Pelagic-benthic coupling in organic matter utilization: the contribution of bacterial communities and benthic suspension feeders to carbon cycling in the Puyuhuapi Fjord ecosystem (Chilean Patagonia)

1. Main oceanographic characteristics of Patagonian fjords The Chilean Patagonia (41°-56°S) encompasses one of the most extensive fjord regions in the world (240000 km2) with oceanographic conditions that can sustain unique ecosystems. The region is made up mainly of fjords and channels, characterized by intertangled geomorphologies where water inputs from terrestrial and marine ecosystems overlap and mix (González et al., 2013). Patagonian fjords are characterized by highly complex geomorphology and hydrographic conditions, besides strong seasonal and latitudinal patterns in precipitation, freshwater discharge, glacier coverage, and light regime (Aracena et al., 2011). These systems receive Sub-Antarctic Water (SAAW) with high loads of nitrate and phosphate from the ocean, and freshwater with high loads of silicic acid from land (Silva, 2008). The surface freshwater layer that is formed by river discharges, high precipitation and glacier melting, gradually mixes with the deeper and salty SAAW layer through estuarine circulation (Chaigneau and Pizarro, 2005; Silva et al., 2009; Schneider et al., 2014). The interplay between oceanic waters and freshwater produces a vertical and horizontal gradient in salinity, nutrients and structure of microplanktonic community, making these fjords highly heterogeneous ecosystems. In addition, this interaction allows the transport and exchange of large amounts of organic matter between terrestrial and open-ocean environments (Sievers and Silva, 2009; González et al., 2011). Patagonian fjords play an important role in biological productivity and in coastal carbon cycling (González et al., 2013; Iriarte et al., 2014). These highly productive ecosystems have a great potential in terms of transfer of food to higher trophic levels, and vertical carbon export (González et al., 2010, 2011; Montero et al., 2011, 2017a,b). Previous studies within this region have highlighted the role of light (Jacob et al., 2014), winds, low-pressure systems, and freshwater discharge in driving cycles of biological productivity and composition of the phytoplankton community at seasonal and shorter time scales (Montero et al., 2011, 2017a,b). 2. Factors that modulate primary production in Chilean Patagonia The hydrodynamic mechanism that usually controls total phytoplankton production is the alternation, in time and/or space, between the destabilization and re-stratification of the water column (Legendre and Razzoulzadegan, 1996). In fjords, fresh water inputs tends to stabilise stratification while wind stress can oppose the stabilising effects of freshwater and is likely to be an important mechanism for mixing the upper water column (Goebel et al., 2005). In general, it is accepted that the interplay between wind-forced vertical mixing, solar radiation, and nutrient availability determines the occurrence of phytoplankton blooms (Sverdrup, 1953). Iriarte and González (2008) have suggested that an improvement in the light regime towards the end of winter is the main factor triggering phytoplankton production in the southern Pacific coastal area. However, this seasonal improvement in light alone may not be enough to cause phytoplankton blooms. Montero et al. (2011) suggests that the annual solar radiation cycle interact with mesoscale patterns of wind variability to modulate primary productivity in fjords. These authors reported that the onset of the productive season in Reloncaví Fjord (41°S; 72°W) coincided with seasonal changes in the direction and intensity of meridional winds. Low-pressure synoptic (LPS) events have also been shown to be important drivers of phytoplankton productivity during winter in Puyuhuapi fjord (44°S; 72°W) (Montero et al., 2017b). In that study, the area was under low surface irradiance levels and was subjected to the passage of several LPS separated by intervals of 2 to 4 days. During these events, strong northern winds (10-20 m s-1) contributed to the mixing of the water column, resulting (subsequent to water column re- stratification) in enhanced phytoplankton productivity at very low irradiance (1-10 μE m-2 s-1) (Montero et al., 2017b). These results are novel and challenge the so far established paradigm of low levels of irradiance as a key factor limiting phytoplankton blooms in fjords ecosystems. This research has also highlighted the importance of LPS events to ecosystem productivity. During these events, periods of intensive freshwater inputs also appear to modulate pulses of primary production (including the onset of productive season) in the Puyuhuapi fjord (44°S; 72°W) (Montero et al., 2017a). Freshwater input stabilizes the water column and increase the concentration of silicic acid in the upper layers, favouring the occurrence of diatom dominated phytoplankton blooms (Montero et al., 2017a). 3. Primary production cycle in Chilean Patagonia The productivity cycle within a number of Patagonian fjords (41-51oS) has been typically described as a two-phase system consisting of a short non-productive winter phase (May to July) and a productive phase extending from late winter (August) to autumn (April) (Iriarte et al., 2007; Montero et al., 2011, 2017a). Nevertheless, more recently, the occurrence of highly productive winter blooms of phytoplankton challenges our earlier perception of winter as a low production season in these fjords (Montero et al., 2017b). The productive season has been characterized by the occurrence of diatom blooms associated with high primary production rates (1-3 g C m-2 d-1), while winter phytoplankton productivity, characterized by the dominance of small phytoplankton cells, has been reported to be a small fraction (<0.5 g C m-2 d-1) of the total annual productivity (Iriarte et al., 2007; Iriarte and González 2008; Czypionka et al. 2011; Montero et al., 2011; Paredes and Montecinos, 2011). Diatoms of the genera Pseudonitzchia, Skeletonema and Chaetoceros usually dominate the phytoplankton community during the productive season, when warmer waters have been associated with high concentrations of Pseudo-nitzschia spp., and silicate-rich waters with Skeletonema spp. and Chaetoceros spp. blooms (Montero et al., 2017a). Diatoms are known to represent a significant component of the overall phytoplankton biomass in Patagonian waters (Iriarte et al. 2001; Cassis et al., 2002; Alves- de-Souza et al., 2008; Iriarte and González, 2008). They are key species that transfer energy efficiently to higher trophic levels and also significantly contribute to the downward transfer of organic carbon (Iriarte et al., 2007; González et al., 2010; Montero et al., 2011; Iriarte et al. 2013). Low rates of primary production and low carbon sedimentation observed in the water column, mainly during winter months, suggests that most of the locally produced organic carbon is recycled within the microbial loop (Montero et al., 2011). Low temperature, and/or unfavourable light conditions, added to the dominance of small flagellates (size range: 2-20 μm) during the non-productive season, have been reported as the key factors depressing phytoplankton activity during the winter in the Patagonian fjord region (Pizarro et al., 2005; Iriarte et al., 2007; Montero et al., 2011; Montero et al., 2017a). Despite these earlier described winter characteristics, a dense bloom of the dinoflagellate Heterocapsa triquetra (32-7900 cells mL-1) was recorded during winter 2015 in the Puyuhuapi Fjord (44°S; 72°W), and was responsible for unusually high levels of winter primary production (1.6 g C m-2 d-1) (Montero et al., 2017b). More recently, we have again recorded winter blooms in the Puyuhuapi Fjord during May/July 2018 and July 2019, this time with a high abundance of diatoms such as Pseudo-nitzschia spp. and Skeletonema spp., associated with high primary production rates (2.3-2.7 g C m-2 d-1) (data unpublished). Thus, our preliminary data appear to suggest that the marked seasonal pattern of the productivity cycle in the Puyuhuapi fjord is changing, and showing every time a less predictable phytoplankton succession model. These findings introduce a novel variability for biological productivity that requires more focused research attention. 4. Bacterial community and dissolved organic matter utilization Dissolved organic matter (DOM) is an important component of the total carbon pool in aquatic ecosystem (Kirchman, 2008) and heterotrophic bacterial community is the main group within the food web involved in the degradation and mineralization of this organic material. Heterotrophic bacterial communities are able to process an important fraction of dissolved organic matter (DOM), offering a permanent and fundamental path for the transfer of matter and energy within the carbon flux (Montero et al., 2011). In the Puyuhuapi Fjord (44°S; 72°W), Reloncaví Fjord (41°S; 72°W) and in some sampling stations next to the fjord area of Southern Ice Field (49°-51°S), we have found significant correlations between primary production and bacterial secondary production rates (Daneri et al., submitted; Montero et al., 2011), reflecting a significant degree of coupling between the primary formation of organic matter and its utilization by the microbial heterotrophic community (Montero et al., 2011). Weak coupling between phytoplankton and bacterial production rates in fjord areas has been reported mainly when bacterial community uses allochthonous organic substrates as a basis for secondary production (Bukaveckas et al., 2002). DOM consists of a heterogeneous mixture of different carbon compounds that vary in chemical quality (Benner, 2002) and include both autochthonous and allochthonous sources. The majority of autochthonous DOM in aquatic ecosystem is produced by phytoplankton (primary production) (Cole et al., 1988; Bianchi, 2007; Nagata, 2008), while allochthonous organic matter is primarily derived from terrestrial vegetation and soils (Bianchi, 2007) that enters the water column mainly through river discharges. In addition of these natural sources of organic matter, coastal environments receive the supply of allochthonous organic substrates derived from anthropogenic activities. In the case of Chilean fjords, the installation of an extensive salmon farming industry in the region can be a significant source of allochthonous inorganic and organic material (Quiñones et al., 2019). While some studies have shown the potential effect of nutrient enrichment associated with salmon industry in Chilean fjords (e.g. Soto and Norambuena 2004; Iriarte et al., 2013), the effect of organic matter releasing remains a main challenge to be unravelled (Quiñones et al., 2019). Different DOM sources provides a series of ecological niches that support the growth of highly diverse bacterial communities (Buchan et al., 2014; Blanchet et al., 2016; Hoikkala et al., 2016). Bacterial community composition and bacterial production rates may vary according to the DOM source (Lucas et al., 2016). Indeed, phytoplankton blooms drive the dynamic of bacterial taxa specialized in processing efficiently photolytically produced organic matter (Buchan et al., 2014). In the case of the DOM derived from salmon farming, it has been indicated that this pool could also be an important source of organic substrates for heterotrophic bacterial community (Yoshikawa et al., 2012; Yoshikawa and Eguchi, 2013; Nimptsch et al., 2015), mainly because their high level of degradability (Nimptsch et al., 2015) and rich content in proteins (Yoshikawa et al., 2017). The responses of bacterial groups to specific DOM compounds may also vary spatially and seasonally (Bunse and Pinhassi, 2017), due to the natural variability in the composition of microbial communities associated with environmental factors (Pinhassi and Hagström, 2000). In Patagonian fjords, temperature, salinity and oxygen (Gutiérrez et al., 2018) and hydrological changes associated with meltwater discharge (Gutiérrez et al., 2015) can modify the diversity of bacterial community in the water column. In the Puyuhuapi fjord (44°S; 72°W) Gutiérrez et al. (2018), reported that the composition of the bacterial community showed a pattern of vertical zonation, where different groups Operational Taxonomic Units (OTUs) were associated to predominant water masses. Bacterial communities represented mainly by Actinomycetales, Rhodobacteraceae, Cryomorphaceae, and Flavobacteriaceae were associated with Estuarine Fresh Waters, whereas Cenarchaeaceae and Oceanospirillales were representative of Modified Sub Antarctic Waters. Salinity and dissolved oxygen concentration were the main factors that explained bacterial segregation in these contrasting water masses (Gutiérrez et al., 2018). In addition, a dramatic reduction of richness and individual abundances within Flavobacteriaceae, Rhodobacteraceae and Cenarchaeaceae families was principally associated by seasonal increase of surface water temperature (Gutiérrez et al., 2018). The composition of phytoplankton can also influence the composition of bacteria in the fjord region of Patagonia (Gutiérrez et al., 2018). Positive correlations of members of Flavobacteriaceae, Alteromonadales, and Verrucomicrobiales with diatoms in subsurface waters and of Flavobacteriales (Cryomorphaceaeand and Flavobacteriaceae), Rhodobacteraceae, and Pelagibacteraceae with dinoflagellates in surface waters suggest that phytoplankton composition could define specific niches for microorganisms in Puyuhuapi fjord waters (Gutiérrez et al., 2018). These observations support previous findings in coastal environments where certain bacterial assemblages have been associated with spring diatom blooms and others with autumn blooms dominated by dinoflagellates (El-Swais et al., 2015). Since diatoms and dinoflagellates are the major primary producers in the Puyuhuapi Fjord (Montero et al., 2017a, b), the study of specific interactions between bacteria and phytoplankton and their role in carbon fluxes certainly merits further analysis. 5. Benthic suspension feeders In aquatic ecosystems, animals that feed on suspended particles in the water are collectively known as filter feeders (Jørgensen, 1990). Benthic communities are generally dominated by suspension feeders animals (Dame et al., 2001), including those actively pumping water, passively encountering particles, or some combination of the two (Petersen, 2004; Sebens et al., 2016). Active suspension feeders produce their own internal currents and pump water through a filtering structure that separates food particles from the water (Riisgård and Larsen, 2000). Contrarily, passive suspension feeders completely rely on the current for food supply (Best, 1988). By filtering water to satisfy their nutritional demands, these animals remove vast quantities of microscopic particles such as bacteria, phytoplankton, detritus, and suspended sediments, which collectively comprise the seston (Wright et al., 1982; Langdon & Newell 1996, Kreeger & Newell 2000, Cranford et al. 2011). Bivalve molluscs such as mussels are active suspension feeders and spatially dominant in benthic communities. Filter feeding behaviour in bivalves is known to be highly responsible to fluctuations in both the abundance and composition of suspended seston (Bayne, 1998; Prins et al., 1998), mainly due they can filter large volumes of water and retain a wide size range of particles (ca. 4-35 μm diameter) (Voudanta et al., 2016). The amount of food consumed by bivalves is determined by the filtration rate and efficiency of particle retention by the gill which has evolved to act as both a water- transporting and a particle-retaining organ (Riisgård, 1988). The filtration rate of a bivalve is a parameter of great ecological importance in aquatic ecosystems, which is critical to understanding the impact of these species on particles fluxes in coastal environments. Having determined the filtration rate and knowing the concentration of suspended particles in the water, it is possible to calculate the amount of food retained by the gills and ingested by the animal, as long as no pseudofaeces are produced (Winter, 1978). Several studies have reported differences in the bivalve filtration rates depending on the composition and quality of suspended particles in the water column (Rosa et al., 2018 and references therein). In addition, seasonal variation of the available food sources has also been demonstrated as a crucial factor in selection of prey and the filtration rate of bivalves (Cranford et al., 2011; Cresson et al., 2016). Bivalves exhibit little or no movement and are dependent upon food sources available in the water column. Vertical flux of organic matter (autochthonous) mainly from phytoplankton has been considered as a main food source for bivalves (Dame, 1996; Arapov et al., 2010; Greene et al., 2011). Several studies have indicated that phytoplankton abundance can be strongly controlled by bivalve grazing in shallow areas (Cloern, 1982; Prins et al., 1998; Lonsdale et al., 2009; Lucas et al., 2016), where even this “cleaning” potential could represent a mechanism to control eutrophication in coastal areas impacted by aquaculture (Officer et al., 1982; Rice, 1999; Rice et al., 2000). Bacteria and detritus have also been described as an important contribution to the diet of bivalve populations, especially in ribbed mussels, whose morphology of gill makes these species a very effective bacterial grazer in comparison to other bivalves (Wright et al., 1982; Stuart and Klumpp, 1984; Langdon and Newell, 1990; Kreeger and Newell, 1996; Gili and Coma, 1998). Blue mussels inhabiting in close vicinity to the fish farms have been described for their capacity to use as a potential food those waste organic material from salmon cages such as uneaten food pellets and fecal particles (Reid et al., 2010; MacDonald et al., 2012; Handa et al., 2012 a, b). Several studies have indicated that bivalves grown adjacent to salmon farming areas remove the organic matter from cages, increase their growth rates (Lander et al., 2004; Peharda et al., 2007; Sarà et al., 2009) and help reduce the ecological impacts of the salmon industry in the water column (Lefebvre et al., 2000; MacDonald et al., 2012). In Chilean Patagonia fjords, salmon farming is the main aquaculture activity that takes place (Buschman et al., 2006), mainly due to favourable physical/chemical water conditions for fish (Katz, 2006). This industry releases a large quantity of organic wastes that modify the load of particulate and dissolved material in the water column (Quiñones et al., 2019), and represent a permanent source of allochthonous organic matter input to the system (Iriarte et al., 2014). These allochthonous materials together with the high amount of autochthonous organic matter produced by phytoplankton in fjords (Montero et al., 2011; Montero et al., 2017a, b) provide a heterogeneous pool of food available to benthic consumers. In this region very little is known about feeding behaviour in bivalves and what are the main components of the diet of these animals, mainly of those dominant species such as ribbed mussel Aulacomya atra present along the protected and semi-protected rocky shores in the Chilean Patagonian fjord (Försterra, 2009). Considering the importance of filter feeding bivalves to the pelagic-benthic coupling is fundamental to know if these organisms are processing both autochthonous and allochthonous organic matter. Knowing the bivalve feeding dynamic will allow a better understanding of carbon fluxes in Chilean fjords. 6. Pelagic-benthic coupling The capture and ingestion of particulate organic matter by benthic suspension feeders is one pathway through which carbon and nutrients are transferred from the water column to the benthos, resulting in their retention, utilization and cycling (Monaghan et al., 2011). Bivalves filter considerable amounts of particles. The amount of material filtered is determined by their biomass, activity and the supply of organic matter to the benthos. In Chilean Patagonian fjords, high rates of primary production (Montero et al., 2017a, b) result in the efficient export of organic carbon to sediments (González et al., 2013). In addition, particulate organic matter from terrestrial origin (Quiroga et al., 2016) together with those derived from salmon cages augments the vertical flux of particles reaching the benthos. The influence of terrestrial and aquaculture derived carbon sources on hard-bottom benthic communities from Chilean fjords has, to date, been poorly studied. Some authors have indicated that allochthonous material from terrestrial origin plays a minor role as a food for suspension feeders (Mayr et al., 2011; Zapata-Hernández et al., 2014). In contrast, autochthonous organic matter from phytoplankton production has been highlighted as one of the main food sources in benthic suspension feeders (bivalves and ascidians) from sub- Antarctic Magellan Strait (Andrade et al., 2016). From our recent studies in Patagonian fjords, we are beginning to better understand carbon fluxes in fjords environments, where bacterioplankton communities are able to process an important fraction of the organic carbon produced by phytoplankton, and classic and microbial food web are coupled playing a significant role in carbon export from surface to the benthos (Montero et al., 2011). However, little is known about the cycling of allochthonous organic matter, mainly those coming from salmon aquaculture and, what is the importance of this allocthonous material in sustaining the bacterial communities (both composition and activity) and benthic suspension feeder. This research describes the main pathways of production and utilization of autochthonous and allochthonous carbon by bacterial communities and their relative importance in sustaining benthic filter feeder bivalves.