Oospore dimensions and morphology in North American Tolypella (Charophyceae,Charophyta)

Characteristics of the oospores have been used to delimit sections and, in some cases, species in the genus Tolypella A. Braun. To test the utility of oospore characters for identifying North American species of Tolypella, we investigated oospores from field‐collected and herbarium specimens. Oospore dimensions (length, width, and length to width ratio) and morphology (color, ridge number and shape, wall ornamentation, and basal impression number) were measured. Oospore dimensions were statistically analyzed and oospore morphology was studied with light and scanning electron microscopy. Statistical analyses showed significant differences in length, width, and length to width ratios among most Tolypella species and populations but there was considerable overlap, which suggested that species identification based on oospore measurements alone is not wholly reliable. In addition, oospore morphology was not unique for every species.     

Phylogeny of North American Tolypella (Charophyceae,Charophyta) based on plastid DNA sequences with a description of Tolypella ramosissima sp. nov.

Characeae (Charophyceae, Charophyta) contains two tribes with six genera: tribe Chareae with four genera and tribe Nitelleae, which includes Tolypella and Nitella. This paper uses molecular and morphological data to elucidate the phylogeny of Tolypella species in North America. In the most comprehensive taxonomic treatment of Characeae, 16 Tolypella species worldwide were subsumed into two species, T. intricata and T. nidifica, in two sections, Rothia and Tolypella respectively. It was further suggested that Tolypella might be a derived group within Nitella. In this investigation into species diversity and relationships in North American Tolypella, sequence data from the plastid genes atpB, psbC, and rbcL were assembled for a broad range of charophycean and land plant taxa. Molecular data were used in conjunction with morphology to test monophyly of the genus and species within it. Phylogenetic analyses of the sequence data showed that Characeae is monophyletic but that Nitelleae is paraphyletic with Tolypella sister to a monophyletic Nitella + Chareae. The results also supported the monophyly of Tolypella and the sections Rothia and Tolypella. Morphologically defined species were supported as clades with little or no DNA sequence differences. In addition, molecular data revealed several lineages and a new species (T. ramosissima sp. nov.), which suggests greater species diversity in Tolypella than previously recognized.     

Effects of salinity on turgor pressure and fertility in Tolypella (Characeae)

We present the first experimental results on salinity tolerance and regulation mechanisms in the genus Tolypella. The two species investigated, T. nidifica and T. glomerata, regulate turgor pressure with almost complete effectiveness by adjustment of K+ and CT concentrations. Sucrose is also involved. The mechanism is basically identical to the mechanism of turgor pressure regulation previously identified in representatives of the genera Chara and Lamprothamnium. Since Chara and Lamprothamnium on the one hand and Tolypella on the other belong to different phylogenetic branches that separated early in the geological history of the Characeae, the K+ regulation mechanism can be assumed to represent an ancient pattern derived from a salt-tolerant common ancestor. Furthermore, our experiments provide evidence that salinity is a limiting factor for fertility in both T. nidifica and T. glomerata. Although the onset of gametangia covers the whole range of salinities tested here (0–29 psu), 12psu was the inhibitory level for the formation of mature oospores. Fertilization is probably disturbed by an increase in salinity. An inability to reproduce sexually under euryhaline conditions could explain why the distribution of the two species is restricted to oligo- and mesohaline environments, despite the wide range of salinity tolerance of their vegetative apparatus.     

Oospore dimensions and wall ornamentation patterns in Swedish charophytes

The ultrastructural features of oospore wall ornamentation patterns revealed by scanning electron microscopy are important taxonomic characters of the Charales. The present study shows inter-and intrapecific variations in 19 species of the genera Chara, Lamprohamnium, Nitella and Tolypella. This is the first time that the oospore wall ornamentation of Swedish Charales has been documented in detail. In the studied Chara species the ornamentation within species was variable, and partly overlapping between species, but only between closely related species. In contrast, the Nitella species showed distinct differences in ornamentation patterns between species, although the same patterns can be found in different species. This study presents for the first time SEM images of the species Chara rudis (pustular ornamentation), Nitella opaca (pitted ornamentation) and Nitella wahlbergiana (anastomosing network ornamentation). The ornamentation pattern in the Nordic species TV. wahlbergiana , supports its separation from Nitella mucronata which has a reticulate ornamentation. The relationship between length and width of the oospores is also of taxonomic significance. Nitella and Tolypella oospores are roundish, whereas those of Chara and Lamprothamnium are elongate. The extent to which environmental and genetic factors can affect oospore size and shape within a species are still unknown, but in the present study both ornamentation pattern and size provided evidence for the distinction between Chara globularis and Chara aspera. Some further taxonomic problems in the Charales are discussed in the light of the results of this study.     

Oospore dimensions and wall ornamentation in <Emphasis Type="Italic">Chara braunii</Emphasis>

Morphological features of oospores of a rarely noted species Chara braunii Gmel. were examined in a local population (fishpond near Tuplice village, Ziemia Lubuska region, mid-western Poland). The largest polar axis (LPA, length), largest equatorial diameter (LED, width), isopolarity index (ISI = LPA/LED* 100), and the number of ridges and width of fossa of 90 oospores were measured. Additionally, the oospore wall ornamentation pattern was examined by means of scanning electron microscopy (SEM). As a result, a cylindrical shape and the lack of so-called “shoulder” were found. Oospore length and width ranged between 466.8–600.1 μm and 250.1–366.7 μm, respectively, with the ISI index values between 148 and 213. The number of ridges was 8–10 and the width of fossa varied between 50.0 and 66.7 μm. The oospore width appeared to be the most changeable feature, whereas, by contrast, the number of ridges was the least variable character. Wall ornamentation can be described as tuberculate and a ribbon was present on the ridges. As a comparative material for the study performed, 9 oospores from Professor Izabela Dąmbska’s Collection of Charophytes of Poland were measured. It was evidenced that variation ranges of most of the features of Chara braunii oospores from Tuplice fishpond are similar to those of Professor Dąmbska’s herbarium materials and to data reported by authors from other countries as well. The morphology of reproductive structures does not follow the variation of thalli characteristics.     

Testing of life history traits of a soilborne pathogen in vitro: Do characteristics of oospores change according the strains of Aphanomyces euteiches and the host plant infected by the pathogen?

Aphanomyces euteiches is a polyphagous, homothallic soilborne pathogen producing asexual (zoospores) and sexual (oospores) spores. Even if oospores are essential for disease development and survival, to date, no study has focused on the production rates of oospores or the quality of the offspring produced by oospores. In this study, a nonabrasive oospore extraction method from infected roots of leguminous species (pea, faba bean and vetch) was developed. This methodology includes steps of grinding and filtration. The quality of oospores (viable, dormant and dead) was assessed with tetrazolium bromide staining, and germination of oospores was tested using exudates of peas, faba bean and vetch. The average yield of the extraction method was approximately 21%. Staining revealed some differences between strains and between leguminous species. The germination percentage of oospores extracted from pea, faba bean and vetch was 25%, 62% and 70%, respectively, and a significant difference was observed according to the origin of A. euteiches‐inoculated strains. Application of exudates seems to stimulate the germination of oospores (2% for the control, 18% for pea exudates and 1% for vetch exudates). Differences observed between A. euteiches strains and leguminous species indicate that more knowledge concerning the biology of oospores is needed. This will help to better estimate evolution process of the pathogen and manage resistance and crop successions.     

Oospore variation in <Emphasis Type="Italic">Nitella gracilis</Emphasis> and <Emphasis Type="Italic">Nitella mucronata</Emphasis> (Charales,Charophyceae) from Poland

The determination of the members of Charales (Charophyceae) is not easy. The main reason of this is a wide range of variation in vegetative characters within most of the species. Many features such as the extent of branchlet cortication, the degree of incrustation, length and number of bract cells, spine cells, stipulodes, diameter of the main axis and oospore size and color do not have universal taxonomic significance. In similar, many Nitella species characters such as: general habitus, branchlets and presence of the top segment (mucro), are helpful in determination, however they are often not developed. In this case, the features of oospore wall ornamentation revealed by light and scanning microscopy can be important taxonomic characters of the Charales. The present study shows interspecific variations of the oospore in two Nitella species from Poland which can be helpful in correct determination of both species. Some of the oospore features like number of striae and fossa width can not be used, as they appeared statistically unimportant, and cannot be used in distinguishing between both species. The presented results are the first detailed report of oospore wall ornamentation pattern from Poland for the species studied. Oospore features are presented in detail on the background of some additional tips and among them particularly: length and width of the oospore, ISI idex, width of fossa, width of striae and number of striae.     

Inhibition of Chara vulgaris oospore germination by sulfidic sediments

The germinability of Chara vulgaris oospores collected from the sediments of four Ontario lakes varies considerably, ranging in germination percentage from 7% to 54%. Chemical analysis of the interstitial water of the sediments indicated that oospores with low germination occurred in lakes which have high acid volatile sulfides (H₂S, FeS, HS⁻) and high soluble Fe²⁺. The inhibitory effects of sediment on oospore germination were demonstrated by transplant experiments, and suggested that sulfide was the toxic agent. Exposure of high-germinating sedimentary oospores to free sulfide concentrations greater than 2.0 mM caused a greater than 30% reduction in oospore germination. The presence of sulfide in sediments was shown to result from sulfate reduction by bacteria in sediment pore water of those lakes where oospore viability was lowest. Differences in oospore germination percentage appear, therefore, to be due to the toxicity of H₂S produced in the sediment, either by a direct effect on the oospore, or on the parent plants.     

Viability Tests for Thick Walled Fungal Spores (ex: Oospores of Peronospora manshurica)

Oospores of Peronospora manshurica, the causal agent of soybean downy mildew, were stained by a variety of techniques. TTC (tetrazolium chloride) and NBT (nitroblue tetrazolium chloride) primarily stained oospores which were cytologically abnormal and appeared degenerating. Cytological normal oospores were not stained by these compounds presumably because the dyes were excluded from the oospore cytoplasm by the oospore wall or the plasmalemma. Strong autofluorescence of dead/degenerating oospores in the FDA test (fluorescein diacetate) made scoring of the oospore viability by this technique unreliable. Phloxine B was found in a consistent way to stain the degenerating oospores and a small proportion of the oospores which by light microscopic, observations could not be scored cytologically abnormal. Control experiments with live and dead cells of yeast (Saccharomyces cerevisiae) confirm that phloxine B is excluded from live cells and dead cells become stained. The presumed mode of action is that the semipermeability of the plasma membrane of live cells excludes the stain. The phloxine B test described here appears a promising technique for the determination of oospore viability of P. manshurica.     

TAXONOMIC REEXAMINATION OF 17 SPECIES OF NITELLA SUBGENUS TIEFFALLENIA (CHARALES,CHAROPHYCEAE) BASED ON INTERNAL MORPHOLOGY OF THE OOSPORE WALL AND MULTIPLE DNA MARKER SEQUENCES1

In an attempt to reconstruct the natural taxonomic system for Nitella, 17 species of Nitella subgenus Tieffallenia were reexamined using SEM observations of the internal morphology of the oospore wall (IMOW) and phylogenetic analyses of 4553 base pairs from multiple DNA markers (atpB, rbcL, psaB, and ITS‐5.8S rRNA genes). Our SEM observations identified three types of IMOW: homogeneous (HG), weakly spongy (W‐SG), and strongly spongy (S‐SG) types. Based on differences in the IMOW, species with reticulate or tuberculate oospore wall ornamentation in the external morphology of the oospore wall (EMOW) were subdivided into two distinct groups (characterized by the HG or S‐SG types of IMOW, respectively), which were robustly separated from each other in our molecular phylogenetic analyses. In our molecular phylogeny, the subgenus Tieffallenia consisted of four robust monophyletic groups—three clades of the HG type and a spongy (S‐SG and W‐SG) type clade—that were characterized by differences in the IMOW and EMOW. In addition, our SEM observations and sequence data verified the distinct status of five species (N. japonica Allen, N. oligospira A. Braun, N. vieillardii stat. nov., N. imperialis stat. nov., and N. morongii Allen) that R. D. Wood had assigned as infraspecific taxa. Moreover, our SEM observations of the IMOW also suggested that N. megaspora (J. Groves) Sakayama originally identified by LM includes at least two distinct species, characterized by W‐SG and S‐SG types of IMOW, respectively.     

Collophora hispanica,a New Pathogen and Potential Threat to the Almond Industry in Iran

Trunk diseases are potential threats for almond productivity and longevity worldwide, including Iran. In a recent survey on fungal species associated with trunk diseases of almonds in north‐western Iran, Collophora isolates (tentatively identified as Collophora hispanica) were recovered with high frequency from wood samples with internal necrosis and brown to black vascular streaking of almond trees showing symptoms of decline. However, the pathogenic potential of Collophora isolates on almond trees in Iran remains unproven. In this study, the identity of the isolates was further confirmed as C. hispanica based on a combination of morphological data and sequence data of ITS‐rDNA region, and pathogenicity of C. hispanica isolates on almond was evaluated using excised shoot method and in greenhouse experiments. Collophora hispanica isolates induced lesions statistically different from the control, in both excised shoot method and greenhouse assays. Significant differences were observed among the isolates in the length of the lesion induced on wood. Collophora hispanica should be considered as the main trunk pathogens of almond trees in north‐western region of Iran. The distribution and host range of this new pathogen on almond remains to be studied.     

Morphological and dietary responses of chipmunks to a century of climate change

Predicting how individual taxa will respond to climatic change is challenging, in part because the impacts of environmental conditions can vary markedly, even among closely related species. Studies of chipmunks (Tamias spp.) in Yosemite National Park provide an important opportunity to explore the reasons for this variation in response. While the alpine chipmunk (T. alpinus) has undergone a significant elevational range contraction over the past century, the congeneric and partially sympatric lodgepole chipmunk (T. speciosus) has not experienced an elevational range shift during this period. As a first step toward identifying the factors underlying this difference in response, we examined evidence for dietary changes and changes in cranial morphology in these species over the past century. Stable isotope analyses of fur samples from modern and historical museum specimens of these species collected at the same localities indicated that signatures of dietary change were more pronounced in T. alpinus, although diet breadth did not differ consistently between the study species. Morphometric analyses of crania from these specimens revealed significant changes in cranial shape for T. alpinus, with less pronounced changes in shape for T. speciosus; evidence of selection on skull morphology was detected for T. alpinus, but not for T. speciosus. These results are consistent with growing evidence that T. alpinus is generally more responsive to environmental change than T. speciosus, but emphasize the complex and often geographically variable nature of such responses. Accordingly, future studies that make use of the taxonomically and spatially integrative approach employed here may prove particularly informative regarding relationships between environmental conditions, range changes, and patterns of phenotypic variation.     

Phylogenetics and comparative analysis of the mitochondrial genomes of three violet‐ringed octopuses

Similar morphological characters and little molecular data of Amphioctopus rex, A. neglectus and A. cf. ovulum resulted in their unknown phylogenetic statuses and equivocal relationships. In this study, the complete mitochondrial genomes of these three species collected in Chinese waters were sequenced and compared with each other to clarify the relationships among them. The lengths of the mitochondrial genomes varied from 15,646 bp to 15,814 bp, and the A + T content and GC skew for protein‐coding genes showed little variation. In contrast, both a dendrogram based on codon usage and the gene arrangements of the three octopuses showed that A. rex was more closely related to A. neglectus than to A. cf. ovulum. Five data sets and two methods (maximum likelihood and Bayesian inference) were utilized for the first time to explore the phylogenetic relationships among these three species in Octopodidae. The results indicated that a data set combining protein‐coding genes and RNA genes (PR) was optimal for analysing the relationships among 43 cephalopods. All of the phylogenetic trees divided the cephalopods into 10 taxa and supported the monophyly of Oegopsida, Myopsida, Sepiidae and Octopodidae. In this study, Idiosepiidae was classified as sister to Sepiolidae. Trees constructed using all data sets robustly supported the monophyly of the genus Amphioctopus. Notably, A. rex was more closely related to A. neglectus than to A. cf. ovulum, although these three species share the characteristic of violet rings on dark ocelli.     

Morphological characterization and phylogenetic relationships of Indochinese box turtles—The Cuora galbinifrons complex

The members of the Indochinese box turtle complex, namely Cuora galbinifrons, Cuora bourreti, and Cuora picturata, rank the most critically endangered turtle species on earth after more than three decades of over‐harvesting for food, traditional Chinese medicine, and pet markets. Despite advances in molecular biology, species boundaries and phylogenetic relationships, the status of the C. galbinifrons complex remains unresolved due to the small number of specimens observed and collected in the field. In this study, we present analyses of morphologic characters as well as mitochondrial and nuclear DNA data to reconstruct the species boundaries and systematic relationships within the C. galbinifrons complex. Based on principal component analysis (PCA) and statistical analysis, we found that phenotypic traits partially overlapped among galbinifrons, bourreti, and picturata, and that galbinifrons and bourreti might be only subspecifically distinct. Moreover, we used the mitochondrial genome, COI, and nuclear gene Rag1 under the maximum likelihood criteria and Bayesian inference criteria to elucidate whether C. galbinifrons could be divided into three separate species or subspecies. We found strong support for a sister relationship between picturata and the other two species, and consequently, we recommend maintaining picturata as a full species, and classifying bourreti and galbinifrons as subspecies of C. galbinifrons. These findings provide evidence for a better understanding of the evolutionary histories of these critically endangered turtles.