Origin and evolution of the colonial volvocales (Chlorophyceae) as inferred from multiple, chloroplast gene sequences

A combined data set of DNA sequences (6021 bp) from five protein-coding genes of the chloroplast genome (rbcL, atpB, psaA, psaB, and psbC genes) were analyzed for 42 strains representing 30 species of the colonial Volvocales (Volvox and its relatives) and 5 related species of green algae to deduce robust phylogenetic relationships within the colonial green flagellates. The 4-celled family Tetrabaenaceae was robustly resolved as the most basal group within the colonial Volvocales. The sequence data also suggested that all five volvocacean genera with 32 or more cells in a vegetative colony (all four of the anisogamous/oogamous genera, Eudorina, Platydorina, Pleodorina, and Volvox, plus the isogamous genus Yamagishiella) constituted a large monophyletic group, in which 2 Pleodorina species were positioned distally to 3 species of Volvox. Therefore, most of the evolution of the colonial Volvocales appears to constitute a gradual progression in colonial complexity and in types of sexual reproduction, as in the traditional volvocine lineage hypothesis, although reverse evolution must be considered for the origin of certain species of Pleodorina. Data presented here also provide robust support for a monophyletic family Goniaceae consisting of two genera: Gonium and Astrephomene.     

Antiquity and evolutionary status of bacterial sulfate reduction: sulfur isotope evidence

The presently available sedimentary sulfur isotope record for the Precambrian seems to allow the following conclusions: (1) In the Early Archaean, sedimentary delta 34S patterns attributable to bacteriogenic sulfate reduction are generally absent. In particular, the delta 34S spread observed in the Isua banded iron formation (3.7 x 10(9) yr) is extremely narrow and coincides completely with the respective spreads yielded by contemporaneous rocks of assumed mantle derivation. Incipient minor differentiation of the isotope pattersn notably of Archaean sulfates may be accounted for by photosynthetic sulfur bacteria rather than by sulfate reducers. (2) Isotopic evidence of dissimilatory sulfate reduction is first observed in the upper Archaean of the Aldan Shield, Siberia (approximately 3.0 x 10(9) yr) and in the Michipicoten and Woman River banded iron formations of Canada (2.75 x 10(9) yr). This narrows down the possible time of appearance of sulfate respirers to the interval 2.8--3.1 x 10(9) yr. (3) Various lines of evidence indicate that photosynthesis is older than sulfate respiration, the SO4(2-) Utilized by the first sulfate reducers deriving most probably from oxidation of reduced sulfur compounds by photosynthetic sulfur bacteria. Sulfate respiration must, in turn, have antedated oxygen respiration as O2-respiring multicellular eucaryotes appear late in the Precambrian. (4) With the bulk of sulfate in the Archaean oceans probably produced by photosynthetic sulfur bacteria, the accumulation of SO4(2-) in the ancient seas must have preceded the buildup of appreciable steady state levels of free oxygen. Hence, the occurrence of sulfate evaporites in Archaean sediments does not necessarily provide testimony of oxidation weathering on the ancient continents and, consequently, of the existence of an atmospheric oxygen reservoir.     

Phototrophs in High-Iron-Concentration Microbial Mats: Physiological Ecology of Phototrophs in an Iron-Depositing Hot Spring

At Chocolate Pots Hot Springs in Yellowstone National Park the source waters have a pH near neutral, contain high concentrations of reduced iron, and lack sulfide. An iron formation that is associated with cyanobacterial mats is actively deposited. The uptake of [¹⁴C]bicarbonate was used to assess the impact of ferrous iron on photosynthesis in this environment. Photoautotrophy in some of the mats was stimulated by ferrous iron (1.0 mM). Microelectrodes were used to determine the impact of photosynthetic activity on the oxygen content and the pH in the mat and sediment microenvironments. Photosynthesis increased the oxygen concentration to 200% of air saturation levels in the top millimeter of the mats. The oxygen concentration decreased with depth and in the dark. Light-dependent increases in pH were observed. The penetration of light in the mats and in the sediments was determined. Visible radiation was rapidly attenuated in the top 2 mm of the iron-rich mats. Near-infrared radiation penetrated deeper. Iron was totally oxidized in the top few millimeters, but reduced iron was detected at greater depths. By increasing the pH and the oxygen concentration in the surface sediments, the cyanobacteria could potentially increase the rate of iron oxidation in situ. This high-iron-content hot spring provides a suitable model for studying the interactions of microbial photosynthesis and iron deposition and the role of photosynthesis in microbial iron cycling. This model may help clarify the potential role of photosynthesis in the deposition of Precambrian banded iron formations.     

PHYLOGENETIC RELATIONSHIPS WITHIN THE COLONIAL VOLVOCALES (CHLOROPHYTA) INFERRED FROM CLADISTIC ANALYSIS BASED ON MORPHOLOGICAL DATA1

A cladistic analysis was used to deduce the phylogenetic relationships within the colonial Volvocales. Forty-one pairs of characters related to gross morphology and ultrastructure of vegetative colonies as well as asexual and sexual reproduction were analyzed based on parsimony, using the PAUP 3.0 computer program, for 25 species belonging to nine volvocacean and goniacean genera of the colonial Volvocales. Chlamydomonas reinhardtii Dangeard was the outgroup. The strict consensus tree indicated the presence of two monophyletic groups, one composed of all the volvocacean species analyzed in this study and the other containing the goniacean species except for the four-celled species Gonium sociale (Dujardin) Warming. In addition, these two groups constitute a large monophyletic group, to which G. sociale is a sister group. A new combination Tetrabaena socialis (Dujardin) Nozaki et Itoh and a new family Tetrabaenaceae Nozaki et Itoh are thus proposed for G. sociale. In addition, the analysis suggests that the volvocacean genera Eudorina and Pleodorina are paraphyletic groups, respectively, and that the monotypic genus Yamagishiella has no autapomorphic characters and represents primitive features of the anisogamous and oogamous genera of the Volvocaceae. Phylogenetic relationships within the Volvocaceae and the Goniaceae, as well as the various modes of sexual reproduction exhibited by these organisms, are discussed on the basis of the analysis.     

Planktonic marine iron oxidizers drive iron mineralization under low‐oxygen conditions

Observations of modern microbes have led to several hypotheses on how microbes precipitated the extensive iron formations in the geologic record, but we have yet to resolve the exact microbial contributions. An initial hypothesis was that cyanobacteria produced oxygen which oxidized iron abiotically; however, in modern environments such as microbial mats, where Fe(II) and O₂ coexist, we commonly find microaerophilic chemolithotrophic iron‐oxidizing bacteria producing Fe(III) oxyhydroxides. This suggests that such iron oxidizers could have inhabited niches in ancient coastal oceans where Fe(II) and O₂ coexisted, and therefore contributed to banded iron formations (BIFs) and other ferruginous deposits. However, there is currently little evidence for planktonic marine iron oxidizers in modern analogs. Here, we demonstrate successful cultivation of planktonic microaerophilic iron‐oxidizing Zetaproteobacteria from the Chesapeake Bay during seasonal stratification. Iron oxidizers were associated with low oxygen concentrations and active iron redox cycling in the oxic–anoxic transition zone (<3 μm O₂, <0.2 μm H₂S). While cyanobacteria were also detected in this transition zone, oxygen concentrations were too low to support significant rates of abiotic iron oxidation. Cyanobacteria may be providing oxygen for microaerophilic iron oxidation through a symbiotic relationship; at high Fe(II) levels, cyanobacteria would gain protection against Fe(II) toxicity. A Zetaproteobacteria isolate from this site oxidized iron at rates sufficient to account for deposition of geologic iron formations. In sum, our results suggest that once oxygenic photosynthesis evolved, microaerophilic chemolithotrophic iron oxidizers were likely important drivers of iron mineralization in ancient oceans.     

Microbes: Mini Iron Factories

Microbes have flourished in extreme habitats since beginning of the Earth and have played an important role in geological processes like weathering, mineralization, diagenesis, mineral formation and destruction. Biotic mineralization is one of the most fascinating examples of how microbes have been influencing geological processes. Iron oxidizing and reducing bacteria are capable of precipitating wide varieties of iron oxides (magnetite), carbonates (siderite) and sulphides (greigite) via controlled or induced mineralization processes. Microbes have also been considered to play an important role in the history of evolution of sedimentary rocks on Earth from the formation of banded iron formations during the Archean to modern biotic bog iron and ochre deposits. Here, we discuss the role that microbes have been playing in precipitation of iron and the role and importance of interdisciplinary studies in the field of geology and biology in solving some of the major geological mysteries.     

Possible ctenophoran affinities of the Precambrian "sea-pen" Rangea

The Namibian Kuibis Quartzite fossils of Rangea are preserved three-dimensionally owing to incomplete collapse of the soft tissues under the load of instantaneously deposited sand. The process of fossilization did not reproduce the original external morphology of the organism but rather the inner surface of collapsed organs, presumably a system of sacs connected by a medial canal. The body of Rangea had tetraradial symmetry, a body plan shared also by the White Sea Russian fossil Bomakellia and possibly some other Precambrian frond-like fossils. They all had a complex internal anatomy, smooth surface of the body, and radial membranes, making their alleged colonial nature unlikely. Despite a different style of preservation, the Middle Cambrian Burgess Shale frond-like Thaumaptilon shows several anatomical similarities to Rangea. The body plan of the Burgess Shale ctenophore Fasciculus, with its numerous, pinnately arranged comb organs, is in many respects transitional between Thaumaptilon and the Early Cambrian ctenophore Maotianoascus from the Chengjiang fauna of South China. It is proposed that the irregularly distributed dark spots on the fusiform units of the petaloid of Thaumaptilon represent a kind of macrocilia and that the units are homologous with the ctenophoran comb organs. These superficial structures were underlain by the complex serial organs, well represented in the fossils of Rangea. The Precambrian "sea-pens" were thus probably sedentary ancestors of the ctenophores.     

The history of atmospheric oxygen

A primeval anoxygenic terrestrial atmosphere having been postulated on astronomical grounds, experiments using simulated conditions have shown that the formation of organic molecules by abiogenic processes with proceed freely in such an environment.Atmospheric oxygen will at first be limited to 0.001 PAL through the Urey mechanism which inhibits further dissociation of water above this level. All atmospheric oxygen exceeding this level must be biogenic and produced by photosynthesis. Molecular fossils prove its existence 2.7 billion years ago. Sedimentary ores, notably pyrite sands of gold-uranium reefs and banded iron formations, attest to the existence of an atmosphere with little oxygen up to 1.8 billion years ago. Geochemistry does not, howerver, supply us with data as to the level of oxygen at that time. The Pasteur Point, on the other hand, at which microbes change from fermentation to respiration and vice versa, is a powerful regulating factor situated at 0.01 PAL of free oxygen.It is postulated that the primeval atmosphere of Lower and Middle Precambrian was limited to this level of free oxygen. At this level pre-life — the formation of organic compounds through inorganic processes — still exists. Pre-life and early life therefore were coexistent for two billion years at least, and were able to influence each, other over all this time. The primeval, atmosphere was definitely superseded by an oxygenic one about 1.45 billion years ago, but the level of 0.1 PAL of free oxygen was only reached during the Ordovician, 0.4–0.5 billion years ago.     

Phototrophs in high-iron-concentration microbial mats: physiological ecology of phototrophs in an iron-depositing hot spring

At Chocolate Pots Hot Springs in Yellowstone National Park the source waters have a pH near neutral, contain high concentrations of reduced iron, and lack sulfide. An iron formation that is associated with cyanobacterial mats is actively deposited. The uptake of [(14)C]bicarbonate was used to assess the impact of ferrous iron on photosynthesis in this environment. Photoautotrophy in some of the mats was stimulated by ferrous iron (1.0 mM). Microelectrodes were used to determine the impact of photosynthetic activity on the oxygen content and the pH in the mat and sediment microenvironments. Photosynthesis increased the oxygen concentration to 200% of air saturation levels in the top millimeter of the mats. The oxygen concentration decreased with depth and in the dark. Light-dependent increases in pH were observed. The penetration of light in the mats and in the sediments was determined. Visible radiation was rapidly attenuated in the top 2 mm of the iron-rich mats. Near-infrared radiation penetrated deeper. Iron was totally oxidized in the top few millimeters, but reduced iron was detected at greater depths. By increasing the pH and the oxygen concentration in the surface sediments, the cyanobacteria could potentially increase the rate of iron oxidation in situ. This high-iron-content hot spring provides a suitable model for studying the interactions of microbial photosynthesis and iron deposition and the role of photosynthesis in microbial iron cycling. This model may help clarify the potential role of photosynthesis in the deposition of Precambrian banded iron formations.     

Bacterial paleontology of the Neoarchean banded iron formations of Karelia and the Kola Peninsula

Probable microfossils, presumably of bacterial origin, were found in the banded iron formations of Karelia and the Kola Peninsula. The age of these formations is 2.7–2.8 Ga. Based on the organic carbon content and balance estimations it was established that these banded iron formations were deposited in environments rich in organic matter. Comparative analysis of the morphology of Recent and Neoarchean microorganisms suggests a bacterial origin for some magnetite in the studied quartzites.     

New data on the stratigraphy of the crystalline complexes of Mongolia

At present, in Mongolia crystalline complexes are reliably established in almost all major structural elements. The largest field of these complexes is recognized in the Early Caledonian structures of the North-Mongolian Folded Belt, in the Tarbagatai and Baidarik blocks. The crystalline complexes of the South Altai Belt have previously been interpreted as fragments of the Precambrian basement. Recent geochronological and isotope geochemical data have shown that pre-Riphean and Early Hercynian formations can be recognized in the Precambrian of Mongolia. These complexes show evidence of similar metamorphic processes, but are different in structural position and geological history. The paper contains new stratigraphic schemes for metamorphic complexes of the Baidarik and Tarbagatai blocks and also the South Altai Metamorphic Belt (SAMB).     

REEXAMINATION OF PHYLOGENETIC RELATIONSHIPS WITHIN THE COLONIAL VOLVOCALES (CHLOROPHYTA): AN ANALYSIS OF atpB AND rbcL GENE SEQUENCES

The chloroplast-encoded atp B gene was sequenced from 33 strains representing 28 species of the colonial Volvocales (the Volvocaceae and its relatives) to reexamine phylogenetic relationships as previously deduced by morphological data and rbc L gene sequence data.1128 base pairs in the coding regions of the atp B gene were analyzed by MP, NJ, and ML analyses. Although supported with relatively low bootstrap values (75% and 65% in the NJ and ML analyses, respectively), three anisogamous/oogamous volvocacean genera— Eudorina, Pleodorina, and Volvox, excluding the section Volvox (= Euvolvox, illegitimate name), constituted a large monophyletic group (Eudorina group). Outside the Eudorina group, a robust lineage composed of three species of Volvox sect. Volvox was resolved as in the rbc L gene trees, rejecting the hypothesis of the previous cladistic analysis based on morphological data that the genus Volvox is monophyletic. In addition, the NJ and ML trees suggested that Eudorina is a nonmonophyletic genus as inferred from the morphological data and rbc L gene sequences. Although phylogenetic status of the genus Gonium is ambiguous in the rbc L gene trees and the paraphyly of this genus is resolved in the cladistic analysis based on morphological data, the atp B gene sequence data suggest monophyly of Gonium with relatively low bootstrap values (56–61%) in the NJ and ML trees. On the basis of the combined sequence data (2256 base pairs) from atp B and rbc L genes, Gonium was resolved as a robust monophyletic genus in the NJ and ML trees (with 68–86% bootstrap values), and Eudorina elegans Ehrenberg represented a paraphyletic species positioned most basally within the Eudorina group. However, phylogenetic status and relationships of the families of the colonial Volvocales were still almost ambiguous even in the combined analysis.     

PHYLOGENETIC RELATIONSHIPS WITHIN THE COLONIAL VOLVOCALES (CHLOROPHYTA) INFERRED FROM rbcL GENE SEQUENCE DATA

The chloroplast-encoded large subunit of the ribulose-1, 5-bisphosphate carboxylase / oxygenase (rbcL) gene was sequenced from 20 species of the colonial Volvocales (the Volvacaceae, Goniaceae, and Tetrabaenaceae) in order to elucidate phylogenetic relationships within the colonial Volvocales. Eleven hundred twenty-eight base pairs in the coding regions of the (rbcL) gene were analyzed by the neighbor-joining (NJ) method using three kinds of distance estimations, as well as by the maximum parsimony (MP) method. A large group comprising all the anisogamous and oogamous volvocacean species was resolved in the MP tree as well as in the NJ trees based on overall and synonymous substitutions. In all the trees constructed, Basichlamys and Tetrabaena (Tetrabaenaceae) constituted a very robust phylogenetic group. Although not supported by high bootstrap values, the MP tree and the NJ tree based on nonsynonymous substitutions indicated that the Tetrabaenaceae is the sister group to the large group comprising the Volvocaceae and the Goniaceae. In addition, the present analysis strongly suggested that Pandorina and Astrephomene are monophyletic genera whereas Eudorina is nonmonophyletic. These results are essentially consistent with the results of the recent cladistic analyses of morphological data. However, the monophyly of the Volvocaceae previously supported by four morphological synapomorphies is found only in the NJ tree based on nonsynonymous substitutions (with very low bootstrap values). The genus Volvox was clearly resolved as a polyphyletic group with V. rousseletii Pocock separated from other species of Volvox in the rbcL gene comparisons, although this genus represents a monophyletic group in the previous morphological analyses. Furthermore, none of the rbcL gene trees supported the monophyly of the Goniaceae; Astrephomene was placed in various phylogenetic positions .     

Antiquity and evolutionary status of bacterial sulfate reduction: Sulfur isotope evidence

The presently available sedimentary sulfur isotope record for the Precambrian seems to allow the following conclusions: (1) In the Early Archaean, sedimentary ^{3 4} patterns attributable to bacteriogenic sulfate reduction are generally absent. In particular, the ^{3 4} spread observed in the Isua banded iron formation (3.7×10⁹ yr) is extremely narrow and coincides completely with the respective spreads yielded by contemporaneous rocks of assumed mantle derivation. Incipient minor differentiation of the isotope patterns notably of Archaean sulfates may be accounted for by photosynthetic sulfur bacteria rather than by sulfate reducers. (2) Isotopic evidence of dissimilatory sulfate reduction is first observed in the upper Archaean of the Aldan Shield, Siberia (3.0×10⁹ yr) and in the Michipicoten and Woman River banded iron formations of Canada (2.75×10⁹ yr). This narrows down the possible time of appearance of sulfate respirers to the interval 2.8–3.1×10⁹ yr. (3) Various lines of evidence indicate that photosynthesis is older than sulfate respiration, the SO ₄ ^2– utilized by the first sulfate reducers deriving most probably from oxidation of reduced sulfur compounds by photosynthetic sulfur bacteria. Sulfate respiration must, in turn, have antedated oxygen respiration as O₂-respiring multicellular eucaryotes appear late in the Precambrian. (4) With the bulk of sulfate in the Archaean oceans probably produced by photosynthetic sulfur bacteria, the accumulation of SO ₄ ^2– in the ancient seas must have preceded the buildup of appreciable steady state levels of free oxygen. Hence, the occurrence of sulfate evaporites in Archaean sediments does not necessarily provide testimony of oxidation weathering on the ancient continents and, consequently, of the existence of an atmospheric oxygen reservoir.Paper presented at the Fourth College Park Colloquium on Chemical Evolution, Limits of Life, October 18–20, 1978.     

Holocene coccidioidomycosis: Valley Fever in early Holocene bison (Bison antiquus)

Early Holocene bison mandibles (Bison antiquus) from Nebraska, ca. 8500 y ago, were examined with a variety of modern histotechnological procedures and staining techniques. A pathological, anatomical diagnosis of moderately severe, locally extensive, mandibular osteomyelitis with intralesional spherules morphologically consistent with fungal pathogens in the genus Coccidioides was made. The modern distribution of the organisms in North America is restricted to the arid Southwest. This implies either the fossil home range of the fungi was larger than it is today or fossil bison migrated between endemic and nonendemic foci during the early Holocene.     

Iron isotope fractionation during microbial dissimilatory iron oxide reduction in simulated Archaean seawater

The largest Fe isotope excursion yet measured in marine sedimentary rocks occurs in shales, carbonates, and banded iron formations of Neoarchaean and Paleoproterozoic age. The results of field and laboratory studies suggest a potential role for microbial dissimilatory iron reduction (DIR) in producing this excursion. However, most experimental studies of Fe isotope fractionation during DIR have been conducted in simple geochemical systems, using pure Fe(III) oxide substrates that are not direct analogues to phases likely to have been present in Precambrian marine environments. In this study, Fe isotope fractionation was investigated during microbial reduction of an amorphous Fe(III) oxide-silica coprecipitate in anoxic, high-silica, low-sulphate artificial Archaean seawater at 30 °C to determine if such conditions alter the extent of reduction or isotopic fractionations relative to those observed in simple systems. The Fe(III)-Si coprecipitate was highly reducible (c. 80% reduction) in the presence of excess acetate. The coprecipitate did not undergo phase conversion (e.g. to green rust, magnetite or siderite) during reduction. Iron isotope fractionations suggest that rapid and near-complete isotope exchange took place among all Fe(II) and Fe(III) components, in contrast to previous work on goethite and hematite, where exchange was limited to the outer few atom layers of the substrate. Large quantities of low-δ(56)Fe Fe(II) (aqueous and solid phase) were produced during reduction of the Fe(III)-Si coprecipitate. These findings shed new light on DIR as a mechanism for producing Fe isotope variations observed in Neoarchaean and Paleoproterozoic marine sedimentary rocks.     

Cyanobacteria(?) in iron banded formations of the Kursk magnetic anomaly

Fossilized cyanobacteria(?) represented by trichomes enclosed in common sheaths were detected in early Proterozoic iron banded formations of the Kursk magnetic anomaly (limonite–martite ores of the Lebedinsky mine and iron banded formations of the Korobkovskoye deposit). These fossils morphologically similar to current representatives of the genus Microcoleus were buried in situ.     

The fossil record and biogeography of the Ciehlidae (Actinopterygii: Labroidei)

The family Ciehlidae is a large group of tropical fishes in the order Perciformes, with an estimated number of living species exceeding 1400. The modern distribution of the family Ciehlidae is predominantly in fresh waters of Central and South America, Africa, Madagascar, India and the Middle East, with fossil members known from Africa, Saudi Arabia, the Levant, Europe, South America and Haiti. Many authors have referred to the distribution as being Gondwanan and have postulated that cichlids originated over 130 million years ago, in the Early Cretaceous. However, the suggested evidence for an Early Cretaceous origin of cichlids is equally or more compatible with a much younger age of origin. Based on the biology and distribution of modern and fossil cichlids, it is more probable that they arose less than 65 million years ago, in the Early Tertiary, and crossed marine waters to attain their current distribution.     

Mineral evolution and processes of ferruginous microbialite accretion – an example from the Middle Eocene stromatolitic and ooidal ironstones of the Bahariya Depression,Western Desert,Egypt

Peritidal ferruginous microbialites form the main bulk of the Middle Eocene ironstone deposits of the Bahariya Depression, Western Desert, Egypt. They include ferruginous stromatolites and microbially coated grains (ferruginous oncoids and ooids). Their internal structures reveal repeated cycles of microbial and Fe oxyhydroxide laminae. The microbial laminae consist of fossilised neutrophilic filamentous iron‐oxidising bacteria. These bacteria oxidised the Fe(II)‐rich acidic groundwater upon meeting the marine water at an approximately neutral pH. The iron oxyhydroxide laminae were initially precipitated as amorphous iron oxhydroxides and subsequently recrystallised into nanocrystalline goethite during early diagenesis. Organic remains such as proteinaceous compounds, lipids, carbohydrates and carotenoids are preserved and can be identified by Raman spectroscopy. The ferruginous microbialites were subjected to post‐depositional subaerial weathering associated with sea‐level retreat and subsurface alteration by continued ascent of the Fe(II)‐rich acidic groundwater. At this stage, another iron‐oxidising bacterial generation prevailed in the acidic environment. The acidity of the groundwater was caused by oxidation of pyrite in the underlying Cenomanian Bahariya formation. The positive iron isotopic ratios and presence of ferrous and ferric iron sulphates may result from partial iron oxidation along the redox boundary in an oxygen‐depleted environment.     

Zygote germination in <Emphasis Type="Italic">Pleodorina starrii</Emphasis> (Volvocaceae,Chlorophyta)

Zygote germination of the anisogamous/oogamous colonial green flagellate Pleodorina starrii was observed. After the zygotes were transferred to the usual, illuminated conditions from the dark treatment on the agar plate, they began to germinate. The germinating zygotes gave rise to one or two viable biflagellate gone cells. This type of zygote germination is rare in the colonial Volvocales and may characterize a certain lineage within the anisogamous/oogamous members of the colonial Volvocales. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.