Biol Invasions (2020) 22:1585–1606 https://doi.org/10.1007/s10530-020-02204-w (0123456789().,-volV)(0123456789().,-volV) ORIGINAL PAPER Preliminary application of DNA barcoding toward the detection of viable plant propagules at an initial, international point-of-entry in Georgia, USA L. E. Whitehurst . C. E. Cunard . J. N. Reed . S. J. Worthy . T. D. Marsico . R. D. Lucardi . K. S. Burgess Received: 13 March 2019 / Accepted: 17 January 2020 / Published online: 27 January 2020 Ó Springer Nature Switzerland AG 2020 Abstract Over 90% of global commercial trade (rbcL ? matK genes) of plant seeds could improve occurs between seaports, which are initial points-of- current processes in the early detection and rapid entry for nonnative, potentially invasive propagules. response to prevent entry/establishment of nonnative As such, there is a need to develop means to both plant species. This research conducted a preliminary rapidly intercept and identify propagules as they foray to evaluate the utility of widely accepted plant arrive. Here, we focus on plant propagules that are plastid DNA barcodes to identify plant propagules assumed to be non-native, in seed form. Because (seeds, hereafter) collected from the air-intake grilles standard morphological techniques alone are laborious of refrigerated shipping containers of a single agricul- and require taxonomic expertise, we sought to address tural commodity arriving at the Port of Savannah, if identification through barcoding of the plastid DNA Georgia, USA. We ask four questions: (1) Can DNA barcoding be used to detect seeds collected from shipping containers at the port? (2) What is the genetic Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10530-020-02204-w) composition of propagules entering the port? (3) How contains supplementary material, which is available to autho- do morphological identifications compare to those rized users. L. E. Whitehurst (&) Á S. J. Worthy Á K. S. Burgess (&) C. E. Cunard Á R. D. Lucardi Department of Biology, Columbus State University, United States Department of Agriculture, US Forest Columbus, GA 31907, USA Service, Southern Research Station, Athens, e-mail: laurenwhitehurst@ufl.edu GA 30602, USA K. S. Burgess S. J. Worthy e-mail: [email protected] University of Maryland, 4094 Campus Drive, College Park, MD 20742, USA Present Address: L. E. Whitehurst Department of Biology, University of Florida, Gainesville, FL 32611, USA C. E. Cunard Á J. N. Reed Á T. D. Marsico Department of Biological Sciences, Arkansas State University, State University, AR 72467, USA 123 1586 L. E. Whitehurst et al. based on genetic analysis? (4) Are nonnative invasive Lockwood et al. 2005; Colautti et al. 2006; Hietala plant species present on shipping containers entering et al. 2018). The magnitude of this propagule pressure the Port of Savannah? This research collected 11,044 and the subsequent success of NNIS propagules can seeds from 628 refrigerated shipping containers depend on the size and quantity of individual propag- between 2015 and 2017. Seeds were then morpholog- ule tissues or individuals and the frequency of ically sorted into Seed Types. Barcoding of the matK introduction events (Lockwood et al. 2005; Catford and rbcL gene regions of the plastid genomes directly et al. 2009). The location of introduction also matters isolated from seeds resulted in poor amplification. because abiotic and biotic factors contribute to the This is likely due to a host of potential confounding success or failure of propagule establishment, and factors. Therefore, we germinated seeds and utilized therefore, nonnative species and invasion success leaf-tissues for sequencing of these two gene regions. (Catford et al. 2009; Cassey et al. 2018). Only recently From BLASTn analyses, results returned top hits for a has the relative importance of propagule pressure on variety of species, with up to 22 possible nonnative the economic and ecological benefits of NNIS pre- plant species and one definite Federal Noxious Weed. vention versus mitigation been considered as part of This work investigates the interception application of invasive species management programs and improve- DNA barcoding to improve agro- and bio-security ment of governmental and/or regulatory interception issues posed by nonnative and invasive species. programs (Colunga-Garcia et al. 2013; Brockerhoff Though this study required the germination of the et al. 2014; Cassey et al. 2018). seeds to obtain leaf-tissue suitable for our DNA Seaports serve as a nexus point of global trade barcoding method, we effectively demonstrated seed between continents for NNIS propagule transport and viability. Our seed identification process was lengthy introduction, in addition to the target trade commodi- and understandably not feasible for real-time applica- ties (Blackburn et al. 2015). As such, seaports tion. Therefore, we seek to improve our methods for experience high quantities of propagule pressure of future applications by testing other approaches that NNIS and other nonnative, but potentially less may better complement morphological identification. impactful species. It has been shown that propagules Next reasonable steps include improved extraction are often passively transported in high frequency and protocols, metabarcoding to generate DNA barcode in large quantities on shipping containers (Epanchin- sequences directly from groups of seeds harvested Niell 2017). The terrestrial habitats associated with from shipping containers and implementing other seaports, referred to here as ‘industrial sites’, experi- next-generation sequencing techniques. ence substantial disturbance and activity for the purpose of trade, making them unique research sites Keywords Biosecurity Á Invasive Á matK Á rbcL Á to understand NNIS biodiversity and abundance at Propagule Á Seed these points-of-entry. These industrial sites may provide a higher probability of introduction and establishment of nonnative propagules, which may then spread to natural and agricultural environments Introduction (Shea and Chesson 2002; MacDougall and Turkington 2005; Bradley et al. 2010; Szymura et al. 2018). Increasing globalization of national economies In the USA, the United States Customs and Border through the trade of commodities and finished prod- Protection (Department of Homeland Security; ucts for consumption is one anthropogenic activity USCBP, hereafter) and the Animal and Plant Health that has been shown to directly increase the abun- Inspection Service-Plant Pest Quarantine (US Depart- dance, diversity, and frequency of nonnative invasive ment of Agriculture; APHIS-PPQ, hereafter) work plant species (NNIS, hereafter) due to their inadvertent together with the port authority to intercept, collect, transport and introduction (Westphal et al. 2008; identify, and decontaminate potentially viable NNIS Hulme 2009; Simberloff et al. 2013). In many cases, of all taxa. These agencies are currently experiencing the movement and subsequent establishment of NNIS reduced personnel and capacity while concurrently plant propagules have a significant and negative experiencing increasing trade volumes (Georgia Ports impact on native plant populations (Lonsdale 1999; Authority 2019). Therefore, we identified a need to 123 Preliminary application of DNA barcoding 1587 find more rapid and/or innovative applications for containers is DNA barcoding. This molecular tech- research to develop and test methods for plant nique, based on sequence variation in short, standard- propagule collection/sampling, detection, and identi- ized regions of the plant genome provides a molecular fication. Additionally, there is a need to make this basis to identify specimens through variation and information more rapidly transmittable to field per- conservation of nucleotides (Hebert et al. 2003; CBOL sonnel. Phytosanitary protocols vary from nation to Plant Working Group 2011; Coissac et al. 2016). For nation, and options for decontamination of potentially terrestrial plant species, a two-locus universal DNA damaging propagules and live organisms are limited barcode composed of the rbcL ? matK gene regions (Lucardi 2015, personal correspondence; Jones and of the plant genome has been successful at the Seghetti 2015; Hallman and Loaharanu 2016). Fur- documentation and identification of species composi- thermore, the collection of plant propagules (seeds, tion and diversity of local (Kress and Erickson 2007; hereafter) is limited, due mostly to their cryptic nature Burgess et al. 2011) and regional (de Vere et al. 2012; (i.e., small size and/or lack of identifying structures), Braukmann et al. 2017) floras. Furthermore, DNA and downstream identification is time-consuming and barcoding has the potential to provide rapid and poses challenges for the regulatory agencies tasked accurate identifications of unknown organisms (Hol- with NNIS prevention and interception inspections at lingsworth 2011; Hollingsworth et al. 2011), which seaports and other international points-of-entry can be very helpful in cases where identification based (USCBP and USDA, APHIS-PPQ). on morphology may be particularly challenging due to Internationally, wealthier nations experience more cryptic and/or ephemeral characteristics (Kress et al. nonnative plant richness due to increased importation 2015; Vassou et al. 2015), such as root-tissue (Hulme 2009). Cargo shipping is often an underesti- (Kesanakurti et al. 2011; Jiao et al. 2018; Meiklejohn mated, extremely large source of products