was confirmed by freeze-substitution methods. Absence of cap membranes in representatives of the Batrachospermales further distinguishes the Batrachospermales from the Nemaliales, the order in which they long resided, and demonstrates the value of this character in elucidating ordinal alliances among the Rhodophyta.     

RHODOPHYCEAN PIT PLUGS: AN ULTRASTRUCTURAL SURVEY WITH TAXONOMIC IMPLICATIONS

The fine structure of pit plugs was examined in 63 species of red algae representing 34 families. The number of plug cap layers was found to be a taxonomically reliable character when tested against a recent revision of the Rhodymeniales which established the order Palmariales. In a broader survey involving all the orders of red algae possessing pit plugs, three basic plug cap configurations were detected. These differ in having one, two, or no plug cap layers. Some taxa have plug caps in which the outer cap layer is greatly elaborated, however, this layer was shown to be positionally and cytochemically identical, thus probably homologous, to the outer cap layer of the typical two-layered plug cap. The pit plug character confirms the validity of the order Palmariales and indicates that the Nemaliales and Cryptonemiales are heterogeneous assemblages. Revision of these groups along lines indicated by the plug character is supported by significant features of morphology and reproduction. These correlations lead us to propose that Rhodophysema and Coriophyllum be transferred to the Palmariales, that the Gelidiales be resurrected, that the Bonnemaisoniales (including the Naccariaceae) is deserving of recognition, that the Batrachospermaceae be raised to a higher rank which will also receive the Lemaneaceae and the Thoreaceae, and that the Corallinaceae and Hildenbrandiaceae both be raised to ordinal status.     

A freeze-etch study of the ultrastructure of red algal pit plugs

Summary Freeze-etch studies indicate that the fully-formed pit plug ofPalmaria palmata consists of a homogeneous plug core, a pair of plug caps, and associated membranes. A single cap membrane separates the two layers of each plug cap and is the only membrane found between the cytoplasm and the plug core. Due to the apparent junction of the plasmalemma and cap membranes, the plug core, along with the adjacent plug cap layer on either face of the core, is entirely membrane-bounded and essentially extracellular. Fracture faces of the cap membrane bear large populations of particles which are inconspicuous due to their low profile. The plasmalemma lining the plug core from cell to cell has numerous, very obvious particles on its fracture faces, more even than does the cytoplasmic plasmalemma.The relationship of the rhodophycean pit plug to the fungal septal pore plug is discussed.This report represents a portion of a thesis submitted in partial fulfillment of the requirements of a Master of Science degree, Cornell University, Ithaca, N.Y.     

ULTRASTRUCTURE OF EARLY DEVELOPMENT IN THE FEMALE REPRODUCTIVE SYSTEM OF POLYSIPHONIA HARVEYI BAILEY (CERAMIALES,RHODOPHYTA)1

Prefertilization and immediate postfertilization development in the female reproductive branch of Polysiphonia harveyi (Bailey) was studied with the electron microscope. Results pertaining to prefertilization morphology and development are consistent with those established in earlier light microscopic studies, but several unexpected ultrastructural characteristics were discovered. The mature carpogonium was found to have double membrane-bound vacuoles of nuclear origin and the carpogonial nucleus contained a nucleolus with a distinctive crystalline lattice. Trichogynes lacked a nucleus. Of particular interest was the discovery of a highly structured channel of smooth endoplasmic reticulum (SER) which extended uninterrupted, except for pit connections, through the carpogonial branch. It is suggested that the message of fertilization is conducted through the SER channel from the carpogonium to the support cell. Few observations were made on postfertilization branches, but evidence of direct fusion between the carpogonium and auxiliary cell was fairly conclusive. Pit plugs in the female branch were found to be of three morphological types differing by the presence and number of pit membranes. We have designated these plugs Type I–III since differential functioning was not fully ascertained. Our data suggest that pit membranes may serve to stabilize the plug and that those plugs without membranes are more readily dismantled to allow cytoplasmic continuity between cells.     

Electron microscopical studies ofThorea ramosissima (Thoreaceae,Rhodophyta): Taxonomic implications ofThorea pit plug ultrastructure

The thallus ofThorea ramosissima was studied electron microscopically. The cells of the medulla, the cortex and the assimilatory hairs differ not only in size and number of plastids and their equipment with thylakoids but also in cell wall structure, the number of mitochondria and the activity of the Golgi apparatus, with dictyosomes transforming complete cisternae into Golgi vesicles with mucilaginous contents in the outer region of the cortex. The pit connections have plugs with a distinct plate—like (not dome-like) outer cap layer. BecauseT. riekei was reported to have dome-like outer cap layers and because this character was the main reason to place theThoreaceae into theBatrachospermales (Pueschel & Cole 1982),T. riekei was reinvestigated, too. A distinct outer cap could not be detected. The reliability of pit plug structure as a taxonomic character and the taxonomic position ofThorea is discussed.     

Affinities of the freshwater red alga Audouinella macrospora (Florideophyceae, Rhodophyta) and related forms based on ssu rrna gene sequence analysis and pit plug ultrastructure

Small subunit rDNA sequencing and transmission electron microscopy were performed to clarify the ordinal affinities of Audouinella macrospora (Wood) Sheath et Burkholder isolates 3394, 3395, and 3603, as well as Chantransia sp. isolate 3585. Culture 3603 is known to produce thalli of Batrachospermum  -like morphology under certain culture conditions. Sequence analyses unequivocally placed the three Audouinella macrospora isolates in a clade with Batrachospermum macrosporum Montagne of the Batrachospermales, and Chantransia sp. was found to have affinities with B. louisianae Skuja and B. virgato-decaisneanum Sirodot. The pit plugs of the Audouinella macrospora cultures 3394 and 3395 were nearly identical in size and structure, having thickened plug caps and no cap membranes. Both of these features agree with those of the Batrachospermaceae, with the latter feature showing batrachospermacean rather than acrochaetioid affinities. Pit plugs in the chantransia phase of 3603 were similar, but the plug caps were less well developed. The Batrachospermum phase generated from 3603 had pit plugs that were variable in diameter, according to location in the thallus, thus reflecting the more variable cell size in this phase. Dome-like outer caps, considered typical of Batrachospermum , were present between cells of the determinate lateral filaments. The pit plugs of Chantransia sp. had prominent, dome-like outer caps, but the plug cores were strikingly and consistently smaller in diameter than those of the A. macrospora chantransia cultures, suggesting that plug diameter may be of systematic value in some contexts.     

A PROPOSAL FOR A NEW RED ALGAL ORDER,THE THOREALES1

Representatives of the freshwater red algal family Thoreaceae were studied to resolve their taxonomic and phylogenetic status. Three specimens of Nemalionopsis and five collections of Thorea were examined for pit plug ultrastructure and analyzed for the sequences of the genes coding for the large subunit of RUBISCO (rbcL) and the small subunit of rRNA (18S rRNA). The phylogenetic trees generated from the two genes, and a combined tree all showed the Thoreaceae to be contained in a well‐supported monophyletic clade that is separate from the other two families currently classified in the Batrachospermales, the Batrachospermaceae and the Lemaneaceae. In addition, secondary structure elements of the 18S rRNA gene were observed at positions 650 and 1145 (Escherichia coli numbering system) that are not present in other members of the Rhodophyta. The pit plugs of the gametophytic and chantransia stages of the Thoreaceae contain two cap layers, the outer one of which is typically plate‐like, though occasionally inflated ones have been seen. No pit plug cap membrane has been observed. These findings indicate the Thoreaceae has been misclassified in the Batrachospermales and should be placed in its own order, the Thoreales. This order is characterized by having freshwater representatives with multiaxial gametophytes, a uniaxial chantransia stage, and pit plugs with two cap layers, the outer one of which is usually plate‐like.     

Force–displacement measurements of earlywood bordered pits using a mesomechanical tester

The elastic properties of pit membranes are reported to have important implications in understanding air‐seeding phenomena in gymnosperms, and pit aspiration plays a large role in wood technological applications such as wood drying and preservative treatment. Here we present force–displacement measurements for pit membranes of circular bordered pits, collected on a mesomechanical testing system. The system consists of a quartz microprobe attached to a microforce sensor that is positioned and advanced with a micromanipulator mounted on an inverted microscope. Membrane displacement is measured from digital image analysis. Unaspirated pits from earlywood of never‐dried wood of Larix and Pinus and aspirated pits from earlywood of dried wood of Larix were tested to generate force–displacement curves up to the point of membrane failure. Two failure modes were observed: rupture or tearing of the pit membrane by the microprobe tip, and the stretching of the pit membrane until the torus was forced out of the pit chamber through the pit aperture without rupture, a condition we refer to as torus prolapse.     

MORPHOLOGY AND DEVELOPMENT OF AHNFELTIA PLICATA (RHODOPHYTA): PROPOSAL OF AHNFELTIALES ORD. NOV.1

Ahnfeltia plicata (Hudson) Fries, the type species of Ahnfeltia Fries, is currently assigned to the Phyllophoraceae (Gigartinales). Several morphological and biochemical characters distance A. plicata from the Phyllophoraceae but, because sexual reproduction has never been demonstrated, an alternative placement has not been possible. A. plicata now is shown to have a heteromorphic sexual life history. Erect branched gametophytes are dioecious. In male sori, spermatangia are cut off transversely from spermatangial mother cells. Female sori form numerous terminal sessile carpogonia. Following fertilization, several zygotes in each sorus fuse facultatively with undifferentiated intercalary cells of the female sorus and cut off gonimoblast initials obliquely outwards. These initials give rise to branching gonimoblast filaments that fuse with apical and intercalary female sorus cells and with each other, then grow radially outward in the compound external carposporophyte and terminate in carposporangia. Carpospores develop in culture into crustose tetrasporophytes identical to Porphyrodiscus simulans Batters. Field-collected P. simulans tetraspores grew into erect A. plicata axes. Tetrasporangia are formed by division and enlargement of crust apical cells followed by sequential enlargement and maturation of tetrasporocytes in an erosive process. Monosporangia are formed in sori on male gametophytes. Pit plugs of both gametophyte and tetrasporophyte phases consist of naked plug cores without cap layers of membranes. Gametophytes exhibit both cell fusions and secondary pit connections whereas tetrasporophytes form cell fusions but lack secondary pit connections. On the basis of the unique female and postfertilization reproductive development and in conjunction with the pit plug structure which is unique among florideophytes, the order Ahnfeltiales, containing the family Ahnfeltiaceae, is proposed.     

PIT MEMBRANE REMNANTS IN PERFORATION PLATES OF PRIMITIVE DICOTYLEDONS AND THEIR SIGNIFICANCE

Perforations of vessel elements characteristically retain remnants of pit membranes (primary walls) in woods of species of more than 30 families of dicotyledons. Scanning electron microscopy is necessary to demonstrate presence and type of membrane remnant. Species with these remnants in perforations given in earlier literature as well as those newly reported here are listed. Perforation membrane remnants may take the form of flakes, strands, or webs, and particular types may characterize particular families (e.g., strands or bands in Illiciaceae). Some families have abundant perforation membrane remnants (e.g., Chloranthaceae, Illiciaceae). Where membranes are nearly intact, they are porose and closely resemble the porose pit membranes on end walls of Tetracentron tracheids. In Tetracentron, however, tracheary elements are monomorphic, so vessel origin cannot yet be said to have occurred. Membrane remnants in perforations are regarded as a relictual primitive feature that should be added to the list of primitive character states claimed for vessel elements in angiosperms; alternative hypotheses are considered and discussed, and evidence from DNA phylogenies is needed. In vessel-bearing dicotyledons with membrane remnants in perforations, many perforations are relatively clear, but an appreciable proportion of perforation plates do have membrane remnants.     

Perforated pit membranes in imperforate tracheary elements of some angiosperms

BACKGROUND AND AIMS: The structure of pit membranes in angiosperms has not been fully examined and our understanding about the structure is incomplete. Therefore, this study aims to illustrate the micromorphology of pit membranes in fibres and tracheids of woody species from various families. METHODS: Specimens from ten species from ten genera and eight families were prepared using two techniques and examined by field-emission scanning electron microscopy. KEY RESULTS: Interfibre pit membranes with an average diameter of <4 microm were frequently perforated or appeared to be very porous. In contrast, pit membranes in imperforate tracheary elements with distinctly bordered pits and an average diameter of >or=4 microm were homogeneous and densely packed with microfibrils. These differences were observed consistently not only among species but also within a single species in which different types of imperforate tracheary elements were present. CONCLUSIONS: This study demonstrates that the structure of interfibre pit membranes differs among cell types and the differences are closely associated with the specialization of the fibre cells. It is suggested that perforated pit membranes between specialized fibres contribute to the dehydration of the fibre cells at or soon after maturation.     

中国裸子植物木材具缘纹孔构造类型的研究

根据100种中国裸子植物木材(隶属4纲、8目、11科,42属)具缘纹孔构造的研究,提出8种不同具缘纹孔类型:1.苏铁型;2.南洋杉 A 型;3.南洋杉 B 型;4.落羽杉 A 型;5.落羽杉 B 型;6.松木 A 型;7.松木 B 型;8.买麻藤型。A 型是指纹孔室内瘤状层缺乏或罕见,B型则具明显的瘤状层。并对具缘纹孔在系统发育中的变化进行了讨论。     

PRESENCE OF VESSELS IN WOOD OF SARCANDRA (CHLORANTHACEAE); COMMENTS ON VESSEL ORIGINS IN ANGIOSPERMS

Sarcandra is the only genus of Chloranthaceae hitherto thought to be vesselless. Study of liquid-preserved material of S. glabra revealed that in root secondary xylem some tracheary elements are wider in diameter and have markedly scalariform end walls combined with circular pits on lateral walls. Examination of these wider tracheary elements with scanning electron microscope (SEM) demonstrated various degrees of pit membrane absence in the end walls. Commonly a few threadlike fibrils traverse the pits (perforations); these as well as intact nature of pit membranes in pits at ends of some perforation plates are evidence that lack of pit membranes does not result from damage during processing. Some perforations lack any remnants of pit membranes. Although perforation plates and therefore vessels are present in Sarcandra roots, no perforations were observed in tracheary elements of stems or lignotubers. Further, stem tracheids do not have the prominently scalariform end walls that the vessel elements in roots do. Presence of vessels in Sarcandra removes at least one (probably several) hypothetical events of vessel origin that must be postulated to account for known patterns of vessel distribution in angiosperms, assuming that they are primitively vesselless. Seven (perhaps fewer) vessel origin events in angiosperms could account for these patterns; two of those events (Nelumbo and monocotyledons) are different from the others in nature. Widely accepted data on trends of vessel specialization in woody dicotyledons yield an unappreciated implication: vessel specialization has happened in a highly polyphyletic manner in dicotyledons, and therefore multiple vessel origins represent a logical extension backward in time. If a group of vesselless dictyoledons ancestral to other angiosperms existed, they can be hypothesized to have had a relatively homogeneous floral plan now that Sarcandra-like plants no longer need be imagined within that group. Sarcandra and other Chloranthaceae show that the borderline between vessel absence and presence is less sharp than generally appreciated.     

Bordered pits in xylem of vesselless angiosperms and their possible misinterpretation as perforation plates

Vesselless wood represents a rare phenomenon within the angiosperms, characterizing Amborellaceae, Trochodendraceae and Winteraceae. Anatomical observations of bordered pits and their pit membranes based on light, scanning and transmission electron microscopy (SEM and TEM) are required to understand functional questions surrounding vesselless angiosperms and the potential occurrence of cryptic vessels. Interconduit pit membranes in 11 vesselless species showed a similar ultrastructure as mesophytic vessel‐bearing angiosperms, with a mean thickness of 245 nm (± 53, SD; n = six species). Shrunken, damaged and aspirated pit membranes, which were 52% thinner than pit membranes in fresh samples (n = four species), occurred in all dried‐and‐rehydrated samples, and in fresh latewood of Tetracentron sinense and Trochodendron aralioides. SEM demonstrated that shrunken pit membranes showed artificially enlarged, > 100 nm wide pores. Moreover, perfusion experiments with stem segments of Drimys winteri showed that 20 and 50 nm colloidal gold particles only passed through 2 cm long dried‐and‐rehydrated segments, but not through similar sized fresh ones. These results indicate that pit membrane shrinkage is irreversible and associated with a considerable increase in pore size. Moreover, our findings suggest that pit membrane damage, which may occur in planta, could explain earlier records of vessels in vesselless angiosperms.     

SEM studies on vessels in ferns. 17. Psilotaceae

Perforation plates are reported in aerial and subaerial axes of Psilotum nudum and in aerial axes of Tmesipteris obliqua. In Psilotum, both perforations lacking pit membranes and perforations with pit membrane remnants were observed. Perforation plates in Psilotum may consist wholly of one type or the other. In Tmespteris, perforations have threadlike pit membranes or consist of porose pit membranes. Wide perforations alternating with narrow pits, a conformation observed in various ferns, were observed in Psilotum (subaerial axes). In Psilotum, perforations are more common in metaxylem than in protoxylem; perforations in protoxylem consist of primary wall areas containing small circular porosities or relatively large circular to oval perforations. There are no modifications in the secondary wall framework of protoxylem or metaxylem in Psilotum or Tmesipteris that would permit one to distinguish presence of perforations or perforation plates with light microscopy, and scanning electron microscopy (SEM) is required for demonstration of porose walls or perforations. The tracheary elements of the Psilotaceae studied have no features not also observed in other ferns with SEM.     

Heterogeneous distribution of pectin epitopes and calcium in different pit types of four angiosperm species

Intervessel pits act as safety valves that prevent the spread of xylem embolism. Pectin-calcium crosslinks within the pit membrane have been proposed to affect xylem vulnerability to cavitation. However, as the chemical composition of pit membranes is poorly understood, this hypothesis has not been verified. Using electron microscopy, immunolabeling, an antimonate precipitation technique, and ruthenium red staining, we studied the distribution of selected polysaccharides and calcium in the pit membranes of four angiosperm tree species. We tested whether shifts in xylem vulnerability resulting from perfusion of stems with a calcium chelating agent corresponded with the distribution of pectic homogalacturonans (HG) and/or calcium within interconduit pit membranes. No HG were detected in the main part of intervessel pit membranes, but were consistently found in the marginal membrane region known as the annulus. Calcium colocalized with HG in the annulus. In contrast to intervessel pits, the membrane of vessel-ray pits showed a high pectin content. The presence of two distinct chemical domains, the annulus and the actual pit membrane, can have substantial implications for pit membrane functioning. We propose that the annulus could affect the observed shift in xylem vulnerability after calcium removal by allowing increased pit membrane deflection.