Use of Multi-Carbon Sources by Zooplankton in an Oligotrophic Lake in the Tibetan Plateau

Abstract

We applied natural abundance stable isotope δ¹³C and radiocarbon ∆¹⁴C analyses to investigate trophic linkages between zooplankton and their potential food sources (phytoplankton, submersed plants, and allochthonous organic carbon) in Lake Nam Co, one of the largest oligosaline and oligotrophic lakes in the Tibetan Plateau, in south-west China. The δ¹³C and ∆¹⁴C levels of the calanoid copepod Arctodiaptomus altissimus pectinatus indicate that it uses different carbon sources. Thus, based on a two-isotope mixing model, our results suggested that recently synthesized but ¹⁴C-depleted primary producers (phytoplankton and submersed plants) were the most important sources of carbon, together contributing 92.2% of the zooplankton biomass. Allochthonous organic carbon and dissolved organic carbon constituted 4.7% and 3.1% of the carbon in the diet of zooplankton, respectively. Our findings from Lake Nam Co suggest that the carbon in the food webs of lakes located in a glaciated environment originates from various sources of different ages.

1. Introduction

Zooplankton have significant impacts on the flow of material and energy in aquatic ecosystems and constitute important links in lake food webs between primary producers, microorganisms, and higher level consumers [1,2]. Traditionally, zooplankton are considered to mainly feed on organic matter derived from contemporary photosynthesis because of its lability and palatability. Phytoplankton is the primary food source and is often grazed upon directly by the zooplankton. In some lakes, macrophytes may also support zooplankton production via bacterioplankton and the microbial food web [3]. Recent research has, however, suggested that zooplankton often exploit a wider variety of food sources, including terrestrial organic carbon (t-OC) [4]. Stable isotope analysis and lake ¹³C labeling techniques have thus demonstrated a significant contribution by t-OC to zooplankton biomass [5,6,7]. Zooplankton are capable of assimilating allochthonous material of terrestrial origin [8,9], either through consumption of micro-heterotrophic organisms using dissolved organic carbon (DOC) or by direct exploitation of particulate organic carbon (POC) [4,6].

Glaciated environments have dynamic and reactive organic carbon systems [10,11,12]. Recent studies have revealed that modern glacier runoffs export organic matter to aquatic ecosystems [13,14,15]. This organic matter is a potentially important carbon source to heterotrophic microbes [16,17], invertebrates [18,19], and some consumers at higher trophic levels such as fish and waterfowl [19,20].

The Tibetan Plateau (TiP) is a source and sink region of some large rivers and lakes. In the past several decades, the impact of climate warming on ecosystems has been more pronounced in TiP than in other regions of the Earth, including phenomena such as increasing/faster retreat of glaciers and a degrading permafrost soil horizon [21,22]. Driven by climate warming, organic materials may be released and transported to the downstream lakes [15]. Given the severe energy limitations in proglacial lakes, t-OC from glaciated watersheds is proposed to be a particularly important cross-ecosystem subsidy that helps buffer energy limitations set by contemporary primary production. Here, we tested the hypothesis that proglacial lake food webs are sustained by t-OC released from glacial ecosystems by using natural abundance stable isotope (δ¹³C) and radiocarbon (Δ¹⁴C) analyses of zooplankton and potential carbon sources in Lake Nam Co, a typical mid-latitude and high-altitude lake in TiP. Moreover, an IsoSource mixing model was used to estimate the relative contribution of potential carbon sources to zooplankton.

2. Materials and Methods

2.1. Study Site Description

Lake Nam Co (Figure 1,

Table 1. Water characteristics of Lake Nam Co.

Altitude	pH	Total Nitrogen	Total Phosphorus	Chlorophyll-a	Salinity	POC	DOC
4730 m a.s.l.	9.9	0.37 mg/L	0.003 mg/L	0.26 μg/L	0.98 g/L	1.32 mg/L	11.93 mg/L

), one of the largest oligosaline and oligotrophic lakes in China, is located in central TiP. It has a surface area of about 1982 km² and a water depth exceeding 90 m [23]. It is a closed lake that is mainly supplied by precipitation and glacier melt water from the south-eastern Nyainqen Tanglha Mountains. The lake is a unique habitat and only insignificantly impacted by human activities due to its harsh, high-altitude natural environment characterized by strong radiation and low temperatures.

2.2. Sample Collection

A 1 L water sample was taken from Lake Nam Co and filtered through pre-combusted Whatman GF/F glass fiber filters (450 °C for 4 h; 0.7 μm nominal pore size) for ¹⁴C analysis of DIC (Dissolved Inorganic Carbon). The DIC samples were stored in pre-cleaned gas-tight polypropylene Nalgene bottles containing 100 μL saturated HgCl₂ solution in the dark at room temperature. POC samples were taken by filtering 10 L of water through pre-combusted Whatman GF/F glass fiber filters for analysis of fatty acids. The filters were subsequently placed in pre-combusted aluminum foil and freeze dried. The submersed plant Ceratophyllum demersum was collected by hand and rinsed with deionized water to remove detritus. Zooplankton (the calanoid copepod Arctodiaptomus altissimus pectinatus, which was the dominant zooplankton species in the lake) were collected by vertical tows through the water column with a 140 μm plankton net and subsequently left for 6 h in filtered lake water to allow gut evacuation. The zooplankton samples were freeze dried and preserved in pre-combusted aluminum foil or pre-cleaned cryogenic vials for further treatment. To account for the fact that terrestrially produced OC could be delivered to the lake during summer flows, POC samples from one main inflowing (Figure 1) stream were also taken to represent allochthonous carbon sources.

2.3. Radiocarbon and Stable Carbon Isotope Analysis

Stable ¹³C isotope analysis was performed using a DeltaPlus Advantage mass spectrometer (Finnigan MAT) connected to a Flash EA1112 elemental analyzer at Nanjing Institute of Geography and Limnology, Nanjing, China. The stable C isotope value is calculated as δ¹³C (‰) = (R_sample/R_standard − 1) × 10³, i.e., parts-per-thousand (‰) deviations from international standards (PeeDee Belemnite for δ¹³C), where R = ¹³C/¹²C. The analytical error between repeated measurements was typically within ±0.1‰ [24].

Radiocarbon ¹⁴C isotope measurements were performed by the Accelerator Mass Spectrometry Facility at the Beta Analytic Radiocarbon Dating Laboratory, which is an ISO 17025-accredited radiocarbon dating laboratory in Miami, Florida. The ∆¹⁴C value is expressed as ∆¹⁴C (‰) = δ¹⁴C − 2(δ¹³C + 25) (1 + δ¹⁴C/1000), i.e., the deviation in parts per thousand (‰) from the ¹⁴C activity of 1950s oxalic acid, where δ¹⁴C (‰) = [(R_sample/R_standard) − 1] × 10³, R = ¹⁴C/¹²C. The analytical precision for ¹⁴C analyses averaged ±3.73‰ [25].

We estimated the δ¹³C value of phytoplankton using phospholipid fatty acids (PLFAs), which represent specific biomarkers for phytoplankton groups [9]. Separation and extraction of main PLFAs from POC samples were performed. In brief, PLFAs were extracted according to a modified Bligh and Dyer procedure in a dichloromethane (DCM)–methanol (MeOH)–phosphate (5:10:4 v/v/v) solution and separated by silica gel column chromatography [26]. PLFAs were derivatized by mild alkaline transmethylation to form FA methyl esters (FAME) [27]. Concurrently, an internal FAME (19:0) standard was added to the extracts. GC-MS (Gas Chromatography–Mass Spectrometer) was used to identify compounds based on retention times and mass spectra. The δ¹³C measurements of individual FAMEs of total PLFA biomarkers were analyzed by GC combustion isotope ratio mass spectrometry coupled to a GC (Gas Chromatography) PAL auto-sampler. The Trace GC Ultra was fitted with an Agilent HP-5 column (50 m, 0.2 mm i.d.). Identification of peaks was based on retention times. In light of PLFA spectra, PLFAs (20:5ω3 and 22:5ω3) were attributed to algae in Lake Nam Co and their concentration-weighted δ¹³C was used as a proxy for the δ¹³C of phytoplankton.

2.4. Isotope Modeling for Multiple Food Sources

Based on δ¹³C and ∆¹⁴C values, the IsoSource model was used to estimate the relative contribution of identifiable sources (autochthonous phytoplankton and submersed plants as well as allochthonous POC and DOC from the inflowing stream) to the biomass of zooplankton in Lake Nam Co. This isotope-mixing model solves iteratively for possible carbon source composition and provides a frequency distribution of the contribution of each carbon source to the consumer [28]. We used tolerance parameters of 1‰–3‰, reflecting the high uncertainty of the sources, no trophic enrichment factor for ∆¹⁴C (because of its definition), and a trophic adjustment of 0.4‰ for δ¹³C [24,25]. The ∆¹⁴C of DIC was used as a surrogate for the ∆¹⁴C of phytoplankton since algae biomass production is based on in situ DIC and therefore tracks that of DIC [29,30,31].

3. Results

3.1. Isotope Signature

The δ¹³C of the potential carbon sources ranged from −29.9‰ to −15.0‰, with phytoplankton and the submersed plant Ceratophyllum demersum having the lowest and highest values, respectively. Bulk lake POC and POC of the inflowing stream had similar δ¹³C values of −25.6‰ and −24.5‰, respectively. The dominant zooplankton species, the calanoid copepod Arctodiaptomus altissimus pectinatus, had δ¹³C levels within the range of these sources (

Table 2. The δ¹³C and ∆¹⁴C of the zooplankton Arctodiaptomus altissimus pectinatus and its potential carbon sources in Lake Nam Co and its watershed. δ¹³C and ∆¹⁴C are given as ‰ deviation from Vienna Pee Dee Belemnite (V-PDB) and oxalic acid II (OX-II), respectively.

Sources	δ13C	∆14C
Zooplankton	−26.3 ± 0.9	−45.3
Phytoplankton	−29.9	−36.1
Submersed plant	−15.0 ± 0.3	−44.1
POC bulk	−25.6 ± 0.6	−247.3
DOC bulk 1	−22.9	−70.2
POC allo	−24.5 ± 1.0	−120.6
DOC allo 1	−23.3	−158.3
DIC	0.6	−36.1

Notes: ¹ Based on the δ¹³C and ∆¹⁴C values of DOC from an inflowing stream to Lake Nam Co [15]. POC _bulk: lake bulk POC; DOC _bulk: lake bulk DOC; POC _allo: allochthonous POC; DOC _allo: allochthonous DOC; DIC: dissolved inorganic carbon.

).

The submersed plant, with a ∆¹⁴C value of −44.1‰, was consistent with the fixation of ¹⁴C depleted dissolved inorganic carbon (DIC) (−36.1‰) in the lake. Lake bulk POC and allochthonous riverine POC had strongly depleted ∆¹⁴C values of −247.3‰ and −120.6‰, respectively. Zooplankton had a negative ∆¹⁴C value of −45.3‰ (Table 2). δ¹³C and ∆¹⁴C values of allochthonous riverine DOC of −23.3‰ and −158.3‰, respectively, have previously been reported for Lake Nam Co [15].

3.2. Contribution of Potential Carbon Sources to Zooplankton

The results of the IsoSource mixing modeling based on δ¹³C and ∆¹⁴C values are shown in

Table 3. IsoSource modeling results, based on ∆¹⁴C and δ¹³C values, showing the proportional % contribution of the four carbon sources to the Lake Nam Co zooplankton given as mean and (1–99) percentiles.

Sources	Proportion (%)
Phytoplankton	73.9 (67–81)
Submersed plant	18.3 (11–25)
POC allo	4.7 (0–10)
DOC allo	3.1 (0–7)

. In Lake Nam Co, recently synthesized in situ but ¹⁴C-depleted primary producers (phytoplankton and submersed plant) emerged as the most important carbon source, constituting 92.2% of the zooplankton biomass. Allochthonous POC made up 4.7% and DOC made up 3.1% of the carbon source.

4. Discussion

The dominant zooplankton in Lake Nam Co, the calanoid copepod Arctodiaptomus altissimus pectinatus, had δ¹³C and ∆¹⁴C levels indicating its use of multiple-carbon sources (Figure 2). Modeling results suggested that its food sources originated mainly from aquatic primary producers (phytoplankton and submersed plants) supplemented with consumption of a fraction of t-OC originating from the glaciated watershed.

Allochthonous OC, including POC and DOC, contributed to ~7.8% of the carbon in the zooplankton biomass. Allochthonous OC becomes available to zooplankton through direct ingestion of detrital particles or through consumption of micro-heterotrophic organisms that have consumed OC [4,6]. Allochthonous OC (in the inflowing stream) was depleted in ∆¹⁴C, indicating that ancient OC was likely released from the glaciated watershed, and zooplankton may become depleted in ¹⁴C through the use of ancient OC [25].

Our modeling results also suggested that ~73.9% of the carbon sources incorporated into zooplankton in Lake Nam Co is derived from phytoplankton, supporting the prevailing view that phytoplankton is the preferred food item. It is also known that submersed plants may support zooplankton production via consumption by bacterioplankton and the microbial food web as well as the release of macrophytes detritus [3]. Our isotope mixing calculations estimated that recently synthesized but ¹⁴C-depleted Ceratophyllum demersum contributed a considerable amount (~18.3%) of the carbon in the diet of zooplankton. Zooplankton might exhibit a ¹⁴C-depleted signature via autotrophic carbon pathways because of reservoir effects of ¹⁴C-depleted DIC in the watershed: i.e., the presence of radiocarbon-depleted DIC serving as a carbon source for the aquatic primary producers.

Both phytoplankton and submerged vegetation in Lake Nam Co demonstrated a depleted ¹⁴C signature. The glacially influenced Lake Nam Co is characterized by a significant radiocarbon reservoir effect as reflected by the ¹⁴C-depleted DIC value. The negative ∆¹⁴C combined with the δ¹³C-DIC of ~0‰ might reflect a contribution of limestone bedrock weathering within the lake watershed [32]. DIC from carbonate rock weathering can then be incorporated into the carbon cycling of the lake food webs via primary production and trophic transfer [33].

5. Conclusions

Though the data reported in this study are limited, our findings showed that zooplankton in the pelagic zone of Lake Nam Co incorporated a fraction of glaciated watershed-derived OC. This suggests that in addition to primary production, OC from glaciated watershesd may be capable of stimulating aquatic productivity and deriving energy in lacustrine pelagic food webs, which has been previously demonstrated for other aquatic ecosystems as well. Future changes of the cryosphere triggered by enhanced warming may be pronounced. A continuing recession of glaciers and degradation of permafrost soil horizon in TiP are expected to transfer OC to the downstream aquatic environment. Therefore, we speculate that the importance of terrestrial organic materials in aquatic ecosystems will become stronger. In the long term, it is important to explore the role of glaciated watersheds as carbon pools for aquatic food web dynamics, a topic not yet well studied in TiP and other similar environments.