The downsizing of gigantic scales and large cells in the genus Mallomonas (Synurales, Chrysophyceae).

Mallomonas is the largest and most speciose genus within the Synurales, a monophyletic clade of siliceous scale-bearing organisms within the class Chrysophyceae. The genus consists of unicellular, motile, photosynthetic organisms found in freshwater localities worldwide. Mallomonas diverged from other synurophytes during the lower Cretaceous at approximately 130 Ma. Recent discoveries of fossil species were used to examine shifts in scale and cell size over geologic time. On average, scales of fossil species were 2.5 times larger than those produced by modern species. However, a smaller subset of extinct fossil taxa lacking modern analogs had scales over four times larger than modern species, and the largest recorded specimens were six times larger. Data from modern species were further used to develop a model relating scale size to cell size, and applied to the fossil specimens. Based on the model, the mean size of fossil cells was almost twice as long and 50% wider compared to modern species, and cells of taxa lacking modern analogs close to three times as large. These large cells, covered with robust siliceous scales, were likely slow swimmers requiring significant energy to maintain their position in the water column, and possibly prone to increased predation.

A first account of the heterotrophic eukaryote Rabdiophrys Rainer from the fossil record and description of a new species from an ancient Eocene Arctic freshwater lake.

Rotosphaerids are unicellular, heterotrophic, eukayotic protists that have filopodia, an exterior covering consisting of highly ornamented siliceous scales, and are classified in the Rotosphaerida within the opistokont lineage. Given their appearance as relatively large spherical cells with protruding filopodia and a silica scale covering, they are often mistaken for centrohelid heliozoans. Even though these organisms are widely distributed in both marine and freshwater environments, many species are rarely reported, and none have been reported from the fossil record. We report extensive remains of a new species of Rabdiophrys, R. giraffensis, from an ancient waterbody that was situated near the Arctic Circle in northern Canada during the Eocene. The new species has both plate and spine scales that are similar in morphology, but significantly larger than its closest modern congeners, R. monopora and R. anulifera. The waterbody in which the new species grew and thrived is inferred to have been a moderately deep, circumneutral pond, with moderate concentrations of nutrients and dissolved humic material.

Morphological evolution of silica scales in the freshwater genus Synura (Stramenopiles).

A high degree of morphological variability is expressed between the ornately sculptured siliceous scales formed by species in the chrysophycean genus, Synura. In this study, we aimed to uncover the general principles and trends underlying the evolution of scale morphology in this genus. We assessed the relationships among thirty extant Synura species using a robust molecular analysis that included six genes, coupled with morphological characterization of the species-specific scales. The analysis was further enriched with addition of morphological information from fossil specimens and by including the unique modern species, Synura punctulosa. We inferred the phylogenetic position of the morphologically unique S. punctulosa, to be an ancient Synura lineage related to S. splendida in the section Curtispinae. Some morphological traits, including development of a keel or a labyrinth ribbing pattern on the scale, appeared once in evolution, whereas other structures, such as a hexagonal meshwork pattern, originated independently several times over geologic time. We further uncovered numerous construction principles governing scale morphology and evolution, as follows: (i) scale roundness and pore diameter decreased during evolution; (ii) elongated scales became strengthened by a higher number of struts or ribs; (iii) as a consequence of scale biogenesis, scales with spines possessed smaller basal holes than scales with a keel and; and (iv) the keel area was proportional to scale area, indicating its potential value in strengthening the scale against breakage.

Remarkably preserved cysts of the extinct synurophyte, Mallomonas ampla, uncovered from a 48 Ma freshwater Eocene lake.

Chrysophyte algae produce a siliceous stage in their life cycle, through either asexual or sexual reproduction, known as a cyst. Cysts form in response to shifts in environmental conditions, population density, or predation pressure, and upon germination provide a seed source for future populations. Cysts are morphologically distinct for each species, and since their remains become part of the sediment or fossil record cysts are valuable tools in ecological and paleolimnological investigations. However, their value as biological indicators is limited because the vast majority of cyst morphotypes have not been linked to specific vegetative species. In the current work, an exquisitely preserved and morphologically complex cyst type is described from a 48 million year old early Eocene fossil site. This finding is remarkable since many of the cysts were still associated with components of the living vegetative cells that produced them, enabling the morphotype to be immediately linked to the synurophyte, Mallomonas ampla. Fusion of identifiable components of the living cell post cyst formation is unknown in modern investigations. The identification of the cyst structure for M. ampla could be valuable in determining cyst morphotypes for other species in the lineage.

Comparing Morphological and Molecular Estimates of Species Diversity in the Freshwater Genus Synura (Stramenopiles): A Model for Understanding Diversity of Eukaryotic Microorganisms.

We performed a comparison of molecular and morphological diversity in a freshwater colonial genus Synura (Chrysophyceae, Stramenopiles), using the island of Newfoundland (Canada) as a case study. We examined the morphological species diversity in collections from 79 localities, and compared these findings to diversity based on molecular characters for 150 strains isolated from the same sites. Of 27 species or species-level lineages identified, only one third was recorded by both molecular and morphological techniques, showing both approaches are complementary in estimating species diversity within this genus. Eight taxa, each representing young evolutionary lineages, were recovered only by sequencing of isolated colonies, whereas ten species were recovered only microscopically. Our complex investigation, involving both morphological and molecular examinations, indicates that our knowledge of Synura diversity is still poor, limited only to a few well-studied areas. We revealed considerable cryptic diversity within the core S. petersenii and S. leptorrhabda lineages. We further resolved the phylogenetic position of two previously described taxa, S. kristiansenii and S. petersenii f. praefracta, propose species-level status for S. petersenii f. praefracta, and describe three new species, S. vinlandica, S. fluviatilis, and S. cornuta. Our findings add to the growing body of literature detailing distribution patterns observed in the genus, ranging from cosmopolitan species, to highly restricted taxa, to species such as S. hibernica found along coastal regions on multiple continents. Finally, our study illustrates the usefulness of combining detailed morphological information with gene sequence data to examine species diversity within chrysophyte algae.

The sponge genus Ephydatia from the high-latitude middle Eocene: environmental and evolutionary significance.

The freshwater sponge species Ephydatia cf. facunda Weltner, 1895 (Spongillida, Spongillidae) is reported for the first time as a fossil from middle Eocene lake sediments of the Giraffe kimberlite maar in northern Canada. The sponge is represented by birotule gemmuloscleres as well as oxea megascleres. Today, E. facunda inhabits warm-water bodies, so its presence in the Giraffe locality provides evidence of a warm climate at high latitudes during the middle Eocene. The morphological similarity of the birotules to modern conspecific forms suggests protracted morphological stasis, comparable to that reported for other siliceous microfossils from the same locality.

Assessing the evolutionary history of the class Synurophyceae (Heterokonta) using molecular, morphometric, and paleobiological approaches.

PREMISE OF THE STUDY: Heterokont algae of the class Synurophyceae, characterized by distinctive siliceous scales that cover the surface of the cell, are ecologically important in inland waters, yet their evolutionary history remains enigmatic. We explore phylogenetic relationships within this group of algae relative to geologic time, with a focus on evolution of siliceous components. METHODS: We combined an expansive five-gene and time-calibrated molecular phylogeny of synurophyte algae with an extensive array of fossil specimens from the middle Eocene to infer evolutionary trends within the group. KEY RESULTS: The group originated in the Jurassic approximately 157 million years ago (Ma), with the keystone genera Mallomonas and Synura diverging during the Early Cretaceous at 130 Ma. Mallomonas further splits into two major subclades, signaling the evolution of the V-rib believed to aid in the spacing and organization of scales on the cell covering. Synura also diverges into two primary subclades, separating taxa with forward-projecting spines on the scale from those with a keel positioned on the scale proper. Approximately one third of the fossil species are extinct, whereas the remaining taxa are linked to modern congeners. CONCLUSIONS: The taxonomy of synurophytes, which relies extensively on the morphology of the siliceous components, is largely congruent with molecular analyses. Scales of extinct synurophytes were significantly larger than those of modern taxa and may have played a role in their demise. In contrast, many fossil species linked to modern lineages were smaller in the middle Eocene, possibly reflecting growth in the greenhouse climatic state that characterized this geologic interval.

Euglyphid testate amoebae (rhizaria: euglyphida) from an arctic eocene waterbody: evidence of evolutionary stasis in plate morphology for over 40 million years.

Well-preserved siliceous plates representing multiple species of euglyphids are described from a Middle Eocene maar lake deposit located near the Arctic Circle in northern Canada. Siliceous plate morphotypes including scutiform, rectangular, hexagonal, oval and circular body forms, six apertural plate types containing from five to thirteen teeth and spine plates, are documented. Many plate types bear resemblance to those found on modern species as well as ones documented from Miocene sites. These findings extend the known geological record for euglyphids and support the concept of evolutionary stasis in regards to plate morphology over much of the Cenozoic. Future use of these euglyphid remains, in conjunction with other microfossil assemblages, for reconstructing historical conditions within the maar lake is discussed.

A time-calibrated multi-gene phylogeny of the diatom genus Pinnularia.

Pinnularia is an ecologically important and species-rich genus of freshwater diatoms (Bacillariophyceae) showing considerable variation in frustule morphology. Interspecific evolutionary relationships were inferred for 36 Pinnularia taxa using a five-locus dataset. A range of fossil taxa, including newly discovered Middle Eocene forms of Pinnularia, was used to calibrate a relaxed molecular clock analysis and investigate temporal aspects of the genus' diversification. The multi-gene approach resulted in a well-resolved phylogeny of three major clades and several subclades that were frequently, but not universally, delimited by valve morphology. The genus Caloneis was not recovered as monophyletic, confirming that, as currently delimited, this genus is not evolutionarily meaningful and should be merged with Pinnularia. The Pinnularia-Caloneis complex is estimated to have diverged between the Upper Cretaceous and the early Eocene, implying a ghost range of at least 10 million year (Ma) in the fossil record.

STUDIES ON ULTRASTRUCTURE AND THREE-GENE PHYLOGENY OF THE GENUS MALLOMONAS (SYNUROPHYCEAE)(1).

The genus Mallomonas, a common and often abundant member of the planktic community in many freshwater habitats worldwide, consists of 180 species divided into 19 sections and 23 series. Classification of species is based largely on ultrastructural characteristics of the siliceous scales and bristles that collectively form a highly organized covering over the cell. However, the relative importance of the different siliceous features of the scales, such as the dome, V rib, and secondary structures, as well as the different types of scales, in understanding the evolution and phylogeny of the genus is little known. In this study, we investigated the scale and bristle ultrastructure, along with sequences of three genes, for 19 isolates (18 species) of Mallomonas (18 isolates were from Korean habitats). The isolates represented nine of the 19 sections. Sequences for both the nuclear SSU and LSU rDNA and plastid LSU of RUBISCO (rbcL) genes for each of the 19 Mallomonas isolates and four outgroups were determined. Bayesian and maximum-likelihood (ML) analyses of the data revealed that Mallomonas consists of two strongly supported clades. Mallomonas bangladeshica (E. Takah. et T. Hayak.) Siver et A. P. Wolfe was at the base of the first clade that included taxa from the sections Planae and Heterospinae, both of which lack a V rib on the shield of the scales. Our results indicated that the sections Planae and Heterospinae should be combined. The second clade, with Mallomonas insignis Penard and Mallomonas punctifera Korshikov at the base, contained taxa from the sections Mallomonas, Striatae, Akrokomae, Annulatae, Torquatae, Punctiferae, and Insignes, all of which have V ribs or well-developed marginal ribs on the scales. Sister relationships between Mallomonas and Striatae were strongly supported, but interrelations among the remaining sections were not resolved, probably due to inclusion of too few species. Our results suggest that the current classification of the genus Mallomonas at the section level will require some revision. Additional species will need to be added in future analyses.

Complex phylogeographic patterns in the freshwater alga Synura provide new insights into ubiquity vs. endemism in microbial eukaryotes.

The global distribution, abundance, and diversity of microscopic freshwater algae demonstrate an ability to overcome significant barriers such as dry land and oceans by exploiting a range of biotic and abiotic colonization vectors. If these vectors are considered unlimited and colonization occurs in proportion to population size, then globally ubiquitous distributions are predicted to arise. This model contrasts with observations that many freshwater microalgal taxa possess true biogeographies. Here, using a concatenated multigene data set, we study the phylogeography of the freshwater heterokont alga Synura petersenii sensu lato. Our results suggest that this Synura morphotaxon contains both cosmopolitan and regionally endemic cryptic species, co-occurring in some cases, and masked by a common ultrastructural morphology. Phylogenies based on both proteins (seven protein-coding plastid and mitochondrial genes) and DNA (nine genes including ITS and 18S rDNA) reveal pronounced biogeographic delineations within phylotypes of this cryptic species complex while retaining one clade that is globally distributed. Relaxed molecular clock calculations, constrained by fossil records, suggest that the genus Synura is considerably older than currently proposed. The availability of tectonically relevant geological time (107-108 years) has enabled the development of the observed, complex biogeographic patterns. Our comprehensive analysis of freshwater algal biogeography suggests that neither ubiquity nor endemism wholly explains global patterns of microbial eukaryote distribution and that processes of dispersal remain poorly understood.

Three extant genera of freshwater thalassiosiroid diatoms from Middle Eocene sediments in northern Canada.

The evolutionary history of diatoms is only constrained partially by the fossil record. The timing of several key events, such as initial colonization of freshwater habitats by marine taxa, remains poorly resolved. Numerous specimens of the genera Cyclotella, Discostella, and Puncticulata (Ochrophyta: Thalassiosirales) have been recovered in Middle Eocene lacustrine sediments from the Giraffe Pipe locality in the Northwest Territories, Canada. These diatoms extend the fossil record of the family Stephanodiscaceae to at least 40 million years before present (Ma) and thus provide a new evolutionary milepost for the thalassiosiroid diatoms, an important clade of centric diatoms with global representation in both marine and freshwater environments. The quality of the fossil material enables detailed investigations of areolae, fultoportulae, and rimoportulae, revealing direct morphological affinities with a number of extant taxa. These observations extend the antiquity of several characters of phylogenetic importance within the thalassiosiroid diatoms, including the fultoportula, and imply that the entire lineage is considerably older than prior constraints from the fossil record, as suggested independently by several recent molecular phylogenies.

THE USE OF INTERFERENCE REFLECTION CONTRAST IN THE EXAMINATION OF DIATOM VALVES.

Even though scanning electron microscopy (SEM) is now needed to identify some species of diatoms, the majority of identifications and quantification of these organisms in ecological works is accomplished with a light microscope, using transmitted light optical methods. In this paper we demonstrate the use of interference reflection contrast (incident light) for the examination of diatoms, a method that significantly improves the resolution of structural detail, and therefore, identification of diatom taxa with light microscopy. Using incident light we were routinely able to distinguish between structures that were close to the theoretical limit of resolution for visible light, and that were not resolvable with such standard transmitted light techniques as phase contrast and differential interference contrast (DIC). Light microscopes with epi-illumination light paths can be easily and inexpensively outfitted to use this simple technique. ABBREVIATIONS: DIC, differential interference contrast; IRC, interference reflection contrast; LM, light microscopy.