Download Conference7 - Zodletone Spring

Abstract
Introduction
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.
Other anaerobic
clone libraries
analysis
Stramenopiles
Sampling sites
Spring Source
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.
•Black, sulfide-saturated slimy-mud.
• Sulfide concentration: 8-10 mM
•Sediments covered with ≈ 50 cm of
water
•High concentration of short chain
alkanes
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.
Viridiplantae Metazoa Alveolata
22%
2%
22%
Stramenopiles
20%
Fungi
34%
N=89
Wastewater digester
Alveolata
Metazoa
Microbial mats
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.
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.
Duck pond
0%
29%
Stramenopiles
0%
Viridiplantae
0%
•Visible along the course of the
spring.
•Gray, green, and purple.
•Highly exposed to sunlight, yet
anoxic
Fungi
71%
N=65
Fort Lupton
Metazoa
22%
Alveolata
5%
Stramenopiles
10%
Viridiplantae
Fungi
19%
Crust
Zodletone clone libraries analysis
Source
20%
Alveolata
12%
2%
Diplomonodida
Diplomonodida
11%
Cercozoa
Fungi
1%
Fungi
1%
18%
Stramenopiles
74%
56%
N=107
Fungi
0%
22%
Stramenopiles
4%
3%
Stramenopiles
Diplomonodida
Cercozoa
Cercozoa
0%
Alveolata
Jakobidae
2%
2%
•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 sulfideoxygen gradient
15%
Jakobidae
Jakobidae
Crust
Mat
Alveolata
N=106
Stramenopiles
44%
N=102
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.
•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.
57%
N=106
 Labyrinthuloides clones represented 11.5% of the stramenopile clones.
Phylogenetic analysis of the Source, Mat, and Crust Zodletone eukaryotic clone libraries
Diplomonadida, Jakobidae, and Cercozoa
Other Anaerobic Environments
Fungi
Alveolata
• 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:
Diplomonadida
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.
Diplomonadida
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.
Ciliophora
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.
Jakobidae
Jakoba
Zeuk2
Conclusions
Dinophyceae
Cercomondida
Cercozoa
Apicomplexa
Euglyphida
Chlorarachniophyceae
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.
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
• 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.
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).
• 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
Future study
• Full sequences of clones belonging to novel phylogenetic groups.
• Ergosterol estimation in sediments used to construct all six clone libraries.
•Q-PCR to quantify microeukaryotic community in situ