SIEVE-ELEMENT ONTOGENY IN THE ROOT OF EQUISETUM HYEMALE

Roots of Equisetum hyemale L. var. affine (Engelm.) A. A. Eat. were fixed in glutaraldehyde, postfixed in osmium tetroxide, and sieve elements of various ages were examined with the electron microscope. Young sieve elements are distinguished by their position within the vascular cylinder and by the presence of numerous refractive spherules, which originate within dilated portions of the endoplasmic reticulum (ER). Early in development, the sieve-element walls undergo a substantial increase in thickness. This is followed by the appearance of massive ER aggregates in the cytoplasm and then by a phase involving stacking and sequestering of the remaining ER. Nuclear degeneration is initiated shortly after the appearance of the ER aggregates. The chromatin condenses into masses of variable size along the inner surface of the nuclear envelope. The envelope then ruptures and chromatin is released into the cytoplasm. During the period of nuclear degeneration, mitochondria and plastids undergo structural modification, while components such as dictyosomes, microtubules, and ribosomes degenerate and disappear. The remaining cytoplasmic components assume a parietal position in the cell, leaving the lumen of the cell clear in appearance. At maturity, the plasmalemma-lined sieve element contains plastids, mitochondria, some ER, and refractive spherules. At this time many of the refractive spherules are discharged into the region of the wall. Pores between sieve elements occur largely on the end walls. During pore development, tubules of ER apparently traverse the pores, but because of the presence of massive callose deposits in the material examined, the true condition of mature pores could not be determined. The connections between mature sieve elements and pericycle cells are characterized by the presence of massive wall thickenings on the pericycle-cell side. Plasmodesmata in the wall thickening are matched by pores on the sieve-element side. Ontogenetic and cytoplasmic factors argue against use of the term “companion cell” for the vascular parenchyma cells associated with the sieve elements.     

GENETIC EVIDENCE FOR DIPLOIDY IN EQUISETUM

Sporophytes and gametophytes of Equisetum arvense, E. laevigatum, and E. telmateia were analyzed using enzyme electrophoresis to estimate isozyme number. Despite their uniformly high chromosome numbers (2n = 216), these three species exhibited isozyme numbers typical of diploid seed plants for the enzymes AAT, ADH, ALD, GDH, [NADP]IDH, LAP, MDH, [NADP]ME, PGI, PGM, SkDH, and 6PGDH. All three species exhibited an additional isozyme for TPI. There is, therefore, no genetic evidence for low base numbers such as x = 9 and x = 12 suggested for Equisetum. Intact chloroplasts were isolated from E. arvense and the chloroplast extract compared electrophoretically to whole plant extracts. The single enzymes observed for LAP, GDH, [NADP]IDH, and [NADP]ME were absent from the chloroplast extract. Isozymes AAT-1, ALD-2, MDH-3, PGI-1, PGM-2, SkDH-2, 6PGDH-2, TPI-2, and TPI-3 were active in the chloroplast fraction; 6PGDH-1, PGI-2, PGM-1, and TPI-1 were lacking from the chloroplast fraction and were considered cytosolic. Isozymes AAT-2, MDH-1, MDH-2, MDH-4, and SkDH-1 were also lacking from the chloroplast fraction but because AAT, MDH, and SkDH have been reported from several subcellular compartments, their localization is unknown. These findings indicate that isozymes in Equisetum species are subcellularly compartmentalized as has also been demonstrated for homosporous ferns, gymnosperms, and angiosperms.     

GROWTH AND DEVELOPMENT IN MICROCULTURE OF GAMETOPHYTES FROM STORED SPORES OF EQUISETUM

Spores of Equisetum telmateia Ehrh. retained 74% viability after two years of storage under glycerine at minus 10 C. Stored spores germinated, grew, and formed mature gametangia on mineral nutrient media in petri plates and in micro-cultures where detailed cytological observations could be made. Growing gametophytes were favorable materials for studies of mitosis, chloroplast replication, gametogenesis, and senescence.     

PHOTOINHIBITION OF TEMPERATE LAKE PHYTOPLANKTON BY NEAR-SURFACE IRRADIANCE: EVIDENCE FROM VERTICAL PROFILES AND FIELD EXPERIMENTS1

To evaluate the in situ occurrence of phytoplankton photoinhibition, the light-mediated depression of chlorophyll in vivo fluorescence (IVF) and of the cellular fluorescence capacity (CFC) of phytoplankton was determined in three southeastern United States reservoirs. Vertical profiles of a fluorescence depression index (FDI) and of the CFC for reservoir phytoplankton showed that near-surface photoinhibition of fluorescence properties occurred in association with high surface irradiance and weak vertical mixing of the water column. To characterize the time scales of photochemical and photosynthetic responses to and recovery from exposure to supraoptimal light intensity, phytoplankton IVF responses and ¹⁴C-fixation rates were measured infield experiments. Phytoplankton chlorophyll IVF, CFC, and photosynthetic ¹⁴C fixation were rapidly (20–40 min) depressed when reservoir phytoplankton were exposed to surface irradiances (1700–2000 μE·m^?2·s^?1). Light-mediated increases in the FDI declined rapidly (20–40 min) to pre-exposure levels during a subsequent low-light (75–200 μE·m^?2·s^?1) period, but CFC and ¹⁴C fixation recovered more slowly (>40 min). Exposure of reservoir phytoplankton to a light-intensity gradient revealed both intensity and time thresholds for IVF and CFC depression. Phytoplankton photochemical responses to bright light operate on time scales that, in conjunction with vertical mixing, should limit the occurrence of photoinhibition to extreme irradiance environments. Our results support the hypothesis that the photoinhibition of phytoplankton productivity occurs less commonly than is indicated by fixed-depth incubation measurements.     

GROWTH IN VITRO OF TISSUES ISOLATED FROM NORMAL STEMS AND INSECT GALLS

Pelet , F., A. C. Hildebrandt , A. J. Riker, and F. Skoog . (U. Wisconsin, Madison.) Growth in vitro of tissues isolated from normal stems and insect galls . Amer. Jour. Bot. 47(3) : 186—195. Illus. 1960.–In a preliminary analysis of the nature of gall formation induced by insects, a comparative study has been made of the in vitro growth and nutrition of plant tissues derived from insect galls and from normal plants. Grape, elm, poplar, and willow tissues were grown on a standard medium, modified White's nutrient medium, with coconut milk and/or various growth factors added. Satisfactory growth was obtained over a temperature range from 16° to 36°C. but was generally optimal at 28°—32°C. The optimum pH was generally 4.0—4.5, but a pH of 6.0 or 7.0 gave better growth when the medium contained 2,4-dichlorophenoxyacetic acid. Detailed nutritional studies were limited to grape tissue. Excised stems and excised galls induced by Phylloxera vastatrix Planch, were grown on the basal medium with vitamins and supplemented with naphthaleneacetic acid, indoleacetic acid, kinetin, casein hydrolysate, yeast extract, adenine and a few amino acids added in various combinations. Growth (fresh weight) was measured after a 6-week growth period. When these substances were added singly the optimal concentrations and the quality of growth of stem explants were as follows: with adenine (40 mg./l.) or kinetin (1 mg./l.), growth poor; with NAA (1 mg./l.) or IAA (2 mg./l.), growth fair; and with the only concentration of a powdered casein hydrolysate (3 g./l.), growth good. Gall explants responded more readily to kinetin or adenine but did not form callus in the presence of casein hydrolysate alone. Stem tissues formed both roots and callus, whereas gall tissues formed only callus. The same substances were tested in various combinations. NAA and kinetin provided for moderate, continuous growth, and excellent growth if casein hydrolysate and adenine also were added to the medium. The NAA requirements were markedly reduced in the grape tissues which had been subcultured for 1 or 4 years on coconut milk medium. Friable tissue types were inhibited by the adenine and casein hydrolysate combinations. They grew through 1 passage only on basal medium and then died if not supplied with NAA and kinetin. Firm tissues responded favorably, although irregularly, to casein hydrolysate and adenine. It was concluded that although nutrient requirements varied with tissues derived from insect galls and from normal plants, they also varied with the time of cultivation in vitro. The induction of galls by Phylloxera was not a permanent change in growth factor requirements comparable to that conferred by the crown gall bacteria. In attempts to grow the insect in sterile culture in vitro 5 successive generations of phylloxera were reared on callus tissue.     

AUTOTETRAPLOIDY IN HAPLOPAPPUS SPINULOSUS HYBRIDS: EVIDENCE FROM NATURAL AND SYNTHETIC TETRAPLOIDS

Haplopappus spinulosus (Asteraceae) is a herbaceous, perennial weed common throughout the western Great Plains of North America and includes both diploid and tetraploid populations. A number of populations in southeastern Colorado were analyzed cytogenetically and morphologically for two reasons. First, initial observations during a routine population survey showed they were morphologically intermediate between the diploid subspecies glaberrimus and spinulosus, suggesting they might have arisen via hybridization. Second, cytological examination revealed that they were tetraploid. Because there was indication of hybrid origin, it was of interest to determine whether the populations were behaving as autopolyploids or segmental allopolyploids. The distinction between these two polyploid types is not easily made since both are likely to form multivalents at meiosis, but equations derived from a model proposed by Jackson and Hauber (1982) have made it possible to determine statistically whether a tetraploid individual is behaving meiotically as an autotetraploid. Meiotic configuration frequencies at diakinesis were determined for each tetraploid plant sampled, and observed frequencies were compared to those expected for an autotetraploid having the same maximum number of chiasmata per bivalent, chiasma frequency and chromosome number. In general, the meiotic behavior of the tetraploids was no different from that expected for autoploids. The initial hypothesis that the populations were derived from hybridization was tested by a detailed cytogenetic and morphological study of the presumed parental subspecies, F, hybrids, and natural putative hybrids. The evidence supports the hypothesis that the natural autotetraploids arose from the hybridization of ssp. glaberrimus and ssp. spinulosus.     

MILDLY DELETERIOUS MUTATIONS IN AVIAN MITOCHONDRIAL DNA: EVIDENCE FROM NEUTRALITY TESTS

To determine whether mildly deleterious mutations (MDMs) are present in nonrecombining genomes such as avian mitochondrial DNA (mtDNA), I analyzed molecular data from 14 studies using the neutrality tests of Tajima (1989a) and McDonald and Kreitman (1991). The presence of MDMs in mtDNA is inferred from trends observed across species in estimates of heterozygosity (θ and π) and by comparisons of polymorphism and divergence using the neutrality index (NI). Assuming neutrality, θ equals π and NI equals one. In this study, however, θ is greater than π more often than expected by chance, which reflects an excess of low-frequency alleles, and NI values presented here and elsewhere are consistently greater than one, which suggests an excess of nonsynonymous mutations within species (polymorphism) relative to between species (divergence). These observations suggest that, within species, there is an excess of rare haplotypes and that these haplotypes are carrying MDMs. The excess rare haplotypes may need to be accounted for when estimating population genetic parameters that assume strict neutrality.     

AN INTERGENERIC HYBRID IN THE SAXIFRAGACEAE: EVIDENCE FROM RIBOSOMAL RNA GENES

The tandemly repeated multigene families encoding the 5S and 18S-25S ribosomal RNAs were studied at the restriction enzyme level in Tolmiea menziesii, Tellima grandiflora, and in a putative intergeneric hybrid. Using restriction endonucleases that cut once per repeat, the repeat lengths of the 5S and 18S-25S ribosomal genes were estimated. The 5S ribosomal gene repeat length is approximately 480 and 450 base pairs, respectively, in Tolmiea and Tellima. The repeat length of the 18S-25S ribosomal genes varied from 11–13 kb in Tolmiea, and was only about 9 kb in Tellima. The putative hybrid combined the repeat lengths of both Tolmiea and Tellima for both the 5S and the 18S-25S ribosomal genes. These data substantiate the occurrence of natural hybridization between Tolmiea and Tellima. For both the 5S and 18S-25S gene experiments, the hybrid appears to contain fewer repeats corresponding to Tolmiea than to Tellima.     

FIXATION OF DELETERIOUS MUTATIONS IN CLONAL LINEAGES: EVIDENCE FROM HYBRIDOGENETIC FROGS

Abstract The hemiclonal waterfrog Rana esculenta (RL genotype), a bisexual hybrid between R. ridibunda (RR) and R. lessonae (LL), eliminates the L genome from its germline and clonally transmits the R genome (hybridogenesis). Matings between hybrids produce R. ridibunda offspring, but they generally die at an early larval stage. Mortality may be due to fixed recessive deleterious mutations in the clonally inherited R genomes that were either acquired through the advance of Muller's ratchet or else frozen in these genomes at hemiclone formation. From this hypothesis results a straightforward prediction: Matings between different hemiclones, that is, between R. esculenta possessing different R genomes of independent origin, should produce viable R. ridibunda offspring because it is unlikely that different clonal lineages have become fixed for the same mutations. I tested this prediction by comparing survival and larval performance of tadpoles from within‐ and between‐population crossings using R. esculenta from Seseglio (Se) in southern, Alpnach (Al) in central, and Elliker Auen (El) in northern Switzerland, respectively. Se is isolated from the other populations by the Alps. Enzyme electrophoresis revealed that parents from Se belonged to a single hemiclone that was different from all hemiclones found north of the Alps. Parents from Al also belonged to one hemiclone, but parents from El belonged to three hemiclones, one of which was indistinguishable from the one in Al. Rana esculenta from Se produced inviable tadpoles when crossed with other hybrids of their own population, but when crossed with R. esculenta from Al and El, tadpoles successfully completed metamorphosis, supporting the hypothesis I tested. Within‐population crosses from Al were also inviable, but some within‐population crosses from El, where three hemiclones were present, produced viable offspring. Only part of the crosses between Al and El were viable, but there was no consistent relationship between hemiclone combination and tadpole survival. When backcrossed with the parental species R. ridibunda, hybrids from all source populations produced viable offspring. Performance of these tadpoles with a sexual and a clonal genome was comparable to that of normal, sexually produced R. ridibunda tadpoles. Thus, in the heterozygous state, the deleterious mutations on the clonal R genomes did not appear to reduce tadpole fitness.     

EVIDENCE OF SIMPLE POLYEMBRYONY IN PERMIAN SEEDS FROM ANTARCTICA

Well-preserved seeds with multiple embryos are described from the Permian of the Beardmore Glacier region, Antarctica. The seeds are platyspermic, winged and contain two archegonia in the micropylar end. Each archegonium contains an undifferentiated, multicellular embryo at a comparable stage of development. Attached to one end of each embryo is an elongate structure similar to the suspensor in extant gymnosperms. This discovery represents the earliest example of simple polyembryony yet recorded from the fossil record.     

HOMOPLOID RETICULATE EVOLUTION IN HELIANTHUS (ASTERACEAE): EVIDENCE FROM RIBOSOMAL GENES

Phylogenetic relationships among the 21 taxa comprising Helianthus sect. Helianthus and three outgroup species were assessed by restriction site mapping of the 18S-25S nuclear ribosomal RNA gene family. Wagner parsimony analysis of the 41 restriction site or length mutations observed produced a single 59-step most parsimonious tree. This tree was then compared to a cytoplasmic-based plastid phylogeny for this group. Several major discrepancies were observed between the two phylogenies suggesting both recent and ancient introgression. Furthermore, three cases of diploid hybrid speciation are unambiguously documented and a fourth case is suggested. These data are interpreted to suggest that evolution in Heliathus is reticulate rather than exclusively dichotomous and branching.     

OCCURRENCE OF INTERCALARY AND UNINTERRUPTED MERISTEMS IN EQUISETUM

Quantitative data on cell length and mitotic index in elongating internodes of seven species of Equisetum are presented as evidence for the occurrence of two patterns of internode development in rhizomes of different species. In rhizomes of three species of subgenus Equisetum (E. arvense, E. diffusum, E. telmateia) uninterrupted meristems are present, characterized by acropetal internode maturation. In rhizomes of four species of subgenus Hippochaetae (E. hyemale, E. variegatum, E. scripoides, and E. laevigatum) intercalary meristems are present. The number and locations of intercalary meristems are described for an aerial shoot of E. diffusum. The absence or diminution of intercalary meristems from rhizomes of a variety of vascular plants with intercalary meristems in aerial shoots is discussed from the standpoint of adaptive significance.     

VASCULAR PATTERNS IN STEMS OF ARACEAE: SUBFAMILY POTHOIDEAE

The stem vasculature of representative species of ten of the 11 genera in the Pothoideae was analyzed with the aid of films of series of transverse sections. In all species a leaf trace typically diverges from a continuing axial bundle before departing to a leaf, with the possible exception of Heteropsis. However, within this common organizational scheme a considerable range of variation exists, e.g., with respect to degree of branching of axial bundles, and distance from a leaf-trace branch to the point of leaf-trace departure to a leaf. In addition, we have found a wide variety of patterns of bud-trace organization in different genera of the Pothoideae. For example, prominent arcs composed of numerous bud traces occur in the central cylinder of Pothos, Pothoidium, and Heteropsis. Comparative anatomy leads to the conclusion that Pothos and Pothoidium more closely resemble Heteropsis than Anadendrum. Anadendrum should be dissociated from the tribe Pothoeae. With respect to other genera in the Pothoideae, our preliminary results suggest that each of the genera Anthurium, Culcasia, Zamioculcas, Acorus, and Gymnostuchys are highly distinct from each other.