Attachment C_FINAL.pdf

Florida Fish and Wildlife Conservation Commission

DETECTION AND MITIGATION PROTOCOLS FOR HYBRID EELGRASS IN RESTORATION

INTRODUCTION & PURPOSE

In 2021, a hybrid species of eelgrass (V. spiralis and V. denseserrulata; hereafter, ‘the hybrid’) was reported for the first time in Florida (Gorham et al. 2021). Both parent species are non-native to North America and their respective natural ranges do not overlap in the wild, suggesting that the hybrid is non-native to Florida and that its origin and mode of introduction might be related to the aquarium pet trade.

Populations of the hybrid have also been found in Japan, Hungary, and Australia (Wasekura et al. 2016);

however, little is known about its distribution in Florida, its competitive potential with native Vallisneria spp., or its effect on oligohaline macrophyte communities and freshwater ecosystems. Limiting the spread of the hybrid is therefore essential to the management of eelgrass habitat in Florida.

Field identification of the hybrid is challenging. As all eelgrass species are phenotypically plastic, eelgrasses are often identified by microscopic traits, and no diagnostic macromorphological characters for the hybrid have yet been published. For now, the only reliable method of screening the hybrid is by genetic testing. To that end, the Florida Fish and Wildlife Research Institute (FWRI) has developed a rapid PCR assay capable of discriminating the hybrid from native eelgrasses using variability in the nrITS region (Tringali et al. 2023). Below are recommended protocols outlining collection and application of this PCR assay for detecting the hybrid in the wild and for limiting hybrid spread through restoration projects.

The purpose of this document is to supplement the Florida Fish and Wildlife Conservation Commission’s Eelgrass Propagation and Restoration Standard Operating Procedures (FWC Eelgrass SOPs) for eelgrass restoration conducted or funded by FWC.

RATIONALE FOR PROTOCOL DEVELOPMENT

The active restoration of eelgrass in Florida generally employs one of two strategies: (1) plants are transplanted directly from natural donor beds to nearby recipient sites or (2) plants are collected, propagated vegetatively in nurseries, and then outplanted into recipient sites. In both cases, eelgrass shoots, connected (or not) by rhizomes, are fashioned into planting units (e.g., bare root, weighted, matted, wrapped, potted, and/or caged) prior to planting. Because the hybrid has been found in nursery stock, and because it is known to occur in mixed-species beds in the wild, strategies to mitigate its spread must address the plant collection step to ensure that the hybrid is not moved as donor material for transplanting or included as broodstock for nursery propagation.

Eelgrasses grow vegetatively by producing new shoots along horizontal rhizomes. All shoots produced in this way will be genetically identical to the parent plant and, collectively, whether the rhizome connection remains intact (i.e., an eelgrass plant) or not, they are referred to as a single clone. Eelgrass clones can grow in isolation or intermixed in beds of varying plant sizes. In natural settings, clonal richness, size, and degree of overlap can vary widely, depending on resource availability, environmental stability, metapopulation structure, seed bank viability, and legacy effects of past disturbance and recruitment events.

Thus, the best strategy to avoid hybrid collection is to maximize the genetic diversity of donor material or broodstock – this avoids oversampling of hybrid plants, assuming they are randomly distributed in the landscape. However, sampling will most often occur without a priori knowledge of hybrid presence, location, prevalence, or population structure (i.e., ages, sizes, and degree of overlap). To address this, sampling programs can use estimates of mean clonal size to set minimum or maximum spacing as appropriate for the field collection of donor and broodstock. For V. americana, clone sizes have been estimated to be 5 m for native populations in the Chesapeake Bay (Engelhardt et al. 2014) and 2-5 m for those in the Detroit River (Lokker et al. 1994). Studies of V. spiralis and V. denseserrulata were comparable at 2 m (Huang et al. 2020, Wang et al. 2022). Until more detailed information is known about clonal structure of Vallisneria spp. in Florida, sampling protocols below recommend collecting shoots at 5-m intervals for maximizing the haphazard selection of unique clones from most eelgrass beds.

The PCR assay can then be used to screen for hybrid presence. Because donor beds for transplanting projects will need to be screened prior to transplanting events (i.e., the collection of donor material), as called for in the FWC Eelgrass SOPs, and because a far greater number of shoots will need to be taken from donor beds than that required for nursery grow-out (i.e., 10s of thousands compared to 100s needed for broodstock), we provide different guidance for each case. A zero-tolerance approach has been adopted by FWC for the agency’s nursery broodstock whereas a probability-based approach will be applied to the collection of plants for direct transplanting conducted or funded by FWC.

METHODS FOR TRANSPLANTING PROJECTS

Collection protocol for transplanting projects:

1. Designate a rectangular site within the proposed donor bed, ideally measuring 40 m x 60 m to provide 2,400 m2 (0.6 acres).

2. Record corner points with D/GPS and mark with temporary markers (e.g., PVC poles).

3. Establish a regular, alternating, or haphazard array of 96 points with a maximum spacing of 5 m

(to reduce the probability of missing a hybrid clone). If haphazard, mark each point with a

D/GPS.

4. Collect a single eelgrass shoot or a leaf from each point in the sampling array where eelgrass is present.

5. Use a genetic assay (the PCR assay or other genetic test that can differentiate the hybrid from the native) to test each sample for hybrid status.

6. Detection of a single hybrid disqualifies the site and its waterbody from subsequent use as donor material for direct transplanting. However, the site may still be used for nursery broodstock collection following methods described below.

7. If no hybrid is detected, donor material may be harvested from within the designated, sampled, and tested area for the duration of the current growing season (i.e., genetic results remain valid for one growing season). Plants outside of the designated plot are not eligible for transplant.

8. If more plants are needed than can be obtained from one 0.6-acre plot, additional sites must be cleared following the protocol above.

METHODS FOR NURSERY OUT-PLANTING PROJECTS

Collection protocol for nursery out-planting projects:

The number of shoots required for propagation will be a function of project size and grow-out duration.

Because the well plate used in most genetic assays holds 96 samples, we recommend targeting multiples of 96 shoots whenever possible. Because every shoot will be tested, it is not necessary to define and mark a sampling site as recommended under Methods for Transplanting Projects; however, doing so will allow for more accurate mapping of the hybrid and may facilitate broodstock collections in the future.

1. Define and mark an area as described above and/or sample 96 eelgrass shoots or whole plants at a minimum spacing of 5 m between plants (to reduce the probability of collecting the same clone twice). An eelgrass plant is operationally defined as all shoots connected by a rhizome.

Connected shoots can be safely considered one plant and will return identical genetic assay results.

2. Plants may be batched to minimize holding space during the hybrid screening process. For example, batch plants into groups of 16 (6 batches of 16 plants will equal 96 samples) and keep batches in separate containers (e.g., 5-gallon buckets) labeled with a batch number (e.g., 1-6) to hold during the hybrid screening process. Batching can be done by any method, but doing so by geographic area (i.e., portion of the sampled site) will help to map the hybrid. Areas where the hybrid has been confirmed in the past can then be avoided during future collection efforts.

3. Containers should be kept watered and in partial shade; plants can be left free-floating.

4. Collect one leaf blade from each plant in each batch and label with the appropriate batch number.

Use the genetic assay to test leaf samples for all plants in every batch.

5. All plants within any batch that includes a hybrid should be discarded. All plants within batches that contain no hybrid plants may be accepted into the nursery and used as broodstock for grow-out. Stock grown from these plants will NOT need to be re-tested prior to out-planting.

METHODS FOR HYBRID DETECTION IN EXISTING NURSERY STOCK

The hybrid has been detected in the long-cultivated stock and among the named cultivars previously used in eelgrass restoration. For nurseries with wild-caught broodstock, the following methods can be used to screen for hybrid presence.

Screening protocols for hybrid detection in existing nursery stock:

1. Establish even intervals at a maximum spacing of 5 m between no less than 16 sampling locations in each raceway, pool, pond, and/or growing tank to be used for out-planting.

2. Collect a single leaf from a shoot at each sampling point. Batch leaves by raceway, pool, pond, or tank.

3. Use the genetic assay to test all leaf samples.

4. Pools, ponds, and/or tanks that do not contain the hybrid can be harvested for out-planting.

REFERENCES

Engelhardt, K. A. M., M. W. Lloyd, and M. C. Neel. 2014. Effects of genetic diversity on conservation and restoration potential at individual, population, and regional scales. Biological Conservation 179:6-16.

This study evaluated nutrient allocation to clone size versus ramet size, and found the two to be inverselty correlated. This information can be useful when developing restoration programs for different systems, such as high or low energy systems. Larger clones with smaller ramets may be better suited for high energy environments, while smaller clones with large ramets may perform better in slower, deeper systems.

Gorham, S. B., S. Seyoum, B. T. Furman, K. M. Darnell, L. K. Reynolds, and M. D. Tringali. 2021.

Molecular detection of a non-native hybrid eelgrass, Vallisneria spiralis Linnaeus (1753) × V.

denseserrulata Makino (1921), in the southeastern United States. Aquatic Botany 175:103445.

During the course of a genetic survey of eel grass in restored systems, the presence of a non-native hybrid species of Vallisneria was discovered to have become naturalized in multiple waterbodies in Florida, Alabama, and Georgia.

Huang, X., X. Xu, B. Guan, S. Liu, H. Xie, Q. Li, and K. Li. 2020. Transformation of Aquatic Plant

Diversity in an Environmentally Sensitive Area, the Lake Taihu Drainage Basin. Front Plant Sci 11:513788.

This study addressed changes in biodiversity and species composition in aquatic pplant communities as a result of anthropogenic influence. They found that from 2010 to 1018, community composition shifted from being dominated by native species to being dominated by invasive species, and biodiversity decreased in the community.

Lokker, C., D. Susko, L. Lovett-Doust, and J. Lovett-Doust. 1994. Population genetic structure of

Vallisneria americana, a dioecious clonal macrophyte. American Journal of Botany 81:1004- 1012.

This study found that environmental conditions can affect the ratios of males:females in a population. This in turn can influence flowering rates where populations with more equal percentages of males and females experience increased flowering and by default greater sexual reproduction and higher genetic diversity.

Tringali, M. D., S. B. Gorham, S. Seyoum, C. Puchulutegui, M. S. Bass, B. T. Furman, and C. Mallison.

2023. A PCR assay for the detection of introduced Vallisneria spiralis, V. denseserrulata and their hybrids. Conservation Genetics Resources https://doi.org/10.1007/s12686-023-01311-9.

Difficulties in distinguishing the native species of Vallisneria in Florida from the non-native hybrid promted the development of a rapid molecular screening assay. This assay will allow high numbers of samples to be processed with lower costs and shorter wait times than traditional sequencing.

Wang, T., L. Fang, C. Wang, C. Liu, D. Yu, and H. Li. 2022. Water depth rather than substrate heterogeneity affects the clonal performance of the stoloniferous submerged plant, Vallisneria spiralis L. Flora 287:151995.

This research measured the response of growth traits (ramet number, leaf length, leaf number, stolon length, spacer thickness, biomass) in Vallisneria spiralis to changes in water depth and substrate. They found that the water depth influenced the growth traits while substrate heterogeneity had only negligible effects. Leaf length, spacer thickness, and biomass increased with depth, while ramet number, leaf number, and stolon length were greater in shallow depths.

Wasekura, H., S. Horie, S. Fujii, and M. Maki. 2016. Molecular identification of alien species of

Vallisneria (Hydrocharitaceae) species in Japan with a special emphasis on the commercially traded accessions and the discovery of hybrid between nonindigenous V. spiralis and native V.

denseserrulata. Aquatic Botany 128:1-6.

This study revealed the presence of naturalized populations of the hybrid Vallisneria denseserrulata and V. spiralis in Japan. This study presents the first formal description of the said hybrid.