The Complete Genome Sequences of Halophila stipulacea and Syringodium filiforme: Advancing Genomic Resources for two Tropical Caribbean Seagrasses

Halophila stipulacea is an invasive seagrass in the Caribbean Sea that also harbors a phytomyxid endoparasite. Phytomyxean parasites are known to cause disease in agricultural crops and are documented to form galls in some seagrass species. Here we make the first report of phytomyxid infection of Halophila stipulacea in the Bahía de Jobos in Salinas, Puerto Rico. We found phytomyxid infected H. stipulacea at 3 of 5 sites examined; expanding the documented range of the Marinomyxa marina phytomyxid infection by almost 400 km from where it was first documented in 2018. Presence of the endoparasite has not impeded H. stipulacea dispersal and continued expansion of H. stipulacea will likely spread both the host seagrass and the endoparasite.

The tropical seagrass species, Halophila stipulacea, originated from the Indian Ocean and the Red Sea, subsequently invading the Mediterranean and has recently established itself in the Caribbean Sea. Due to its invasive nature, there is growing interest in understanding this species’ capacity to adapt to new conditions. One approach to understanding the natural tolerance of a plant is to compare the tolerant species with a closely related non-tolerant species. We compared the physiological responses of H. stipulacea exposed to different salinities, with that of its nearest freshwater relative, Vallisneria americana. To achieve this goal, H. stipulacea and V. americana plants were grown in dedicated microcosms, and exposed to the following salt regimes: (i) H. stipulacea: control (40 PSU, practical salinity units), hyposalinity (25 PSU) and hypersalinity (60 PSU) for 3 weeks followed by a 4-week recovery phase (back to 40 PSU); (ii) V. americana: control (1 PSU), and hypersalinity (12 PSU) for 3 weeks, followed by a 4-week recovery phase (back to 1 PSU). In H. stipulacea, leaf number and chlorophyll content showed no significant differences between control plants and plants under hypo and hypersalinities, but a significant decrease in leaf area under hypersalinity was observed. In addition, compared with control plants, H. stipulacea plants exposed to hypo and hypersalinity were found to have reduced below-ground biomass and C/N ratios, suggesting changes in the allocation of resources in response to both stresses. There was no significant effect of hypo/hypersalinity on dark-adapted quantum yield of photosystem II (Fv/Fm) suggesting that H. stipulacea photochemistry is resilient to hypo/hypersalinity stress. In contrast to the seagrass, V. americana exposed to hypersalinity displayed significant decreases in above-ground biomass, shoot number, leaf number, blade length and Fv/Fm, followed by significant recoveries of all these parameters upon return of the plants to non-saline control conditions. These data suggest that H. stipulacea shows remarkable tolerance to both hypo and hypersalinity. Resilience to a relatively wide range of salinities may be one of the traits explaining the invasive nature of this species in the Mediterranean and Caribbean Seas.

Photo 1. On the Caribbean island of Bonaire, we observed that invasive seagrass Halophila stipulacea often uses bioturbation mounds as start locations to settle and then spread into the native Thalassia testudinum meadow. The bioturbation mounds provide space and light for this plant to succeed in colonizing new habitat. Photo credit: Fee Smulders. Photo 2. On Curacao, we observed that the invasive seagrass may be in competition with native upside-down jellyfish that prefers the same habitat. The author Naomi Slikboer, using SCUBA, is counting the number of invasive Halophila stipulacea shoots and native upside-down jellyfish individuals in control plots in Spanish water bay, Curaçao. Photo credit: Erik Wurz. Photo 3. The native upside-down jellyfish with its photosynthesizing symbionts is swimming through the water column before selecting a space to settle. In the background, the researcher and author Naomi Slikboer is taking pictures of the experimental plot. Photo credit: Erik Wurz. Photo 4. End points of two different plots. (A) After 45 days, plots where no invasive seagrass shoots had started growing throughout the experiment, were often occupied fully by native upside-down jellyfish. (B) When invasive seagrass shoots did start growing into the plot, as was the case for the majority of the plots, upside-down jellyfish were pushed out and only invasive seagrass remained. Photo credit: Naomi Slikboer. Photo 5. The apparent competition between the upside-down jellyfish and invasive seagrass leads to many interesting follow-up questions. Will the outcome always be a single-species equilibrium or can the two species cycle or co-exist on these bioturbation mounds? And how will this impact the patch dynamics in the ecosystem on the landscape scale? Photo credit: Erik Wurz. These photographs illustrate the article “Battle for the mounds: Niche competition between upside-down jellyfish and invasive seagrass” by Fee O. H. Smulders, Naomi Slikboer, Marjolijn J. A. Christianen, and Jan Arie Vonk published in Ecology. https://doi.org/10.1002/ecy.3980

Seagrass ecosystems provide essential nursery habitat to numerous coral reef fishes. Native Caribbean seagrasses Syringodium filiforme and Thalassia testudinum provide several juvenile fish species with foraging habitat and protection during this vulnerable stage of life. In 2002, an invasive seagrass Halophila stipulacea was discovered off the coast of Grenada and has since rapidly spread throughout the Caribbean. Halophila stipulacea has been shown to displace native seagrass species and may pose additional threats to juvenile reef fish populations that depend on native seagrass habitats as nurseries. The purpose of this study was to determine the effects of H. stipulacea on the early life history of yellowtail snapper (Ocyurus chrysurus). Settlement, mortality, and condition of juvenile yellowtail snapper were compared among native and invasive seagrass habitats around southern St. Thomas, United States Virgin Islands. From May 2020 to June 2021, monthly surveys within fixed plots during new moon periods provided information on settlement rates and survivorship among seagrass types. Length and weight of 260 juvenile yellowtail snapper were measured to derive condition factor among seagrass species. Significantly higher settlement and similar trends in mortality were seen in H. stipulacea when compared to native seagrasses. No significant difference was observed in the condition factor of juvenile yellowtail snapper among seagrass species, although trends demonstrated a higher condition in individuals from native seagrass species. This study is the first of its kind to identify the direct effects of H. stipulacea during the early life history stages of a commercially important fish species in the Caribbean.

The loss of biodiversity by the replacement of invasive species could lead to the loss of functional traits that maintain certain ecosystem services (ES). The ES method provides a conceptual framework to value changes of functional traits related to this loss of biodiversity. The Caribbean Sea offers a multifaceted seascape to evaluate this approach as native seagrass species (Thalassia testudinum, Syringodium filiforme or Halodule wrightii) cohabit this region together with the invasive seagrass Halophila stipulacea, native to the Indian Ocean. The functional traits of native seagrass species in the Caribbean are compared to different traits of H. stipulacea observed worldwide with the aim of evaluating the dimensions of this change in terms of the ES that seagrass meadows provide in the Caribbean. Under a changing seascape due to climate change and anthropogenic pressures that have driven the disappearance of most seagrass meadows in the Caribbean, we explore how this invasive seagrass could play a role in restoration attempts as a pioneer species where native species have been lost. The potential unintended consequences of the presence of H. stipulacea to replace services of native species are also noted.

Seagrasses provide an important ecosystem service by creating a stable erosion‐resistant seabed that contributes to effective coastal protection. Variable morphologies and life‐history strategies, however, are likely to impact the sediment stabilization capacity of different seagrass species. We question how opportunistic invasive species and increasing grazing by megaherbivores may alter sediment stabilization services provided by established seagrass meadows, using the Caribbean as a case study. Utilizing two portable field‐flumes that simulate unidirectional and oscillatory flow regimes, we compared the sediment stabilization capacity of natural seagrass meadows in situ under current‐ and wave‐dominated regimes. Monospecific patches of a native (Thalassia testudinum) and an invasive (Halophila stipulacea) seagrass species were compared, along with the effect of three levels of megaherbivore grazing on T. testudinum: ungrazed, lightly grazed and intensively grazed. For both hydrodynamic regimes, the long‐leaved, dense meadows of the climax species, T. testudinum provided the highest stabilization. However, the loss of above‐ground biomass by intensive grazing reduced the capacity of the native seagrass to stabilize the surface sediment. Caribbean seagrass meadows are presently threatened by the rapid spread of the invasive opportunistic seagrass, H. stipulacea. The dense meadows of H. stipulacea were found to accumulate fine sediment, and thereby, appear to be effective in reducing bottom shear stress during calm periods. This fine sediment within the invasive meadows, however, is easily resuspended by hydrodynamic forces, and the low below‐ground biomass of H. stipulacea make it susceptible to uprooting during storm events, potentially leaving large regions vulnerable to erosion. Overall, this present study highlights that intensive megaherbivore grazing and opportunistic invasive species threaten the coastal protection services provided by mildly grazed native species. Synthesis. Seagrass meadows of dense, long‐leaved species stabilize the sediment surface and maintain the seabed integrity, thereby contributing to coastal protection. These services are threatened by intensive megaherbivore grazing, which reduces the stability of the surface sediment, and opportunistic invasive species, which are susceptible to uprooting in storms and thereby can leave the seabed vulnerable to erosion.

Halophila stipulacea is a small tropical seagrass, native to the Red Sea, Persian Gulf, and the Indian Ocean. It invaded the Mediterranean Sea 150 years ago as a Lessepsian migrant, but so far has remained in insulated, small populations across this basin. Surprisingly, in 2002 it was reported in the Caribbean Sea, where within a decade it spread to most of the Caribbean Island nations and reaching the South American continent. Unlike its invasion of Mediterranean, in the Caribbean H. stipulacea creates large, continuous populations in many areas. Reports from the Caribbean demonstrated the invasiveness of H. stipulacea by showing that it displaces local Caribbean seagrass species. The motivation for this review comes from the necessity to unify the existing knowledge on several aspects of this species in its native and invasive habitats, identify knowledge gaps and develop a critical strategy to understand its invasive capacity and implement an effective monitoring and conservation plan to mitigate its potential spread outside its native ranges. We systematically reviewed 164 studies related to H. stipulacea to create the “Halophila stipulacea database”. This allowed us to evaluate the current biological, ecological, physiological, biochemical, and molecular knowledge of H. stipulacea in its native and invasive ranges. Here we i) discuss the possible environmental conditions and plant mechanisms involved in its invasiveness, ii) assess the impact of H. stipulacea on native seagrasses and ecosystem functions in the invaded regions, iii) predict the ability of this species to invade European and transoceanic coastal waters, iv) identify knowledge gaps that should be addressed to better understand the biology and ecology of this species both in its native and non-native habitats, which would improve our ability to predict H. stipulacea’s potential to expand into new areas in the future Considering the predicted climate change scenarios and exponential human pressures on coastal areas, we stress the need for coordinated global monitoring and mapping efforts that will record changes in H. stipulacea and its associated communities over time, across its native, invasive and prospective distributional ranges. This will require the involvement of biologists, ecologists, economists, modellers, managers and local stakeholders.

Battle for the mounds: Niche competition between upside-down jellyfish and invasive seagrass.

Halophila stipulacea is a small tropical seagrass species. It is the dominant seagrass species in the Gulf of Aqaba (GoA; northern Red Sea), where it grows in both shallow and deep environments (1–50 m depth). Native to the Red Sea, Persian Gulf, and Indian Ocean, this species has invaded the Mediterranean and has recently established itself in the Caribbean Sea. Due to its invasive nature, there is growing interest to understand this species’ capacity to adapt to new conditions, which might be attributed to its ability to thrive in a broad range of ecological niches. In this study, a multidisciplinary approach was used to depict variations in morphology, biochemistry (pigment and phenol content) and epiphytic bacterial communities along a depth gradient (4–28 m) in the GoA. Along this gradient, H. stipulacea increased leaf area and pigment contents (Chlorophyll a and b, total Carotenoids), while total phenol contents were mostly uniform. H. stipulacea displayed a well conserved core bacteriome, as assessed by 454-pyrosequencing of 16S rRNA gene reads amplified from metagenomic DNA. The core bacteriome aboveground (leaves) and belowground (roots and rhizomes), was composed of more than 100 Operational Taxonomic Units (OTUs) representing 63 and 52% of the total community in each plant compartment, respectively, with a high incidence of the classes Alphaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria across all depths. Above and belowground communities were different and showed higher within-depth variability at the intermediate depths (9 and 18 m) than at the edges. Plant parts showed a clear influence in shaping the communities while depth showed a greater influence on the belowground communities. Overall, results highlighted a different ecological status of H. stipulacea at the edges of the gradient (4–28 m), where plants showed not only marked differences in morphology and biochemistry, but also the most distinct associated bacterial consortium. We demonstrated the pivotal role of morphology, biochemistry (pigment and phenol content), and epiphytic bacterial communities in helping plants to cope with environmental and ecological variations. The plant/holobiont capability to persist and adapt to environmental changes probably has an important role in its ecological resilience and invasiveness.

The nature and strength of interactions between native and invasive species can determine invasion success. Species interactions can drive, prevent or facilitate invasion, making understanding the nature and outcome of these interactions critical. We conducted mesocosm experiments to test the outcome of interactions between Halophila stipulacea, a seagrass that invaded the Mediterranean and Caribbean Seas, and native seagrasses (Cymodocea nodosa and Syringodium filiforme, respectively) to elucidate mechanisms explaining the successful invasions. Mesocosms contained intact cores with species grown either mixed or alone. Overall, in both locations, there was a pattern of the invasive growing faster with the native than when alone, while also negatively affecting the native, with similar patterns for shoot density, aboveground and belowground biomass. In the Caribbean, H. stipulacea increased by 5.6 ± 1.0 SE shoots in 6 weeks when grown with the native while, when alone, there was a net loss of −0.8 ± 1.6 SE shoots. The opposite pattern occurred for S. filiforme, although these differences were not significant. While the pattern in the Mediterranean was the same as the Caribbean, with the invasive grown with the native increasing shoots more than when it grew alone, these differences for shoots were not significant. However, when measured as aboveground biomass, H. stipulacea had negative effects on the native C. nodosa. Our results suggest that a seagrass that invaded two seas may drive its own success by both negatively affecting native seagrasses and benefiting from that negative interaction. This is a novel example of a native seagrass species facilitating the success of an invasive at its own cost, providing one possible mechanism for the widespread success of this invasive species.

The spread of invasive species is a major component of global ecological change and how and when to manage particular species is a difficult empirical question. Ideally, these decisions should be based on the specific impacts of invading species including both their effects on native competitors and how they may or may not play similar roles in broader ecosystem functioning. Halophila stipulacea is an invasive seagrass currently spreading through the Caribbean, and as seagrasses are foundation species, the effects of invasion have the potential to be particularly far-reaching. To evaluate the impacts of H. stipulacea we quantified spread and potential for displacement of native seagrasses as well as the effects of invasion on multiple ecosystem processes, particularly resource support for higher trophic levels and habitat creation. Long-term monitoring suggested that H. stipulacea likely displaces some native seagrasses (Syringodium filiforme and Halodule wrightii), but not others. Halophila stipulacea had lower N and protein levels and higher C:N ratios than native seagrasses, and as such is a poorer quality resource for consumers. We also observed significantly lower consumption of H. stipulacea than the native S. filiforme but limited differences compared to Thalassia testudinum. We found H. stipulacea created a more nutrient limited environment than T. testudinum and there were significantly distinct invertebrate assemblages in native- and invasive-dominated seagrass beds, but no difference in species richness or invertebrate biomass. These results suggest that the spread of H. stipulacea would impact a variety of ecological processes, potentially restructuring seagrass ecosystems through both direct impacts on environmental conditions (e.g., nutrient availability) and indirect food web interactions.

Seasonal distribution and variation in the nutritional quality of different fractions of two seagrass species from Aqaba (Red Sea), Jordan

The spatial scale and the magnitude of carbon and nitrogen translocation was examined in 5 tropical (Cymodocea serrulata, Halophila stipulacea, Halodule uninervis, Thalassodendron cilia- tum, Thalassia hemprichii) and 3 temperate (Cymodocea nodosa, Posidonia oceanica, Zostera noltii) seagrass species using 13 Carbon ( 13 C) and 15 Nitrogen ( 15 N) as tracers in experiments conducted in situ. Seagrass leaf and rhizome production during the study period varied from <0.001 to 0.015 g DW shoot -1 d -1 and 0.002 to 0.017 g DW rhizome apex -1 d -1 , respectively. Based on measured leaf and rhi- zome growth rates, the demand of resources for leaf production varied from 0.19 to 4.99 mgC shoot -1 d -1 , and from 0.01 to 0.24 mgN shoot -1 d -1 , while the demand for rhizome production varied from 0.62 to 5.57 mgC rhizome apex -1 d -1 and from 0.02 to 0.12 mgN rhizome apex -1 d -1 . Seagrass leaves incor- porated the isotopes at rates ranging from 0.04 to 0.63 µg 13 C g DW -1 h -1 , and <0.01 to 0.35 µg 15 N g DW -1 h -1 . After 4 d, all incubated shoots had shared part of the incorporated 13 C and 15 N with ram- ets placed at maximum distances ranging from 2.7 (H. stipulacea) to 81 cm (C. nodosa), indicating that seagrass clonal integration may be maintained between 1.6 d (H. stipulacea) and 5.4 yr (P. ocean- ica). Resource translocation within seagrass clones was stimulated towards horizontal rhizome apices. Seagrass ramets, in 4 d, shared with their neighbours between 0.37 and 390 µg 13 C and between 0.02 and 178 µg 15 N. During the study period, resource translocation would supply <5% and up to 40% of the leaf carbon and nitrogen required by a neighbouring developing ramet, respec- tively, and <5% and up to 36% of the carbon and nitrogen required for rhizome growth; provided that the incorporated resources over 1 d were mobilised at similar rates over 4 d. These results con- clusively demonstrate physiological integration between seagrass ramets, and that resource translo- cation may be an important mechanism for young seagrass ramets to acquire resources and for sea- grass clones to expand and persist.

Invasive plants, including marine macrophytes, are one of the most important threats to biodiversity by displacing native species and organisms depending on them. Invasion success is dependent on interactions among living organisms, but their study has been mostly limited to negative interactions while positive interactions are mostly underlooked. Recent studies suggested that microorganisms associated with eukaryotic hosts may play a determinant role in the invasion process. Along with the knowledge of their structure, taxonomic composition, and potential functional profile, understanding how bacterial communities are associated with the invasive species and the threatened natives (species-specific/environmentally shaped/tissue-specific) can give us a holistic insight into the invasion mechanisms. Here, we aimed to compare the bacterial communities associated with leaves and roots of two native Caribbean seagrasses (Halodule wrightii and Thalassia testudinum) with those of the successful invader Halophila stipulacea, in the Caribbean island Curaçao, using 16S rRNA gene amplicon sequencing and functional prediction. Invasive seagrass microbiomes were more diverse and included three times more species-specific core OTUs than the natives. Associated bacterial communities were seagrass-specific, with higher similarities between natives than between invasive and native seagrasses for both communities associated with leaves and roots, despite their strong tissue differentiation. However, with a higher number of OTUs in common, the core community (i.e., OTUs occurring in at least 80% of the samples) of the native H. wrightii was more similar to that of the invader H. stipulacea than T. testudinum, which could reflect more similar essential needs (e.g., nutritional, adaptive, and physiological) between native and invasive, in contrast to the two natives that might share more environment-related OTUs. Relative to native seagrass species, the invasive H. stipulacea was enriched in halotolerant bacterial genera with plant growth-promoting properties (like Halomonas sp. and Lysinibacillus sp.) and other potential beneficial effects for hosts (e.g., heavy metal detoxifiers and quorum sensing inhibitors). Predicted functional profiles also revealed some advantageous traits on the invasive species such as detoxification pathways, protection against pathogens, and stress tolerance. Despite the predictive nature of our findings concerning the functional potential of the bacteria, this investigation provides novel and important insights into native vs. invasive seagrasses microbiome. We demonstrated that the bacterial community associated with the invasive seagrass H. stipulacea is different from native seagrasses, including some potentially beneficial bacteria, suggesting the importance of considering the microbiome dynamics as a possible and important influencing factor in the colonization of non-indigenous species. We suggest further comparison of H. stipulacea microbiome from its native range with that from both the Mediterranean and Caribbean habitats where this species has a contrasting invasion success. Also, our new findings open doors to a more in-depth investigation combining meta-omics with bacterial manipulation experiments in order to confirm any functional advantage in the microbiome of this invasive seagrass.

Halophila stipulacea (Forsskål and Niebuhr) Ascherson is a small marine seagrass that belongs to the Hydrocharitaceae family. It is native to the Red Sea, Persian Gulf, and Indian Ocean and has successfully invaded the Mediterranean and Caribbean Seas. This article summarizes the pharmacological activities and phytochemical content of H. stipulacea, along with its botanical and ecological characteristics. Studies have shown that H. stipulacea is rich in polyphenols and terpenoids. Additionally, it is rich in proteins, lipids, and carbohydrates, contributing to its nutritional value. Several biological activities are reported by this plant, including antimicrobial, antioxidant, anticancer, anti-inflammatory, anti-metabolic disorders, and anti-osteoclastogenic activities. Further research is needed to validate the efficacy and safety of this plant and to investigate the mechanisms of action underlying the observed effects.