Litter decomposition fuels food webs in forest streams. Thus, understanding the effects of water nutrients and temperature on litter decomposition and decomposers is key for predicting stream functioning under environmental change. We incubated leaf litter of woody species in streams to assess the effects of nutrient concentrations and temperature on microbial-driven litter decomposition and microbial activities. There was a hump-shaped relationship between biotic variables and nitrate concentrations and a U-shaped relationship between biotic variables and temperature. The stimulatory effect of high nutrient availability was limited by low temperature, while high nutrient availability alleviated low-temperature effects on litter decomposition and decomposers.
","acknowledgements":"We thank Ana Pereira and Ana Mafalda Cruz for technical work, IMAR – Institute of Marine Research for ergosterol quantification, and INOVA – Instituto de Inovação Tecnológica dos Açores for water nutrient determinations.
","authors":[{"affiliations":["MARE – Marine and Environmental Sciences Centre","ARNET – Aquatic Research Network","University of Coimbra"],"departments":["","","Department of Life Sciences"],"credit":["conceptualization","fundingAcquisition","project","resources","dataCuration","formalAnalysis","validation","investigation","writing_originalDraft"],"email":"veronica@ci.uc.pt","firstName":"Verónica","lastName":"Ferreira","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-7688-2626"},{"affiliations":["University of the Azores","CIBIO – Research Centre in Biodiversity and Genetic Resources","InBIO Associate Laboratory","BIOPOLIS Program in Genomics, Biodiversity and Land Planning – UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands"],"departments":["","","",""],"credit":["investigation","writing_reviewEditing"],"email":"pedro.mv.raposeiro@uac.pt","firstName":"Pedro M.","lastName":"Raposeiro","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7461-0851"},{"affiliations":["University of the Azores","CIBIO – Research Centre in Biodiversity and Genetic Resources","InBIO Associate Laboratory","BIOPOLIS Program in Genomics, Biodiversity and Land Planning – UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands"],"departments":["","","",""],"credit":["project","resources","writing_reviewEditing"],"email":"vitor.mc.goncalves@uac.pt","firstName":"Vítor","lastName":"Gonçalves","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-5737-296X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/fa9c8bb02525601704721d091b3e8c13.csv"},"extendedData":[],"funding":"This study was financed by the European Fund for Economic and Regional Development (FEDER) through the Program Operational Factors of Competitiveness (COMPETE; FCOMP-01-0124-FEDER-041055) and by National Funds through the Portuguese Foundation of Science and Technology (FCT; SFRH/BPD/76482/2011, EXPL/AAG-GLO/0189/2013, UID/MAR/04292/2013, SFRH/BPD/99461/2014, IF/00129/2014, CEECIND/02484/2018, UIDB/50027/2020, UID/50027, UID/50027/2025, UID/04292/2025, and LA/P/0069/2020).
","image":{"url":"https://portal.micropublication.org/uploads/da86d51ca26771b39443630e5a8ae417.jpg"},"imageCaption":"Figure. Relationships between biotic variables [microbial-driven litter decomposition rates (A, B), fungal biomass (C, D), and cumulative conidial production (E, F)] associated with leaf litter of three woody species (Acacia melanoxylon, Clethra arborea, and Pittosporum undulatum) and water nitrate concentration (left column) and temperature (right column) in six Azorean streams.
Table. Relationships between biotic variables (microbial-driven litter decomposition rates, fungal biomass, and cumulative conidial production) associated with leaf litter of three woody species (Acacia melanoxylon, Clethra arborea, and Pittosporum undulatum) and water nitrate concentration and temperature in six Azorean streams. In most cases, data were fitted to a quadratic function: y = ax2 + bx + c, where y is the biotic variable, x is the abiotic variable and a, b, and c are constants; a > 0 indicates a U-shaped curve and a < 0 indicates a hump-shaped curve. The curve vertex (i.e. the minimum value in a U-shaped curve or the maximum value in a hump-shaped curve) and the coefficient of determination of the model (R2) are shown. The relationship between decomposition rates and nitrate concentration for C. arborea was fitted to a linear model (y = 0.00001x + 0.0078).
","imageTitle":"Relationships between biotic variables associated with leaf litter decomposition of three woody species and water nitrate concentration and temperature in six Azorean streams
","methods":"Leaf litter incubation was carried out in six streams on São Miguel island, Azores archipelago, North Atlantic Ocean. Streams had similar geomorphology, were not visibly affected by direct human activities, were slightly alkaline (pH 7.7 – 8.1) and well oxygenated (8.4 – 9.1 mg O2/L), and varied in water temperature (13.4 – 15.6 ºC) and nutrient concentrations (28 – 1033 μg NO3–/L and 30 – 175 μg PO43–/L). Water temperature was recorded hourly over the 56-days incubation period during Spring and Summer 2014 using data loggers (Hobo Pendant UA-001-08, Onset Computer Corp., Massachusetts, USA) and hourly values were used to estimate daily means, which were averaged for each stream. On five occasions, stream water was filtered (fiberglass filters, 47-mm diameter, GF/C, Whatman, GE Healthcare Europe GmbH, Little Chalfont, UK) and used to determine nitrate concentration (ion chromatography; APHA 1995). A detailed description of the study area and streams is provided in Ferreira et al. (2016).
Air-dry leaves of three evergreen woody species commonly found in the riparian vegetation of Azorean streams were used to provide a gradient of litter palatability, in increasing order: Acacia melanoxylon (R. Br.) < Clethra arborea (Aiton) < Pittosporum undulatum (Vent.) (Ferreira et al., 2016). Leaves were weighed (2.90 – 3.10 g) and enclosed in fine-mesh bags (10 × 15 cm, 0.5-mm mesh), which prevent invertebrate access and where decomposition is mostly driven by microbial decomposers. Twelve litter bags per species were deployed in each stream on 11 or 12 June, 2014. Three replicate litter bags were recovered from each stream after 7, 21, 35 and 56 days, and transported cold to the laboratory. In the laboratory, litter was used to determine fungal biomass (Gessner 2020), conidia production by aquatic hyphomycetes (Bärlocher 2020), and litter mass remaining (Ferreira et al., 2016). For determination of litter mass remaining, litter was oven-dried (70 ºC, 48 h) and weighed to assess dry mass (DM) remaining. DM remaining was ignited (500 ºC, 8 h) and weighed to assess ash mass. Ash-free dry mass (AFDM) remaining was estimated as the difference between DM and ash mass, and the fraction of AFDM remaining was estimated as the ratio between AFDM remaining and initial AFDM. Initial AFDM of samples was estimated as the product of initial air-dry mass by a conversion factor derived from extra sets of five fine-mesh bags per species. These extra bags, were prepared as the samples, taken to the field on day 0, immersed in water for ~ 10 min, and returned to the laboratory for determination of DM and AFDM as described above. The conversion factor was estimated as the ratio between initial AFDM and initial air-dry mass. Fraction AFDM remaining was used to estimate exponential decomposition rates (k, /days) for each species and stream as the slope of a linear regression between ln(fraction of AFDM remaining) and time (days).
The relationships between biological variables [microbial-driven litter decomposition rates, fungal biomass (average across sampling dates), and cumulative conidial production (sum of daily values that were estimated by linear interpolation of the values of the adjacent sampling dates)] of the three leaf litter species and abiotic variables [nitrate concentration (average across sampling dates) and water temperature (average across 56 days)] in six streams were analyzed by fitting the data to a quadratic function. The relationship between litter decomposition rates and nitrate concentration for C. arborea was fitted to a linear model. Models were derived using Statistica 7 software (StatSoft Inc., Tulsa, Oklahoma, USA).
","reagents":"","patternDescription":"Leaf litter decomposition is a key ecosystem process in forest streams, where litter inputs from the riparian vegetation are the main source of carbon and nutrients for aquatic food webs (Wallace et al., 1997). In island streams, where invertebrate shredders are generally rare (Benstead et al., 2009; MacKenzie et al., 2013; Raposeiro et al., 2014), litter decomposition is mostly driven by microbial decomposers, aquatic hyphomycetes in particular (Benstead et al., 2009; Larned 2000; MacKenzie et al., 2013; Raposeiro et al., 2014; Ferreira et al., 2016). Microbial decomposers are highly responsive to water temperature, with increases in temperature within organisms’ thermal ranges stimulating metabolism rates, and consequently the mineralization of litter carbon into CO2 through respiration (Ferreira and Chauvet 2011; Pérez et al., 2023). Microbial decomposers are also highly sensitive to nutrient availability, with increases in dissolved nutrient concentrations stimulating the use of litter as a source of carbon (Gulis and Suberkorpp 2003; Ferreira and Chauvet 2011). Consequently, increases in water temperature and nutrient availability generally translate into accelerated microbial-driven litter decomposition rates (Ferreira and Chauvet 2011; Fernandes et al., 2014). However, in island streams, variation in water temperature and nutrient concentrations, which may occur over a short distance due to steep changes in elevation and geology, do not necessary occur in the same direction, which complicates predictions of litter decomposition rates.
In this study, we incubated leaf litter of three woody species [Acacia melanoxylon (R. Br.), Clethra arborea (Aiton), and Pittosporum undulatum (Vent.)] in 0.5-mm mesh bags, for up to 56 days (with four sampling dates), in six streams on São Miguel island, North Atlantic Ocean, to assess microbial-driven litter decomposition rates, fungal biomass, and conidial production by aquatic hyphomycetes. The temporal dynamics of the biotic variables over the incubation period can be found in Ferreira et al. (2016), while here we report on the relationships between the biotic variables and water nitrate concentrations and temperature to assess the interaction between these two abiotic variables in moderating biotic responses under natural settings.
The relationship between biotic variables and nitrate concentration followed a hump-shaped curve, with maximum decomposition rates (across species) of 0.0082 – 0.0460/d estimated at 500 – 714 μg NO3–/L, maximum fungal biomass of 50 – 73 mg/g AFDM estimated at 623 – 663 μg NO3–/L, and maximum cumulative conidial production of 3.8 million – 6.9 million conidia/5 discs estimated at 565 – 639 μg NO3–/L (Figure, Table). The relationship between decomposition rates and nitrate concentration was linear for C. arborea (Figure, Table). The relationship between biotic variables and water temperature followed a U-shaped curve, with minimum decomposition rates (across species) of 0.0047 – 0.0259/d estimated at 14.0 – 14.7 ºC, minimum fungal biomass of 15 – 42 mg/g AFDM estimated at 14.3 – 14.4 ºC, and minimum cumulative conidial production of –28219 – 384022 conidia/5 discs estimated at 14.0 – 14.3 ºC (Figure, Table).
Litter decomposition and associated microbial activities were stimulated when nutrient availability and water temperature increased simultaneously across the group of streams including Lom1, Lom2, Lom 4, AFG1, and AFG2, as anticipated (Ferreira and Chauvet 2011; Fernandes et al., 2014). This stimulation was more evident for the fast-decomposing P. undulatum litter than for the other two species. This suggests that litter is more sensitive to increases in nutrient availability and temperature at more advanced stages of decomposition, which can be due to better microbial colonization of the litter and to the litter increased recalcitrance and thus higher dependence of decomposers on dissolved nutrients. Consistent with this interpretation, other studies found stronger differences in litter mass remaining across streams mostly at later sampling dates (Gulis and Suberkropp 2003; Ferreira et al., 2016, 2021).
However, when an increase in nutrient concentrations was accompanied by a decrease in water temperature (stream Lom3), litter decomposition and associated microbial activities were lower compared with what was expected from the increase in nutrient concentrations alone (see hump-shaped relationships) and higher when compared with what was expected from the decrease in water temperature alone (see U-shaped relationships). This suggests that water temperature was the limiting factor for maximum litter decomposition rates and associated microbial activities in this stream. Prescott (2010) has proposed the “concept of thresholds” for the control of litter decomposition in terrestrial ecosystems, where “inadequate conditions with respect to any of these factors (in her case, temperature, moisture, lignin concentration, and nutrient concentrations) can divert litter into the realm of slower decomposition, regardless of the adequacy of the other factors”. This concept can also apply to litter decomposition in streams. Interestingly is that the role of temperature as a limiting factor was observed when comparing temperatures that cannot be considered extreme (13.4ºC – 15.5ºC) and are within a short interval (2.1ºC). Contrarily, higher nutrient availability may alleviate the effect of low water temperature as an increase in nutrient availability stimulated litter decomposition and associated microbial activities at lower temperature, although generally not at levels observed when temperature was higher. This agrees with Fernandes et al. (2014), who showed that higher nitrogen concentrations are needed to achieve maximum microbial activities at lower temperature.
","references":[{"reference":"Bärlocher F. 2020. Sporulation by Aquatic Hyphomycetes. Methods to Study Litter Decomposition : 241-245.
","pubmedId":"","doi":"10.1007/978-3-030-30515-4_26"},{"reference":"Benstead JP, March JG, Pringle CM, Ewel KC, Short JW. 2009. Biodiversity and ecosystem function in species-poor communities: community structure and leaf litter breakdown in a Pacific island stream. Journal of the North American Benthological Society 28: 454-465.
","pubmedId":"","doi":"10.1899/07-081.1"},{"reference":"Fernandes I, Seena S, Pascoal C, Cássio F. 2014. Elevated temperature may intensify the positive effects of nutrients on microbial decomposition in streams. Freshwater Biology 59: 2390-2399.
","pubmedId":"","doi":"10.1111/fwb.12445"},{"reference":"Ferreira V, Chauvet E. 2010. Synergistic effects of water temperature and dissolved nutrients on litter decomposition and associated fungi. Global Change Biology 17: 551-564.
","pubmedId":"","doi":"10.1111/j.1365-2486.2010.02185.x"},{"reference":"Ferreira V, Raposeiro PM, Pereira A, Cruz AM, Costa AC, Graça MAS, Gonçalves V. 2016. Leaf litter decomposition in remote oceanic island streams is driven by microbes and depends on litter quality and environmental conditions. Freshwater Biology 61: 783-799.
","pubmedId":"","doi":"10.1111/fwb.12749"},{"reference":"Ferreira V, Silva J, Cornut J, Sobral O, Bachelet Q, Bouquerel J, Danger M. 2021. Organic-matter decomposition as a bioassessment tool of stream functioning: A comparison of eight decomposition-based indicators exposed to different environmental changes. Environmental Pollution 290: 118111.
","pubmedId":"","doi":"10.1016/j.envpol.2021.118111"},{"reference":"Gessner MO. 2020. Ergosterol as a Measure of Fungal Biomass. Methods to Study Litter Decomposition : 247-255.
","pubmedId":"","doi":"10.1007/978-3-030-30515-4_27"},{"reference":"Gulis V, Suberkropp K. 2002. Leaf litter decomposition and microbial activity in nutrient‐enriched and unaltered reaches of a headwater stream. Freshwater Biology 48: 123-134.
","pubmedId":"","doi":"10.1046/j.1365-2427.2003.00985.x"},{"reference":"Larned ST. 2000. Dynamics of coarse riparian detritus in a Hawaiian stream ecosystem: a comparison of drought and post-drought conditions. Journal of the North American Benthological Society 19: 215-234.
","pubmedId":"","doi":"10.2307/1468066"},{"reference":"MacKenzie RA, Wiegner TN, Kinslow F, Cormier N, Strauch AM. 2013. Leaf-litter inputs from an invasive nitrogen-fixing tree influence organic-matter dynamics and nitrogen inputs in a Hawaiian river. Freshwater Science 32: 1036-1052.
","pubmedId":"","doi":"10.1899/12-152.1"},{"reference":"Pérez J, Cornejo A, Alonso A, Guerra A, García G, Nieto C, et al., Boyero. 2023. Warming overrides eutrophication effects on leaf litter decomposition in stream microcosms. Environmental Pollution 332: 121966.
","pubmedId":"","doi":"10.1016/j.envpol.2023.121966"},{"reference":"Prescott CE. 2010. Litter decomposition: what controls it and how can we alter it to sequester more carbon in forest soils?. Biogeochemistry 101: 133-149.
","pubmedId":"","doi":"10.1007/s10533-010-9439-0"},{"reference":"Raposeiro PM, Martins GM, Moniz I, Cunha A, Costa AC, Gonçalves V. 2014. Leaf litter decomposition in remote oceanic islands: The role of macroinvertebrates vs. microbial decomposition of native vs. exotic plant species. Limnologica 45: 80-87.
","pubmedId":"","doi":"10.1016/j.limno.2013.10.006"},{"reference":"Wallace JB, Eggert SL, Meyer JL, Webster JR. 1997. Multiple Trophic Levels of a Forest Stream Linked to Terrestrial Litter Inputs. Science 277: 102-104.
","pubmedId":"","doi":"10.1126/science.277.5322.102"}],"title":"Water nutrient concentration and temperature control microbial-driven decomposition of leaf litter in oceanic island streams
","reviews":[{"reviewer":{"displayName":"Matthew Wilson"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"644775db-47ac-4bf9-8508-38bb3b9ca1ad","decision":"revise","abstract":"Litter decomposition fuels food webs in forest streams. Thus, understanding the effects of water nutrients and temperature on litter decomposition and decomposers is key for predicting stream functioning under environmental change. We incubated leaf litter of woody species in streams to assess the effects of nutrient concentrations and temperature on microbial-driven litter decomposition and microbial activities. The relationship between biotic and abiotic variables was generally best described by a quadratic model: there was a hump-shaped relationship between biotic variables and nitrate concentrations and a U-shaped relationship between biotic variables and temperature. The stimulatory effect of high nutrient availability was limited by low temperature, while high nutrient availability alleviated low-temperature effects on litter decomposition and decomposers.
","acknowledgements":"We thank Ana Pereira and Ana Mafalda Cruz for technical work, IMAR – Institute of Marine Research for ergosterol quantification, and INOVA – Instituto de Inovação Tecnológica dos Açores for water nutrient determinations.
","authors":[{"affiliations":["MARE – Marine and Environmental Sciences Centre","ARNET – Aquatic Research Network","University of Coimbra"],"departments":["","","Department of Life Sciences"],"credit":["conceptualization","fundingAcquisition","project","resources","dataCuration","formalAnalysis","validation","investigation","writing_originalDraft"],"email":"veronica@ci.uc.pt","firstName":"Verónica","lastName":"Ferreira","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-7688-2626"},{"affiliations":["University of the Azores","CIBIO – Research Centre in Biodiversity and Genetic Resources","InBIO Associate Laboratory","BIOPOLIS Program in Genomics, Biodiversity and Land Planning – UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands"],"departments":["","","",""],"credit":["investigation","writing_reviewEditing"],"email":"pedro.mv.raposeiro@uac.pt","firstName":"Pedro M.","lastName":"Raposeiro","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7461-0851"},{"affiliations":["University of the Azores","CIBIO – Research Centre in Biodiversity and Genetic Resources","InBIO Associate Laboratory","BIOPOLIS Program in Genomics, Biodiversity and Land Planning – UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands"],"departments":["","","",""],"credit":["project","resources","writing_reviewEditing"],"email":"vitor.mc.goncalves@uac.pt","firstName":"Vítor","lastName":"Gonçalves","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-5737-296X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/fa9c8bb02525601704721d091b3e8c13.csv"},"extendedData":[{"description":"Revised manuscript
","doi":null,"resourceType":"Text","name":"Ferreira&al_microPubBiol_paper rev clean.docx","url":"https://portal.micropublication.org/uploads/ff142d049da532f80b04dc57d6b0a928.docx"}],"funding":"This study was financed by the European Fund for Economic and Regional Development (FEDER) through the Program Operational Factors of Competitiveness (COMPETE; FCOMP-01-0124-FEDER-041055) and by National Funds through the Portuguese Foundation of Science and Technology (FCT; SFRH/BPD/76482/2011, EXPL/AAG-GLO/0189/2013, UID/MAR/04292/2013, SFRH/BPD/99461/2014, IF/00129/2014, CEECIND/02484/2018, UIDB/50027/2020, UID/50027, UID/50027/2025, UID/04292/2025, and LA/P/0069/2020).
","image":{"url":"https://portal.micropublication.org/uploads/da86d51ca26771b39443630e5a8ae417.jpg"},"imageCaption":"Figure. Relationships between biotic variables [microbial-driven litter decomposition rates (A, B), fungal biomass (C, D), and cumulative conidial production (E, F)] associated with leaf litter of three woody species (Acacia melanoxylon, Clethra arborea, and Pittosporum undulatum) and water nitrate concentration (left column) and temperature (right column) in six Azorean streams during Spring/Summer 2014. Stream labels: Lom1 – Lom4, streams located in Lombadas valley; AFG1 and AFG2, tributaries of Fogo lagoon.
Table. Relationships between biotic variables (microbial-driven litter decomposition rates, fungal biomass, and cumulative conidial production) associated with leaf litter of three woody species (Acacia melanoxylon, Clethra arborea, and Pittosporum undulatum) and water nitrate concentration and temperature in six Azorean streams. In most cases, data were fitted to a quadratic function: y = ax2 + bx + c, where y is the biotic variable, x is the abiotic variable and a, b, and c are constants; a > 0 indicates a U-shaped curve and a < 0 indicates a hump-shaped curve. The curve vertex (i.e. the minimum value in a U-shaped curve or the maximum value in a hump-shaped curve) was calculated as: x = – (b / 2a) and y = ax2 + bx + c. The coefficient of determination of the models (R2) is also shown. The relationship between decomposition rates and nitrate concentration for C. arborea was fitted to a linear model (y = 0.00001x + 0.0078).
Note: The table loses formats when uploaded as CSV. Can I submit the table in a different format (Excel, Word) to keep formats?
","imageTitle":"Relationships between biotic variables associated with leaf litter decomposition of three woody species and water nitrate concentration and temperature in six Azorean streams
","methods":"Leaf litter incubation was carried out in six small streams in the central massif of São Miguel island, Azores archipelago, North Atlantic Ocean. Four streams were tributaries of Ribeira Grande stream in Lombadas valley (Lom1 – Lom4) and two streams were tributaries of Fogo Lagoon (AFG1 and AFG2). Streams had similar geomorphology, were not visibly affected by direct human activities, were slightly alkaline (pH 7.7 – 8.1) and well oxygenated (8.4 – 9.1 mg O2/L), and varied in water temperature (13.4 – 15.6 ºC) and nutrient concentrations (28 – 1033 μg NO3–/L and 30 – 175 μg PO43–/L). Water temperature was recorded hourly over the 56-days incubation period during Spring and Summer 2014 using data loggers (Hobo Pendant UA-001-08, Onset Computer Corp., Massachusetts, USA) and hourly values were used to estimate daily means, which were averaged for each stream. On five occasions, stream water was filtered (fiberglass filters, 47-mm diameter, GF/C, Whatman, GE Healthcare Europe GmbH, Little Chalfont, UK) and used to determine nitrate concentration (ion chromatography; APHA 1995). A detailed description of the study area and streams is provided in Ferreira et al. (2016).
Air-dried leaves of three evergreen woody species commonly found in the riparian vegetation of Azorean streams were used to provide a gradient of litter palatability, in increasing order: Acacia melanoxylon (R. Br.) < Clethra arborea (Aiton) < Pittosporum undulatum (Vent.) (Ferreira et al., 2016). Leaves were weighed (2.90 – 3.10 g) and enclosed in fine-mesh bags (10 × 15 cm, 0.5-mm mesh), which prevent invertebrate access and where decomposition is mostly driven by microbial decomposers. Twelve litter bags per species were deployed in each stream on 11 or 12 June, 2014. Three replicate litter bags were recovered from each stream after 7, 21, 35 and 56 days, and transported cold to the laboratory. In the laboratory, litter was rinsed with distilled water and two sets of five leaf discs were extracted with a cork borer (12-mm diameter): one set was frozen at – 18 ºC for later determination of fungal biomass (Gessner 2020) and one set was used fresh to induce conidia production by aquatic hyphomycetes (Bärlocher 2020). The bulk litter mass was used to determine litter mass remaining (Ferreira et al., 2016).
For determination of fungal biomass, five frozen leaf discs were lyophilized (LY3TE, Snijders Scientific, Tilburg, The Netherlands), weighed (Kern 870, Kern & Sohn GmbH, Balingen, Germany) to determine dry mass, and incubated in alkaline methanol (8 g KOH/L, 30 min at 80 ºC) to extract ergosterol. The extract was purified using solid phase extraction cartridges (Waters Sep-Pak® Vac RC tC18 cartridges; Waters Corp., Massachusetts, USA) and eluted with isopropanol. Ergosterol was quantified by high-performance liquid chromatography (Dionex DX-120, California, USA), using a Thermo Scientific Syncronis C18 column and a Thermo Universal Uniguard holder 4/4.6 mm ID3 + Syncronis C18 drop in guard pre-column (Thermo, Waltham, Massachusetts, USA); the mobile phase was 100% methanol, kept at 33ºC and flowing at 1.4 mL/min. Ergosterol was detected by reading absorbance at 282 nm with a UV detector and absorbance was converted into ergosterol concentration using a standard curve of ergosterol in isopropanol. Ergosterol concentration was converted into fungal biomass considering 5.5 mg ergosterol/mg fungal dry mass (Gessner and Chauvet 1993) and fungal biomass was expressed as mg/g AFDM, averaged across sampling dates.
Conidia production by aquatic hyphomycetes was determined after incubating five fresh leaf discs in 25 mL of filtered stream water (48h at 13 ºC, 10h light:14 h dark photoperiod, and 100 rpm). The conidia suspension was preserved with 2 mL of formalin and leaf discs were processed as described below for determination of discs ash-free dry mass. Suspensions were homogenized with 150 mL Triton X-100 and a magnetic stirring bar, and aliquots were filtered (nitrocellulose filters, 25-mm diameter, 5-mm pore size; Sartorius Stedim Biotech GmhH, Göttingen, Germany). Filters were stained with trypan blue in lactic acid and mounted on a slide. Conidia were counted at 320´ magnification (DM1000 microscope, Leica, Wetzlar, Germany). Rates of conidia production were expressed as no. conidia/mg leaf AFDM/day, and cumulative (total) conidial production over the incubation period was estimated as the sum of daily values, which were derived by linear interpolation of the values of the adjacent sampling dates, and expressed as no. conidia/5 discs.
For determination of litter mass remaining, litter (after extraction of leaf discs) was oven-dried (70 ºC, 48 h) and weighed (Kern 870, Kern & Sohn GmbH, Balingen, Germany) to assess dry mass (DM) remaining. DM remaining was ignited (500 ºC, 8 h) and weighed to assess ash mass. Ash-free dry mass (AFDM) remaining was estimated as the difference between DM and ash mass (after accounting for the extracted discs), and the fraction of AFDM remaining was estimated as the ratio between AFDM remaining and initial AFDM. Initial AFDM of samples was estimated as the product of initial air-dry mass by a conversion factor derived from extra sets of five fine-mesh bags per species. These extra bags, were prepared as the samples, taken to the field on day 0, immersed in water for ~ 10 min, and returned to the laboratory for determination of DM and AFDM as described above. The conversion factor was estimated as the ratio between initial AFDM and initial air-dry mass. Fraction AFDM remaining across the incubation times was used to estimate the overall exponential decomposition rate (k, /days) for each species and stream as the slope of a linear regression between ln(fraction of AFDM remaining) and time (days).
The relationships between biological variables (microbial-driven litter decomposition rates, average fungal biomass, and cumulative conidial production) of the three leaf litter species and abiotic variables [nitrate concentration (average across sampling dates) and water temperature (average across 56 days)] in six streams were analyzed by fitting the data to a quadratic function. The relationship between litter decomposition rates and nitrate concentration for C.arborea was fitted to a linear model. Models were derived using Statistica 7 software (StatSoft Inc., Tulsa, Oklahoma, USA).
","reagents":"","patternDescription":"Leaf litter decomposition is a key ecosystem process in forest streams, where litter inputs from the riparian vegetation are the main source of carbon and nutrients for aquatic food webs (Wallace et al., 1997). In island streams, where invertebrate shredders are generally rare (Benstead et al., 2009; MacKenzie et al., 2013; Raposeiro et al., 2014), litter decomposition is mostly driven by microbial decomposers, aquatic hyphomycetes in particular (Benstead et al., 2009; Larned 2000; MacKenzie et al., 2013; Raposeiro et al., 2014; Ferreira et al., 2016). Microbial decomposers are highly responsive to water temperature, with increases in temperature within organisms’ thermal ranges stimulating metabolism rates, and consequently the mineralization of litter carbon into CO2 through respiration (Ferreira and Chauvet 2011; Pérez et al., 2023). Microbial decomposers are also highly sensitive to nutrient availability, with increases in dissolved nutrient concentrations stimulating the use of litter as a source of carbon (Gulis and Suberkorpp 2003; Ferreira and Chauvet 2011). Consequently, increases in water temperature and nutrient availability generally translate into accelerated microbial-driven litter decomposition rates (Ferreira and Chauvet 2011; Fernandes et al., 2014). However, in island streams, variation in water temperature and nutrient concentrations, which may occur over a short distance due to steep changes in elevation and geology, do not necessary occur in the same direction, which complicates predictions of litter decomposition rates.
In this study, we incubated leaf litter of three woody species [Acacia melanoxylon (R. Br.), Clethra arborea (Aiton), and Pittosporum undulatum (Vent.)] in 0.5-mm mesh bags, for up to 56 days (with four sampling dates), in six streams on São Miguel island, North Atlantic Ocean, to assess microbial-driven litter decomposition rates, fungal biomass, and conidia production by aquatic hyphomycetes. The temporal dynamics of the biotic variables over the incubation period can be found in Ferreira et al. (2016), while here we report on the relationships between the biotic variables and water nitrate concentration and temperature to assess the interaction between these two abiotic variables in moderating biotic responses under natural settings.
The relationship between biotic variables and nitrate concentration generally followed a hump-shaped curve, with maximum decomposition rates (across species) of 0.0082 – 0.0460/d estimated at 500 – 714 μg NO3–/L, maximum fungal biomass of 50 – 73 mg/g AFDM estimated at 623 – 663 μg NO3–/L, and maximum cumulative conidial production of 3.8 million – 6.9 million conidia/5 discs estimated at 565 – 639 μg NO3–/L (Figure, Table). The relationship between decomposition rates and nitrate concentration was linear for C. arborea (Figure, Table). The relationship between biotic variables and water temperature followed a U-shaped curve, with minimum decomposition rates (across species) of 0.0047 – 0.0259/d estimated at 14.0 – 14.7 ºC, minimum fungal biomass of 15 – 42 mg/g AFDM estimated at 14.3 – 14.4 ºC, and minimum cumulative conidial production of –28219 – 384022 conidia/5 discs estimated at 14.0 – 14.3 ºC (Figure, Table).
Litter decomposition and associated microbial activities were stimulated when nutrient availability and water temperature increased simultaneously across the group of streams including Lom1, Lom2, Lom4, AFG1, and AFG2 (Figure), as anticipated (Ferreira and Chauvet 2011; Fernandes et al., 2014). This stimulation was more evident for the fast-decomposing P. undulatum litter than for the other two species. This suggests that litter is more sensitive to increases in nutrient availability and temperature at more advanced stages of decomposition, which can be due to better microbial colonization of the litter and to the litter increased recalcitrance and thus higher dependence of decomposers on dissolved nutrients. Consistent with this interpretation, other studies found stronger differences in litter mass remaining across streams mostly at later sampling dates (Gulis and Suberkropp 2003; Ferreira et al., 2016, 2021).
However, when an increase in nutrient concentrations was accompanied by a decrease in water temperature (stream Lom3), litter decomposition and associated microbial activities were lower compared with what was expected from the increase in nutrient concentrations alone (see hump-shaped relationships) and higher when compared with what was expected from the decrease in water temperature alone (see U-shaped relationships) (Figure). This suggests that water temperature was the limiting factor for maximum litter decomposition rates and associated microbial activities in this stream. The role of temperature as a limiting factor was observed when comparing temperatures that cannot be considered extreme (13.4ºC – 15.5ºC) and are within a short interval (2.1ºC), suggesting that even small changes in temperature may have strong effects on decomposer activity and litter decomposition. Contrarily, higher nutrient availability may alleviate the effect of low water temperature as an increase in nutrient availability stimulated litter decomposition and associated microbial activities at lower temperature, although generally not at levels observed when temperature was higher. This agrees with Fernandes et al. (2014), who showed that higher nitrogen concentrations are needed to achieve maximum microbial activities at lower temperature.
Although the relationship between biotic variables and water temperature was described by a U-shaped curve, there was high variability at the highest temperatures with stream Lom2 showing higher litter decomposition (1.1 – 2.3-fold), fungal biomass (1.5 – 4.6-fold), and cumulative conidial production (1.9 – 5.9-fold) than stream Lom1 (Figure). This variation in biotic variables between both streams with similar water temperature (mean ± SE: 15.6 ± 0.1 ºC in Lom1 and 15.5 ± 0.1 ºC in Lom2) can be attributed to differences in dissolved nutrient concentration, which was 2.5-fold higher in stream Lom2 (588 ± 19 mg/L) than in stream Lom1 (238 ± 24 mg/L). This suggests that lower nutrient concentration was the limiting factor for maximum litter decomposition rates and associated microbial activities in stream Lom1.
","references":[{"reference":"Bärlocher F. 2020. Sporulation by Aquatic Hyphomycetes. Methods to Study Litter Decomposition : 241-245.
","pubmedId":"","doi":"10.1007/978-3-030-30515-4_26"},{"reference":"Benstead JP, March JG, Pringle CM, Ewel KC, Short JW. 2009. Biodiversity and ecosystem function in species-poor communities: community structure and leaf litter breakdown in a Pacific island stream. Journal of the North American Benthological Society 28: 454-465.
","pubmedId":"","doi":"10.1899/07-081.1"},{"reference":"Fernandes I, Seena S, Pascoal C, Cássio F. 2014. Elevated temperature may intensify the positive effects of nutrients on microbial decomposition in streams. Freshwater Biology 59: 2390-2399.
","pubmedId":"","doi":"10.1111/fwb.12445"},{"reference":"Ferreira V, Chauvet E. 2010. Synergistic effects of water temperature and dissolved nutrients on litter decomposition and associated fungi. Global Change Biology 17: 551-564.
","pubmedId":"","doi":"10.1111/j.1365-2486.2010.02185.x"},{"reference":"Ferreira V, Raposeiro PM, Pereira A, Cruz AM, Costa AC, Graça MAS, Gonçalves V. 2016. Leaf litter decomposition in remote oceanic island streams is driven by microbes and depends on litter quality and environmental conditions. Freshwater Biology 61: 783-799.
","pubmedId":"","doi":"10.1111/fwb.12749"},{"reference":"Ferreira V, Silva J, Cornut J, Sobral O, Bachelet Q, Bouquerel J, Danger M. 2021. Organic-matter decomposition as a bioassessment tool of stream functioning: A comparison of eight decomposition-based indicators exposed to different environmental changes. Environmental Pollution 290: 118111.
","pubmedId":"","doi":"10.1016/j.envpol.2021.118111"},{"reference":"Gessner MO. 2020. Ergosterol as a Measure of Fungal Biomass. Methods to Study Litter Decomposition : 247-255.
","pubmedId":"","doi":"10.1007/978-3-030-30515-4_27"},{"reference":"Gessner MO, Chauvet E. 1993. Ergosterol-to-Biomass Conversion Factors for Aquatic Hyphomycetes. Applied and Environmental Microbiology 59: 502-507.
","pubmedId":"","doi":"10.1128/aem.59.2.502-507.1993"},{"reference":"Gulis V, Suberkropp K. 2002. Leaf litter decomposition and microbial activity in nutrient‐enriched and unaltered reaches of a headwater stream. Freshwater Biology 48: 123-134.
","pubmedId":"","doi":"10.1046/j.1365-2427.2003.00985.x"},{"reference":"Larned ST. 2000. Dynamics of coarse riparian detritus in a Hawaiian stream ecosystem: a comparison of drought and post-drought conditions. Journal of the North American Benthological Society 19: 215-234.
","pubmedId":"","doi":"10.2307/1468066"},{"reference":"MacKenzie RA, Wiegner TN, Kinslow F, Cormier N, Strauch AM. 2013. Leaf-litter inputs from an invasive nitrogen-fixing tree influence organic-matter dynamics and nitrogen inputs in a Hawaiian river. Freshwater Science 32: 1036-1052.
","pubmedId":"","doi":"10.1899/12-152.1"},{"reference":"Pérez J, Cornejo A, Alonso A, Guerra A, García G, Nieto C, et al., Boyero. 2023. Warming overrides eutrophication effects on leaf litter decomposition in stream microcosms. Environmental Pollution 332: 121966.
","pubmedId":"","doi":"10.1016/j.envpol.2023.121966"},{"reference":"Prescott CE. 2010. Litter decomposition: what controls it and how can we alter it to sequester more carbon in forest soils?. Biogeochemistry 101: 133-149.
","pubmedId":"","doi":"10.1007/s10533-010-9439-0"},{"reference":"Raposeiro PM, Martins GM, Moniz I, Cunha A, Costa AC, Gonçalves V. 2014. Leaf litter decomposition in remote oceanic islands: The role of macroinvertebrates vs. microbial decomposition of native vs. exotic plant species. Limnologica 45: 80-87.
","pubmedId":"","doi":"10.1016/j.limno.2013.10.006"},{"reference":"Wallace JB, Eggert SL, Meyer JL, Webster JR. 1997. Multiple Trophic Levels of a Forest Stream Linked to Terrestrial Litter Inputs. Science 277: 102-104.
","pubmedId":"","doi":"10.1126/science.277.5322.102"}],"title":"Water nutrient concentration and temperature control microbial-driven decomposition of leaf litter in oceanic island streams","reviews":[],"curatorReviews":[]},{"id":"eef60c9b-ee9d-4d9c-b07b-865ff80209ec","decision":"edit","abstract":"Litter decomposition fuels food webs in forest streams. Thus, understanding the effects of water nutrients and temperature on litter decomposition and decomposers is key for predicting stream functioning under environmental change. We incubated leaf litter of woody species in streams to assess the effects of nutrient concentrations and temperature on microbial-driven litter decomposition and microbial activities. The relationship between biotic and abiotic variables was generally best described by a quadratic model: there was a hump-shaped relationship between biotic variables and nitrate concentrations and a U-shaped relationship between biotic variables and temperature. The stimulatory effect of high nutrient availability was limited by low temperature, while high nutrient availability alleviated low-temperature effects on litter decomposition and decomposers.
","acknowledgements":"We thank Ana Pereira and Ana Mafalda Cruz for technical work, IMAR – Institute of Marine Research for ergosterol quantification, and INOVA – Instituto de Inovação Tecnológica dos Açores for water nutrient determinations.
","authors":[{"affiliations":["MARE – Marine and Environmental Sciences Centre","ARNET – Aquatic Research Network","University of Coimbra"],"departments":["","","Department of Life Sciences"],"credit":["conceptualization","fundingAcquisition","project","resources","dataCuration","formalAnalysis","validation","investigation","writing_originalDraft"],"email":"veronica@ci.uc.pt","firstName":"Verónica","lastName":"Ferreira","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-7688-2626"},{"affiliations":["University of the Azores","CIBIO – Research Centre in Biodiversity and Genetic Resources","InBIO Associate Laboratory","BIOPOLIS Program in Genomics, Biodiversity and Land Planning – UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands"],"departments":["","","",""],"credit":["investigation","writing_reviewEditing"],"email":"pedro.mv.raposeiro@uac.pt","firstName":"Pedro M.","lastName":"Raposeiro","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7461-0851"},{"affiliations":["University of the Azores","CIBIO – Research Centre in Biodiversity and Genetic Resources","InBIO Associate Laboratory","BIOPOLIS Program in Genomics, Biodiversity and Land Planning – UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands"],"departments":["","","",""],"credit":["project","resources","writing_reviewEditing"],"email":"vitor.mc.goncalves@uac.pt","firstName":"Vítor","lastName":"Gonçalves","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-5737-296X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/fa9c8bb02525601704721d091b3e8c13.csv"},"extendedData":[{"description":"Revised manuscript
","doi":null,"resourceType":"Text","name":"Ferreira&al_microPubBiol_paper rev clean.docx","url":"https://portal.micropublication.org/uploads/ff142d049da532f80b04dc57d6b0a928.docx"}],"funding":"This study was financed by the European Fund for Economic and Regional Development (FEDER) through the Program Operational Factors of Competitiveness (COMPETE; FCOMP-01-0124-FEDER-041055) and by National Funds through the Portuguese Foundation of Science and Technology (FCT; SFRH/BPD/76482/2011, EXPL/AAG-GLO/0189/2013, UID/MAR/04292/2013, SFRH/BPD/99461/2014, IF/00129/2014, CEECIND/02484/2018, UIDB/50027/2020, UID/50027, UID/50027/2025, UID/04292/2025, and LA/P/0069/2020).
","image":{"url":"https://portal.micropublication.org/uploads/da86d51ca26771b39443630e5a8ae417.jpg"},"imageCaption":"Figure. Relationships between biotic variables [microbial-driven litter decomposition rates (A, B), fungal biomass (C, D), and cumulative conidial production (E, F)] associated with leaf litter of three woody species (Acacia melanoxylon, Clethra arborea, and Pittosporum undulatum) and water nitrate concentration (left column) and temperature (right column) in six Azorean streams during Spring/Summer 2014. Stream labels: Lom1 – Lom4, streams located in Lombadas valley; AFG1 and AFG2, tributaries of Fogo lagoon.
Table. Relationships between biotic variables (microbial-driven litter decomposition rates, fungal biomass, and cumulative conidial production) associated with leaf litter of three woody species (Acacia melanoxylon, Clethra arborea, and Pittosporum undulatum) and water nitrate concentration and temperature in six Azorean streams streams during Spring/Summer 2014. In most cases, data were fitted to a quadratic function: y = ax2 + bx + c, where y is the biotic variable, x is the abiotic variable and a, b, and c are constants; a > 0 indicates a U-shaped curve and a < 0 indicates a hump-shaped curve. The curve vertex (i.e. the minimum value in a U-shaped curve or the maximum value in a hump-shaped curve) was calculated as: x = – (b / 2a) and y = ax2 + bx + c. The coefficient of determination of the models (R2) is also shown. The relationship between decomposition rates and nitrate concentration for C. arborea was fitted to a linear model (y = 0.00001x + 0.0078).
","imageTitle":"Relationships between biotic variables associated with leaf litter decomposition of three woody species and water nitrate concentration and temperature in six Azorean streams
","methods":"Leaf litter incubation was carried out in six small streams in the central massif of São Miguel island, Azores archipelago, North Atlantic Ocean. Four streams were tributaries of Ribeira Grande stream in Lombadas valley (Lom1 – Lom4) and two streams were tributaries of Fogo Lagoon (AFG1 and AFG2). Streams had similar geomorphology, were not visibly affected by direct human activities, were slightly alkaline (pH 7.7 – 8.1) and well oxygenated (8.4 – 9.1 mg O2/L), and varied in water temperature (13.4 – 15.6 ºC) and nutrient concentrations (28 – 1033 μg NO3–/L and 30 – 175 μg PO43–/L). Water temperature was recorded hourly over the 56-days incubation period during Spring and Summer 2014 using data loggers (Hobo Pendant UA-001-08, Onset Computer Corp., Massachusetts, USA) and hourly values were used to estimate daily means, which were averaged for each stream. On five occasions, stream water was filtered (fiberglass filters, 47-mm diameter, GF/C, Whatman, GE Healthcare Europe GmbH, Little Chalfont, UK) and used to determine nitrate concentration (ion chromatography; APHA 1995). A detailed description of the study area and streams is provided in Ferreira et al. (2016).
Air-dried leaves of three evergreen woody species commonly found in the riparian vegetation of Azorean streams were used to provide a gradient of litter palatability, in increasing order: Acacia melanoxylon (R. Br.) < Clethra arborea (Aiton) < Pittosporum undulatum (Vent.) (Ferreira et al., 2016). Leaves were weighed (2.90 – 3.10 g) and enclosed in fine-mesh bags (10 × 15 cm, 0.5-mm mesh), which prevent invertebrate access and where decomposition is mostly driven by microbial decomposers. Twelve litter bags per species were deployed in each stream on 11 or 12 June, 2014. Three replicate litter bags were recovered from each stream after 7, 21, 35 and 56 days, and transported cold to the laboratory. In the laboratory, litter was rinsed with distilled water and two sets of five leaf discs were extracted with a cork borer (12-mm diameter): one set was frozen at – 18 ºC for later determination of fungal biomass (Gessner 2020) and one set was used fresh to induce conidia production by aquatic hyphomycetes (Bärlocher 2020). The bulk litter mass was used to determine litter mass remaining (Ferreira et al., 2016).
For determination of fungal biomass, five frozen leaf discs were lyophilized (LY3TE, Snijders Scientific, Tilburg, The Netherlands), weighed (Kern 870, Kern & Sohn GmbH, Balingen, Germany) to determine dry mass, and incubated in alkaline methanol (8 g KOH/L, 30 min at 80 ºC) to extract ergosterol. The extract was purified using solid phase extraction cartridges (Waters Sep-Pak® Vac RC tC18 cartridges; Waters Corp., Massachusetts, USA) and eluted with isopropanol. Ergosterol was quantified by high-performance liquid chromatography (Dionex DX-120, California, USA), using a Thermo Scientific Syncronis C18 column and a Thermo Universal Uniguard holder 4/4.6 mm ID3 + Syncronis C18 drop in guard pre-column (Thermo, Waltham, Massachusetts, USA); the mobile phase was 100% methanol, kept at 33ºC and flowing at 1.4 mL/min. Ergosterol was detected by reading absorbance at 282 nm with a UV detector and absorbance was converted into ergosterol concentration using a standard curve of ergosterol in isopropanol. Ergosterol concentration was converted into fungal biomass considering 5.5 mg ergosterol/mg fungal dry mass (Gessner and Chauvet 1993) and fungal biomass was expressed as mg/g AFDM, averaged across sampling dates.
Conidia production by aquatic hyphomycetes was determined after incubating five fresh leaf discs in 25 mL of filtered stream water (48h at 13 ºC, 10h light:14 h dark photoperiod, and 100 rpm). The conidia suspension was preserved with 2 mL of formalin and leaf discs were processed as described below for determination of discs ash-free dry mass. Suspensions were homogenized with 150 mL Triton X-100 and a magnetic stirring bar, and aliquots were filtered (nitrocellulose filters, 25-mm diameter, 5-mm pore size; Sartorius Stedim Biotech GmhH, Göttingen, Germany). Filters were stained with trypan blue in lactic acid and mounted on a slide. Conidia were counted at 320´ magnification (DM1000 microscope, Leica, Wetzlar, Germany). Rates of conidia production were expressed as no. conidia/mg leaf AFDM/day, and cumulative (total) conidial production over the incubation period was estimated as the sum of daily values, which were derived by linear interpolation of the values of the adjacent sampling dates, and expressed as no. conidia/5 discs.
For determination of litter mass remaining, litter (after extraction of leaf discs) was oven-dried (70 ºC, 48 h) and weighed (Kern 870, Kern & Sohn GmbH, Balingen, Germany) to assess dry mass (DM) remaining. DM remaining was ignited (500 ºC, 8 h) and weighed to assess ash mass. Ash-free dry mass (AFDM) remaining was estimated as the difference between DM and ash mass (after accounting for the extracted discs), and the fraction of AFDM remaining was estimated as the ratio between AFDM remaining and initial AFDM. Initial AFDM of samples was estimated as the product of initial air-dry mass by a conversion factor derived from extra sets of five fine-mesh bags per species. These extra bags, were prepared as the samples, taken to the field on day 0, immersed in water for ~ 10 min, and returned to the laboratory for determination of DM and AFDM as described above. The conversion factor was estimated as the ratio between initial AFDM and initial air-dry mass. Fraction AFDM remaining across the incubation times was used to estimate the overall exponential decomposition rate (k, /days) for each species and stream as the slope of a linear regression between ln(fraction of AFDM remaining) and time (days).
The relationships between biological variables (microbial-driven litter decomposition rates, average fungal biomass, and cumulative conidial production) of the three leaf litter species and abiotic variables [nitrate concentration (average across sampling dates) and water temperature (average across 56 days)] in six streams were analyzed by fitting the data to a quadratic function. The relationship between litter decomposition rates and nitrate concentration for C.arborea was fitted to a linear model. Models were derived using Statistica 7 software (StatSoft Inc., Tulsa, Oklahoma, USA).
","reagents":"","patternDescription":"Leaf litter decomposition is a key ecosystem process in forest streams, where litter inputs from the riparian vegetation are the main source of carbon and nutrients for aquatic food webs (Wallace et al., 1997). In island streams, where invertebrate shredders are generally rare (Benstead et al., 2009; MacKenzie et al., 2013; Raposeiro et al., 2014), litter decomposition is mostly driven by microbial decomposers, aquatic hyphomycetes in particular (Benstead et al., 2009; Larned 2000; MacKenzie et al., 2013; Raposeiro et al., 2014; Ferreira et al., 2016). Microbial decomposers are highly responsive to water temperature, with increases in temperature within organisms’ thermal ranges stimulating metabolism rates, and consequently the mineralization of litter carbon into CO2 through respiration (Ferreira and Chauvet 2011; Pérez et al., 2023). Microbial decomposers are also highly sensitive to nutrient availability, with increases in dissolved nutrient concentrations stimulating the use of litter as a source of carbon (Gulis and Suberkorpp 2003; Ferreira and Chauvet 2011). Consequently, increases in water temperature and nutrient availability generally translate into accelerated microbial-driven litter decomposition rates (Ferreira and Chauvet 2011; Fernandes et al., 2014). However, in island streams, variation in water temperature and nutrient concentrations, which may occur over a short distance due to steep changes in elevation and geology, do not necessary occur in the same direction, which complicates predictions of litter decomposition rates.
In this study, we incubated leaf litter of three woody species [Acacia melanoxylon (R. Br.), Clethra arborea (Aiton), and Pittosporum undulatum (Vent.)] in 0.5-mm mesh bags, for up to 56 days (with four sampling dates), in six streams on São Miguel island, North Atlantic Ocean, to assess microbial-driven litter decomposition rates, fungal biomass, and conidia production by aquatic hyphomycetes. The temporal dynamics of the biotic variables over the incubation period can be found in Ferreira et al. (2016), while here we report on the relationships between the biotic variables and water nitrate concentration and temperature to assess the interaction between these two abiotic variables in moderating biotic responses under natural settings.
The relationship between biotic variables and nitrate concentration generally followed a hump-shaped curve, with maximum decomposition rates (across species) of 0.0082 – 0.0460/d estimated at 500 – 714 μg NO3–/L, maximum fungal biomass of 50 – 73 mg/g AFDM estimated at 623 – 663 μg NO3–/L, and maximum cumulative conidial production of 3.8 million – 6.9 million conidia/5 discs estimated at 565 – 639 μg NO3–/L (Figure, Table). The relationship between decomposition rates and nitrate concentration was linear for C. arborea (Figure, Table). The relationship between biotic variables and water temperature followed a U-shaped curve, with minimum decomposition rates (across species) of 0.0047 – 0.0259/d estimated at 14.0 – 14.7 ºC, minimum fungal biomass of 15 – 42 mg/g AFDM estimated at 14.3 – 14.4 ºC, and minimum cumulative conidial production of –28219 – 384022 conidia/5 discs estimated at 14.0 – 14.3 ºC (Figure, Table).
Litter decomposition and associated microbial activities were stimulated when nutrient availability and water temperature increased simultaneously across the group of streams including Lom1, Lom2, Lom4, AFG1, and AFG2 (Figure), as anticipated (Ferreira and Chauvet 2011; Fernandes et al., 2014). This stimulation was more evident for the fast-decomposing P. undulatum litter than for the other two species. This suggests that litter is more sensitive to increases in nutrient availability and temperature at more advanced stages of decomposition, which can be due to better microbial colonization of the litter and to the litter increased recalcitrance and thus higher dependence of decomposers on dissolved nutrients. Consistent with this interpretation, other studies found stronger differences in litter mass remaining across streams mostly at later sampling dates (Gulis and Suberkropp 2003; Ferreira et al., 2016, 2021).
However, when an increase in nutrient concentrations was accompanied by a decrease in water temperature (stream Lom3), litter decomposition and associated microbial activities were lower compared with what was expected from the increase in nutrient concentrations alone (see hump-shaped relationships) and higher when compared with what was expected from the decrease in water temperature alone (see U-shaped relationships) (Figure). This suggests that water temperature was the limiting factor for maximum litter decomposition rates and associated microbial activities in this stream. The role of temperature as a limiting factor was observed when comparing temperatures that cannot be considered extreme (13.4ºC – 15.5ºC) and are within a short interval (2.1ºC), suggesting that even small changes in temperature may have strong effects on decomposer activity and litter decomposition. Contrarily, higher nutrient availability may alleviate the effect of low water temperature as an increase in nutrient availability stimulated litter decomposition and associated microbial activities at lower temperature, although generally not at levels observed when temperature was higher. This agrees with Fernandes et al. (2014), who showed that higher nitrogen concentrations are needed to achieve maximum microbial activities at lower temperature.
Although the relationship between biotic variables and water temperature was described by a U-shaped curve, there was high variability at the highest temperatures with stream Lom2 showing higher litter decomposition (1.1 – 2.3-fold), fungal biomass (1.5 – 4.6-fold), and cumulative conidial production (1.9 – 5.9-fold) than stream Lom1 (Figure). This variation in biotic variables between both streams with similar water temperature (mean ± SE: 15.6 ± 0.1 ºC in Lom1 and 15.5 ± 0.1 ºC in Lom2) can be attributed to differences in dissolved nutrient concentration, which was 2.5-fold higher in stream Lom2 (588 ± 19 mg/L) than in stream Lom1 (238 ± 24 mg/L). This suggests that lower nutrient concentration was the limiting factor for maximum litter decomposition rates and associated microbial activities in stream Lom1.
","references":[{"reference":"Bärlocher F. 2020. Sporulation by Aquatic Hyphomycetes. Methods to Study Litter Decomposition : 241-245.
","pubmedId":"","doi":"10.1007/978-3-030-30515-4_26"},{"reference":"Benstead JP, March JG, Pringle CM, Ewel KC, Short JW. 2009. Biodiversity and ecosystem function in species-poor communities: community structure and leaf litter breakdown in a Pacific island stream. Journal of the North American Benthological Society 28: 454-465.
","pubmedId":"","doi":"10.1899/07-081.1"},{"reference":"Fernandes I, Seena S, Pascoal C, Cássio F. 2014. Elevated temperature may intensify the positive effects of nutrients on microbial decomposition in streams. Freshwater Biology 59: 2390-2399.
","pubmedId":"","doi":"10.1111/fwb.12445"},{"reference":"Ferreira V, Chauvet E. 2010. Synergistic effects of water temperature and dissolved nutrients on litter decomposition and associated fungi. Global Change Biology 17: 551-564.
","pubmedId":"","doi":"10.1111/j.1365-2486.2010.02185.x"},{"reference":"Ferreira V, Raposeiro PM, Pereira A, Cruz AM, Costa AC, Graça MAS, Gonçalves V. 2016. Leaf litter decomposition in remote oceanic island streams is driven by microbes and depends on litter quality and environmental conditions. Freshwater Biology 61: 783-799.
","pubmedId":"","doi":"10.1111/fwb.12749"},{"reference":"Ferreira V, Silva J, Cornut J, Sobral O, Bachelet Q, Bouquerel J, Danger M. 2021. Organic-matter decomposition as a bioassessment tool of stream functioning: A comparison of eight decomposition-based indicators exposed to different environmental changes. Environmental Pollution 290: 118111.
","pubmedId":"","doi":"10.1016/j.envpol.2021.118111"},{"reference":"Gessner MO. 2020. Ergosterol as a Measure of Fungal Biomass. Methods to Study Litter Decomposition : 247-255.
","pubmedId":"","doi":"10.1007/978-3-030-30515-4_27"},{"reference":"Gessner MO, Chauvet E. 1993. Ergosterol-to-Biomass Conversion Factors for Aquatic Hyphomycetes. Applied and Environmental Microbiology 59: 502-507.
","pubmedId":"","doi":"10.1128/aem.59.2.502-507.1993"},{"reference":"Gulis V, Suberkropp K. 2002. Leaf litter decomposition and microbial activity in nutrient‐enriched and unaltered reaches of a headwater stream. Freshwater Biology 48: 123-134.
","pubmedId":"","doi":"10.1046/j.1365-2427.2003.00985.x"},{"reference":"Larned ST. 2000. Dynamics of coarse riparian detritus in a Hawaiian stream ecosystem: a comparison of drought and post-drought conditions. Journal of the North American Benthological Society 19: 215-234.
","pubmedId":"","doi":"10.2307/1468066"},{"reference":"MacKenzie RA, Wiegner TN, Kinslow F, Cormier N, Strauch AM. 2013. Leaf-litter inputs from an invasive nitrogen-fixing tree influence organic-matter dynamics and nitrogen inputs in a Hawaiian river. Freshwater Science 32: 1036-1052.
","pubmedId":"","doi":"10.1899/12-152.1"},{"reference":"Pérez J, Cornejo A, Alonso A, Guerra A, García G, Nieto C, et al., Boyero. 2023. Warming overrides eutrophication effects on leaf litter decomposition in stream microcosms. Environmental Pollution 332: 121966.
","pubmedId":"","doi":"10.1016/j.envpol.2023.121966"},{"reference":"Prescott CE. 2010. Litter decomposition: what controls it and how can we alter it to sequester more carbon in forest soils?. Biogeochemistry 101: 133-149.
","pubmedId":"","doi":"10.1007/s10533-010-9439-0"},{"reference":"Raposeiro PM, Martins GM, Moniz I, Cunha A, Costa AC, Gonçalves V. 2014. Leaf litter decomposition in remote oceanic islands: The role of macroinvertebrates vs. microbial decomposition of native vs. exotic plant species. Limnologica 45: 80-87.
","pubmedId":"","doi":"10.1016/j.limno.2013.10.006"},{"reference":"Wallace JB, Eggert SL, Meyer JL, Webster JR. 1997. Multiple Trophic Levels of a Forest Stream Linked to Terrestrial Litter Inputs. Science 277: 102-104.
","pubmedId":"","doi":"10.1126/science.277.5322.102"}],"title":"Water nutrient concentration and temperature control microbial-driven decomposition of leaf litter in oceanic island streams","reviews":[],"curatorReviews":[]},{"id":"2659ba03-2b48-4f1d-b39d-145015bb4365","decision":"accept","abstract":"Litter decomposition fuels food webs in forest streams. Thus, understanding the effects of water nutrients and temperature on litter decomposition and decomposers is key for predicting stream functioning under environmental change. We incubated leaf litter of woody species in streams to assess the effects of nutrient concentrations and temperature on microbial-driven litter decomposition and microbial activities. The relationship between biotic and abiotic variables was generally best described by a quadratic model: there was a hump-shaped relationship between biotic variables and nitrate concentrations and a U-shaped relationship between biotic variables and temperature. The stimulatory effect of high nutrient availability was limited by low temperature, while high nutrient availability alleviated low-temperature effects on litter decomposition and decomposers.
","acknowledgements":"We thank Ana Pereira and Ana Mafalda Cruz for technical work, IMAR – Institute of Marine Research for ergosterol quantification, and INOVA – Instituto de Inovação Tecnológica dos Açores for water nutrient determinations.
","authors":[{"affiliations":["University of Coimbra, Coimbra, 06, Portugal"],"departments":["MARE-Marine and Environmental Sciences Centre, ARNET-Aquatic Research Network, Department of Life Sciences,Department of Life Sciences"],"credit":["conceptualization","fundingAcquisition","project","resources","dataCuration","formalAnalysis","validation","investigation","writing_originalDraft"],"email":"veronica@ci.uc.pt","firstName":"Verónica","lastName":"Ferreira","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-7688-2626"},{"affiliations":["University of the Azores, Ponta Delgada, 20, Portugal","University of the Azores, Ponta Delgada, 20, Portugal"],"departments":["Faculty of Sciences and Technology","CIBIO – Research Centre in Biodiversity and Genetic Resources, InBIO Associate Laboratory, BIOPOLIS Program in Genomics, Biodiversity and Land Planning – UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands"],"credit":["investigation","writing_reviewEditing"],"email":"pedro.mv.raposeiro@uac.pt","firstName":"Pedro M.","lastName":"Raposeiro","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7461-0851"},{"affiliations":["University of the Azores, Ponta Delgada, 20, Portugal","University of the Azores, Ponta Delgada, 20, Portugal"],"departments":["Faculty of Sciences and Technology","CIBIO – Research Centre in Biodiversity and Genetic Resources, InBIO Associate Laboratory, BIOPOLIS Program in Genomics, Biodiversity and Land Planning – UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands"],"credit":["project","resources","writing_reviewEditing"],"email":"vitor.mc.goncalves@uac.pt","firstName":"Vítor","lastName":"Gonçalves","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-5737-296X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/fa9c8bb02525601704721d091b3e8c13.csv"},"extendedData":[{"description":"Revised manuscript
","doi":null,"resourceType":"Text","name":"Ferreira&al_microPubBiol_paper rev clean.docx","url":"https://portal.micropublication.org/uploads/ff142d049da532f80b04dc57d6b0a928.docx"}],"funding":"This study was financed by the European Fund for Economic and Regional Development (FEDER) through the Program Operational Factors of Competitiveness (COMPETE; FCOMP-01-0124-FEDER-041055) and by National Funds through the Portuguese Foundation of Science and Technology (FCT; SFRH/BPD/76482/2011, EXPL/AAG-GLO/0189/2013, UID/MAR/04292/2013, SFRH/BPD/99461/2014, IF/00129/2014, CEECIND/02484/2018, UIDB/50027/2020, UID/50027, UID/50027/2025, UID/04292/2025, and LA/P/0069/2020).
","image":{"url":"https://portal.micropublication.org/uploads/da86d51ca26771b39443630e5a8ae417.jpg"},"imageCaption":"Figure. Relationships between biotic variables [microbial-driven litter decomposition rates (A, B), fungal biomass (C, D), and cumulative conidial production (E, F)] associated with leaf litter of three woody species (Acacia melanoxylon, Clethra arborea, and Pittosporum undulatum) and water nitrate concentration (left column) and temperature (right column) in six Azorean streams during Spring/Summer 2014. Stream labels: Lom1 – Lom4, streams located in Lombadas valley; AFG1 and AFG2, tributaries of Fogo lagoon.
Table. Relationships between biotic variables (microbial-driven litter decomposition rates, fungal biomass, and cumulative conidial production) associated with leaf litter of three woody species (Acacia melanoxylon, Clethra arborea, and Pittosporum undulatum) and water nitrate concentration and temperature in six Azorean streams streams during Spring/Summer 2014. In most cases, data were fitted to a quadratic function: y = ax2 + bx + c, where y is the biotic variable, x is the abiotic variable and a, b, and c are constants; a > 0 indicates a U-shaped curve and a < 0 indicates a hump-shaped curve. The curve vertex (i.e. the minimum value in a U-shaped curve or the maximum value in a hump-shaped curve) was calculated as: x = – (b / 2a) and y = ax2 + bx + c. The coefficient of determination of the models (R2) is also shown. The relationship between decomposition rates and nitrate concentration for C. arborea was fitted to a linear model (y = 0.00001x + 0.0078).
","imageTitle":"Relationships between biotic variables associated with leaf litter decomposition of three woody species and water nitrate concentration and temperature in six Azorean streams
","methods":"Leaf litter incubation was carried out in six small streams in the central massif of São Miguel island, Azores archipelago, North Atlantic Ocean. Four streams were tributaries of Ribeira Grande stream in Lombadas valley (Lom1 – Lom4) and two streams were tributaries of Fogo Lagoon (AFG1 and AFG2). Streams had similar geomorphology, were not visibly affected by direct human activities, were slightly alkaline (pH 7.7 – 8.1) and well oxygenated (8.4 – 9.1 mg O2/L), and varied in water temperature (13.4 – 15.6 ºC) and nutrient concentrations (28 – 1033 μg NO3–/L and 30 – 175 μg PO43–/L). Water temperature was recorded hourly over the 56-days incubation period during Spring and Summer 2014 using data loggers (Hobo Pendant UA-001-08, Onset Computer Corp., Massachusetts, USA) and hourly values were used to estimate daily means, which were averaged for each stream. On five occasions, stream water was filtered (fiberglass filters, 47-mm diameter, GF/C, Whatman, GE Healthcare Europe GmbH, Little Chalfont, UK) and used to determine nitrate concentration (ion chromatography; APHA 1995). A detailed description of the study area and streams is provided in Ferreira et al. (2016).
Air-dried leaves of three evergreen woody species commonly found in the riparian vegetation of Azorean streams were used to provide a gradient of litter palatability, in increasing order: Acacia melanoxylon (R. Br.) < Clethra arborea (Aiton) < Pittosporum undulatum (Vent.) (Ferreira et al., 2016). Leaves were weighed (2.90 – 3.10 g) and enclosed in fine-mesh bags (10 × 15 cm, 0.5-mm mesh), which prevent invertebrate access and where decomposition is mostly driven by microbial decomposers. Twelve litter bags per species were deployed in each stream on 11 or 12 June, 2014. Three replicate litter bags were recovered from each stream after 7, 21, 35 and 56 days, and transported cold to the laboratory. In the laboratory, litter was rinsed with distilled water and two sets of five leaf discs were extracted with a cork borer (12-mm diameter): one set was frozen at – 18 ºC for later determination of fungal biomass (Gessner 2020) and one set was used fresh to induce conidia production by aquatic hyphomycetes (Bärlocher 2020). The bulk litter mass was used to determine litter mass remaining (Ferreira et al., 2016).
For determination of fungal biomass, five frozen leaf discs were lyophilized (LY3TE, Snijders Scientific, Tilburg, The Netherlands), weighed (Kern 870, Kern & Sohn GmbH, Balingen, Germany) to determine dry mass, and incubated in alkaline methanol (8 g KOH/L, 30 min at 80 ºC) to extract ergosterol. The extract was purified using solid phase extraction cartridges (Waters Sep-Pak® Vac RC tC18 cartridges; Waters Corp., Massachusetts, USA) and eluted with isopropanol. Ergosterol was quantified by high-performance liquid chromatography (Dionex DX-120, California, USA), using a Thermo Scientific Syncronis C18 column and a Thermo Universal Uniguard holder 4/4.6 mm ID3 + Syncronis C18 drop in guard pre-column (Thermo, Waltham, Massachusetts, USA); the mobile phase was 100% methanol, kept at 33ºC and flowing at 1.4 mL/min. Ergosterol was detected by reading absorbance at 282 nm with a UV detector and absorbance was converted into ergosterol concentration using a standard curve of ergosterol in isopropanol. Ergosterol concentration was converted into fungal biomass considering 5.5 mg ergosterol/mg fungal dry mass (Gessner and Chauvet 1993) and fungal biomass was expressed as mg/g AFDM, averaged across sampling dates.
Conidia production by aquatic hyphomycetes was determined after incubating five fresh leaf discs in 25 mL of filtered stream water (48h at 13 ºC, 10h light:14 h dark photoperiod, and 100 rpm). The conidia suspension was preserved with 2 mL of formalin and leaf discs were processed as described below for determination of discs ash-free dry mass. Suspensions were homogenized with 150 mL Triton X-100 and a magnetic stirring bar, and aliquots were filtered (nitrocellulose filters, 25-mm diameter, 5-mm pore size; Sartorius Stedim Biotech GmhH, Göttingen, Germany). Filters were stained with trypan blue in lactic acid and mounted on a slide. Conidia were counted at 320´ magnification (DM1000 microscope, Leica, Wetzlar, Germany). Rates of conidia production were expressed as no. conidia/mg leaf AFDM/day, and cumulative (total) conidial production over the incubation period was estimated as the sum of daily values, which were derived by linear interpolation of the values of the adjacent sampling dates, and expressed as no. conidia/5 discs.
For determination of litter mass remaining, litter (after extraction of leaf discs) was oven-dried (70 ºC, 48 h) and weighed (Kern 870, Kern & Sohn GmbH, Balingen, Germany) to assess dry mass (DM) remaining. DM remaining was ignited (500 ºC, 8 h) and weighed to assess ash mass. Ash-free dry mass (AFDM) remaining was estimated as the difference between DM and ash mass (after accounting for the extracted discs), and the fraction of AFDM remaining was estimated as the ratio between AFDM remaining and initial AFDM. Initial AFDM of samples was estimated as the product of initial air-dry mass by a conversion factor derived from extra sets of five fine-mesh bags per species. These extra bags, were prepared as the samples, taken to the field on day 0, immersed in water for ~ 10 min, and returned to the laboratory for determination of DM and AFDM as described above. The conversion factor was estimated as the ratio between initial AFDM and initial air-dry mass. Fraction AFDM remaining across the incubation times was used to estimate the overall exponential decomposition rate (k, /days) for each species and stream as the slope of a linear regression between ln(fraction of AFDM remaining) and time (days).
The relationships between biological variables (microbial-driven litter decomposition rates, average fungal biomass, and cumulative conidial production) of the three leaf litter species and abiotic variables [nitrate concentration (average across sampling dates) and water temperature (average across 56 days)] in six streams were analyzed by fitting the data to a quadratic function. The relationship between litter decomposition rates and nitrate concentration for C.arborea was fitted to a linear model. Models were derived using Statistica 7 software (StatSoft Inc., Tulsa, Oklahoma, USA).
","reagents":"","patternDescription":"Leaf litter decomposition is a key ecosystem process in forest streams, where litter inputs from the riparian vegetation are the main source of carbon and nutrients for aquatic food webs (Wallace et al., 1997). In island streams, where invertebrate shredders are generally rare (Benstead et al., 2009; MacKenzie et al., 2013; Raposeiro et al., 2014), litter decomposition is mostly driven by microbial decomposers, aquatic hyphomycetes in particular (Benstead et al., 2009; Larned 2000; MacKenzie et al., 2013; Raposeiro et al., 2014; Ferreira et al., 2016). Microbial decomposers are highly responsive to water temperature, with increases in temperature within organisms’ thermal ranges stimulating metabolism rates, and consequently the mineralization of litter carbon into CO2 through respiration (Ferreira and Chauvet 2011; Pérez et al., 2023). Microbial decomposers are also highly sensitive to nutrient availability, with increases in dissolved nutrient concentrations stimulating the use of litter as a source of carbon (Gulis and Suberkorpp 2003; Ferreira and Chauvet 2011). Consequently, increases in water temperature and nutrient availability generally translate into accelerated microbial-driven litter decomposition rates (Ferreira and Chauvet 2011; Fernandes et al., 2014). However, in island streams, variation in water temperature and nutrient concentrations, which may occur over a short distance due to steep changes in elevation and geology, do not necessary occur in the same direction, which complicates predictions of litter decomposition rates.
In this study, we incubated leaf litter of three woody species [Acacia melanoxylon (R. Br.), Clethra arborea (Aiton), and Pittosporum undulatum (Vent.)] in 0.5-mm mesh bags, for up to 56 days (with four sampling dates), in six streams on São Miguel island, North Atlantic Ocean, to assess microbial-driven litter decomposition rates, fungal biomass, and conidia production by aquatic hyphomycetes. The temporal dynamics of the biotic variables over the incubation period can be found in Ferreira et al. (2016), while here we report on the relationships between the biotic variables and water nitrate concentration and temperature to assess the interaction between these two abiotic variables in moderating biotic responses under natural settings.
The relationship between biotic variables and nitrate concentration generally followed a hump-shaped curve, with maximum decomposition rates (across species) of 0.0082 – 0.0460/d estimated at 500 – 714 μg NO3–/L, maximum fungal biomass of 50 – 73 mg/g AFDM estimated at 623 – 663 μg NO3–/L, and maximum cumulative conidial production of 3.8 million – 6.9 million conidia/5 discs estimated at 565 – 639 μg NO3–/L (Figure, Table). The relationship between decomposition rates and nitrate concentration was linear for C. arborea (Figure, Table). The relationship between biotic variables and water temperature followed a U-shaped curve, with minimum decomposition rates (across species) of 0.0047 – 0.0259/d estimated at 14.0 – 14.7 ºC, minimum fungal biomass of 15 – 42 mg/g AFDM estimated at 14.3 – 14.4 ºC, and minimum cumulative conidial production of –28219 – 384022 conidia/5 discs estimated at 14.0 – 14.3 ºC (Figure, Table).
Litter decomposition and associated microbial activities were stimulated when nutrient availability and water temperature increased simultaneously across the group of streams including Lom1, Lom2, Lom4, AFG1, and AFG2 (Figure), as anticipated (Ferreira and Chauvet 2011; Fernandes et al., 2014). This stimulation was more evident for the fast-decomposing P. undulatum litter than for the other two species. This suggests that litter is more sensitive to increases in nutrient availability and temperature at more advanced stages of decomposition, which can be due to better microbial colonization of the litter and to the litter increased recalcitrance and thus higher dependence of decomposers on dissolved nutrients. Consistent with this interpretation, other studies found stronger differences in litter mass remaining across streams mostly at later sampling dates (Gulis and Suberkropp 2003; Ferreira et al., 2016, 2021).
However, when an increase in nutrient concentrations was accompanied by a decrease in water temperature (stream Lom3), litter decomposition and associated microbial activities were lower compared with what was expected from the increase in nutrient concentrations alone (see hump-shaped relationships) and higher when compared with what was expected from the decrease in water temperature alone (see U-shaped relationships) (Figure). This suggests that water temperature was the limiting factor for maximum litter decomposition rates and associated microbial activities in this stream. The role of temperature as a limiting factor was observed when comparing temperatures that cannot be considered extreme (13.4ºC – 15.5ºC) and are within a short interval (2.1ºC), suggesting that even small changes in temperature may have strong effects on decomposer activity and litter decomposition. Contrarily, higher nutrient availability may alleviate the effect of low water temperature as an increase in nutrient availability stimulated litter decomposition and associated microbial activities at lower temperature, although generally not at levels observed when temperature was higher. This agrees with Fernandes et al. (2014), who showed that higher nitrogen concentrations are needed to achieve maximum microbial activities at lower temperature.
Although the relationship between biotic variables and water temperature was described by a U-shaped curve, there was high variability at the highest temperatures with stream Lom2 showing higher litter decomposition (1.1 – 2.3-fold), fungal biomass (1.5 – 4.6-fold), and cumulative conidial production (1.9 – 5.9-fold) than stream Lom1 (Figure). This variation in biotic variables between both streams with similar water temperature (mean ± SE: 15.6 ± 0.1 ºC in Lom1 and 15.5 ± 0.1 ºC in Lom2) can be attributed to differences in dissolved nutrient concentration, which was 2.5-fold higher in stream Lom2 (588 ± 19 mg/L) than in stream Lom1 (238 ± 24 mg/L). This suggests that lower nutrient concentration was the limiting factor for maximum litter decomposition rates and associated microbial activities in stream Lom1.
","references":[{"reference":"Bärlocher F. 2020. Sporulation by Aquatic Hyphomycetes. Methods to Study Litter Decomposition : 241-245.
","pubmedId":"","doi":"10.1007/978-3-030-30515-4_26"},{"reference":"Benstead JP, March JG, Pringle CM, Ewel KC, Short JW. 2009. Biodiversity and ecosystem function in species-poor communities: community structure and leaf litter breakdown in a Pacific island stream. Journal of the North American Benthological Society 28: 454-465.
","pubmedId":"","doi":"10.1899/07-081.1"},{"reference":"Fernandes I, Seena S, Pascoal C, Cássio F. 2014. Elevated temperature may intensify the positive effects of nutrients on microbial decomposition in streams. Freshwater Biology 59: 2390-2399.
","pubmedId":"","doi":"10.1111/fwb.12445"},{"reference":"Ferreira V, Chauvet E. 2010. Synergistic effects of water temperature and dissolved nutrients on litter decomposition and associated fungi. Global Change Biology 17: 551-564.
","pubmedId":"","doi":"10.1111/j.1365-2486.2010.02185.x"},{"reference":"Ferreira V, Raposeiro PM, Pereira A, Cruz AM, Costa AC, Graça MAS, Gonçalves V. 2016. Leaf litter decomposition in remote oceanic island streams is driven by microbes and depends on litter quality and environmental conditions. Freshwater Biology 61: 783-799.
","pubmedId":"","doi":"10.1111/fwb.12749"},{"reference":"Ferreira V, Silva J, Cornut J, Sobral O, Bachelet Q, Bouquerel J, Danger M. 2021. Organic-matter decomposition as a bioassessment tool of stream functioning: A comparison of eight decomposition-based indicators exposed to different environmental changes. Environmental Pollution 290: 118111.
","pubmedId":"","doi":"10.1016/j.envpol.2021.118111"},{"reference":"Gessner MO. 2020. Ergosterol as a Measure of Fungal Biomass. Methods to Study Litter Decomposition : 247-255.
","pubmedId":"","doi":"10.1007/978-3-030-30515-4_27"},{"reference":"Gessner MO, Chauvet E. 1993. Ergosterol-to-Biomass Conversion Factors for Aquatic Hyphomycetes. Applied and Environmental Microbiology 59: 502-507.
","pubmedId":"","doi":"10.1128/aem.59.2.502-507.1993"},{"reference":"Gulis V, Suberkropp K. 2002. Leaf litter decomposition and microbial activity in nutrient‐enriched and unaltered reaches of a headwater stream. Freshwater Biology 48: 123-134.
","pubmedId":"","doi":"10.1046/j.1365-2427.2003.00985.x"},{"reference":"Larned ST. 2000. Dynamics of coarse riparian detritus in a Hawaiian stream ecosystem: a comparison of drought and post-drought conditions. Journal of the North American Benthological Society 19: 215-234.
","pubmedId":"","doi":"10.2307/1468066"},{"reference":"MacKenzie RA, Wiegner TN, Kinslow F, Cormier N, Strauch AM. 2013. Leaf-litter inputs from an invasive nitrogen-fixing tree influence organic-matter dynamics and nitrogen inputs in a Hawaiian river. Freshwater Science 32: 1036-1052.
","pubmedId":"","doi":"10.1899/12-152.1"},{"reference":"Pérez J, Cornejo A, Alonso A, Guerra A, García G, Nieto C, et al., Boyero. 2023. Warming overrides eutrophication effects on leaf litter decomposition in stream microcosms. Environmental Pollution 332: 121966.
","pubmedId":"","doi":"10.1016/j.envpol.2023.121966"},{"reference":"Prescott CE. 2010. Litter decomposition: what controls it and how can we alter it to sequester more carbon in forest soils?. Biogeochemistry 101: 133-149.
","pubmedId":"","doi":"10.1007/s10533-010-9439-0"},{"reference":"Raposeiro PM, Martins GM, Moniz I, Cunha A, Costa AC, Gonçalves V. 2014. Leaf litter decomposition in remote oceanic islands: The role of macroinvertebrates vs. microbial decomposition of native vs. exotic plant species. Limnologica 45: 80-87.
","pubmedId":"","doi":"10.1016/j.limno.2013.10.006"},{"reference":"Wallace JB, Eggert SL, Meyer JL, Webster JR. 1997. Multiple Trophic Levels of a Forest Stream Linked to Terrestrial Litter Inputs. Science 277: 102-104.
","pubmedId":"","doi":"10.1126/science.277.5322.102"}],"title":"Water nutrient concentration and temperature control microbial-driven decomposition of leaf litter in oceanic island streams","reviews":[],"curatorReviews":[]},{"id":"ce54e9c6-add1-4263-a4f2-2bde84b47d98","decision":"publish","abstract":"Litter decomposition fuels food webs in forest streams. Thus, understanding the effects of water nutrients and temperature on litter decomposition and decomposers is key for predicting stream functioning under environmental change. We incubated leaf litter of woody species in streams to assess the effects of nutrient concentrations and temperature on microbial-driven litter decomposition and microbial activities. The relationship between biotic and abiotic variables was generally best described by a quadratic model: there was a hump-shaped relationship between biotic variables and nitrate concentrations and a U-shaped relationship between biotic variables and temperature. The stimulatory effect of high nutrient availability was limited by low temperature, while high nutrient availability alleviated low-temperature effects on litter decomposition and decomposers.
","acknowledgements":"We thank Ana Pereira and Ana Mafalda Cruz for technical work, IMAR – Institute of Marine Research for ergosterol quantification, and INOVA – Instituto de Inovação Tecnológica dos Açores for water nutrient determinations. We also thank the comments from anonymous reviewers on an earlier version of the manuscript.
","authors":[{"affiliations":["MARE – Marine and Environmental Sciences Centre, ARNET – Aquatic Research Network, Department of Life Sciences, University of Coimbra, Coimbra, Portugal"],"departments":[""],"credit":["conceptualization","fundingAcquisition","project","resources","dataCuration","formalAnalysis","validation","investigation","writing_originalDraft"],"email":"veronica@ci.uc.pt","firstName":"Verónica","lastName":"Ferreira","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-7688-2626"},{"affiliations":["University of the Azores, Faculty of Sciences and Technology, Ponta Delgada, Portugal","CIBIO – Research Centre in Biodiversity and Genetic Resources, InBIO Associate Laboratory, BIOPOLIS Program in Genomics, Biodiversity and Land Planning – UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands, University of the Azores, Ponta Delgada, Portugal"],"departments":["",""],"credit":["investigation","writing_reviewEditing"],"email":"pedro.mv.raposeiro@uac.pt","firstName":"Pedro M.","lastName":"Raposeiro","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7461-0851"},{"affiliations":["University of the Azores, Faculty of Sciences and Technology, Ponta Delgada, Portugal","CIBIO – Research Centre in Biodiversity and Genetic Resources, InBIO Associate Laboratory, BIOPOLIS Program in Genomics, Biodiversity and Land Planning – UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands, University of the Azores, Ponta Delgada, Portugal"],"departments":["",""],"credit":["project","resources","writing_reviewEditing"],"email":"vitor.mc.goncalves@uac.pt","firstName":"Vítor","lastName":"Gonçalves","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-5737-296X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/fa9c8bb02525601704721d091b3e8c13.csv"},"extendedData":[{"description":"Revised manuscript
","doi":null,"resourceType":"Text","name":"Ferreira&al_microPubBiol_paper rev clean.docx","url":"https://portal.micropublication.org/uploads/ff142d049da532f80b04dc57d6b0a928.docx"}],"funding":"This study was financed by the European Fund for Economic and Regional Development (FEDER) through the Program Operational Factors of Competitiveness (COMPETE; FCOMP-01-0124-FEDER-041055) and by National Funds through the Portuguese Foundation of Science and Technology (FCT; SFRH/BPD/76482/2011, EXPL/AAG-GLO/0189/2013, UID/MAR/04292/2013, SFRH/BPD/99461/2014, IF/00129/2014, CEECIND/02484/2018, UIDB/50027/2020, UID/50027, UID/50027/2025, UID/04292/2025, and LA/P/0069/2020).
","image":{"url":"https://portal.micropublication.org/uploads/da86d51ca26771b39443630e5a8ae417.jpg"},"imageCaption":"Figure 1. Relationships between biotic variables [microbial-driven litter decomposition rates (A, B), fungal biomass (C, D), and cumulative conidial production (E, F)] associated with leaf litter of three woody species (Acacia melanoxylon, Clethra arborea, and Pittosporum undulatum) and water nitrate concentration (left column) and temperature (right column) in six Azorean streams during Spring/Summer 2014. Stream labels: Lom1 – Lom4, streams located in Lombadas valley; AFG1 and AFG2, tributaries of Fogo lagoon.
Table 1. Relationships between biotic variables (microbial-driven litter decomposition rates, fungal biomass, and cumulative conidial production) associated with leaf litter of three woody species (Acacia melanoxylon, Clethra arborea, and Pittosporum undulatum) and water nitrate concentration and temperature in six Azorean streams streams during Spring/Summer 2014. In most cases, data were fitted to a quadratic function: y = ax2 + bx + c, where y is the biotic variable, x is the abiotic variable and a, b, and c are constants; a > 0 indicates a U-shaped curve and a < 0 indicates a hump-shaped curve. The curve vertex (i.e. the minimum value in a U-shaped curve or the maximum value in a hump-shaped curve) was calculated as: x = – (b / 2a) and y = ax2 + bx + c. The coefficient of determination of the models (R2) is also shown. The relationship between decomposition rates and nitrate concentration for C. arborea was fitted to a linear model (y = 0.00001x + 0.0078).
","imageTitle":"Relationships between biotic variables associated with leaf litter decomposition of three woody species and water nitrate concentration and temperature in six Azorean streams
","methods":"Leaf litter incubation was carried out in six small streams in the central massif of São Miguel island, Azores archipelago, North Atlantic Ocean. Four streams were tributaries of Ribeira Grande stream in Lombadas valley (Lom1 – Lom4) and two streams were tributaries of Fogo Lagoon (AFG1 and AFG2). Streams had similar geomorphology, were not visibly affected by direct human activities, were slightly alkaline (pH 7.7 – 8.1) and well oxygenated (8.4 – 9.1 mg O2/L), and varied in water temperature (13.4 – 15.6 ºC) and nutrient concentrations (28 – 1033 μg NO3–/L and 30 – 175 μg PO43–/L). Water temperature was recorded hourly over the 56-days incubation period during Spring and Summer 2014 using data loggers (Hobo Pendant UA-001-08, Onset Computer Corp., Massachusetts, USA) and hourly values were used to estimate daily means, which were averaged for each stream. On five occasions, stream water was filtered (fiberglass filters, 47-mm diameter, GF/C, Whatman, GE Healthcare Europe GmbH, Little Chalfont, UK) and used to determine nitrate concentration (ion chromatography; APHA 1995). A detailed description of the study area and streams is provided in Ferreira et al. (2016).
Air-dried leaves of three evergreen woody species commonly found in the riparian vegetation of Azorean streams were used to provide a gradient of litter palatability, in increasing order: Acacia melanoxylon (R. Br.) < Clethra arborea (Aiton) < Pittosporum undulatum (Vent.) (Ferreira et al., 2016). Leaves were weighed (2.90 – 3.10 g) and enclosed in fine-mesh bags (10 × 15 cm, 0.5-mm mesh), which prevent invertebrate access and where decomposition is mostly driven by microbial decomposers. Twelve litter bags per species were deployed in each stream on 11 or 12 June, 2014. Three replicate litter bags were recovered from each stream after 7, 21, 35 and 56 days, and transported cold to the laboratory. In the laboratory, litter was rinsed with distilled water and two sets of five leaf discs were extracted with a cork borer (12-mm diameter): one set was frozen at – 18 ºC for later determination of fungal biomass (Gessner 2020) and one set was used fresh to induce conidia production by aquatic hyphomycetes (Bärlocher 2020). The bulk litter mass was used to determine litter mass remaining (Ferreira et al., 2016).
For determination of fungal biomass, five frozen leaf discs were lyophilized (LY3TE, Snijders Scientific, Tilburg, The Netherlands), weighed (Kern 870, Kern & Sohn GmbH, Balingen, Germany) to determine dry mass, and incubated in alkaline methanol (8 g KOH/L, 30 min at 80 ºC) to extract ergosterol. The extract was purified using solid phase extraction cartridges (Waters Sep-Pak® Vac RC tC18 cartridges; Waters Corp., Massachusetts, USA) and eluted with isopropanol. Ergosterol was quantified by high-performance liquid chromatography (Dionex DX-120, California, USA), using a Thermo Scientific Syncronis C18 column and a Thermo Universal Uniguard holder 4/4.6 mm ID3 + Syncronis C18 drop in guard pre-column (Thermo, Waltham, Massachusetts, USA); the mobile phase was 100% methanol, kept at 33 ºC and flowing at 1.4 mL/min. Ergosterol was detected by reading absorbance at 282 nm with a UV detector and absorbance was converted into ergosterol concentration using a standard curve of ergosterol in isopropanol. Ergosterol concentration was converted into fungal biomass considering 5.5 mg ergosterol/mg fungal dry mass (Gessner and Chauvet 1993) and fungal biomass was expressed as mg/g litter AFDM, averaged across sampling dates.
Conidia production by aquatic hyphomycetes was determined after incubating five fresh leaf discs in 25 mL of filtered stream water (48 h at 13 ºC, 10 h light:14 h dark photoperiod, and 100 rpm). The conidia suspension was preserved with 2 mL of formalin and leaf discs were processed as described below for determination of discs ash-free dry mass. Suspensions were homogenized with 150 mL Triton X-100 and a magnetic stirring bar, and aliquots were filtered (nitrocellulose filters, 25-mm diameter, 5-mm pore size; Sartorius Stedim Biotech GmhH, Göttingen, Germany). Filters were stained with trypan blue in lactic acid and mounted on a slide. Conidia were counted at 320× magnification (DM1000 microscope, Leica, Wetzlar, Germany). Rates of conidia production were expressed as no. conidia/mg leaf AFDM/day, and cumulative (total) conidial production over the incubation period was estimated as the sum of daily values, which were derived by linear interpolation of the values of the adjacent sampling dates, and expressed as no. conidia/5 leaf discs.
For determination of litter mass remaining, litter (after extraction of leaf discs) was oven-dried (70 ºC, 48 h) and weighed (Kern 870, Kern & Sohn GmbH, Balingen, Germany) to assess dry mass (DM) remaining. DM remaining was ignited (500 ºC, 8 h) and weighed to assess ash mass. Ash-free dry mass (AFDM) remaining was estimated as the difference between DM and ash mass (after accounting for the extracted discs), and the fraction of AFDM remaining was estimated as the ratio between AFDM remaining and initial AFDM. Initial AFDM of samples was estimated as the product of initial air-dry mass by a conversion factor derived from extra sets of five fine-mesh bags per species. These extra bags, were prepared as the samples, taken to the field on day 0, immersed in water for ~ 10 min, and returned to the laboratory for determination of DM and AFDM as described above. The conversion factor was estimated as the ratio between initial AFDM and initial air-dry mass. Fraction AFDM remaining across the incubation times was used to estimate the overall exponential decomposition rate (k, /days) for each species and stream as the slope of a linear regression between ln(fraction of AFDM remaining) and time (days).
The relationships between biological variables (microbial-driven litter decomposition rates, average fungal biomass, and cumulative conidial production) of the three leaf litter species and abiotic variables [nitrate concentration (average across sampling dates) and water temperature (average across 56 days)] in six streams were analyzed by fitting the data to a quadratic function. The relationship between litter decomposition rates and nitrate concentration for C.arborea was fitted to a linear model. Models were derived using Statistica 7 software (StatSoft Inc., Tulsa, Oklahoma, USA).
","reagents":"","patternDescription":"Leaf litter decomposition is a key ecosystem process in forest streams, where litter inputs from the riparian vegetation are the main source of carbon and nutrients for aquatic food webs (Wallace et al., 1997). In island streams, where invertebrate shredders are generally rare (Benstead et al., 2009; MacKenzie et al., 2013; Raposeiro et al., 2014), litter decomposition is mostly driven by microbial decomposers, aquatic hyphomycetes in particular (Benstead et al., 2009; Larned 2000; MacKenzie et al., 2013; Raposeiro et al., 2014; Ferreira et al., 2016). Microbial decomposers are highly responsive to water temperature, with increases in temperature within organisms’ thermal ranges stimulating metabolic rates, and consequently the mineralization of litter carbon into CO2 through respiration (Ferreira and Chauvet 2011; Pérez et al., 2023). Microbial decomposers are also highly sensitive to nutrient availability, with increases in dissolved nutrient concentrations stimulating the use of litter as a source of carbon (Gulis and Suberkorpp 2003; Ferreira and Chauvet 2011). Consequently, increases in water temperature and nutrient availability generally translate into accelerated microbial-driven litter decomposition rates (Ferreira and Chauvet 2011; Fernandes et al., 2014). However, in island streams, variation in water temperature and nutrient concentrations, which may occur over a short distance due to steep changes in elevation and geology, do not necessary occur in the same direction, which complicates predictions of litter decomposition rates.
In this study, we incubated leaf litter of three woody species [Acacia melanoxylon (R. Br.), Clethra arborea (Aiton), and Pittosporum undulatum (Vent.)] in 0.5-mm mesh bags, for up to 56 days (with four sampling dates), in six streams on São Miguel island, North Atlantic Ocean, to assess microbial-driven litter decomposition rates, fungal biomass, and conidia production by aquatic hyphomycetes. The temporal dynamics of the biotic variables over the incubation period can be found in Ferreira et al. (2016), while here we report on the relationships between the biotic variables and water nitrate concentration and temperature to assess the interaction between these two abiotic variables in moderating biotic responses under natural settings.
The relationship between biotic variables and nitrate concentration generally followed a hump-shaped curve, with maximum decomposition rates (across species) of 0.0082 – 0.0460/d estimated at 500 – 714 μg NO3–/L, maximum fungal biomass of 50 – 73 mg/g AFDM estimated at 623 – 663 μg NO3–/L, and maximum cumulative conidial production of 3.8 million – 6.9 million conidia/5 leaf discs estimated at 565 – 639 μg NO3–/L (Figure 1, Table 1). The relationship between decomposition rates and nitrate concentration was linear for C. arborea (Figure 1, Table 1). The relationship between biotic variables and water temperature followed a U-shaped curve, with minimum decomposition rates (across species) of 0.0047 – 0.0259/d estimated at 14.0 – 14.7 ºC, minimum fungal biomass of 15 – 42 mg/g AFDM estimated at 14.3 – 14.4 ºC, and minimum cumulative conidial production of –28219 – 384022 conidia/5 leaf discs estimated at 14.0 – 14.3 ºC (Figure 1, Table 1).
Litter decomposition and associated microbial activities were stimulated when nutrient availability and water temperature increased simultaneously across the group of streams including Lom1, Lom2, Lom4, AFG1, and AFG2 (Figure 1), as anticipated (Ferreira and Chauvet 2011; Fernandes et al., 2014). This stimulation was more evident for the fast-decomposing P. undulatum litter than for the other two species. This suggests that litter is more sensitive to increases in nutrient availability and temperature at more advanced stages of decomposition, which can be due to better microbial colonization of the litter and to the litter increased recalcitrance and thus higher dependence of decomposers on dissolved nutrients. Consistent with this interpretation, other studies found stronger differences in litter mass remaining across streams mostly at later sampling dates (Gulis and Suberkropp 2003; Ferreira et al., 2016, 2021).
However, when an increase in nutrient concentrations was accompanied by a decrease in water temperature (stream Lom3), litter decomposition and associated microbial activities were lower compared with what was expected from the increase in nutrient concentrations alone (see hump-shaped relationships) and higher when compared with what was expected from the decrease in water temperature alone (see U-shaped relationships) (Figure 1). This suggests that water temperature was the limiting factor for maximum litter decomposition rates and associated microbial activities in this stream. The role of temperature as a limiting factor was observed when comparing temperatures that cannot be considered extreme (13.4 – 15.5 ºC) and are within a short interval (2.1 ºC), suggesting that even small changes in temperature may have strong effects on decomposer activity and litter decomposition. Contrarily, higher nutrient availability may alleviate the effect of low water temperature as an increase in nutrient availability stimulated litter decomposition and associated microbial activities at lower temperature, although generally not at levels observed when temperature was higher. This agrees with Fernandes et al. (2014), who showed that higher nitrogen concentrations are needed to achieve maximum microbial activities at lower temperature.
Although the relationship between biotic variables and water temperature was described by a U-shaped curve, there was high variability at the highest temperatures with stream Lom2 showing higher litter decomposition rates (1.1 – 2.3-fold), fungal biomass (1.5 – 4.6-fold), and cumulative conidial production (1.9 – 5.9-fold) than stream Lom1 (Figure 1). This variation in biotic variables between both streams with similar water temperature (mean ± SE: 15.6 ± 0.1 ºC in Lom1 and 15.5 ± 0.1 ºC in Lom2) can be attributed to differences in dissolved nutrient concentration, which was 2.5-fold higher in stream Lom2 (588 ± 19 mg/L) than in stream Lom1 (238 ± 24 mg/L). This suggests that lower nutrient concentration was the limiting factor for maximum litter decomposition rates and associated microbial activities in stream Lom1.
","references":[{"reference":"Bärlocher F. 2020. Sporulation by Aquatic Hyphomycetes. Methods to Study Litter Decomposition : 241-245.
","pubmedId":"","doi":"10.1007/978-3-030-30515-4_26"},{"reference":"Benstead JP, March JG, Pringle CM, Ewel KC, Short JW. 2009. Biodiversity and ecosystem function in species-poor communities: community structure and leaf litter breakdown in a Pacific island stream. Journal of the North American Benthological Society 28: 454-465.
","pubmedId":"","doi":"10.1899/07-081.1"},{"reference":"Fernandes I, Seena S, Pascoal C, Cássio F. 2014. Elevated temperature may intensify the positive effects of nutrients on microbial decomposition in streams. Freshwater Biology 59: 2390-2399.
","pubmedId":"","doi":"10.1111/fwb.12445"},{"reference":"Ferreira V, Chauvet E. 2010. Synergistic effects of water temperature and dissolved nutrients on litter decomposition and associated fungi. Global Change Biology 17: 551-564.
","pubmedId":"","doi":"10.1111/j.1365-2486.2010.02185.x"},{"reference":"Ferreira V, Raposeiro PM, Pereira A, Cruz AM, Costa AC, Graça MAS, Gonçalves V. 2016. Leaf litter decomposition in remote oceanic island streams is driven by microbes and depends on litter quality and environmental conditions. Freshwater Biology 61: 783-799.
","pubmedId":"","doi":"10.1111/fwb.12749"},{"reference":"Ferreira V, Silva J, Cornut J, Sobral O, Bachelet Q, Bouquerel J, Danger M. 2021. Organic-matter decomposition as a bioassessment tool of stream functioning: A comparison of eight decomposition-based indicators exposed to different environmental changes. Environmental Pollution 290: 118111.
","pubmedId":"","doi":"10.1016/j.envpol.2021.118111"},{"reference":"Gessner MO. 2020. Ergosterol as a Measure of Fungal Biomass. Methods to Study Litter Decomposition : 247-255.
","pubmedId":"","doi":"10.1007/978-3-030-30515-4_27"},{"reference":"Gessner MO, Chauvet E. 1993. Ergosterol-to-Biomass Conversion Factors for Aquatic Hyphomycetes. Applied and Environmental Microbiology 59: 502-507.
","pubmedId":"","doi":"10.1128/aem.59.2.502-507.1993"},{"reference":"Gulis V, Suberkropp K. 2002. Leaf litter decomposition and microbial activity in nutrient‐enriched and unaltered reaches of a headwater stream. Freshwater Biology 48: 123-134.
","pubmedId":"","doi":"10.1046/j.1365-2427.2003.00985.x"},{"reference":"Larned ST. 2000. Dynamics of coarse riparian detritus in a Hawaiian stream ecosystem: a comparison of drought and post-drought conditions. Journal of the North American Benthological Society 19: 215-234.
","pubmedId":"","doi":"10.2307/1468066"},{"reference":"MacKenzie RA, Wiegner TN, Kinslow F, Cormier N, Strauch AM. 2013. Leaf-litter inputs from an invasive nitrogen-fixing tree influence organic-matter dynamics and nitrogen inputs in a Hawaiian river. Freshwater Science 32: 1036-1052.
","pubmedId":"","doi":"10.1899/12-152.1"},{"reference":"Pérez J, Cornejo A, Alonso A, Guerra A, García G, Nieto C, et al., Boyero. 2023. Warming overrides eutrophication effects on leaf litter decomposition in stream microcosms. Environmental Pollution 332: 121966.
","pubmedId":"","doi":"10.1016/j.envpol.2023.121966"},{"reference":"Prescott CE. 2010. Litter decomposition: what controls it and how can we alter it to sequester more carbon in forest soils?. Biogeochemistry 101: 133-149.
","pubmedId":"","doi":"10.1007/s10533-010-9439-0"},{"reference":"Raposeiro PM, Martins GM, Moniz I, Cunha A, Costa AC, Gonçalves V. 2014. Leaf litter decomposition in remote oceanic islands: The role of macroinvertebrates vs. microbial decomposition of native vs. exotic plant species. Limnologica 45: 80-87.
","pubmedId":"","doi":"10.1016/j.limno.2013.10.006"},{"reference":"Wallace JB, Eggert SL, Meyer JL, Webster JR. 1997. Multiple Trophic Levels of a Forest Stream Linked to Terrestrial Litter Inputs. Science 277: 102-104.
","pubmedId":"","doi":"10.1126/science.277.5322.102"}],"title":"Water nutrient concentration and temperature control microbial-driven decomposition of leaf litter in oceanic island streams","reviews":[],"curatorReviews":[]},{"id":"1cf3f7d5-e621-4bbc-83ab-320b8eca8522","decision":"publish","abstract":"Litter decomposition fuels food webs in forest streams. Thus, understanding the effects of water nutrients and temperature on litter decomposition and decomposers is key for predicting stream functioning under environmental change. We incubated leaf litter of woody species in streams to assess the effects of nutrient concentrations and temperature on microbial-driven litter decomposition and microbial activities. The relationship between biotic and abiotic variables was generally best described by a quadratic model: there was a hump-shaped relationship between biotic variables and nitrate concentrations and a U-shaped relationship between biotic variables and temperature. The stimulatory effect of high nutrient availability was limited by low temperature, while high nutrient availability alleviated low-temperature effects on litter decomposition and decomposers.
","acknowledgements":"We thank Ana Pereira and Ana Mafalda Cruz for technical work, IMAR – Institute of Marine Research for ergosterol quantification, and INOVA – Instituto de Inovação Tecnológica dos Açores for water nutrient determinations. We also thank the comments from anonymous reviewers on an earlier version of the manuscript.
","authors":[{"affiliations":["MARE – Marine and Environmental Sciences Centre, ARNET – Aquatic Research Network, Department of Life Sciences, University of Coimbra, Coimbra, Portugal"],"departments":[""],"credit":["conceptualization","fundingAcquisition","project","resources","dataCuration","formalAnalysis","validation","investigation","writing_originalDraft"],"email":"veronica@ci.uc.pt","firstName":"Verónica","lastName":"Ferreira","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-7688-2626"},{"affiliations":["University of the Azores, Faculty of Sciences and Technology, Ponta Delgada, Portugal","CIBIO – Research Centre in Biodiversity and Genetic Resources, InBIO Associate Laboratory, BIOPOLIS Program in Genomics, Biodiversity and Land Planning – UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands, University of the Azores, Ponta Delgada, Portugal"],"departments":["",""],"credit":["investigation","writing_reviewEditing"],"email":"pedro.mv.raposeiro@uac.pt","firstName":"Pedro M.","lastName":"Raposeiro","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7461-0851"},{"affiliations":["University of the Azores, Faculty of Sciences and Technology, Ponta Delgada, Portugal","CIBIO – Research Centre in Biodiversity and Genetic Resources, InBIO Associate Laboratory, BIOPOLIS Program in Genomics, Biodiversity and Land Planning – UNESCO Chair – Land Within Sea: Biodiversity & Sustainability in Atlantic Islands, University of the Azores, Ponta Delgada, Portugal"],"departments":["",""],"credit":["project","resources","writing_reviewEditing"],"email":"vitor.mc.goncalves@uac.pt","firstName":"Vítor","lastName":"Gonçalves","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-5737-296X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/fa9c8bb02525601704721d091b3e8c13.csv"},"extendedData":[{"description":"Revised manuscript
","doi":null,"resourceType":"Text","name":"Ferreira&al_microPubBiol_paper rev clean.docx","url":"https://portal.micropublication.org/uploads/ff142d049da532f80b04dc57d6b0a928.docx"}],"funding":"This study was financed by the European Fund for Economic and Regional Development (FEDER) through the Program Operational Factors of Competitiveness (COMPETE; FCOMP-01-0124-FEDER-041055) and by National Funds through the Portuguese Foundation of Science and Technology (FCT; SFRH/BPD/76482/2011, EXPL/AAG-GLO/0189/2013, UID/MAR/04292/2013, SFRH/BPD/99461/2014, IF/00129/2014, CEECIND/02484/2018, UIDB/50027/2020, UID/50027, UID/50027/2025, UID/04292/2025, and LA/P/0069/2020).
","image":{"url":"https://portal.micropublication.org/uploads/da86d51ca26771b39443630e5a8ae417.jpg"},"imageCaption":"Figure 1. Relationships between biotic variables [microbial-driven litter decomposition rates (A), fungal biomass (B), and cumulative conidial production (C)] associated with leaf litter of three woody species (Acacia melanoxylon, Clethra arborea, and Pittosporum undulatum) and water nitrate concentration (left column) and temperature (right column) in six Azorean streams during Spring/Summer 2014. Stream labels: Lom1 – Lom4, streams located in Lombadas valley; AFG1 and AFG2, tributaries of Fogo lagoon.
Table 1. Relationships between biotic variables (microbial-driven litter decomposition rates, fungal biomass, and cumulative conidial production) associated with leaf litter of three woody species (Acacia melanoxylon, Clethra arborea, and Pittosporum undulatum) and water nitrate concentration and temperature in six Azorean streams streams during Spring/Summer 2014. In most cases, data were fitted to a quadratic function: y = ax2 + bx + c, where y is the biotic variable, x is the abiotic variable and a, b, and c are constants; a > 0 indicates a U-shaped curve and a < 0 indicates a hump-shaped curve. The curve vertex (i.e. the minimum value in a U-shaped curve or the maximum value in a hump-shaped curve) was calculated as: x = – (b / 2a) and y = ax2 + bx + c. The coefficient of determination of the models (R2) is also shown. The relationship between decomposition rates and nitrate concentration for C. arborea was fitted to a linear model (y = 0.00001x + 0.0078).
","imageTitle":"Relationships between biotic variables associated with leaf litter decomposition of three woody species and water nitrate concentration and temperature in six Azorean streams
","methods":"Leaf litter incubation was carried out in six small streams in the central massif of São Miguel island, Azores archipelago, North Atlantic Ocean. Four streams were tributaries of Ribeira Grande stream in Lombadas valley (Lom1 – Lom4) and two streams were tributaries of Fogo Lagoon (AFG1 and AFG2). Streams had similar geomorphology, were not visibly affected by direct human activities, were slightly alkaline (pH 7.7 – 8.1) and well oxygenated (8.4 – 9.1 mg O2/L), and varied in water temperature (13.4 – 15.6 ºC) and nutrient concentrations (28 – 1033 μg NO3–/L and 30 – 175 μg PO43–/L). Water temperature was recorded hourly over the 56-days incubation period during Spring and Summer 2014 using data loggers (Hobo Pendant UA-001-08, Onset Computer Corp., Massachusetts, USA) and hourly values were used to estimate daily means, which were averaged for each stream. On five occasions, stream water was filtered (fiberglass filters, 47-mm diameter, GF/C, Whatman, GE Healthcare Europe GmbH, Little Chalfont, UK) and used to determine nitrate concentration (ion chromatography; APHA 1995). A detailed description of the study area and streams is provided in Ferreira et al. (2016).
Air-dried leaves of three evergreen woody species commonly found in the riparian vegetation of Azorean streams were used to provide a gradient of litter palatability, in increasing order: Acacia melanoxylon (R. Br.) < Clethra arborea (Aiton) < Pittosporum undulatum (Vent.) (Ferreira et al., 2016). Leaves were weighed (2.90 – 3.10 g) and enclosed in fine-mesh bags (10 × 15 cm, 0.5-mm mesh), which prevent invertebrate access and where decomposition is mostly driven by microbial decomposers. Twelve litter bags per species were deployed in each stream on 11 or 12 June, 2014. Three replicate litter bags were recovered from each stream after 7, 21, 35 and 56 days, and transported cold to the laboratory. In the laboratory, litter was rinsed with distilled water and two sets of five leaf discs were extracted with a cork borer (12-mm diameter): one set was frozen at – 18 ºC for later determination of fungal biomass (Gessner 2020) and one set was used fresh to induce conidia production by aquatic hyphomycetes (Bärlocher 2020). The bulk litter mass was used to determine litter mass remaining (Ferreira et al., 2016).
For determination of fungal biomass, five frozen leaf discs were lyophilized (LY3TE, Snijders Scientific, Tilburg, The Netherlands), weighed (Kern 870, Kern & Sohn GmbH, Balingen, Germany) to determine dry mass, and incubated in alkaline methanol (8 g KOH/L, 30 min at 80 ºC) to extract ergosterol. The extract was purified using solid phase extraction cartridges (Waters Sep-Pak® Vac RC tC18 cartridges; Waters Corp., Massachusetts, USA) and eluted with isopropanol. Ergosterol was quantified by high-performance liquid chromatography (Dionex DX-120, California, USA), using a Thermo Scientific Syncronis C18 column and a Thermo Universal Uniguard holder 4/4.6 mm ID3 + Syncronis C18 drop in guard pre-column (Thermo, Waltham, Massachusetts, USA); the mobile phase was 100% methanol, kept at 33 ºC and flowing at 1.4 mL/min. Ergosterol was detected by reading absorbance at 282 nm with a UV detector and absorbance was converted into ergosterol concentration using a standard curve of ergosterol in isopropanol. Ergosterol concentration was converted into fungal biomass considering 5.5 mg ergosterol/mg fungal dry mass (Gessner and Chauvet 1993) and fungal biomass was expressed as mg/g litter AFDM, averaged across sampling dates.
Conidia production by aquatic hyphomycetes was determined after incubating five fresh leaf discs in 25 mL of filtered stream water (48 h at 13 ºC, 10 h light:14 h dark photoperiod, and 100 rpm). The conidia suspension was preserved with 2 mL of formalin and leaf discs were processed as described below for determination of discs ash-free dry mass. Suspensions were homogenized with 150 mL Triton X-100 and a magnetic stirring bar, and aliquots were filtered (nitrocellulose filters, 25-mm diameter, 5-mm pore size; Sartorius Stedim Biotech GmhH, Göttingen, Germany). Filters were stained with trypan blue in lactic acid and mounted on a slide. Conidia were counted at 320× magnification (DM1000 microscope, Leica, Wetzlar, Germany). Rates of conidia production were expressed as no. conidia/mg leaf AFDM/day, and cumulative (total) conidial production over the incubation period was estimated as the sum of daily values, which were derived by linear interpolation of the values of the adjacent sampling dates, and expressed as no. conidia/5 leaf discs.
For determination of litter mass remaining, litter (after extraction of leaf discs) was oven-dried (70 ºC, 48 h) and weighed (Kern 870, Kern & Sohn GmbH, Balingen, Germany) to assess dry mass (DM) remaining. DM remaining was ignited (500 ºC, 8 h) and weighed to assess ash mass. Ash-free dry mass (AFDM) remaining was estimated as the difference between DM and ash mass (after accounting for the extracted discs), and the fraction of AFDM remaining was estimated as the ratio between AFDM remaining and initial AFDM. Initial AFDM of samples was estimated as the product of initial air-dry mass by a conversion factor derived from extra sets of five fine-mesh bags per species. These extra bags, were prepared as the samples, taken to the field on day 0, immersed in water for ~ 10 min, and returned to the laboratory for determination of DM and AFDM as described above. The conversion factor was estimated as the ratio between initial AFDM and initial air-dry mass. Fraction AFDM remaining across the incubation times was used to estimate the overall exponential decomposition rate (k, /days) for each species and stream as the slope of a linear regression between ln(fraction of AFDM remaining) and time (days).
The relationships between biological variables (microbial-driven litter decomposition rates, average fungal biomass, and cumulative conidial production) of the three leaf litter species and abiotic variables [nitrate concentration (average across sampling dates) and water temperature (average across 56 days)] in six streams were analyzed by fitting the data to a quadratic function. The relationship between litter decomposition rates and nitrate concentration for C.arborea was fitted to a linear model. Models were derived using Statistica 7 software (StatSoft Inc., Tulsa, Oklahoma, USA).
","reagents":"","patternDescription":"Leaf litter decomposition is a key ecosystem process in forest streams, where litter inputs from the riparian vegetation are the main source of carbon and nutrients for aquatic food webs (Wallace et al., 1997). In island streams, where invertebrate shredders are generally rare (Benstead et al., 2009; MacKenzie et al., 2013; Raposeiro et al., 2014), litter decomposition is mostly driven by microbial decomposers, aquatic hyphomycetes in particular (Benstead et al., 2009; Larned 2000; MacKenzie et al., 2013; Raposeiro et al., 2014; Ferreira et al., 2016). Microbial decomposers are highly responsive to water temperature, with increases in temperature within organisms’ thermal ranges stimulating metabolic rates, and consequently the mineralization of litter carbon into CO2 through respiration (Ferreira and Chauvet 2011; Pérez et al., 2023). Microbial decomposers are also highly sensitive to nutrient availability, with increases in dissolved nutrient concentrations stimulating the use of litter as a source of carbon (Gulis and Suberkorpp 2003; Ferreira and Chauvet 2011). Consequently, increases in water temperature and nutrient availability generally translate into accelerated microbial-driven litter decomposition rates (Ferreira and Chauvet 2011; Fernandes et al., 2014). However, in island streams, variation in water temperature and nutrient concentrations, which may occur over a short distance due to steep changes in elevation and geology, do not necessary occur in the same direction, which complicates predictions of litter decomposition rates.
In this study, we incubated leaf litter of three woody species [Acacia melanoxylon (R. Br.), Clethra arborea (Aiton), and Pittosporum undulatum (Vent.)] in 0.5-mm mesh bags, for up to 56 days (with four sampling dates), in six streams on São Miguel island, North Atlantic Ocean, to assess microbial-driven litter decomposition rates, fungal biomass, and conidia production by aquatic hyphomycetes. The temporal dynamics of the biotic variables over the incubation period can be found in Ferreira et al. (2016), while here we report on the relationships between the biotic variables and water nitrate concentration and temperature to assess the interaction between these two abiotic variables in moderating biotic responses under natural settings.
The relationship between biotic variables and nitrate concentration generally followed a hump-shaped curve, with maximum decomposition rates (across species) of 0.0082 – 0.0460/d estimated at 500 – 714 μg NO3–/L, maximum fungal biomass of 50 – 73 mg/g AFDM estimated at 623 – 663 μg NO3–/L, and maximum cumulative conidial production of 3.8 million – 6.9 million conidia/5 leaf discs estimated at 565 – 639 μg NO3–/L (Figure 1, Table 1). The relationship between decomposition rates and nitrate concentration was linear for C. arborea (Figure 1, Table 1). The relationship between biotic variables and water temperature followed a U-shaped curve, with minimum decomposition rates (across species) of 0.0047 – 0.0259/d estimated at 14.0 – 14.7 ºC, minimum fungal biomass of 15 – 42 mg/g AFDM estimated at 14.3 – 14.4 ºC, and minimum cumulative conidial production of –28219 – 384022 conidia/5 leaf discs estimated at 14.0 – 14.3 ºC (Figure 1, Table 1).
Litter decomposition and associated microbial activities were stimulated when nutrient availability and water temperature increased simultaneously across the group of streams including Lom1, Lom2, Lom4, AFG1, and AFG2 (Figure 1), as anticipated (Ferreira and Chauvet 2011; Fernandes et al., 2014). This stimulation was more evident for the fast-decomposing P. undulatum litter than for the other two species. This suggests that litter is more sensitive to increases in nutrient availability and temperature at more advanced stages of decomposition, which can be due to better microbial colonization of the litter and to the litter increased recalcitrance and thus higher dependence of decomposers on dissolved nutrients. Consistent with this interpretation, other studies found stronger differences in litter mass remaining across streams mostly at later sampling dates (Gulis and Suberkropp 2003; Ferreira et al., 2016, 2021).
However, when an increase in nutrient concentrations was accompanied by a decrease in water temperature (stream Lom3), litter decomposition and associated microbial activities were lower compared with what was expected from the increase in nutrient concentrations alone (see hump-shaped relationships) and higher when compared with what was expected from the decrease in water temperature alone (see U-shaped relationships) (Figure 1). This suggests that water temperature was the limiting factor for maximum litter decomposition rates and associated microbial activities in this stream. The role of temperature as a limiting factor was observed when comparing temperatures that cannot be considered extreme (13.4 – 15.5 ºC) and are within a short interval (2.1 ºC), suggesting that even small changes in temperature may have strong effects on decomposer activity and litter decomposition. Contrarily, higher nutrient availability may alleviate the effect of low water temperature as an increase in nutrient availability stimulated litter decomposition and associated microbial activities at lower temperature, although generally not at levels observed when temperature was higher. This agrees with Fernandes et al. (2014), who showed that higher nitrogen concentrations are needed to achieve maximum microbial activities at lower temperature.
Although the relationship between biotic variables and water temperature was described by a U-shaped curve, there was high variability at the highest temperatures with stream Lom2 showing higher litter decomposition rates (1.1 – 2.3-fold), fungal biomass (1.5 – 4.6-fold), and cumulative conidial production (1.9 – 5.9-fold) than stream Lom1 (Figure 1). This variation in biotic variables between both streams with similar water temperature (mean ± SE: 15.6 ± 0.1 ºC in Lom1 and 15.5 ± 0.1 ºC in Lom2) can be attributed to differences in dissolved nutrient concentration, which was 2.5-fold higher in stream Lom2 (588 ± 19 mg/L) than in stream Lom1 (238 ± 24 mg/L). This suggests that lower nutrient concentration was the limiting factor for maximum litter decomposition rates and associated microbial activities in stream Lom1.
","references":[{"reference":"Bärlocher F. 2020. Sporulation by Aquatic Hyphomycetes. Methods to Study Litter Decomposition : 241-245.
","pubmedId":"","doi":"10.1007/978-3-030-30515-4_26"},{"reference":"Benstead JP, March JG, Pringle CM, Ewel KC, Short JW. 2009. Biodiversity and ecosystem function in species-poor communities: community structure and leaf litter breakdown in a Pacific island stream. Journal of the North American Benthological Society 28: 454-465.
","pubmedId":"","doi":"10.1899/07-081.1"},{"reference":"Fernandes I, Seena S, Pascoal C, Cássio F. 2014. Elevated temperature may intensify the positive effects of nutrients on microbial decomposition in streams. Freshwater Biology 59: 2390-2399.
","pubmedId":"","doi":"10.1111/fwb.12445"},{"reference":"Ferreira V, Chauvet E. 2010. Synergistic effects of water temperature and dissolved nutrients on litter decomposition and associated fungi. Global Change Biology 17: 551-564.
","pubmedId":"","doi":"10.1111/j.1365-2486.2010.02185.x"},{"reference":"Ferreira V, Raposeiro PM, Pereira A, Cruz AM, Costa AC, Graça MAS, Gonçalves V. 2016. Leaf litter decomposition in remote oceanic island streams is driven by microbes and depends on litter quality and environmental conditions. Freshwater Biology 61: 783-799.
","pubmedId":"","doi":"10.1111/fwb.12749"},{"reference":"Ferreira V, Silva J, Cornut J, Sobral O, Bachelet Q, Bouquerel J, Danger M. 2021. Organic-matter decomposition as a bioassessment tool of stream functioning: A comparison of eight decomposition-based indicators exposed to different environmental changes. Environmental Pollution 290: 118111.
","pubmedId":"","doi":"10.1016/j.envpol.2021.118111"},{"reference":"Gessner MO. 2020. Ergosterol as a Measure of Fungal Biomass. Methods to Study Litter Decomposition : 247-255.
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","pubmedId":"","doi":"10.1128/aem.59.2.502-507.1993"},{"reference":"Gulis V, Suberkropp K. 2002. Leaf litter decomposition and microbial activity in nutrient‐enriched and unaltered reaches of a headwater stream. Freshwater Biology 48: 123-134.
","pubmedId":"","doi":"10.1046/j.1365-2427.2003.00985.x"},{"reference":"Larned ST. 2000. Dynamics of coarse riparian detritus in a Hawaiian stream ecosystem: a comparison of drought and post-drought conditions. Journal of the North American Benthological Society 19: 215-234.
","pubmedId":"","doi":"10.2307/1468066"},{"reference":"MacKenzie RA, Wiegner TN, Kinslow F, Cormier N, Strauch AM. 2013. Leaf-litter inputs from an invasive nitrogen-fixing tree influence organic-matter dynamics and nitrogen inputs in a Hawaiian river. Freshwater Science 32: 1036-1052.
","pubmedId":"","doi":"10.1899/12-152.1"},{"reference":"Pérez J, Cornejo A, Alonso A, Guerra A, García G, Nieto C, et al., Boyero. 2023. Warming overrides eutrophication effects on leaf litter decomposition in stream microcosms. Environmental Pollution 332: 121966.
","pubmedId":"","doi":"10.1016/j.envpol.2023.121966"},{"reference":"Prescott CE. 2010. Litter decomposition: what controls it and how can we alter it to sequester more carbon in forest soils?. Biogeochemistry 101: 133-149.
","pubmedId":"","doi":"10.1007/s10533-010-9439-0"},{"reference":"Raposeiro PM, Martins GM, Moniz I, Cunha A, Costa AC, Gonçalves V. 2014. Leaf litter decomposition in remote oceanic islands: The role of macroinvertebrates vs. microbial decomposition of native vs. exotic plant species. Limnologica 45: 80-87.
","pubmedId":"","doi":"10.1016/j.limno.2013.10.006"},{"reference":"Wallace JB, Eggert SL, Meyer JL, Webster JR. 1997. Multiple Trophic Levels of a Forest Stream Linked to Terrestrial Litter Inputs. Science 277: 102-104.
","pubmedId":"","doi":"10.1126/science.277.5322.102"}],"title":"Water nutrient concentration and temperature control microbial-driven decomposition of leaf litter in oceanic island streams","reviews":[],"curatorReviews":[]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon chilense","label":"Adenocaulon 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