Difference in antifungal activity of morphotypes of clavicipitaceous endophytes within and between species

In a previous study, a total of 484 endophytic fungi were isolated and purified from seven populations of Achnatherum sibiricum (L.) Keng collected at six geographical locations in Inner Mongolia, China. Based on growth rates as well as morphological characteristics, the isolates were classified into five morphotypes. Among them, morphotypes A, B and C were ascribed to the same species, Neotyphodium chisosum, based on ITS sequences. Morphotype E was identified as Epichloë amarillans. In the present study, four morphotypes, A, B, C and E, belonging to two species, were chosen for an in vitro pathogen trial. The results showed that both endophyte colonies and endophyte filtrate of all morphotypes could inhibit the mycelia growth and spore germination of the pathogen fungi tested. The magnitude of inhibition varied not only between species, but also among morphotypes of the same species. Overall, the antifungal ability of E. amarillans (morphotype E) was higher than that of N. chisosum. Within N. chisosum, the antifungal ability was highest in morphotype C, followed by morphotype A, and lowest in morphotype B. This variability suggests that different morphotypes might represent different genotypes of endophyte. The effect of endophyte infection on the host grass should be examined not only on the species level but also on the morphotype level to determine the possible interactions.     

Genetic and morphologic variability of annual glassworts (Salicornia L.) from the Gulf of Trieste (Northern Adriatic)

The genetic variability of four pre-determined morphotypes of Salicornia (S. patula, S. emerici, S. veneta and the “saline type”) from 10 locations on the Gulf of Trieste coast were studied by means of ploidy level estimation using flow cytometry and by molecular DNA analysis of ITS regions of nrDNA and cpDNA. Two groups, the diploids and tetraploids, with matching nrDNA sequences, were recognized. Two types of cpDNA emerged among the diploids; one the same as in tetraploids. This incongruence between nrDNA and cpDNA sequences indicates a hybridization with tetraploid maternal progenitors and demonstrates the evidence for reticulate evolution. The morphometry, based on generative morphological traits, did not clearly separate the four morphotypes. However, the most important characters—length of the middle fertile segment, length of the lateral flower, width of the scarious margin of the fertile segment in the floral region, conform to two genetically recognized types: diploid S. patula and the widely distributed tetraploid S. emerici, also comprising the “saline type” and morphotype, known as a charismatic endemic S. veneta, a flagship species for nature conservation. Other discriminative traits for diploid and tetraploid morphotypes are parameters of the flowers (comparison of length of the central vs. lateral flower) and stomatal index. The determination key is also given. The tetraploid S. emerici is by far the most common species of annual glassworts in the area, occupying more extreme habitats than a diploid S. patula, which mostly forms monodominate stands.     

Raphidiopsis mediterranea Skuja represents non-heterocytous life-cycle stages of Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju in Lake Kastoria (Greece), its type locality: Evidence by morphological and phylogenetic analysis

The taxonomical relationship of Cylindrospermopsis raciborskii and Raphidiopsis mediterranea was studied by morphological and 16S rRNA gene diversity analyses of natural populations from Lake Kastoria, Greece. Samples were obtained during a bloom (23,830 trichomes mL⁻¹) in August 2003. A high diversity of apical cell, trichome, heterocyte and akinete morphology, trichome fragmentation and reproduction was observed. Trichomes were grouped into three dominant morphotypes: the typical and the non-heterocytous morphotype of C. raciborskii and the typical morphotype of R. mediterranea. A morphometric comparison of the dominant morphotypes showed significant differences in mean values of cell and trichome sizes despite the high overlap in the range of the respective size values. Additionally, two new morphotypes representing developmental stages of the species are described while a new mode of reproduction involving a structurally distinct reproductive cell is described for the first time in planktic Nostocales. A putative life-cycle, common for C. raciborskii and R. mediterranea is proposed revealing that trichome reproduction of R. mediterranea gives rise both to R. mediterranea and C. raciborskii non-heterocytous morphotypes. The phylogenetic analysis of partial 16S rRNA gene (ca. 920 bp) of the co-existing Cylindrospermopsis and Raphidiopsis morphotypes revealed only one phylotype which showed 99.54% similarity to R. mediterranea HB2 (China) and 99.19% similarity to C. raciborskii form 1 (Australia). We propose that all morphotypes comprised stages of the life cycle of C. raciborkii whereas R. mediterranea from Lake Kastoria (its type locality) represents non-heterocytous stages of Cylindrospermopsis complex life cycle.     

Genetic divergence between and within two color morphotypes of <Emphasis Type="Italic">Parapercis sexfasciata</Emphasis> (Perciformes: Pinguipedidae) from Tosa Bay,southern Japan

The anterior half of the mitochondrial DNA control region (mtCR) sequence (ca. 400 base pairs) was compared between two color morphotypes (A, B) of Parapercis sexfasciata from Tosa Bay, southern Japan, using 16 and 21 specimens, respectively. Intramorphotypic mtCR divergences were only 0.0–0.5% and 1.0–2.5% for morphotypes A and B, respectively. In contrast, intermorphotypic mtCR divergence was much greater, 12.7–14.0%. Furthermore, phylogenetic analysis using a neighbor-joining algorithm, with P. multifasciata as an outgroup, showed that each morphotype was reciprocally monophyletic. These results and the distinct coloration and overlapping distribution indicate that the two color morphotypes of P. sexfasciata represent two distinct species. Mismatch distribution analysis suggested that both morphotypes had undergone population expansion; however, estimates of initial population sizes and mutational timescales suggested that morphotype B comprises historically larger and older populations than morphotype A.     

Morphological differences among three color morphotypes of Sebastes inermis (Scorpaenidae)

 Morphological differences among three color morphotypes occurring almost sympatrically (A, reddish type; B, blackish type; C, brownish type) of Sebastes inermis are clarified. Twenty-six morphometric characters and seven meristic characters were examined on about 20 specimens of each morphotype, 15 of the morphometric characters differing significantly. Principal component analysis (PCA) of the morphometric characters showed morphotype C to differ from the other two morphotypes. Canonical discriminant analysis (CDA) of the morphometric characters showed clear separation of each morphotype. Four meristic characters also differed significantly. Modes of pectoral fin ray counts of morphotypes A, B, and C were 15, 16, and 17, respectively, and the modes of anal fin ray counts of morphotypes B and C were 7 and 8, respectively. Many individuals of morphotype A had seven or eight anal fin rays. These differences suggested that the three morphotypes may be reproductively isolated from each other. Received: October 15, 2001 / Revised: March 14, 2002 / Accepted: April 9, 2002     

A GENETIC MARKER TO SEPARATE EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) MORPHOTYPES1

Emiliania huxleyi (Lohm.) Hay and Mohler is a ubiquitous unicellular marine alga surrounded by an elaborate covering of calcite platelets called coccoliths. It is an important primary producer involved in oceanic biogeochemistry and climate regulation. Currently, E. huxleyi is separated into five morphotypes based on morphometric, physiological, biochemical, and immunological differences. However, a genetic marker has yet to be found to characterize these morphotypes. With the use of sequence analysis and denaturing gradient gel electrophoresis, we discovered a genetic marker that correlates significantly with the separation of the most widely recognized A and B morphotypes. Furthermore, we reveal that the A morphotype is composed of a number of distinct genotypes. This marker lies within the 3′ untranslated region of a coccolith associated protein mRNA, which is implicated in regulating coccolith calcification. Consequently, we tentatively termed this marker the coccolith morphology motif.     

North American black-fruited hawthorns. I. Variation in floral construction,breeding system correlates,and their possible evolutionary significance inCrataegus sect.Douglasii London

Differences in the numbers of stamens and styles per flower are conspicuous features of variation in North American hawthorns (Crataegus L.). Variation in stamen number between individuals is discontinuous, with modes of approximately 20 and 10 (or fewer). In North American black-fruited sectionDouglasii Loudon the 10-stamen morphotype is exclusively polyploid, whereas the 20-stamen morphotype comprises both diploids and polyploids. Polyploidy is associated with apospory, self-fertility, and varying degrees of pollen sterility. Variation in stamen number may also be correlated with variation in distribution, phenology, leaf shape, and other features of floral morphology, leading to recognition of taxa at the specific or infraspecific level. Comparable variation in stamen number is virtually unknown in Eurasian hawthorns, as in the majority of flowering plants. In sectionDouglasii stamen number morphotypes have been recognized as either varieties or species; although correlations between stamen number and other features are not as straightforward as was previously surmised, the higher rank appears to be appropriate. These data on breeding system and morphological variation may be explainable in terms of hypotheses linking gametophytic apomixis, polyploidization, and optimal strategies for pollen dispersal.     

Echinochara triplicata sp. nov. (Clavatoraceae,fossil Charophyta) from the Lower Albian of Tunisia

Echinochara Peck, 1957 is a poorly known charophyte genus from the Upper Jurassic and Lower Cretaceous of the United States and Europe. Its fossil record is currently limited to two species, Echinochara spinosa Peck, 1957 and Echinochara peckii (Mädler, 1952) nov. comb. Grambast, 1956 emend. Schudack, 1993, which have obscure phylogenetic relationships. A third species of this genus, Echinochara triplicata nov. sp., is described here from the Lower Albian of Jebel Koumine (Central Tunisia) and is hypothesized to derive from the Barremian-Aptian morphotypes of Echinochara peckii. The new species represents the first record of the genus in Africa and its more recent record worldwide. It occurs along with the clavatoraceans: Clavator harrisii zavialensis and Atopochara trivolvis trivolvis and provides an additional tool for the biostratigraphic characterization of non-marine Albian of Europe and North Africa.     

Taxonomie der gattungchiropteridium gocht (dinophyceae) im rupelium des mainzer beckens (mitteloligozän,SW-Deutschland)

Five morphotypes of the dinoflagellate cyst genusChiropteridium Gocht from Middle Oligocene sediments in the Mainz Basin (SW-Germany) are described. Two of the morphotypes are grouped with the speciesChiropteridium aspinatum and C.lobospmosum. For another morphotype the new speciesChiropteridium brevifolium is proposed.     

Simple Sequence Repeat Markers Distinguish among Morphotypes of Sphaeropsis sapinea

Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that can cause disease following predisposition of trees by biotic or abiotic stresses. Four morphotypes of S. sapinea have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pines in the Southern Hemisphere. The aim of this study was to develop robust polymorphic markers that could be used in both taxonomic and population studies. Inter-short-sequence-repeat primers containing microsatellite sequences and degenerate anchors at the 5′ end were used to target microsatellite-rich areas in an S. sapinea isolate. PCR amplification using an annealing temperature of 49°C resulted in profiles containing 5 to 10 bands. These bands were cloned and sequenced, and new short-sequence-repeat (SSR) primer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of S. sapinea representing different morphotypes as well as on 2 isolates of the closely related species Botryosphaeria obtusa. The putative I morphotype was found to be identical to B. obtusa. Otherwise, the markers clearly distinguished the remaining three morphotypes and, furthermore, showed that the C morphotype was more closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locus showed that the most polymorphic markers had mutations within a microsatellite sequence.     

NEW EVIDENCE FOR MORPHOLOGICAL AND GENETIC VARIATION IN THE COSMOPOLITAN COCCOLITHOPHORE EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) FROM THE COX1b‐ATP4 GENES1

Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler is a cosmopolitan coccolithophore occurring from tropical to subpolar waters and exhibiting variations in morphology of coccoliths possibly related to environmental conditions. We examined morphological characters of coccoliths and partial mitochondrial sequences of the cytochrome oxidase 1b (cox1b) through adenosine triphosphate synthase 4 (atp4) genes of 39 clonal E. huxleyi strains from the Atlantic and Pacific Oceans, Mediterranean Sea, and their adjacent seas. Based on the morphological study of culture strains by SEM, Type O, a new morphotype characterized by coccoliths with an open central area, was separated from existing morphotypes A, B, B/C, C, R, and var. corona, characterized by coccoliths with central area elements. Molecular phylogenetic studies revealed that E. huxleyi consists of at least two mitochondrial sequence groups with different temperature preferences/tolerances: a cool‐water group occurring in subarctic North Atlantic and Pacific and a warm‐water group occurring in the subtropical Atlantic and Pacific and in the Mediterranean Sea.     

Mitochondrial phylogeography,contact zones and taxonomy of grass snakes (Natrix natrix,N. megalocephala)

Grass snakes (Natrix natrix) represent one of the most widely distributed snake species of the Palaearctic region, ranging from the North African Maghreb region and the Iberian Peninsula through most of Europe and western Asia eastward to the region of Lake Baikal in Central Asia. Within N. natrix, up to 14 distinct subspecies are regarded as valid. In addition, some authors recognize big‐headed grass snakes from western Transcaucasia as a distinct species, N. megalocephala. Based on phylogenetic analyses of a 1984‐bp‐long alignment of mtDNA sequences (ND4+tRNAs, cyt b) of 410 grass snakes, a nearly range‐wide phylogeography is presented for both species. Within N. natrix, 16 terminal mitochondrial clades were identified, most of which conflict with morphologically defined subspecies. These 16 clades correspond to three more inclusive clades from (i) the Iberian Peninsula plus North Africa, (ii) East Europe and Asia and (iii) West Europe including Corso‐Sardinia, the Apennine Peninsula and Sicily. Hypotheses regarding glacial refugia and postglacial range expansions are presented. Refugia were most likely located in each of the southern European peninsulas, Corso‐Sardinia, North Africa, Anatolia and the neighbouring Near and Middle East, where the greatest extant genetic diversity occurs. Multiple distinct microrefugia are inferred for continental Italy plus Sicily, the Balkan Peninsula, Anatolia and the Near and Middle East. Holocene range expansions led to the colonization of more northerly regions and the formation of secondary contact zones. Western Europe was invaded from a refuge within southern France, while Central Europe was reached by two distinct range expansions from the Balkan Peninsula. In Central Europe, there are two contact zones of three distinct mitochondrial clades, and one of these contact zones was theretofore completely unknown. Another contact zone is hypothesized for Eastern Europe, which was colonized, like north‐western Asia, from the Caucasus region. Further contact zones were identified for southern Italy, the Balkans and Transcaucasia. In agreement with previous studies using morphological characters and allozymes, there is no evidence for the distinctiveness of N. megalocephala. Therefore, N. megalocephala is synonymized with N. natrix.     

Merychippus (Mammalia, Perissodactyla, Equidae) from the Middle Miocene of state of Oaxaca, southeastern Mexico

Mexican material referable to Merychippus from two localities in eastern Oaxaca was described first nearly 50 years ago. Subsequent work there and in Central Oaxaca, spanning some 30 years, has allowed to establish the detail stratigraphy in both regions, and assembled a collection of merychippine material from the Matatlán (Central Oaxaca) and El Camarón (eastern Oaxaca) Formations, both K-Ar dated ~15 Ma (late early Barstovian). Detailed taxonomic analysis of this collection indicate the presence of two subhypsodont horse species referable to “Merychippus” cf. “M.” primus and “M.” cf. “M.” sejunctus in both regions. These records document the coexistence in tropical southern North America of basal and hipparionine affinity merychippine grade species, and provide a glimpse in to the diversity of subhypsodont equids in this region.     

Biostratigraphical value of some Strunian (Devonian, uppermost Famennian) Productidina, Rhynchonellida, Spiriferida, Spiriferinida brachiopods

The space of Devonian time considered in this paper corresponds to the uppermost part of the Famennian or the latest Famennian as a fourfold subdivision of the stage, usually called by many authors Strunian in neritic facies and Wocklumeria Stufe or Wocklumian in pelagic facies. Here, we examine the biostratigraphical value of certain brachiopod genera and species as bio-markers of the uppermost Famennian throughout the world (Europe, the former USSR, Middle East, Asia, North America, North Africa, and Australia). We have focused our study for species which stratigraphical range has been firmly established in correlation with the conodont biozones (Upper expansa, Early, Middle and Late praesulcata), the ammonoid biozones (do VI = “Wocklumeria Stufe”), or the foraminifera biozones (kobeitusana Zone). Other data for which the stratigraphical range is not based on standard biozonation are provided in the annex. The brachiopod bio-markers discussed in this paper belong to 14 Productidina genera (Acanthatia, Araksalosia, Ericiata, Hamlingella, Mesoplica, Nigerinoplica, Orbinaria, Ovatia, Rugauris, Semiproductus, Sentosia, Spinocarinifera, Steinhagella, Whidbornella), 7 Rhynchonellida genera (Araratella, Centrorhynchus, Hadyrhyncha, Megalopterorhynchus, Novaplatirostrum, Rozmanaria, Tchanakhtchirostrum), 13 Spiriferida genera (Brachythyris, Cyrtospirifer, Dichospirifer, Eochoristites, Imbrexia, Parallelora, Prospira, Rigauxia, Sphenospira, Tenisia, Toryniferella, Tylothyris, Voiseyella) and one Spiriferinida genus (Syringothyris). Other orders have not been studied in this paper. The main features of the uppermost Famennian brachiopod taxa represented in this paper are commented. All these taxa are listed as completely as possible throughout the world (with complements in the annex). A quick summary of the geographic distribution of the more represented taxa is given in conclusion.     

Size analyses of the coccolith species Biscutum constans and Watznaueria barnesiae from the Late Albian “Niveau Breistroffer” (SE France): taxonomic and palaeoecological implications

The size variability of the coccolith species Biscutum constans and Watznaueria barnesiae has been studied in 50 samples of the Late Albian “Niveau Breistroffer” black shales (Col de Palluel section, SE France). For each species, length and width of the total coccolith and the central unit have been measured in 60 specimens per sample. In addition, ellipticity and central unit length/total coccolith length ratio have been calculated. This study aims to improve our understanding of the taxonomic concepts of B. constans and W. barnesiae, and to test whether the size changes correspond to palaeoceanographic changes interpreted from other proxies. Two morphotypes (varieties) were differentiated for each of the two taxa studied. B. constans includes morphotypes with a narrow (B. constans var. constans) and a wide central unit (B. constans var. ellipticum). Between these two morphotypes no significant size differentiation has been observed. They seem to represent end members of a size continuum, relating to both the total coccolith- and central unit size distribution. The two morphotypes of W. barnesiae differ only in the absence (W. barnesiae var. barnesiae) and the presence of a narrow central opening (W. barnesiae var. fossacincta). No significant size differences have been observed between these two morphotypes, and both show similar distributions of the measured characteristics. We recommend that the studied morphotypes of both B. constans and W. barnesiae should not be assigned to different morphospecies. Throughout the studied black shale succession the measured parameters show mostly statistically insignificant, short-term fluctuations, but significant long-term trends have been observed for B. constans. These show a trend to smaller forms with a narrower central unit in the upper part of the succession. This change coincides with a cooling trend, as indicated by a nannofossil based temperature index. Therefore the two morphotypes of B. constans are interpreted to represent ecophenotypic varieties rather than two different morphospecies. No clear relationship has been recognized between the size of W. barnesiae and the palaeoenvironmental conditions.     

Direct Electron Microscopy Study on the Morphological Diversity of Bacteriophage Populations in Lake Plußsee

Direct electron microscopy of bacteriophages adsorbed to a carbon film without prior enrichment by specific host strains or concentration by physical or chemical methods was used to study the morphological diversity of natural bacteriophage assemblages in a North German lake. All samples contained a mixture of morphologically different tailed viruses, which were regarded as bacteriophages. Most of them had isometric heads and long noncontractile tails, belonging to morphotype B1 (Siphoviridae). In addition, members of morphotypes A1 (Myoviridae), B2 (Siphoviridae with elongated heads), and C1 (Podoviridae) were present in lower numbers. Only one cubic virus was detected, while no filamentous or pleomorphic phages were found. Up to 11 different phages per sample, and a total of 39 phages when all samples were considered together, could be distinguished by morphological criteria. The total number of phages was estimated to be on the order of 10⁸/ml.     

Courtship in Two Morphotypes of the <Emphasis Type="Italic">Anastrepha fraterculus</Emphasis> (Diptera: Tephritidae) Cryptic Species Complex and their Implications for Understanding Mate Recognition

Ritualized courtship behaviors are used to recognize potential mates and behavioral patterns are inevitably different among populations that demonstrate reproductive incompatibility. We characterized and compared the courtship behaviors of two morphotypes of the cryptic species complex Anastrepha fraterculus: Brazil-1 morphotype and Brazil-3 morphotype. Courtship behaviors were filmed to analyze the behavioral sequences of these two morphotypes during homotypic crossings. The behavioral units Alignment (AL) and Abdominal movements (AB and AB-call) were newly recognized in the courtship ethogram of Anastrepha fraterculus males. The two morphotypes show distinct behavioral sequences leading up to copulation. Some behaviors were repeated frequently during the courtship process, while others were more restricted to the final moments of courtship. The three behavioral units that contributed most to copulation success were Contact, Alignment, and Arrowhead 1 in the Brazil-1 morphotype and Alignment, Arrowhead 1, and Fanning in the Brazil-3 morphotype. Some behavioral routines differed across the two morphotypes. Significant differences were also noted between the frequencies of the behavioral units displayed during courtship in the two morphotypes. The relationships between the pre-zygotic incompatibilities of the Brazil-1 and Brazil-3 morphotypes and the differences between the courtship behaviors of their males are discussed. Our results indicate that behavioral isolation is involved in the process of pre-zygotic reproductive isolation of Brazil-1 and Brazil-3 morphotypes.     

The b chromosome polymorphism in Armenian and Turkey populations of the grasshopper Eyprepocnemis plorans plorans (Charpentier) (Orthoptera, Acrididae, Eyprepocnemidinae)

Karyotype analysis of the grasshopper Eyprepocnemis plorans samples derived from Armenian and Turkey populations was performed using C-banding technique. Chromosome polymorphism was associated only with B chromosomes that were revealed in all studied populations. Six new B chromosome morphotypes were described. Four morphotypes were found only in Armenian populations. One morphotype was revealed only in Turkey populations. One B chromosome morphotype was present in Armenian and Turkey populations. B chromosomes derived from Asian populations consisted mostly from C-positive regions. In some of the B chromosomes small C-negative regions were also registered. Morphotypes of the B chromosomes derived from Armenian and Turkey populations drastically differed from the B chromosomes described in the Iberian Peninsula and North African populations. In contrast to the B chromosomes from Armenian and Turkey populations the B chromosomes from Spain populations contained C-positive and C-negative regions alternated in their arms.