Phytoplankton benthic propagule (PBP) viability and community in the estuaries
The present study demonstrated that the PBP present in the sediments representing the upper, middle, and lower regions of the Zuari and Mandovi rivers are photosynthetically inactive. The variable fluorescence (Fv), an indicator for cell viability [36], suggested that the PPB was photosynthetically not viable at the start of the experiment. However, they were capable of reviving viability and photosynthetic activity, i.e., increase in Fv and Fv/Fm due to vegetative growth after germination under l2h light: dark photocycle and macronutrients (Figs. 2 to 5; Suppl Fig. 1). Interestingly, the revival of PBP was also observed from those samples representing the middle and upper regions of both the rivers when incubated in the nutrient-enriched 35 PSU seawater. Further, this study also demonstrates the utility of the variable fluorescence technique in the resurrection studies (detection of photosynthetic viability and responses to environmental cues). Resurrection ecologists have long recognized sediments as sources of viable propagules (“seed or egg banks”), including diatoms, with which to explore questions of community ecology, ecological response, and evolutionary ecology [38]. Generally, diatom benthic stages and dinoflagellate cysts represent PBP. Still, in this study, only the response of diatoms was observed, indicating their dominance to the viable PBP pool present in the stations representing upper, middle, and lower regions of the Zuari and Mandovi rivers. The available information from the area suggests dinoflagellate cyst abundance is very low (a few hundred per gram of dry sediment), which could be one reason for their no response in the incubation. Nevertheless, the focus is on the PBP, which responded to the treatments and contributed significantly to the region’s phytoplankton dynamics.
Altogether, 21 diatom species were recorded after incubation (Figs. 6 and 8), and the following species Skeletonema, Thalassionema, Thalassiosira, Navicula, Grammatophora, and Coscinodiscus dominated the BP community. The occurrence pattern of these forms in the estuarine system is as follows: 1. the propagules of Skeletonema, a bloom-causing species along the west coast of India, including the present estuarine system, are present from downstream to upstream up to several kilometers in Zuari and Mandovi rivers respectively. In Zuari, the cell abundance of Skeletonema after incubation is higher compared to Mandovi; 2. The presence of Thalassionema propagules is restricted from downstream to midstream in both Zuari (Z1 and Z2) and Mandovi (M1 and M2) rivers; 3. The propagules of Thalassiora are present only in the stations along Mandovi, and it's high cell abundance after incubation was observed in midstream and upstream sediments (M2 and M3) compared to downstream (M1); 4. Navicula, Grammatophora, and Coscinodiscus are present in the upper, middle, and lower Zuari and Mandovi rivers regions. The cell abundance of these species after incubation is low compared to the three dominant species. Nevertheless, the dominant species’ presence from the down to upstream stations can be attributed to tides. During non-monsoon, tidal advection from the sea to the upstream is dominant than river discharge. It has been observed that during the spring tide of the season, the salinity of ~1.5 is observed at the upstream location i.e., ~40 Km from the mouth [39], corroborating the role of tides in the widespread distribution of the PBP in the region. However, this aspect needs to detailed investigation.
The resting stages are formed to overcome unfavorable environmental conditions such as nutrient limitation, light, low temperature, salinity fluctuation, and internal regulation [25, 40, 41]. Recent findings also indicated a link between cell density and spore formation in the diatom Chaetoceros socialis [41]. Several studies demonstrated that nitrogen depletion is the most important environmental variable for forming resting stages in several microalgae including diatoms [25,]. A field-based study in Zuari estuary also revealed that the bloom’s termination coincided with nitrate depletion and a corresponding increase in benthic propagules [18, 31]. However, the onset of favorable conditions such as non-limiting nutrient concentration, appropriate combinations of temperature and light conditions (duration, quality, and quantity) seem to favor resting stages to germinate [23, 25, 42-44]. In tropical regions, water temperature variations are minimum compared to temperate areas, and therefore temperature may not be a significant factor. Previous field studies indicated that the peaks in chlorophyll coincided with improved underwater light conditions (except during peak monsoon discharges and periods of the high suspended load during non-monsoon), nutrients influx, and relatively high salinity (except during peak discharges) [18, 30, 31]. Given these specific experiments were performed and are discussed subsequently.
Phytoplankton benthic propagule (PBP) responses to macro-nutrients
The availability of nutrients (such as nitrate, phosphate, silicate) in addition to carbon dioxide, sunlight, and micronutrient is a known fact affecting the phytoplankton life cycle (including resting stage formation and germination). Nutrient enrichment experiment indicated that the maximum growth was observed when the macronutrients were supplied in conjunction (NPSi) compared to the availability of individual nutrient sources. This was supported by the fluorescence (both raw and variable fluorescence) as well as abundance data. The species composition in each treatment was the same, and this was observed in both Zuari and Mandovi sediments. For Zuari samples, significant PBP growth was observed from day seven onwards. In Mandovi, significant PBP growth was observed from day seven (for downstream and midstream stations) and day thirteen for the upstream station. Previous studies also reported a 1 to 8 days lag period compared to vegetative cells, which can grow almost immediately [27, 29, 42]. Even the photosynthetic efficiency remained low during the lag period but showed a drastic improvement upon the commencement of the growth after the end of the respective lag period (Fig. 3 and 4). Such a delayed lag period is because the benthic propagules first need to germinate, followed by asexual growth, whereas the vegetative cells just need to grow asexually. Another reason for prolonging the lag period could be fewer resting stages in the inoculum [29]. However, this was not the case in this study as the sediment samples harbored sufficient propagules, but there could be other limiting factors (e.g., light) for the long lag period. Further, the prolonged lag period for the PBP growth was almost the same irrespective of nutrient sources, indicating that the nutrients might be playing a minor role in the germination of diatoms. However, upon germination, subsequent growth success depends on nutrients availability, which was evident irrespective of the sample's origin. The experimental data further indicated that enhanced growth was observed even when supplied with any macronutrients compared to control. Still, the highest increase will be observed when all macronutrients (NPSi) are available.
Phytoplankton benthic propagule (PBP) responses to light intensities
Germination of individual cells appears to occur whenever sediments are exposed to a well-lit zone. Several studies have indicated that the photoperiod i.e. day length [23], intensity/irradiance [45-47], and quality such as spectral wavelength, e.g., blue, light [44] are known to influence the germination. The day length does not vary significantly in the tropical region, unlike the high latitude and temperate areas. Montresor et al. [47] indicated the day length theory holds the key for the germination of high latitude species but does not seems to be affected by changes in the day length for temperate species. Therefore it is presumed that the day length, which varies between 12 to 14 h depending upon the season, should not be a limiting factor in the Indian subcontinent and most probably for tropical species.
Laboratory experiments showed that a threshold irradiance is required to induce germination and subsequent growth [45, 46] but not mandatory as the resting stages of a diatom Aulacosira can germinate in dark conditions [65]. Earlier works have indicated that diatom resting stages can germinate within few days when exposed to light conditions [25, 40, 45, 49] and can have fully developed plastids after four days [50]. Research indicated an increase in the germination rate concerning increasing light intensity from 300 to 4000 lux, which was also evident in the present study [47]. Fluorescence (both raw and variable fluorescence) and abundance data indicated that the maximum growth of PBP was observed under HL (300 µmol photons m-2s-1) followed by ML (70 µmol photons m-2s-1) and LL (25 µmol photons m-2 s-1). Even the photosynthetic efficiency also followed a similar trend initially but remained maximum until the end of the experiment. However, the species composition remained the same under all light intensities for the respective samples. The experimental results also indicated that the light intensities play a major role in deciding the lag periods duration required for the germination and subsequent growth of PBP, i.e., higher light intensities affect a shorter lag period compared with lower light intensities. The lag period for Zuari and Mandovi sediments was same; the lag period in HL, ML, and LL was four, seven, and thirteen. It should also be noted that germination rates showed species-specific responses to light intensity and could affect species succession patterns when species were exposed to light. In this study, the germination rates of the dominant diatom species were more under HL conditions. Skeletonema and Thalassiosira, common planktonic species, were more sensitive to light, and both responded similarly to the light levels.
Previous studies revealed that both Skeletonema and Thalassiosira are dominant in the sediments during all the seasons and were reported to form blooms in the region but on different occasions [18]. Skeletonema blooms were observed under lower (during monsoon as single species) and higher (during monsoon break or non-monsoon mixed species) salinities. In contrast, Thalassiosira blooms were observed mainly during the non-monsoon season at higher salinity [18, 31]. However, the Skeletonema population outbreak’s magnitude was more significant than Thalassiosira [18]. Although blooms of these species occurred on different occasions, the bloom-forming mechanism for both is the same. For instance, the physical disturbances inducing benthic resuspension causing lower water transparency, nutrients influx, propagule transfer to the surface (photic zone) followed by germination when exposed to light, and the vegetative cells that arise from the sediment inoculum proliferate under nutrient-replete conditions. However, the nature of physical forcings inducing benthic resuspension is different during monsoon and non-monsoon seasons. The physical forcings associated before Skeletonema and Thalassiosira blooms are the freshwater discharge (during monsoon) and the local processes (during non-monsoon), respectively. Although the species' benthic propagules get resuspended, the species’ tolerance capabilities to salinity could be the dominant factor that led to causing the population increase not to co-occur in the region. Further, both the species lag period depends on the magnitude and duration of the factors such as benthic resuspension and cloud cover affecting the light availability but not to the extent of growth-limiting levels. During the initiation of Skeletonema bloom, light availability is governed by both benthic resuspension and cloud cover (only during monsoon). In contrast, for Thalassiosira blooms, only benthic resuspension will influence light availability.