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Although most of the tens of thousands of diatom species are obligate photoautotrophs, many mixotrophic species can also use extracellular organic carbon for growth, and a small number of obligate heterotrophs have lost photosynthesis entirely. We sequenced the genome of a nonphotosynthetic diatom, Nitzschia sp. strain Nitz4, to determine how carbon metabolism was altered in the wake of this rare and radical trophic shift in diatoms. Like other groups that have lost photosynthesis, the genomic consequences were most evident in the plastid genome, which is exceptionally AT-rich and missing photosynthesis-related genes. The relatively small (27 Mb) nuclear genome did not differ dramatically from photosynthetic diatoms in gene or intron density. Genome-based models suggest that central carbon metabolism, including a central role for the plastid, remains relatively intact in the absence of photosynthesis. All diatom plastids lack an oxidative pentose phosphate pathway (PPP), leaving photosynthesis as the main source of plastid NADPH. Consequently, nonphotosynthetic diatoms lack the primary source of NADPH required for essential biosynthetic pathways that remain in the plastid. Genomic models highlighted similarities between nonphotosynthetic diatoms and apicomplexan parasites for provisioning NADPH in their plastids. The ancestral absence of a plastid PPP might constrain loss of photosynthesis in diatoms compared to Archaeplastida, whose plastid PPP continues to produce reducing cofactors following loss of photosynthesis. Finally, Nitzschia possesses a complete β-ketoadipate pathway. Previously known only from fungi and bacteria, this pathway may allow mixotrophic and heterotrophic diatoms to obtain energy through the degradation of abundant plant-derived aromatic compounds.

The salinity gradient separating marine and freshwater environments is a major ecological divide, and the mechanisms by which most organisms adapt to new salinity environments are poorly understood. Diatoms are a lineage of ancestrally marine microalgae that have repeatedly colonized and diversified in freshwaters. Cyclotella cryptica is a euryhaline diatom that naturally tolerates a broad range of salinities, thus providing a powerful system for understanding the genomic mechanisms for mitigating and acclimating to low salinity. To understand how diatoms mitigate acute hypoosmotic stress, we abruptly shifted C. cryptica from seawater to freshwater and performed transcriptional profiling during the first 10 hours. Freshwater shock dramatically remodeled the transcriptome, with ~50% of the genome differentially expressed in at least one time point. The peak response occurred within 1 hour, with strong repression of genes involved in cell growth and osmolyte production, and strong induction of specific stress defense genes. Transcripts largely returned to baseline levels within 4–10 hours, with growth resuming shortly thereafter, suggesting that gene expression dynamics may be useful for predicting acclimation. Moreover, comparison to a transcriptomics study of C. cryptica following months-long acclimation to freshwater revealed little overlap between the genes and processes differentially expressed in cells exposed to acute stress versus fully acclimated conditions. Altogether, this study highlights the power of time-resolved transcriptomics to reveal fundamental insights into how cells dynamically respond to an acute environmental shift and provides new insights into how diatoms mitigate natural salinity fluctuations and have successfully diversified across freshwater habitats worldwide.

Despite the obstacles facing marine colonists, most lineages of aquatic organisms have colonized and diversified in freshwaters repeatedly. These transitions can trigger rapid morphological or physiological change and, on longer timescales, lead to increased rates of speciation and extinction. Diatoms are a lineage of ancestrally marine microalgae that have diversified throughout freshwater habitats worldwide. We generated a phylogenomic dataset of genomes and transcriptomes for 59 diatom taxa to resolve freshwater transitions in one lineage, the Thalassiosirales. Although most parts of the species tree were consistently resolved with strong support, we had difficulties resolving a Paleocene radiation, which affected the placement of one freshwater lineage. This and other parts of the tree were characterized by high levels of gene tree discordance caused by incomplete lineage sorting and low phylogenetic signal. Despite differences in species trees inferred from concatenation versus summary methods and codons versus amino acids, traditional methods of ancestral state reconstruction supported six transitions into freshwaters, two of which led to subsequent species diversification. Evidence from gene trees, protein alignments, and diatom life history together suggest that habitat transitions were largely the product of homoplasy rather than hemiplasy, a condition where transitions occur on branches in gene trees not shared with the species tree. Nevertheless, we identified a small set of putatively hemiplasious genes, many of which have been associated with shifts to low salinity, indicating that hemiplasy played a small but potentially important role in freshwater adaptation. Accounting for differences in evolutionary outcomes, in which some taxa became locked into freshwaters while others were able to return to the ocean or become salinity generalists, might help further distinguish different sources of adaptive mutation in freshwater diatoms.

A strong consensus has emerged that taxonomic classifications should be based on an underlying phylogenetic hypothesis. According to this view, named groups should be monophyletic, ensuring that a ...

The large population sizes and high dispersal potential of microbes suggests that a given microbial species should be found in all suitable habitats worldwide. Consequently, microbes should not exhibit the kinds of biogeographic patterns seen in macroorganisms. This paradigm is challenged by a growing list of exotic microbes with biogeographic disjunctions that instead promotes microbial dispersal as inherently limited. We sampled water bodies in the United States and compiled records from the literature and public databases to characterize the distribution of the freshwater planktonic diatom, Discostella asterocostata (Xie, Lin, and Cai) Houk and Klee. Discostella asterocostata was thought to be restricted to the Far East, but we report its presence in ecologically similar water bodies across the eastern United States. Populations from the U.S. and China are indistinguishable morphometrically, suggesting they may be recently separated—a hypothesis supported by paleolimnological data, which support an introduction of D. asterocostata into the U.S. as recently as the mid-1980s. The overlapping distributions of D. asterocostata and invasive carp species, in both their native and nonnative ranges, highlighted Asian carp as a possible vector for introduction of the diatom in the U.S. The existence of exotic diatoms underscores natural constraints on microbial dispersal, resulting in biogeographic distributions that can be upended through human activity.

The diatom, Cyclotella cryptica, is a well-established model species for physiological studies and biotechnology applications of diatoms. To further facilitate its use as a model diatom, we report an improved reference genome assembly and annotation for C. cryptica strain CCMP332. We used a combination of long- and short-read sequencing to assemble a high-quality and contaminant-free genome. The genome is 171 Mb in size and consists of 662 scaffolds with a scaffold N50 of 494 kb. This represents a 176-fold decrease in scaffold number and 41-fold increase in scaffold N50 compared to the previous assembly. The genome contains 21,250 predicted genes, 75% of which were assigned putative functions. Repetitive DNA comprises 59% of the genome, and an improved classification of repetitive elements indicated that a historically steady accumulation of transposable elements has contributed to the relatively large size of the C. cryptica genome. The high-quality C. cryptica genome will serve as a valuable reference for ecological, genetic, and biotechnology studies of diatoms.

We used light and electron microscopy to characterize a Late Miocene freshwater diatomite deposit in the Chalk Hills Formation, near Payette, Idaho, USA. The diatom assemblage from this sample was ...

Intricate networks of single-celled eukaryotes (protists) dominate carbon flow in the ocean. Their growth, demise, and interactions with other microorganisms drive the fluxes of biogeochemical elements through marine ecosystems. Mixotrophic protists are capable of both photosynthesis and ingestion of prey and are dominant components of open-ocean planktonic communities. Yet, the role of mixotrophs in elemental cycling is obscured by their capacity to act as primary producers or heterotrophic consumers depending on factors that remain largely uncharacterized. Here we introduce a machine learning model that can predict thein situnutritional mode of aquatic protists based on their patterns of gene expression. This approach leverages a public collection of protist transcriptomes as a training set to identify a subset of gene families whose transcriptional profiles predict trophic status. We applied our model to nearly 100 metatranscriptomes obtained during two oceanographic cruises in the North Pacific and found community-level and population-specific evidence that abundant open-ocean mixotrophic populations shift their predominant mode of nutrient and carbon acquisition in response to natural gradients in nutrient supply and sea surface temperature. In addition, metatranscriptomic data from ship-board incubation experiments revealed that abundant mixotrophic prymnesiophytes from the oligotrophic North Pacific subtropical gyre rapidly remodelled their transcriptome to enhance photosynthesis when supplied with limiting nutrients. Coupling the technique introduced here with experiments designed to reveal the mechanisms driving mixotroph physiology is a promising approach for understanding the ecology of mixotrophic populations in the natural environment.Significance statementMixotrophy is a ubiquitous nutritional strategy in marine ecosystems. Although our understanding of the distribution and abundance of mixotrophic plankton has improved significantly, the functional roles of mixotrophs are difficult to pinpoint, as mixotroph nutritional strategies are flexible and form a continuum between heterotrophy and phototrophy. We employ a machine learning-driven metatranscriptomic technique to assess the nutritional strategies of abundant planktonic populationsin situand demonstrate that mixotrophic populations play varying functional roles along physico-chemical gradients in the North Pacific Ocean, revealing a degree of physiological plasticity unique to aquatic mixotrophs. Our results highlight mechanisms that may dictate the flow of biogeochemical elements and the ecology of the North Pacific Ocean, one of the largest biogeographical provinces on Earth.

In contrast to surveys based on a few genes that often provide limited taxonomic resolution, transcriptomes provide a wealth of genomic loci that can resolve relationships among taxonomically challenging lineages. Diatoms are a diverse group of aquatic microalgae that includes important bioindicator species and many such lineages. One example is Nitzschia palea, a widespread species complex with several morphologically based taxonomic varieties, some of which are critical pollution indicators. Morphological differences among the varieties are subtle and phylogenetic studies on a few genes fail to resolve their evolutionary relationships. We conducted morphometric and transcriptome analyses of 10 Nitzschia palea strains to resolve the relationships among strains and taxonomic varieties. Nitzschia palea was resolved into three clades, one of which corresponds to a group of strains with narrow linear-lanceolate valves. The other morphological group recovered in the shape outline analysis was not monophyletic and consisted of two clades. Subsequent gene-tree concordance analyses and phylogenetic network estimations revealed patterns of incomplete lineage sorting and gene flow between intraspecific lineages. We detected reticulated evolutionary patterns among lineages with different morphologies and a resulting putative recent hybrid. Our study shows that phylogenomic analyses of many unlinked nuclear loci, complemented with morphometrics, can resolve complex evolutionary histories of recently diverged species complexes.

The salinity gradient separating marine and freshwater environments represents a major ecological divide for microbiota, yet the mechanisms by which marine microbes have adapted to and ultimately diversified in freshwater environments are poorly understood. Here, we take advantage of a natural evolutionary experiment: the colonization of the brackish Baltic Sea by the ancestrally marine diatom Skeletonema marinoi. To understand how diatoms respond to low salinity, we characterized transcriptomic responses of S. marinoi grown in a common garden. Our experiment included eight genotypes from source populations spanning the Baltic Sea salinity cline. Changes in gene expression revealed a shared response to salinity across genotypes, where low salinities induced profound changes in cellular metabolism, including upregulation of carbon fixation and storage compound biosynthesis, and increased nutrient demand and oxidative stress. Nevertheless, the genotype effect overshadowed the salinity effect, as genotypes differed significantly in their response, both in the magnitude and direction of gene expression. Intraspecific differences included regulation of transcription and translation, nitrogen metabolism, cell signaling, and aerobic respiration. The high degree of intraspecific variation in gene expression observed here highlights an important but often overlooked source of biological variation associated with how diatoms respond and adapt to environmental change.

We report draft genomes of two bacterial strains in the genera Hyphobacterium and Reichenbachiella, which are associated with the diatom Cyclotella cryptica strain CCMP332. Genomes from these strains were 2,691,501 bp and 3,325,829 bp in size, respectively, and will be useful for understanding interactions between diatoms and bacteria., We report draft genomes of two bacterial strains in the genera Hyphobacterium and Reichenbachiella, which are associated with the diatom Cyclotella cryptica strain CCMP332. Genomes from these strains were 2,691,501 bp and 3,325,829 bp, respectively, and will be useful for understanding interactions between diatoms and bacteria.

The diatom,Cyclotella cryptica, is a well-established experimental model for physiological studies and, more recently, biotechnology applications of diatoms. To further facilitate its use as a model diatom species, we report an improved reference genome assembly and annotation forC. crypticastrain CCMP332. We used a combination of long- and short-read sequencing to assemble a high-quality and contaminant-free genome. The genome is 171 Mb in size and consists of 662 scaffolds with a scaffold N50 of 494 kb. This represents a 176-fold decrease in scaffold number and 41-fold increase in scaffold N50 compared to the previous assembly. The genome contains 21,250 predicted genes, 75% of which were assigned putative functions. Repetitive DNA comprises 59% of the genome, and an improved classification of repetitive elements indicated that a historically steady accumulation of transposable elements has contributed to the relatively large size of theC. crypticagenome. The high-qualityC. crypticagenome will serve as a valuable reference for ecological, genetic, and biotechnology studies of diatoms.Data available fromNCBI BioProjects PRJNA628076 and PRJNA589195

We sequenced mitochondrial genomes from five diverse diatoms (Toxarium undulatum, Psammoneis japonica, Eunotia naegelii, Cylindrotheca closterium, and Nitzschia sp.), chosen to fill important phylogenetic gaps and help us characterize broadscale patterns of mitochondrial genome evolution in diatoms. Although gene content was strongly conserved, intron content varied widely across species. The vast majority of introns were of group II type and were located in the cox1 or rnl genes. Although recurrent intron loss appears to be the principal underlying cause of the sporadic distributions of mitochondrial introns across diatoms, phylogenetic analyses showed that intron distributions superficially consistent with a recurrent-loss model were sometimes more complicated, implicating horizontal transfer as a likely mechanism of intron acquisition as well. It was not clear, however, whether diatoms were the donors or recipients of horizontally transferred introns, highlighting a general challenge in resolving the evolutionary histories of many diatom mitochondrial introns. Although some of these histories may become clearer as more genomes are sampled, high rates of intron loss suggest that the origins of many diatom mitochondrial introns are likely to remain unclear.

Summary Patterns of species richness are commonly linked to life history strategies. In diatoms, an exceptionally diverse lineage of photosynthetic heterokonts important for global photosynthesis and burial of atmospheric carbon, lineages with different locomotory and reproductive traits differ dramatically in species richness, but any potential association between life history strategy and diversification has not been tested in a phylogenetic framework.We constructed a time‐calibrated, 11‐gene, 1151‐taxon phylogeny of diatoms – the most inclusive diatom species tree to date. We used this phylogeny, together with a comprehensive inventory of first–last occurrences of Cenozoic fossil diatoms, to estimate ranges of expected species richness, diversification and its variation through time and across lineages.Diversification rates varied with life history traits. Although anisogamous lineages diversified faster than oogamous ones, this increase was restricted to a nested clade with active motility in the vegetative cells.We propose that the evolution of motility in vegetative cells, following an earlier transition from oogamy to anisogamy, facilitated outcrossing and improved utilization of habitat complexity, ultimately leading to enhanced opportunity for adaptive divergence across a variety of novel habitats. Together, these contributed to a species radiation that gave rise to the majority of present‐day diatom diversity.

Diatoms (Bacillariophyta) are a species-rich group of eukaryotic microbes diverse in morphology, ecology, and metabolism. Previous reconstructions of the diatom phylogeny based on one or a few genes have resulted in inconsistent resolution or low support for critical nodes. We applied phylogenetic paralog pruning techniques to a data set of 94 diatom genomes and transcriptomes to infer perennially difficult species relationships, using concatenation and summary-coalescent methods to reconstruct species trees from data sets spanning a wide range of thresholds for taxon and column occupancy in gene alignments. Conflicts between gene and species trees decreased with both increasing taxon occupancy and bootstrap cutoffs applied to gene trees. Concordance between gene and species trees was lowest for short internodes and increased logarithmically with increasing edge length, suggesting that incomplete lineage sorting disproportionately affects species tree inference at short internodes, which are a common feature of the diatom phylogeny. Although species tree topologies were largely consistent across many data treatments, concatenation methods appeared to outperform summary-coalescent methods for sparse alignments. Our results underscore that approaches to species-tree inference based on few loci are likely to be misled by unrepresentative sampling of gene histories, particularly in lineages that may have diversified rapidly. In addition, phylogenomic studies of diatoms, and potentially other hyperdiverse groups, should maximize the number of gene trees with high taxon occupancy, though there is clearly a limit to how many of these genes will be available.

How diatoms respond to fluctuations in osmotic pressure is important from both ecological and applied perspectives. It is well known that osmotic stress affects photosynthesis and can result in the accumulation of compounds desirable in pharmaceutical and alternative fuel industries. Gene expression responses to osmotic stress have been studied in short-term trials, but it is unclear whether the same mechanisms are recruited during long-term acclimation. We used RNA-seq to study the genome-wide transcription patterns in the euryhaline diatom, Cyclotella cryptica, following long-term acclimation to salinity that spanned the natural range of fresh to oceanic water. Long-term acclimated C. cryptica exhibited induced synthesis or repressed degradation of the osmolytes glycine betaine, taurine and dimethylsulfoniopropionate (DMSP). Although changes in proline concentration is one of the main responses in short-term osmotic stress, we did not detect a transcriptional change in proline biosynthetic pathways in our long-term experiment. Expression of membrane transporters showed a general tendency for increased import of potassium and export of sodium, consistent with the electrochemical gradients and dependence on co-transported molecules. Our results show substantial between-genotype differences in growth and gene expression reaction norms and suggest that the regulation of proline synthesis important in short-term osmotic stress might not be maintained in long-term acclimation. Further examination using time-course gene expression experiments, metabolomics and genetic validation of gene functions would reinforce patterns inferred from RNA-seq data.

PREMISE OF THE STUDY Loss of photosynthesis is a common and often repeated trajectory in nearly all major groups of photosynthetic eukaryotes. One small subset of "apochloritic" diatoms in the genus Nitzschia have lost their ability to photosynthesize and require extracellular carbon for growth. Similar to other secondarily nonphotosynthetic taxa, apochloritic diatoms maintain colorless plastids with highly reduced plastid genomes. Although the narrow taxonomic breadth of apochloritic Nitzschia suggests a single loss of photosynthesis in their common ancestor, previous phylogenetic analyses suggested that photosynthesis was lost multiple times. METHODS We analyzed genes from the nuclear, plastid, and mitochondrial genomes for a broad set of taxa to test whether photosynthesis was lost one or multiple times in Bacillariales. We also sequenced and characterized the plastid genome of a nonphotosynthetic Nitzschia species. KEY RESULTS Phylogenetic analyses showed that genes from all three genetic compartments either supported or failed to reject monophyly of apochloritic Nitzschia species, consistent with a single loss of photosynthesis in this group. The plastid genomes of two apochloritic Nitzschia are highly similar in all respects, indicating streamlining of the plastid genome before the split of these two species. CONCLUSIONS A better understanding of the phylogeny and ecology of apochloritic Nitzschia, together with emerging genomic resources, will help identify the factors that have driven and maintained the loss of photosynthesis in this group of diatoms. Finally, some habitats host diverse communities of co-occurring nonphotosynthetic diatoms, reflecting resource abundance or resource partitioning in ecologically favorable habitats.

Many clades that span the marine-freshwater boundary are disproportionately more diverse in the younger, shorter-lived, and scarcer freshwater environments than they are in the marine realm. This disparity is thought to be related to differences in diversification rates between marine and freshwater lineages. However, marine and freshwaters are not ecologically homogeneous, so the study of diversification across the salinity divide should also account for other potentially interacting variables. In diatoms, freshwater and substrate-associated (benthic) lineages are several-fold more diverse than their marine and suspended (planktonic) counterparts. These imbalances provide an excellent system to understand whether these variables interact with diversification. Using multistate hidden-state speciation and extinction models we found that freshwater lineages diversify faster than marine lineages regardless of whether they inhabit the plankton or the benthos. Freshwater lineages also had higher turnover rates (speciation + extinction), suggesting that habitat transitions impact speciation and extinction rates jointly. The plankton-benthos contrast was also consistent with state-dependent diversification, but with modest differences in diversification and turnover rates. Asymmetric, and bidirectional transitions rejected hypotheses about the plankton and freshwaters as absorbing, inescapable habitats. Our results further suggest that the high turnover rate of freshwater diatoms is related to high turnover of freshwater systems themselves.

Loss of photosynthesis is a common and often repeated trajectory in nearly all major groups of photosynthetic eukaryotes. One small subset of ‘apochloritic’ diatoms in the genus Nitzschia have lost their ability to photosynthesize and require extracellular carbon for growth. Similar to other secondarily nonphotosynthetic taxa, apochloritic diatoms maintain colorless plastids with highly reduced plastid genomes. Although the narrow taxonomic breadth of apochloritic diatoms suggests a single loss of photosynthesis in the common ancestor of these species, previous phylogenetic analyses suggested that photosynthesis was lost multiple times. We sequenced additional phylogenetic markers from the nuclear and mitochondrial genomes for a larger set of taxa and found that the best trees for datasets representing all three genetic compartments provided low to moderate support for monophyly of apochloritic Nitzschia, consistent with a single loss of photosynthesis in diatoms. We sequenced the plastid genome of one apochloritic species and found that it was highly similar in all respects to the plastid genome of another apochloritic Nitzschia species, indicating that streamlining of the plastid genome had completed prior to the split of these two species. Finally, it is increasingly clear that some locales host relatively large numbers apochloritic Nitzschia species that span the phylogenetic diversity of the group, indicating that these species co-exist because of resource abundance or resource partitioning in ecologically favorable habitats. A better understanding of the phylogeny and ecology of this group, together with emerging genomic resources, will help identify the factors that have driven and maintained the loss of photosynthesis in this group, a rare event in diatoms.

Metabarcoding has offered unprecedented insights into microbial diversity. In many studies, short DNA sequences are binned into consecutively lower Linnaean ranks, and ranked groups (e.g., genera) are the units of biodiversity analyses. These analyses assume that Linnaean ranks are biologically meaningful and that identically ranked groups are comparable. We used a metabarcode dataset for marine planktonic diatoms to illustrate the limits of this approach. We found that the 20 most abundant marine planktonic diatom genera ranged in age from 4 to 134 million years, indicating the non-equivalence of genera because some have had more time to diversify than others. However, species richness was largely independent of genus age, suggesting that disparities in species richness among genera were better explained by variation in rates of speciation and extinction. Taxonomic classifications often do not reflect phylogeny, so genus-level analyses can include phylogenetically nested genera, further confounding rank-based analyses. These results underscore the indispensable role of phylogeny in understanding patterns of microbial diversity.

Using a broadly sampled, time-calibrated phylogeny of Cyanobacteria and photosynthetic eukaryotes, Sanchez-Baracaldo et al. (1) report that the most-recent common ancestor (MRCA) of Archaeplastida likely inhabited a low-salinity (freshwater) habitat, providing an advance on a longstanding debate about the ecological context for the origin of plastids (2). The freshwater ancestry for Cyanobacteria and Archaeplastida was based on reconstructions that assumed equal transition rates among marine, brackish, and freshwater habitats (1), which likely oversimplified the 2.5-billion-year history of these groups. We relaxed the assumption of a single transition rate by fitting a model with additional parameters for independent transition rates among marine, brackish, and freshwater habitats. This model provided a … [↵][1]1To whom correspondence should be addressed. Email: tnakov{at}uark.edu. [1]: #xref-corresp-1-1

Premise of the studyDiatoms are one of the most species-rich lineages of microbial eukaryotes. Similarities in clade age, species richness, and contributions to primary production motivate comparisons to flowering plants, whose genomes have been inordinately shaped by whole genome duplication (WGD). These events that have been linked to speciation and increased rates of lineage diversification, identifying WGDs as a principal driver of angiosperm evolution. We synthesized a relatively large but scattered body of evidence that, taken together, suggests that polyploidy may be common in diatoms.MethodsWe used data from gene counts, gene trees, and patterns of synonymous divergence to carry out the first large-scale phylogenomic analysis of genome-scale duplication histories for a phylogenetically diverse set of 37 diatom taxa.Key resultsSeveral methods identified WGD events of varying age across diatoms, though determining the exact number and placement of events and, more broadly, inferences of WGD at all, were greatly impacted by gene-tree uncertainty. Gene-tree reconciliations supported allopolyploidy as the predominant mode of polyploid formation, with particularly strong evidence for ancient allopolyploid events in the thalassiosiroid and pennate diatom clades.ConclusionsWhole genome duplication appears to have been an important driver of genome evolution in diatoms. Denser taxon sampling will better pinpoint the timing of WGDs and likely reveal many more of them. We outline potential challenges in reconstructing paleopolyploid events in diatoms that, together with these results, offer a framework for understanding the evolutionary roles of genome duplication in a group that likely harbors substantial genomic diversity.

Metabarcoding has offered unprecedented insights into microbial diversity. In many studies, short DNA sequences are binned into consecutively higher Linnaean ranks, and ranked groups (e.g., genera) are the units of biodiversity analyses. These analyses assume that Linnaean ranks are biologically meaningful and that identically ranked groups are comparable. We used a meta-barcode dataset for marine planktonic diatoms to illustrate the limits of this approach. We found that the 20 most abundant marine planktonic diatom genera ranged in age from 4 to 134 million years, indicating the non-equivalence of genera because some had more time to diversify than others. Still, species richness was only weakly correlated with genus age, highlighting variation in rates of speciation and/or extinction. Taxonomic classifications often do not reflect phylogeny, so genus-level analyses can include phylogenetically nested genera, further confounding rank-based analyses. These results underscore the indispensable role of phylogeny in understanding patterns of microbial diversity.

Photosynthesis by diatoms accounts for roughly one-fifth of global primary production, but despite this, relatively little is known about their plastid genomes. We report the completely sequenced plastid genomes for eight phylogenetically diverse diatoms and show them to be variable in size, gene and foreign sequence content, and gene order. The genomes contain a core set of 122 protein-coding genes, with 15 additional genes exhibiting complex patterns of 1) gene losses at varying phylogenetic scales, 2) functional transfers to the nucleus, 3) gene duplication, divergence, and differential retention of paralogs, and 4) acquisitions of putatively functional recombinase genes from resident plasmids. The newly sequenced genomes also contain several previously unreported genes, highlighting how poorly characterized diatom plastid genomes are overall. Genome size variation reflects major expansions of the inverted repeat region in some cases but, more commonly, large-scale expansions of intergenic regions, many of which contain unique open reading frames of likely foreign origin. Although many gene clusters are conserved across species, rearrangements appear to be frequent in most lineages.

With species found throughout both marine and fresh waters, the diatom order Thalassiosirales is one of the most phylogenetically and ecologically diverse lineages of planktonic diatoms. A clear understanding of the timescale of Thalassiosirales evolution would provide novel insights into the rates and patterns of species diversification associated with major habitat shifts, as well as provide valuable context for understanding the age and evolutionary history of the model species, Cyclotella nana (= Thalassiosira pseudonana). The freshwater fossil record for Thalassiosirales is extensive, well characterized, and generally supportive of a Miocene origin for the major freshwater lineages. The marine record is, by comparison, more sparse and in many cases, unverified. The discovery of freshwater thalassiosiroids in Eocene sediments pushed the freshwater fossil record considerably further back in time, highlighting an apparent gap of some 30 million years. An alternative interpretation is that the Miocene and Eocene reports represent competing hypotheses. In the absence of additional independent and decisive fossil data, I explored the relative plausibility of these two scenarios with Bayesian relaxed molecular clock methods under a range of fossil calibration schemes. Although I found no support for the Eocene fossil dates, the two major freshwater colonization events probably occurred much earlier than previously thought—as early as the Paleocene for Cyclotella, followed by an Eocene origin for the cyclostephanoid lineage. Much of the extant freshwater diversity in both lineages traces back to the Miocene, however, giving the impression of a single Miocene origin. Efforts to infer the timescale of Thalassiosirales evolution more accurately would benefit from a systematic reevaluation of the marine fossil record and formal integration of fossil species into existing phylogenetic hypotheses.

Strategies for optimizing fitness in a dilute, competitive, and changing environment are thought to underlie cell size evolution in phytoplankton. Support for cell size as an adaptive trait comes from observed shifts in cell size distributions in response to environmental cues at geologic time scales and across environmental gradients. Physicochemical differences between marine and freshwater environments are thought to drive diatom cell size evolution in opposite directions, with larger sizes conferring benefits in marine habitats and small sizes in freshwater habitats. We tested this hypothesis in one lineage of diatoms, the Thalassiosirales, which spans marine and freshwater habitats, has a well-supported phylogeny, and whose members are relatively homogenous with respect to cell shape, growth habit, and habitat preference. A comparison of adaptive models for cell size evolution supports the hypothesis for different cell size optima between marine and freshwater habitats. The data are best explained by a model with separate selective regimes for marine and freshwater lineages. However, a scenario of stabilizing selection towards a single global cell size optimum irrespective of habitat cannot be completely discounted. Understanding of the processes that shape cell size evolution in phytoplankton would benefit from models that incorporate phylogeny, intrinsic properties of species (e.g., cell shape, colony formation, and motility), and more specific habitat characterization, as well as genetic and genomic properties of different phytoplankton groups.

Light-harvesting antennae are critical for collecting energy from sunlight and providing it to photosynthetic reaction centers. Their abundance and composition are tightly regulated to maintain efficient photosynthesis in changing light conditions. Many cyanobacteria alter their light-harvesting antennae in response to changes in ambient light-color conditions through the process of chromatic acclimation. The control of green light induction (Cgi) pathway is a light-color-sensing system that controls the expression of photosynthetic genes during chromatic acclimation, and while some evidence suggests that it operates via transcription attenuation, the components of this pathway have not been identified. We provide evidence that translation initiation factor 3 (IF3), an essential component of the prokaryotic translation initiation machinery that binds the 30S subunit and blocks premature association with the 50S subunit, is part of the control of green light induction pathway. Light regulation of gene expression has not been previously described for any translation initiation factor. Surprisingly, deletion of the IF3-encoding gene infCa was not lethal in the filamentous cyanobacterium Fremyella diplosiphon, and its genome was found to contain a second, redundant, highly divergent infC gene which, when deleted, had no effect on photosynthetic gene expression. Either gene could complement an Escherichia coli infC mutant and thus both encode bona fide IF3s. Analysis of prokaryotic and eukaryotic genome databases established that multiple infC genes are present in the genomes of diverse groups of bacteria and land plants, most of which do not undergo chromatic acclimation. This suggests that IF3 may have repeatedly evolved important roles in the regulation of gene expression in both prokaryotes and eukaryotes.

The hybrid pigeonpea (Cajanus cajan) breeding technology based on cytoplasmic male sterility (CMS) is currently unique among legumes and displays major potential for yield increase. CMS is defined as a condition in which a plant is unable to produce functional pollen grains. The novel chimeric open reading frames (ORFs) produced as a results of mitochondrial genome rearrangements are considered to be the main cause of CMS. To identify these CMS-related ORFs in pigeonpea, we sequenced the mitochondrial genomes of three C. cajan lines (the male-sterile line ICPA 2039, the maintainer line ICPB 2039, and the hybrid line ICPH 2433) and of the wild relative (Cajanus cajanifolius ICPW 29). A single, circular-mapping molecule of length 545.7 kb was assembled and annotated for the ICPA 2039 line. Sequence annotation predicted 51 genes, including 34 protein-coding and 17 RNA genes. Comparison of the mitochondrial genomes from different Cajanus genotypes identified 31 ORFs, which differ between lines within which CMS is present or absent. Among these chimeric ORFs, 13 were identified by comparison of the related male-sterile and maintainer lines. These ORFs display features that are known to trigger CMS in other plant species and to represent the most promising candidates for CMS-related mitochondrial rearrangements in pigeonpea.

The genus Cucurbita (Cucurbitaceae) includes five species that were domesticated independently in the Americas, giving rise to an immense diversity of squashes, pumpkins, and gourds. To gain an improved understanding of the evolution of Cucurbita and its domesticated taxa, we used four chloroplast loci to estimate the phylogeny of 23 taxa that represent the broad-level diversity within Cucurbita. Our results provide a strongly supported framework hypothesis for the phylogeny of the genus, robustly confirming the basal position of the C. digitata group of xerophytic perennials and the monophyly of a large group of mesophytic annuals that represent most of the known diversity in the genus, both wild and domesticated. The chloroplast evidence provides strong support for a novel grouping of the mesophytic annual C. ficifolia (known only from cultivation) with the xerophytic perennials C. foetidissima and C. pedatifolia. This study also provides the first DNA-based evidence in support of the isozyme-based hypothesis that C. pepo subsp. ovifera var. ovifera (represented by most ornamental gourds and several squashes) was domesticated from the wild taxon C. pepo subsp. ovifera var. ozarkana. This lends support to the hypothesis that var. ovifera was domesticated in the eastern United States and that this region served as one of about 10 independent centers of origin of human agriculture. Although the level of bootstrap support for this and certain other peripheral relationships in Cucurbita is low, definitive resolution of these issues is within reach, as next-generation sequencing should soon deliver entire organelle genome sequences from a comprehensive sampling of the genus.

Although the plastid genomes of diatoms maintain a conserved architecture and core gene set, considerable variation about this core theme exists and can be traced to several different processes. Gene duplication, pseudogenization, and loss, as well as intracellular transfer of genes to the nuclear genome, have all contributed to variation in gene content among diatom species. In addition, some noncoding sequences have highly restricted phylogenetic distributions that suggest a recent foreign origin. We sequenced the plastid genome of the marine diatom, Toxarium undulatum, and found that the genome contains three genes (chlB, chlL, and chlN) involved in light-independent chlorophyll a biosynthesis that were not previously known from diatoms. Phylogenetic and syntenic data suggest that these genes were differentially retained in this one lineage as they were repeatedly lost from most other diatoms. Unique among diatoms and other heterokont algae sequenced so far, the genome also contains a large group II intron within an otherwise intact psaA gene. Although the intron is most similar to one in the plastid-encoded psaA gene of some green algae, high sequence divergence between the diatom and green algal introns rules out recent shared ancestry. We conclude that the psaA intron was likely introduced into the plastid genome of T. undulatum, or some earlier ancestor, by horizontal transfer from an unknown donor. This genome further highlights the myriad processes driving variation in gene and intron content in the plastid genomes of diatoms, one of the world's foremost primary producers.

The Surirellales and Rhopalodiales are large, widespread, and morphologically diverse groups of raphid pennate diatoms (Bacillariphyta) whose raphe, a structure that facilitates active motility, opens internally into a siliceous canal. We collected 202 representatives of the lineage and sequenced genes from the nuclear, plastid, and mitochondrial genomes to infer phylogenetic relationships as a basis for comparative study of ecology and morphological evolution as well as reclassification. The lineage was ancestrally marine, and we report the first evidence for a ‘stepping stone’ model of marine–freshwater transitions in which freshwater invasions were preceded by adaptation to intermediate brackish habitats. Phylogenetic comparative analyses also showed that the shift from an apical (e.g., Entomoneis) to transapical major axis of development (e.g., Surirella) did not have to proceed through subcircular intermediate forms (i.e., Campylodiscus). Rather, subcircular forms evolved both within lineages with longer apical axis or longer transapical axis. We also used the inferred phylogeny as a basis for genus-level reclassification of the lineage. Campylodiscus now includes the fastuosoid members of Surirella and Campylodiscus, but excludes other marine Campylodiscus which are now classified as Coronia. Surirella includes the Surirella striatula clade, Surirella Pinnatae group, and species formerly classified as Cymatopleura. We resurrected the genus Iconella to accommodate Stenopterobia and the robustoid members of Surirella and Campylodiscus. We broadened Epithemia to include members of the paraphyletic genus Rhopalodia. Finally, we discuss the challenges of constructing a classification that best leverages available phylogenetic data, while minimizing disruption to the research community and recognizing practical considerations stemming from the slow rate of progress on systematic studies of understudied organisms.

The angiosperm genus Silene exhibits some of the most extreme and rapid divergence ever identified in mitochondrial genome architecture and nucleotide substitution rates. These patterns have been considered mitochondrial specific based on the absence of correlated changes in the small number of available nuclear and plastid gene sequences. To better assess the relationship between mitochondrial and plastid evolution, we sequenced the plastid genomes from four Silene species with fully sequenced mitochondrial genomes. We found that two species with fast-evolving mitochondrial genomes, S. noctiflora and S. conica, also exhibit accelerated rates of sequence and structural evolution in their plastid genomes. The nature of these changes, however, is markedly different from those in the mitochondrial genome. For example, in contrast to the mitochondrial pattern, which appears to be genome wide and mutationally driven, the plastid substitution rate accelerations are restricted to a subset of genes and preferentially affect nonsynonymous sites, indicating that altered selection pressures are acting on specific plastid-encoded functions in these species. Indeed, some plastid genes in S. noctiflora and S. conica show strong evidence of positive selection. In contrast, two species with more slowly evolving mitochondrial genomes, S. latifolia and S. vulgaris, have correspondingly low rates of nucleotide substitution in plastid genes as well as a plastid genome structure that has remained essentially unchanged since the origin of angiosperms. These results raise the possibility that common evolutionary forces could be shaping the extreme but distinct patterns of divergence in both organelle genomes within this genus.

Theoretical arguments suggest that mutation rates influence the proliferation and maintenance of RNA editing. We identified RNA editing sites in five species within the angiosperm genus Silene that exhibit highly divergent mitochondrial mutation rates. We found that mutational acceleration has been associated with rapid loss of mitochondrial editing sites. In contrast, we did not find a significant difference in the frequency of editing in chloroplast genes, which lack the mutation rate variation observed in the mitochondrial genome. As found in other angiosperms, the rate of substitution at RNA editing sites in Silene greatly exceeds the rate at synonymous sites, a pattern that has previously been interpreted as evidence for selection against RNA editing. Alternatively, we suggest that editing sites may experience higher rates of C-to-T mutation than other portions of the genome. Such a pattern could be caused by gene conversion with reverse-transcribed mRNA (i.e., retroprocessing). If so, the genomic distribution of RNA editing site losses in Silene suggests that such conversions must be occurring at a local scale such that only one or two editing sites are affected at a time. Because preferential substitution at editing sites appears to occur in angiosperms regardless of the mutation rate, we conclude that mitochondrial rate accelerations within Silene have “fast-forwarded” a preexisting pattern but have not fundamentally changed the evolutionary forces acting on RNA editing sites.

The mitochondrial genomes of seed plants are unusually large and vary in size by at least an order of magnitude. Much of this variation occurs within a single family, the Cucurbitaceae, whose genomes range from an estimated 390 to 2,900 kb in size. We sequenced the mitochondrial genomes of Citrullus lanatus (watermelon: 379,236 nt) and Cucurbita pepo (zucchini: 982,833 nt)--the two smallest characterized cucurbit mitochondrial genomes--and determined their RNA editing content. The relatively compact Citrullus mitochondrial genome actually contains more and longer genes and introns, longer segmental duplications, and more discernibly nuclear-derived DNA. The large size of the Cucurbita mitochondrial genome reflects the accumulation of unprecedented amounts of both chloroplast sequences (>113 kb) and short repeated sequences (>370 kb). A low mutation rate has been hypothesized to underlie increases in both genome size and RNA editing frequency in plant mitochondria. However, despite its much larger genome, Cucurbita has a significantly higher synonymous substitution rate (and presumably mutation rate) than Citrullus but comparable levels of RNA editing. The evolution of mutation rate, genome size, and RNA editing are apparently decoupled in Cucurbitaceae, reflecting either simple stochastic variation or governance by different factors.

Diatoms are unicellular heterokont algae known most notably for their elaborately ornamented cell walls of opaline silica. The scanning electron microscope (SEM) revolutionized diatom systematics, revealing taxonomically important ultrastructural features that were otherwise hidden from the light microscope. The SEM quickly became an important tool for taxon delimitation, and SEM data are now foundational to nearly all levels of the diatom classification system (Round et al. 1990). The introduction of multivariate statistical methods to diatom studies represented a similar advance, revealing complex sets of continuous morphometric characters to taxonomists for the first time. Although less commonly employed than the SEM, morphometric analysis has facilitated the discovery of numerous previously undescribed taxa (e.g., Mann et al. 2004; Theriot and Stoermer 1984). Most recently, the application of molecular biological techniques to systematic studies of diatoms has revealed still more variation, and these methods now play an important role in the discovery and delimitation of diatom species. Each of these tools enhanced our ability to detect and extract subtle differences, and in each case the diatom species category became effectively smaller and more exclusive. This is evident in the increasingly common discovery of "cryptic" species—morphologically similar but genetically distinct species that, at some point, shared the same specific epithet. In some cases, DNA sequence data have indeed provided compelling evidence for the existence of cryptic species, in which the rates of morphological and molecular evolution have apparently been decoupled. In other cases, DNA sequence data retrained the eye to reveal subtle morphological differences, so there was corroborating evidence from morphology and molecules to differentiate multiple species from what previously had been considered one. These species are sometimes referred to as "semi-" or "pseudo-cryptic," but the term "previously undetected" seems to apply equally well. The power of the molecular systematic approach to species research, though fully established (Avise 2000), is just being realized for diatoms. This article focuses on the burgeoning use of molecular data to discover and delimit diatom species. The reader is referred to Mann (1999) for valuable historical context and a more thorough account of the traditional approaches to diatom taxonomy. This review instead will focus on studies that use phylogenetic analysis of DNA sequences to identify and delimit species of diatoms. The approach has a proven history of success, particularly in animal systems. A simple glance at the animal literature on species-level molecular systematics, and the closely related field of phylogeography, demonstrates the great potential of this approach to transform species research in diatoms. The molecular systematic approach is not without its challenges, however, as is clear from the general literature (e.g., Funk and Omland 2003) and the handful of diatom studies undertaken so far. This review will draw from those examples to highlight both the promises and problems of the molecular systematic approach to species delimitation. First, the extent to which common diatom species concepts influence or constrain species delimitation, and the advantages of adopting a lineage-based species concept, will be discussed. Second, different genetic markers can provide conflicting evidence about species boundaries, highlighting the importance of marker choice, particularly for single-locus studies. Several advantageous properties of mitochondrial DNA (mtDNA) for species-level studies are reviewed, which together recommend mtDNA as a better "first pass" molecular marker over the traditional nuclear ribosomal DNA (rDNA). Finally, the importance of species monophyly is discussed. To date, most species-level molecular systematic studies of diatoms have focused on the detection of cryptic species, which are, with rare exceptions (Slapeta et al. 2006), very likely to be recently diverged and therefore unlikely to exhibit reciprocal monophyly in their gene trees. This underscores the need to develop and evaluate numerous low-copy nuclear markers for systematics research in diatoms.

Horizontal gene transfer is surprisingly common among plant mitochondrial genomes. The first well-established case involves a homing group I intron in the mitochondrial cox1 gene shown to have been frequently acquired via horizontal transfer in angiosperms. Here, we report extensive additional sampling of angiosperms, including 85 newly sequenced introns from 30 families. Analysis of all available data leads us to conclude that, among the 640 angiosperms (from 212 families) whose cox1 intron status has been characterized thus far, the intron has been acquired via roughly 70 separate horizontal transfer events. We propose that the intron was originally seeded into angiosperms by a single transfer from fungi, with all subsequent inferred transfers occurring from one angiosperm to another. The pattern of angiosperm-to-angiosperm transfer is biased toward exchanges between plants belonging to the same family. Illegitimate pollination is proposed as one potential factor responsible for this pattern, given that aberrant, cross-species pollination is more likely between close relatives. Other potential factors include shared vectoring agents or common geographic locations. We report the first apparent cases of loss of the cox1 intron; losses are accompanied by retention of the exonic coconversion tract, which is located immediately downstream of the intron and which is a product of the intron's self-insertion mechanism. We discuss the many reasons why the cox1 intron is so frequently and detectably transferred, and rarely lost, and conclude that it should be regarded as the “canary in the coal mine” with respect to horizontal transfer in angiosperm mitochondria.

Marine and freshwater diatoms show several important differences in silicon physiology. In addition to containing an order of magnitude more silica in their cell walls, freshwater diatoms also appear to have a less efficient silicic acid uptake mechanism. A novel set of silicon transporters (SITs), encoded by a small gene family, import silicic acid from the environment into the diatom cell. Some evidence suggests that the disparity in uptake efficiency between marine and freshwater diatoms might be attributable to different demands on SITs in the two environments. To test this hypothesis, partial SIT genes were cloned and sequenced from 45 species of Thalassiosirales, a diatom lineage with high diversity in marine and freshwaters. Phylogenetically based codon substitution models were used to test whether SITs from marine and freshwater taxa were under similar selective constraints and whether codons in different structural locations of the protein were under similar functional constraints. Purifying selection is the predominant evolutionary force acting on SITs, irrespective of location in the protein, and differences in efficiency of silicic acid uptake between marine and freshwater diatoms are not due to sequence differences in SITs.

The centric diatom order Thalassiosirales includes all diatoms with a fultoportula (strutted process), a feature now recognized as a synapomorphy for the lineage. Within Thalassiosirales, Thalassiosira is perhaps the most taxonomically and morphologically diverse genus, and at least two distinct morphological groups have been recognized within it. Group “A” Thalassiosira species, which include the type species, T. nordenskioeldii, have short inward and long outward extensions of the strutted processes and a labiate process on the valve mantle. Group “B” species have exceptionally long inward and reduced outward extensions of the strutted processes, and a labiate process on the valve face. We collected and cultured Thalassiosira ritscheri, which has a combination of group A and B characters. It has a labiate process on the valve face and reduced outward extensions of the strutted processes. We show for the first time that T. ritscheri has short inward, A-type extensions of the strutted processes. A phyloge...

Diatoms have been classified historically as either centric or pennate based on a number of features, cell outline foremost among them. The consensus among nearly every estimate of the diatom phylogeny is that the traditional pennate diatoms (Pennales) constitute a well-supported clade, whereas centric diatoms do not. The problem with the centric‐pennate classification was highlighted by some recent analyses concerning the phylogenetic position of Toxarium, whereby it was concluded that this ‘‘centric’’ diatom independently evolved several pennate-like characters including an elongate, pennate-like cell outline. We performed several phylogenetic analyses to test the hypothesis that Toxarium evolved its elongate shape independently from Pennales. First, we reanalyzed the original data set used to infer the phylogenetic position of Toxarium and found that a more thorough heuristic search was necessary to find the optimal tree. Second, we aligned 181 diatom and eight outgroup SSU rDNA sequences to maximize the juxtapositioning of similar primary and secondary structure of the 18S rRNA molecule over a much broader sampling of diatoms. We then performed a number of phylogenetic analyses purposely based on disparate sets of assumptions and found that none of these analyses supported the conclusion that Toxarium acquired its pennate-like outline independently from Pennales. Our results suggest that elongate outline is congruent with SSU rDNA data and may be synapomorphic for a larger, more inclusive clade than the traditional Pennales.

Morphological features of the siliceous cell wall traditionally have been used to diagnose and classify species of diatoms, though an increasing number of studies distinguish new species, in part, by phylogenetic analysis of rDNA sequences. Intragenomic sequence variation is common among the hundreds to thousands of rDNA cistrons present within a genome, and this variation has strong potential to obscure species boundaries based on rDNA sequences. We screened six Skeletonema culture strains for intragenomic nucleotide polymorphisms in the small subunit (SSU) rDNA gene and found that all strains had polymorphic sites, with proportions ranging from 0.57% to 1.81%. In all cases, transitions accounted for more than 70% of nucleotide differences at polymorphic sites. Polymorphic sites were split nearly evenly in the SSU rRNA molecule between the base-paired regions of helices (52%) and the unpaired regions of loops and bulges (48%). Phylogenetic analysis showed that SSU rDNA genotypes were monophyletic for two of the six culture strains examined. Genotypes from the other four culture strains either showed little or no phylogenetic structure compared with genotypes of other conspecific culture strains or had phylogenetic structure that was incongruent with existing species boundaries. Moderate to strong support for monophyly was recovered for four of the seven species included in the analysis. Phylogenetic results combined with the low sequence divergence of SSU rDNA genotypes within species suggest that concerted evolution has not proceeded to completion in these species and/or that the rate at which variation is being generated exceeds the rate at which concerted evolution is expunging variation.

Much attention is being directed toward understanding and co-opting the mechanisms by which diatoms control patterning of silica for nanotechnology applications. Given the enormous diversity of cell-wall patterns among diatom taxa, a complete understanding of these mechanisms will require comparative analysis of nanopatterning strategies from a diversity of diatom taxa. A well-supported phylogenetic tree provides the best basis for such comparisons because closely related taxa will have many similar attributes (morphological, physiological, ecological, etc.) simply because they share a recent common ancestor. Phylogenetic information is exploited in as diverse a set of fields as pharmacology, epidemiology, and geology. Nanotechnologists can use the phylogenetic tree of diatoms to help select exemplar taxa and to streamline the search for alternative nanopatterning strategies. We review the progress made thus far in reconstructing the phylogeny of diatoms. Most analyses have been based on small subunit ribosomal DNA sequences, and inferences have varied substantially depending on the number and diversity of taxa included in the analyses. We review several seminal studies on diatom phylogeny in light of theoretical and empirical investigations that have emphasized the critical importance of taxonomic sampling on phylogenetic inference. One consistent result is that centric diatoms grade into araphid pennates, and araphid pennates grade the raphe-bearing pennate diatoms, which are a natural group. Including more taxa and more characters (molecular and morphological) should yield a better supported hypothesis of diatom relationships.

Applied algal studies typically require enumeration of preserved cells. As applications of algal assessments proliferate, understanding sources of variability inherent in the methods by which abundance and species composition data are obtained becomes even more important for precision of measurements. We performed replicate counts of diatoms on permanently fixed coverglasses and all algae in Palmer–Maloney chambers to assess precision and accuracy of measurements derived from common counting methods. We counted diatoms and all algae with transects and random fields. Variability estimates (precision) of diatom density, species diversity, and species composition on permanent coverglasses were low between replicate subsamples and between replicate transects. However, average density estimates of diatoms settled on coverglasses determined with transect methods were 42–52% greater than density estimates made with random fields. This bias was due to a predictable, nonrandom distribution of diatoms on the coverglass with few diatoms near edges. Despite bias in density when counting diatoms along coverglass transects, no bias was observed in estimates of species composition. Estimates of density and taxa richness of all-algae in Palmer–Maloney chambers also had low variability among multiple transects and high similarity in species composition between transects. In addition, counting method in Palmer–Maloney chambers did not affect estimates of algal cell density, taxa richness, and species composition, which suggested that counting units were distributed randomly in the chambers. Thus, most sources of variability in sample preparation and analysis are small; however, transect counts should not be used to estimate cell density, and sufficient numbers of random fields must be counted to account for edge effects on cell distribution with material settled on permanently fixed coverglasses.

SUMMARY 1. Monthly patterns of diatom ingestion by Blepharicera magna and B. similans (Diptera: Blephariceridae) larvae were examined to determine whether dietary attributes fluctuate within the final-instar stadium. The two species are isolated temporally: B. magna diets were examined from October to December, and B. similans diets were examined from June to August. Each month, the diatom assemblage of larval diets was assessed and compared with that of the forage base. In addition, total diatom ingestion was estimated. 2. Larval gut samples were ordinated on the basis of relative abundance of common diatom taxa, and month‐month comparisons of larval diets were made, which revealed that (i) B. magna dietary assemblages did not vary significantly from October to December; (ii) diatom species composition of B. magna diets generally resembled that of the forage base; and (iii) B. similans dietary assemblages varied significantly from June to August, with few changes attributable to seasonality of ambient diatom flora. 3. Total diatom ingestion by B. magna was relatively stable from October to December. For B. similans, total diatom ingestion was greatest in July, when larvae were midstage of instar IV. The latter result was accompanied by increased ingestion of grazer-resistant diatom taxa (e.g. Achnanthes deflexa var. alpestris, Achnanthidium minutissimum, and Cocconeis placentula var. lineata), which can be more digestible than grazer-susceptible taxa (e.g. chain-forming Fragilaria and rosette-forming Synedra species). This result suggests demand for a large quantity of nutritionally rich food at midstage of instar IV, which could represent a period of critical growth for B. similans.

Diatom algae are single-celled, photosynthetic organisms with a cell wall called a frustule—a periodically patterned nano-structure made of silica. Throughout the last decade, diatom frustules have been studied for their potential uses as photonic crystals and biomimetic templates for artificially developed metamaterials. A MATLAB program characterizing their pore structure as a function of angle was developed, potentially giving insight into how their geometric characteristics determine their optical properties.