PRELIMINARY INVESTIGATION OF THE DOUBLE-DIFFUSION TECHNIQUE AS A TOOL IN DETERMINING RELATIONSHIPS AMONG SOME MYXOMYCETES,ORDER PHYSARALES

Double-diffusion technique was used to investigate myxomycete relationships within the order Physarales. Extracts of plasmodia of 22 slime mold isolates were reacted with five antisera produced to Plasmodia of Didymium nigripes, Physarella oblonga, Physarum polycephalum, Physarum gyrosum and Fuligo septica. Two isolates of Fuligo septica tested alike. Four isolates of Physarum pusillum did not test alike, and no valid conclusion of the relationship of this species was possible. These isolates showed strong serological affinity: (1) Physarum gyrosum, Physarella oblonga, two isolates of Fuligo septica, and possibly two isolates of Physarum pusillum, and Physarum tenerum; (2) Physarum polycephalum and Physarum flavicomum; (3) Fuligo septica and many of the species tested; (4) Didymium nigripes and at least one isolate of Didymium iridis. In most cases serologial relationships among species tested did not coincide with current taxonomy based on morphology of fructification.     

TAXONOMIC SIGNIFICANCE OF INTRASPECIFIC ISOZYME PATTERNS OF THE SLIME MOLD FULIGO SEPTICA PRODUCED BY DISC ELECTROPHORESIS

Plasmodial extracts of 45 isolates of Fuligo septica (L.) Wiggers selected from diverse geographical areas were subjected to acrylamide disc electrophoresis. Profiles for ten enzymes and general protein were determined. Profile variability among isolates occurred. Indices of similarity calculated for each isolate indicated that the variations of the yellow and white plasmodial isolates fell within different ranges. Determination of the total range of variation for the species must wait on the analysis of more white isolates. The extreme sensitivity of disc electrophoresis in detecting color forms suggests it can be useful as a tool in myxomycete taxonomy.     

Spore germination and swarm cell morphogenesis in the acellular slime mold Fuligo septica

This report describes conditions under which spores of the acellular slime mold Fuligo septica underwent a very rapid, synchronous and complete (100%) germination followed by morphogenesis of motile, flagellated swarm cells from the released protoplasts. This developmental sequence was initiated immediately upon wetting the spores with a surfactant and was completed within 40–50 min in the absence of any exogenous nutrient other than sodium phosphate buffer. Oxygen was required for germination. The rate and percentage of germination diminished with increasing spore concentration suggesting the existence of an autoinhibitor. The morphological sequence of events in the differentiation process was examined by scanning electron microscopy.     

煤绒菌Fuligo septica显型原质团细胞核及菌核的超微结构

从煤绒菌显型原质团中提纯细胞核、核仁,诱导原质团形成菌核,并在透射电镜下观察。研究结果表明:细胞核具有中央核仁,核仁可以看到明显的纤维中心、致密纤维中心和颗粒结构。原质团中存在大量的黏液颗粒。菌核具有双层膜,内含有细胞器及脂滴。     

煤绒菌Fuligo septica显型原质团细胞核及菌核的超微结构

从煤绒菌显型原质团中提纯细胞核、核仁,诱导原质团形成菌核,并在透射电镜下观察。研究结果表明:细胞核具有中央核仁,核仁可以看到明显的纤维中心、致密纤维中心和颗粒结构。原质团中存在大量的黏液颗粒。菌核具有双层膜,内含有细胞器及脂滴。     

FIBRILLAR DIFFERENTIATION IN MYXOMYCETE PLASMODIA

Myxomycete plasmodia of four different types (not including Physarum polycephalum) were studied in thin sections viewed in the electron microscope. In the cytoplasm of the protoplasmodia of Clastoderma debaryanum and the phaneroplasmodia of Fuligo septica fixed in situ, fibrillar differentiations of three rather distinct kinds were observed. One of these is filamentous and closely resembles the filaments (or "microtubules") of the mitotic apparatus of other species. The larger phaneroplasmodia of two species belonging to the Physarales and the plasmodium of Hemitrichia vesparium showed fewer and less well defined fibrils, and no fibrils were seen in the aphanoplasmodium of Stemonitis fusca. Good stabilization of such fibrils in larger plasmodia may require fixation methods more rigidly controlled than those which succeed with microscopic protoplasmodia. The function of the observed fibrils cannot yet be determined. Their presence in cytoplasm fixed in situ, however, lends support to those theories of protoplasmic movement which are dependent on integral cross-bonding of one or a few molecular species.     

Pattern of Substrate Preferences of Free Living Protists (Myxomycetes) on Decaying Wood

We have studied the influence of the stage of decomposition and acidity of wood, as well as the illumination of the microhabitat on the species composition, abundance, and occurrence of slime molds (Myxomycetes) of the xylobiontic (inhabitants of the wood) substrate complex of forest communities in Siberia (Altai krai, Altai Republic, and Novosibirsk oblast). This work is based on a study of 1777 samples of fruit bodies (sporophores) of myxomycetes. In the analysis of data for communities of Myxomycetes of the xylobiontic substrate complex, we recognize a successional series which clearly correlates with the stage of wood decomposition. The study of the distribution of the species composition of the slime mold on wood of various stages of decomposition shows that the maximum number of species is observed on dead trunks where the wood is of medium density and the bark can easily be separated (W3). The lowest specificity and diversity of species composition is observed on the fallen trunks of trees with very dense wood and dense bark (W1). These dead trees have low water-retaining capacity, tight bark, and almost intact wood containing large amounts of lignin, preventing the invasion of plasmodia and myxamoebae of Myxomycetes into the trunk. A study of the location of slime-mold colonies in relation to the illumination level shows that the greatest number of species is found on the front, side, and bottom parts of dead trunks of woody plants. The least specificity and the least variety of Myxomycetes species are found on apical parts of dead tree trunks facing the sun. The exception are species with large fruit bodies such as Fuligo septica and Reticularia splendens, which are most often observed on the upper parts of the sun facing trunks of woody plants. In the research area, the complex of species typical for wood of coniferous trees is described. It is noted that acidophilic slime molds of the genera Comatricha and Cribraria can be traced in xylobiontic and epiphytic–corticuloid substrate complexes.     

Phylogenetic position of the enigmatic myxomycete genus Kelleromyxa revealed by SSU rDNA sequences

The coprophilous myxomycete Kelleromyxa fimicola (Dearn. & Bisby) Eliasson was first described in 1929 as Licea fimicola Dearn. & Bisby. Based on the superficial resemblance of its sporocarps to those of Licea biforis, the species was placed within the Liceales, an order assigned by molecular phylogenies to the bright-spored clade of myxomycetes. However, detailed studies of the morphology and life cycle of K. fimicola revealed several characters of the dark-spored order Physarales. To elucidate the systematic position of Kelleromyxa, we obtained three partial and one complete sequence of the SSU rDNA, which demonstrated a clear relationship of K. fimicola to the order Physarales. However, the obtained sequences are not closely related to any of the two known families of Physarales, supporting the erection of a monotypic family for this species. Along with morphological observations, our data support the exclusion of K. fimicola from the order Liceales and placement among the order Physarales within the dark-spored clade of myxomycetes.     

Genetic Variation in Nacobbus aberrans: An Approach toward Taxonomic Resolution

Biochemical and molecular analyses of genetic variation were evaluated to address the taxonomic status of Nacobbus aberrans. Isolates from Mexico, Peru, and Argentina, cultured on tomato in the greenhouse, were analyzed with respect to isozyme and DNA marker variation. Although acid phosphatase and malate dehydrogenase revealed distinct profiles for each isolate, non-specific esterases revealed possible affinities between the Peruvian isolates and between the isolates from Mexico and Peru. Two of l 0 RAPD primers revealed affinities suggested by esterase profiles. RFLP analysis of the rDNA repeating unit with six restriction enzymes revealed identical cleavage patterns between the Peru isolates and a distinct profile shared by isolates from Mexico and Argentina. Nucleotide sequence analysis of the 5.8S rRNA coding region revealed differences among the four isolates at eight of 157 positions; sequences of the Peruvian isolates differed from each other at only one position, whereas the Mexican and Argentine isolates were identical and could be distinguished from the Peruvian isolates. A distance matrix from unweighted pairwise comparisons of the 5.8S rDNA revealed apparent elevated intraspecific divergence in N. aberrans comparable to intergeneric divergence between Heterodera and Globodera. Analysis of additional N. aberrans isolates from throughout the distribution range should help determine the full extent of intraspecific genetic variation that underlies the phenotypic and morphologic diversity of the genus.     

Geographical distribution of myxomycetes living on Cryptomeria japonica bark in Japan

There is little available data on the biogeography of myxomycetes at the regional scale within any given climate zone. To investigate the geographical distribution of these protozoans, we focused on corticolous myxomycetes associated with the bark of Cryptomeria japonica trees, which we sampled extensively throughout Japan. Myxomycete sporophores developed in 73% of 2244 moist-chamber cultures of 188 bark samples from 24 regions, including 31 species. The most abundant species were Paradiacheopsis rigida and Cribraria confusa, which accounted for over 20% of all myxomycetes sampled. Non-metric multidimensional scaling was used to analyze the distribution of myxomycetes in relation to geographical variables and bark pH. The distribution of myxomycetes was influenced by altitude, temperature and bark pH. Temperature gradients and relative abundance of species were negatively correlated in P. rigida and Comatricha laxa and positively correlated in C. confusa, Macbrideora cornea, and Diderma chondrioderma. Bark pH was also positively correlated with the relative abundance of C. confusa, D. chondrioderma, and Physarum nutans and negatively correlated with that of P. rigida, P. cribrata, Enerthnema papillatum, and E. melanospermum. Geographical distribution of corticolous myxomycete communities was determined based on temperature and bark pH, which acted as local barriers in our study area.     

Estimation of Genetic Divergence in Meloidogyne Mitochondrial DNA

Restriction fragments from purified mitochondrial DNA can be readily detected following rapid end-labeling with [α-³²]nucleoside triphosphates and separation by gel electrophoresis. Mitochondrial DNA from 12 populations of Meloidogyne species was digested with 12 restriction enzymes producing more than 60 restriction fragments for each species. The mitochondrial genome of M. arenaria is the most genetically distinct of the four species compared. M. arenaria shows approximately 2.1-3.1% nucleotide sequence divergence from the mitochondrial genomes of M. javanica, M. incognita, and M. hapla. Among the latter three species, interspecific estimates of sequence divergence range from 0.7 to 2.3%. Relatively high intraspecific variation in mitochondrial restriction fragment patterns was observed in M. hapla. Intraspecific variation in M. incognita resulted in sequence divergence estimates of 0.5-1.0%. Such polymorphisms can serve as genetic markers for discerning mitochondrial DNA genotypes in nematode populations in the same way that allozymes have been used to discern nuclear DNA genotypes.     

The Identity of Specimens of the Anastrepha fraterculus Complex (Diptera, Tephritidae) with Atypical Aculeus Tip

Several specimens collected in Paraguay along with Anastrepha fraterculus (sensu lato) have an aculeus tip similar to species from the fraterculus complex, but the teeth of the aculeus of these specimens are poorly defined. As Anastrepha species identification is based mostly on subtle differences in the aculeus tip, we studied these specimens with atypical aculeus tips (with poorly defined teeth) that slightly differs from the aculeus tip of species of the fraterculus complex (with well-developed blunt teeth), to determine if this is due to intraspecific variation or if it can characterize a full species. The Paraguayan specimens were separated in six groups under stereomicroscope according to variation in their aculeus tip. Specimens within each group were studied by means of morphometrics (traditional and geometric) and gene sequence analysis (COI and ITS1). Morphometric analyses were significant, but no clear groups were formed by the discriminant analyses of the aculeus and wing, and the COI and ITS1 sequence analysis clustered specimens with all six aculeus variations. Therefore, the subtle morphological differences observed in the aculeus tip of Paraguayan specimens are intraspecific variations and the Paraguayan specimens were more genetically closely related to Anastrepha sp. 3 from the fraterculus complex.     

Non-transfer of materials between plasmodia of different species of myxomycetes

Summary Under the experimental conditions of this study no transfer of radiophosphorus occurred from one living plasmodium to another when a radioactive plasmodium ofPhysarum polycephalum and a non-radioactive plasmodium ofPhysarum gyrosum orFuligo septica were in intimate contact for 24 hours.This research constitutes a part of a program Experimental Approach to the Taxonomy of the Myxomycetes, supported by National Science Foundation Grant G-6382.     

Assessing Nuclear and Mitochondrial DNA Sequence Variation Within Steinernema (Rhabditida: Steinernematidae)

DNA sequence analysis was used to characterize the nuclear ribosomal DNA ITS1 region and a portion of the COII and 16S rDNA genes of the mitochondrial genome from Steinernema entomopathogenic nematodes. Nuclear ITS1 nucleotide divergence among seven Steinernema spp. ranged from 6 to 22%, and mtDNA divergence among five species ranged from 12 to 20%. No intraspecific variation was observed among three S. feltiae strains. Phylogenetic analysis of both nuclear and mitochondrial DNA sequences confirms the existing morphological relationships of several Steinernema species. Both the rDNA ITS1 and mtDNA sequences were useful for resolving relationships among Steinernema taxa.     

Intraspecific variation of Eolophiodon laboriense,a basal Lophiodontidae (Mammalia,Perissodactyla) from the early Eocene of Southern France

The Lophiodontidae is an emblematic and well-documented Eocene family of perissodactyls from Western Europe. However, after more than a century and a half of studies, lophiodontids still display a complex systematics associated with blurry intraspecific variation and a poorly known early radiation. The locality of La Borie, located near the city of Toulouse, France, has yielded numerous remains of Eolophiodon laboriense. This abundance of remains allows for the first time the study of the intraspecific variation of a basal lophiodontid. The variation has been investigated for dental and cranio-mandibular characters, notably dental polymorphism, size variation and sexual dimorphism. The intraspecific variation of E. laboriense is high with more than 20 polymorphic characters of the dentition, including many additional crests and conules. This dental polymorphism is similar to the one observed in the Bartonian lophiodontid Lophiodon lautricense. E. laboriense also displays an important degree of sexual dimorphism, with male specimens having broader and longer mandibles with larger canines than females. Despite this high intraspecific variation, the low size variation of teeth and the consistency of diagnostic characters strengthen the validity of the genus Eolophiodon and does not impact the previous lophiodontid phylogeny.     

DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes

Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5ʹ (DNA barcode) region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN) system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27%) at COI, while intraspecific distances averaged 0.6% (range = 0.0% - 7.6%). BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci), and one predatory thrips (Aeolothrips intermedius) showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips.     

Molecular identification of seven Sarcocystis species in red deer (Cervus elaphus) from Lithuania

The diaphragm muscles of 77 free-ranging red deer (Cervus elaphus) were examined for Sarcocystis species in Lithuania. Sarcocysts were detected in 61 out of 77 (79.2%) animals investigated. A total of 60 isolated sarcocysts were identified to species using subunit I of cytochrome c oxidase (cox1) sequence analysis. Overall, seven species, S. entzerothi, S. hjorti, S. iberica, S. linearis, S. pilosa, S. truncata and S. venatoria, were confirmed in Lithuanian red deer. Sarcocystis entzerothi was reported in red deer for the first time. Previously this species was shown to use sika deer as well as roe deer and fallow deer as an intermediate host. Based on cox1, with the addition of the current data, altogether 13 Sarcocystis species have so far been shown to use red deer as an intermediate host. Species detected in red deer demonstrated considerable differences in intraspecific genetic variation at cox1. Genetic distances between different samples of S. hjorti and S. linearis were calculated using principal coordinates analysis (PCoA), implying molecular divergence of same Sarcocystis species using different hosts in the same geographical area and divergence of those employing same intermediate host species from different areas.     

Intraspecific sequence variation of chloroplast DNA among the component species of evergreen broad-leaved forests in Japan

For the purpose of phylogeographic study of lucidophyllous (evergreen broad-leaved) forests in Japan, we surveyed intraspecific chloroplast DNA (cpDNA) variation in 41 component species of such forests. Intraspecific cpDNA variations were detected in 14 species. In 15 species and one species group, 16 non-coding cpDNA regions were examined to find intraspecific sequence variation. The extent of variation in these regions was compared. The largest amount of intraspecific variation was detected in the rps16 region. A relatively large amount of intraspecific variation was detected in the petD-rpoA, rpl16, and trnL-F regions. It is suggested that these regions of cpDNA would be useful for detecting intraspecific variation in plant species, and could provide valuable information for various research purposes.     

Investigation of nuclear and mitochondrial DNA polymorphism in closely related species of endemic Baikal sponges

In order to investigate the phylogenetic relationships among endemic Baikal sponges of the family Lubomirskiidae and to clarify their taxonomy, the sequence variation at the silicatein-encoding gene and the mtDNA intergenic region was examined. Phylogenetic analysis of the silicatein α1 gene exonic regions in six freshwater sponge species revealed considerable interspecific variability of this region. The analysis performed did not support the monophyly of the genera Lubomirskia and Baikalospongia. The mtDNA region between the COX2 and ATP6 genes was examined in five species from the Lubomirskiidae family, including multiple samples for analyzing intraspecific variations. According to the data obtained, the genus Baikalospongia was paraphyletic with respect to Lubomirskia, while B. bacilifera and B. recta did not form monophyletic groups. Molecular data indicate that taxonomy of Lubomirskiidae should be revised. It was demonstrated that, in endemic Baikal sponges, the accelerated evolution was accompanied by an increase in the length of the non-coding regions in both mitochondrial and nuclear genes.