Thalassia testudinum

MSc Research Report 

Abstract Seagrass beds are among the world’s most productive systems with many ecosystem services such as coastal protection. The global rate of seagrass loss is high and despite legislation, the rate of loss has not slowed down substantially. A significant stressor on the Caribbean coast is the Sargassum mats that wash ashore. The coastal waters become enriched in nutrients and oxygen-depleted, releasing toxic hydrogen sulfide gas. The sediment becomes hereby uninhabitable for the native seagrass Thalassia testudinum.  

This study aimed to investigate whether there is a facilitating effect of Holophila stipulacea and Halodule wrightii on T. testudinum through sediment oxidizing properties. The following sub-questions were raised: (1) “Is there a difference in the growth rate of the seagrasses H. stipulacea, H. wrightii, and T. testudinum when they are transplanted to a post-SBT sulfide-rich environment?”, (2) “Is there a difference in sulfide levels in the sediment next to transplanted H. stipulacea, H. wrightii and T. testudinum cores?”, and lastly (3) “Is there a difference in the growth of individual T. testudinum shoots next to these transplanted cores?”.

To test this, cores of all three species were taken from healthy seagrass beds and transplanted to a sulfide-rich area. (1) The relative growth rate of the cores was measured, and, (2) sulfide levels in the sediment next to the transplanted cores compared. Lastly, (3) the growth of individual T. testudinum shoots was measured when placed next to the transplanted cores.

This study showed (1) no difference in growth rates of the transplanted cores, and (2) temporarily elevated pore-water sulfide levels in the immediate surroundings of the transplanted cores. Lowest sulfide levels near the H. wrightii transplantation and (3) no difference in the growth of the individual T. testudinum shoots between treatments. Concludingly, it is unlikely that there is a plant-plant facilitating effect of H. stipulacea, T. testudinum and H. wrightii on T. testudinum. Transplantation of T. testudinum cores with H. stipulacea or H. wrightii is not a restoration measure that may facilitate T. testudinum in bare areas with high pore-water sulfide levels. Investigating the behavior of the sulfide levels over a longer period is advised since the duration of the experiment was too short to observe the equilibrium that arose. 

Abstract
Seagrass is of great importance worldwide for coastal protection, carbon sequestration and as a nursery and feeding habitat for various species. However, due to climate change, eutrophication, turtle grazing and anthropogenic activities seagrass meadows are declining globally. Seagrass restoration might be a tool to restore the seagrass ecosystem and bring back the linked ecosystem services. In the case the area is ought to be suitable for restoration, different restoration methods can be used. This study will focus on the importance of sediment stabilisation for seagrass restoration of the native seagrass Thalassia testudinum and the invasive Halophila stipulacea, using biodegradable sheets that mimic the sediment stabilizing function of seagrass meadows. This study is executed in Lac Bay, Bonaire. It is expected that by using these stabilizing sheets, the balance between native and invasive seagrass can be shifted towards native seagrass occurrence. During this research we found that using sediment stabilizing root mats can improve restoration of the native seagrass T. testudinum, especially in environments with high wave action and currents. Sediment stability is provided and fragments are held in place by the use of these biodegradable sheets, which prevents fragments from washing away. However, for the long-term these biodegradable sheets are possibly negatively affecting seagrass growth, likely due to interference with rhizome growth. This should, however, be researched into further detail. The invasive seagrass species H. stipulacea does not experience advantages in terms of growth when using these root mats. Fragments of H. stipulacea are fragile and possibly suffer from different kinds of stress when implementing in between the sheets. It could be stated that by using the sediment stabilizing sheets, the balance between native and invasive species can be shifted towards native seagrass in this research. This will benefit the seagrass ecosystem and its ecosystem services. In general it can be stated that the effect of using these biodegradable sheets differs depending on the seagrass species and various environmental factors such as hydrodynamics. There is also an indication of a difference in efficiency of the use of these sheets between the short-term and long-term growth. Furthermore bioturbation is likely to influence seagrass expansion and the functionality of these biodegradable sheets, therefore further research is advised.

Abstract
Seagrass meadows provide essential ecosystem services. However, seagrass cover has decreased the past decades, due to climate change and other disturbing factors. To ascertain coastal stability, biodiversity and ecological well-faring, seagrass meadows need to be restored. In this thesis, we unfold complex interactions that need to be taken into account during such restoration projects.

In particular, this study explores the spatial distribution and factors influencing seagrass cover in Lac Bay, Bonaire, with a focus on the interactions between turtle grazing, bioturbation, invasive seagrass (Halophila stipulacea), and native seagrass (Thalassia testudinum). The observational study reveals competition between the two seagrass species, as well as the negative impact of high grazing pressure and bioturbation on T. testudinum. A linear mixed model identifies significant predictors for T. testudinum cover, including macroalgae cover, turtle grazing pressure, H. stipulacea cover, and mound cover. Additionally, a negative influence between H. stipulacea and T. testudinum is observed. Bioturbation is found to negatively affect T. testudinum but does not significantly impact H. stipulacea. The experimental study investigates the effect of bioturbation on seagrass growth using different planting techniques, indicating that transplanting T. testudinum fragments can be successful for restoration. However, no significant differences are found between lattice and mesh treatments. The study concludes that the complex interactions between these factors contribute to the decline of T. testudinum and the proliferation of H. stipulacea. Future research is suggested to further investigate these interactions and evaluate the long-term effects of bioturbation and planting techniques on seagrass growth. The findings emphasize the importance of considering multiple variables when studying seagrass ecosystems and propose transplanting as a potential restoration measure in areas with high bioturbation.

4. Discussion and Recommendations
Seagrass

Overall, there has been a decrease in the native species of T. testudinum and an increase in the invasive species H. stipulacea. S. filliforme populations appear to be stable, with a slight increase in coverage. Native seagrass Thalassia testudinum (Tt) has had an overall decrease in coverage from 48.78% in 2011 to 20.61% in 2022 . Over this same time period there has been a slight
increase in native seagrass Syringodium filiforme, from 3.85% in 2011 to 6.44% in 2022.

Lastly, there has been an alarming increase in the invasive seagrass Halophila stipulacea, growing from 6.01% in 2011 to 35.24% in 2022. A table with the annual averages for the three seagrasses can be found below in Table 1.

Sargassum has been an issue within Lac Bay, with several of the survey sites being locations where decaying sargassum has created a thick mat, which in most cases was slowly removed with the tide. Physical impact of the sargassum landings can be seen by the seagrass dieback all along the mangrove border from the south until just north of Punto Kalbas. This is noticeable at G.
Additionally, at location E, a very fluffy sediment was found to be covering the substrate. A likely explanation is that this is the result of decomposed sargassum settling at this site. The overall cover by all species together seems to be stable, but in terms of biomass it would appear to be lower. The ecosystem services provided by Halophila stipulacea are significantly lower than those of
Thalassia testudinum due to its shallow root structure (Smulders et al., 2017) and the fact that it is less nutrient rich than native seagrass species (Boman et al., 2019). The shift towards this nonnative species is of concern and should be closely monitored.

Benthic Species
Since 2018 Halimeda species and in 2022 bioturbation observations were added to the methodology of these surveys. Although bioturbators were noted in 2020, they were not quantified in such a way to allow objective, quantitative comparisons moving forward. Overall cover by Halimeda seems to have decreased but a longer time series is required to draw more definitive conclusions.
Two students have looked into carbonate sand production by Halimeda during the Lac Ecological Restoration project: Laura Timmermans (2018) and Valeria Pesch (2019). Results from these studies were inconclusive, highlighting the need for additional research to fully understand the contribution of Halimeda to carbonate sands and infilling of the bay.

In addition, more information is needed on the influence of eutrophication (Slijkerman et al., 2011) on this process. Table 2 below shows the overall averages for both species of Halimeda from 2018 to 2022. Table 2: Overall Halimeda averages between 2018 and 2022.

Sand particles size in Lac was measured during the Conch Stock Restoration project. Largerfractions often show Halimeda segments next to small shells and other carbonate particles(Figure 5). For this reason, it is believed thatHalimeda sp.are a major contributor of sandwithin the bay.

Sediments have been analyzed for carbonate content in several other studies such as theEHLZK projectand duringthe baseline surveys conducted in 2012 (Debrot et al, 2012).Although the findings have not been published, the data showed that sediments towards thecenter of the bay have a higher CaCO3content, and the distribution sand, silt, clay changes(Appendix VI). In addition, it was foundthat terrigenous sediments were most prevalent alongthe borders of Lac-mainly in the northwestern sector, whereas endogenous sediments werefound in the central part of the bay and towards the reef. 

Bioturbators have also been added to recent surveys as it is believed to be important as itmay cause a loss of sequestered carbon, and new sediment may facilitate settlement ofH.stipulacea. Bioturbators are considered to be ecosystem engineers, changing the substratelandscape.Common bioturbators are callianassid (burrowing ghost) shrimp, the lugworm,mantis shrimp and the burrowing sea cucumber.

Historical Data and Background

The first known qualitative seagrass survey in Lac Bay was conducted in 1969. Sincethen, additional quantitative surveys were conducted in 1999 and 2007 and on a regularbasis since 2011 (2011, 2013, 2015, 2017, 2018,2019, 2020).After each survey, raw dataforseagrass observationswereuploaded to the DutchCaribbeanBiodiversityDatabase (DCBD)and have beenreferenced innumerouspublications, most notably the seagrassandsea turtle research byChristianen andSmulders from Wageningen University and Research (WUR).In 2010, the invasive speciesHalophila stipulaceawas first documented in Lac Bay, andhas continued to spread since. This is a concern as this species is fast growing andhas been known to outcompete local species of seagrasses. If left unchecked,Halophila stipulaceais a threat to native seagrasses and the species of which dependon it (such as green turtle and queen conch).

Discussion

In recent monitoring (2019, 2020) pictures were taken of all quadrants surveyed as wellas of the surroundingarea.SeagrassOverall, there has been a decrease in the native species ofT. testudinumand anincrease in the invasive speciesH. stipulacea. S. filliformepopulations appeartobe stable, with a slight increase in coverage.Sargassum has been an issue within Lac Bay, with several of the survey sites beinglocations where decaying sargassum has created a thick mat, which in most cases wasslowly removed with the tide. Two ofthese sites, Locations A and G were completelycovered by sargassum. Physical repercussions of the sargassum landings can be seenby the seagrass dieback all along the mangrove border from the south until just north ofPunto Kalba.Additionally, at location E, a very fluffy sediment was found to be covering the substrate.A likely explanation is that this is the result of decomposed sargassum settling at thissite.The overall cover by all species together seems to be stable, but in terms of biomass itwould appear to be lower. The ecosystem services provided byHalophila stipulaceaaresignificantly lower than those ofThalassia testudinumdue to its shallow root structure(Smulders et al., 2017) and the fact that it is less nutrient rich than native seagrassspecies (Boman et al., 2019).The shift towards this nonnative species is of concern andshould be closely monitored.

Halimeda

Since 2018 Halimeda species and bioturbation observations were added to themethodology of these surveys. 2020Data onHalimeda sp.occurrence can be found inAppendix III.Overall cover by Halimeda seems to have decreasedbut a longer time series isrequired to draw more definitive conclusions. Two students have looked into carbonatesand production by Halimeda during theLac Ecological Restoration project: LauraTimmermans (2018)and Valeria Pesch (2019). Results from these studies wereinconclusive, highlighting the need for additional research to fully understand thecontribution of Halimeda to carbonate sands and infilling of the bay. In addition, moreinformation is needed on the influence of eutrophication (Slijkerman et al., 2011) on thisprocess.

Sand particles size in Lac was measured during the Conch Stock Restoration project.Larger fractions often show Halimeda segments next to small shells and othercarbonate particles (Figure 5). For this reason, it is believed thatHalimeda sp.are amajorcontributor of sand within the bay.Sediments have been analyzed for carbonate content in several other studies such asthe EHLZK project and during the baseline surveys conducted in 2012 (Debrot et al,2012). Although the findings have not been published, the data showed that sedimentstowards the center of the bay have a higher CaCO3content, and the distribution sand,silt, clay changes (Appendix V). In addition, it was found that terrigenous sedimentswere most prevalent along the borders of Lac-mainly in the northwestern sector,whereas endogenous sediments were found in the central part of the bay and towardsthe reef.

Bioturbators

Bioturbators have also been added to recent surveys as it is believed to be important asit may cause a loss of sequestered carbon, and new sediment may facilitate settlementofH. stipulacea. Bioturbators are considered to be ecosystem engineers, changingthesubstrate landscape. Common bioturbators are callianassid (burrowing ghost) shrimp,the lugworm, mantis shrimp and the burrowing sea cucumber. To date, surveys haveonly included rough estimates as to the number of mounds per square meter.After discussing this issue among local researchers, it has been decided to amend theprotocol for next year to count all mounds in the same quadrant as the seagrass cover.If more than half of the mound falls out of the quadrant the mound will not be counted

Summary
Thalassia testudinum seagrass is defined as important foundation species by serving as nursery area, giving coastal protection, maintaining high water qualities and their function as carbon sink. In the east Caribbean Sea, T.testudinum beds are under threat caused by the decomposition of excessive amounts of holo-pelagic sargassum blooms (Sargassum natans and Sargassum fluitans), creating anoxic conditions and causing the production of hydrogen sulfide( H2S) in the porewater. Hence, sulfide intrusion and toxicity can occur in T.testudinum causing lower plant performances and big scale seagrass mortalities. Positive effects of bioturbation were thought to relieve sulfide stress from T.testudinum by modifying the geo-chemistry by a process called ‘bio-irrigation’. This research assessed the effects of Bio-irrigating Upogebia shrimps on the porewater sulfide and sulfide plant intrusion within T.testudinum habitat. Firstly, a monitoring study was carried out to determined the effects of habitat and sargassum decomposition on Upogebia hole densities(indicator for Upogebia densities). Secondly, an Upogebia hole manipulation experiment was setup to investigate the effect of adding and removing bio-irrigating shrimps(Upogebia affinis) on porewater sulfide and sulfide intrusion within T.testudinum seagrass. In addition to the experimental effects, the overall effects of Upogebia hole densities on porewater sulfide and sulfide intrusion taken over the whole experimental period were investigated to account for possible delayed effects on sulfide intrusion within T.testudinum. Our results showed highest Upogebia hole densities in bare habitat with lower sargassum decomposition. Besides the possible negative effects of T.testudinum habitat reducing burrowing space for Upogebia, the bare habitat mainly consisted of Halimeda calcified sediment which likely facilitated the Upogebia burrow construction. Within the overall effects, Upogebia hole density showed negative effects on both porewater sulfide and total sulfur within T.testudinum leaf suggesting some possible bio-irrigation. Within the treatment effects, the removal intervention caused higher TS and lower δ34S in the T.testudinum leaf, but this was just visible after 16 days, indicating delayed effects of the removal intervention. Future mesocosm experiments are recommended to account for side effects and for more accurate sulfide measurements. This will fill more scientific knowledge gaps on the role of Upogebia shrimps as potential sulfide stress relievers for T.testudinum seagrass.

MSc thesis

The economy of Bonaire is highly dependent on tourism. Tourists are drawn to Bonaire because of the beautiful nature and biodiversity in the coastal ecosystems, e.g, mangrove forests, seagrass meadows, coral reefs. Therefore, it is important to protect these coastal ecosystems. Currently, eutrophication and pollution are serious threats to Bonaire’s mangrove forests and seagrass meadows through terrestrial run-off and influx of Sargassum. Seagrasses are known to be sensitive to local environmental changes by bioaccumulation of chemicals and nutrients through absorption in their tissues. In this study, we used turtle grass (Thalassia testudinum) as a bioindicator of spatial and temporal variation in eutrophication and pollution in five different bays on Bonaire. We found that T. testudinum is a good bioindicator of eutrophication and pollution. Analysis of stable isotope signatures (δ13C, δ15N), nutrient (%N, %P, %S) and trace metal content (%As, %Cd, %Co, %Fe, %Mn, and %Ni) in T. testudinum leaf samples
revealed that Lagun was the most eutrophic and polluted bay on Bonaire. The high eutrophic and polluted state in Lagun is mainly due to a nearby landfill, large catchment area, and influxes of pelagic Sargassum spp.. In Lac Bay, higher sulfide stress was observed in T. testudinum leaf tissues in 2019 compared to 2015, which may have hindered the uptake of N and P by T. testudinum. The difference in sulfide stress between 2015 and 2019 is due to the massive influx of Sargassum that occurred in 2018. However, we suggest a possible recovery given better uptake of N and P and lower sulfur content in T. testudinum leaf tissues in 2022. This may indicate less sulfide stress in 2022 compared to 2019. In Lac Bay in 2022, biochemical content of T. testudinum leaf tissues collected at fixed sampling sites where the direct cumulative effect of Sargassum influxes was assumed to be highest (i.e., west of Lac Bay), were similar to tissues collected in areas with no or intermediate direct impact of Sargassum. This may also suggest recovery of T. testudinum that has survived the most severe influx in 2018. We showed that Bonaire’s coastal ecosystems are threatened by eutrophication and pollution through land-based run-off and the influx of pelagic Sargassum. Hence, we want to encourage the local government with this study that nature restoration measures need to be taken immediately to protect their coastal ecosystems.
 

The success of invasive macrophytes can depend on local nutrient availability and consumer pressure, which may interact. We therefore experimentally investigated the interacting effects of nutrient (nitrogen and phosphorus) addition, the exclusion of large herbivorous fishes and mimicked grazing on the expansion rates of the invasive seagrass Halophila stipulacea. The experiments were established on Bonaire and Aruba, two islands in the southern Caribbean, which differ in fish community structure. We observed that multiple Caribbean fish species feed on H. stipulacea. At both study sites, nutrient enrichment decreased invasive leaf carbon:nitrogen ratios. However only on Bonaire, where herbivore fish abundance was 7 times higher and diversity was 4.5 times higher, did nutrient enrichment result in a significant reduction of H. stipulacea expansion into native Thalassia testudinum meadows. This effect was likely due to increased herbivory on nutrient enriched seagrass leaves, as we found that excluding large herbivorous fish (e.g. parrotfish) doubled invasive expansion rates in bare patches on Bonaire. On Aruba, H. stipulacea expansion rates were higher overall, which coincided with lower abundances and diversity of native fishes, and were limited by mimicked fish grazing. We suggest that top-down control by the native fish community may counteract eutrophication effects by increased grazing pressure on nutrient-rich invasive seagrass leaves. We conclude that diverse and abundant herbivore communities likely play an important role in limiting invasion success and their conservation and restoration may serve as a tool to slow down seagrass invasions.

Seagrasses comprise 78 species and are rarely invasive. But the seagrass Halophila stipulacea, firstly recorded in the Caribbean in the year 2002, has spread quickly throughout the region. Previous works have described this species as invasive in the Caribbean, forming dense mats that exclude native seagrass species. During a reconnaissance field survey of Caribbean seagrass meadows at the islands of Bonaire and Sint Maarten in 2013, we observed that this species was only extremely dense at 5 out of 10 studied meadows. Compared to areas with sparse growth of H. stipulacea, these dense meadows showed consistently higher nutrient concentrations, as indicated by higher leaf tissue N contents of the seagrass Thalassia testudinum (dense when C:N < 22.5) and sediments (dense when %N > 11.3). Thus, the potential invasiveness of this non-native seagrass most likely depends on the environmental conditions, especially the nutrient concentrations.

Although reef corals are dependent of the di- noflagellate Symbiodinium, the large majority of corals spawn gametes that do not contain their vital symbiont. This suggests the existence of a pool of Symbiodinium in the environment, of which surprisingly little is known. Reefs around Curac ̧ao (Caribbean) were sampled for free- living Symbiodinium at three time periods (summer 2009, summer 2010, and winter 2010) to characterize different habitats (water column, coral rubble, sediment, the macroalgae Halimeda spp., Dictyota spp., and Lobophora variegata, and the seagrass Thalassia testudinum) that could serve as environmental sources of symbionts for corals. We detected the common clades of Symbiodinium that engage in symbiosis with Caribbean coral hosts A, B, and C using Symbiodinium-specific primers of the hyper- variable region of the chloroplast 23S ribosomal DNA gene. We also discovered clade G and, for the first time in the Caribbean, the presence of free-living Symbiodinium clades F and H. Additionally, this study expands the habitat range of free-living Symbiodinium as environmental Symbiodinium was detected in T. testudinum seagrass beds. The patterns of association between free-living Symbio- dinium types and habitats were shown to be complex. An interesting, strong association was seen between some clade A sequence types and sediment, suggesting that sediment could be a niche where clade A radiated from a free-living ancestor. Other interesting relationships were seen between sequence types of Symbiodinium clade C with Halimeda spp. and clades B and F with T. testudinium. These relationships highlight the importance of some macroalgae and seagrasses in hosting free-living Symbio- dinium. Finally, studies spanning beyond a 1-yr cycle are needed to further expand on our results in order to better understand the variation of Symbiodinium in the environ- ment through time. All together, results presented here showed that the great diversity of free-living Symbiodinium has a dynamic distribution across habitats and time.