Ovalopodium desertum n. sp. and the phylogenetic relationships of Cochliopodiidae (Amoebozoa)

An amoeba isolated from a weakly saline semi-desert pond in Kazakhstan (Central Asia) resembles a small Cochliopodium in the light microscope, but has a dorsal fibrous cell coat without scales. Thus it can be identified morphologically as a new species of Ovalopodium Sawyer, 1980, and it is herein named O. desertum. Phylogenetic analysis of the SSU rRNA gene sequences of the new species and four Cochliopodium spp. sequenced additionally shows that Ovalopodium desertum is a sister clade to a robustly monophyletic Cochliopodium. The close relationship between Ovalopodium and Cochliopodium is also confirmed by the analysis of SSU rRNA secondary structure showing the specific helices in the region V5 in all species of both genera. Analysis of actin gene sequences fails to resolve the position of Ovalopodium but demonstrates that Parvamoeba Rogerson, 1993 is probably related to Cochliopodium. The position of Cochliopodiidae within Amoebozoa remains unresolved, despite our efforts to resolve it using broader taxonomic sampling of Amoebozoa, testing alternative tree topologies and removing the fast-evolving sites. Among sequenced genera, Parvamoeba and Endostelium Olive et al., 1984 are probable relatives to Cochliopodiidae. Molecular trees weakly support an inclusion of the family in Flabellinia (Discosea), but more phylogenomic data are necessary to test this hypothesis.     

18S ribosomal RNA gene sequences of Cochliopodium (Himatismenida) and the phylogeny of Amoebozoa

Cochliopodium is a very distinctive genus of discoid amoebae covered by a dorsal tectum of carbohydrate microscales. Its phylogenetic position is unclear, since although sharing many features with naked "gymnamoebae", the tectum sets it apart. We sequenced 18S ribosomal RNA genes from three Cochliopodium species (minus, spiniferum and Cochliopodium sp., a new species resembling C. minutum). Phylogenetic analysis shows Cochliopodium as robustly holophyletic and within Amoebozoa, in full accord with morphological data. Cochliopodium is always one of the basal branches within Amoebozoa but its precise position is unstable. In Bayesian analysis it is sister to holophyletic Glycostylida, but distance trees mostly place it between Dermamoeba and a possibly artifactual long-branch cluster including Thecamoeba. These positions are poorly supported and basal amoebozoan branching ill-resolved, making it unclear whether Discosea (Glycostylida, Himatismenida, Dermamoebida) is holophyletic; however, Thecamoeba seems not specifically related to Dermamoeba. We also sequenced the small-subunit rRNA gene of Vannella persistens, which constantly grouped with other Vannella species, and two Hartmannella strains. Our trees suggest that Vexilliferidae, Variosea and Hartmannella are polyphyletic, confirming the existence of two very distinct Hartmannella clades: that comprising H. cantabrigiensis and another divergent species is sister to Glaeseria, whilst Hartmannella vermiformis branches more deeply.     

Cochliopodium arabianum n. sp. (Amorphea,Amoebozoa)

A new species of Cochliopodium isolated from freshwater at Arabia Lake in Lithonia, GA, USA is described based on light microscopic morphology, fine structure, and molecular genetic evidence. Cochliopodium arabianum n. sp., previously labeled as “isolate Con1” in prior publications, has been shown to group within the genus Cochliopodium in our molecular phylogenetic analysis. Light microscopy and fine structure evidence indicates the new isolate not only shares characters of the genus but also unique distinctive features. Cochliopodium arabianum n. sp. is typically round when stationary; or oval to sometimes broadly flabellate or triangular in shape during locomotion, with average length of 35 μm and breadth of 51 μm. Fine structure evidence indicates C. arabianum n. sp. has tower‐like scales, lacking a terminal spine, sharing high similarity with its closest relative C. actinophorum. However, the scales of C. arabianum n. sp. are unique in height and the breadth of the base plate. Both morphological and molecular data, including SSU‐rDNA and COI, indicate that this new species falls in a clade sufficiently different from other species to suggest that it is a valid new species.     

A New Freshwater Amoeba: Cochliopodium pentatrifurcatum n. sp. (Amoebozoa,Amorphea)

Cochliopodium pentatrifurcatum n. sp. (ATCC^© 30935^TM) is described based on light microscopic morphology, fine structure, and molecular genetic evidence. Cochliopodium pentatrifurcatum n. sp. (length ~ 25 μm) is characterized by surface microscales (0.3 μm tall) containing a circular porous base (~ 0.6 μm diam.) with a thin peripheral rim. Five radially arranged feet, emanating from the base, support a short central column terminating apically as a funnel‐shaped collar (~ 0.5 μm diam.) composed of five radial, trifurcate rays extending from the center toward a thin peripheral rim. The central spine is 0.5–0.6 μm long. The comparative morphologies and combined molecular genetic evidence, SSU‐rDNA and COI, indicate that the new species falls in a clade sufficiently different from other species to suggest that it is a valid new species.     

Techniques to preserve soluble surface components in birch pollen wall: a scanning and transmission electron microscopic study

The exine of birch pollen was examined by scanning and transmission electron microscopy in the native state and after fixation in different aqueous fixatives: glutaraldehyde + OsO4; glutaraldehyde + cetylpyridinium chloride (CPC) + OsO4; glutaraldehyde + cuprolinic blue (CB); and periodate + lysine + paraformaldehyde (PLP). The native pollen exine showed a thin (3-5-nm) border of electron-dense material lining the tectum and electron-dense material within microchannels and bacula cavities. Fixation with the addition of CPC resulted in a voluminous surface coat surrounding the pollen grain, but empty microchannels and bacula cavities. After fixation with the addition of CB, there was a thin surface coat, whereas microchannels and bacula cavities were partially filled with electron-dense material. The other fixatives led to empty microchannels and bacula cavities. There was no surface coat on the pollen grain. However, after all fixation procedures, a thin electron-dense border of the tectum remained visible. Concerning the electron-dense material filling microchannels and bacula cavities in the native pollen grain, the results obtained in the present study suggest that it is either completely lost (after conventional and PLP fixation) or, after fixation with a precipitating additive, partially (CB) or completely (CPC) solubilized and precipitated on the surface of the pollen grain as a surface coat.     

Stimulatory effect of cooling tower biocides on amoebae.

Two species of amoebae were isolated from the cooling tower of an air-conditioning system and examined for effects of exposure to four cooling tower biocides, a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds, another quaternary ammonium compound alone, and an isothiazolin derivative. The amoebae isolated were Acanthamoeba hatchetti and a Cochliopodium species. Two other amoeba cultures, an A. hatchetti culture and Cochliopodium bilimbosum, were obtained from the American Type Culture Collection (ATCC) and were also tested. The cooling tower isolates were more resistant to most of the biocides than the ATCC isolates were. The isothiazolin derivative was the least inhibitory to all four amoeba isolates, and tributyltin neodecanoate mixed with quaternary ammonium compounds was the most inhibitory to three of the four isolates. After exposure to lower concentrations of the biocides, including for one strain the manufacturer's recommended concentration of one biocide, the cooling tower amoeba populations increased significantly compared with unexposed controls, whereas the ATCC isolates were not stimulated at any of the concentrations tested. In some cases, concentrations which stimulated cooling tower amoebae inhibited the growth of the ATCC isolates. These results suggest that cooling tower amoebae may adapt to biocides, underscoring the need to use freshly isolated cooling tower organisms rather than organisms from culture collections for testing the efficacy of such biocides. The stimulatory effect of biocides on amoeba populations is an alarming observation, since these organisms may be reservoirs for legionellae. Biocides used to control microbial growth may actually enhance populations of host organisms for pathogenic bacteria.     

Taxonomy of the Genus Oreocharis (Gesneriaceae)

The genus Oreocharis as circumscribed here consists of 27 species including 5 varieties, of which 5 species and 4 varieties are described as new in the present paper. In the work analysed were the external morphology and geographic distribution and examined under SEM were pollen exine of 22 species and seed coat of 16 species. As a result, three types of the corolla, two types of the anther, three types of the pollen exine and three types of the seed coat are distinguished here in the paper. It is discovered that the corolla in the genus is relatively stable, though diverse, and highly correlated with the characters of pollen grains and seeds. The corolla clearly bilabiate but constricted at the throat, occurring in O. auricula, O. cordatula, O. aurantiaca, etc., for an example, is correlated with smooth, reticulate pollen exine and partial tectum and the reticulate and smooth seed coat. For this reason the subdivision of the genus in the paper is mainly based on the characters of the corolla, but combined with those of the anther, pollen and seed coat. The genus is divided into four sections in the present classification. Dasydesmus Craib, based on a single species. O. bodinieri, is reduced here, and the reasons are given. The genus is distributed mainly in the subtropics, and less frequently in the tropics, of China south of 32.5°N and east of 98.5°E, with only two species beyond the border, O. hirsuta in Thailand (only a single locality in Chiengmai) and O. aurea also found in north Vietnam (see Fig. 1, Table 3). Sect. 1. Stomactin (Clarke) Fritsch. Corolla urceolate-tubular, constricted at the throat, with limb distinctly bilabiate; anthers broad-oblong; seed coat reticulate, smooth, rarely minutely tuberculate; pollen exine fine-reticulate, tectum partial and smooth, luminae slightly unequal in size. Sect. 2. Orthanthera K. Y. Pan Corolla campanulate or campanulate-tubular; anthers broad-oblong; seed coat reticulate, muri smooth, rarely spiny-processed; pollen exine fine-reticulate, with partial and smooth tectum and luminae slightly unequal in size, rarely exine insular and fine-tuberculate, tectum perforate. Setc. 3. Oreocharis Corolla thin-tubular; anthers broad-oblong; seed coat densely spinyprocessed, rarely fine-tuberculate; pollen exine insular, densely spiny-processed, rarely finereticulate and smooth, luminae unequal in size. Sect. 4. Platyanthera K. Y. Pan Corolla campanulate; anthers hippocrepiform; seed coat densely spiny-processed; pollen exine fine-reticulate, tectum perforate, luminae small, nearly equal in size. In the section Stomactin, although the constriction of corolla at its throat is a specialized character, the characters of seed coat, pollen grains and anthers are apparently primitive. Therefore it may be said at least that more primitive characters are preserved in the section. In the section Oreocharis, on the contrary, the characters of corolla, seed coat and pollen exine are all advanced. And in the section Platyanthera, the seed coat, pollen (with perforate tectum) and anthers have developed rather specialized characters.     

Cell proliferation and migration in the roof of the mesencephalon (tectum) in Xenopus laevis tadpoles and adult frogs normally and in brain injury. II. Cell proliferation and differentiation of the tectum in frogs]

The proliferation and directions of cell differentiation in tectum opticum were studied in the young frogs under the conditions of normal development and upon brain trauma by means of 3H-thymidine autoradiography. The same types of cells were shown to be able of proliferation in both the cases: cells of the ventricle zone and glioblasts (gliocytes) in all other tectum layers. A study of directions of the tectum proliferating cells' differentiation in the frogs has shown that the proliferating cells differentiate mainly in the ependyme tanicytes in the ventricle layer 1 and gliocytes in the other tectum layers. The trauma did not change the direction of proliferating cells' differentiation towards the formation of neurons. The complete regeneration is observed in the tectum layer 1 only.     

Acanthodasys paurocactus sp. n., a new species of Thaumastodermatidae (Gastrotricha, Macrodasyida) with multiple scale types from Capron Shoal, Florida

A new species of Acanthodasys (Gastrotricha, Macrodasyida, Thaumastodermatidae) is described from sublittoral sediments off the Atlantic coast of Florida. Acanthodasys paurocactussp. n. is a relatively small species (to 450 μm long) with a strap-shaped outline, a series of anterior, lateral, and ventrolateral adhesive tubes, paired caudal pedicles with posterior adhesive tubes, and a morphologically diverse cuticle. The cuticle contains both spined and unspined scales. Unspined scales are present in two general shapes: lanceolate and eye shaped, with some transitional shapes. All scales have a thickened rim and depressed central region; some scales of both shapes bear either one or more central bumps, a parallel ridge, or a perpendicular ridge that gives the appearance of a cross-shaped pattern under transmitted light. Spined scales are somewhat quadrangular in shape and bear uniancres to 15 μm long with a cross-shaped sectional profile. The new species is now one of five described species to possess both spined and spineless scales, and only one of two species to possess two types of spineless scales (the second species is an incompletely described specimen from Norway).     

几种含笑属(木兰科)植物的花粉形态

含笑属 ( Michelia) 4种花粉光镜下形态相似。SEM下观察 ,黄兰 ( M.champaca)和金叶含笑 ( M.foveolata)表面雕纹为小穿孔 ,苦梓含笑 ( M.balansae)为小穴 ,野含笑 ( M.skinner-iana)为细皱纹 ,较粗糙。 TEM下观察 ,4种花粉外壁均可分为覆盖层、柱状层和基层。其中黄兰的柱状层结构较原始 ,金叶含笑的较进化。花粉形态在一定程度上可反映类群的系统演化。     

An Empirical Evaluation of Genetic Distance Statistics Using Microsatellite Data from Bear (Ursidae) Populations

A large microsatellite data set from three species of bear (Ursidae) was used to empirically test the performance of six genetic distance measures in resolving relationships at a variety of scales ranging from adjacent areas in a continuous distribution to species that diverged several million years ago. At the finest scale, while some distance measures performed extremely well, statistics developed specifically to accommodate the mutational processes of microsatellites performed relatively poorly, presumably because of the relatively higher variance of these statistics. At the other extreme, no statistic was able to resolve the close sister relationship of polar bears and brown bears from more distantly related pairs of species. This failure is most likely due to constraints on allele distributions at microsatellite loci. At intermediate scales, both within continuous distributions and in comparisons to insular populations of late Pleistocene origin, it was not possible to define the point where linearity was lost for each of the statistics, except that it is clearly lost after relatively short periods of independent evolution. All of the statistics were affected by the amount of genetic diversity within the populations being compared, significantly complicating the interpretation of genetic distance data.     

Histochemical localization of acetylcholinesterase in the cerebellum and optic tectum of four freshwater teleosts.

A histochemical study has been carried out on the localization of acetylcholinesterase (AChE) in the cerebellum and optic tectum of four species of freshwater teleosts. AChE distribution in the cerebellar cortex of teleosts shows differences among the species examined and, in the trout, also differences between different cerebellar areas. This uneven kind of enzyme distribution corresponds to a similar variety of AChE patterns noticed in other vertebrates, especially mammals. AChE distribution in the optic tectum shows a prevalent pattern characterized by precise laminar distribution of enzymatic activity which is alternatively strong, weak or absent in the different tectal layers. The results suggest that most of sensitive imput and many systems of stimuli propagation may be mediated by cholinergic mechanisms in the optic tectum of telecosts.     

Three New Freshwater Cochliopodium Species (Himatismenida,Amoebozoa) from the Southeastern United States

Cochliopodium is a lens‐shaped genus of Amoebozoa characterized by a flexible layer of microscopic dorsal scales. Recent taxonomic and molecular studies reported cryptic diversity in this group and suggested that the often‐used scale morphology is not a reliable character for species delineation in the genus. Here, we described three freshwater Cochliopodium spp. from the southeastern United States based on morphological, immunocytochemistry (ICC), and molecular data. A maximum‐likelihood phylogenetic analysis and pairwise comparison of COI sequences of Cochliopodium species showed that each of these monoclonal cultures were genetically distinct from each other and any described species with molecular data. Two of the new isolates, “crystal UK‐YT2” (Cochliopodium crystalli n. sp.) and “crystal‐like UK‐YT3” (C. jaguari n. sp.), formed a clade with C. larifeili, which all share a prominent microtubule organizing center (MTOC) and have cubical‐shaped crystals. The “Marrs Spring UK‐YT4” isolate, C. marrii n. sp., was 100% identical to “Cochliopodium sp. SG‐2014 KJ569724 .” These sequences formed a clade with C. actinophorum and C. arabianum. While the new isolates can be separated morphologically, most of the taxonomic features used in the group show plasticity; therefore, Cochliopodium species can only be reliably identified with the help of molecular data.     

Evolutionary evaluation of reciprocity of connections in the turtle tectofugal visual system

In two turtle species—Emys orbicularis and Testudo horsfieldi—by the method of anterograde and retrograde traicing at the light and electron microscopy level, the existence is proven of direct descending projections from the thalamic nucleus of the tectofugal visual system n. rotunds (Rot) to the optic tectum. After injection of tracers into Rot alone and into Rot with involvement of the tectothalamic tract (Trtth), occasional labeled fibers with varicosities and terminals are revealed predominantly in the deep sublayers of SGFS of the rostral optic tectum, while in the lower amount—in other tectal layers. After the tracer injections into the optic tectum, a few retrogradely labeled neurons were found mainly in the Rot ventral parts and within Trtth. Their localization coincides with that of GABA-immunoreactive cells. Electron microscopy showed the existence of many retrogradely labeled dendrites throughout the whole Rot; a few labeled cell bodies were also present there, some of them being also GABA-immunoreactive. These results allow us to conclude about the existence of reciprocal connections between the optic tectum and Rot in turtles, these connections being able to affect processing of visual information in tectum. We suggest that reciprocity of tectothalamic connections might be the ancestral feature of the vertebrate brain; in the course of amniote evolution the functional significance of this feature can be decreased and even lost in parallel with a rise of the role of direct corticotectal projections.     

Requirement for the zebrafish mid-hindbrain boundary in midbrain polarisation, mapping and confinement of the retinotectal projection.

The organizer at the midbrain-hindbrain boundary (MHB organizer) has been proposed to induce and polarize the midbrain during development. We investigate the requirement for the MHB organizer in acerebellar mutants, which lack a MHB and cerebellum, but retain a tectum, and are mutant for fgf8, a candidate inducer and polarizer. We examine the retinotectal projection in the mutants to assay polarity in the tectum. In mutant tecta, retinal ganglion cell (RGC) axons form overlapping termination fields, especially in the ventral tectum, and along both the anterior-posterior and dorsal-ventral axis of the tectum, consistent with a MHB requirement in generating midbrain polarity. However, polarity is not completely lost in the mutant tecta, in spite of the absence of the MHB. Moreover, graded expression of the ephrin family ligand Ephrin-A5b is eliminated, whereas Ephrin-A2 and Ephrin-A5a expression is leveled in acerebellar mutant tecta, showing that ephrins are differentially affected by the absence of the MHB. Some RGC axons overshoot beyond the mutant tectum, suggesting that the MHB also serves a barrier function for axonal growth. By transplanting whole eye primordia, we show that mapping defects and overshooting largely, but not exclusively, depend on tectal, but not retinal genotype, and thus demonstrate an independent function for Fgf8 in retinal development. The MHB organizer, possibly via Fgf8 itself, is thus required for midbrain polarisation and for restricting axonal growth, but other cell populations may also influence midbrain polarity.     

The geographic scaling of biotic interactions

A central tenet of ecology and biogeography is that the broad outlines of species ranges are determined by climate, whereas the effects of biotic interactions are manifested at local scales. While the first proposition is supported by ample evidence, the second is still a matter of controversy. To address this question, we develop a mathematical model that predicts the spatial overlap, i.e. co‐occurrence, between pairs of species subject to all possible types of interactions. We then identify the scale of resolution in which predicted range overlaps are lost. We found that co‐occurrence arising from positive interactions, such as mutualism (+/+) and commensalism (+/0), are manifested across scales. Negative interactions, such as competition (?/?) and amensalism (?/0), generate checkerboard patterns of co‐occurrence that are discernible at finer resolutions but that are lost and increasing scales of resolution. Scale dependence in consumer–resource interactions (+/?) depends on the strength of positive dependencies between species. If the net positive effect is greater than the net negative effect, then interactions scale up similarly to positive interactions. Our results challenge the widely held view that climate alone is sufficient to characterize species distributions at broad scales, but also demonstrate that the spatial signature of competition is unlikely to be discernible beyond local and regional scales.     

Marking zebrafish, Danio rerio (cyprinidae), using scale regeneration

Tagging or marking small laboratory-bred fish species is not an easy task. This also holds for the zebrafish, Danio rerio, which is widely used throughout the world as a model organism for genetics, developmental biology, etc. We present a simple marking technique based on scale regeneration. A comparative morphological study of various types of zebrafish scales indeed shows that regenerated scales are easily distinguishable from nonregenerated ones. We propose to take advantage of this typical morphology to mark a single or several individuals. This technique, based on a natural biological process, is easy to perform and does not enhance fish mortality in laboratory breeding conditions. It permits assembly of several specimens in a single tank with the possibility of identifying each of them by regenerated-scale coding. Nevertheless, a prerequisite is that the species does not lose and regenerate scales in large numbers in laboratory breeding conditions. To check this, 5,200 scales were removed from a large region of the left flank in 100 zebrafish and the number and position of regenerated scales were statistically analysed. Our results indicate that (1) laboratory-bred zebrafish have only a few regenerated scales (7.48%), (2) the probability of finding a regenerated scale is similar whatever its position in a row (antero-posterior axis), but (3) it differs from one row to another (scales from the back are more frequently lost than those from the pectoral region). This paper presents a procedure to mark small breeding colonies of zebrafish using scale regeneration with the number and position of the scales to be removed with high probability of marking success. J. Exp. Zool. 286:297-304, 2000.     

五种木兰属植物的花粉形态

对木兰属(Magnolia)5种花粉进行了光学显微镜观察,形态相似。扫描电镜下观察,花粉外壁均为小穴状纹饰,荷花玉兰(M．grandiflora)略为粗糙。透射电镜下观察,5种花粉外壁均可分为覆盖层、柱状层和基层。覆盖层不连续,有小穿孔。在远极面萌发沟区域,外壁逐渐减薄,最后覆盖层和柱状层消失,仅残留基层。柱状层内部空隙较小,多由颗粒构成,处于小柱发育的初级阶段。荷花玉兰和玉兰萌发沟区域下有明显的外壁-2。木兰属花粉属于原始类型的代表。     

The palynology of the genus Rondeletia L. (Rubiaceae-Cinchonoideae-Rondeletieae)

Pollen of 20 species of Rondeletia, covering three subgenera and seven sections or “species groups” of the genus, was studied using light and scanning microscopy. Rondeletia, characterized by binucleate mature pollen grains like numerous other Rubiaceae, was found to be eurypalynous. The pollen is isopolar and small-sized (average equatorial diameters 11.4–22.8 μm) and 3-colporate (rarely 4-colporate). In equatorial view, the pollen grains are mostly ± spheroidal (rarely suboblate); in polar view (ambitus), they are mainly circular or semiangular (intersemiangular in one species only). Three different exine types are distinguishable: exine with 1) tectum minutely perforate, perforations usually isodiametric, 2) tectum with comparatively large, not isodiametric perforations or 3) coarsely reticulate; supratectal sculpturing elements are lacking; the width of the perforations and lumina is either equal or different at the apo- and mesocolpia; in a few species rod-like luminal processes are present. Palynological features of most of the investigated taxa tend to overlap so that palynology is not very helpful in supporting the subgeneric division of Rondeletia. It was, however, noted that pollen morphology, to some extent, supports the sections of subgen. Arachnothrix. Palynologically, the subgen. Rogiera was found to be heterogeneous in that each of the three investigated species is characterized by a different exine type; one of these, R. strigosa, differs from all other Rondeletia species investigated in having a coarsely reticulate exine. R. odorata (subgen. Rondeletia) differs from all other investigated species in its intersemiangular pollen shape.