Contrasted Micro-Eukaryotic Diversity Associated with Sphagnum Mosses in Tropical, Subtropical and Temperate Climatic Zones.

Sphagnum-dominated ecosystem plays major roles as carbon sinks at the global level. Associated microbial communities, in particular, eukaryotes, play significant roles in nutrient fixation and turnover. In order to understand better the ecological processes driven by these organisms, the first step is to characterise these associated organisms. We characterised the taxonomic diversity, and from this, inferred the functional diversity of microeukaryotes in Sphagnum mosses in tropical, subtropical and temperate climatic zones through an environmental DNA diversity metabarcoding survey of the V9 region of the gene coding for the RNA of the small subunit of the ribosomes (SSU rRNA). As microbial processes are strongly driven by temperatures, we hypothesised that saprotrophy would be highest in warm regions, whereas mixotrophy, an optimal strategy in oligotrophic environments, would peak under colder climates. Phylotype richness was higher in tropical and subtropical climatic zones than in the temperate region, mostly due to a higher diversity of animal parasites (i.e. Apicomplexa). Decomposers, and especially opportunistic yeasts and moulds, were more abundant under warmer climates, while mixotrophic organisms were more abundant under temperate climates. The dominance of decomposers, suggesting a higher heterotrophic activity under warmer climates, is coherent with the generally observed faster nutrient cycling at lower latitudes; this phenomenon is likely enhanced by higher inputs of nutrients most probably brought in the system by Metazoa, such as arthropods.

What is the optimum sample size for the study of peatland testate amoeba assemblages?

Testate amoebae are widely used in ecological and palaeoecological studies of peatlands, particularly as indicators of surface wetness. To ensure data are robust and comparable it is important to consider methodological factors which may affect results. One significant question which has not been directly addressed in previous studies is how sample size (expressed here as number of Sphagnum stems) affects data quality. In three contrasting locations in a Russian peatland we extracted samples of differing size, analysed testate amoebae and calculated a number of widely-used indices: species richness, Simpson diversity, compositional dissimilarity from the largest sample and transfer function predictions of water table depth. We found that there was a trend for larger samples to contain more species across the range of commonly-used sample sizes in ecological studies. Smaller samples sometimes failed to produce counts of testate amoebae often considered minimally adequate. It seems likely that analyses based on samples of different sizes may not produce consistent data. Decisions about sample size need to reflect trade-offs between logistics, data quality, spatial resolution and the disturbance involved in sample extraction. For most common ecological applications we suggest that samples of more than eight Sphagnum stems are likely to be desirable.

Re-assessing the vertical distribution of testate amoeba communities in surface peats: Implications for palaeohydrological studies.

Testate amoeba-derived transfer functions are frequently used in peatland palaeohydrological studies and involve the development of training sets from surficial peats. However, within acrotelmic peats, considerable vertical variation in assemblage composition can occur, particularly along Sphagnum stems, which may limit the representation of the associated 'contemporary' testate amoeba samples as analogues for the peatland surface. This paper presents contiguous testate amoeba assemblage data from nine monoliths collected from different peatland microforms (hummock, hollow, lawn) in three Sphagnum dominated ombrotrophic peatlands in Ontario and Quebec, eastern Canada. The aim is to: (i) gain a greater understanding of the vertical distribution of xerophilous/hygrophilous taxa along Sphagnum stems; (ii) determine the vertical extent of live/encysted taxa along this gradient; and (iii) assess the significance of this distribution on surface sampling protocols. The results show that testate amoeba communities in the uppermost acrotelmic peat layers display considerable variability. This may reflect a complex interplay of abiotic and biotic controls, including moisture, temperature, light and other characteristics, food availability, and mineral particle availability for test construction. These findings underline the complexity of testate amoeba community structure and highlight the importance of analysing both living and dead Sphagnum stem sections when developing calibration sets.

Origin and diversity of testate amoebae shell composition: Example of Bullinularia indica living in Sphagnum capillifolium.

Testate amoebae are free-living shelled protists that build a wide range of shells with various sizes, shapes, and compositions. Recent studies showed that xenosomic testate amoebae shells could be indicators of atmospheric particulate matter (PM) deposition. However, no study has yet been conducted to assess the intra-specific mineral, organic, and biologic grain diversity of a single xenosomic species in a natural undisturbed environment. This study aims at providing new information about grain selection to develop the potential use of xenosomic testate amoebae shells as bioindicators of the multiple-origin mineral/organic diversity of their proximal environment. To fulfil these objectives, we analysed the shell content of 38 Bullinularia indica individuals, a single xenosomic testate amoeba species living in Sphagnum capillifolium, by scanning electron microscope (SEM) coupled with X-ray spectroscopy. The shells exhibited high diversities of mineral, organic, and biomineral grains, which confirms their capability to recycle xenosomes. Mineral grain diversity and size of B. indica matched those of the atmospheric natural mineral PM deposited in the peatbog. Calculation of grain size sorting revealed a discrete selection of grains agglutinated by B. indica. These results are a first step towards understanding the mechanisms of particle selection by xenosomic testate amoebae in natural conditions.

Contrasting diversity of testate amoebae communities in Sphagnum and brown-moss dominated patches in relation to shell counts.

Ecological studies of peatland testate amoebae are generally based on totals of 150 individuals per sample. However, the suitability of this standard has never been assessed for alkaline habitats such as spring fens. We explored the differences in testate amoeba diversity between Sphagnum and brown-moss microhabitats at a mire site with a highly diversified moss layer which reflects the small-scale heterogeneity in groundwater chemistry. Relationships between sampling efficiency and sample completeness were explored using individual-based species accumulation curves and the effort required to gain an extra species was assessed. Testate amoeba diversity differed substantially between microhabitats, with brown mosses hosting on average twice as many species and requiring greater shell totals to reach comparable sample analysis efficiency as for Sphagnum. Thus, for samples from alkaline conditions an increase in shell totals would be required and even an overall doubling up to 300 individuals might be considered for reliable community description. Our small-scale data are likely not robust enough to provide an ultimate solution for the optimization of shell totals. However, the results proved that testate amoebae communities from acidic and alkaline environments differ sharply in both species richness and composition and they might call for different methodological approaches.

Nebela jiuhuensis nov. sp. (Amoebozoa; Arcellinida; Hyalospheniidae): A New Member of the Nebela saccifera - equicalceus - ansata Group Described from Sphagnum Peatlands in South-Central China.

Hyalospheniids are among the most common and conspicuous testate amoebae in high-latitude peatlands and forest humus. These testate amoebae were widely studied as bioindicators and are increasingly used as models in microbial biogeography. However, data on their diversity and ecology are still very unevenly distributed geographically: notably, data are lacking for low-latitude peatlands. We describe here a new species, Nebela jiuhuensis, from peatlands near the Middle Yangtze River reach of south-central China with characteristic morphology. The test (shell) has hollow horn-like lateral extensions also found in N. saccifera, N. equicalceus (=N. hippocrepis), and N. ansata, three large species restricted mostly to Sphagnum peatlands of Eastern North America. Mitochondrial cytochrome oxidase (COI) data confirm that N. jiuhuensis is closely related to the morphologically very similar North American species N. saccifera and more distantly to N. ansata within the N. penardiana group. These species are all found in wet mosses growing in poor fens. Earlier reports of morphologically similar specimens found in South Korea peatlands suggest that N. jiuhuensis may be distributed in comparable peatlands in Eastern Asia (China and Korea). The discovery of such a conspicuous new species in Chinese peatlands suggests that many new testate amoebae species are yet to be discovered, including potential regional endemics. Furthermore, human activities (e.g., drainage, agriculture, and pollution) have reduced the known habitat of N. jiuhuensis, which can thus be considered as locally endangered. We, therefore, suggest that this very conspicuous micro-organism with a probably limited geographical distribution and specific habitat requirement should be considered as a flagship species for microbial biogeography as well as local environmental conservation and management.

Two new species of Milnesium (Tardigrada: Milnesiidae).

Two new species of Milnesium are described: Milnesium vorax sp. nov from Sicily and Milnesium tumanovi sp. nov. from Crimea. Milnesium vorax sp. nov. is uncoloured, with eye spots, smooth cuticle, wide buccal tube, stylet supports inserted on the buccal tube at almost 62 % of its length; claws with configuration [2-3]-[3-2]; accessory points present on the main branches. Milnesium tumanovi sp. nov. is also uncoloured, has eye spots, smooth cuticle, and wide buccal tube, but it has the stylet supports inserted on the buccal tube at about 52 % of its length, and claws with configuration [3-3]-[3-3]; the accessory points are present on the main branches.

Testing the Effect of Refrigerated Storage on Testate Amoeba Samples.

Samples for analysis of testate amoebae and other protists frequently need to be stored for many months before microscopy. This storage commonly involves refrigeration, but we know that testate amoebae can live and reproduce in these conditions. This raises the question: do communities change during storage and how might this effect the data produced? We analysed Sphagnum samples over a 16-week period to address this question. Our results show no evidence for detectable change. This is a reassuring result supporting much current practice although we suggest that frozen storage or the addition of a fixative may be worthwhile precautions where feasible.

To what extent do food preferences explain the trophic position of heterotrophic and mixotrophic microbial consumers in a Sphagnum peatland?

Although microorganisms are the primary drivers of biogeochemical cycles, the structure and functioning of microbial food webs are poorly studied. This is the case in Sphagnum peatlands, where microbial communities play a key role in the global carbon cycle. Here, we explored the structure of the microbial food web from a Sphagnum peatland by analyzing (1) the density and biomass of different microbial functional groups, (2) the natural stable isotope (δ(13)C and δ(15)N) signatures of key microbial consumers (testate amoebae), and (3) the digestive vacuole contents of Hyalosphenia papilio, the dominant testate amoeba species in our system. Our results showed that the feeding type of testate amoeba species (bacterivory, algivory, or both) translates into their trophic position as assessed by isotopic signatures. Our study further demonstrates, for H. papilio, the energetic benefits of mixotrophy when the density of its preferential prey is low. Overall, our results show that testate amoebae occupy different trophic levels within the microbial food web, depending on their feeding behavior, the density of their food resources, and their metabolism (i.e., mixotrophy vs. heterotrophy). Combined analyses of predation, community structure, and stable isotopes now allow the structure of microbial food webs to be more completely described, which should lead to improved models of microbial community function.

COI barcoding of Nebelid testate amoebae (Amoebozoa: Arcellinida): extensive cryptic diversity and redefinition of the Hyalospheniidae Schultze.

We used Cytochrome Oxidase Subunit 1 (COI) to assess the phylogenetic relationships and taxonomy of Nebela sensu stricto and similar taxa (Nebela group, Arcellinida) in order to clarify the taxonomic validity of morphological characters. The COI data not only successfully separated all studied morphospecies but also revealed the existence of several potential cryptic species. The taxonomic implications of the results are: (1) Genus Nebela is paraphyletic and will need to be split into at least two monophyletic assemblages when taxon sampling is further expanded. (2) Genus Quadrulella, one of the few arcellinid genera building its shell from self-secreted siliceous elements, and the mixotrophic Hyalosphenia papilio branch within the Nebela group in agreement with the general morphology of their shell and the presence of an organic rim around the aperture (synapomorphy for Hyalospheniidae). We thus synonymise Hyalospheniidae and Nebelidae. Hyalospheniidae takes precedence and now includes Hyalosphenia, Quadrulella (previously in the Lesquereusiidae) and all Nebelidae with the exception of Argynnia and Physochila. Leptochlamys is Arcellinida incertae sedis. We describe a new genus Padaungiella Lara et Todorov and a new species Nebela meisterfeldi n. sp. Heger et Mitchell and revise the taxonomic position (and rank) of several taxa. These results show that the traditional morphology-based taxonomy underestimates the diversity within the Nebela group, and that phylogenetic relationships are best inferred from shell shape rather than from the material used to build the shell.

Sphagnum-dwelling testate amoebae in subarctic bogs are more sensitive to soil warming in the growing season than in winter: the results of eight-year field climate manipulations.

Sphagnum-dwelling testate amoebae are widely used in paleoclimate reconstructions as a proxy for climate-induced changes in bogs. However, the sensitivity of proxies to seasonal climate components is an important issue when interpreting proxy records. Here, we studied the effects of summer warming, winter snow addition solely and winter snow addition together with spring warming on testate amoeba assemblages after eight years of experimental field climate manipulations. All manipulations were accomplished using open top chambers in a dry blanket bog located in the sub-Arctic (Abisko, Sweden). We estimated sensitivity of abundance, diversity and assemblage structure of living and empty shell assemblages of testate amoebae in the living and decaying layers of Sphagnum. Our results show that, in a sub-arctic climate, testate amoebae are more sensitive to climate changes in the growing season than in winter. Summer warming reduced species richness and shifted assemblage composition towards predominance of xerophilous species for the living and empty shell assemblages in both layers. The higher soil temperatures during the growing season also decreased abundance of empty shells in both layers hinting at a possible increase in their decomposition rates. Thus, although possible effects of climate changes on preservation of empty shells should always be taken into account, species diversity and structure of testate amoeba assemblages in dry subarctic bogs are sensitive proxies for climatic changes during the growing season.

Characterizing the feeding habits of the testate amoebae Hyalosphenia papilio and Nebela tincta along a narrow "fen-bog" gradient using digestive vacuole content and 13C and 15N isotopic analyses.

Population dynamics and feeding habits of the testate amoebae Nebela tincta and Hyalosphenia papilio were studied along a short "fen" to "bog" gradient in a Sphagnum-dominated mire (Jura, France). Samples were collected in living "top segments" (0-3 cm) and early declining "bottom segments" (3-6 cm) of Sphagnum fallax peat. Observations of digestive vacuole content and stable isotope analyses ((13)C and (15)N) were used to establish the feeding behavior of both testate amoeba species. Owing to their vertical distribution, the feeding habit of H. papilio was described from top segments, and that of N. tincta from bottom segments. Among identified food sources, those most frequently ingested by N. tincta were spores and mycelia of fungi (55%), microalgae (25%) and cyanobacteria (8.5%). For H. papilio, the most frequently ingested prey were ciliates (55%) and microalgae (35%). Nonmetric Multidimensional Scaling analysis clearly demonstrated that the two species did not have the same feeding habit along the "fen-bog" gradient, and furthermore that a significant spatial split exists in the feeding behavior of H. papilio. Additionally, isotope analyses suggested that H. papilio and N. tincta did not have the same trophic position in the microbial food web, probably resulting from their different feeding strategies.

The potential influence of short-term environmental variability on the composition of testate amoeba communities in Sphagnum peatlands.

Testate amoebae are a group of moisture-sensitive, shell-producing protozoa that have been widely used as indicators of changes in mean water-table depth within oligotrophic peatlands. However, short-term environmental variability (i.e., sub-annual) also probably influences community composition. The objective of this study was to assess the potential influence of short-term environmental variability on the composition of testate amoeba communities in Sphagnum-dominated peatlands. Testate amoebae and environmental conditions, including hourly measurements of relative humidity within the upper centimeter of the peatland surface, were examined throughout the 2008 growing season at 72 microsites within 11 peatlands of Pennsylvania and Wisconsin, USA. Relationships among testate amoeba communities, vegetation, depth to water table, pH, and an index of short-term environmental variability (EVI), were examined using nonmetric multidimensional scaling and correlation analysis. Results suggest that EVI influences testate amoeba communities, with some taxa more abundant under highly variable conditions (e.g., Arcella discoides, Difflugia pulex, and Hyalosphenia subflava) and others more abundant when environmental conditions at the peatland surface were relatively stable (e.g., Archerella flavum and Bullinularia indica). The magnitude of environmental variability experienced at the peatland surface appears to be primarily controlled by vegetation composition and density. In particular, sites with dense Sphagnum cover had lower EVI values than sites with loose-growing Sphagnum or vegetation dominated by vascular plants and/or non-Sphagnum bryophytes. Our results suggest that more environmental information may be inferred from testate amoebae than previously recognized. Knowledge of relationships between testate amoebae and short-term environmental variability should lead to more detailed and refined environmental inferences.

Description and phylogenetic relationships of Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. (Amoebozoa, Arcellinida).

Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. were isolated and described from dry epiphytic moss. The morphology and ultrastructure of both species clearly demonstrate that they belong to the genus Spumochlamys (family Microchlamyiidae). They differ from its only described member, Spumochlamys iliensis (as well as from species of Microchlamys), in the relief of the dorsal surface of the test, revealed by scanning electron microscopy, which can represent a good characteristic for species identification. They also differ in the structure of the dorsal part of the test wall (especially S. perforata). Small subunit ribosomal DNA-based molecular phylogenetic analyses show that Spumochlamys is a deeply branching lineage of the Arcellinida, without any close affinities. Actin gene sequence analysis places this genus within the Tubulinea, close to two other arcellinid lineages but without forming a monophyletic group with them. These data together strongly suggest that the lack of resolution in the arcellinid molecular phylogenies is due to serious undersampling of taxa, a limited number of sequence data, and high divergence rates in most of the species.

Form variation in Puytoracia bergeri Bonnet and a description of P. bonneti n. sp. (Rhizopoda, Filosea).

Since its description 35 years ago from Quebec, Canada, the poorly known monospecific testate rhizopod genus Puytoracia (type species, Puytoracia bergeri) has been reported only from the Himalayan region of Nepal. This species was recently found at 13 locations in Ontario, Canada, and the range of form variation of the species from widely separated sites is described. At two of these sites, boreal forest bogs in north-western Ontario, a second, previously unidentified, Puytoracia species was also found and described (as Puytoracia bonneti n. sp.). Differences between the two species were clearly demonstrated statistically using morphometric variables. The large elliptical scales covering the test of P. bonneti (easily seen with relatively low-power microscopy) provide the most important criterion for separating this species from some Trinema species having superficially similar test morphology (e.g. Trinema penardi Thomas & Chardez, or Trinema galeata (Penard) Jung.). On the basis of test structure, it is suggested that P. bonneti occupies a taxonomic position relative to P. bergeri that is similar to that occupied by T. penardi relative to Trinema enchelys (Ehr.) Leidy. Results of this study further confirm the logical position of Puytoracia within the family Trinematidae, close to, but distinct from, the genera Corythion and Trinema.

Vertical micro-distribution and response to nitrogen deposition of testate amoebae in Sphagnum.

Previous studies have shown the existence of a vertical micro-distribution of testate amoebae in the first centimeters of Sphagnum and their response to nutrient enrichment. In order to test the response of testate amoebae to depth and N addition in dry moss carpets recolonizing cutover peatlands, we sampled Sphagnum that had received 0, 1, 3, or 10g N m(-2) yr(-1) for three years. The mosses were cut into three segments: 0-1cm, 1-3cm and 3-5cm and analyzed for testate amoebae. The overall diversity (22 taxa) was high considering the dryness of the site, but the species richness of individual samples was low (mean 6.6). The presence of several species characteristic of wetter conditions suggests that they have a broader tolerance than usually believed and/or have a high colonization potential. Species richness increased with depth. Assulina muscorum was most abundant in the top segment, while Phryganella acropodia, Heleopera rosea and Nebela militaris were most abundant in the deepest segment. Neither the metabolism type nor the shell characteristics significantly explained the vertical distribution of species. There was no overall response of testate amoebae to N, although one species, Bullinularia indica, was significantly more abundant in the fertilized than in the control plots.