Abstract Zodletone spring in Southwestern Oklahoma is a mesophilic spring characterized by high dissolved sulfide concentration (8-10 mM), an abundance of microbial mats, and high concentrations of short chain gaseous alkanes (methane, ethane, and propane). As a result, parts of the spring are anaerobic and highly reduced. Molecular characterization of the prokaryotic community at the Zodletone spring has revealed a highly diverse population with novel division-level diversity within bacterial and archaeal communities. To identify the Eukaryotic community present in the spring, we amplified, cloned, and sequenced 18S ribosomal RNA genes from bulk DNA extracted from sediments. Three clone libraries were generated from different locations in the spring and 319 clones were sequenced and analyzed. 63% of the sequences belonged to the Stramenopiles, 17% belonged to the Fungi, 14% belonged to the Alveolata, and 2% belonged to the Cercozoa. Stramenopile clones were mainly within the known lineages of diatoms and biflagellates. Surprisingly, 39% of the Stramenopile clones were closely related to members of the genus Cafeteria, which,-until now- has been retrieved only from marine environments. The majority of fungal clones were closely related to the Ascomycetous and Basidiomycetous yeasts, or belonged to a novel uncultured fungal group (Zeuk1 group), members of which have so far been retrieved from anaerobic habitats. Alveolata clones were mainly related to bacterivorous ciliates. Cercozoa clones belonged to flagellated Cercomonadida. In addition, two sequences belong to the early branching amitochondrial group Diplomonadida, and four clones belong to the early diverging mitochondrial protist Jakobidae have been encountered. Other anaerobic environments were sampled (waste water digester, gas condensate contaminated sediments, and an anaerobic freshwater pond) and investigated by 18S rRNA gene cloning and sequencing. Surprisingly, fungal clones (either Ascomycetous yeasts, Chytrids, or the novel Zeuk1 group comprised a large percentage of the clones sequenced (between 34 and 71%). This work supports the view that microeukaryotes in anaerobic environments are more diverse than previously assumed, and documents the importance of fungi in anaerobic habitats. Characteristics of the Spring North of Zodletone mountain in the Anadarko basin of Southwestern Oklahoma. High levels of dissolved sulfide (8-10 mM) and sulfur in the emergent spring water maintains anoxic, conditions in the water and underlying sediments. Abundance of mesophilic, phototrophic microbial mats. The spring flows approximately 20 m before discharging into a nearby creek. Goals of the Project To investigate micro-eukaryotic community in this extreme environment. Detect novel groups. 18S ribosomal RNA genes were amplified from DNA extracted from sediment Clones were constructed, including 384 clones from a source sample, 196 clones from a mat sample, and 196 clones from a crust sample. Introduction Sampling sites Black, sulfide-saturated slimy-mud. Sulfide concentration: 8-10 mM Sediments covered with ≈ 50 cm of water High concentration of short chain alkanes Visible along the course of the spring. Gray, green, and purple. Highly exposed to sunlight, yet anoxic Develops on the banks of the stream 5mm thick, light gray with underlying purple color Surface exposed to air, underlying soft rock saturated with sulfide, leading to an extremely steep sulfide- oxygen gradient Spring Source Microbial mats Crust Stramenopiles Fig. 4 Distance dendrogram based on the 18s sequences of Eukaryotic Stramenopile clones encountered in Zodletone source, mat, and crust clone libraries. Numbers in parentheses represents the frequency of occurrence of a specific OTU in the source, mat, and crust clone libraries, respectively. Fungi 11% Stramenopiles 74% Cercozoa 0% Diplomonodida 1% Jakobidae 2% Alveolata 12% Fungi 18% 56% Cercozoa 3% Diplomonodida 1% Jakobidae 2% Alveolata 20% Stramenopiles Cercozoa Diplomonodida Jakobidae Fungi 22% 57% 4% 0% 2% Alveolata 15% Stramenopiles Zodletone clone libraries analysis Source Mat Crust Diplomonadida Jakobidae Cercozoa Cercomondida Euglyphida Chlorarachniophyceae Jakoba Diplomonadida Diplomonadida, Jakobidae, and Cercozoa Fig. 1. Distance dendrogram based on the 18s sequences of eukaryotic clones (Diplomonadida, Jackobidae, and Cercozoa) encountered in Zodletone source, mat, and crust clone libraries. Bootstrap values (in percent) are based on 1000 replicates and are shown for branches with more than 50% bootstrap support. Numbers in parentheses represent the frequency of occurrence of a specific OTU in the source, mat, and crust clone libraries, respectively. Alveolata Fig. 2. Distance dendrogram based on the 18s sequences of eukaryotic Alveolata clones encountered in Zodletone source, mat, and crust clone libraries. Numbers in parentheses represent the frequency of occurrence of a specific OTU in the source, mat, and crust clone libraries, respectively. All Zodletone clones belonged to the family Ciliophora, members of which are universally distributed in freshwater, marine, aerobic, and anaerobic habitats A cluster of 7 OTUs potentially have low sequence similarity (93%) to previously described cultured ciliates and environmental clones and potentially represents a novel group within the Ciliophora Ciliophora Zeuk2 Dinophyceae Apicomplexa Fig. 3 Distance dendrogram based on the 18s sequences of eukaryotic fungal clones encountered in Zodletone source, mat, and crust clone libraries. Numbers in parentheses represents the frequency of occurrence of a specific OTU in the source, mat, and crust clone libraries, respectively. In spite of the prevalent anaerobic conditions in Zodletone spring, members of the kingdom fungi represented 17% of the clones in Zodletone libraries. Several fungal clones belonged to known Yeast groups (order Saccharomycetes) members of which are known for their ability for oxygen-independent fermentative metabolism 11 fungal clones from the mat and crust clone libraries formed a novel fungal group together with several other clones encountered in a variety of anaerobic environments (from Lake Ketelmeer, Netherlands, and Berkeley Aquatic Park sediments, Berkeley, CA). Other Anaerobic Environments Conclusions Future study Full sequences of clones belonging to novel phylogenetic groups. Ergosterol estimation in sediments used to construct all six clone libraries. A molecular survey of the microeukaryotic community of Zodletone spring suggested a thriving microeukaryotic population.This population is extremely diverse, capable of surviving in an highly- reduced, sulfide-saturated environment. Some of the clones detected belonged to groups never before isolated from anaerobic habitats (e.g. Jackoba ), or which have been isolated only from marine environments (e.g. Cafeteria ) Fungal clones represented a significant fraction in all six clone libraries, indicating that the importance of fungi is underestimated in anaerobic settings. A novel group of fungi was identified, and it appears to be globally distributed in anaerobic habitats Anaerobic samples from anaerobic fresh water pond sediments (Norman duck pond, Norman, OK), Norman wastewater treatment plant (Norman, OK), and gas-condensate contaminated aquifer (Fort Lupton, CO) were collected. 3 clone libraries were constructed and a total of 288 clones from the three libraries were partially or completely sequenced. Preliminary analysis indicated:  No Diplomonadidae, Jakobidae, and Cercozoa clones were detected in any of the three libraries.  Compared to Zodletone, the three libraries had a lower representation of Alveolata and Stramenopiles and higher representation of Fungi. In addition, Metazoa and Viridiplantae represented a significant fraction of the library.  The majority of stramenopile clones belonged mainly to photosynthetic lineages (Chrysophyta, Bacillariophyceae, and Raphidophyceae). No stramenopile clones were encountered in anaerobic waste water treatment samples, probably due to the aphotic nature of the ecosystem.  Fungal clones represented a significant fraction of the three clone libraries (46,46, and 29 clones for Waster water, Fort Lupton, and Duck pond respectively). Fungal clones were either Sacharomycetous yeasts, Chytrids, or members of the novel Zeuk1 group, and Zeuk1 group has been identified in all three environments. Phylogenetic analysis of the Source, Mat, and Crust Zodletone eukaryotic clone libraries N=107N=106 Duck pond Wastewater digester Fort Lupton Alveolata 0% Stramenopiles 0% Fungi 71% Viridiplantae 0% Metazoa 29% N=65 Alveolata 5% Stramenopiles 10% Fungi 44% Viridiplantae 19% Metazoa 22% N=102 Alveolata 22% Stramenopiles 20% Fungi 34% Viridiplantae 22% Metazoa 2% N=89 About 63% of Zodletone clones clones belonged to the Stramenopiles. Three major groups of Stramenopiles from our clones were detected  Diatoms of the family Bacillariophyceae represented 31% of the total Stramenopile clones.  Bicosoecida clones closely related to genus Cafeteria, members of which are marine heterotrophic flagellates represented 40.5% of the stramenopile clones.  Labyrinthuloides clones represented 11.5% of the stramenopile clones. Other anaerobic clone libraries analysis Q-PCR to quantify microeukaryotic community in situ Fungi