Using isotope labeling to partition sources of CO2 efflux in newly established mangrove seedlings

Carbon dioxide (CO2) flux is a critical component of the global C budget. While CO2 flux has been increasingly studied in mangroves, better partitioning of components contributing to the overall flux will be useful in constraining C budgets. Little information is available on how CO2 flux may vary with forest age and conditions. We used a combination of C stable isotope labeling and closed chambers to partition CO2 efflux from the seedlings of the widespread mangrove Avicennia marina in laboratory microcosms, with a focus on sediment CO2 efflux in establishing forests. We showed that (1) above-ground part of plants were the chief component of overall CO2 efflux; and (2) the degradation of sediment organic matter was the major component of sediment CO2 efflux, followed by root respiration and litter decomposition, as determined using isotope mixing models. There was a significant relationship between C isotope values of CO2 released at the sediment–air interface and both root respiration and sediment organic matter decomposition. These relative contributions of different components to overall and sediment CO2 efflux can be used in partitioning of the sources of overall respiration and sediment C mineralization in establishing mangroves. Mangroves contain variably thick organic sediments and are the most carbon (C) rich forests (Donato et al. 2011; Sanders et al. 2016). The high C accumulation capacity of mangroves has been recognized, and termed “blue C,” along with saltmarsh and seagrasses (Mcleod et al. 2011; Duarte et al. 2013; Ouyang and Lee 2014). However, studies of mangrove carbon dioxide (CO2) flux vary in the precision of their partitioning. CO2 flux in mangroves may originate from the canopy, woody debris, root, litter and sediment organic matter (SOM), and is collectively called ecosystem respiration (Ee), which has been usually studied separately as canopy (above-ground parts, Ec) and sediment respiration (the other components, Es). Mangrove organic material such as leaf litter, if not exported, becomes incorporated in the sediment through decay and chemically modified by microbes inhabiting the mangrove forest floor (Kristensen et al. 2008). In contrast to the intensively studied and relatively established pattern of C exchange between mangroves and nearshore ecosystems (Lee 1995), the pattern of C gas flux released from mangrove sediment is less clear, although there is an increasing interest in this topic and C gas flux at the ecosystem scale (Lovelock 2008; Barr et al. 2010; Chen et al. 2010, 2012; Livesley and Andrusiak 2012; Barr 2013; Leopold et al. 2013, 2015, 2016; Bulmer et al. 2015). A key but poorly known aspect is the partitioning of Es attributable to various components, i.e., root, litter, and SOM (including the microphytobenthos). Laboratory microcosms have been used effectively in studies of mangrove energy pathways. For example, Bui and Lee (2014) evaluated relative contributions of organic matter from mangrove leaf litter and sediment to crab’s diet via laboratory microcosms. Zhu et al. (2014) conducted a microcosm study to investigate the fate of two abundant congeners in polluted mangrove sediment. We use laboratory microcosms to partition different sources of Ee, and in particular Es. The microcosms emulate field conditions with seedlings and sediments collected from mangrove forests, and then growing seedlings in the sediments. The study expands the horizon of current studies (e.g., Lovelock et al. 2015), which measure the portions of Ee in mature mangroves and do not completely partition Es. *Correspondence: [email protected]; [email protected] Present address: Simon S.F. Li Marine Science Laboratory, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1 LIMNOLOGY and OCEANOGRAPHY Limnol. Oceanogr. 00, 2017, 00–00 VC 2017 The Authors Limnology and Oceanography published by Wiley Periodicals, Inc. on behalf of Association for the Sciences of Limnology and Oceanography doi: 10.1002/lno.10663 Isotopic (dC) values can be used to distinguish photosynthetic pathways, shifts of vegetation and C sources supporting food chains (O’Leary 1981; Ouyang et al. 2015). Further, there is evidence that dC values can differ among mangrove tissues, although no consistent patterns of variation has yet been demonstrated (Bouillon et al. 2008a). There is also evidence that the SOM pool in mangroves was consistently enriched in C in relation to the mangrove litter in sites where litter was expected to be the sole input (Lallier-Verges et al. 1998). This difference is likely due to a rise in microbial and fungal residues (Ehleringer et al. 2000). However, the d C values of mangrove live tissues and litter are usually not distinguished. Boon et al. (1997) documented that the dC values of the pneumatophores of Avicennia, a widely distributed species, were on some occasions depleted in dC in relation to leaves by up to 3.1&, while Vane et al. (2013) stated that the difference between leaves and pneumatophores was<2&. Rao et al. (1994) noted little difference in dC values (< 1&) between fresh and senescent leaves for five tree species of Kenyan mangroves, but for four other species, senescent leaves were significantly depleted in relation to fresh ones. However, Lee (2000) suggested that the direction and magnitude of this difference was opposite. Natural C isotope signals, therefore, may not be able to differentiate sources from roots and litter, suggesting that isotopic labeling might be preferable. The enriched C isotope technique has been used to identify food sources with similar C signatures in food web research to overcome the drawback of natural C (Lee et al. 2011), and been used in other ecosystems (Galv an et al. 2008; Luo and Zhou 2010; Lee et al. 2012; Oakes et al. 2012). Similarly, it may be applicable in partitioning the sources of CO2 flux if combined with the closed chamber technique (Luo and Zhou 2010; Ouyang et al. 2017), which has been used to measure CO2 flux. The microcosms outweigh field experiments, for which it is difficult to perform isotopic enrichment in leaf litter and sediments under field conditions. It is suggested that a relatively low proportion of the organic matter in leaves of Avicennia is lost by leaching, while most of the labile portion is present as non-leachable but easily decayed organic material. Avicennia leaves tended to be decayed through microbial action relative to crab consumption (Robertson 1988). Although decomposition rates of mangrove litter vary (Lee 1999), much of the important biochemical action occurs relatively quickly, with half-life period of just 10.5 d for Avicennia (Sessegolo and Lana 1991). The relatively short half-life period for Avicennia has been attributed to lower tannin content and higher initial N concentrations (Alongi 2009). Hence, it takes a short time to investigate the composition of Es, attributable to leaf litter and their incorporated fraction into sediment for Avicennia. This study aims to distinguish Ec and Es, and focuses on partitioning Es attributable to different components using laboratory microcosms. As Es occurs at the sediment–air interface, tides were not set as a controlling factor in our laboratory microcosms. C enrichment combined with the closed chamber technique was used to partition different sources of CO2 efflux in microcosms with Avicennia marina seedlings simulating newly established stands. Our proposed method has the advantage of partitioning Es without disturbing the sediment compared with directly measuring different components of Es, e.g., the measurement of root respiration from detached roots (Lovelock et al. 2015). Experimental materials and methods Laboratory microcosms Seeds of A. marina (a cryptoviviparous species) and sediments were collected in June 2015 from the mangrove forest on Tallebudgera Creek (288602200S, 15382604900E) in southeast Queensland, Australia. The developing seedlings comprise cotyledons with fine roots at one side but no branching stems. Ninety healthy seeds were picked and planted in six glass chambers (40 3 30 3 50 cm) containing local sediment of 10 cm depth (see Fig. 1) and maintained at 248C ( mean local ambient temperature) under fluorescent lighting in a constant temperature room. Another chamber just contained sediment without seedlings, established for the measurement of ESOM. From the mangrove forest where the seedlings grew, sediments were collected, mixed and then put in the chambers. The initial volumetric water content of sediment is 32.6%64.1% (mean6 SD), and sediment chlorophyll a concentration is 845.76212.4 lg L (mean6 SD). Seawater was collected near the mangrove forest and injected in each chamber in equal quantities every 2 d to keep the sediment moist but not flooded. After injection, water either evaporated, or percolated through the sediment and could be absorbed by the seedlings for growth. After 1 month when leaves grew out of the cotyledons, polypropylene nets (1 cm mesh size) were hung in three of the chambers (over the sediment but under the cotyledons) to collect leaf litter. The netting was not set in the other three chambers. This net design prevented incorporation of leaf litter into the sediment, thus allowing separation of the contribution of leaf litter from Es. When seedlings had 4–6 leaves by August 2015, they were enriched with C using methods modified from Bui and Lee (2014) and Bromand et al. (2001). A bottle containing 25 mL of 1 M NaHCO3 (99 atom% C, Cambridge Isotope Laboratories) was put in each chamber before the chamber lid was tightly sealed. One milliliters of 1 M HCl acid was added to the bottle every 2 d for 45 d through a glass pipette passing through the lid of the chamber to generate CO2 in situ. A small fan (D58 cm) was turned on for 30 min after the addition of acid to promote even dispersion of CO2 within the growth chamber. At the end of the experiment, the seedlings grew to near the top of the chambers and were 40 cm height and the diameter of stems was 0.5 cm. After sampling at the end of the experiment, the plots were dug up and the roots were found to grow to the bottom of the chambers and some roots continued to extend horizontally in the sediments. Ouyang et al. Partitioning CO2 efflux in mangrove seedlings