Abundant Citizen Science Data Reveal That the Peacock Butterfly Aglais io Recently Became Bivoltine in Belgium.

The peacock butterfly is abundant and widespread in Europe. It is generally believed to be univoltine (one generation per year): adults born in summer overwinter and reappear again in spring to reproduce. However, recent flight patterns in western Europe mostly show three peaks during the year: a first one in spring (overwintering butterflies), a second one in early summer (offspring of the spring generation), and a third one in autumn. It was thus far unclear whether this autumn flight peak was a second new generation or consisted of butterflies flying again in autumn after a summer rest (aestivation). The life cycle of one of Europe's most common butterflies is therefore still surprisingly inadequately understood. We used hundreds of thousands of observations and thousands of pictures submitted by naturalists from the public to the online portal observation.orgin Belgium and analyzed relations between flight patterns, condition (wear), reproductive cycles, peak abundances, and phenology to clarify the current life history. We demonstrate that peacocks have shifted towards two new generations per year in recent decades. Mass citizen science data in online portals has become increasingly important in tracking the response of biodiversity to rapid environmental changes such as climate change.

Global radiation in a rare biosphere soil diatom.

Soil micro-organisms drive the global carbon and nutrient cycles that underlie essential ecosystem functions. Yet, we are only beginning to grasp the drivers of terrestrial microbial diversity and biogeography, which presents a substantial barrier to understanding community dynamics and ecosystem functioning. This is especially true for soil protists, which despite their functional significance have received comparatively less interest than their bacterial counterparts. Here, we investigate the diversification of Pinnularia borealis, a rare biosphere soil diatom species complex, using a global sampling of >800 strains. We document unprecedented high levels of species-diversity, reflecting a global radiation since the Eocene/Oligocene global cooling. Our analyses suggest diversification was largely driven by colonization of novel geographic areas and subsequent evolution in isolation. These results illuminate our understanding of how protist diversity, biogeographical patterns, and members of the rare biosphere are generated, and suggest allopatric speciation to be a powerful mechanism for diversification of micro-organisms.

Urbanization drives cross-taxon declines in abundance and diversity at multiple spatial scales.

The increasing urbanization process is hypothesized to drastically alter (semi-)natural environments with a concomitant major decline in species abundance and diversity. Yet, studies on this effect of urbanization, and the spatial scale at which it acts, are at present inconclusive due to the large heterogeneity in taxonomic groups and spatial scales at which this relationship has been investigated among studies. Comprehensive studies analysing this relationship across multiple animal groups and at multiple spatial scales are rare, hampering the assessment of how biodiversity generally responds to urbanization. We studied aquatic (cladocerans), limno-terrestrial (bdelloid rotifers) and terrestrial (butterflies, ground beetles, ground- and web spiders, macro-moths, orthopterans and snails) invertebrate groups using a hierarchical spatial design, wherein three local-scale (200 m × 200 m) urbanization levels were repeatedly sampled across three landscape-scale (3 km × 3 km) urbanization levels. We tested for local and landscape urbanization effects on abundance and species richness of each group, whereby total richness was partitioned into the average richness of local communities and the richness due to variation among local communities. Abundances of the terrestrial active dispersers declined in response to local urbanization, with reductions up to 85% for butterflies, while passive dispersers did not show any clear trend. Species richness also declined with increasing levels of urbanization, but responses were highly heterogeneous among the different groups with respect to the richness component and the spatial scale at which urbanization impacts richness. Depending on the group, species richness declined due to biotic homogenization and/or local species loss. This resulted in an overall decrease in total richness across groups in urban areas. These results provide strong support to the general negative impact of urbanization on abundance and species richness within habitat patches and highlight the importance of considering multiple spatial scales and taxa to assess the impacts of urbanization on biodiversity.

A genomics approach reveals the global genetic polymorphism, structure, and functional diversity of ten accessions of the marine model diatom Phaeodactylum tricornutum.

Diatoms emerged in the Mesozoic period and presently constitute one of the main primary producers in the world's ocean and are of a major economic importance. In the current study, using whole genome sequencing of ten accessions of the model diatom Phaeodactylum tricornutum, sampled at broad geospatial and temporal scales, we draw a comprehensive landscape of the genomic diversity within the species. We describe strong genetic subdivisions of the accessions into four genetic clades (A-D) with constituent populations of each clade possessing a conserved genetic and functional makeup, likely a consequence of the limited dispersal of P. tricornutum in the open ocean. We further suggest dominance of asexual reproduction across all the populations, as implied by high linkage disequilibrium. Finally, we show limited yet compelling signatures of genetic and functional convergence inducing changes in the selection pressure on many genes and metabolic pathways. We propose these findings to have significant implications for understanding the genetic structure of diatom populations in nature and provide a framework to assess the genomic underpinnings of their ecological success and impact on aquatic ecosystems where they play a major role. Our work provides valuable resources for functional genomics and for exploiting the biotechnological potential of this model diatom species.

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta).

Diatoms are one of the most abundant and arguably the most species-rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon-rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single-locus automated species delimitation methods. We compared the DNA-based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.

Incomplete Reproductive Isolation Between Genetically Distinct Sympatric Clades of the Pennate Model Diatom Seminavis robusta.

Incomplete reproductive isolation between genetically distinct taxa provides an interesting opportunity for speciation and adaptation studies. This phenomenon is well-described in macro-organisms, but less experimental evidence is available for unicellular eukaryotes. Here, we document the sympatric occurrence of genetically differentiated populations of the pennate model diatom Seminavis robusta in coastal subtidal biofilm communities and show widespread potential for gene flow between them. Based on sequence variation in the plastid-encoded rbcL gene, three distinct clades were identified. Morphological variation between the clades reflected their phylogenetic relationships, with subtle differences in valve morphology in the most distant clade compared to the other two clades, which were indistinguishable. Using a large number of experimental crosses we showed that, although reproductive output was significantly lower compared to the majority of within-clade crosses, approximately 34.5% of the inter-clade crosses resulted in viable and fertile progeny. While the nature of the incomplete reproductive isolation remains unknown, its occurrence in natural diatom populations represents an additional mechanism contributing to population genetic structuring and adaptation and can spur further research into the mechanisms of species divergence and the maintenance of species identity in the presence of gene flow.

Nutrient removal from horticultural wastewater by benthic filamentous algae Klebsormidium sp., Stigeoclonium spp. and their communities: From laboratory flask to outdoor Algal Turf Scrubber (ATS).

Benthic filamentous algae have evident advantages in wastewater treatment over unicellular microalgae, including the ease in harvesting and resistance to predation. To assess the potentials of benthic filamentous algae in treating horticultural wastewater under natural conditions in Belgium, three strains and their mixture with naturally wastewater-borne microalgae were cultivated in 250 ml Erlenmeyer flasks in laboratory as well as in 1 m(2) scale outdoor Algal Turf Scrubber (ATS) with different flow rates. Stigeoclonium competed well with the natural wastewater-borne microalgae and contributed to most of the biomass production both in Erlenmeyer flasks and outdoor ATS at flow rates of 2-6 L min(-1) (water velocity 3-9 cm s(-1)), while Klebsormidium was not suitable for growing in horticultural wastewater under the tested conditions. Flow rate had great effects on biomass production and nitrogen removal, while phosphorus removal was less influenced by flow rate due to other mechanisms than assimilation by algae.

Fatty acid profiles of four filamentous green algae under varying culture conditions.

Although benthic filamentous algae are interesting targets for wastewater treatment and biotechnology, relatively little is known about their biochemical composition and variation in response to growth conditions. Fatty acid composition of four benthic filamentous green algae was determined in different culture conditions. Although the response was partly species-dependent, increasing culture age, nitrogen deprivation and dark exposure of stationary phase greatly increased both total fatty acid content (TFA) from 12-35 to 40-173mgg(-1) dry weight (DW) and the relative proportion of polyunsaturated fatty acids (PUFAs) from 21-58% to 55-87% of TFA, with dark exposure having the greatest effect. However, the main variation in fatty acid composition was between species, with Uronema being rich in C16:0 (2.3% of DW), Klebsormidium in C18:2ω6 (5.4% of DW) and Stigeoclonium in C18:3ω3 (11.1% of DW). This indicates the potential of the latter two species as potential sources of these PUFAs.

The common bloom-forming cyanobacterium Microcystis is prone to a wide array of microbial antagonists.

Many degraded waterbodies around the world are subject to strong proliferations of cyanobacteria - notorious for their toxicity, high biomass build-up and negative impacts on aquatic food webs - the presence of which puts serious limits on the human use of affected water bodies. Cyanobacterial blooms are largely regarded as trophic dead ends since they are a relatively poor food source for zooplankton. As a consequence, their population dynamics are generally attributed to changes in abiotic conditions (bottom-up control). Blooms however generally contain a vast and diverse community of micro-organisms of which some have shown devastating effects on cyanobacterial biomass. For Microcystis, one of the most common bloom-forming cyanobacteria worldwide, a high number of micro-organisms (about 120 taxa) including viruses, bacteria, microfungi, different groups of heterotrophic protists, other cyanobacteria and several eukaryotic microalgal groups are currently known to negatively affect its growth by infection and predation or by the production of allelopathic compounds. Although many of these specifically target Microcystis, sharp declines of Microcystis biomass in nature are only rarely assigned to these antagonistic microbiota. The commonly found strain specificity of their interactions may largely preclude strong antagonistic effects on Microcystis population levels but may however induce compositional shifts that can change ecological properties such as bloom toxicity. These highly specific interactions may form the basis of a continuous arms race (co-evolution) between Microcystis and its antagonists which potentially limits the possibilities for (micro)biological bloom control.

Genotypic diversity and differentiation among populations of two benthic freshwater diatoms as revealed by microsatellites.

Given their large population sizes and presumed high dispersal capacity, protists are expected to exhibit homogeneous population structure over large spatial scales. On the other hand, the fragmented and short-lived nature of the lentic freshwater habitats that many protists inhabit promotes strong population differentiation. We used microsatellites in two benthic freshwater diatoms, Eunotia bilunaris 'robust' and Sellaphora capitata, sampled from within a pond and connected ponds, through isolated ponds from the same region to western Europe to determine the spatial scale at which differentiation appears. Because periods of low genotypic diversity contribute to population differentiation, we also assessed genotypic diversity. While genotypic diversity was very high to maximal in most samples of both species, some had a markedly lower diversity, with up to half (Eunotia) and over 90% (Sellaphora) of the strains having the same multilocus genotype. Population differentiation showed an isolation-by-distance pattern with very low standardized FST values between samples from the same or connected ponds but high values between isolated ponds, even when situated in the same region. Partial rbcL sequences in Eunotia were consistent with this pattern as isolated ponds in the same region could differ widely in haplotype composition. Populations identified by Structure corresponded to the source ponds, confirming that 'pond' is the main factor structuring these populations. We conclude that freshwater benthic diatom populations are highly fragmented on a regional scale, reflecting either less dispersal than is often assumed or reduced establishment success of immigrants, so that dispersal does not translate into gene flow.

Expansion of the 'Reticulosphere': Diversity of Novel Branching and Network-forming Amoebae Helps to Define Variosea (Amoebozoa).

Amoebae able to form cytoplasmic networks or displaying a multiply branching morphology remain very poorly studied. We sequenced the small-subunit ribosomal RNA gene of 15 new amoeboid isolates, 14 of which are branching or network-forming amoebae (BNFA). Phylogenetic analyses showed that these isolates all group within the poorly-known and weakly-defined class Variosea (Amoebozoa). They are resolved into six lineages corresponding to distinct new morphotypes; we describe them as new genera Angulamoeba (type species Angulamoeba microcystivorans n. gen., n. sp.; and A. fungorum n. sp.), Arboramoeba (type species Arboramoeba reticulata n. gen., n. sp.), Darbyshirella (type species Darbyshirella terrestris n. gen., n. sp.), Dictyamoeba (type species Dictyamoeba vorax n. gen., n. sp.), Heliamoeba (type species Heliamoeba mirabilis n. gen., n. sp.), and Ischnamoeba (type species Ischnamoeba montana n. gen., n. sp.). We also isolated and sequenced four additional variosean strains, one belonging to Flamella, one related to Telaepolella tubasferens, and two members of the cavosteliid protosteloid lineage. We identified a further 104 putative variosean environmental clone sequences in GenBank, comprising up to 14 lineages that may prove to represent additional novel morphotypes. We show that BNFA are phylogenetically widespread in Variosea and morphologically very variable, both within and between lineages.

Sensitivity of the green alga Pediastrum duplex Meyen to allelochemicals is strain-specific and not related to co-occurrence with allelopathic macrophytes.

Interspecific differences in the response of microalgae to stress have numerous ecological implications. However, little is known of intraspecific sensitivities and the potential role of local genetic adaptation of populations. We compared the allelochemical sensitivity of 23 Pediastrum duplex Meyen strains, a common component of the freshwater phytoplankton. In order to test for local genetic adaptation, strains were isolated from water bodies with and without the allelopathically-active submerged macrophyte Myriophyllum. Strains were assigned to P. duplex on the basis of cell shape and colony morphology and only P. duplex strains that belonged to the same lineage in an ITS rDNA phylogeny were used. Inhibition of strain growth rates and maximum quantum yields of photosystem II were measured after exposure to tannic acid (TA) and co-culture with Myriophyllum spicatum. Growth rate inhibition varied over one order of magnitude between the P. duplex strains. There was no correlation between the presence of Myriophyllum in the source location and the sensitivity of the strains to TA or the presence of Myriophyllum, suggesting that at least strong unidirectional local adaptation to Myriophyllum had not taken place in the studied water bodies. The maximum quantum yield of photosystem II of TA exposed algae decreased, whereas the yield of algae exposed to M. spicatum was slightly higher than that of the controls. The ranking of P. duplex strain sensitivities differed between the types of exposure (single additions of TA versus co-existence with M. spicatum) and the parameter measured (growth rate versus maximum quantum yield), emphasizing the importance of measuring multiple traits when analysing strain-specific sensitivities towards allelochemicals. The observation that sensitivities to allelochemicals vary widely among strains of a single freshwater algal species should be taken into account if evaluating ecological consequences of allelopathic interactions.

Planktonic ciliate community structure in shallow lakes of lowland Western Europe.

Temperate shallow meso- to eutrophic lakes can exist in one of two alternative states with contrasting foodwebs, referred to as the clear-water and the turbid state. We describe the planktonic ciliate communities of such lakes based on a survey of 66 northwestern European lakes. Ciliates were enumerated and identified to species level according to the quantitative protargol staining technique. Ciliate biomass was on average twice as high in the turbid than in the clear-water lakes. The ciliate communities were dominated by oligotrichs and protostomatids, and no differences in functional composition or α-diversity could be detected between turbid and clear-water lakes, although ß-diversity tended to be higher in the latter. At the species level, however, community structure strongly differed between turbid and clear-water lakes, and several indicator species could be identified for the different lake categories. Variation partitioning showed that nutrient status did not explain ciliate community structure independent of the alternative states, while lake area was identified as an additional structuring factor for the ciliate communities. These results stress the importance of the ecosystem structure in shaping ciliate communities in temperate shallow lakes and suggest that nutrient status has little direct effect on ciliate community structure in such lakes.

An inordinate fondness? The number, distributions, and origins of diatom species.

The number of extant species of diatoms is estimated here to be at least 30,000 and probably ca. 100,000, by extrapolation from an eclectic sample of genera and species complexes. Available data, although few, indicate that the pseudocryptic species being discovered in many genera are not functionally equivalent. Molecular sequence data show that some diatom species are ubiquitously dispersed. A good case can be made that at least some diatom species and even a few genera are endemics, but many such claims are still weak. The combination of very large species numbers and relatively rapid dispersal in diatoms is inconsistent with some versions of the "ubiquity hypothesis" of protist biogeography, and appears paradoxical. However, population genetic data indicate geographical structure in all the (few) marine and freshwater species that have been examined in detail, sometimes over distances of a few tens of kilometres. The mode of speciation may often be parapatric, in the context of a constantly shifting mosaic of temporarily isolated (meta) populations, but if our "intermediate dispersal hypothesis" is true (that long-distance dispersal is rare, but not extremely rare), allopatric speciation could also be maximized.

Linkage mapping identifies the sex determining region as a single locus in the Pennate diatom Seminavis robusta.

The pennate diatom Seminavis robusta, characterized by an archetypical diatom life cycle including a heterothallic mating system, is emerging as a model system for studying the molecular regulation of the diatom cell and life cycle. One of its main advantages compared with other diatom model systems is that sexual crosses can be made routinely, offering unprecedented possibilities for forward genetics. To date, nothing is known about the genetic basis of sex determination in diatoms. Here, we report on the construction of mating type-specific linkage maps for S. robusta, and use them to identify a single locus sex determination system in this diatom. We identified 13 mating type plus and 15 mating type minus linkage groups obtained from the analysis of 463 AFLP markers segregating in a full-sib family, covering 963.7 and 972.2 cM, respectively. Five linkage group pairs could be identified as putative homologues. The mating type phenotype mapped as a monogenic trait, disclosing the mating type plus as the heterogametic sex. This study provides the first evidence for a genetic sex determining mechanism in a diatom.

Molecular evidence for distinct Antarctic lineages in the cosmopolitan terrestrial diatoms Pinnularia borealis and Hantzschia amphioxys.

Recent morphology-based studies indicate that freshwater diatom floras in the Antarctic comprise a significant share of endemics among a majority of apparently cosmopolitan species. Given the widespread (pseudo)cryptic species diversity in diatoms, we assessed the molecular divergence and temperature-dependent growth characteristics between Antarctic and non-Antarctic strains for two presumed species with a cosmopolitan distribution, namely Pinnularia borealis and Hantzschia amphioxys. Molecular phylogenies based on the plastid gene rbcL and the nuclear 28S rDNA (D1-D3 region) revealed that both taxa consist of multiple lineages, each including a distinct Antarctic lineage. A molecular clock estimates the origin of P. borealis at 35.8 (30-47) million years (Ma) ago, making this the oldest known diatom species complex. The Antarctic P. borealis lineage is estimated to have diverged 7.8 (2-15) Ma ago, after the geographical and thermal isolation of the Antarctic continent. Despite not being psychrophilic, the Antarctic lineages of P. borealis and H. amphioxys have a lower optimal growth temperature and upper lethal temperature than most lineages from more temperate regions, indicating niche differentiation. Together, this suggests that many presumed cosmopolitan Antarctic diatom species are in fact species complexes, possibly containing Antarctic endemics with low temperature preferences.

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.

Lack of phylogeographic structure in the freshwater cyanobacterium Microcystis aeruginosa suggests global dispersal.

BACKGROUND: Free-living microorganisms have long been assumed to have ubiquitous distributions with little biogeographic signature because they typically exhibit high dispersal potential and large population sizes. However, molecular data provide contrasting results and it is far from clear to what extent dispersal limitation determines geographic structuring of microbial populations. We aimed to determine biogeographical patterns of the bloom-forming freshwater cyanobacterium Microcystis aeruginosa. Being widely distributed on a global scale but patchily on a regional scale, this prokaryote is an ideal model organism to study microbial dispersal and biogeography. METHODOLOGY/PRINCIPAL FINDINGS: The phylogeography of M. aeruginosa was studied based on a dataset of 311 rDNA internal transcribed spacer (ITS) sequences sampled from six continents. Richness of ITS sequences was high (239 ITS types were detected). Genetic divergence among ITS types averaged 4% (maximum pairwise divergence was 13%). Preliminary analyses revealed nearly completely unresolved phylogenetic relationships and a lack of genetic structure among all sequences due to extensive homoplasy at multiple hypervariable sites. After correcting for this, still no clear phylogeographic structure was detected, and no pattern of isolation by distance was found on a global scale. Concomitantly, genetic differentiation among continents was marginal, whereas variation within continents was high and was mostly shared with all other continents. Similarly, no genetic structure across climate zones was detected. CONCLUSIONS/SIGNIFICANCE: The high overall diversity and wide global distribution of common ITS types in combination with the lack of phylogeographic structure suggest that intercontinental dispersal of M. aeruginosa ITS types is not rare, and that this species might have a truly cosmopolitan distribution.

Evaluation of ionic liquid stationary phases for one dimensional gas chromatography-mass spectrometry and comprehensive two dimensional gas chromatographic analyses of fatty acids in marine biota.

Ionic liquid stationary phases were tested for one dimensional gas chromatography-mass spectrometry (GC-MS) and comprehensive two dimensional gas chromatography (GC×GC) of fatty acid methyl esters from algae. In comparison with polyethylene glycol and cyanopropyl substituted polar stationary phases, ionic liquid stationary phases SLB-IL 82 and SLB-IL 100 showed comparable resolution, but lower column bleeding with MS detection, resulting in better sensitivity. The selectivity and polarity of the ionic liquid phases are similar to a highly polar biscyanopropyl-silicone phase (e.g. HP-88). In GC×GC, using an apolar polydimethyl siloxane×polar ionic liquid column combination, an excellent group-type separation of fatty acids with different carbon numbers and number of unsaturations was obtained, providing information that is complementary to GC-MS identification.

Strong effects of amoebae grazing on the biomass and genetic structure of a Microcystis bloom (Cyanobacteria).

Despite its importance for bloom toxicity, the factors determining the population structure of cyanobacterial blooms are poorly understood. Here, we report the results of a two-year field survey of the population dynamics of Microcystis blooms in a small hypertrophic urban pond. Microscopic enumeration of Microcystis and its predators and parasites was combined with pigment and microcystin analysis and denaturing gradient gel electrophoresis of the ITS rDNA region to assess population dynamics and structure. Two main Microcystis morpho- and ITS types were revealed, corresponding to M. aeruginosa and M. viridis. In both years, high population densities of naked amoebae grazing on Microcystis coincided with rapid decreases in Microcystis biomass. In one year, there was a shift from heavily infested M. aeruginosa to the less-infested M. viridis, allowing the bloom to rapidly recover. The preference of amoebae for M. aeruginosa was confirmed by grazing experiments, in which several amoeba strains were capable of grazing down a strain of M. aeruginosa, but not of M. viridis. Zooplankton and chytrid parasites appeared to be of minor importance for these strong and fast reductions in Microcystis biomass. These findings demonstrate a strong impact of small protozoan grazers on the biomass and genetic structure of Microcystis blooms.