Unsuspected diversity of <Emphasis Type="Italic">Niphargus</Emphasis> amphipods in the chemoautotrophic cave ecosystem of Frasassi,central Italy

Background  

The sulfide-rich Frasassi caves in central Italy contain a rare example of a freshwater ecosystem supported entirely by chemoautotrophy. Niphargus ictus, the sole amphipod species previously reported from this locality, was recently shown to host the first known case of a freshwater chemoautotrophic symbiosis. Since the habitat of N. ictus is highly fragmented and is comprised of streams and lakes with various sulfide concentrations, we conducted a detailed study to examine the potential genetic diversity of this species within Frasassi.     

Molecular phylogeny and taxonomy of colorless, filamentous sulfur bacteria of the genus Thiothrix

Comprehensive investigation combining molecular genetic techniques and comparative studies of morphological and physiological properties made it possible to resolve the disputed issue of the taxonomic status of the groups ??T. nivea?? and ??Eikelboom type 021N?? of the genus Thiothrix. The phylogenetic trees constructed on the basis of 16S rRNA and gyrB gene sequences demonstrated that members of the genus Thiothrix formed a cluster within the order Thiotrichales. According to the ??ribosomal?? tree, the cluster of the genus Thiothrix was divided into two main groups, I and II, corresponding to the groups ??T. nivea?? and ??Eikelboom type 021N??. The levels of similarity between the 16S rRNA gene sequences of Thiothrix species reached 88.9?C100%. On the contrary, in the ??gyrase?? tree, these species were not divided into ??T. nivea?? and ??Eikelboom type 021N?? groups. The levels of similarity between the amino acid sequences of the gyrB gene fragments of Thiothrix species varied from 74.5 to 99.2%. Importantly, members of the groups ??T. nivea?? and ??Eikelboom type 021N?? formed very similar 16S rRNA secondary structures in the variable region V3, where a 30-nucleotide deletion characteristic of all Thiothrix species was detected. Phenotypic analysis of the studied bacteria revealed some morphological and physiological properties shared by the groups ??T. nivea?? and ??Eikelboom type 021N??. The data obtained indicate that members of the groups ??T. nivea?? and ??Eikelboom type 021N?? are phenotypically and genetically heterogeneous species within the single monophyletic genus Thiothrix..     

Phylogenetic Position and In Situ Identification of Ectosymbiotic Spirochetes on Protists in the Termite Gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.     

Dominant Microbial Populations in Limestone-Corroding Stream Biofilms, Frasassi Cave System, Italy

Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. β-, γ-, δ-, and -proteobacteria in sulfur-cycling clades accounted for ≥75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating δ-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous γ-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of -proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.     

The Motility Symbiont of the Termite Gut Flagellate Caduceia versatilis Is a Member of the “Synergistes” Group

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the “Synergistes” phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the “Synergistes” phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The “Synergistes” phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, “Candidatus Tammella caduceiae,” for this unique motility ectosymbiont of Caducaia versatilis.     

Can Environment Predict Cryptic Diversity? The Case of Niphargus Inhabiting Western Carpathian Groundwater

In the last decade, several studies have shown that subterranean aquatic habitats harbor cryptic species with restricted geographic ranges, frequently occurring as isolated populations. Previous studies on aquatic subterranean species have implied that habitat heterogeneity can promote speciation and that speciation events can be predicted from species’ distributions. We tested the prediction that species distributed across different drainage systems and karst sectors comprise sets of distinct species. Amphipods from the genus Niphargus from 11 caves distributed along the Western Carpathians (Romania) were investigated using three independent molecular markers (COI, H3 and 28S). The results showed that: 1) the studied populations belong to eight different species that derive from two phylogenetically unrelated Niphargus clades; 2) narrow endemic species in fact comprise complexes of morphologically similar species that are indistinguishable without using a molecular approach. The concept of monophyly, concordance between mitochondrial and nuclear DNA, and the value of patristic distances were used as species delimitation criteria. The concept of cryptic species is discussed within the framework of the present work and the contribution of these species to regional biodiversity is also addressed.     

Capacity for nitrate respiration as a new aspect of metabolism of the filamentous sulfur bacteria of the genus Thiothrix

Capacity of Thiothrix species (T. lacustris strains AS and BL^T, T. caldifontis G1^T, T. unzii A1^T, and T. eikelboomii AR3^T) for anaerobic respiration in the presence of nitrate was discovered. The dynamics of nitrate reduction to nitrite was studied and the coupling of this process to thiosulfate oxidation was shown. The investigated Thiothrix representatives performed anaerobic thiosulfate-dependent reduction of nitrate only to nitrite. The presence of the narG gene, encoding the α-subunit of respiratory nitrate reductase NarGHI, was revealed in the cells. The induction of this gene expression was shown for the T. lacustris strain AS under anaerobic conditions of growth. The activity of several enzymes involved in the conversion of reduced sulfur compounds was determined.     

Evidence for speciation underground in diving beetles (Dytiscidae) from a subterranean archipelago

Most subterranean animals are assumed to have evolved from surface ancestors following colonization of a cave system; however, very few studies have raised the possibility of “subterranean speciation” in underground habitats (i.e., obligate cave‐dwelling organisms [troglobionts] descended from troglobiotic ancestors). Numerous endemic subterranean diving beetle species from spatially discrete calcrete aquifers in Western Australia (stygobionts) have evolved independently from surface ancestors; however, several cases of sympatric sister species raise the possibility of subterranean speciation. We tested this hypothesis using vision (phototransduction) genes that are evolving under neutral processes in subterranean species and purifying selection in surface species. Using sequence data from 32 subterranean and five surface species in the genus Paroster (Dytiscidae), we identified deleterious mutations in long wavelength opsin (lwop), arrestin 1 (arr1), and arrestin 2 (arr2) shared by a sympatric sister‐species triplet, arr1 shared by a sympatric sister‐species pair, and lwop and arr2 shared among closely related species in adjacent calcrete aquifers. In all cases, a common ancestor possessed the function‐altering mutations, implying they were already adapted to aphotic environments. Our study represents one of the first confirmed cases of subterranean speciation in cave insects. The assessment of genes undergoing pseudogenization provides a novel way of testing modes of speciation and the history of diversification in blind cave animals.     

Extremely acidic, pendulous cave wall biofilms from the Frasassi cave system, Italy

The sulfide-rich Frasassi cave system hosts an aphotic, subsurface microbial ecosystem including extremely acidic (pH 0-1), viscous biofilms (snottites) hanging from the cave walls. We investigated the diversity and population structure of snottites from three locations in the cave system using full cycle rRNA methods and culturing. The snottites were composed primarily of bacteria related to Acidithiobacillus species. Other populations present in the snottites included Thermoplasmata group archaea, bacteria related to Sulfobacillus, Acidimicrobium, and the proposed bacterial lineage TM6, protists, and filamentous fungi. Based on fluorescence in situ hybridization population counts, Acidithiobacillus are key members of the snottite communities, accompanied in some cases by smaller numbers of archaea related to Ferroplasma and other Thermoplasmata. Diversity estimates show that the Frasassi snottites are among the lowest-diversity natural microbial communities known, with one to six prokaryotic phylotypes observed depending on the sample. This study represents the first in-depth molecular survey of cave snottite microbial diversity and population structure, and contributes to understanding of rapid limestone dissolution and cave formation by microbially mediated sulfuric acid speleogenesis.     

Identification,enrichment, and isolation ofThiothrix spp. from environmental samples

We have developed a method to enrich, isolate, and identifyThiothrix spp. in environmental samples. This procedure employs low concentrations of organic compounds, the addition of reduced sulfur compounds (sulfide or thiosulfate), and preparation with spring water that containsThiothrix spp. The enrichment enhanced identification ofThiothrix spp. by promoting deposition of intracellular sulfur granules and inhibiting overgrowth by other bacteria. The relatively high calcium content of the spring water contributed to the culture procedure. With this technique,Thiothrix spp. were observed in two activated sludge systems, a municipal water storage tank, three springs, and four underground freshwater caves in the phreatic zone of the Floridan aquifer. Two differentThiothrix cultures have been isolated from a freshwater cave and a water storage tank by this procedure. It appears that media prepared with spring water known to supportThiothrix spp. can be designed to provide highly selective methods for isolation ofThiothrix spp. from a wide range of environments.Florida Agricultural Experimentation Station, Journal Series Number R-03446.     

Trophic plasticity among spring vs. cave populations of <Emphasis Type="Italic">Gammarus minus:</Emphasis> examining functional niches using stable isotopes and C/N ratios

In some environments, species may exhibit trophic plasticity, which allows them to extend beyond their assigned functional group. For Gammarus minus, a freshwater amphipod classified as a shredder or detritivore, cave populations have been observed consuming heterotrophs as well as shredding leaves, and therefore may be exhibiting trophic plasticity. To test this possibility, we examined the C and N stable isotope and C/N ratios for cave and spring populations of G. minus. A 15-day feeding experiment using leaves and G. minus from a spring population established that the diet-tissue discrimination factor was 3.2 ‰ for δ¹⁵N. Cave G. minus were 8 ‰ higher in δ¹⁵N relative to cave leaves, indicating they did not derive nitrogen from leaves, whereas field collected spring populations were 2–3 ‰ higher than spring leaves, indicating that they did. Cave G. minus were 2.6 ‰ higher in δ¹⁵N than the cave isopod, Caecidotea holsingeri. Relative to spring populations, Organ Cave G. minus were ¹⁵N enriched by 6 ‰, suggesting they occupied a different trophic level, or incorporated an isotopically distinct N source. While stable isotopes cannot tell what the cave G. minus are eating, the isotopes certainly show that G. minus are not eating leaves and are trophically distinct form the surface populations. Differences in C/N ratios were observed, but reflect the size of the G. minus examined and not feeding group or habitat. The isotope data strongly support the hypothesis that cave populations of G. minus have become generalist or omnivorous by including animal protein in their diet.     

Community genomic analysis of an extremely acidophilic sulfur-oxidizing biofilm

Highly acidic (pH 0–1) biofilms, known as ‘snottites'', form on the walls and ceilings of hydrogen sulfide-rich caves. We investigated the population structure, physiology and biogeochemistry of these biofilms using metagenomics, rRNA methods and lipid geochemistry. Snottites from the Frasassi cave system (Italy) are dominated (>70% of cells) by Acidithiobacillus thiooxidans, with smaller populations including an archaeon in the uncultivated ‘G-plasma'' clade of Thermoplasmatales (>15%) and a bacterium in the Acidimicrobiaceae family (>5%). Based on metagenomic evidence, the Acidithiobacillus population is autotrophic (ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), carboxysomes) and oxidizes sulfur by the sulfide–quinone reductase and sox pathways. No reads matching nitrogen fixation genes were detected in the metagenome, whereas multiple matches to nitrogen assimilation functions are present, consistent with geochemical evidence, that fixed nitrogen is available in the snottite environment to support autotrophic growth. Evidence for adaptations to extreme acidity include Acidithiobacillus sequences for cation transporters and hopanoid synthesis, and direct measurements of hopanoid membrane lipids. Based on combined metagenomic, molecular and geochemical evidence, we suggest that Acidithiobacillus is the snottite architect and main primary producer, and that snottite morphology and distributions in the cave environment are directly related to the supply of C, N and energy substrates from the cave atmosphere.     

Groundwater biodiversity in a chemoautotrophic cave ecosystem: how geochemistry regulates microcrustacean community structure

The Frasassi cave system in central Italy hosts one of the few known examples of a groundwater metazoan community that is supported by sulfur-based lithoautotrophic microbes. Despite the challenging conditions represented by high concentrations of H₂S and low concentrations of O₂, this cave system is home to many invertebrate species. Here, we analyzed the copepods inhabiting sulfidic lakes and non-sulfidic dripping pools in order to investigate how environmental conditions in sulfidic waters regulate the spatial distribution of the cave microcrustacean community over time. We also sampled copepod assemblages of sulfidic lakes under conditions of both high and low H₂S concentration. Cluster analysis and canonical correspondence analysis separated the copepod assemblages inhabiting dripping pools from those of sulfidic lakes. H₂S concentration, pH and O₂ concentration were identified as the main factors regulating community structure. These results indicate that the distribution of groundwater copepods within the cave system is ecologically and spatially structured. Sulfidic lakes showed lower Simpson dominance, higher Shannon diversity and higher Pielou equitability at higher H₂S concentrations. The complex community structure of the copepods of the Frasassi cave system suggests that a chemosynthetically produced food source facilitated the colonization of stygobionts in sulfidic groundwater due to their tolerance to the environmental conditions.     

Novel Epibiotic Thiothrix Bacterium on a Marine Amphipod

Comparative analysis of the 16S rRNA gene and fluorescent in situ hybridization (FISH) was used to identify epibiotic filamentous bacteria living on the marine amphipod crustacean Urothoe poseidonis. The epibionts belong to the gamma proteobacteria and represent a novel marine phylotype within the genus Thiothrix. FISH and denaturing gradient gel electrophoresis revealed that the Thiothrix filaments are present on the majority of the amphipods examined.     

Phylogenetic in situ/ex situ analysis of a sulfur mat microbial community from a thermal sulfide spring in the north Caucasus

A phylogenetic in situ/ex situ analysis of a sulfur mat formed by colorless filamentous sulfur bacteria in a thermal sulfide spring (northern spur of the main Caucasian ridge) was carried out. Nine phylotypes were revealed in the mat. Thiothrix sp. and Sphaerotilus sp. were the dominant phylotypes (66.3% and 26.3%, respectively). The 16S rRNA gene nucleotide sequence of Sphaerotilus sp. phylotype from the clone library was identical to the sequences of the seven Sphaerotilus strains isolated from the same source. A very high degree of similarity of Sphaerotilus strains revealed by ERIC-PCR fingerprints indicated little or no population diversity of this species in the mat. Thiothrix phylotype from the clone library and two Thiothrix strains isolated from the same mat sample differed in one to three nucleotides of 16S rRNA genes; this is an indication of this organism’s population variability in the mat. 16S rRNA genes of the strains and clones of Thiothrix sp. exhibited the highest similarity (ca. 99%) with Thiothrix unzii; the strains and clones of Sphaerotilus had 99% similarity with the type species Sphaerotilus natans (the only species of this genus) and therefore can be assigned to this species. The minor seven components belong to the phylotypes from the Proteobacteria (3%), as well as the Chlorobia, Cyanobacteria, Clostridia, and Bacteroidetes phylogenetic groups, each of them constituting not more than 1%. Intracellular accumulation of elemental sulfur by Sphaerotilus similar to other filamentous sulfur bacteria was demonstrated for the first time (both in the population of the sulfur spring and in cultures with sulfide). Although mass growth of Sphaerotilus and Thiothrix is typical of bacterial populations of anthropogenic ecosystems (the activated sludge of treatment facilities), stable communities of these bacteria have not been previously found in the sulfur mats or “threads” of natural sulfide springs.     

Biodiversity and monitoring of colorless filamentous bacteitrtn sulfide aquatic systems of North Caucasus region

Bacterial mats in sulfide aquatic systems of North Caucasus are basically composed by the species of genera Thiothrix and Sphaerotilus. Additionally, several non-filamentous sulfur-oxidizing bacteria were isolated from the mats and several minor 16S rRNA phylotypes were found in clone libraries from these mats. The minor components were affiliated with Proteobacteria, Chlorobia, Cyanobacteria and Firmicutes. Even in an individual mat population heterogeneity of Thiothrix spp. was revealed by analysis of 16S rRNA gene and RAPD-PCR. Five Thiothrix isolates were described as new species Thiothrix caldifontis sp. nov. and Thiothrix lacustris sp. nov. In the Thiothrix-Sphaerotilus type of bacterial mat the proportion of dominant organisms might be influenced by sulfide concentration in the spring water. The higher sulfide concentration (more than 10 mg/1) in the spring water is more favorable for the development of bacterial mats with dominant Thiothrix organisms than for Thiothrix-Sphaerotilus type of sulfur mat.     

Symbiotic Relationship of Thiothrix spp. with an Echinoderm

Immunoassay procedures were used to investigate the symbiotic relationship of Thiothrix spp. in the intestinal cecum of the spatangoid species Echinocardium cordatum. Thiothrix spp. were identified in nodule samples from E. cordatum digestive tubes based on microscopic examination, enzyme-linked immunosorbent assay, and indirect immunofluorescence. Thiothrix spp. protein made up as much as 84% of the total protein content of the nodules. This is the first identification of Thiothrix spp. internally symbiotic with marine invertebrates.     

A Novel Role for Mc1r in the Parallel Evolution of Depigmentation in Independent Populations of the Cavefish Astyanax mexicanus

The evolution of degenerate characteristics remains a poorly understood phenomenon. Only recently has the identification of mutations underlying regressive phenotypes become accessible through the use of genetic analyses. Focusing on the Mexican cave tetra Astyanax mexicanus, we describe, here, an analysis of the brown mutation, which was first described in the literature nearly 40 years ago. This phenotype causes reduced melanin content, decreased melanophore number, and brownish eyes in convergent cave forms of A. mexicanus. Crosses demonstrate non-complementation of the brown phenotype in F₂ individuals derived from two independent cave populations: Pachón and the linked Yerbaniz and Japonés caves, indicating the same locus is responsible for reduced pigmentation in these fish. While the brown mutant phenotype arose prior to the fixation of albinism in Pachón cave individuals, it is unclear whether the brown mutation arose before or after the fixation of albinism in the linked Yerbaniz/Japonés caves. Using a QTL approach combined with sequence and functional analyses, we have discovered that two distinct genetic alterations in the coding sequence of the gene Mc1r cause reduced pigmentation associated with the brown mutant phenotype in these caves. Our analysis identifies a novel role for Mc1r in the evolution of degenerative phenotypes in blind Mexican cavefish. Further, the brown phenotype has arisen independently in geographically separate caves, mediated through different mutations of the same gene. This example of parallelism indicates that certain genes are frequent targets of mutation in the repeated evolution of regressive phenotypes in cave-adapted species.