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A PCR assay for the detection of introduced Vallisneria spiralis, V. denseserrulata and their hybrids

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Abstract

The southern varieties of the American eelgrass, Vallisneria americana, are vital contributors to aquatic ecosystems throughout Florida and other areas within the southeastern United States. Recent discovery of cryptic non-native Vallisneria in natural waterbodies of central and southeast Florida has raised concerns for the biosecurity of native American eelgrass populations, which have been lost from many historical locations and are the subject of multiple, intensive restoration/reintroduction efforts throughout the state. The non-native grasses were found to be derivatives of a hybrid V. spiralis × V. denseserrulata, which is ubiquitous within the global aquarium trade and represents a worldwide invasive threat. Concern for the native taxon and difficulties in visually distinguishing it from non-native taxa prompted the development of a molecular screening assay. The method leverages parsimony-informative insertion/deletion-based variation within a fluorescently labeled amplified segment of the nuclear ribosomal internal transcribed spacer region. Based on comparisons to sequenced specimens (n = 96), fragment length polymorphisms for the amplicon, visualized by capillary electrophoresis, reliably diagnosed the Florida non-native grasses with 100% accuracy, sensitivity, and specificity. With additional ground-truthing to confirm intraspecific stability, this assay has the potential for broader application within the genus, including detection of the invasive V. australis (cf. V. gigantea), another ubiquitous aquarium product, as well as identification of first-generation hybrid forms.

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Data availability

Sequence alignments for accessioned nrITS region data in FASTA format are given in Supplementary Material Seq File S1. All nrITS ‘region of interest’ sequences generated in house and analyzed for assay performance (n = 96) are available from the corresponding author on reasonable request.

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Acknowledgements

We thank Caroline Gorga for project guidance and Jamie Richardson, Amanda Christiansen, Angela Wilder, Brittany Lay, Carter Henne, Charles Thompson, Chris Boever, Christopher Haggerty, Courtney Milloway, Dan Kolterman, Dennis Giardina, Ed Harris, Eric Latimer, Eric Lund, Stephanie Szura, Geoff Lokuta, Hunter King, John Snow, Kristine Campbell, Lorne Malo, Michael Sowinski, Robert Lovestrand, Victoria Congdon, Zack Whalen, and Dan Roberts for their assistance in sample collection. Liz Wallace and Brandon Barthel provided helpful feedback on an earlier draft.

Funding

Research funding was provided by the Invasive Plant Management Section within the FL Fish and Wildlife Conservation Commission’s Division of Habitat and Species Conservation.

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Authors and Affiliations

  1. FL Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 Eighth Avenue S.E., Saint Petersburg, FL, 33701, USA

    Michael D. Tringali, Siobhan B. Gorham, Seifu Seyoum, Cecilia Puchulutegui, Margaret S. Bass, Bradley T. Furman & Craig Mallison

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  1. Michael D. Tringali
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  2. Siobhan B. Gorham
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  3. Seifu Seyoum
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  4. Cecilia Puchulutegui
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  7. Craig Mallison
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Contributions

MDT: conceptualization, visualization, methodology, formal analysis, resources, writing—original draft. SBG: conceptualization, investigation, data curation, resources, writing—review & editing. SS: methodology, formal analysis, investigation. CP: investigation, writing—review & editing. MSB: investigation, data curation. btf: conceptualization, resources, writing—review & editing. CM: conceptualization, funding acquisition, writing—review & editing.

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Correspondence to Michael D. Tringali.

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Tringali, M.D., Gorham, S.B., Seyoum, S. et al. A PCR assay for the detection of introduced Vallisneria spiralis, V. denseserrulata and their hybrids. Conservation Genet Resour 15, 125–133 (2023). https://doi.org/10.1007/s12686-023-01311-9

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