Ancient European lakes are clustered within a radius of 300 km around Lake Ohrid. Information concerning microbial diversity in these lakes is limited. We studied diversity of the dominant prokaryotic phylotypes in the sediments in one of these lakes, known as Lake Pamvotis. The analysis was performed in samples from two stations for four seasons of the same year. DNA extraction followed by PCR amplification (16S rDNA), Denaturing Gradient Gel Electrophoresis, cloning and sequencing w as applied in order to reveal the sequence signatures of the dominant bacterial and archaeal phylotypes. Bacterial and archaeal cell numbers were quantified by real-time PCR. Several environmental variables measured in parallel, including pH, Nickel, Chromium, Arsenic, Calcium, Total Nitrogen and Total Carbon, were found to affect strongly the prokaryotic abundances. Most of the identified sequences of Bacteria belong to Proteobacteria and most of the sequences of Archaea belong to Euryarchaeota. The great majority of these bacterial (84.21%) and archaeal sequences (95.65%) have no cultivated counterparts in the databases. In addition, many of these bacterial (50.88%) and archaeal sequences (20.65%) correspond to potentially new species. Six of the bacterial sequences constitute a new class of Cyanobacteria which we have named “Lake Pamvotis cluster” (LPC). Our findings highlight Lake Pamvotis as a habitat for several previously unidentified species of Bacteria and Archaea.
Worldwide ancient lakes such as Baikal, Tanganyika, Victoria, Titicaca represent “natural laboratories’’ for evolutionary research and major hotspots of biological diversity [
Lake Pamvotis has been in existence throughout the Plio-Pleistocene period, as shown by the identification of several endemic mollusc taxa which are known to be 500,000 years old [
Unfortunately, microbial diversity has not been extensively studied either in Lake Pamvotis or in other lakes of the wider region. The few studies conducted were mainly focusing on the problems of gradual eutrophication and urbanization in some of these lakes [
interesting. Molecular data reveal that the population of the filamentous Cyanobacteria from Lake Pamvotis is homogeneous, but divergent from other populations worldwide [
Our study addresses three important questions on the organization of this aquatic microbial ecosystem: 1) Are there novel, previously unidentified, bacterial and archaeal species among the dominant phylotypes? 2) Are archaeal communities a quantitatively important component of microbial communities inhabiting this environment? 3) Is there a correlation between physicochemical variables, prokaryotic abundance and diversity of the dominant phylotypes?
Lake Pamvotis is a closed hydrological system. It lies approximately at 39˚40'N, 20˚53'E, at 470 meters above sea-level in the mountainous region of the Pindus. It is a shallow lake (4.23 m average depth) and has a surface area of about 22.8 km2 [
Sediment samples (top 5 - 10 cm) were collected using a grab sampler at two sampling stations (SS): SS1 and SS2. SS1 is situated approximately in the middle of the lake (depth 6.5 to 7.5 m depending on the season) and SS2 is a station where the maximum depth of the lake was measured (8.5 to 9.5 m depending on the season) (
Sediment samples were dried at 70˚C for 24 h upon arrival to the laboratory.
For the pH measurements, sediment samples were diluted in 1M KCl (1:2 sediment to solution ratio) and a Hanna pH meter was used (Hanna Instruments pΗ211) [
Two grams of each sample were extracted twice with 20 mL of bidistilled water, for anions (Cl−, SO42−) analysis, and 20 mL of 40 mM nitric acid aqueous solution, for cations (Na+, K+, Ca2+, Mg2+) analysis, in an ultrasonic bath for 30 min. The extracts were centrifuged, combined and diluted in bidistilled water to a volume of 50 mL. 20 μL of each sample were injected in HPLC equipped with a conductivity detector (Shimadzu CDD-10A VP). For the determination of cations IC YK-421 column with a Shodex IC YK-G column guard and anions IC NI-424 column with IC NI-G column guard in a Shimadzu CTO-10AC column oven were used with shipping solvent. Standard solutions of the above ions at concentrations ranging from 1 to 100 mg/L in seven levels were analyzed as external calibration basis quantification [
Total carbon (TC) and total organic carbon (TOC) were analyzed with a Shimadzu TOC-VCPH carbon analyzer (Shimadzu, Japan), coupled to a solid state combustion unit (model SSM-5000A). One gram of dried sample was inserted in solid state combustion unit. For TC the unit uses catalytically aided combustion oxidation at 900˚C method and for inorganic carbon (IC) pre-acidification, with oven temperature 250˚C. After the treatment in the solid state combustion unit, samples were automatically inserted directly in the carbon analyzer, which measures the TC and IC. TOC was derived by subtracting the IC from the TC.
The total nitrogen in the sediment (TN) was determined spectrophotometrically by Total Kjeldahl (Nessler method) after digestion by the HACH Digesdahl Apparatus together with 3 mL H2SO4 (98% v/v) at 450˚C, while for the amendment of the digest the HACH method 8075 was used. The concentration of TN within the sample was measured in a HACH DR/2010 Spectrophotometer at the wavelength of 460 nm. The total phosphorous content (TP) in the sediment was determined by the molybdenum blue method (HACH) [
Heavy metals Sb, Ni, Hg, Se, Cd, Mn, Pb, Fe, Cu, Cr, Zn and As were determined using ICP-AES (Thermo Scientific iCAP 6300 ICP Spectrometer) according to the methodology described by Ashley et al. [
For the isolation of culturable bacterial species, R2A plates (LABM, United Kingdom) were prepared according to the manufacturer’s instructions. R2A medium was used for a general view of culturable freshwater Bacteria. Ten grams of sediment samples taken during summer from both stations were suspended in sterile water. A series of 10-fold dilutions were prepared. R2A medium plates were inoculated with 100 μL aliquots from different dilutions as described earlier [
DNA was extracted from the sediment samples using an UltraClean soil DNA isolation kit from MoBio Laboratories (PowerSoil DNA Isolation kit, Carlsbad, CA 92010) in accordance with the manufacturer’s instructions.
PCR amplification was performed in a Biorad iCycler in a 50 μL reaction volume. For archaeal 16S rDNA amplification, a 344F-GC and 915R primer set was used and a touchdown PCR was performed as described earlier [
For bacterial 16S rDNA amplification a 341F-GC and 907R primer set was used and a touchdown PCR was performed as described earlier [
PCR products for both Archaea and Bacteria 16S rDNA were evaluated in a 1% (w/v) agarose gel electrophoresis and subsequently used for Denaturing Gradient Gel Electrophoresis (DGGE).
For quantification of archaeal and bacterial 16S rRNA genes in our samples, serial 10-fold dilutions of recombinant plasmids containing a partial fragment of an archaeal and a bacterial 16S rDNA respectively were used as external standards, to obtain a reference curve. The standard dilutions ranged from 103 to 105 and from 104 to 1010 for archaeal and bacterial reference curves, respectively.
The real-time PCR was performed in a LightCycler 480 (Roche) instrument using the LightCycler 480 SYBR Green Master I (Roche) following the manufacturer’s instructions. The final 20 μL reaction mix contained 10 μL of the SYBR Green Master Mix I, the original primer set (in case of Forward primers without the GC clamp) for Bacteria and Archaea and an appropriate dilution of the DNA samples were initially incubated at 95˚C for 5 min followed by 40 cycles of a 3-step cycling at 95˚C for 45 s (denaturation), 61˚C for 45 s for Archaea or 60˚C for 45 s for Bacteria (annealing), 72˚C for 45 s (extension) and a final extension for 10 min at 72˚C. All samples, standards and negative controls were tested in triplicates. Finally, we used CT values to determine the 16S rDNA copy numbers in our samples and we converted them into cell numbers assuming that archaeal cells contain 2 and bacterial cells contain 3.8 16S rDNA copies per cell [
DGGE for Archaea and Bacteria was performed as described earlier by Muyzer et al. [
Depth (m) | T˚C | pH | TC (mg/g) | TOC (mg/g ) | TP (mg/g) | TN (mg/g) | |
---|---|---|---|---|---|---|---|
Spring, SS1 | 8.40 ± 0.20 | 20 ± 0.50 | 6.96 ± 0.02 | 67.54 ± 7.26 | 60.69 ± 3.13 | 3.02 ± 0.02 | 2.94 ± 0.01 |
Spring, SS2 | 9.30 ± 0.30 | 19 ± 0.40 | 6.30 ± 0.01 | 99.79 ± 3.53 | 87.31 ± 3.99 | 4.05 ± 0.02 | 5.03 ± 0.11 |
Summer, SS1 | 7.30 ± 0.15 | 26 ± 0.70 | 7.07 ± 0.01 | 68.58 ± 2.31 | 65.00 ± 2.01 | 4.83 ± 0.05 | 3.91 ± 0.03 |
Summer, SS2 | 8.20 ± 0.18 | 24 ± 0.50 | 6.31 ± 0.01 | 99.53 ± 2.07 | 93.70 ± 4.09 | 4.13 ± 0.02 | 3.33 ± 0.02 |
Autumn, SS1 | 8.10 ± 0.25 | 11 ± 0.50 | 7.08 ± 0.01 | 67.39 ± 2.42 | 63.54 ± 2.56 | 10.01 ± 0.09 | 3.99 ± 0.03 |
Autumn, SS2 | 9.00 ± 0.20 | 12 ± 0.50 | 6.23 ± 0.02 | 65.60 ± 1.01 | 61.30 ± 1.96 | 9.69 ± 0.08 | 5.01 ± 0.05 |
Winter, SS1 | 8.40 ± 0.10 | 6 ± 0.10 | 7.18 ± 0.20 | 59.73 ± 3.60 | 58.19 ± 2.12 | 10.75 ± 0.42 | 3.94 ± 0.03 |
Winter, SS2 | 9.30 ± 0.40 | 6 ± 0.20 | 6.45 ± 0.02 | 79.80 ± 11.14 | 79.60 ± 1.02 | 8.78 ± 0.05 | 5.80 ± 0.01 |
Na+ (mg/kg) | K+ (mg/kg) | Ca2+ (mg/kg) | Mg2+ (mg/kg) | Cl− (mg/kg) | SO42- (mg/kg) | |
---|---|---|---|---|---|---|
Spring, SS1 | 4.96 ± 0.01 | 4.17 ± 0.01 | 37.69 ± 0.02 | 13.06 ± 0.04 | 64.34 ± 0.02 | 296.62 ± 0.04 |
Spring, SS2 | 6.09 ± 0.01 | 5.41 ± 0.01 | 72.15 ± 0.26 | 17.83 ± 0.01 | 117.82 ± 0.70 | 537.42 ± 0.10 |
Summer, SS1 | 4.87 ± 0.01 | 4.34 ± 0.01 | 50.04 ± 0.02 | 14.48 ± 0.03 | 64.66 ± 0.02 | 554.17 ± 0.03 |
Summer, SS2 | 5.54 ± 0.01 | 5.98 ± 0.01 | 76.34 ± 0.79 | 19.41 ± 0.04 | 103.95 ± 0.18 | 386.02 ± 0.12 |
Autumn, SS1 | 4.91 ± 0.01 | 5.57 ± 0.03 | 49.19 ± 0.12 | 18.73 ± 0.02 | 70.17 ± 0.01 | 431.68 ± 0.02 |
Autumn, SS2 | 6.33 ± 0.01 | 5.86 ± 0.01 | 70.98 ± 0.02 | 18.95 ± 0.03 | 109.87 ± 0.09 | 589.16 ± 0.03 |
Winter, SS1 | 3.39 ± 0.01 | 5.56 ± 0.01 | 57.28 ± 0.02 | 16.33 ± 0.02 | 35.35 ± 0.02 | 450.01 ± 0.03 |
Winter, SS2 | 3.62 ± 0.02 | 5.02 ± 0.02 | 51.26 ± 0.73 | 16.32 ± 0.02 | 51.16 ± 0.04 | 428.55 ± 0.07 |
Sb (mg/kg) | Ni (mg/kg) | Hg (mg/kg) | Se (mg/kg) | Cd (mg/kg) | Mn (mg/kg) | |
---|---|---|---|---|---|---|
Spring, SS1 | 5.10 ± 0.08 | 132.00 ± 0.25 | 1.13 ± 0.01 | <6.00 | <4.00 | 1090.00 ± 22.00 |
Spring, SS2 | 2.43 ± 0.02 | 98.00 ± 1.75 | 1.59 ± 0.01 | <6.00 | <4.00 | 959.00 ± 10.00 |
Summer, SS1 | 2.85 ± 0.02 | 126.00 ± 0.20 | 0.16 ± 0.01 | <6.00 | <4.00 | 1130.00 ± 35.00 |
Summer, SS2 | 2.78 ± 0.01 | 97.10 ± 1.53 | 0.84 ± 0.02 | <6.00 | <4.00 | 843.00 ± 21.00 |
Autumn, SS1 | 2.89 ± 0.01 | 123.00 ± 0.17 | 0.44 ± 0.01 | <6.00 | <4.00 | 1330.00 ± 76.00 |
Autumn, SS2 | 2.72 ± 0.02 | 96.50 ± 1.20 | 0.95 ± 0.01 | <6.00 | <4.00 | 923.00 ± 15.00 |
Winter, SS1 | 3.32 ± 0.02 | 135.00 ± 0.22 | 0.18 ± 0.01 | <6.00 | <4.00 | 950.00 ± 15.00 |
Winter, SS2 | 3.52 ± 0.02 | 88.50 ± 2.70 | <0.10 | <6.00 | <4.00 | 999.00 ± 32.00 |
Pb (mg/kg) | Fe (mg/kg) | Cu (mg/kg) | Cr (mg/kg) | Zn (mg/kg) | As (mg/kg) | |
---|---|---|---|---|---|---|
Spring, SS1 | <30.0 | 25200.00 ± 58.00 | 31.40 ± 1.10 | 83.10 ± 2.87 | 81.30 ± 1.99 | 2.76 ± 0.01 |
Spring, SS2 | <30.0 | 25500.00 ± 61.00 | 31.80 ± 0.90 | 59.40 ± 2.12 | 89.90 ± 3.22 | 4.58 ± 0.02 |
Summer, SS1 | <30.0 | 27600.00 ± 32.00 | 32.10 ± 0.70 | 83.60 ± 2.66 | 93.00 ± 1.42 | 1.88 ± 0.01 |
Summer, SS2 | <30.0 | 26400.00 ± 59.00 | 32.10 ± 1.20 | 66.00 ± 1.89 | 91.50 ± 2.89 | 4.44 ± 0.03 |
Autumn, SS1 | <30.0 | 28100.00 ± 45.00 | 30.60 ± 0.50 | 81.70 ± 1.57 | 90.10 ± 1.08 | 2.40 ± 0.02 |
Autumn, SS2 | <30.0 | 23600.00 ± 52.00 | 31.00 ± 0.20 | 63.60 ± 1.75 | 97.00 ± 3.55 | 4.37 ± 0.01 |
Winter, SS1 | <30.0 | 28200.00 ± 42.00 | 34.00 ± 0.90 | 79.20 ± 1.28 | 97.00 ± 2.80 | 2.14 ± 0.01 |
Winter, SS2 | <30.0 | 21000.00 ± 20.00 | 37.00 ± 0.40 | 58.40 ± 1.97 | 78.70 ± 1.43 | 4.80 ± 0.02 |
Physicochemical properties of the sediments in Lake Pamvotis sample station 1 (SS1) and 2 (SS2). (a) Depth, T, pH, Carbon, Nitrogen and Phosphorous contents; (b) Major anions and cations; c) Heavy metals. Heavy metal concentrations exceeding the PEC or TEC limits are indicated in bold (Ni PEC: 48.6 mg/kg, Hg PEC: 1.06 mg/kg, Hg TEC: 0.18 mg/kg, Cr TEC: 43.4 mg/kg, Cu TEC: 31.6 mg/kg) [
afterwards they were cloned using a TOPO TA cloning Kit (Invitrogen, USA) according to the manufacturer’s instructions. Subsequently, ten recombinant clones from each library (corresponding to each DGGE band) were randomly picked for further analysis. Inserts were digested with restriction enzyme HaeIII (HT Biotechnology Ltd, Cambridge, United Kingdom) in order to identify different Restriction Fragment Length Polymorphisms (RFLPs) [
The final sequences were deposited at GenBank and were assigned accession numbers KC510289-KC510380 for Archaea, KP244158-KP244214 for Bacteria and KU862661-KU862683 for cultured isolates.
All sequences were compared against GenBank using BLAST in order to obtain their phylogenetic affiliation. Phylogenetic analyses were performed with MEGA6.1 software. Trees were constructed using the Neighbor-Joining method with Jukes-Cantor distance correction [
Spearman’s correlation coefficient was used to investigate possible relationships among bacterial and archaeal abundances and the physicochemical variables. All statistical analyses were conducted with STATISTICA 7 (Tulsa, OK, USA).
Total Carbon (TC), Total Organic Carbon (TOC), Total Nitrogen (TN) and Total Phosphorus (TP) concentrations (
Concerning heavy metal concentrations, according to the Sediment Quality Guidelines (SQGs) [
In a previous study conducted between 1991-1993 heavy metal concentrations had been measured in surface sediment samples from Lake Pamvotis stations SS1 and SS2 [
Nickel and Cr input in lake sediments are possibly enhanced either by mining activities [
Mercury (Hg) concentrations in Lake Pamvotis sediments remains stable relative to the concentrations measured previously (1991-1993) [
In a recent study [
The prokaryotic community in the Lake Pamvotis sediments was found to be dominated by Bacteria. Archaea accounted for 6.17% to 14.09% of the total prokaryotic 16S rDNA copy number (
Our data are in agreement with previously published studies on other lakes suggesting that Archaea are not the dominant component of the prokaryotic community in freshwater sediments. In sediments of Lake Pavin, qPCR analysis
Bacterial 16S rDNA copies/g sediment | Archaeal 16S rDNA copies/g sediment | %Archaeal 16S rDNA copies | Bacteria estimated cell number/g sediment | Archaea estimated cell number/g sediment | %Archaea cell number | |
---|---|---|---|---|---|---|
Spring, SS1 | 3.16 ± 0.29 × 109 | 2.08 ± 0.21 × 108 | 6.17% | 0.83 × 109 | 1.04 × 108 | 11.13% |
Summer, SS1 | 4.52 ± 0.24 × 109 | 5.24 ± 0.18 × 108 | 10.38% | 1.18 × 109 | 2.62 × 108 | 18.16% |
Autumn, SS1 | 4.04 ± 0.22 × 109 | 3.94 ± 0.23 × 108 | 8.88% | 1.06 × 109 | 1.97 × 108 | 15.30% |
Winter, SS1 | 4.76 ± 0.26 × 109 | 3.25 ± 0.20 × 108 | 6.39% | 1.25 × 109 | 1.62 × 108 | 11.47% |
Spring, SS2 | 4.32 ± 0.21 × 109 | 6.32 ± 0.19 × 108 | 12.76% | 1.13 × 109 | 3.16 × 108 | 21.85% |
Summer, SS2 | 5.24 ± 0.23 × 109 | 8.60 ± 0.24 × 108 | 14.09% | 1.37 × 109 | 4.30 × 108 | 23.88% |
Autumn, SS2 | 5.68 ± 0.28 × 109 | 8.12 ± 0.26 × 108 | 12.54% | 1.49 × 109 | 4.06 × 108 | 21.41% |
Winter, SS2 | 5.64 ± 0.27 × 109 | 7.68 ± 0.22 × 108 | 11.98% | 1.48 × 109 | 3.84 × 108 | 20.96% |
Quantification of both bacterial and archaeal 16S rDNA gene copies in Lake Pamvotis sediments, as determined by quantitative PCR assays. Bacterial and archaeal cell numbers have been estimated assuming 3.8 and 2 copies of the 16S rDNA per bacterial and archaeal cell, respectively [
revealed that Archaea accounted for 5% - 18% of the prokaryotic community [
Based on our results, SS2 displays higher abundances for both bacterial and archaeal communities. Spring is the period of the year where both bacterial and archaeal numbers are lower, whereas the highest abundances are recorded in summer (
A total of 153 DGGE bands were identified (
Is this bacterial diversity recognizable also with common cultivating techniques? To address this question, R2A plates were inoculated as described in Methods. A total of fifty randomly selected bacterial colonies were grown and characterized further. Of these 50 colonies, 23 different bacterial phylotypes were identified based on 16S rDNA sequences. Interestingly, 13.04% of these sequences, were found to have <97% identity to already deposited Genbank entries (
Based on the constructed phylogenetic tree (Figures 2(a)-(c)), the DGGE-retrieved sequences (BacPamv; red symbols in
More specifically, most of the DGGE-retrieved Proteobacterial sequences are contained in the class β-Proteobacteria (9 sequences). Four of them have low identity to any known bacterial sequences (<94%) (
Cyanobacterial clones were identified in both stations and during all seasons (
In lake Pamvotis, two distinct planktonic cyanobacterial populations had been identified previously, based on internal transcribed spacer (ITS) analysis. One of them was defined as Microcystis sp. and the other one consisted of various filamentous Cyanobacteria which comprise a phylogenetically diverse group unprecedented by other populations worldwide [
Nitrospirae-like and Acidobacteria-like BacPamv sequences were difficult to be phylogenetically affiliated into the general bacterial phylogenetic tree, mainly due to their low homologies to known Nitrospirae and Acidobacterial sequences (sequence identity 89% - 96%) [
Overall, we detected 13 bacterial phyla in Lake Pamvotis sediments. Proteobacteria, Bacteroidetes, Planctomycetes, Actinobacteria, Firmicutes, Acidobacteria and Nitrospirae, have also been observed in other lakes and rivers [
Relative to Bacteria, fewer DGGE bands were identified for Archaea (130 in total) but the banding pattern of Archaea was more variable (
Nineteen of the 92 archaeal sequences (20.65%) were found to have <97% identity to any already known GenBank entry. When comparing with already known cultivated archaeal species, 88 of these sequences (95.65%) were found to have <97% identity to any sequence from cultured Archaea (
Methanogenic Archaea of the Methanomicrobiales, Methanocellales and Methanosarcinales lineages were predominant in our samples, suggesting that the main archaeal metabolic function in the surface sediment of Lake Pamvotis is methane production. These lineages are frequently observed in the superficial zone of freshwater sediments [
Uncultured archaeal lineages appear to be ubiquitous in Lake Pamvotis as also observed in other freshwater sediments. Interestingly, we found that the phylogenetic cluster containing the most ArcPamv phylotypes coincides with a previously reported [
The numbers of ArcPamv sequences belonging to the Marine Benthic Group-D (MBG-D) and Rice Cluster V (RC-V) are comparable to those in the “unknown cluster I”. MBG-D represents a highly common fraction of the prokaryotic community in hypersaline sediments and along with RC-V and Lake Dagow Sediment (LDS) lineages represents the most widely distributed uncultured lineages in freshwater sediments [
Based on our phylogenetic tree, the LDS cluster was revealed to be more closely related to Candidatus Parvarchaeum acidiphilum (ARMAN-4) [
ARMANS, are nanosized Archaea which have been discovered in chemoautotrophic biofilms of the acidic metal rich Richmond Mine of Iron Mountain California [
Could LDS or the “unknown cluster V” represent acidophilic nanosized symbionts of archaeal lineages related to Thermoplasmatales? This remains to be elucidated. Based on the available 16S rDNA fragments, the representatives of both the LDS and the “unknown cluster V” are characterized by high AT contents comparable to the ones of ARMANS.
The four other Euryarchaeotal rare sequences (ArcPamv36, ArcPamv21C, ArcPamv71 and ArcPamv3A) were found to be related to Thermoplasmatales. Finally, three archaeal sequences (ArcPamv54, ArcPamv45 and ArcPamv114) fall into three robust closely related but distinct clusters with external sequences which have been previously characterized as Miscellaneous Crenarchaeota Group (MCG) [
In any case, MCG is a cosmopolitan group, frequently identified in anoxic habitants [
Regarding nutrient loads, TN was positively correlated with bacterial and, to a lesser extent, with archaeal abundances, whereas TOC was found to affect mainly the archaeal abundances (
Bacteria | Archaea | |
---|---|---|
Depth | 0.29 | 0.34 |
T | −0.34 | 0.07 |
pH | −0.57 | −0.85 |
TC | 0.12 | 0.55 |
TOC | 0.27 | 0.65 |
TP | 0.44 | 0.01 |
TN | 0.61 | 0.54 |
Na | −0.02 | 0.39 |
K | 0.56 | 0.54 |
Ca | 0.58 | 0.75 |
Mg | 0.54 | 0.64 |
Cl | 0.16 | 0.59 |
SO4 | 0.46 | 0.43 |
Sb | −0.53 | −0.61 |
Ni | −0.69 | −0.91 |
Hg | −0.32 | 0.07 |
Mn | −0.02 | −0.08 |
Fe | −0.46 | −0.52 |
Cu | 0.40 | 0.19 |
Cr | −0.66 | −0.81 |
Zn | 0.20 | 0.09 |
As | 0.55 | 0.80 |
Results of correlation analysis between physicochemical variables and bacterial/archaeal abundances. Spearman’s correlation coefficients are shown. Statistically significant correlations are indicated in yellow (p < 0.05) or in red (p < 0.001).
the major players in the recycling of nitrogen and Archaea might be more important for carbon mineralization. In SS2, which is more heavily loaded with TC and TOC, methanogenic phylotypes are more common than in SS1 (
Calcium concentration levels were correlated positively with both bacterial and archaeal cell numbers, suggesting a possible adaptation of the prokaryotic populations to a calcareous environment. Such an environment has been established in the sediments of the lake from ancient years, since the surrounding mountains consist mainly of lime bedrocks.
Concerning heavy metals, As had a strong positive effect on archaeal and a mild positive effect on bacterial cell abundances. In contrast, Ni and Cr seem to affect negatively both bacterial and archaeal abundances and, again, the effect is stronger on Archaea. Given that genes for metabolism, resistance and detoxification of metals are widespread throughout the archaeal and the bacterial domains [
In our study, pH was found to affect negatively the abundances of both Archaea and Bacteria, but the most significant effect was found for Archaea (
Soil pH affects the chemical form, concentration and availability of different substrates [
Concerning diversity, there are no obvious differences between the two sample stations with respect to the dominant bacterial phylotypes (
From the relatively limited available literature, numerical differences between bacterial and archaeal diversities in lake sediments remain unclear. Some previously published studies indicate a higher bacterial over archaeal diversity in lake sediments [
A number of environmental factors such as pH [
To our knowledge, this is the first study on both bacterial and archaeal abundances, diversity and community structure in the sediments of an ancient lake within the major European freshwater biodiversity hotspot.
Ni and Cr affect negatively both bacterial and archaeal abundances while Ca concentrations were found to have a positive effect. pH affects negatively mainly the archaeal abundance. TN has a strong positive effect on bacterial abundance, whereas As and TOC affect mainly Archaea.
Based on molecular characterization of the microbial communities, several new prokaryotic species were identified. A new class of Cyanobacteria was discovered in Lake Pamvotis sediments and termed “Lake Pamvotis cluster” (LPC). Concerning Archaea, most of the sequences retrieved from the sediments were affiliated to Euryarchaeota (dominated by Methanogenic Archaea). Interestingly, the widespread uncultivated cluster LDS was found to be phylogenetically related to ARMAN-4 lineage suggesting an unprecedented ecological role for this cluster.
We would like to acknowledge the late professor Evangelos Briasoulis for his critical reading of the paper, his valuable corrections and helpful discussion. We would like to acknowledge Mr. Sotiris Simos for his help during sampling in Lake Pamvotis.
The authors declare no conflict of interest.
Touka, A., Vareli, K., Igglezou, M., Monokrousos, N., Alivertis, D., Halley, J.M., Hadjikakou, S., Frillingos, S. and Sainis, I. (2018) Ancient European Lakes: Reservoirs of Hidden Microbial Diversity? The Case of Lake Pamvotis (NW Greece). Open Journal of Ecology, 8, 537-578. https://doi.org/10.4236/oje.2018.810033
Clone Affiliation of Bacteria
Clone name | Accession number | % query, % identity culture collection | % query, % identity cultured species |
---|---|---|---|
BacPamv1 | KP244158 | 100% 99% HM153665.1 | 100% 99% NR029024.1 Hydrogenophaga defluvii BSB 9.5 |
BacPamv2 | KP244159 | 99% 98% FQ659268.1 | 99% 88% NR074757.1 Treponema caldaria DSM 7334 |
BacPamv3 | KP244160 | 100% 93% GQ472421.1 | 100% 83% NR075001.1 Moorella thermoacetica ATCC 39073 |
BacPamv4 | KP244161 | 100% 99% HM243914.1 | 100% 86% NR074330.1 Nitrosococcus oceani ATCC 19707 |
BacPamv5 | KP244162 | 100% 96% JN805711.1 | 100% 92% NR075002.1 Syntrophobacter fumaroxidans MPOB |
BacPamv6 | KP244163 | 100% 87% JQ516335.1 | 100% 78% NR075009.1 Geobacter sulfurreducens PCA |
BacPamv7 | KP244164 | 100% 95% HQ910926.1 | 100% 85% NR028695.1 Lewinella nigricans SS-2 |
BacPamv8 | KP244165 | 100% 99% KC432448.1 | 100% 86% NR104911.1 Vampirovibrio chlorellavorus ICPB 3707 |
BacPamv9A | KP244166 | 100% 99% KC432448.1 | 100% 85% NR043559.1 Gracilibacter thermotolerans JW/YJL-S1 |
BacPamv9B | KP244167 | 100% 100% JF265807.1 | 100% 96% NR029287.1 Nitrospira moscoviensis NSP M-1 |
BacPamv10 | KP244168 | 100% 99% HM346679.1 | 100% 94% NR042824.1 Collimonas arenae NCCB 100031 |
BacPamv11 | KP244169 | 100% 92% EF203209.1 | 100% 85% NR036977.1 Thiococcus pfennigii 4250 |
BacPamv12 | KP244170 | 100% 99% AB661525.1 | 100% 84% NR109681.1 Thermoanaerobaculum aquaticum MP-01 |
BacPamv13A | KP244171 | 100% 99% AB196055.1 | 100% 92% NR044309.1 Steroidobacter denitrificans F5 |
---|---|---|---|
BacPamv13B | KP244172 | 99% 93% HF677528.1 | 98% 85% NR075001.1 Moorella thermoacetica ATCC 39073 |
BacPamv13C | KP244173 | 100% 99% KC989704.1 | 100% 99% NR074314.1 Microcystis aeroginosa NIES-843 |
BacPamv14 | KP244174 | 100% 90% JN473052.1 | 100% 78% NR075001.1 Moorella thermoacetica ATCC 39073 |
BacPamv15 | KP244175 | 100% 99% HM346679.1 | 100% 93% NR042824.1 Collimonas arenae NCCB 100031 |
BacPamv16 | KP244176 | 100% 99% JN868188.1 | 100% 92% NR043249.1 Denitratisoma oestradiolicum AcBE2-1 |
BacPamv17A | KP244177 | 98% 96% JQ583178.1 | 97% 94% NR074351.1 Candidatus solibcter Ellin 6076 |
BacPamv17B | KP244178 | 100% 96% HQ904418.1 | 100% 85% NR102459.1 Chamaesiphon minutes PCC 6605 |
BacPamv18 | KP244179 | 100% 99% KF287757.1 | 100% 99% NR025816.1 Porphyrobacter donghaensis SW-132 |
BacPamv19 | KP244180 | 100% 99% KC248046.1 | 100% 98% NR042941.1 Paucibacter toxinivorans 2C20 |
BacPamv20A | KP244181 | 100% 97% HQ661184.1 | 100% 84% NR102468.1 Stanieria cyanospaera PCC 7437 |
BacPamv20B | KP244182 | 100% 89% EU376186.1 | 100% 79% NR102456.1 Leptolyngbya PCC 7376 |
BacPamv21 | KP244183 | 100% 99% EU104276.1 | 100% 89% NR040990.1 Owenweeksia hongkongensis UST 20020801 |
BacPamv22 | KP244184 | 100% 96% HQ661184.1 | 100% 84% NR102456.1 Leptolyngbya PCC 7376 |
BacPamv23 | KP244185 | 100% 96% KF939466.1 | 100% 94% NR102987.1 Clostridium clariflavum DSM 19732 |
BacPamv24A | KP244186 | 100% 99% KC541335.1 | 100% 93% NR044309.1 Steroidobacter denitrificans FS |
---|---|---|---|
BacPamv24B | KP244187 | 99% 97% AY693835.1 | 99% 83% NR025079.1 Desulfomonile limimaris DSB-M |
BacPamv25 | KP244188 | 100% 99% HM243891.1 | 100% 88% NR074345.1 Thermodesulfovibrio yellowstonii DSM 11347 |
BacPamv26 | KP244189 | 100% 99% KC666549.1 | 100% 97% NR043993.1 Rheinheimera tangshanensis JA3-B52 |
BacPamv27 | KP244190 | 100% 93% HQ246251.1 | 100% 93% NR029024.1 Hydrogenophaga defluvii BSB 9.5 |
BacPamv28 | KP244191 | 100% 91% AB722172.1 | 100% 84% NR037137.1 Treponema medium G7201 |
BacPamv29 | KP244192 | 99% 91% AB661540.1 | 99% 86% NR074757.1 Treponema caldaria DSM 7334 |
BacPamv30 | KP244193 | 100% 99% JN257048.1 | 100% 95% NR074317.1 Nostoc punctiforme PCC 73102 |
BacPamv31 | KP244194 | 100% 91% GQ356966.1 | 100% 84% NR025150.1 Desulfobulbus mediterraneus 86FS1 |
BacPamv32 | KP244195 | 100% 99% KF556697.1 | 100% 98% NR074760.1 Albidiferax ferrireducens T118 |
BacPamv33 | KP244196 | 100% 99% AB793710.1 | 100% 97% NR026102.1 Clostridium papyrosolvens DSM 2792 |
BacPamv34 | KP244197 | 100% 92% GU208417.1 | 100% 85% NR028745.1 Thioalkalivibrio denitrificans ALJD |
BacPamv35 | KP244198 | 100% 89% AM181924.1 | 100% 84% NR043929.1 Skermanella aerolata 5416T-32 |
BacPamv36 | KP244199 | 100% 99% HG792168.1 | 100% 99% NR036911.2 Aeromonas media RM |
BacPamv37 | KP244200 | 100% 99% KC815481.1 | 100% 99% NR102447.1 Cyanobium gracile PCC 6307 |
BacPamv38 | KP244201 | 100% 99% LK054500.1 | 100% 99% NR075062.2 Micrococcus luteus NCTC 2665 |
---|---|---|---|
BacPamv39 | KP244202 | 100% 92% KF384384.1 | 100% 87% NR104682.1 Marinilabilia salmonicolor JCM 21150 NBRC 15946 |
BacPamv40 | KP244203 | 100% 98% EU376186.1 | 100% 83% NR102456.1 Leptolyngbya PCC 7376 |
BacPamv41 | KP244204 | 100% 93% DQ642331.1 | 100% 90% NR041306.1 Syntrophorhabdus aromaticivorans U1 |
BacPamv42 | KP244205 | 100% 96% GU197631.1 | 100% 92% NR074317.1 Nostoc punctiforme PCC 73102 |
BacPamv43 | KP244206 | 100% 88% AB186797.1 | 100% 81% NR043385.1 Dictyoglomus turgidum DSM 6724 |
BacPamv44 | KP244207 | 100% 95% AB486150.1 | 100% 89% NR075011.1 Geobacter metallireducens GS-15 |
BacPamv45 | KP244208 | 100% 99% KC432448.1 | 100% 85% NR043559.1 Gracilibacter thermotolerans JW/YJL-S1 |
BacPamv46 | KP244209 | 100% 96% GU454906.1 | 100% 87% NR074757.1 Treponema caldaria DSM 7334 |
BacPamv47 | KP244210 | 100% 99% KC432448.1 | 100% 84% NR043559.1 Gracilibacter thermotolerans JW/YJL-S1 |
BacPamv48 | KP244211 | 99% 92% AB240355.1 | 99% 84% NR044075.1 Thermodesulfovibrio hydrogeniphiles HbrS |
BacPamv49 | KP244212 | 100% 93% JN397726.1 | 100% 91% NR028715.1 Acidovorax temperans PHL |
BacPamv50 | KP244213 | 100% 99% JX223096.1 | 100% 99% NR040800.1 Vogesella indigofera ATCC 19706 |
BacPamv51 | KP244214 | 100% 96% JQ624950.1 | 100% 92% NR026102.1 Clostridium papyrosolvens DSM 2792 |
Clone affiliation of culturable Bacteria
Clone name | Accession number | % query, % identity culture collection | % query, % identity cultured species |
---|---|---|---|
PamvBac iso.1 | KU862661 | 100% 100% KF481602.1 | 100% 99% NR042502.1 Massilia aurea AP13 |
PamvBac iso.2 | KU862662 | 100% 99% KF556686.1 | 100% 97% NR043699.1 Rheinheimera chironomi K19414 |
PamvBac iso.3 | KU862663 | 100% 99% EF471218.1 | 100% 99% NR042596.1 Cryseobacterium luteum P456/04 |
PamvBac iso.4 | KU862664 | 100% 99% JX223096.1 | 100% 99% NR040800.1 Vogesella indigofera ATCC 19706 |
PamvBac iso.5 | KU862665 | 100% 99% KF556697.1 | 100% 98% NR114646.1 Rhodoferax ferrireducens T118 |
PamvBac iso.6 | KU862666 | 100% 99% HG792168.1 | 100% 99% NR036911.2 Aeromonas media RM |
PamvBac iso.7 | KU862667 | 100% 99% KF555636.1 | 100% 99% NR041057.1 Flavobacterium frigidimaris KUC-1 |
PamvBac iso.8 | KU862668 | 100% 99% JF145482.1 | 100% 99% NR044292.1 Flavobacterium resistens BD-b365 |
PamvBac iso.9 | KU862669 | 100% 99% KC666807.1 | 100% 99% NR025425.1 Acinetobacteria parvus LUH 4616 |
PamvBac iso.10 | KU862670 | 100% 100% JX657101.1 | 100% 98% NR108576.1 Flavobacterium compostarboris 15C3 |
PamvBac iso.11 | KU862671 | 100% 99% KC666549.1 | 100% 97% NR043993.1 Rheinheimera tangshanensis JA3-B52 |
PamvBac iso.12 | KU862672 | 100% 99% KC294042.1 | 100% 99% NR029319.1 Pseudomonas anguilliseptica S1 |
PamvBac iso.13 | KU862673 | 100% 99% GU291856.1 | 100% 98% NR109728.1 Flavobacterium cutihirudinis E89 |
PamvBac iso.14 | KU862674 | 97% 99% JQ317797.1 | 97% 98% NR029319.1 Pseudomonas anguilliseptica S1 |
PamvBac iso.15 | KU862675 | 100% 99% HM149209.1 | 100% 99% NR044581.1 Flavobacterium chungangense CJ7 |
---|---|---|---|
PamvBac iso.16 | KU862676 | 99% 99% KF894688.1 | 99% 98% NR115957.1 Chryseobacterium flavum strain CW-E2 |
PamvBac iso.17 | KU862677 | 100% 99% KC248046.1 | 100% 98% NR042941.1 Paucibacter toxinivorans 2C20 |
PamvBac iso.18 | KU862678 | 100% 93% HQ246251.1 | 100% 93% NR029024.1 Hydrogenophaga defluvii BSB 9.5 |
PamvBac iso.19 | KU862679 | 100% 100% KC294042.1 | 100% 100% NR029319.1 Pseudomonas anguilliseptica S1 |
PamvBac iso.20 | KU862680 | 100% 99% HM153665.1 | 100% 99% NR029024.1 Hydrogenophaga defluvii BSB 9.5 |
PamvBac iso.21 | KU862681 | 100% 99% NR109522.1 | 100% 99% NR109522.1 Flavobacterium fontis MIC 3010 |
PamvBac iso.22 | KU862682 | 100% 99% KF287757.1 | 100% 99% NR025816.1 Porphyrobacter donghaensis SW-132 |
PamvBac iso.23 | KU862683 | 100% 99% LK054500.1 | 100% 99% NR075062.2 Micrococcus luteus NCTC 2665 |
clone name | SS1 | SS2 | Sp. | Su. | Au. | Wi. | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
SS1 | SS2 | SS1 | SS2 | SS1 | SS2 | SS1 | SS2 | ||||
γ-Proteobacteria | BacPamv26 | √ | √ | - | √ | - | - | √ | - | - | - |
BacPamv36 | √ | - | - | - | - | - | - | - | √ | - | |
BacPamv13A | √ | - | √ | - | - | - | - | - | - | - | |
BacPamv24A | √ | √ | - | - | √ | √ | √ | - | - | - | |
BacPamv4 | √ | √ | √ | √ | - | - | √ | √ | √ | √ | |
BacPamv35 | √ | - | - | - | - | - | - | - | √ | - | |
BacPamv11 | √ | - | √ | - | - | - | - | - | - | - | |
TOTAL SEQUENCES | 7 | 3 | 3 | 2 | 1 | 1 | 3 | 1 | 3 | 1 | |
β-Proteobacteria | BacPamv10 | √ | - | √ | - | √ | - | - | - | - | - |
BacPamv15 | √ | √ | √ | - | - | √ | - | √ | - | √ | |
BacPamv50 | - | √ | - | - | - | √ | - | - | - | - | |
BacPamv16 | √ | √ | √ | √ | - | - | √ | - | - | - |
BacPamv27 | √ | √ | - | - | - | √ | √ | - | - | - | |
---|---|---|---|---|---|---|---|---|---|---|---|
BacPamv49 | - | √ | - | - | - | √ | - | √ | - | - | |
BacPamv19 | √ | √ | - | - | √ | √ | - | √ | √ | √ | |
BacPamv1 | √ | √ | √ | √ | √ | √ | √ | √ | √ | √ | |
BacPamv32 | √ | - | - | - | - | - | - | - | √ | - | |
TOTAL SEQUENCES | 7 | 7 | 4 | 2 | 3 | 6 | 3 | 4 | 3 | 3 | |
α-Proteobacteria | BacPamv18 | √ | - | - | - | √ | - | - | - | - | - |
TOTAL SEQUENCES | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | |
Bacteroidetes | BacPamv7 | √ | √ | √ | - | √ | √ | √ | √ | √ | √ |
BacPamv21 | √ | - | - | - | √ | - | √ | - | - | - | |
BacPamv39 | - | √ | - | √ | - | - | - | - | - | √ | |
TOTAL SEQUENCES | 2 | 2 | 1 | 1 | 2 | 1 | 2 | 1 | 1 | 2 | |
δ-Proteobacteria | BacPamv24B | √ | √ | - | √ | √ | √ | - | √ | - | √ |
BacPamv44 | - | √ | - | √ | - | √ | - | √ | - | √ | |
BacPamv41 | - | √ | - | √ | - | - | - | - | - | √ | |
TOTAL SEQUENCES | 1 | 3 | 0 | 3 | 1 | 2 | 0 | 2 | 0 | 3 | |
Actinobacteria | BacPamv38 | - | √ | - | √ | - | - | - | - | - | - |
TOTAL SEQUENCES | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |
Gemmatimonadetes | BacPamv34 | √ | √ | - | - | - | √ | - | - | √ | - |
TOTAL SEQUENCES | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | |
Spirochaetes | BacPamv2 | √ | √ | √ | - | - | - | - | √ | - | - |
BacPamv29 | √ | - | - | - | - | - | √ | - | - | - | |
BacPamv28 | √ | √ | - | √ | - | - | √ | - | - | - | |
TOTAL SEQUENCES | 3 | 2 | 1 | 1 | 0 | 0 | 2 | 1 | 0 | 0 | |
Planctomycetes | BacPamv14 | √ | √ | √ | - | - | √ | - | - | √ | √ |
BacPamv6 | √ | √ | √ | √ | - | √ | √ | - | √ | - | |
TOTAL SEQUENCES | 2 | 2 | 2 | 1 | 0 | 2 | 1 | 0 | 2 | 1 | |
Cyanobacteria | BacPamv20A | √ | √ | - | √ | √ | - | √ | - | - | - |
BacPamv40 | - | √ | - | √ | - | √ | - | - | - | √ | |
BacPamv22 | √ | √ | - | √ | √ | √ | - | - | √ | - | |
BacPamv20B | - | √ | - | √ | - | √ | - | √ | - | √ | |
BacPamv17B | - | √ | - | √ | - | - | - | √ | - | √ | |
BacPamv30 | √ | - | - | - | - | - | √ | - | √ | - | |
BacPamv42 | - | √ | - | √ | - | - | - | - | - | - | |
BacPamv37 | √ | - | - | - | - | - | - | - | √ | - | |
BacPamv13C | √ | √ | - | √ | - | √ | √ | √ | √ | √ |
TOTAL SEQUENCES | 5 | 7 | 0 | 7 | 2 | 4 | 3 | 3 | 4 | 4 | |
---|---|---|---|---|---|---|---|---|---|---|---|
Firmicutes | BacPamv33 | √ | √ | - | - | - | √ | - | - | √ | - |
BacPamv51 | - | √ | - | - | - | - | - | - | - | √ | |
BacPamv23 | √ | - | - | - | √ | - | - | - | - | - | |
TOTAL SEQUENCES | 2 | 2 | 0 | 0 | 1 | 1 | 0 | 0 | 1 | 1 | |
Nitrospirae | BacPamv25 | √ | √ | - | √ | √ | √ | √ | √ | - | √ |
BacPamv31 | √ | - | - | - | - | - | √ | - | - | - | |
BacPamv48 | - | √ | - | - | - | √ | - | √ | - | √ | |
BacPamv12 | √ | - | √ | - | - | - | - | - | - | - | |
BacPamv9B | √ | - | √ | - | √ | - | - | - | - | - | |
TOTAL SEQUENCES | 4 | 2 | 2 | 1 | 2 | 2 | 2 | 2 | 0 | 2 | |
Acidobacteria | BacPamv5 | √ | √ | √ | √ | √ | - | √ | √ | √ | √ |
BacPamv3 | √ | √ | √ | - | √ | - | √ | √ | - | - | |
BacPamv13B | √ | - | - | - | √ | - | √ | - | √ | - | |
BacPamv17A | √ | √ | - | √ | √ | - | - | √ | - | - | |
TOTAL SEQUENCES | 4 | 3 | 2 | 2 | 4 | 0 | 3 | 3 | 2 | 1 | |
Unclassified cluster I | BacPamv43 | - | √ | - | √ | - | √ | - | √ | - | √ |
TOTAL SEQUENCES | 0 | 1 | 0 | 1 | 0 | 1 | 0 | 1 | 0 | 1 | |
Unclassified cluster II | BacPamv45 | - | √ | - | √ | - | - | - | - | - | - |
BacPamv47 | - | √ | - | - | - | √ | - | - | - | - | |
BacPamv9A | √ | √ | √ | √ | - | √ | √ | √ | √ | √ | |
BacPamv8 | √ | - | √ | - | √ | - | - | - | - | - | |
TOTAL SEQUENCES | 2 | 3 | 2 | 2 | 1 | 2 | 1 | 1 | 1 | 1 | |
Unclassified cluster III | BacPamv46 | - | √ | - | - | - | √ | - | - | - | - |
TOTAL SEQUENCES | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | |
SS1 | SS2 | ||||||||||
TOTAL SEQUENCES | 41 | 40 | |||||||||
SS1: Sample Station 1 | |||||||||||
SS2: Sample Station 2 | |||||||||||
Sp.: Spring | |||||||||||
Su.: Summer | |||||||||||
Au.: Autumn | |||||||||||
Wi.: Winter |
Clone Affiliation of Archaea
Clone name | Accession number | % query, % identity culture collection | % query, % identity cultured species |
---|---|---|---|
ArcPamv1 | KC510289 | 100% 96% JN617408 | 100% 95% NR028163 Methanolinea tarda NOBI-1 |
ArcPamv2 | KC510290 | 100% 99% JN617359 | 100% 95% NR 044422 Methanosphaerula palustris strain E1-9c |
ArcPamv3A | KC510291 | 100% 94% JX196214 | 100% 79% NR028646 Methanotorris formicicus strain Mc-S-70 |
ArcPamv3B | KC510292 | 99% 89% JN853647 | 100% 72% NR029140 Methanococcus aeolicus Nankai-3 |
ArcPamv4A | KC510293 | 100% 99% HM244131 | 100% 95% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv4B | KC510294 | 100% 99% JQ795001 | 100% 80% NR042784 Methanobrevibacter ruminantium strain M1 |
ArcPamv4C | KC510295 | 100% 99% JX426833 | 100% 96% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv5 | KC510296 | 100% 99% JN 617444 | 100% 97% NR028242 Methanosaeta concilii strain Opfikon |
ArcPamv6 | KC510297 | 100% 98% DQ301909 | 100% 95% NRO44422 Methanosphaerula palustris strain E1-9c |
ArcPamv7 | KC510298 | 99% 99% JQ794950 | 99% 97% NR028163 Methanolinea tarda NOBI-1 |
ArcPamv8A | KC510299 | 100% 99% JF431625 | 100% 78% NR029059 Palaeococcus helgesonii strain PI1 |
ArcPamv8B | KC510300 | 100% 99% FJ755715 | 88% 95% NR028179 Thermococcus thioreducens OGL-20P |
ArcPamv9 | KC510301 | 100% 99% JQ245676 | 100% 94% NR028163 Methanolinea tarda NOBI-1 |
ArcPamv10 | KC510302 | 100% 99% EF639431 | 100% 96% NR028163 Methanolinea tarda NOBI-1 |
ArcPamv11 | KC510303 | 100% 99 JX426833 | 100% 96% NR044422 Methanosphaerula palustris strain E1-9c |
---|---|---|---|
ArcPamv12 | KC510304 | 99% 99% AY125724 | 99% 96% NR028163 Methanolinea tarda NOBI-1 |
ArcPamv13 | KC510305 | 99% 99% DQ785302 | 99% 95% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv14 | KC510306 | 100% 99% HQ330724 | 100% 95% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv15 | KC510307 | 100% 98% HQ330702 | 100% 91% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv16 | KC510308 | 100% 99% JQ794997 | 100% 96% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv17 | KC510309 | 100% 99% AM503280 | 100% 95% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv18A | KC510310 | 100% 98% EF639431 | 100% 96% NR028163 Methanolinea tarda NOBI-1 |
ArcPamv18B | KC510311 | 100% 99% JX426828 | 100% 80% NR042784 Methanobrevibacter ruminantium M1 strain M1 |
ArcPamv20 | KC510312 | 100% 99% FM165672 | 100% 96% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv21A | KC510313 | 100% 99% JQ795001 | 100% 80% NR042784 Methanobrevibacter ruminantium M1 strain M1 |
ArcPamv21B | KC510314 | 100% 99% JQ794995 | 100% 80% NR042784 Methanobrevibacter ruminantium M1 strain M1 |
ArcPamv21C | KC510315 | 100% 99% JF431702 | 100% 78% NR029055 Thermococcus aegaeus |
ArcPamv22 | KC510316 | 100% 99% FJ755715 | 85% 80% NR028179 Thermococcus thioreducens OGL-20P |
ArcPamv23 | KC510317 | 100% 99% HM244131 | 100% 95% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv24 | KC510318 | 100% 99% HQ330702 | 100% 93% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv25 | KC510319 | 100% 99% JQ245676 | 100% 94% NR044422 Methanosphaerula palustris strain E1-9c |
---|---|---|---|
ArcPamv28 | KC510320 | 100% 99% DQ676243 | 100% 95% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv29 | KC510321 | 100% 99% HQ330690 | 90% 92% NR042740 Thermococcus hydrothermalis strain AL662 |
ArcPamv30 | KC510322 | 100% 99% HQ330690 | 85% 81% NR028179 Thermococcus thioreducens OGL-20P |
ArcPamv31 | KC510323 | 100% 99% DQ785302 | 100% 94% NR028163 Methanolinea tarda NOBI-1 |
ArcPamv33 | KC510324 | 100% 98% JF262336 | 100% 96% NR028163 Methanolinea tarda NOBI-1 |
ArcPamv35 | KC510325 | 100% 97% JQ792848 | 100% 78% NR028248 Methanothermobacter defluvii |
ArcPamv36 | KC510326 | 100% 92% JF853612 | 100% 78% NR043089 Methanomethylovorans thermophila |
ArcPamv37 | KC510327 | 100% 99% FJ755715 | 90% 92% NR028179 Thermococcus thioreducens OGL-20P |
ArcPamv38 | KC510328 | 100% 100% FJ755715 | 90% 92% NR028179 Thermococcus thioreducens OGL-20P |
ArcPamv39 | KC510329 | 100% 99% JQ079951 | 100% 98% NR028242 Methanosaeta concilii strain Opfikon |
ArcPamv42 | KC510330 | 99% 99% JQ794950 | 99% 97% NR028163 Methanolinea tarda NOBI-1 |
ArcPamv43 | KC510331 | 99% 99% JX426879 | 99% 96% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv44 | KC510332 | 99% 98% LN896671 | 99% 76% NR029140 Methanococcus aeolicus NanKai-3 |
ArcPamv45 | KC510333 | 94% 98% AJ240005 97% 96% AF005766 | 94% 100% NR029214 Thermofilum pendens strain Hvv3, DSM 2474 94% 100% NR028877 Staphylothermus hellenicus DSM 12710 strain P8 |
ArcPamv49 | KC510334 | 100% 98% JF980361 | 100% 95% NR044422 Methanosphaerula palustris strain E1-9c |
---|---|---|---|
ArcPamv51A | KC510335 | 100% 99% JF431901 | 100% 80% NR042784 Methanobrevibacter ruminantium M1 strain M1 |
ArcPamv51B | KC510336 | 99% 98% JQ794997 | 99% 94% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv52 | KC510337 | 100% 99% HM244091 | 100% 80% NR042784 Methanobrevibacter ruminantium M1 strain M1 |
ArcPamv54 | KC510338 | 100% 99% JF431775 | 99% 83% NR043512 Ignisphaera aggregans DSM 17230 strain AQ1.S1 |
ArcPamv55 | KC510339 | 100% 99% FM165672 | 100% 96% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv57 | KC510340 | 99% 99% JQ794950 | 99% 96% NR028163 Methanolinea tarda NOBI-1 |
ArcPamv58A | KC510341 | 99% 99% JN649164 | 99% 95% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv58B | KC510342 | 99% 96% FN432722 | 99% 78% NR042734 Thermococcus barophilus MP strain DSM 11836 |
ArcPamv59 | KC510343 | 99% 97% HQ330736 | 100% 78% NR025718 Methanococcus vannielii strain 5B |
ArcPamv60 | KC510344 | 100% 99% DQ310455 | 100% 77% NR028210 Ferroplasma cupricumulans BH2 |
ArcPamv65A | KC510345 | 100% 90% HM004825 | 100% 78% NR029140 Methanococcus aeolicus NanKai-3 |
ArcPamv65B | KC510346 | 100% 97% FJ755715 | 100% 78% NR042781 Methanobacterium bryantii strain MOH |
ArcPamv66 | KC510347 | 98% 93% AB653407 | 100% 78% NR041513 Thermogymnomonas acidicola strain JCM 13583 |
ArcPamv67A | KC510348 | 100% 99% HE796161 | 100% 78% NR028701 Methanocaldococcus vulcanius M7 |
ArcPamv67B | KC510349 | 100% 99% HQ404340 | 100% 77% NR028646 Methanotorris formicicus strain Mc-S-70 |
ArcPamv69 | KC510350 | 100% 99% JN853654 | 100% 80% NR042784 Methanobrevibacter ruminantinum M1 |
---|---|---|---|
ArcPamv70 | KC510351 | 99% 100% AB652545 | 100% 93% NR028164 Methanocella paludicola SANAE |
ArcPamv71 | KC510352 | 100% 90% EF639526 | 100% 80% NR044786 Methanobrevibacter smithii ATCC 35061 |
ArcPamv72A | KC510353 | 100% 98% JQ595987 | 100% 95% NR042789 Methanospirillum hungatei JF-1, strain JF1 |
ArcPamv72B | KC510354 | 100% 99% DQ785302 | 100% 94% NR044422 Methanosphaerula palustris strain E1-9c |
ArcPamv75 | KC510355 | 100% 96% JN649130 | 100% 94% NR043961 Methanoculleus receptaculi |
ArcPamv76 | KC510356 | 100% 99% AB775723 | 100% 97% NR028163 Methanolinea tarda NOBI-1 |
ArcPamv77A | KC510357 | 100% 99% JQ794950 | 100% 96% NR028163 Methanolinea tarda NOBI-1 |
ArcPamv77B | KC510358 | 99% 97% FN646492 | 99% 92% NR044422 Methanosphaerula palustris E1-9c |
ArcPamv79A | KC510359 | 100% 96% JN853749 | 100% 78% NR029059 Palaeococcus helgesonii |
ArcPamv79B | KC510360 | 100% 99% JX426833 | 100% 96% NR044422 Methanosphaerula palustris E1-9c |
ArcPamv79C | KC510361 | 100% 99% JQ792430 | 100% 96% NR044422 Methanosphaerula palustris E1-9c |
ArcPamv79D | KC510362 | 97% 99% HQ330660 | 97% 95% NR044422 Methanosphaerula palustris E1-9c |
ArcPamv82 | KC510363 | 100% 99% JX426833 | 100% 96% NR044422 Methanosphaerula palustris E1-9c |
ArcPamv83A | KC510364 | 99% 98% EF639443 | 99% 93% NR044422 Methanosphaerula palustris E1-9c |
ArcPamv83B | KC510365 | 100% 99% JQ245676 | 100% 94% NR028163 Methanolinea tarda NOBI-1 |
---|---|---|---|
ArcPamv84A | KC510366 | 100% 99% JQ245676 | 100% 94% NR0128163 Methanolinea tarda NOBI-1 |
ArcPamv84B | KC510367 | 87% 87% HQ330736 97% 76% EU983178 | 82% 76% NR102915 Methanothermococcus okinawensis IH 1 |
ArcPamv86A | KC510368 | 100% 99% JN617381 | 100% 80% NR074217 Aciduliprofundum boonei T469 strain T469 |
ArcPamv86B | KC510369 | 99% 97% EU519275 | 99% 95% NR044422 Methanosphaerula palustris E1-9c |
ArcPamv88 | KC510370 | 98% 99% JF431886 | 98% 98% NR028242 Methanosaeta concilii strain Opfikon |
ArcPamv89 | KC510371 | 100% 96% JQ792848 | 100% 78% NR116289 Methanobacterium movens strain TS-2 |
ArcPamv90 | KC510372 | 100% 97% FN646483 | 100% 93% NR028242 Methanosaeta concilii Opfikon |
ArcPamv92 | KC510373 | 100% 99% JQ079951 | 100% 98% NR028242 Methanosaeta concilii Opfikon |
ArcPamv96 | KC510374 | 100% 99% HE964957 | 100% 94% NR028242 Methanosaeta concilii Opfikon |
ArcPamv108A | KC510375 | 100% 99% HQ330667 | 100% 94% NR044422 Methanosphaerula palustris E1-9c |
ArcPamv108B | KC510376 | 99% 99% JX426828 | 99% 80% NR042784 Methanobrevibacter ruminantium M1 |
ArcPamv109 | KC510377 | 100% 99% HQ330667 | 100% 95% NR044422 Methanosphaerula palustris E1-9c |
ArcPamv112 | KC510378 | 100% 99% HQ330702 | 100% 93% NR044422 Methanosphaerula palustris E1-9c |
ArcPamv114 | KC510379 | 100% 99% HM244128 | 100% 84% NR028877 Staphylothermus hellenicus DSM 12710 strain P8 |
ArcPamv115 | KC510380 | 99% 98% FN646492 | 99% 92% NR04442 Methanosphaerula palustris E1-9c |
clone name | SS1 | SS2 | Sp. | Su. | Au. | Wi. | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
SS1 | SS2 | SS1 | SS2 | SS1 | SS2 | SS1 | SS2 | ||||
Methanomicrobiales | ArcPamv9 | √ | √ | √ | √ | √ | √ | √ | √ | √ | √ |
ArcPamv84A | - | √ | - | - | - | √ | - | - | - | - | |
ArcPamv25 | √ | √ | - | - | √ | √ | - | - | - | - | |
ArcPamv83B | - | √ | - | - | - | √ | - | √ | - | - | |
ArcPamv13 | √ | √ | - | √ | √ | √ | √ | √ | - | - | |
ArcPamv31 | √ | √ | - | - | - | - | √ | √ | √ | - | |
ArcPamv72B | - | √ | - | √ | - | - | - | - | - | - | |
ArcPamv58A | - | √ | - | √ | - | √ | - | √ | - | √ | |
ArcPamv33 | √ | √ | - | √ | - | - | √ | - | √ | - | |
ArcPamv49 | √ | - | - | - | - | - | - | - | √ | - | |
ArcPamv28 | √ | - | - | - | - | - | √ | - | - | - | |
ArcPamv75 | - | √ | - | √ | - | √ | - | √ | - | √ | |
ArcPamv77B | - | √ | - | √ | - | - | - | - | - | - | |
ArcPamv115 | - | √ | - | - | - | - | - | √ | - | - | |
ArcPamv51B | √ | - | - | - | - | - | - | - | √ | - | |
ArcPamv16 | √ | √ | - | √ | √ | √ | √ | √ | √ | √ | |
ArcPamv4C | √ | √ | √ | √ | - | √ | - | √ | √ | - | |
ArcPamv79B | - | √ | - | - | - | √ | - | - | - | - | |
ArcPamv43 | √ | √ | - | - | - | - | √ | √ | - | - | |
ArcPamv11 | √ | √ | √ | √ | √ | √ | √ | √ | - | √ | |
ArcPamv82 | - | √ | - | - | - | √ | - | - | - | - | |
ArcPamv6 | √ | - | √ | - | - | - | - | - | - | - | |
ArcPamv14 | √ | √ | - | - | √ | √ | - | - | - | - | |
ArcPamv79D | - | √ | - | - | - | √ | - | - | - | - | |
ArcPamv108A | - | √ | - | - | - | - | - | √ | - | - | |
ArcPamv109 | - | √ | - | - | - | - | - | √ | - | - | |
ArcPamv23 | √ | √ | - | √ | √ | √ | √ | √ | - | √ | |
ArcPamv4A | √ | √ | √ | √ | √ | √ | √ | √ | √ | - | |
ArcPamv17 | √ | √ | - | √ | √ | √ | √ | √ | √ | - | |
ArcPamv55 | √ | - | - | - | - | - | - | - | √ | - | |
ArcPamv20 | √ | √ | - | √ | √ | √ | √ | - | - | - | |
ArcPamv2 | √ | - | √ | - | - | - | - | - | - | - | |
ArcPamv79C | - | √ | - | - | - | √ | - | - | - | - | |
ArcPamv18A | √ | - | - | - | √ | - | √ | - | - | - | |
ArcPamv86B | - | √ | - | - | - | √ | - | - | - | - |
ArcPamv76 | - | √ | - | √ | - | - | - | - | - | - | |
---|---|---|---|---|---|---|---|---|---|---|---|
ArcPamv7 | √ | √ | √ | √ | √ | √ | √ | √ | √ | √ | |
ArcPamv12 | √ | - | - | - | √ | - | - | - | - | - | |
ArcPamv42 | √ | - | - | - | - | - | √ | - | - | - | |
ArcPamv57 | √ | - | - | - | - | - | - | - | √ | - | |
ArcPamv10 | √ | √ | √ | √ | √ | √ | √ | √ | √ | √ | |
ArcPamv77A | - | √ | - | √ | - | - | - | - | - | - | |
ArcPamv1 | √ | - | √ | - | - | - | - | - | - | - | |
ArcPamv83A | - | √ | - | - | - | √ | - | - | - | - | |
ArcPamv24 | √ | √ | - | √ | √ | √ | √ | √ | √ | √ | |
ArcPamv15 | √ | √ | - | √ | √ | √ | √ | √ | √ | √ | |
ArcPamv12 | √ | - | - | - | √ | - | - | - | - | - | |
ArcPamv72A | - | √ | - | √ | - | - | - | - | - | - | |
ArcPamv90 | - | √ | - | - | - | √ | - | - | - | - | |
ArcPamv70 | - | √ | - | √ | - | - | - | √ | - | - | |
ArcPamv96 | - | √ | - | - | - | √ | - | - | - | - | |
ArcPamv5 | √ | √ | √ | √ | √ | √ | √ | √ | √ | √ | |
ArcPamv92 | - | √ | - | - | - | √ | - | √ | - | √ | |
ArcPamv39 | √ | - | - | - | - | - | √ | - | - | - | |
ArcPamv88 | - | √ | - | - | - | √ | - | √ | - | - | |
TOTAL SEQUENCES | 32 | 42 | 10 | 23 | 18 | 30 | 20 | 24 | 16 | 12 | |
Miscellaneous Crenarchaeota | ArcPamv54 | √ | - | - | - | - | - | - | - | √ | - |
ArcPamv45 | √ | - | - | - | - | - | - | - | √ | - | |
ArcPamv114 | - | √ | - | - | - | - | - | √ | - | - | |
TOTAL SEQUENCES | 2 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 2 | 0 | |
Unknown cluster I | ArcPamv35 | √ | - | - | - | - | - | √ | - | - | - |
ArcPamv89 | - | √ | - | - | - | √ | - | √ | - | √ | |
ArcPamv29 | √ | - | - | - | - | - | √ | - | - | - | |
ArcPamv58B | - | √ | - | √ | - | √ | - | - | - | - | |
ArcPamv79A | - | √ | - | - | - | √ | - | - | - | - | |
ArcPamv30 | √ | - | - | - | - | - | √ | - | - | - | |
ArcPamv65B | - | √ | - | √ | - | - | - | - | - | - | |
ArcPamv8B | √ | - | √ | - | - | - | - | - | √ | - | |
ArcPamv22 | √ | - | - | - | √ | - | √ | - | - | - | |
ArcPamv37 | √ | - | - | - | - | - | √ | - | - | - | |
ArcPamv38 | √ | √ | - | √ | - | √ | √ | - | - | - |
TOTAL SEQUENCES | 7 | 5 | 1 | 3 | 1 | 4 | 6 | 1 | 1 | 1 | |
---|---|---|---|---|---|---|---|---|---|---|---|
Unknown cluster II | ArcPamv36 | √ | - | - | - | - | - | √ | - | - | - |
TOTAL SEQUENCES | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | |
Unknown cluster III | ArcPamv21C | √ | - | - | - | √ | - | √ | - | - | - |
TOTAL SEQUENCES | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | |
Unidentified | ArcPamv71 | - | √ | - | √ | - | - | - | - | - | - |
TOTAL SEQUENCES | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |
Unknown cluster IV | ArcPamv3A | √ | - | √ | - | - | - | - | - | - | - |
TOTAL SEQUENCES | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
MBG-D | ArcPamv86A | - | √ | - | - | - | √ | - | √ | - | √ |
ArcPamv69 | - | √ | - | √ | - | - | - | - | - | - | |
ArcPamv52 | √ | - | - | - | - | - | - | - | √ | - | |
ArcPamv108B | - | √ | - | - | - | - | - | √ | - | - | |
ArcPamv18B | √ | - | - | - | √ | - | - | - | - | - | |
ArcPamv21B | √ | √ | - | √ | √ | - | √ | - | - | - | |
ArcPamv51A | √ | - | - | - | - | - | - | - | √ | - | |
ArcPamv4B | √ | - | √ | √ | - | √ | - | - | - | ||
ArcPamv21A | √ | √ | - | √ | √ | - | √ | - | - | - | |
TOTAL SEQUENCES | 6 | 5 | 1 | 3 | 4 | 1 | 3 | 2 | 2 | 1 | |
Rice cluster V | ArcPamv8A | √ | - | √ | - | - | - | - | - | - | - |
ArcPamv67B | - | √ | - | √ | - | - | - | √ | - | - | |
ArcPamv65A | - | √ | - | √ | - | - | - | √ | - | - | |
ArcPamv60 | - | √ | - | √ | - | - | - | - | - | - | |
ArcPamv66 | - | √ | - | √ | - | - | - | - | - | - | |
ArcPamv44 | √ | √ | - | - | - | √ | - | √ | √ | √ | |
ArcPamv59 | - | √ | - | √ | - | - | - | - | - | - | |
ArcPamv84B | - | √ | - | - | - | √ | - | - | - | - | |
TOTAL SEQUENCES | 2 | 7 | 1 | 5 | 0 | 2 | 0 | 3 | 1 | 1 | |
Unknown cluster V | ArcPamv67A | - | √ | - | √ | - | - | - | √ | - | - |
TOTAL SEQUENCES | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | |
LDS | ArcPamv3B | √ | - | √ | - | - | - | - | - | - | - |
TOTAL SEQUENCES | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
SS1 | SS2 | ||||||||||
TOTAL SEQUENCES | 53 | 62 | |||||||||
SS1: Sample Station 1 | |||||||||||
SS2: Sample Station 2 | |||||||||||
Sp.: Spring | |||||||||||
Su.: Summer | |||||||||||
Au.: Autumn | |||||||||||
Wi.: Winter |