Biogeosciences, 18, 4369–4388, 2021 https://doi.org/10.5194/bg-18-4369-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License.

Carbon sources of benthic fauna in temperate lakes across multiple trophic states

Annika Fiskal1, Eva Anthamatten1, Longhui Deng1, Xingguo Han1, Lorenzo Lagostina1, Anja Michel1, Rong Zhu1, Nathalie Dubois2,3, Carsten J. Schubert1,4, Stefano M. Bernasconi3, and Mark A. Lever1 1Institute of Biogeochemistry and Pollutant Dynamics (IBP), ETH Zurich, Universitätstrasse 16, 8092 Zurich, Switzerland 2Department Surface Waters – Research and Management, Eawag, Swiss Federal Institute of Aquatic Science and Technology, Überlandstrasse 133, 8600 Dübendorf, Switzerland 3Department of Earth Sciences, ETH Zurich, Sonneggstrasse 5, 8092 Zurich, Switzerland 4Department of Surface Waters – Research and Management, Swiss Federal Institute of Aquatic Science and Technology (EAWAG), Seestrasse 79, 6047 Kastanienbaum, Switzerland

Correspondence: Annika Fiskal (annikafi[email protected]) and Mark A. Lever ([email protected])

Received: 26 August 2020 – Discussion started: 11 September 2020 Revised: 2 June 2021 – Accepted: 21 June 2021 – Published: 28 July 2021

Abstract. Previous studies have shown that microbially pro- in so far uncharacterized relationships with detrital organic- duced methane can be a dominant carbon source of lacus- matter-degrading bacterial endosymbionts. trine sedimentary macrofauna in eutrophic lakes, most likely through grazing on methane-oxidizing bacteria. Here we in- vestigate the contributions of different carbon sources to macrofaunal biomass across five lakes in central Switzerland 1 Introduction that range from oligotrophic to highly eutrophic. Macrofau- nal communities change with trophic state, with chironomid Lake sediments are globally important organic C sinks (Ein- larvae dominating oligotrophic and tubificid oligochaetes sele et al., 2001; Mendonça et al., 2017) and sources of dominating eutrophic lake sediments. The 13C-isotopic data the greenhouse gas methane (CH4) (Bastviken et al., 2004; suggest that the average contribution of methane-derived car- Raymond et al., 2013; Holgerson and Raymond, 2016). bon to the biomass of both macrofaunal groups is similar Overall the burial of organic carbon is usually higher in but consistently remains minor, ranging from only ∼ 1 % eutrophic compared to oligotrophic lakes due to high nu- in the oligotrophic lake to at most 12 % in the eutrophic trient loads which increase primary production (Dean and lakes. The remaining biomass can be explained by the assim- Gorham, 1998; Maerki et al., 2009; Heathcote and Down- ilation of detritus-derived organic carbon. Low abundances ing, 2012; Anderson et al., 2013; Anderson et al., 2014). The of methane-cycling microorganisms in macrofaunal speci- resulting increases in aerobic respiration lead to O2 deple- mens, burrows, and surrounding sediment based on 16S ri- tion and increased organic matter deposition to sediments bosomal RNA (rRNA) gene sequences and copy numbers (Hollander et al., 1992; Steinsberger et al., 2017) where of genes involved in anaerobic and aerobic methane cycling this increased organic matter stimulates microbial methane (mcrA, pmoA) support the interpretation of isotopic data. No- production (Fiskal et al., 2019). The combination of in- tably, 16S rRNA gene sequences of macrofauna, including creased methane production in sediments and decreased aer- macrofaunal guts, are highly divergent from those in tubes obic methane consumption in overlying water then results in or sediments. Many macrofaunal specimens are dominated higher methane emissions from eutrophic lakes (DelSontro by a single 16S rRNA phylotype of Fusobacteria, α-, β-, et al., 2016). γ -, or ε-Proteobacteria, Bacteroidetes, or Parcubacteria. This In addition to trophic state, the presence of macrofauna, raises the question of whether dominant lake macrofauna live which physically mix sediments, mechanically break down organic particles, or pump O2 into deeper, otherwise anoxic

Published by Copernicus Publications on behalf of the European Geosciences Union. 4370 A. Fiskal et al.: Carbon sources of benthic fauna layers, influences O2 and C cycle dynamics in sediments (Meysman et al., 2006; White and Miller, 2008; Kristensen et al., 2012). While most research on macrofaunal effects on organic carbon burial and respiration reactions have been on marine sediments, there have also been numerous studies on freshwater sediments. These studies suggest that macrofauna can be present in high abundances (up to 11 000 individu- als per square meter) (Armitage et al., 1995; Mousavi, 2002) and significantly influence nutrient fluxes and sedimentary matrices in lake sediments (Stief, 2013; Holker et al., 2015). Insects, in particular tube-dwelling chironomid larvae, can cause oxic–anoxic oscillations around their burrows through their pumping activity (Lewandowski et al., 2007; Roskosch et al., 2012; Baranov et al., 2016; Hupfer et al., 2019) These redox fluctuations affect the sedimentary cycles of nitro- gen (Pelegri et al., 1994; Jeppesen et al., 1998; Stief et al., 2009; Stief, 2013), phosphorus (Andersson et al., 1988; Kat- sev et al., 2006), iron (Hupfer and Lewandowski, 2008), and methane (Deines et al., 2007b; Gentzel et al., 2012). Worms, especially tubificid oligochaetes, can also increase oxygena- tion and O2 uptake in surface sediments (Lagauzère et al., + Figure 1. Map of the study area from Fiskal et al., (2019). The 2009) and influence the release of ammonium (NH4 ), ni- sampling stations within each of the five lakes are indicated by red − 3− trate (NO3 ), and phosphate (PO4 ) (Svensson et al., 2001; dots and numbered 1 to 3. Color indicates trophic state from light Mermillod-Blondin et al., 2005; Gautreau et al., 2020) from blue (oligotrophic) to dark blue (eutrophic). The map is based on surface sediments. Many tubificids are moreover head-down aerial images from DigitalGlobe (CO) and CNES/Airbus (France) deposit feeders that defecate on the sediment surface (Mc- as provided by Google (CA) and was created with the software R Call and Tevesz, 1982). This upward movement of reduced (South, 2011). The small insert map is from d-maps (https://www. sediment can cause significant reworking and alter the redox d-maps.com/carte.php?num_car=2648&lang=en). potential in surface sediment (Davis, 1974). The community composition of lacustrine sedimentary locally or seasonally high contributions of methane-derived macrofauna varies in response to trophic state (Aston, 1973; carbon (Premke et al., 2010). Verdonschot, 1992; Nicacio and Juen, 2015) in part due to Despite these past studies, the conditions under which differences in hypoxia/anoxia tolerance among macrofaunal methane-derived carbon becomes an important C source to species (Chapman et al., 1982). Different lacustrine macro- chironomid larvae or oligochaetes are not well understood. faunal species, moreover, vary in their impact on methane Furthermore, the main pathways of methane-derived carbon cycling in sediments (Bussmann, 2005; Figueiredo-Barros incorporation into macrofaunal biomass, e.g., selective graz- et al., 2009). methane oxidation in surface sediments is of- ing or gardening of methane-oxidizing bacteria or carbon ten stimulated by chironomid larval O2 input, which enriches transfer from methane-oxidizing bacteria gut symbionts, re- populations of methane-oxidizing bacteria in larval tubes and main unclear. Here we analyze shallow sublittoral to profun- surrounding sediment (“microbial gardening”) (Kajan and dal sediments of five temperate lakes in central Switzerland Frenzel, 1999). As a result, methane-oxidizing bacteria can that differ strongly in trophic state and macrofaunal com- become an important food source, and in some cases the munity composition. We analyze the community structures main C source, of chironomid larvae (Kankaala et al., 2006; of chironomid larvae and oligochaetes and compare their C- Deines et al., 2007a; Jones et al., 2008; Jones and Grey, isotopic compositions to those of total organic C (TOC), dis- 2011). High contributions of methane-derived carbon via solved organic C (DOC), and methane to investigate how C grazing on methane-oxidizing bacteria are typically found in sources vary across dominant macrofaunal groups in relation profundal regions of eutrophic lakes with seasonal stratifica- to trophic state and water depth. In addition, we analyze mi- tion and low O2 concentrations (Hershey et al., 2006; Jones crobial community structure based on 16S rRNA gene se- and Grey, 2011). Yet, variable isotopic values of chironomid quences and quantify functional genes involved in aerobic biomass, even within the same location, suggest that diets of and anaerobic methane oxidation in macrofaunal specimens, chironomid larvae vary greatly (Kiyashko et al., 2001; Reuss macrofaunal burrows, and surrounding sediment to elucidate et al., 2013). The limited C-isotopic data on tubificid worms the potential for microbial gardening or symbiotic associa- suggest that worm C sources also vary from detritus-based to tions between macrofauna and microbiota.

Biogeosciences, 18, 4369–4388, 2021 https://doi.org/10.5194/bg-18-4369-2021 A. Fiskal et al.: Carbon sources of benthic fauna 4371

Table 1. Overview of sampled lakes, their trophic status, and maximum water depths, as well as the geographic coordinates, water depths, and bottom water dissolved O2 concentrations (ranges are O2 concentrations over the time course of 1 year) of the stations that were sampled. O2 concentrations ≤ 15.6 µM are termed “hypoxic”. All data are from Fiskal et al., (2019). Trophic status and O2 concentrations are taken from Swiss Federal Office of the Environment (BAFU) (https://www.bafu.admin.ch/bafu/de/home/themen/wasser/fachinformationen/ zustand-der-gewaesser/zustand-der-seen/wasserqualitaet-der-seen.html).

Trophic Max. depth Station no. Latitude Longitude Waterdepth (m) O2 (µM) status (m) (◦ N) (◦ E) Lake Greifen Eutrophic 32 1 47◦ 21.134 8◦ 40.511 15 Seasonally Hypoxic 2 47◦ 21.118 8◦ 40.484 32 3 47◦ 21.038 8◦ 40.185 24 Lake Baldegg Eutrophic 66 1 47◦ 11.929 8◦ 15.613 66 15.6–125 125–250 15.6–125 2 47◦ 11.759 8◦ 15.392 45 3 47◦ 11.649 8◦ 15.417 21 Lake Zug Eutrophic 198 1 47◦ 10.272 8◦ 30.036 25 125–250 2 47◦ 10.104 8◦ 29.946 35 125–250 3 47◦ 09.834 8◦ 29.814 50 125–250 Lake Zurich Mesotrophic 137 1 47◦ 16.995 8◦ 35.624 137 Hypoxic 2 47◦ 16.708 8◦ 35.033 45 125–250 3 47◦ 16.395 8◦ 35.195 25 15.6–125 Lake Lucerne Oligotrophic 214 1 47◦ 00.051 8◦ 20.218 24 > 250 2 46◦ 59.812 8◦ 20.820 93 > 250 3 46◦ 59.915 8◦ 20.413 45 > 250

2 Material and methods

2.1 Sampling and site description

Sediment cores were obtained from three different water depths in the oligotrophic Lake Lucerne, the mesotrophic Lake Zurich, and the eutrophic Lake Zug, Lake Baldegg, and Lake Greifen in central Switzerland in June and July 2016 (Fig. 1, Table 1; for further information on trophic histories, please see Fiskal et al., 2019). Sediment cores were taken using gravity cores with 60 cm long liners that had an inner diameter of 150 mm (UWITEC, AT) from boats or motorized platforms. The four sediment cores per station were used as Figure 2. Average abundances of macrofauna in each lake. Error follows: the most undisturbed core was used for microsen- bars indicate standard deviations of three stations per lake, except sor measurements (O2, pH) and afterwards for macrofaunal for Lake Zurich where the macrofauna-free deep station was not community sampling. The second core was used for analyses considered, and error bars indicate the range of the two shallower of DNA sequences, methane concentrations, δ13C-methane, stations. The degree of eutrophication is based on water column TOC content, and δ13C-TOC. The remaining cores were used phosphorous concentrations measured by the Swiss Federal Office for porewater sampling using rhizons (0.2 µm pore size, Rhi- of the Environment (BAFU), which uses the OECD model (Vollen- 13 weider and Kerekes, 1982) to declare trophic state. According to zosphere), with DOC and δ C-DOC sampling being done the OECD model, lakes with average total P concentration values on a separate core than that for dissolved inorganic carbon 13 of ≤ 15 mgm−3 are oligotrophic, lakes with 15–45 mgPm−3 are (DIC) and δ C-DIC sampling. A wide range of additional mesotrophic, and lakes with > 45 mgPm−3 are eutrophic. porewater geochemical analyses were performed on the core used for DIC sampling (including concentrations of nitrate, sulfate, hydrogen sulfide, Fe2+, Mn2+, and ammonium; for further details, see Fiskal et al., 2019). In all cores, the top 4 cm was sampled in 0.5 to 1 cm depth intervals, samples from 4 to 20 cm sediment depth in 2 cm depth intervals, and https://doi.org/10.5194/bg-18-4369-2021 Biogeosciences, 18, 4369–4388, 2021 4372 A. Fiskal et al.: Carbon sources of benthic fauna

Figure 3. Depth distributions of oligochaetes and chironomid larvae at each station. Water depths of each station are indicated in each subplot. Horizontal lines indicate depth distributions of laminated sediment layers. all deeper layers in 4 cm depth intervals. Cores were typi- alytical details, see Fiskal et al., 2019). Cores for macrofau- cally ∼40–50 cm long; however, the lowermost 5–10 cm was nal community analyses were extruded and macrofauna col- discarded due to contamination with lake water during core lected by sieving sediments through 400 and 200 µm mesh retrieval. An additional, narrow core 6 cm in diameter was sieves. Three stations (two in Lake Lucerne, one in Lake obtained for radionuclide (210Pb and 137Cs) analyses (for an- Baldegg) were revisited in November 2017 and October 2018

Biogeosciences, 18, 4369–4388, 2021 https://doi.org/10.5194/bg-18-4369-2021 A. Fiskal et al.: Carbon sources of benthic fauna 4373

Figure 4. Pie charts of taxonomic analyses on oligochaetes (a) and chironomid larvae (b) in each lake (Ntax = number of taxonomically identified specimens, Ntotal = total number of specimens). No chironomid larvae were found in Lake Greifen. In Lake Lucerne only four oligochaetes were found of which one was taxonomically analyzed (Potamothrix vejdovskyi; not shown). Numbers show relative abundances in percentages. to collect additional macrofaunal specimens and chironomid 2.3 Stable carbon isotope analyses larval tubes for DNA analyses. Carbon isotope analyses were performed on DOC, methane, 2.2 Macrofaunal abundance and taxonomy TOC, macrofaunal specimens, or separately on guts and re- maining bodies of macrofaunal specimens. Values are given For each depth interval, specimen numbers of oligochaetes in the δ notation, i.e., and chironomid larvae were carefully picked with tweez- ers, counted, and preserved in 70 % ethanol for taxonomic   13 13 12  13 12  13 δ C = C C C C . and C-isotopic analyses or frozen on dry ice for DNA sample standard extractions. Detailed taxonomic analyses to the genus and, when possible, species level were performed on a subset For δ13C-DOC, porewater samples were analyzed as de- of oligochaetes and chironomid larvae. Oligochaete speci- scribed in Lang et al. (2012). Briefly, 2–7 mL of sample was mens were sent to AquaLytis (Wildau, Germany), where they added to 12 mL Vacutainers®. After removal of dissolved in- were embedded in epoxy resin and identified by light mi- organic C by addition of 85 % phosphoric acid and bubbling croscopy. Chironomid larvae were microscopically identified with high purity He, DOC was oxidized to CO2 using persul- by AquaDiptera (Emmendingen, Germany). ◦ fate (1 h at 100 C). The evolved CO2 was analyzed on a Gas- Bench II coupled to a Delta V mass spectrometer (Thermo Fisher Scientific, Bremen). Water soluble organic standards of known isotope composition (phthalic acid and sucrose) were used as standards. https://doi.org/10.5194/bg-18-4369-2021 Biogeosciences, 18, 4369–4388, 2021 4374 A. Fiskal et al.: Carbon sources of benthic fauna

For δ13C-Methane, methane was extracted by creating a 2.6 Quantitative polymerase chain reaction sediment slurry with Milli-Q water under saturating NaCl concentrations (∼ 6.3 M). A total of 2 cm3 of sediment was Quantitative polymerase chain reaction (qPCR) was per- transferred to 20 mL crimp vials containing 2.514 g NaCl formed to quantify bacterial and archaeal 16S rRNA genes, and 5 mL Milli-Q water, crimped, mixed, and stored on ice as well as genes encoding particulate methane monooxyge- or at +4 ◦C until analysis using a trace gas (Isoprime) cou- nase of aerobic methanotrophic bacteria (pmoA) and methyl pled to an isotope ratio mass spectrometer (GC-IRMS; Iso- coenzyme M reductase of methanogenic and anaerobic prime, Manchester). Separation was performed through a methane-oxidizing archaea (mcrA) (Table S1 in the Sup- GC-column (PoraPLOT Q 30 m column). The precision of plement). Standards consisting of plasmids containing 16S the method was ±0.7 %. After every sixth sample we in- rRNA, pmoA, or mcrA genes from specific organisms (Ta- cluded a standard with a known δ13C value (standards: L- ble S1 in the Supplement) were run in 10-fold dilutions of 13 1 7 iso1 with 2500 ppmv CH4 at −66.5 % δ C-methane and T- ∼ 10 to ∼ 10 gene copies per qPCR reaction. All sample 13 iso3 with 250 ppmv CH4 at −38.3 % δ C-CH4; Air Liq- DNA extracts and standard dilutions were run in duplicate. uide). The qPCR protocols are shown in Table S2 in the Sup- For δ13C-TOC analyses, 5–10 g of frozen sediment was plement. For each qPCR reaction, 2 µL of DNA extract was freeze-dried in glass vials and quantified using an elemen- mixed with 1 µL of molecular grade water, 1 µL of bovine tal analyzer (Thermo Fisher Flash EA 1112) coupled to serum albumin (10 mgmL−1; New England Biolabs, USA), an isotope-ratio mass spectrometer (Thermo Fisher Delta V 0.5 µL each of forward and reverse primers (10 µM), and Plus) (EA-IRMS) as outlined in Fiskal et al. (2019). 5 µL LightCycler® 480 SYBR Green I Master Mix (Roche, For δ13C-Macrofauna, δ13C-analyses were performed on Switzerland). All standards and samples were kept on ice macrofaunal biomass according to the same method used for throughout the preparations and run immediately after in δ13C-TOC. Single specimens were cleaned with molecular transparent 96-well plates on a Roche LightCycler® 480. grade water to remove sediment. Whole organisms, or sep- arated guts and residual bodies, were placed in tin foil cap- 2.7 Next generation sequencing (NGS) and sules, which were mounted to 96-well plates. The 96-well bioinformatics analyses plates were sealed using plastic seal foil, the foil above each well was pierced, and the whole plate was freeze dried. After- Libraries of bacterial and archaeal communities were pro- ward, the foil was removed, the tin foil capsules were closed, duced using the universal 16S rRNA primer pair Univ519F and the δ13C of macrofaunal biomass was measured. and Univ802R (Claesson et al., 2009; Wang and Qian, 2009). After library preparation DNA was pooled and sequenced us- 2.4 Two end-member mixing model ing a MiSeq (Illumina Inc., USA). Library preparations and subsequent data processing, including 97 % zero-radius op- Assuming TOC and methane as the only carbon sources, a erational taxonomic unit (ZOTU) clustering, were done as two end-member mixing model was used to estimate the con- outlined in Han et al. (2020) (for polymerase chain reaction tribution of methane to biomass C of macrofauna. mixtures and cycler conditions, see Table S3 in the Supple-  ment). Briefly, raw sequences were initially quality trimmed  13 13  using seqtk (https://github.com/lh3/seqtk) and paired-end CH4-Contribution (%) = 100 − δ Cfauna − δ CCH 4 reads were merged using flash (Magocˇ and Salzberg, 2011).    This was followed by a final quality filtering using prinseq δ13C − δ13C × 100 TOC CH4 (Schmieder and Edwards, 2011). Sequences were then used to generate ZOTUs with USEARCH unoise3 using a 97 % 2.5 DNA extraction from macrofauna, macrofaunal clustering identity (Edgar, 2016). tubes, and sediment 2.8 Statistical analyses DNA was extracted according to lysis protocol II of the mod- ular DNA extraction method of Lever et al. (2015) following Statistical differences between C isotope signatures of the exact procedure outlined in Han et al. (2020). While we macrofauna and C pools, as well as of percentages of bac- used existing sediment DNA extracts from the latter study, terial 16S rRNA, mcrA, and pmoA gene copy numbers rel- DNA from empty larval tubes and from macrofauna were ative to total 16S rRNA gene copy numbers across macro- newly extracted. To remove sediment, macrofaunal speci- fauna, larval tubes, and sediment, were determined using mens were rinsed with molecular grade water. DNA was then Wilcoxon signed rank tests for paired data. All tests were extracted from entire specimens, or separately on guts and performed in R (Team, 2018) using the following command: the remaining body, after being cut into three to four pieces wilcox.test (A, B, paired = TRUE, alternative = “two.sided” to increase extraction efficiency using a sterile scalpel. All for (a), “greater/less” for (b), mu = 0.0, exact = TRUE, cor- DNA extracts were stored at −80 ◦C. rect = TRUE, conf.int = TRUE, conf.level = 0.95). Principal

Biogeosciences, 18, 4369–4388, 2021 https://doi.org/10.5194/bg-18-4369-2021 A. Fiskal et al.: Carbon sources of benthic fauna 4375 coordinates analysis (PCoA) on bacterial communities at the phylum, class, order, family, and genus levels was performed using Bray–Curtis distances in R (Team, 2018).

3 Results

3.1 Macrofaunal distribution in relation to lake trophic state

Macrofauna are present at all stations except the hy- poxic deep station of Lake Zurich and are dominated by oligochaetes and chironomid larvae. While oligochaetes are present in all lakes, no chironomid larvae were found in Lake Greifen. Oligochaete densities increase with trophic state, from 75 ± 86 individuals per square meter in Lake Lucerne to 4849±4443 individuals per square meter in Lake Baldegg (number of individuals are expressed as averages per lake with standard deviations of three stations). Numbers of chi- ronomid larvae show the opposite trend, decreasing from 641 ± 346 individuals per square meter in Lake Lucerne and 849 ± 160 individuals per square meter in Lake Zurich to less than 75 ± 86 individuals per square meter in the three eutrophic lakes (Fig. 2, Table S5 in the Supplement). Other macrofauna, e.g., copepods, Daphnia, and leeches, were only occasionally found and will not be discussed further. The depth distributions of oligochaetes and chironomid larvae follow different trends (Fig. 3). Chironomid larvae are most abundant in surface sediment (0–5 cmblf, centimeters below lake floor), while oligochaetes occur over a greater depth interval (Fig. 3). In Lake Greifen and Lake Baldegg, oligochaetes are present in high numbers to 12 and 15 cm sediment depth, respectively, including layers that are dis- tinctly laminated (see horizontal lines in Fig. 3). In Lake Zug, oligochaetes are present to even greater depths (22 cm). In sediments of Lake Zurich, where oligochaetes and chirono- mids occur in similar abundances, chironomids dominate the top ∼2–3 cm, whereas oligochaetes dominate below. Despite depth ranges extending significantly below the sediment sur- face, macrofaunal sediment reworking is minimal based on radionuclide measurements. These show 137Cs peaks that match the 1986 (Chernobyl) and 1963 (bomb test) time Figure 5. Boxplots of 13C isotopic compositions of CH , TOC, 210 4 markers, and clear Pbunsupported decreases from the top DOC, oligochaetes, and chironomid larvae for each lake (note: 2 cm downward at all faunated stations (Fig. S6 in the Sup- no larvae were found in Lake Greifen). Boxes show 75 % and plement; data analyzed but not shown in Fiskal et al., 2019). 25 % quartiles. Whiskers show minimum and maximum values. Light microscopic images of the two dominant macrofau- Wilcoxon signed rank tests were applied to check whether 13C- nal groups and depth distributions of individual macrofaunal isotopic signatures of macrofauna and TOC were significantly species can be found in Fig. S1 in the Supplement. different (ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001). For each Wilcoxon test, macrofaunal specimens 3.2 Macrofaunal community structure and diversity were paired with TOC isotopic signatures from the same depth across lakes (±2 cm), and only data were included for which there were data macrofauna and TOC data from matching depths. Samples with Oligochaetes and chironomid larvae were assigned to 9 N < 5 are displayed as individual data points. N indicates the num- and 14 different taxonomic groups, respectively (Fig. 4; ber of data points for each variable above. for station-specific data, see Fig. S2 in the Supplement). All oligochaetes belong to the family Naididae (Syn. Tubi- https://doi.org/10.5194/bg-18-4369-2021 Biogeosciences, 18, 4369–4388, 2021 4376 A. Fiskal et al.: Carbon sources of benthic fauna

Table 2. Contributions of TOC and methane to oligochaete and chironomid larval biomass C based on a two end-member mixing model. Estimates outside of the parentheses are maximum values as they assume no isotopic fractionation during aerobic methane oxidation. Values within parentheses are more conservative and assume a fractionation factor that is in the upper range previously determined for freshwater sediments and pure-culture incubations (−39 %) (Kruger et al., 2002; Templeton et al., 2006; Kankaala et al., 2007). For the calculations, only macrofaunal specimens were included that could be paired with TOC and methane isotopic values from the same sediment depth (±2 cm); values display averages ± standard deviation.

Contribution of TOC (%) Contribution of methane (%) Oligochaetes Chironomid larvae Oligochaetes Chironomid larvae Lake Lucerne – 97.3 ± 4.1 (98.6 ± 2.0) – 2.7 ± 4.1 (1.5 ± 2.0) Lake Zurich 98.5 ± 3.9 (99.2 ± 1.5) 99.1 ± 4.3 (99.5 ± 2.4) 1.5 ± 3.9 (0.8 ± 1.5) 0.9 ± 4.3 (0.5 ± 2.4) Lake Zug 88.3 ± 3.3 (94.0 ± 1.7) – 11.7 ± 3.3 (6.0 ± 1.7) – Lake Greifen 93.1 ± 7.6 (96.5 ± 3.5) – 6.9 ± 7.6 (3.5 ± 3.5) – Lake Baldegg 88.2 ± 2.8 (93.9 ± 1.5) 87.9 ± 1.6 (93.9 ± 0.8) 11.8 ± 2.8 (6.2 ± 1.5) 12.1 ± 1.6 (6.3 ± 0.8)

Table 3. Results of Wilcoxon sign rank test (one-sided) to examine whether the ratios of bacterial 16S rRNA gene (BAC), mcrA, and pmoA to total 16S rRNA gene copy numbers differ significantly between oligochaete, chironomid larval, and chironomid larval tube samples relative to surrounding sediment (ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001), and actual p values can be found in brackets below. Only data were included for which matching values existed from the same sediment depth (±2 cm).

% Oligochaetes Larvae Tubes vs. sediment vs. sediment vs. sediment BAC of total 16S *** ** ** Oligochaetes higher Larvae higher Tubes higher (p = 0.000002) (p = 0.006) (p = 0.001953) mcrA of total 16S ** *** ** Oligochaetes lower Larvae lower Tubes lower (p = 0.002921) (p = 0.000008) (p = 0.004002) pmoA of total 16S ** ns ns Oligochaetes higher Larvae higher Tubes higher (p = 0.005212) (p = 0.9998) (p = 0.1792)

ficidae) and all chironomid larvae to the family Chirono- partially overlap. Lake Zurich sediment is dominated by Mi- midae. Two oligochaete morphotypes, Tubificidae + bristles cropsectra sp., Tanytarsus sp., Chironomus riparius, Chi- and Tubificidae − bristles, could not be assigned to a known ronomus piger gr., and Sergentia coracina, whereas Lake genus. Lucerne is dominated by Procladius sp., Micropsectra sp., For Oligochaete group overlap between lakes, four of the Macropelopia fehlmanni Kieffer 1911, Tanytarsus sp., and S. nine groups (Tubificidae + bristles, Tubificidae − bristles, P. coracina. Micropsectra sp., Tanytarsus sp., and S. coracina hammoniensis, L. hoffmeisteri) occur in four of the five lakes. are mainly sedimentary detritus feeders, whereas Chirono- E. velutinus (Lake Zurich), L. profundicula (Lake Baldegg), mus riparius and C. piger gr. are known to mainly filter and P. vejdovskyi (Lake Lucerne) were the only species feed. Both Procladius sp. and M. fehlmanni are predators that were only found in one lake. Comparing the domi- (Table S6 in the Supplement). nant oligochaete groups reveals the dominance of unchar- acterized Tubificidae (+ bristles) in Lake Zurich, Lake Zug, 3.3 C isotope composition of macrofauna and bulk C and Lake Greifen but very different communities in Lake pools Baldegg, which is dominated by uncharacterized Tubificidae (− bristles) and L. hoffmeisteri. All identified tubificids ex- Average C isotope compositions of macrofaunal speci- cept E. velutinus are subsurface deposit feeders that are be- mens are displayed with those of the potential C sources lieved to mainly feed on sedimentary bacteria, whereas E. methane, TOC, and DOC in Fig. 5 (for depth profiles, see velutinus is a surface deposit feeder (Table S6 in the Supple- Fig. S3 in the Supplement). Macrofaunal values are lowest ment). in Lake Baldegg (oligochaetes: −36.7 ± 3.3 %, N = 14; lar- Chironomid larval communities in Lake Zurich and Lake vae: −37.6 ± 1.9 %, N = 4) and Lake Greifen (oligochaetes: Lucerne share many members, but the dominant groups only −37.6 ± 2.5 %, N = 12; no larvae found) and highest in

Biogeosciences, 18, 4369–4388, 2021 https://doi.org/10.5194/bg-18-4369-2021 A. Fiskal et al.: Carbon sources of benthic fauna 4377

Figure 6. Relative abundances of Bacteria at the phylum level (Proteobacteria at class level) based on 16S rRNA gene sequences. Sequences were obtained from 17 sediment, 10 chironomid larval tube, 26 chironomid larvae (Nbody = 7, Ngut = 7, Nwhole = 12), and 36 oligochaete (Nbody = 5, Ngut = 6, Nwhole = 25) samples. Station and sample IDs are indicated by sample names, which indicate station water depth (m), sediment depth (cm), and portion of macrofaunal body analyzed (w = whole specimen, g = gut, b = body). Bodies and guts of the same specimens are marked by the same symbols. All sediment 16S rRNA gene sequence data are from Han et al. (2020).

https://doi.org/10.5194/bg-18-4369-2021 Biogeosciences, 18, 4369–4388, 2021 4378 A. Fiskal et al.: Carbon sources of benthic fauna

3.4 Average contributions of methane-derived carbon and TOC to macrofaunal biomass C

A two end-member mixing model suggests that on average ≥ 88 % of macrofaunal biomass-C can be explained with as- similation of detrital organic C (TOC) (Table 2). By contrast, methane-derived carbon accounts for ≤ 12.1 % or ≤ 6.3 % of biomass-C depending on the assumed isotopic fractionation factor during aerobic methane oxidation (for further details, see Table 1 caption). Chironomid larvae and oligochaetes from the same lakes have highly similar average methane- derived carbon contributions to biomass. Consistent with past studies (Hershey et al., 2006; Jones and Grey, 2011), the contribution of methane-derived carbon to macrofaunal Figure 7. PCoA analysis of bacterial community structure at the order level using Bray–Curtis distances. All sediment 16S rRNA biomass increases with trophic state, with the lowest contri- gene sequence data are from Han et al. (2020). butions in Lake Zurich and Lake Lucerne and highest con- tributions in Lake Baldegg, followed by Lake Zug and Lake Greifen. Lake Lucerne (oligochaetes: −31.7 ± 0.4 %, N = 2; larvae: −31.5 ± 2.2 %, N = 24) and Lake Zurich (oligochaetes: 3.5 Microbial communities of macrofauna, larval −32.8±0.9 %, N = 5; larvae: −32.5±2.1 %, N = 24). There tubes, and surrounding sediments was no apparent trend between δ13C values of macrofauna and sediment depth (Fig. S3 in the Supplement). To investigate the nature of macrofauna–microbiota associa- Average δ13C-methane values are in all cases ∼ 35 % to tions, e.g., with respect to microbial gardening or grazing of 50 % more negative than those of macrofauna. The most neg- methane-cycling microorganisms or symbiotic relationships, ative methane values are present in Lake Lucerne (−78.8 ± we studied 16S rRNA gene sequences of macrofauna (whole 4.3 %, N = 18) and Lake Zurich (−76.7 ± 2.4 %, N = 25), organisms, guts, residual body without guts) and chironomid followed by Lake Baldegg (−74.3 ± 2.6 %, N = 20), Lake larval tubes and compared these to those in surrounding sed- Greifen (−73.6 ± 3.7 %, N = 21), and Lake Zug (−70.1 ± iments (Fig. 6). 4.5 %, N = 23). All stations except the middle station in Lake Baldegg have 13C-methane increases indicative of 3.5.1 Bacteria methane oxidation in surface layers (Fig. S3 in the Supple- ment). Sediment and tube samples share similar bacterial com- The δ13C values of TOC are much closer to those of munities across all lakes, stations, and sediment depths macrofauna (Fig. 5; Fig. S3 in the Supplement), with av- (Fig. 6). Both sample types are dominated by β-, δ- and erages ranging from equal (Lake Zurich) to ∼ 5 % higher γ -Proteobacteria, Chloroflexi (mainly Anaerolineae), Aci- (Lake Baldegg). The lowest average δ13C-TOC was mea- dobacteria, Bacteroidetes (dominated by Sphingobacteriia), sured in Lake Greifen (−34.5 ± 1.5 %, N = 35), followed Planctomycetes, and Verrucomicrobia. Furthermore, sedi- by Lake Baldegg (−32.4 ± 1.2 %, N = 37), Lake Zurich ments and tubes from Lake Zurich and Lake Lucerne share (−32.2±1.9 %, N = 29), Lake Zug (−30.8±1.3 %, N = 35), elevated fractions of Nitrospirae. Conspicuous differences and Lake Lucerne (−29.7 ± 1.2 %, N = 32). Isotopic values are the higher fractions of δ-Proteobacteria in sediments and of TOC increase by 4 %–6 % with sediment depth at all sites of Chloroflexi, Actinobacteria, Gemmatimonadetes, and Ig- (Fig. S3 in the Supplement). Despite the small differences navibacteriae in tubes, as well as the virtual absence of between δ13C-TOC and δ13C-macrofauna, δ13C-TOC values Aminicenantes in tubes. By comparison, chironomid lar- are significantly higher than those of oligochaetes and larvae vae and oligochaetes have very different bacterial commu- in all lakes except Lake Zurich (Fig. 5). Average δ13C-DOC nities, which moreover vary greatly between and within both is slightly higher than δ13C-TOC in all lakes and significantly macrofaunal groups. higher than the δ13C of macrofaunal biomass (Fig. 5). Ad- Depending on the specimens, bacterial communities ditional analyses on water column algal material and algal of chironomid larvae are dominated by γ -, β-, and α- bloom layers in sediment (Fig. S3 and Table S4 in the Sup- Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, plement) suggest δ13C values similar to those of TOC. and/or Fusobacteria. Many larval specimens are dominated (> 50 % of reads) by a single group of α-, β-, or γ - Proteobacteria or Firmicutes, and guts of two specimens from Lake Lucerne contain ≥ 99 % γ -Proteobacteria. With respect to dominant groups or ZOTUs, there is no clear trend

Biogeosciences, 18, 4369–4388, 2021 https://doi.org/10.5194/bg-18-4369-2021 A. Fiskal et al.: Carbon sources of benthic fauna 4379

Figure 8. Ratios (expressed in %) of bacterial (BAC) 16S rRNA gene copy numbers (left panel), mcrA copy numbers (middle panel), and pmoA copy numbers (right panel) to total 16S rRNA gene copy numbers (sum of bacterial and archaeal 16S rRNA gene copy numbers). The three x axes differ in ranges and scales (linear and log). All sediment 16S rRNA gene values are from Han et al. (2020). Values on the lower limit of the x axis in the middle and right panels indicate samples in which mcrA or pmoA were below qPCR detection. in relation to lake, trophic state, or water depth. Yet, gut, and the body were detected in oligochaetes. This could, however, to a lesser extent body, bacterial communities from the same be due to the greater difficulty of separating guts from the samples are sometimes highly similar. Furthermore, bacte- rest of the body in oligochaetes. rial communities in guts often differ clearly from those in Ordination plots based on PCoA at the order level (Fig. 7) the remaining body. For instance, Firmicutes in several spec- and at the phylum, class, family, and genus levels (Fig. S5 imens dominate larval guts but are virtually absent from the in the Supplement) confirm the trends observed in Fig. 6. rest of the body. By contrast, the fractions of α- and β- Sediment and tube samples from all lakes and sediment Proteobacteria are often lower in guts than the remaining depths are highly similar and form tight clusters, which only body. Compared to tubes, chironomid larvae generally have become separated at the order level and below. Chirono- lower abundances of Chloroflexi (nearly absent), Verrucomi- mid larvae and oligochaetes are phylogenetically very dif- crobia, Gemmatimonadetes, Nitrospirae, and/or Ignavibacte- ferent from sediments and tubes and phylogenetically highly ria. heterogeneous due to the dominance of Fusobacteria or α- Bacterial communities of oligochaetes are also variable Proteobacteria or the varying relative abundances of diverse and differ clearly from those in chironomid larvae. As for proteobacterial classes and orders. chironomid larvae, these bacterial communities do not fol- low clear trends related to lake, trophic state, or water depth. 3.5.2 Archaea About half of all specimens are strongly dominated (≥ 80 % of 16S reads) by Fusobacteria (Fusobacteriales), a phylum Archaea only account for low percentages (< 10 %) of ± that accounts for on average only 0.01 0.02 % of total 16S prokaryotic 16S rRNA gene sequences in chironomid larvae, ∼ reads in sediment samples and was only detected in 20 % larval tubes, and oligochaetes and were even below detection of larval specimens. Several other oligochaete specimens are in 69 % of chironomid larval and 39 % of oligochaete sam- dominated (> 50 %) by single groups of α-, β-, δ-, and ε- ples analyzed (Fig. S4 in the Supplement; also see following Proteobacteria, or Parcubacteria, or have elevated relative section). Yet, distinct trends are evident. Larval tubes have a abundances of Spirochaetae or Cyanobacteria. Most phyla lower diversity than sediments, being dominated by Woese-, that are abundant in sediment and/or larval tubes (Chlo- Pace-, and Thaumarchaeota and to a lesser degree Dia- roflexi, Acidobacteria, Gemmatimonadetes, Nitrospirae, Ver- pherotrites. In sediments, Eury- and Bathyarchaeota were ad- rucomicrobiae, Aminicenantes) are less common or nearly ditionally present in high percentages along with low per- absent from oligochaetes. Unlike chironomid larvae, no sys- centages of Altiarchaeales, Lokiarchaeota, and an unclassi- tematic phylogenetic differences between guts and the rest of fied phylum-level cluster of Asgardarchaeota. The archaeal https://doi.org/10.5194/bg-18-4369-2021 Biogeosciences, 18, 4369–4388, 2021 4380 A. Fiskal et al.: Carbon sources of benthic fauna community of larvae was highly variable and dominated by samples. While pmoA contributions decrease with depth in Pace-, Eury- and Woesearchaeota, with typically only one to sediments, there is no clear depth trend in oligochaete or two phyla present per sample. The oligochaete archaeal com- larval tube samples. The 16S rRNA gene sequences indi- munity was more diverse and dominated by essentially the cate that all methane-oxidizing bacteria are γ -Proteobacteria, same groups as sediments, i.e. Woese-, Pace-, Bathy-, Eury-, dominated by Crenothrix (Methylococcales). Crenothrix are and/or Thaumarchaeota and to a lesser degree Lokiarchaeota, moreover the only methane-oxidizing bacterium detected in Altiarchaeales, and Diapherotrites. oligochaetes, whereas low read percentages of Methylococ- caceae (Methylobacter, Methylocaldum, Methylococcus, and 3.6 Abundance analysis of Bacteria, Archaea, and Methyloparacoccus) were detected in larvae, larval tubes, functional genes related to methane cycling and sediments. In addition, the denitrifying methanotroph Methylomirabilis (candidate phylum NC10) was detected To further investigate potential interactions between macro- in low read numbers in several tube and sediment sam- fauna and microorganisms in general, and methane-cycling ples (mostly from Lake Lucerne). Despite the significantly microorganisms in particular, we compared the contribu- higher calculated abundance of methane-oxidizing bacteria tions of Bacteria, methane-cycling archaea, and methane- in oligochaetes based on ratios of pmoA to total 16S rRNA oxidizing bacteria across sample types. Trends related to gene copy numbers, we did not detect significantly different lake trophic state and gardening of or preferential grazing on 16S read percentages between larvae, tubes, oligochaetes, or methane-cycling microorganisms are largely absent, but we sediments (data not shown). observe other trends. Bacteria account for > 80 % of total 16S gene copies in all samples (Fig. 8, left panel). Significantly higher pro- 4 Discussion portions are present in oligochaetes, larvae, and tubes rela- tive to sediments (Table 3). The contribution of Bacteria de- Methane has been indicated as an important C source to creases from 94 %–98 % in surface sediments to 82 %–86 % lacustrine sedimentary macrofauna (Kankaala et al., 2006; below 12 cmblf. By comparison, Bacteria contribute ≥ 99 % Deines et al., 2007a; Jones et al., 2008; Jones and Grey, in most macrofauna samples. The lowest bacterial contribu- 2011). Yet, open questions remain regarding the conditions tions are ∼ 98 % in chironomid larvae, 90 % in oligochaetes, under which this methane-derived carbon is an important C and 96 % in tubes. source or how it is incorporated into macrofaunal biomass. In the vast majority of samples, mcrA gene copy numbers We investigate these questions by analyzing macrofaunal are ≥ 100 times lower than total 16S rRNA gene copy num- community structure, isotopic compositions of macrofauna bers (range: below the detection limit of ∼ 0.0001 % to 2 %) and possible C sources, and microbial community struc- (Fig. 8, middle panel), suggesting very low contributions ture across five temperate lakes with widely differing trophic of methanogenic and/or anaerobic methanotrophic archaea. states. The mcrA contributions are significantly higher in sediments We observe a clear macrofaunal community shift, with compared to oligochaetes, larvae, and tubes (Table 2) and oligochaetes dominating eutrophic lakes, chironomid larvae are even below qPCR detection in all but one larval speci- dominating the oligotrophic lake, and similar abundances of men. While the contribution of mcrA increases with depth both in the mesotrophic lake (Fig. 2). Maximum abundances in larval tubes, oligochaetes and sediments show no depth- of oligochaetes are higher than those of chironomid larvae, related trends. The 16S rRNA genes of methane-cycling Ar- and oligochaetes extend deeper into sediments than chirono- chaea were found in sediments (mainly Methanobacteria and mid larvae, matching the different feeding behaviors of the M. fastidiosa) and at very low read numbers in a few tubes two groups (Fig. 3). Taxonomic analyses reveal overlaps but (M. fastidiosa) and oligochaetes (M. fastidiosa, M. peredens) also clear differences in oligochaete and chironomid larval but not in larvae. communities between lakes (Fig. 4). The pmoA contributions range from below detection (≤∼ While chironomid communities vary strongly with water 0.001 %) to ∼ 15 % (Fig. 8, right panel) and are – com- depth in the same lakes, oligochaete communities are more pared to sediments – significantly elevated in oligochaetes similar across different locations within the same lake. This but not in larval specimens or larval tubes (Table 3). This suggests that chironomid larval and oligochaete communities suggests the potential for preferential grazing by, or ele- are controlled by different environmental factors. vated populations of symbiotic aerobic methanotrophic bac- Comparing 13C isotopic compositions, 13C-methane is al- teria within, oligochaetes. Nonetheless, it is worth men- ways far more negative (−35 to −50 %), while 13C-TOC tioning that the median calculated pmoA percentage in is similar or slightly enriched (+0.3 to +5.2 %) relative to oligochaetes was only ∼ 1 % and that based on the max- macrofaunal biomass. This suggests that detrital organic mat- imum calculated value of 15 % methane-oxidizing bacteria ter is the main C source of macrofauna (Fig. 5). Estimated in no case dominated oligochaete bacterial communities. As contributions of methane-derived carbon range from statisti- for mcrA, pmoA was only detected in very few (2) larval cally insignificant to at most 12 % and increase with trophic

Biogeosciences, 18, 4369–4388, 2021 https://doi.org/10.5194/bg-18-4369-2021 A. Fiskal et al.: Carbon sources of benthic fauna 4381 state (Table 2). Despite differences in feeding behavior and tain species of chironomid larvae (Pinder, 1995). Additional environmental drivers of their species compositions, the cal- reasons could be the superior ability of oligochaetes to ex- culated contribution of methane-derived carbon is highly ploit high organic matter supplies or that deeper burrows of similar across chironomid larval and oligochaete specimens oligochaetes provide better protection from benthic preda- from the same lakes, suggesting an important role of lake- tors, such as bottom-feeding fish, which are abundant in eu- specific variables. trophic lakes (Scheffer et al., 1993). Bacterial communities of macrofauna differ clearly from While most oligochaete specimens could only be clas- those in chironomid tubes or sediments. The majority of sified to the family level – Tubificidae (+bristles); Tubi- reads in many macrofaunal specimens belong to single ZO- ficidae (− bristles); Fig. 4; Table S7 in the Supplement – TUs, implying potential symbiotic relations with their hosts distributions of those that were taxonomically classifiable (Figs. 6 and 7; discussed in detail later). Consistent with to the species level match published distributions. On one the calculated minor contributions of methane-derived C hand, subsurface deposit feeders known to rely on bacte- to macrofaunal biomass, pmoA copy numbers indicate that ria and algae as food sources dominated eutrophic lakes. methane-oxidizing bacteria are minor, yet significant, com- L. hoffmeisteri, an indicator species of eu- to hypertrophic ponents of bacterial communities in numerous macrofaunal lakes (Brinkhurst, 1982), occurs in high abundances in Lake specimens (∼1 %–10 %; Fig. 8, right panel; Table 3). This Baldegg (Table S7 in the Supplement). P. hammoniensis and is not the case for methane-cycling archaea, whose con- T. tubifex, which frequently co-occur in high abundances in tribution, based on mcrA copy numbers, was always small mesotrophic to eutrophic lakes (Lang, 1990; Timm, 1996), (≤ 1 %) and significantly lower in oligochaetes, chironomid dominate Lake Zurich, Lake Zug, and Lake Greifen. On the larvae, and chironomid larval tubes than in surrounding sed- other hand, surface-deposit-feeding E. velutinus, which indi- iment (Fig. 8, middle panel; Table 3). cates oligo- to mesotrophic conditions (Martin et al., 2008), In the following sections, we discuss in detail the potential was only found in Lake Zurich. drivers of macrofaunal community structure, the likely car- Even though many tubificids are subsurface conveyor bon sources of macrofauna, and the potential trophic roles feeders, the lakes investigated show little evidence of sed- of observed (endo-)symbiotic bacteria in their macrofaunal iment mixing. We observed clear laminations at the deep hosts. station in Lake Baldegg and the deep and middle station in Lake Greifen in sediments that were being deposited until 4.1 Abundance and taxonomy of macrofauna along the mid-1980s and ∼ 2010, respectively (Fig. 3, Fig. S7 in trophic state the Supplement; Fiskal et al., 2019), so until the onset of ar- tificial water column mixing and oxygenation in these lakes Oligochaete abundances follow the environmental index pro- (Lake Baldegg in 1984 and Lake Greifen in 2009; Fiskal posed previously by Milbrink (1983) which predicts a strong et al., 2019). While the subsequent disappearance of lami- rise in worm abundance with increasing trophic state. Chi- nae suggests rapid re-colonization by macrofauna, it appears ronomid abundances are also within the range previously re- that mixing has remained limited to surface sediments even ported for lakes (Mousavi, 2002). While chironomid larvae though burrows of tubificids extend far into laminated lay- show typical depth distributions (e.g., Panis et al., 1996), ers. Depth profiles of radionuclides confirm this interpreta- oligochaetes have unusually deep ranges, with high abun- tion and even indicate minimal sediment mixing in the pres- dances to 10–14 cm in eutrophic lakes. By contrast, most ence of macrofauna (Fig. S7 in the Supplement). Indepen- publications report that oligochaetes are mainly present at 2– dent of faunal presence, 137Cs peaks that match the 1986 8 cm sediment depth (reviewed in McCall and Tevesz, 1982). (Chernobyl) and 1963 (bomb test) time markers, and clear 210 The observed shift in dominance from chironomid larvae Pbunsupported decreases from the top 2 cm downward, are to tubificids with increasing trophic state (Fig. 2, Fig. 3) present at all stations. These findings contrast with the rapid matches past studies reporting the dominance of oligochaetes sediment homogenization to 10 cm by tubificids in the labo- in eutrophic lakes (Saether, 1980; Lang, 1985; Timm, 1996; ratory (Fisher et al., 1980; Matisoff et al., 1999) and homo- Bürgi and Stadelmann, 2002) and changes from chironomid- geneous radionuclide profiles to 6 cm in tubificid-dominated larva- to oligochaete-dominated communities as the first natural lake sediments (Robbins et al., 1977; Krezoski et al., signs of eutrophication (Saether, 1979). This dominance 1978). Similar to tubificids, chironomid larval communities of oligochaetes in eutrophic lakes is possibly related to change in relation to trophic state (Fig. 4; Table S7 in the an overall higher tolerance of low O2 conditions as many Supplement). Large free-living and predatory larvae account oligochaetes feed in anoxic parts of sediments (McCall and for half of the specimens in Lake Lucerne, whereas tube- Tevesz, 1982) and efficiently exchange gases through their building herbivorous, surface detritus-feeding, and garden- body walls (Martin et al., 2008). Longer survivorship of ing larvae dominate Lake Zurich and the small sample sizes anoxic conditions among oligochaetes is also possible (Ham- in eutrophic lakes. The shift in diet at higher trophic levels burger et al., 1998), though anaerobic respiration and tol- matches the higher input of algae and algal detritus (Fiskal erance of extended anoxic periods is also known for cer- et al., 2019), whereas the potential increase in microbial gar- https://doi.org/10.5194/bg-18-4369-2021 Biogeosciences, 18, 4369–4388, 2021 4382 A. Fiskal et al.: Carbon sources of benthic fauna dening matches observed increases in gardening by C. ripar- growth of methane-oxidizing bacteria is mainly promoted ius and other Chironomus spp. under hypoxic or eutrophic at narrow oxic–anoxic (high O2-methane) interfaces pro- conditions (Stief et al., 2005; Yasuno et al., 2013). By con- duced by ventilating and tube-building chironomid larvae trast, the reasons for the high abundances of predatory larvae (Brune et al., 2000). Tubificids, which dominated our eu- in Lake Lucerne are unclear. Possible reasons are the low trophic lakes, do not produce such stable oxic–anoxic inter- hypoxia tolerance of large predatory Macropelopia and Pro- faces and also perform less burrow ventilation than chirono- cladius spp. (Hamburger et al., 1998; Brodersen et al., 2008), mid larvae (Gautreau et al., 2020 and references within). Yet, higher availability of zooplankton food in oligotrophic lakes the fact that all three identified larvae from Lake Baldegg be- (Jeppesen et al., 1990; Jeppesen et al., 1999), and/or stronger long to tube-building taxa and that the four isotopically ana- predation pressure in mesotrophic and eutrophic lakes, which lyzed larvae from this lake only had minor methane-derived often have high populations of bottom-feeding fish (Scheffer carbon contributions suggests that yet unknown factors con- et al., 1993). tribute to the enrichment of methane-oxidizing bacteria by tube-building chironomids in surface sediment. 4.2 Carbon sources of lake sedimentary macrofauna Instead of methane-derived carbon, our C-isotopic data in- dicate that algal or detrital organic carbon, or microorgan- Similar to previous studies (e.g., Grey et al., 2004; Jones isms that have assimilated the isotopic signatures of algal or et al., 2008) we calculate an increase in the contribution of detrital organic carbon, is the main food source of dominant methane-derived carbon with increasing trophic state (Fig. 5; macrofauna (Table 2). Rather than methane-derived carbon, Table 2). Yet, this contribution is at most 12 %, even in selective feeding on isotopically depleted subportions of the the highly eutrophic lakes. Other studies have estimated TOC pool could even, in principle, explain the minor isotopic methane-derived carbon contributions of > 40 % for chi- depletions of oligochaete and chironomid larval biomass in ronomid larvae in eutrophic lakes (e.g., Deines and Grey, eutrophic lakes. Yet, our limited data on algal bloom layers in 2006; Eller et al., 2007; Jones et al., 2008) and reported sediments and phytoplankton from overlying water indicate strong δ13C depletions in oligochaete specimens from pro- similar 13C values relative to TOC (Fig. S5 in the Supple- fundal sediment (Premke et al., 2010). Yet, minor contribu- ment). Preferential feeding on organic C from surface sedi- tions of methane-derived carbon to the biomass of benthic in- ments, which in many cases has the lowest C-isotopic values, vertebrates are not new. A survey of 87 lakes suggested that or isotopic fractionations during C-assimilation and biosyn- marked 13C depletions were only present in chironomid lar- thesis are also not plausible. As bottom-up conveyor feed- vae from lakes with seasonal stratification and bottom water ers, tubificids feed mostly at several centimeters depth (Mc- anoxia (Jones et al., 2008). Moreover, the limited published Call and Tevesz, 1982), and C-isotopic fractionation during δ13C data on lake oligochaetes are mostly similar to those of biosynthesis of bulk animal biomass is typically low (Fry and TOC (Kiyashko et al., 2001; Premke et al., 2010). Sherr, 1989). In support of C-isotopic interpretations, DNA-based anal- yses indicate that neither methane-oxidizing bacteria nor 4.3 Potential diet and host–microorganism interactions methanogens are dominant microorganisms in surface sed- in tubificid worms iments or chironomid larval tubes. Thus, strong enrichment or gardening of methane-oxidizing bacteria or methanogens Minimal sediment reworking and deep sedimentary distribu- as observed elsewhere in chironomid tubes (e.g., Kajan and tions of tubificids suggest that shallow subsurface deposit Frenzel, 1999; Kelly et al., 2004) or surface sediments (e.g., feeding may not be the main dietary mode of these worms Eller et al., 2005; Deines et al., 2007a) is absent for rea- in the lakes studied, raising questions concerning their main sons that are unclear. Despite being artificially oxygenated, foraging strategy. One possibility is that oligochaetes selec- bottom water in Lake Baldegg and Lake Greifen experi- tively graze on microbial biofilms inhabiting the walls of −1 ences seasonally low O2 conditions (0.5–4 mgL ) or hy- their deep and extensive gallery-type burrow networks. Un- poxic conditions (< 0.5 mgL−1), respectively (Fiskal et al., der this scenario one might expect large amounts of DNA 2019). These values are within or below the seasonal O2 of sediment microorganisms in oligochaete intestines. This threshold (2–4 mgL−1) that is characteristic of lakes with is not the case, however, suggesting that grazed communities marked 13C depletions in chironomid biomass (Jones et al., are very different from those in sediments or their DNA is 2008). Jones et al. (2008) argued that the contribution of rapidly digested. Another foraging strategy may not involve methane-derived carbon increases inversely with the depth ingestion via the oral cavity but diffusive uptake. T. tubifex of the oxic–anoxic interface. In June 2016, this interface can actively take up short-chain organic acids, such as acetate was ≤ 1 mm at all stations in Lake Baldegg and ≤ 2 mm and propionate, through their body wall (Hipp et al., 1985; at those in Lake Greifen, while methanogenesis occurred in Sedlmeier and Hoffmann, 1989). The subsequent respiration the top 1 cm of sediment (Fiskal et al., 2019). Thus, condi- of these organic acids can account for up to 40 % of T. tubifex tions were potentially well-suited for the strong enrichment energy turnover (Hipp et al., 1986). Other species of tubifi- of methane-cycling microorganisms. It is possible that the cids take up amino acids through the body wall (Brinkhurst

Biogeosciences, 18, 4369–4388, 2021 https://doi.org/10.5194/bg-18-4369-2021 A. Fiskal et al.: Carbon sources of benthic fauna 4383 and Chua, 1969). Tubificid body walls are also permeable to fit hosts under low O2 conditions given that succinate is the dissolved gases, which is why tubificids acquire O2 by undu- main intermediate during anaerobic metabolism of tubificids lating movements of their tail ends in oxic water above sed- (Seuß et al., 1983). ZOTU8 falls into the facultatively aer- iments (Brinkhurst, 1996). Permeability to gases could also obic β-proteobacterial genus Deefgea (order Neisseriales), provide energy if, for example, methane or H2 diffusing from members of which ferment carbohydrates to organic acids pore water into worms supports symbiotic microorganisms. (Stackebrandt et al., 2007) and could benefit hosts as pro- Matching the slight increase in methane-derived carbon in posed for Fusobacteriaceae. The α-proteobacterial ZOTU4 oligochaetes from eutrophic lakes, we observe higher con- falls into the family Holosporaceae, members of which are tributions of pmoA in oligochaetes compared to surround- obligately intracellular, potentially parasitic symbionts of cil- ing sediment. Assuming that these pmoA belong to living iates (Santos and Massard, 2014). ZOTU4 could derive from methane-oxidizing bacteria, movement of oligochaetes be- commensal ciliates, which often inhabit guts of freshwa- tween methane-rich, deeper layers and the oxic sediment sur- ter oligochaetes (Falls, 1972). Alternatively, given the high face could favor their growth and result in an endosymbi- percentage of 16S reads in one specimen (93 %), a novel otic relationship. The potential for annelid hindguts to make form of (intracellular) symbiosis with tubificids cannot be excellent microbial habitats was previously demonstrated in discounted. Similarly unclear is the host relationship with the polychaete Abarenicola vagabunda (Plante et al., 1989). ZOTU199, which belongs to the candidate phylum Parcubac- How this methane-derived carbon would be assimilated is teria of the Candidate Phyla Radiation (Brown et al., 2015). unclear, however. Potential mechanisms include uptake of Members of this phylum have been retrieved from diverse, organic intermediates of methane oxidation, e.g., methanol, mostly anoxic habitats, have genes linked to carbohydrate through the hindgut or ingestion of faeces that are enriched fermentation, and have been implicated in ectosymbiotic or in methane-oxidizing bacteria. parasitic lifestyles (Wrighton et al., 2012; Nelson and Ste- The overwhelming majority of microbial DNA from gen, 2015). The remaining ZOTUs fall into an unclassified oligochaetes, however, belongs to Bacteria, often single ZO- genus-level subcluster of β-Proteobacteria (ZOTU6; order TUs, which are not linked to methane oxidation. In 22 Rhodocyclales) and an unclassified order-level cluster of ε- of the 30 specimens sequenced, a single ZOTU accounted Proteobacteria (ZOTU9). Based on existing knowledge, it for > 50 % of the total reads (Table S8 in the Supplement). is not possible to infer the potential roles of these ZOTUs In 15 specimens, this dominant ZOTU belonged to a sin- within their hosts. gle genus-level cluster of unclassified Fusobacteriaceae (Fu- sobacteriaceae Cluster I) that was previously found in earth- 4.4 Potential host–microorganism interactions in worm and aquatic vertebrate intestines, anaerobic sediments, chironomid larvae bioreactors, soil, and diverse water samples (Fig. S7A in the Supplement). The high percentages of this cluster are Similar to tubificids, most chironomid larvae (12 of 19 se- striking considering that Fusobacteriaceae account for on quenced specimens) are dominated by single ZOTUs (Ta- average only 0.01 ± 0.02 % of total 16S reads in the sur- ble S8 in the Supplement). Interestingly, more specimens rounding sediments. All cultivated members of Fusobacte- are dominated by single ZOTUs in Lake Lucerne (9 of 10) riaceae are anaerobes that fermentatively degrade polymeric than in Lake Zurich (three of seven) or Lake Baldegg (zero organic compounds, in particular proteins and carbohydrates, of two), suggesting that the frequency and/or importance of with acetate, butyrate, and other short-chain organic acids as these associations is linked to trophic state. These single main end products (Olsen, 2014). Given previous evidence dominant ZOTUs are mostly Proteobacteria (10 of 12; α, β, for the preference of proteinaceous organic matter by tubifi- and γ classes). In addition, single specimens were dominated cids (de Valk et al., 2017), these Fusobacteriaceae could be by the same unclassified Fusobacteriaceae (Fusobacteriaceae primary degraders of proteins within the digestive tracts of Cluster I) that dominate tubificids and an unclassified sister oligochaetes. This relationship could be mutually beneficial, group of Bacteroides, Bacteroidetes), which we call “Unclas- commensal, or parasitic. A mutually beneficial relationship sified Wastewater and Gut Group” based on reported occur- could entail symbionts gaining energy by fermenting pro- rences. teins that are not digestible by the host and through the host Two proteobacterial groups most commonly dominate chi- respiring the resulting fermentation products. ronomid larvae. ZOTU2 of the α-proteobacterial genus Wol- The remaining seven dominant ZOTUs belong to the phyla bachia (Rickettsiales) dominates four specimens. Members Proteobacteria (α, β, and ε classes), Bacteroidetes, and Par- of this genus are widespread intracellular symbionts of in- cubacteria (Fig. S8, Table S8 in the Supplement). ZOTU18 sects whose relationships with their hosts range from par- falls into the anaerobic ε-proteobacterial genus Wolinella (or- asitic to mutualistic (Correa and Ballard, 2016), though, to der Campylobacterales), isolates of which use H2 or formate our knowledge, dietary contributions have not been demon- as electron donors and fumarate and nitrate as electron ac- strated. ZOTU3 and ZOTU21 of the γ -proteobacterial genus ceptors (Tanner and Paster, 1992). Succinate is the main end Aeromonas dominate three specimens. Members of this fac- product of fumarate reduction by Wolinella and could bene- ultatively anaerobic genus are widespread in aquatic habi- https://doi.org/10.5194/bg-18-4369-2021 Biogeosciences, 18, 4369–4388, 2021 4384 A. Fiskal et al.: Carbon sources of benthic fauna tats (Huys, 2014) and were previously found in aquatic in- Code and data availability. Biogeochemical data will be made vertebrates, including chironomid larvae (Eller et al., 2007). available after publication of the manuscript on Pangaea. The se- Aeromonads can ferment carbohydrates to organic acids, quence data have been deposited at DDBJ/EMBL/GenBank under which might supplement the diet of chironomid larvae, but the accession KDVU00000000. The version described in this paper they have also been shown to degrade the egg masses of is the first version, KDVU01000000. chironomids (Senderovich et al., 2008). Similar functions, ranging from mutualistic to detrimental, are likely for the γ - proteobacterial genus Serratia (γ -Proteobacteria) and an un- Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/bg-18-4369-2021-supplement. classified cluster of Moraxellaceae (Pseudomonadales) and for the Unclassified Wastewater and Gut Group. ZOTUs of these groups each dominate one larval specimen (ZOTU11, Author contributions. AF, AM, EA, LD, XH, RZ, LL, ND, CJS, ZOTU26, and ZOTU28, respectively). All three groups de- SMB, and MAL helped with sample collection and/or measure- grade carbohydrates anaerobically (Serratia, Bacteroidetes) ments. AF, SMB, and MAL substantially contributed to the interpre- or aerobically/facultatively anaerobically (Moraxellaceae) to tation of data. AF and MAL wrote the manuscript. MAL designed organic acids, which may provide energy to larvae but can the study and acquired the funding for the project. All authors com- also be pathogenic or mutualistic in ways unrelated to diet mented on and approved the final version of the manuscript. (Grimont and Grimont, 2006; Sabri et al., 2011; Teixeira and Merquior, 2014; Wexler, 2014). The remaining ZO- TUs belong to unclassified genus-level subclusters of β- Competing interests. The authors declare that they have no conflict proteobacterial Rhodocyclales (ZOTU6) and Burkholderi- of interest. ales (ZOTU12). Due to the very diverse ecophysiologies of Rhodocyclales and Burkholderiales the potential roles of these ZOTUs within their hosts are highly uncertain. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 5 Conclusions

Our study indicates clear changes in lacustrine sedimen- Acknowledgements. We thank the Genetic Diversity Centre (GDC) tary macrofaunal communities with increasing trophic state, at ETH Zurich for help with next generation sequencing and anal- including a shift in dominance from chironomid larvae to ysis. We are thankful to AquaDiptera and AquaLytis for the taxo- tubificid oligochaetes. Carbon isotopic and genetic analy- nomic analysis of the macrofauna samples, especially to Susanne Michiels and Ute Michels. We thank Madalina Jaggi for technical ses show that, independent of faunal group or trophic state, 13 detritus-derived organic carbon rather than methane-derived support of macrofaunal C analysis and Serge Robert for technical support during δ13C-methane measurements. carbon is the main carbon source of these animals. Yet, the exact carbon sources remain unclear and may include actual detritus, detrital carbon-assimilating microorganisms, and/or Financial support. This research has been supported by waste products of microbial detritus degradation. Thus bac- the Schweizerischer Nationalfonds zur Förderung der Wis- terial symbionts that are abundant within tubificids and chi- senschaftlichen Forschung (grant no. 205321_163371). ronomids but rare in surrounding sediment could be im- portant. Known carbon sources of these symbionts pro- vide potential clues to predominant tubificid and larval food Review statement. This paper was edited by Aninda Mazumdar and sources. Given that most tubificid specimens are dominated reviewed by two anonymous referees. by known protein-degrading bacteria (Fusobacteriaceae), se- lective feeding on protein-rich organic matter fractions, such as microbial cells, is likely for these specimens. Similarly, given that half of the dominant ZOTUs in chironomid lar- vae belong to carbohydrate-degrading taxa (Aeromonas, Ser- References ratia, Moraxellaceae, Bacteroidales), preferential feeding on algal detritus in surface sediments is plausible for these chi- Anderson, N. J., Dietz, R. D., and Engstrom, D. R.: Land- ronomid taxa. Though more research is needed, both macro- use change, not climate, controls organic carbon burial in faunal groups may benefit from their endosymbionts through lakes, P. Roy. Soc. B-Biol. Sci., 280, ARTN 20131278 the production of short-chain organic acids, which can be https://doi.org/10.1098/rspb.2013.1278, 2013. taken up through the hindgut wall and subsequently used for Anderson, N. J., Bennion, H., and Lotter, A. F.: Lake eu- energy conservation or biosynthesis. trophication and its implications for organic carbon seques- tration in Europe, Global Change Biol., 20, 2741–2751, https://doi.org/10.1111/gcb.12584, 2014.

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