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

The 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.     

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.     

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 rbc L 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 rbc L 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 rbc L 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 .     

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.     

Oospore Wall Ornamentation in the Genus Tolypella (Charales,Charophyceae)

The ultrastructural features of oospore wall ornamentation in the genus Tolypella were examined using scanning electron microscopy (SEM). The taxonomic relationships among several species were discussed on the basis of oospore ultrastructure and measurements. In the case of T. glomerata and T. nidifica, our results support the status of separate species. Close relationships and transitional forms may exist between T. nidifica and T. normaniana, and not only in oospore wall ornamentation. Oospores of T. hispanica exhibited the same distinct type of reticulate oospore wall as previously reported, but our results do not support the recognition of T. hispanica as a separate species. Ultrastructure of the oospore walls of T. prolifera and T. intricata was almost identical, suggesting that these species are closely related. We therefore reject previous suggestions that morphological characteristics of oospores as observed in SEM are sufficient for identification of individual species. Although significant differences were found among oospores in individual species of Tolypella, large variation among populations, and among individuals belonging to the same population, caused substantial overlap among species.     

New finds of charophytes in Iceland with an update on the distribution of the charophyte flora

Charophyte species new to the flora of Iceland are reported and an update on the distribution of the previously known species is given. Four species are new to Iceland; Chara aspera Willdenow, Chara contraria Braun ex Kützing, Tolypella canadensis Sawa and Tolypella glomerata (Desvaux) Leonhardi, while four species were previously known: Chara globularis Thuiller, Chara virgata Kützing, Nitella flexilis (L.) Agardh and Nitella opaca (Agardh ex Bruzelius) Agardh. The finding of the species new to Iceland add to the hitherto known worldwide distribution of those species, including a significant extension to the north.     

Structure and development of the sex organs in Tolypella nidifica (O. Müll.) A.Br. (Characeae)

The occurrence of Tolypella (Characeae) in Libya is recorded for the first time. Also for the first time, the structural development of the male and female fructifications (the globule (antheridium) and the nucule (oogonium) respectively) in Tolypella nidifca (O. Müll.) A.Br. is described. Both the globule and the nucule show the same course of development as is seen in Chara. In the final structure of the globule, however, there are two stalk cells in Tolypella , while only a single cell is present in Chava and Nitella. The corona has ten cells in two tiers of five each, as in Nitella , but in Chara there is only a single tier of five cells forming the corona.     

Reconstruction of the former charophyte community out of the fructifications identified in Albufera de València lagoon sediments

Several decades ago, due to eutrophication, Albufera de València lagoon lost the charophyte meadows that covered the bottom in the past but had never been studied in detail. An area close to the lagoon will be restored now to improve water quality and to recover submerged vegetation. To have information on the background of the charophytes from this area, four 80-87 cm long sediment cores were extracted from the lagoon. The subfossil charophyte fructifications (oospores and gyrogonites) were isolated and identified in two of the four cores. Moreover, sediment core fractions from the third and fourth cores were allowed to germinate under several conditions. The study of the lagoon sediments has revealed that at least 11 morphologies of charophyte fructifications existed in the past. Ten of these were determined at species level: Chara vulgaris, Chara hispida, Chara aspera, Chara baltica, Chara tomentosa, Chara globularis, Lamprothamnium papulosum, Nitella hyalina, Tolypella glomerata and Tolypella hispanica. However, one form of Chara gyrogonite did not fit in any known species. L. papulosum oospores and gyrogonites were the most abundant and concentrated in the deepest sediment layers when the lagoon held brackish water. Large changes in salinity due to freshwater input drove charophyte species substitution (disappearance of L. papulosum). During the oligohaline period C. vulgaris sexual propagules were the most abundant. The last charophyte community before its disappearance due to eutrophication was composed mainly of C. hispida, C. tomentosa and N. hyalina (the two latter with small numbers of oospores in the sediment). To characterize the morphology of the unidentified gyrogonite species, 100 specimens were studied. This type could be a local variety of a more common species, probably C. vulgaris, grown under particular ecological conditions. Old collected oospores (more than 45 years old) germinated and could be identified as C. vulgaris and C. hispida.     

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.     

巴尔干半岛现生轮藻植物综述

欧洲东南部巴尔干轮藻植物由于其产地生境的差异及以镶嵌状分布的模式,具有较高的生物分异度,计有44种,归属于现生属Nitella(10),Tolypella(4),Nitellopsis(1),Lychnothamnus(1),Lamprothamnium(1) and Chara(27).巴尔干半岛产有4种土著类型Chara rohlenae,Chara ohridana,Chara corfuensis and Chara visianii.建立1新种Chara hydropitys.该地区轮藻植物分布不均衡,部分类型例如Chara strigosa,Chara kokeilii,Chara muscosa,Chara fragifera,Chara imperfecta,Lychnothamnus barbatus为稀有品种,仅在少数地点发现.巴尔干地区轮藻植物之丰富多彩远远超过已知的程度,有待于深入研究.     

Some New Taxa and Records of Characeae in Hunan,China

In this paper, one new species, four new varieties and thirteen new records of Characeae from Hunan, China, are reported. They are Chara quadriscutulum, sp. nov., Nitella axilliformis var. biformis, var. nov., N. microcarpa var. unicarpa, var. nov., Chara braunii var. cylindrospora, var. nov., C. hydropitys var. hunanensis, var. nov., Nitella microcarpa Braun, N. leptoclada Braun, N. annandalei Pal, N. confervacea A. Braun, N. gracilliformis J. Groves, N. inversa Imahori, N. musashiensis Morioka, N. paucicostata T. F. Allen, N. acuminata var. gollmeriana (A. Br.) Zanev. et Wood, Tolypella boldii Sawa. Chara longifolia Robinson, C. pistianensis J. Vilhelm, C. grovesii Pal.     

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.     

Occurrence,composition, and structure of microbody crystalloids in charophycean gametangial sheath cells

Summary An ultrastructural study was made of the cellular sheaths surrounding the sexual organs of five species of algae in the three genera ofCharophyceae: Nitella flexilis, N. mirabilis, Chara brattnii, Tolypella boldii andT. intricata. Microbodies similar in appearance, with crystalline nucleoids, were present in the sheath cells of all five species. The microbodies resembled in size and topographical associations those of other green algae. The hexagonal-shaped crystalloids consisted of parallel arrays of fine tubules of about 15 nm in diameter arranged parallel to the long axis of the crystalloid. In cross sections of the crystalloid, the close packing of the tubules showed hexagonal arrays. The intertubular distance is about 7 nm. At higher magnification there is a suggestion that the walls of these tubules are themselves constructed of smaller tubules. Further electron microscopic observations of diaminobenzidine (DAB)-treated preparations revealed pronounced deposition of reaction product in the microbodies, particularly on the crystalloids. The reaction was completely blocked by the catalase inhibitor, aminotriazole. These results strongly suggest that catalase is involved in this reaction and that catalase is located in the crystalloids.     

Research Note: Observations on a terrestrial charophyte in a temperate environment

This study describes the unusual growth forms of Chara (Charales) in a natural terrestrial environment in a temperate region (southern Siberia). Chara vulgaris had a moss‐like habit and grew on wet soil in small compact turfs probably germinated from oospores. The upper branchlets were curved in a hook‐liked manner and convergent above the apex of the thallus. The lowest internode and branchlets of fertile plants were ecorticate, whereas all other parts were normally corticated; the completely ecorticate plants were sterile. The terrestrial growth of charophytes had an irregular appearance but was successfully maintained in culture, providing a potential model of the first steps of land colonization by the ancestors of embryophytes.     

Oospores progenies from Phytophthora ramorum

Oospores of Phytophthora ramorum were produced from intraspecific pairings between a European A1 and European or American A2 strains. Their viability was evaluated through colouration with tetrazolium bromide. The distribution of oospores in the different classes of colouration was similar to that found in other Phytophthora species (homothallic and heterothallic): most of the oospores stained purple, which corresponds to spores in dormancy. In order to produce single-oospore cultures, a method was developed to separate oospores from mycelium and chlamydospores. Germination of oospores was observed in 110, 250, 350 and 500-d-old cultures at a low proportion. Microsatellite marker analyses on oospore progenies revealed that the oospores resulted from hybridisation. More than 50 oospore progenies were characterised in terms of mating type, aggressiveness on Rhododendron leaves, and growth rate on two different media. The results are discussed in the context of pest risk analysis.     

ANNUAL GERMINATION WINFOW IN OOSPORES OF NITELLA FURCATA (CHAROPHYCEAE)1

Oospores of Nitella furcata subsp. megacarpa (Allen emend. Wood) were collected from an oospore bank in the sediments of Lake George, New York. Incubated at constant temperatures, all or nearly all of the oospores germinated when exposed to a brief pulse of red light when the annual window of germinability was open. The window seems related to the annual cycle of sediment temperatures. It is open in spring and closes wit the onset of a secondary dormacncy in the summer. Oospores in storage follow a parallel path if held at 18°C, a summer equivalent temperature; the window remains open indefinitely if the oospores are held at 4°C. Attention is drawn to the similarity if the cyclic window of germinability in seeds of summer annuals and oospores of N. furcata.