First record of Phytomyxid infection of the non-native seagrass Halophila stipulacea in Puerto Rico

Abstract

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.

Seagrasses, marine angiosperms, are ecosystem engineers and foundation species that provide a wide array of ecosystem services including habitat and food for higher trophic level organisms including humans. Worldwide, the decline of seagrass beds continues unabated and has been a topic of concern for resource managers and scientists. In a few notable locations there have been changes in the distribution of seagrass species related to colonization by non-native species. Two seagrasses, Halophila stipulacea (Forssk.) Ascher. and Zostera japonica Ascher. et Graeb are widely considered to be invasive in the Caribbean Sea and the Pacific Northwest of North America, respectively. The ecology and invasion history of Z. japonica have previously been reviewed by Shafer et al. (2013). Similarly, the ecology and colonization history of Halophila stipulacea was reviewed by Winters et al. (2020). Maitz et al. (2021) reported a phytomyxid infection of H. stipulacea collected from St. Eustatius in the Caribbean; Kolátková et al. (2022) confirmed the presence of the species-specific intracellular phytomyxid parasite, Marinomyxa marina in H. stipulacea populations from several locations in the Caribbean. Phytomyxid parasites are well known from agricultural crops and are generally regarded as pathogens although processes associated with infection and how they influence seagrass fitness are poorly understood (Kolátková et al. 2020). Here, we report the presence of phytomyxid nodules in the petioles of H. stipulacea from several locations within the Jobos Bay National Estuarine Research Reserve (NERR) in Salinas-Guayama, Puerto Rico.

During June 7–14, 2022, we collected 18 core samples (15 cm diameter) from monotypic stands of H. stipulacea at 4 sites in the Jobos Bay NERR and one site in Guánica Bay, Puerto Rico (Table 1). Samples in Jobos were obtained in about 1.5 m depth, while those from Guánica were at about 0.3 m depth. Core samples were sieved in the field over 5-mm mesh and all plant material was placed in a labeled plastic bag and stored on ice for transport to the lab. In the lab, plant samples were rinsed with distilled water, and counted for shoot density. Subsequently, plants were dried and weighed for biomass. During the processing procedure, small black nodules at the petioles of several plants were observed (Fig. 1). Our initial impression was that the nodules could be seeds or flowers; male flowers have been reported for H. stipulacea in the Caribbean, but seeds have not been observed (Chiquillo et al. 2018, Smulders et al. 2020, Vera et al. 2014). However, observations under a dissecting microscope indicated that they were not seagrass reproductive structures (e.g. not flowers). A representative sample of H. stipulacea with nodules was pressed onto an herbarium sheet and is maintained in the Pacific Ecological Systems Division, Pacific Coastal Ecology Branch Herbarium in Newport, OR. Several additional fragments were preserved in 70% isopropyl alcohol for three months and transferred to 50% EtOH and are retained by the lead author.

Table 1.

Sampling locations (decimal degree coordinates) and core details for Halophila stipulacea samples that included fragments infected with phytomyxid. Shoot density and biomass are mean ± SD calculated from replicate core samples at a site (n = 3–4).

Bay	Site	Latitude (decimal degrees N)	Longitude (decimal degrees W)	Temp (⁰C)	Salinity	# cores sampled	# cores with infected plants	H. stipulacea density (shoots m−2)	H. stipulacea Biomass (gdw m−2)
Jobos	NERR dock	17.955181969	−66.218064584	30.8	36.76	4	0	3070 ± 1190	65 ± 34
	St. 10	17.938785547	−66.257828794	30.4	37.68	3	1	5753 ± 2611	73 ± 61
	SE of St. 20	17.929570079	−66.210612609	29.1	35.29	4	1	6309 ± 250	155 ± 26
	Caya Barca	17.917732687	−66.231452361	28.9	35.38	4	1	10752 ± 2449	193 ± 41
Guánica	Gilligans	17.950035411	−66.877844670	28.6	35.19	3	0	11148 ± 3578	266 ± 90

Figure 1.

Photograph of Halophila stipulacea with phytomyxid nodules at the base of the leaf. Photo by J.E. Kaldy

Infected plants had a phytomyxid nodule associated with each leaf along the rhizome (Fig. 1). Dissecting the nodules out and examination under low power dissecting scope indicated that these were not seagrass reproductive structures or seeds (Fig. 2). We consulted with experts on Halophila stipulacea (G. Winters) and seagrass phytomyxid infections (V. Kolátková) to evaluate photographs of the observed nodules. They confirmed that the observed structures were a phytomyxid parasite, most likely the newly described species Marinomyxa marina since it is a species-specific obligate parasite infecting H. stipulacea (Kolátková et al. 2021). Phytomyxean species are endobiotic parasites infecting a wide range of organisms, including brown algae and seagrasses (Neuhauser et al. 2011). Phytomyxean parasites are well known agents of plant disease or viral vectors in economically important agricultural crops such as potatoes and cereals (Neuhauser et al. 2011). Twelve species of phytomyxids that infect seagrasses or other marine macrophytes have been described and phytomyxids have species-specific hosts including Zostera marina, Halodule wrightii, Halophila ovalis, Halophila stipulacea and Ruppia spp. (Kolátková et al. 2021). The first phytomyxid infection of the genus Halophila was described over 100 years ago from H. ovalis collected in Indonesia, while the first infection of H. stipulacea was found in the Red Sea in 1961 (Kolátková et al. 2021). Further, Kolátková et al. (2021) suggest that the host seagrass and endoparasite likely comigrated into the Mediterranean Sea which may also be the case for the Caribbean although additional genetic work will be required to confirm that hypothesis. Although ubiquitous in marine environments, little to no work has been conducted to evaluate the pathogenic potential of marine phytomyxids on their host physiology or ecology (Kolátková et al. 2022, Neuhauser et al. 2011). However, several recent reports indicate that infected shoots are morphologically dwarfed relative to uninfected shoots (Kolátková et al. 2022, Maitz et al. 2021, Neuhauser et al. 2011). Our observations are consistent with the pattern of infected shoot dwarfism. Visual estimates from photographs with scale bars of infected H. stipulacea fragments, indicate leaf lengths were on the order of about 3 cm and width of about 5 mm (Fig.1). In comparison, uninfected H. stipulacea leaves were generally 4–6 cm long by 6–9 mm wide. Similarly, infected plant rhizome internode distance appears to be compressed compared to uninfected plants, however, these were not quantified. Additional research will be required to quantify any physiological or ecological impacts from the endoparasite infection of H. stipulacea and subsequent dwarfism.

Figure 2.

Photograph of dissected phytomyxid nodule removed from H. stipulacea plant showing the spores. Scale at bottom 1 mm between tick marks. Photo by J.E. Kaldy.

Of the 18 Halophila stipulacea cores sampled in Puerto Rico, we found infected shoots from three different sites within the Jobos Bay NERR (Table 1). Our finding expands the documented presence of phytomyxid infected H. stipulacea almost 400 km to the north-east of St. Eustatius. Previous reports of phytomyxid infected H. stipulacea in the Caribbean include Bonaire, Martinique and Saba (Kolátková et al. 2022, Maitz et al. 2021). The weight of evidence from these recent reports supports the hypothesis that Marinomyxa marina is likely widespread throughout the Caribbean (Maitz et al. 2021). Field conditions during the Puerto Rico sampling were typical for summer; all sites were hypersaline ranging between 35.2 and 37.7 salinity, while water temperatures ranged between 28.6 and 30.8 °C (Table 1) consistent with previous reports (Winters et al. 2020). It is unclear how long the phytomyxid endoparasite has been in Puerto Rico. In February 2019 Jobos Bay NERR staff had noted the presence of nodules on H. stipulacea (A. Dieppa, pers. obs.) but at the time it was not investigated further. Although infected fragments were present in the cores, most shoots within an individual core were not infected with the parasite. Endoparasite transmission in H. stipulacea has not been resolved. Low density infection of H. stipulacea is consistent with previous observations in the Caribbean (Kolátková et al. 2022). Sampled H. stipulacea monotypic stand shoot densities at the sampling sites ranged between about 3,000 and 11,000 shoots m⁻². The presence of the phytomyxid infection and its genetic fingerprints may provide additional insight to the source of the original invasion. Further, research is needed to evaluate the physiological impact of Marinomyxa marina infection on H. stipulacea, especially with respect to plant carbon balance and nutrient requirements, as well as on the prevalence and infection vectors of the endoparasite. Recognition of this parasitic infection highlights that two distinct species, the seagrass Halophila stipulacea and its endoparasite Marinomyxa marina have invaded Puerto Rico estuaries. Since H. stipulacea spread is believed to occur via vessel and storm driven fragment transport (Winters et al. 2020), it is likely that both the host seagrass and endoparasite will continue to expand their range in the Caribbean and could also spread to systems in the Gulf of Mexico and other areas along the Gulf Stream (e.g. Bahamas, Bermuda and southeastern US estuaries).