2.1 Study sites and sampling
The 13 sub-alpine and alpine lakes in our study are located in Jämtland in western Sweden (Fig. 1), including 4 lakes in Edsåsdalen (63.3°N and 12.9°E) and 9 lakes in Skalstugan (63.5–63.7°N and 12.1–12.2°E). The lakes in Edsåsdalen are at or above present-day treeline, whereas the lakes in Skalstugan are at or below treeline. Intensive field surveys were conducted during June-September 2017, which included lake morphology, gill-net fishing, and water and surface sediment sampling. The current fish species in the lakes are Arctic char (Salvelinus alpinus) and/or Brown trout (Salmo trutta) (Table 1). The relative abundance of fish is measured as catch per unit of effort [CPUE, expressed as gram per net; see Capo et al.22 for detailed description of the fish sampling].
Table 1
Lake properties, water chemistry, and fish abundance in the 13 study lakes in Jämtland. All the water chemistry data was presented as mean ± SD.
Lake | Area | Depth | pH | DOC | NO3-N | NH4-N | PO4-P | TN | TP | Fish CPUE | Current fish species |
ha | m | mg/L | µg/L | µg/L | µg/L | µg/L | µg/L | g/net |
ZF08 | 4.8 | 14 | 6.40 | 3.98 ± 0.22 | 5.20 ± 1.63 | 7.88 ± 1.65 | 0.97 ± 1.09 | 154.3 ± 55.3 | 4.2 ± 0.9 | 440.8 | S. alpinus, S. trutta |
ZF09 | 2.7 | 8 | 6.00 | 4.51 ± 0.49 | 2.80 ± 1.38 | 6.28 ± 1.20 | 0.90 ± 0.48 | 162.6 ± 42.4 | 4.0 ± 0.9 | 305.4 | S. trutta |
ZF10 | 10.2 | 23 | 6.30 | 2.05 ± 0.13 | 13.15 ± 5.98 | 6.38 ± 1.32 | 0.51 ± 0.48 | 146.5 ± 70.5 | 4.1 ± 1.0 | 300.3 | S. alpinus, S. trutta |
ZF11 | 3.9 | 14 | 6.27 | 6.23 ± 1.10 | 3.85 ± 1.30 | 9.38 ± 3.27 | 0.73 ± 0.38 | 190.2 ± 21.8 | 4.3 ± 0.3 | 1171.5 | S. alpinus, S. trutta |
ZF12 | 3 | 9 | 6.13 | 5.10 ± 1.43 | 2.60 ± 0.72 | 7.82 ± 0.93 | 0.62 ± 0.30 | 165.0 ± 43.9 | 3.9 ± 0.3 | 749.5 | S. alpinus, S. trutta |
ZF13 | 3.7 | 9 | 6.03 | 7.52 ± 1.08 | 5.24 ± 1.34 | 7.16 ± 0.69 | 1.20 ± 0.29 | 188.4 ± 22.7 | 4.8 ± 0.6 | 394.9 | S. trutta |
ZF14 | 2.7 | 8 | 5.76 | 4.90 ± 1.23 | 2.09 ± 0.97 | 7.25 ± 0.77 | 0.73 ± 0.52 | 183.1 ± 37.0 | 3.8 ± 0.4 | 335.8 | S. trutta |
ZF15 | 1.3 | 3.5 | 6.04 | 7.13 ± 1.63 | 2.76 ± 0.81 | 7.19 ± 0.74 | 0.65 ± 0.06 | 199.3 ± 27.9 | 5.3 ± 1.2 | 550.4 | S. trutta |
ZF16 | 1.8 | 4.5 | 6.11 | 7.50 ± 1.25 | 2.85 ± 0.29 | 7.55 ± 0.78 | 1.33 ± 0.41 | 193.9 ± 45.1 | 5.0 ± 0.7 | 518.4 | S. trutta |
ZF17 | 2.5 | 8 | 5.93 | 4.76 ± 0.14 | 1.36 ± 0.53 | 5.73 ± 1.27 | 1.33 ± 0.53 | 163.6 ± 18.8 | 4.4 ± 0.7 | --* | S. alpinus, P. phoxinus† |
ZF19 | 5.7 | 14 | 6.16 | 4.48 ± 0.26 | 3.46 ± 3.36 | 4.98 ± 2.43 | 0.58 ± 0.09 | 136.0 ± 46.9 | 4.5 ± 0.3 | 506.6 | S. alpinus |
ZF20 | 2.7 | 7 | 6.05 | 3.23 ± 0.22 | 0.73 ± 0.47 | 4.67 ± 1.02 | 1.07 ± 0.35 | 152.0 ± 59.0 | 4.0 ± 0.9 | 2113.4 | S. alpinus |
ZF21 | 3.8 | 12 | 5.99 | 2.73 ± 0.08 | 2.94 ± 0.50 | 7.31 ± 2.60 | 1.02 ± 0.21 | 171.9 ± 66.0 | 5.1 ± 1.6 | 312.8 | S. trutta |
--*: only one S. alpinus (1603.35g) was caught over 9 nets. |
†: Phoxinus phoxinus. |
Surface sediments were collected from the deepest basin in all 13 lakes, and for 11 lakes also a shallow site close to shore to assess potential spatial variations in fish DNA due to different preferred habitats (i.e., 24 core sites in total). For each site, we collected the uppermost 1–2 cm of undisturbed sediment from three sediment cores that were transferred immediately into DNA-free Falcon tubes. The triplicate surface sediment samples from each core site were treated individually for the DNA analyses, i.e., 72 samples in total. Sample tubes were cleaned with 10% bleach prior to entering a dedicated ancient DNA lab at Umeå University and were stored at -20°C until DNA analysis.
2.2 Water chemistry and sediment geochemistry
Water samples were collected on several occasions during the survey period, and consisted of integrated samples collected from several depths. The samples for dissolved organic carbon (DOC) analysis were acidified in the field with 500 µL 1.2M HCl. In the lab, water samples were filtered through 0.45 µm filters before analysis of nitrate (NO3-N), ammonium (NH4+-N), phosphate (PO42-P) and dissolved organic carbon (DOC). Lake-water DOC was analyzed using an infrared gas analyzer, Formacs HT-I (Skalar). Dissolved nutrients (NO3-N, NH4+-N, PO42-P) were analyzed using a spectrophotometer with a segmented flow analyzer, according to methods in MT3B (QuAAtro Multi-test Methods): for nitrate, method Q-035-04 using a copperized Cd reduction coil to form an azo dye; for ammonia, salicylate method Q-033-04; and for phosphate, molybdenum blue method Q-037-05. For total phosphorus (TP) analysis, filtered samples were passed through an online digestion step, alkaline acidic persulfate method, at 110°C and 0.9MPa and then analyzed using a QuAAtro 39 (Seal Analytical). Total nitrogen (TN) was analyzed on an ND25 unit connected to the Fomacs using a chemiluminescent detector. We also include data (winter data) of lake-water TN and TP analyzed earlier in the same year by the County Administrative Board of Jämtland in order to obtain annual lake nutrient information.
Surface sediments were freeze-dried prior to geochemical analysis. Organic content was estimated via loss on ignition (LOI) at 550°C, and total concentrations of major and trace elements were measured on 200 mg sediment samples by wavelength dispersive X-ray fluorescence spectrometry (XRF) using a Bruker S8 Tiger spectrometer [see full method description in Rydberg23]. Principal component analysis (PCA) was used for the characterizing the sediment properties. Prior to PCA, data that was not normally distributed was log-transformed.
2.3 Test of sed-eDNA yields from DNeasy PowerSoil DNA Kit
To test the efficacy of the PowerSoil Kit to extract sufficient sed-eDNA, we measured the quantities of extracted DNA from the PowerSoil Kit in comparison to the three lysis buffers. To assess potential differences in DNA extraction due to varying organic matter content, we used three samples previously collected from other sites that had very different organic matter contents (measured as LOI), ranging from low (< 5%), medium (ca. 30%) to high (ca. 60%). Lysis buffers and their ingredients used for this test were: Buffer 1 contained 1% sodium dodecyl sulfate (SDS), 100mM Tris-HCl, 200 mM EDTA, and 500 mM Na2HPO4 (pH 8.6)24; Buffer 2 contained 4% SDS, 100 mM Tris-HCl, 200 mM EDTA, and 500 mM Na2HPO4; Buffer 3 contained 30 mM Tris-HCl, 30 mM EDTA, 800 mM quanidium hydrochloride, and 0.5% Triton X-10011; and the Buffer from the PowerSoil Kit, which was the C1 solution (lysis buffer) with the buffer in bead tubes. Each sediment was subjected to a quick centrifuge to remove most of the pore water. For each sediment type, three replicate subsamples of 0.5 g wet sediment were mixed with each of the four buffers for DNA extraction. For buffers 1,2 and 3, subsamples were mixed with 500 µL buffer solution. The mixtures were incubated at 70°C for 30 min with interval vortex, and the supernatant collected after 2 min centrifuge at 13000 rpm. The DNA in the supernatant was mixed with an equal volume of 20% PEG8000/2.5M NaCl for DNA precipitation and concentration, and the DNA was finally resuspended in 50 µL TE buffer for quantification. The quantities of extracted DNA were evaluated by Qubit with a dsDNA HS Assay Kit. Two-way ANOVA was used to test the effects of DNA lysis buffers on DNA yields from sediments with varied LOI, by using the software MedCalc.
The extracted DNA from the PowerSoil Kit was subsequently purified with the kit and the final DNA recovery after purification was analyzed in order to quantify the DNA loss during purification. We subsequently tested the amplifiability of the extracted DNA. Due to the fact that the teleo primers were not used and tested on sed-eDNA previously and thus the performance remained uncertain, we selected the widely used and reliable trnL c and h primers for plant sed-eDNA amplification24 in conventional PCR as described in the Sect. 2.5.
2.4 DNA extraction from lake sediment for fish DNA metabarcoding
DNA isolation from sediments were performed in a dedicated ancient DNA lab at Umeå University that is isolated from other labs and has a positive air pressure system accompanied by HEPA air filter system. Before extraction, samples were placed in a 4°C fridge for 1 hour for sediment precipitation followed by water removal by pipetting and homogenization by mixing, because eDNA is adsorbed on sediment but not in pore water 6. Each sediment was extracted once. Because the precipitated sediments remained high in moisture content, we used 0.5 g of sediment for DNA extraction in order to increase the volume of actual sediment and the likelihood of fish DNA recovery. From each sample, approximately 0.5 g of condensed sediment was processed using the PowerSoil Kit by following the manufacturer’s protocol with a modification that the samples were incubated overnight on a shaker at 60°C for the lysis step (after adding C1 buffer). Together with the samples, each extraction batch included one extraction blank to assess lab contamination and cross contamination between samples during extraction. In total, 6 extraction blanks and 72 sediment extracts were prepared.
2.5 DNA amplification and sequencing
We used two PCR methods, conventional PCR and digital droplet PCR (ddPCR), with a pair of general fish DNA metabarcodes – teleo_F and teleo_R primers14, to determine the presence of amplifiable fish DNA in the sediment extracts. These teleo primers were designed based on mitochondrial 12S gene of 64 European freshwater fish species and the resulting reference database tested on water samples from 62 sites corresponding to a wide range of aquatic ecosystems14. Moreover, Valentini et al.14 and primer BLAST against GenBank both suggested that the teleo primers amplify S. alpinus and S. trutta (expected amplicon of approx. 100 bp including primers), which are found in our Swedish lakes, but no other non-fish taxa, and therefore, these teleo primers were selected for this study. To avoid contamination, PCR preparations for both conventional PCR and ddPCR were done in the ancient DNA lab.
PCRs were set up in 25 µL reaction containing 1.25 U of HotStartTaq DNA polymerase (QIAGEN), 1x PCR buffer, 2 mM Mg2+, 0.2 mM dNTPs, 4 µg of Bovine Serum Albumin (BSA), 0.4 µM teleo_F and teleo_R primers, and 2 µL DNA isolate. Each PCR batch had at least one PCR negative control (no DNA template) and one PCR positive control (extracted Salvelinus alpinus DNA) to assess PCR performance. The prepared PCR mixtures were brought to a PCR lab for amplification under the condition of denaturation for 15 min at 95°C, followed by 35 cycles of 30s at 94°C, 30s at 56°C, and 1 min at 72°C, and then a final elongation for 7 min at 72°C.
The ddPCR assays were performed using QX200™ ddPCR™ EvaGreen Supermix (Bio-Rad, CA, USA), following the manufacturer’s instructions, and the details are provided in Supplementary Materials. Each ddPCR reaction contains 1x ddPCR EvaGreen Supermix, 0.2 µM teleo_F and teleo_R primers, and DNA isolate of 3 µL in 20 µL reaction. The PCR program for the ddPCR assay was: denature at 95°C for 5 min, followed by 35 cycles of 30s at 95°C and 1 min at 56°C with ramp rate of 2°C per sec (note that ddPCR with EvaGreen Supermix does not require elongation process), and finally 5 min at 4°C and 5 min at 90°C for signal stabilization and then hold at 4°C. We ran triplicate PCR reactions for each DNA extract. The PCR plate was then scanned by the QX200 Droplet Reader (Bio-Rad) to read fluorescence from each droplet, and the Bio-Rad QuantaSoft software version 1.7.4 was used to calculate target DNA concentration. The PCR triplicates were averaged and the final DNA count was shown as number of copies per µL in a PCR reaction.
For the proof of concept, we collected the amplified ddPCR droplets from one sample, which had the highest number of positive droplets, for cloning and DNA sequencing to verify the amplicon sequences. The droplets from transferred to a 1.5 mL tube with 20 µL of TE (Tris-EDTA) buffer and 70 µL of chloroform, and the sample was mixed for 1 min. After centrifuge at 13,000 rpm for 10 min, the upper aqueous phase containing purified amplicon DNA was cloned using CloneJet PCR Kit (ThermoScientific) at the Protein Expertise Platform at Umeå University. Twenty colonies were picked and sequenced at Eurofins. DNA sequences were analyzed using Geneious Prime© 2021.0.3 version.