Seagrasses are a critical foundation species in coastal habitats that create a globally significant carbon stock (Fourqurean et al., 2012), habitat and nurseries for fish and invertebrates, coastal protection, sediment stabilization, and improved water clarity (Mtwana Nordlund et al., 2016). Unfortunately, global seagrass declines threaten to decrease the abundance of associated and dependent species (Hughes et al., 2009; FFWCC, 2021). Infectious disease is a major cause of seagrass decline, namely mortality and pathology from seagrass wasting disease (SWD). In the 1930s, SWD was the primary cause of mass mortality of the seagrass Zostera marina along both coasts of the Atlantic Ocean, resulting in total loss of seagrass beds and their ecosystem functions in some areas (Short et al., 1987). This infectious disease continues to affect seagrasses globally at local to regional scales (Sullivan et al., 2013). Though outbreaks are observed globally, we know relatively little about the broad-scale diversity and biogeography of Labyrinthula species, the etiological agent of SWD.

The Labyrinthulomycetes are classified under the Super-group Chromalveolata, first rank Stramenopiles, and second rank Labyrinthulomycetes (Adl et al., 2012), clearly distinguishing them from “true fungi”; however, they do have characteristics similar to fungi, particularly in their use of a saprotrophic mode of nutrition (Tsui et al., 2009). Though many slime nets are decomposers in marine environments (Raghukumar and Damare, 2011), some taxa have adopted a different strategy as parasites. Phylogenetic work of the Labyrinthulomycetes showed that this group consists of three distinct lineages including the thraustochytrids, labyrinthulids, and aplanochytrids (Tsui et al., 2009; Leander and Porter, 2011), all of which contain organisms associated with diseases of various hosts (Burge et al., 2013). Culture independent examinations of diversity across the Labyrinthulomycetes show these taxa are incredibly diverse and ubiquitous within aquatic habitats globally (Pan et al., 2017; Xie et al., 2018; Menning et al., 2021).

Parasites in the genus Labyrinthula comprise the monotypic family Labyrinthulaceae (Stramenopiles, Labyrinthulomycetes) (Handley et al., 2007). Currently, there are only three described species of Labyrinthula: L. zosterae (Muehlstein et al., 1991), L. terrestris (Bigelow et al., 2005), and L. diatomea (Popova et al., 2020). However, further culture independent surveys indicate many additional presumed terrestrial and marine species (Chitrampalam et al., 2015; Martin et al., 2016; Sullivan et al., 2017; Trevathan-Tackett et al., 2018; Popova et al., 2020), and genetic methods are increasingly being used to delineate species and strain-level taxonomy of Labyrinthula across hosts and geographic regions. For example, Chitrampalam et al. (2015) reported four phylogenetically distinct and thus potentially separate, yet unidentified species of Labyrinthula from turfgrass collected in Arizona, USA and New Mexico, USA. Douhan et al. (2009) found geographically separated isolates from turfgrass consisted of likely additional unidentified species of Labyrinthula. Surveys of seagrass species have also yielded evidence of new species of Labyrinthula. A global survey to assess and delineate species of Labyrinthula revealed between 15–20 phylogenetically distinct and likely different species of Labyrinthula (Martin et al., 2016). One major clade in this study consisted of at least 5–7 species of Labyrinthula that appeared to be pathogenic and specific to seagrasses (Martin et al., 2016). In a survey of five seagrass species from Australian waters, Trevathan-Tackett et al. (2018) used phylogenetic analyses to differentiate five presumed species of Labyrinthula. Additionally, Pagenkopp Lohan et al. (2020) previously reported eight presumptive species of Labyrinthula associated with multiple species of seagrass in the Indian River Lagoon in Florida. Clearly, the species-level diversity within these taxa remains mostly unexplored.

Much less is known about the sub-species or strain-level genetic variation within Labyrinthula species. Douhan et al. (2009) reported multiple genotypes of Labyrinthula terrestris from turf grass, indicating high strain-level variation likely exists within this species. From seagrass hosts, Trevathan-Tackett et al. (2018) reported 2–3 strains for multiple presumed species of Labyrinthula from Australia waters. Similarly, Pagenkopp Lohan et al. (2020) recovered multiple presumed strains (2–5 zero-radius operational taxonomic units or ZOTUs) from two of the presumed Labyrinthula species detected in Florida waters. Additional strains of L. zosterae associated with Z. marina are also suspected in the North Pacific, but no phylogenetic evidence exists to confirm strain delineation (Dawkins et al., 2018). Examining the genetic diversity of this parasite at the species and strain levels is important for linking this sub-species genetic diversity with traits, particularly host specificity and pathogenicity, which experimental data has shown can vary across strains and species of Labyrinthula (Martin et al., 2016; Dawkins et al., 2018; Trevathan-Tackett et al., 2018).

We used targeted amplicon-based high throughput sequencing (HTS) of the first internal transcribed spacer region (ITS1) to examine the phylogeography of Labyrinthula species and strains associated with two seagrass species collected from both coasts of North America, including the USA and Mexico. Our objectives were to (1) identify species of Labyrinthula in North American waters, (2) explore the diversity of Labyrinthula species and strains within and across locations and hosts, and (3) examine the genetic connectivity of these species across locations and seagrass hosts. We expected to find high diversity of Labyrinthula species within and across locations. We also expected to find high connectivity across these parasites at the species and strain levels across broad geographic locations and hosts.

In total, we analyzed 189 seagrass blades from 16 locations across five US and one Mexican state ( Table 1 ). Labyrinthula spp. were not detected at two locations: Landscape Lab in Shinnecock Bay, New York and Estero Punta Banda in the Punta Banda Estuary, Mexico. Where Labyrinthula spp. were detected, PCR prevalence from targeted collection of potentially diseased leaves ranged from 20% (SMS Dock & Big Island, Florida; Tiona Beach, New York) to 100% (Fleming Key, Florida). In general, PCR prevalence was lowest in sites in Virginia and New York, while highest in sites in Florida (Florida Keys and Dry Tortugas), Massachusetts, and central California.

We successfully amplified and sequenced the ITS1 region from 115 blades. In total, 14,206,358 raw reads were generated, which was reduced to 10,936,646 reads after initial filtering, merging, and chimera removal. With the removal of 1) negative control samples, and 2) sequences not identified as Labyrinthula, 10,936,628 sequences remained, which after curation through LULU yielded 25 unique Labyrinthula ASVs.

Our phylogenetic analyses indicate that these 25 unique ASVs fell into three clades ( Figure 1 ). Eight ASVs were assigned to a clade indicating these are strains of Labyrinthula zosterae. Two ASVs were identified as Labyrinthula sp. “D”, while 15 ASVs were identified as Labyrinthula sp. “E”. All three of these species of Labyrinthula were previously identified as pathogenic (Martin et al., 2016). Labyrinthula zosterae had the highest relative abundance in every location except in Florida, with 97.8% of sequences assigned to this species detected from Z. marina from locations outside of Florida ( Figure 2A ; Table 1 ). Both Labyrinthula sp. “E” (99.8%, n=211,545 of 211,919 reads) and “D” (99.8%, n= 6,597,883 of 6,605,370 reads) had the most reads recovered in Florida ( Figure 2A ). This coincides with locations where Thalassia testudinum was sampled ( Table 1 ).

Narrowing down to the strain level, of the 25 ASVs detected, 13 ASVs were detected in only a single location. Of the 12 ASVs detected in multiple locations ( Figure 3 ), for 7 of those ASVs (i.e., 15, 7, 1, 19, 27, 34, 9), 99% of the sequences were detected in a single location. While Labyrinthula sp. “E” had the highest genetic diversity detected ( Figure 1 ; Figure 3 ), 87% of the ASVs (n = 13) were primarily detected in Florida waters (with >99% of sequences of these ASVs detected only in Florida). For the other two ASVs identified as Labyrinthula sp. “E”, most of the sequences (61-81%) were detected from Z. marina in California. Labyrinthula zosterae had the second highest genetic diversity detected ( Figures 1 , 3 ), with the most variability in the distribution of ASVs. Two ASVs were detected only from T. testudinum in Florida (ASV531 and ASV11 in Man of War Harbor), while two others were detected only from Z. marina in California (ASV663 in San Francisco Bay, ASV745 in Tomales Bay). For the other four ASVs, two were most abundant in Massachusetts, one in California, and one in New York ( Figure 3 ). The ASVs most frequently detected were ASV1 (n = 79 detections), ASV4 (n = 49), ASV3 (n = 40), and ASV9 (n = 30). The Chao 1 and Shannon alpha diversity estimates show similar overall patterns, with the highest ASV richness estimates found in Florida and Massachusetts ( Supplementary Figure 1 ). While the relative abundance of these ASVs shows much higher abundance in few locations, the ASV network ( Figure 4 ) shows how these ASVs occur across geographic areas. The ASV network shows 12 haplotypes, as those ASVs with minor differences (e.g., sequence length, gaps) were collapsed for this analysis ( Figure 4 ). Thus, though one Labyrinthula species and some strains appear to dominate within each state sampled based on relative abundance, all species occurred in all states sampled with a single exception, Labyrinthula sp. “D” was not detected on Z. marina from NY ( Figures 2B , 4 ). Additionally, the haplotype network shows two ASVs (one from Labyrinthula sp. “E” and one from Labyrinthula zosterae) that occurred in all six regions, indicating high genetic connectivity, though their relative abundance differs across these areas ( Figure 3 ).

Community composition of Labyrinthula ASVs appears driven by a combination of host species and sampling region ( Figure 5 ). Communities of Labyrinthula from some blades of Florida T. testudinum appear to separate from all other blades along Axis 1, while communities from other T. testudinum blades group with communities on Z. marina blades collected from all other North American locations. Upon examining the ASV scores, four ASVs were identified as significantly contributing to the ordination results (ASV1, ASV4, ASV3, ASV2; p<0.01). Despite the apparent delineation of these communities from the Florida blades along Axis 1, further analyses exploring the Labyrinthula communities on the blades collected in Florida show that these communities do not consistently group by sampling location in Florida, as blades collected from Key West and the Indian River Lagoon spread across Axis 1, while those from Bush Key are more similar and only spread across Axis 2 ( Supplementary Figure 2 ). Communities of Labyrinthula from Z. marina blades vary across region and coast primarily along Axis 2, with communities from some locations being highly similar (e.g., Virginia, Massachusetts) while others are less similar (e.g., Mexico, California) ( Figure 4 , Supplementary Figure 2 ). Though three different Bays were sampled in California, the Labyrinthula communities on the blades show lots of overlap, particularly communities on blades from Tomales and Bodega Bays, with there being more differentiation between blades from these Bays and those collected in San Francisco Bay ( Supplementary Figure 2 ). The variation in Labyrinthula ASVs across seagrass host species (PERMANOVA: df = 1, sum of squares = 5.3, F _model = 18.98, R ² = 0.14, p = 0.001) and across geographic regions (PERMANOVA: df = 5, sum of squares = 9.8, F _model = 7.8, R ² = 0.26, p = 0.001) were significantly different, with these two parameters explaining ~58% of the variation. Dispersion was significant for host species (F_species = 24.48, p_species <0.0001) and geographic regions (F_region = 18.4, p_region < 0.0001).

Rarefaction curves generated to assess the limits of the current sampling indicate that none of the sampling in any region was sufficient to detect all the ASVs likely present ( Figure 6A ). While some of the curves appear close to an asymptote (e.g., New York), the small sample sizes collected in each region require interpreting these curves with caution. Upon extrapolating these curves, most regions require collection of >90 blades to fully assess the ASV diversity within the region ( Figure 6B ).

This is the first North American wide assessment of the phylogeography of Labyrinthula species and strains. We detected three species of Labyrinthula, represented by 25 ASVs (or strains) at 14 of 16 sites sampled across North America from two seagrass hosts. We detected the highest strain level variation in Florida, which is likely due to larger sample sizes from Florida compared to other sampling locations, but could be a result of higher parasite richness in tropical locations. All three species detected were previously identified as pathogenic, indicating that Labyrinthula species capable of causing wasting disease exist across the regions where seagrasses were sampled for this study. While assessing the genetic connectivity among regions was limited by sample sizes, some strains and species appeared more geographically isolated than others, likely due to limited connectivity or rarity of certain strains across sites.

Since the massive epizootic in the Atlantic Ocean during the 1930s (Renn, 1936), smaller-scale epizootics of SWD have continued to occur around the globe, impacting many seagrass species (Sullivan et al., 2013). Critical knowledge gaps remain regarding the underlying mechanisms that resulted in such a widespread and devastating epizootic and whether something similar could occur in the future (Sullivan et al., 2018). Much research has explored the various factors influencing the timing and severity of SWD epizootics. Currently, host demography (Groner et al., 2014), dosage and strain of parasite (Dawkins et al., 2018), higher temperature (Bishop et al., 2017; Groner et al., 2021; Aoki et al., 2022), salinity (Trevathan et al., 2011; Bishop et al., 2017), hypoxia and sulfide (Bishop et al., 2017), host immunity (Duffin et al., 2020; Schenck et al., 2023), and parasite diversity (Pagenkopp Lohan et al., 2020; Schenck et al., 2023) all appear to be important factors to consider for this disease, though some of these factors are more thoroughly studied than others, particularly across geographic locations and host species. Most of these studies used either a single strain (Trevathan et al., 2011; Bishop et al., 2017) or did not assess if the disease examined was caused by one or more species or strains of the parasite (Groner et al., 2014; Duffin et al., 2020; Groner et al., 2021). Thus, critical knowledge gaps for understanding this pathosystem include the lack of information about the genetic diversity, phylogeography, and connectivity of these parasites so that the potential variation in disease severity within and across hosts, as well as how these vary across different locations, can be examined.

We detected pathogenic species of Labyrinthula in every region and in most sites where seagrasses were sampled, indicating that these parasites are geographically widespread. Our analyses demonstrate that the richness of ASVs detected was significantly correlated with the number of blades tested (R² = 0.62, F = 19.8, p = 0.0007; Supplementary Figure 3 ). Additionally, our rarefaction and network analyses indicate that our data likely underestimate the richness of pathogenic Labyrinthula strains infecting Z. marina and T. testudinum across their ranges. Though these parasites are present, major epizootics have not occurred in many of these locations in decades, or are not known to ever have occurred. Rather, some seagrass beds have experienced localized, smaller scale disease events (Trevathan-Tackett et al., 2018; Duffin et al., 2021; Groner et al., 2021), or lesions are present without evidence of damage to the beds. While many environmental factors are known to impact the severity of seagrass wasting disease, experimental work has shown that many environmental conditions appear to favor the seagrass host while negatively impacting the parasite. For example, Trevathan et al. (2011) demonstrated that hypersaline conditions had little impact on T. testudinum, but negatively impacted the growth of the parasite. While multi-stressor experiments by Bishop et al. (2017) showed that environmental stressors can more negatively impact the parasite than the seagrass host particularly under ambient conditions, Schenck et al. (2023) and DuBois et al. (2020) found that temperature enhanced disease severity. Thus, how biotic and abiotic factors combine to create disease and potentially lead to future epizootics appear context dependent, making understanding how these factors vary across the diversity of species and strains of Labyrinthula even more critical to understanding this pathosystem.

The host specificity of Labyrinthula spp. has been explored previously and, like the data presented here, prior studies indicated that these parasites are host generalists. For example, Martin et al. (2016) was able to experimentally inoculate and cause disease in seagrasses of the same species from different geographic locations as well as different seagrass species from different geographic locations from where Labyrinthula isolates were obtained, indicating that at least some strains can infect multiple genera of seagrass hosts. Trevathan-Tackett et al. (2018) similarly experimentally inoculated and caused disease in two different seagrass hosts (Zostera muelleri and Heterozostera nigricaulis). Additionally, Garcias-Bonet et al. (2011) experimentally demonstrated that different strains and species of the parasite can infect seagrass hosts across different genera. Similar to Trevizan Segovia et al. (2021), we report high connectivity within strains and species, but low overall species diversity of Labyrinthula, which is consistent with a parasite generalist. Additionally, though Z. marina and T. testudinum are ecologically and evolutionarily distinct seagrass species (Larkum et al., 2006), many Labyrinthula ASVs were detected from both seagrass hosts. Despite this high connectivity, one Labyrinthula species dominated in relative abundance within each region sampled, further indicating that abiotic factors may be differentially favorable to Labyrinthula strains.

In this study, we used a targeted metabarcode approach to assess the intra- and inter-specific diversity of pathogenic Labyrinthula species infecting seagrass blades. Labyrinthula spp. lack defining morphological characteristics, so previous attempts to define species turned to a phylogenetic approach to tease apart suitable species level delineations (Martin et al., 2016). We chose to use the same approach here, which has limitations given the lack of a suitable outgroup resulting in an odd tree topology creating a polytomy for L. zosterae sequences. More detailed analyses are warranted to truly delineate species across this genus, perhaps even using genome-scale analyses (Tan et al., 2021). At the intra-specific level, there was a relatively high potential for cross contamination, both in the processing of the blades in the lab and in the library preparation steps. Thus, to mitigate any impacts of this potential contamination, we took several approaches. First, seagrass blades were collected and initially processed (e.g., lesions were removed from blades) primarily by the labs who collected them, making cross-contamination from blades collected from disparate regions extremely unlikely. Next, we used strict laboratory methods to reduce contamination, such as using spin-column extractions and strip tubes with lids, only opening one sample at a time, and working in separate pre- and post-PCR spaces. We also utilized dual-indexing of our amplicons, which can also decrease the likelihood of cross-contamination (MacConaill et al., 2018). Then, after our initial processing with DADA2, we ended up with 1,283 ASVs. As it was virtually impossible for us to determine if these ASVs resulted from sequencing error or from the hypervariable ITS1 region that was amplified, we included a second denoising step, utilizing the LULU package to compare sequence similarity and occurrence patterns to remove likely erroneous sequences. Given these precautions, we have confidence that our results showing high connectivity and strain and species dominance across geographic locations are unlikely to be solely attributable to sequencing error or cross-contamination.

We present a broad-scale phylogeographic examination of Labyrinthula strain and species diversity across North America, providing additional evidence regarding the host generality of this parasite. Yet many questions remain, and future research is needed to examine how the pathogenicity of the Labyrinthula species found here vary across these two seagrass hosts. Additionally, future research will need to explore if different strains or species of Labyrinthula respond differently in terms of pathogenicity or prevalence under different environmental conditions. Finally, it is unclear how Labyrinthula strains and species could be spread across these vast geographic distances, though it is possible that maritime shipping, and specifically ballast water, could play a role in the long-distance dispersal of this parasite. Previous studies have detected labyrinthulids in ships’ ballast water (Pagenkopp Lohan et al., 2016, 2022), yet it is unknown whether the parasite survives and is infective following discharge.