Oospore Wall Research Articles

Morphology and Molecular Phylogeny of Chara altaica (Charales, Charophyceae), a Monoecious Species of the Section Desvauxia

Chara altaica A. Braun (Charales, Charophyceae), a monoecious species of the section Desvauxia, was newly found in Japan and studied by light and scanning electron microscopy (SEM) as well as gene sequence analysis of the large subunit of Rubisco (rbcL). Our first SEM observations of C. altaica oospores revealed that small papillae were scattered on the fossa wall, an SEM oospore wall ornamentation that is essentially different from that of the monoecious species C. (sect. Desvauxia) evoluta T. F. Allen. Phylogenetic analysis demonstrated that C. altaica and 10 European samples of the dioecious species C. (sect. Desvauxia) canescens Desvaux & Loiseleur-Deslongchamps formed a robust monophyletic group, in which the rbcL gene sequence from C. altaica is identical to those from 4 parthenogenetic samples, but different from 6 other bisexual and parthenogenetic samples of C. canescens.

TAXONOMIC REEXAMINATION OF CHARA GLOBULARIS (CHARALES, CHAROPHYCEAE) FROM JAPAN BASED ON OOSPORE MORPHOLOGY AND rbcL GENE SEQUENCES, AND THE DESCRIPTION OF C. LEPTOSPORA SP. NOV.(1).

Chara globularis Thuillier (=f. globularis sensu R. D. Wood) is a widespread species of the genus and inhabits fresh- and brackish-water environments. In an attempt to reexamine the taxonomic status of C.globularis collected from Japan, we reassessed vegetative and oospore morphology of Japanese material and herbarium specimens originating from Europe (including the type specimen) and conducted molecular phylogenetic analyses based on rbcL gene sequences. Although the other vegetative morphologies were consistent with the description of C.globularis f. globularis sensu R. D. Wood, we identified two types of branchlets within the Japanese materials: one has elongate end segments (EL type), and the other has short end segments (SH type) corresponding to the type material. Moreover, the oospore wall of the EL type was different from that in the SH type. The oospores of the EL type were dark brown to reddish brown and had a spongy pattern with the pusticular elevations on the fossa wall, whereas the fossa wall of the SH type was black with a granulate to papillate or fine pusticular pattern. In addition, our sequence data demonstrated that these two types are separated phylogenetically from each other. Therefore, we describe the EL type as a new species, C.leptospora sp. nov.

Phytophthora cactorum causing bleeding canker of common beech, horse chestnut, and white poplar in the Czech Republic

In 2007, declining mature trees of common beech (Fagus sylvatica), horse chestnut (Aesculus hippocastanum), and white poplar (Populus alba) with bleeding cankers were found in several locations in the Central Bohemia region. The morphological and cultural characteristics of isolates from diseased tissues were consistent with those of Phytophthora cactorum (Erwin & Ribeiro, 1996). The colonies were stellate to rosaceous, appressed with slight aerial mycelium on V8 agar. Radial growth was 8–14 mm day−1 at 20°C. Optimum growth temperature was 23–25°C, minimum 3–8°C and maximum 30–33°C. Strains were homothallic, produced abundant terminal, smooth-walled, spherical oogonia (20–35 µm in diameter). Oospores were plerotic or nearly plerotic, 22–31 µm in diameter and the oospore wall 1·0–2·2 µm thick. Antheridia were paragynous, but amphigynous antheridia also occurred in up to a quarter of cases. Sporangiophores were sympodial, sporangia ellipsoidal, ovoid to subglobose, papillate and measured 17–38 × 14–30 µm (L:B ratio 1·05–1·80). Comparison of DNA sequences of ITS region of representative isolates from all hosts (GenBank Acc. Nos. EU562207–EU562209) gave 99% identity to P. cactorum. Isolates were deposited at Culture Collection of Fungi, Prague under CCF Nos. 3757, 3758, 3762. The pathogenicity was tested using 20, 1-year-old saplings of all three hosts. Wounds (5 mm diam.) on stems of ten saplings of each host were artificially inoculated by plugs of V8 agar from the actively growing colony margins of isolates and the wounds were sealed by Parafilm. The characteristic stem necroses developed after 1–2 weeks on all plants. Stems of many beech and horse chestnut saplings were girdled in 2 to 3 months and in c. 33% of poplars, with the majority of poplar saplings forming callus and surviving the infection. The pathogen was successfully reisolated from plants showing symptoms. The control plants inoculated in the same way with sterile agar plugs remained healthy. Phytophthora cactorum is well known pathogen of beech and horse chestnut (Brasier & Strouts, 1976; Jung et al., 2005), but not reported on any poplar species. This is the first report of P. cactorum causing bleeding canker of common beech and horse chestnut in the Czech Republic and the first record of it on white poplar. The work was supported by project no. QH71273 of the Ministry of Agriculture of the Czech Republic.

Oospore variation in Nitella gracilis and Nitella mucronata (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.

Review: Taxonomy of Nitella (Charales, Charophyceae) based on comparative morphology of oospores and multiple DNA marker phylogeny using cultured material

SUMMARYThe genus Nitella is the largest group in the Charales and has the highest diversity of vegetative and oospore morphologies. In his worldwide monograph on the Charales, R. D. Wood characterized the sections and species of Nitella mainly on the basis of vegetative morphology and treated oospore wall morphology as diagnostic at the infraspecific level. Therefore, many species of Nitella exhibiting distinct external morphology of the oospore wall (EMOW) were reduced to infraspecific rank and only 53 of 204 species previously described were recognized within Nitella. However, recent morphological and molecular phylogenetic studies have demonstrated the phylogenetic validity of using EMOW for diagnosing some species of Nitella. More recently, a scanning electron microscopy (SEM) study of internal morphology of the oospore wall (IMOW) and multiple DNA marker analyses using both nuclear and chloroplast gene sequences were conducted to improve our understanding of the taxonomy of Nitella at the species level, on the basis of cultured material of a large number of species. Multiple DNA marker analyses resolved detailed and robust phylogenetic relationships within the genus and demonstrated the taxonomic and phylogenetic significance of IMOW. In addition, they supported the taxonomic decisions based on differences in oospore morphology, suggesting that an integrated approach, involving both SEM studies of the EMOW and IMOW and multiple DNA marker analyses, is appropriate to address problems at lower taxonomic levels within the genus, as well as to construct a natural taxonomic system for the genus. In this paper, recent morphological and molecular phylogenetic studies are reviewed and recent improvements in taxonomy of Nitella are summarized. Moreover, the evolution of morphological features and phylogenetic relationships within Nitella are discussed, focusing especially on oospore morphology.

Examinations of nuclear behaviour during sexual cycle in the diplontic oomycete Phytophthora palmivora (Butl.) Butl.

Nuclear behaviour during gametangial- and oospore development in Phytophthora palmivora was studied by light-, fluorescence-, and electron microscopy. After contact between oogonial and anther-idial initials, the antheridial initial is penetrated by the oogonial initial. The latter expands and nuclei and cytoplasm stream into it. After the delimitation of gametangia, a number of nuclei degenerate, the rest proceed through two gametic meiotic divisions both in the antheridium and the oogonium. Three of the meiotic products from each nucleus degenerate. The oogonium is penetrated by the antheridial fertilization tube and nuclear fusion occurs. The oosphere forms, then the periplasm degenerates, and the oospore wall builds up. Upon release of zoospores diploid mycelia are produced.

Oospore dimensions and wall ornamentation in Chara braunii

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.

Evidence for uncharted biodiversity in the Albugo candida complex, with the description of a new species

During the past two years the integrity of Albugo candida as the only species of Albugo parasitic to Brassicaceae has been challenged. The existence of two distinct species parasitic to Brassicaceae has been confirmed, to which a third species was added. For the purpose of further exploring the diversity of the A. candida complex, eight Albugo specimens on Draba lasiocarpa, D. nemorosa, and D. verna ( Brassicaceae) were morphologically and molecularly compared with other Albugo species. Based on sequence comparisons and thorough investigation of the characteristics of the oospores, especially surface ornamentation, Albugo voglmayrii sp. nov., parasitic to Draba nemorosa, is described from five specimens collected in Korea and China. It differs from the previously described species, A. candida, A. koreana, and A. lepidii, by its oospore wall ornamentation. The morphological discrepancy is supported by high genetic distances to other species of Albugo in ITS rDNA and cox2 mtDNA. Albugo specimens from D. lasiocarpa and D. verna were grouped with A. candida, revealing that two distinct species may cause white blister rust on the genus Draba. Therefore, the paradigms that: (1) there is only a single species parasitic to Brassicaceae, that (2) oospore morphology is useful only for distinguishing between largely unrelated species; and (3) in general only one species of Albugo may occur on a single host genus need to be discarded.

A new species ofAlbugoonPterostylis(Orchidaceae) from Australia: confirmation of the genusAlbugoon a monocotyledonous host

Albugo macalpineana sp. nov., causing white blister disease of terrestrial orchids in the genus Pterostylis, is described from Victorian and South Australian specimens more than 50 years old. Subglobose sporangia have uniformly thickened walls, and oospore walls show a raised cerebriform ridge pattern. This rare fungus has not been collected since 1954. The work reported confirms earlier entries in South Australian and Victorian plant disease lists of the occurrence of the genus Albugo on a monocotyledonous host.

Concerted evolution of a tandemly arrayed family of mating-specific genes in Phytophthora analyzed through inter- and intraspecific comparisons

Multigene families are features of most eukaryotic genomes, which evolve through a variety of mechanisms. This study describes the structure, expression, and evolution of a novel family in the oomycete Phytophthora. In the heterothallic species P. infestans, M96 is expressed specifically during sexual sporogenesis, and encodes a low-complexity extracellular protein that may be a component of oospore walls. Intriguingly, M96 exists in P. infestans as 22 relatively homogeneous loci tandemly repeated at a single site, which is partitioned by inversions and retroelements into subclusters exhibiting semi-independent evolution. M96 relatives were detected in other heterothallic and homothallic oomycetes including species closely (P. mirabilis, P. phaseoli) or distantly (P. ramorum, P. sojae) related to P. infestans. Those M96 relatives also exhibit oosporogenesis-specific expression and are arrayed multigene families. Nucleotide changes and repeat expansion diversify M96 in each species, however, paralogues are more related than orthologues. Concerted evolution through gene conversion and not strong purifying selection appears to be the major contributor to intraspecific homogenization. Divergence and concerted evolution was also detected between isolates of P. infestans. The divergence of M96 proteins between P. infestans, P. ramorum, and P. sojae exceeds that of typical proteins, reflecting trends in reproductive proteins from other kingdoms.

Taxonomic re‐examination of 17 species of Nitella subgenus tieffallenia (Charales, Charophyceae) based on internal morphology of the oospore wall and multiple DNA marker sequences (Vol. 41:195–211)

Taxonomic re‐examination of 17 species of <i>Nitella</i> subgenus <i>tieffallenia</i> (Charales, Charophyceae) based on internal morphology of the oospore wall and multiple DNA marker sequences (Vol. 41:195–211)

First Report of Downy Mildew of Opium Poppy Caused by Peronospora arborescens in Spain.

Opium poppy (Papaver somniferum) is an economically important pharmaceutical crop in Spain with approximately 7,400 ha cultivated annually. In the spring of 2004, severe attacks by a new foliar disease were observed approximately 500 km apart in commercial opium poppy fields in the Castilla-La Mancha and Andalusia regions of central and southern Spain, respectively. The incidence of affected fields ranged from 40 to 50%, and incidence of diseased plants ranged from 20 to 30%. Initial disease symptoms included irregularly shaped, chlorotic-to-light yellow leaf lesions (ranging in size from 0.5 to 4 cm). Affected tissues curled, thickened, and became deformed and necrotic as disease developed. Lesions expanded in size and often coalesced, eventually giving rise to large necrotic areas in leaves or death of entire leaves. In wet weather or conditions of high relative humidity, a dense felt of sporangiophores with sporangia was produced on the abaxial leaf surface and occasionally on the adaxial surface. Microscopic observations revealed sporangiophores branching dichotomically at least four to six times, ending with sterigmata bearing single sporangia. Sporangia were hyaline, elliptical to spherical in shape, and measured 18 to 24 × 14 to 18 μm (average 19 ± 1.2 × 15 ± 1.6 μm). Occasionally, oospores formed in necrotic leaf tissues. Oospores were dark brown (the surface was irregularly ridged) and measured 36 to 46 μm in diameter (average 39 ± 4.4 μm). The oospore wall was 3 to 11 μm thick. On the basis of the observed morphological features of six symptomatic plant samples from fields at Castilla-La Mancha and Andalusia regions, we identified the pathogen as Peronospora arborescens (1). Pathogenicity was confirmed by inoculating 4- to 6-week-old opium poppy plants (cv. nigrum) with an isolate collected from a field in Ecija, Andalusia. Seed of test plants was surface disinfested and germinated under sterile conditions. Plants were sprayed with a suspension of 1 to 5 × 105 sporangia per ml in sterile distilled water. Plants sprayed with sterile water served as controls. There were five replicate plants per treatment. Plants were enclosed in sealed plastic bags and kept in the dark for 24 h. This was followed by incubation in a growth chamber at 21°C, 60 to 90% relative humidity, and a 12-h photoperiod (fluorescent light: 360 μE·m-2·s-1). After 5 to 7 days, typical downy mildew symptoms developed in inoculated plants. All control plants remained symptomless. Sporulation by the pathogen on symptomatic leaves occurred when affected plants were sprayed with water, enclosed in sealed plastic bags, and incubated at 21°C in the dark for 24 h. To our knowledge, this is the first report of P. arborescens infecting opium poppy in Spain. Infestations of poppy weeds (Papaver rhoeas) and wild Papaver somniferum were also observed in affected opium poppy fields, which may bear importance in the epidemiology of the disease as alternative hosts for inoculum increase and survival of P. arborescens under field conditions.

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

In an attempt to reconstruct the natural taxonomic system forNitella, 17 species ofNitellasubgenusTieffalleniawere 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 subgenusTieffalleniaconsisted 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.japonicaAllen,N.oligospiraA. Braun,N.vieillardiistat. nov.,N.imperialisstat. nov., andN.morongiiAllen) that R. D. Wood had assigned as infraspecific taxa. Moreover, our SEM observations of the IMOW also suggested thatN.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.

Taxonomic significance of micromorphology and dimensions of oospores in the genus Chara L., Charales, Chlorophyta

Oospore wall ornamentation patterns as revealed by scanning electron microscopy (SEM) provide important taxonomic characters in the Charales. The present study illustrates inter- and intra-specific variations in the oospore wall ornamentation of five species of Chara, including eight intra-specific taxa. The type variety and form of C. globularis shows pusticular elevations with pores at the base of the elevation. The variety and form aspera of C. globularis shows a porous oospore wall, and the oospore is provided with a ribbon which bears nearly granular ornamentation. The type form of C. setosa has a granular oospore wall. The three forms of C. braunii show a similar type of ornamentation and support their placement under a single species. Chara corallina has a roughened oospore wall. Oospore dimensions studied at the population level are another feature included in the present study. The oospore dimension data (length, width fossa-breadth, number of striae) at the population level were analyzed statistically, and the results show species-specificity of these parameters. The significance of oospore wall ornamentation and dimensions are discussed simultaneously with morphological features.

Taxonomic re-examination of six species of Nitella (Charales, Charophyceae) from Asia, and phylogenetic relationships within the genus based on rbcL and atpB gene sequences

Six taxa of Nitella (Charales, Charophyceae), collected from Asia, were investigated using light and scanning electron microscopy (SEM) for the oospores, and sequencing of the gene encoding the large subunit of Rubisco (rbcL), in order to improve our understanding of their taxonomic status. Our SEM observations demonstrated that the oospore morphology of four taxa belonging to the subgenus Tieffa1lenia [N. megaspora (J. Groves) Sakayama, comb. nov. (= N. pseudoflabellata f. megaspora), N. tumulosa (= N. furcata f. tumulosa), N. gracillima (= N. gracilis f. gracillima) and N. axilliformis (= N. translucens f. axilliformis)] is distinctly different from that of the species N. pseudoflabellata, N. furcata, N. gracilis and N. translucens, respectively, to which R.D. Wood assigned them as infraspecific taxa. Our rbcL sequence data showed that N. megaspora is separated phylogenetically from N. pseudoflabellata, N. tumulosa from N. furcata and N. axilliformis from N. translucens. In addition, to re-examine the taxonomic system of Nitella proposed by R.D. Wood, who treated oospore wall ornamentations as diagnostic at the infraspecific level, we carried out molecular phylogenetic analyses using the combined sequence data set for the gene encoding the beta subunit of ATP synthase (atpB) and the rbcL gene of these six species, as well as eleven species of Nitella studied previously. The combined sequence data resolved fiverobust clades within the subgenus Tieffallenia that were characterized by differences in oospore wall ornamentation. However, the species and sectional diagnoses of R.D. Wood, such as the form and cell number of dactyls in vegetative thalli, were variable within the clades. Therefore, R.D. Wood's taxonomic system appears unnatural, at least within the subgenus Tieffallenia.

First Report of Stem Rot of Guiana Chestnut (Pachira aquatica) Caused by Pythium splendens.

Guiana chestnut is a perennial tropical plant that has recently become popular as a potted ornamental in Japan. In October 2001, severe stem rot occurred on Guiana chestnut plants grown in a greenhouse in Mie Prefecture, Japan. Water-soaked lesions appeared initially at the base of the stems and enlarged gradually toward the tops of plants. The affected tissues were softened and turned dark brown. Rotting was observed in the vascular bundles with advanced disease development. Globose hyphal swellings were numerous on diseased stems. Sections from diseased stems were cleaned by washing with running tap-water, placed on water agar, and incubated at 25°C. A species of Pythium was identified on the basis of morphological and cultural characteristics (1) and isolated consistently from the rotted stems of diseased plants. All isolates produced abundant hyphal swellings that were globose, smooth, 12 to 39 μm in diameter, mostly terminal, dark colored, and with dense granulated contents. Zoospores were absent. All isolates were of the compatibility '+ type' with production of sexual organs when paired with cultures of the '- type' tester isolate of Pythium splendens Braun (CBS462.48). Oogonia produced by crossings between Guiana chestnut isolates and isolate CBS462.48 were terminal or intercalary, globose, smooth-walled, and 32 to 38 μm in diameter. Antheridia were terminal, one to three per oogonium, sac-like, and diclinous. Oospores were single, aplerotic, globose, and 28 to 32 μm in diameter. The thickness of the oospore wall ranged from 1 to 2 μm. The internal transcribed spacer rDNA sequences of representative isolate OPU591 from Guiana chestnut matched those of CBS462.48 (similarity 99.2%) and have been deposited in GenBank under the Accession No. AY375242. Pathogenicity tests were conducted on potted Guiana chestnut plants (30 cm high and 7 to 10 cm in diameter at base of the stem) using isolate OPU591. A mycelial suspension from one culture, grown at 25°C for 7 days on water agar, was inoculated onto a single plant. Prior to inoculation, a wound (10 mm deep and 30 mm long) was made on the surface at the stem base on five plants. The mycelial suspension was poured onto the base of the stems of five wounded and five nonwounded plants. In addition, five wounded and five nonwounded, noninoculated plants were used as controls. Plants were maintained in a greenhouse for 8 weeks after inoculation. The temperature and relative humidity in the greenhouse ranged from 25 to 30°C and 65 to 75%, respectively. Dark-brown rotting developed on the stems of all wounded, inoculated plants by 20 days after inoculation. P. splendens was isolated from diseased tissues and found to be morphologically identical to the original isolate. This confirmed P. splendens as the causal agent of the disease. Disease did not develop on nonwounded inoculated plants or noninoculated plants. To our knowledge, this is the first report of P. splendens on Guiana chestnut. Potted plants of Guiana chestnut are often injured by frequent transplanting and transferring. Such injuries may have predisposed the plant to infection by P. splendens. Reference: (1) A. J. van der Plaats-Niterink. Page 1 in: Monograph of the Genus Pythium. Studies in Mycology Vol. 21, Centraalbureau Voor Schimmelcultures, Baarn, the Netherlands, 1981.

139 Taxonomic Re‐Examination of Eight Species of Nitella (Charales, Charophyceae) from Asia, And Phylogenetic Relationships Within the Genus Based on rbcL and Atpb Gene Sequences

Eight taxa of Nitella, collected from Asia, were investigated using LM and SEM of the oospores and sequencing of rbcL gene to improve understanding of their taxonomic status. Our SEM observations demonstrated that the oospore morphology of six taxa –N. capitulifera (=N. acuminata f. capitulifera), N. japonica (=N. furcata f. japonica), N. tumulosa (=N. furcata f. tumulosa), N. megaspora comb. nov. (=N. pseudoflabellata f. megaspora), N. gracillima (=N. gracilis f. gracillima), and N. axilliformis (=N. translucens f. axilliformis) – is distinctly different from that of the species to which R.D. Wood assigned them as infraspecific taxa. Our rbcL sequence data showed that N. japonica is separated phylogenetically from N. furcata, N. tumulosa from N. furcata, N. megaspora from N. pseudoflabellata, and N. axilliformis from N. translucens. In addition, to re‐examine the taxonomic system of Nitella proposed by R.D. Wood, who treated oospore wall ornamentations as a diagnosis at the infraspecific level, we carried out molecular phylogenetic analyses using the combined sequence dataset for atpB and rbcL genes of these eight species, as well as twelve species of Nitella previously studied. The combined sequence data resolved five robust clades within the subgenus Tieffallenia that were characterized by differences in oospore wall ornamentation. However, the species and sectional diagnoses of R.D. Wood, such as the form and cell number of dactyls in vegetative thalli, were variable within the clades. Therefore, R.D. Wood's taxonomic system appears unnatural, at least within the subgenus Tieffallenia.

Taxonomic re-examination of Nitella (Charales, Charophyceae) from Japan, based on microscopical studies of oospore wall ornamentation and rbcL gene sequences

Nine taxa of Nitella (Charales, Charophyceae) from Japan, including five Japanese or east Asian endemics, were examined to improve understanding of their taxonomic status. The approaches used were light and scanning electron microscopy (SEM) of the oospores and sequencing of the gene (rbcL) encoding the large subunit of Rubisco. The species delineated were N. pulchella (= N. dualis f. pulchella), N. furcala, N. inversa (= N. furcata f. inversa), N. gracilens (= N. furcata f. gracilens), N. pseudoflabellata, N. hyalina, N. spieiformis (= N. gracilis f. spiciformis), N. moriokae (= N. rigida f. moriokae) and N. axillaris (= N. translucens var. axillaris). SEM observations showed that the oospore morphology of four taxa – N. inversa, N. gracilens, N. spiciformis and N. axillaris – was distinctly different from that of the species (N. furcata, N. furcata, N. gracilis and N. translucens, respectively) to which R.D. Wood assigned them as infraspecific taxa. Furthermore, the rbcL data showed that N. gracilens was separated phylogenetically from N. furcata and N. axillaris from N. translucens. This is the first integration of SEM oospore morphology and molecular phylogenetics in charalean taxonomy, demonstrating the efficiency of both approaches to address problems at lower taxonomic levels.

SEM study of compound oospore wall ornamentation of some members of Charales from Yugoslavia, Croatia and Slovenia

The outermost layer of the oospore wall of the members of Chorales shows various ornamentation patterns, formed due to differential deposition of wall material. These ornamentations are very conservative in nature and used as taxonomic criteria in identifying species and intraspecies taxa in Chorales. In the present investigation, Scanning Electron Microscopy (SEM) was used to study the compound oospore wall ornamentations of twelve species belonging to the genera Chara, Nitella, Lamprothamnium and Lychnothamnus from Yugoslavia, Croatia and Slovenia. The taxonomic identification and systematic position of these taxa are discussed in the light of these characteristic ornamentation patterns revealed by SEM.

Biometry of oospores and intraspecific variation of four Pythium species

Evidence that the biometric characters of the oogonial unit of Pythium are of significant taxonomic value, allowing for a good species separation, has been presented by other workers, based on 40 species, each represented by fairly few isolates. Since broad intraspecific variation and interspecific overlapping of biometric parameters was observed in isolates of four Pythium species from Denmark, it was suspected that a such wide variability would weaken the conclusions obtained by previous biometric approaches. Species to be studied should be represented by higher numbers of isolates to reflect intraspecific variation. In the present biometric study 49 isolates were used to test the robustness of the biometric approach and to examine the taxonomic value of biometric characters from a practical perspective. Good species separation was obtained despite intraspecific variation. The biometric parameter set for the oogonium, consisting of oogonial, oospore and ooplast diameters, supplemented with the thickness of the oospore wall or the protoplast diameter and with derived indices of volume or linear ratios of the measured parameters were all of taxonomic value for the separation of species, as shown from canonical variate plots after linear discriminant analysis. More detailed information on species separation, isolate-species or isolate—isolate associations could be obtained by separation based on the Mahalanobis distance function in the linear discriminant analysis. Both the derived volume and linear indices contributed significantly to species separation and appeared to be equally satisfactory. The substrates used for oogonium production may influence the biometric parameters to an extent which may seriously affect the results of the biometric approach to taxonomy. These results stress the importance of standardizing substrates in morphological taxonomy and identification work.