Molecular confirmation of hybridization with invasive curly-leaf pondweed (Potamogeton crispus) in the Sacramento–San Joaquin Delta, California

Abstract

Weed managers recognize that hybridization can influence invasiveness in target weeds. As such, the identification of hybridization in target weeds has become of fundamental interest. Curly-leaf pondweed (Potamogeton crispus) is a heavily managed invasive aquatic weed in the United States. The genus is known for extensive interspecific hybridization, but the extent to which invasive P. crispus in the United States hybridizes is unknown. In October 2018, an aquatic vegetation survey in the California Sacramento-San Joaquin river delta identified plants that were suspected as P. crispus hybrids. These plants closely resembled P. crispus but differed in several ways, including having smaller, finer leaves and lacking the presence of true turions. We performed genetic analysis on these plants by comparing the internal transcribed spacer (ITS) DNA sequences from the putative hybrids to those identified as pure P. crispus and to Potamogeton accessions retrieved from GenBank. The putative hybrids had two divergent ITS sequences, one of which corresponded to sequences from P. crispus, and the other of which corresponded to sequences from P. pusillus, providing strong evidence of interspecific hybridization between these two species. Further, we identified genetic diversity even among pure P. crispus in North America. The extent of genetic diversity and the relevance to P. crispus ecology or management are currently unknown. Given the extent of management of P. crispus in North America, and the recognition that hybridization and genetic diversity can impact management outcomes, a geographic survey of genetic diversity and hybridization in P. crispus is warranted.

Headnote

ABSTRACT

Weed managers recognize that hybridization can influence invasiveness in target weeds. As such, the identification of hybridization in target weeds has become of fundamental interest. Curly-leaf pondweed (Potamogeton crispus) is a heavily managed invasive aquatic weed in the United States. The genus is known for extensive interspecific hybridization, but the extent to which invasive P. crispus in the United States hybridizes is unknown. In October 2018, an aquatic vegetation survey in the California Sacramento-San Joaquin river delta identified plants that were suspected as P. crispus hybrids. These plants closely resembled P. crispus but differed in several ways, including having smaller, finer leaves and lacking the presence of true turions. We performed genetic analysis on these plants by comparing the internal transcribed spacer (ITS) DNA sequences from the putative hybrids to those identified as pure P. crispus and to Potamogeton accessions retrieved from GenBank. The putative hybrids had two divergent ITS sequences, one of which corresponded to sequences from P. crispus, and the other of which corresponded to sequences from P. pusillus, providing strong evidence of interspecific hybridization between these two species. Further, we identified genetic diversity even among pure P. crispus in North America. The extent of genetic diversity and the relevance to P. crispus ecology or management are currently unknown. Given the extent of management of P. crispus in North America, and the recognition that hybridization and genetic diversity can impact management outcomes, a geographic survey of genetic diversity and hybridization in P. crispus is warranted.

Key words: GenBank, Pondweed hybrid, Potamogeton berchtoldi, Potamogeton pusillus, putative hybrid.

INTRODUCTION

Aquatic plant managers increasingly recognize that genetic variation can impact aquatic plant management outcomes. For example, fluridone efficacy in Florida populations of hydrilla (Hydrilla verticillata) is influenced by DNA substitutions in the phytoene desaturase gene (Michel et al. 2004), which can be detected by genetic screening (Benoit and Les 2013). Similarly, different genotypes of Eurasian (Myriophyllum spicatum) and hybrid water milfoil (M. spicatum X M. sibiricum) vary in their growth and response to several herbicides (e.g., Glomski and Netherland 2009, Berger et al. 2012, Thum et al, 2012, LaRue et al. 2013, Taylor et al. 2017, Netherland and Willey 2018), and distinct phenotypes of fanwort (Cabomba caroliniana) differ in their response to several herbicides (Bultemeier et al. 2009).

Hybridization between invasive species and their native relatives is one source of genetic variation that can influence invasiveness (Ellstrand and Schierenbeck 2000). For example, introduced Eurasian watermilfoil frequently hybridizes with native northern watermilfoil (M. sibiricum) in North America (Moody and Les 2002, 2007, Zuellig and Thum 2012). Hybridization is associated with increased invasiveness in milfoils (Moody and Les 2002, LaRue et al. 2012), and different hybrid genotypes exhibit different responses to several commonly used herbicides (Poovey et al. 2007, Glomski and Netherland 2009, Berger et al. 2012, 2015, Thum et al. 2012, LaRue et al. 2013, Taylor et al. 2017, Netherland and Willey 2018). The identification of hybrids frequently requires molecular genetic data because hybrids can be difficult to distinguish from parental species, especially when parental species themselves are difficult to distinguish from closely related species.

Potamogeton crispus is a frequently managed invasive aquatic plant in the United States and Canada, causing a multitude of problems such as outcompeting native plants, nutrient loading after senescence, and reducing recreation by blocking waterways (Jones et al. 2012, James et al. 2009, Parkinson and Mangold, 2016). Hybridization among Potamogeton pondweeds is common in their native range. For example, Zalewska (2002) identified 78 hybrids out of 5,000 herbarium specimens of Potamogeton. Invasive P. crispus has been documented on the basis of morphological analysis in many places; however, genetic data collection has not been routinely incorporated into management projects for P. crispus, and to our knowledge, no genetic survey of the species has been conducted in North America. Therefore, the extent to which P. crispus may hybridize is unknown.

In the Sacramento-San Joaquin River Delta, P. crispus is typically managed by morphological assessment using point intercept surveys (Madsen 1999) along with sonar/gps Biobase® (Howell and Richardson 2017). Following species identification and confirmation of biovolume, infested areas of P. crispus are treated with fluridone pellets (Sonar Q®, Sonar One®, and Sonar PR®), dipotassium salt of endothall (Aquathol K®), or diquat. Follow up surveys of biovolume are done in the fall to ensure reduction of the invasive plant. These monitoring and treatment actions are largely conducted by the California Division of Boating and Waterways (California Department of Parks and Recreation-Division of Boating and Water Ways 2017).

In this article, we document hybrid P. crispus in a California population using molecular methods. During an aquatic vegetation survey as part of a collaboration of California Division of Boating and Waterways and SePRO Corporation in October 2018, we identified a Potamogeton with an unusual phenotype in several locations, which we suspected as hybrid P. crispus. The stems of the putative hybrid were thinner and darker green than that of P. crispus, with a more fusiform cross section around 2 mm in diameter. The leaves of the plant were dark green, softer, lorate, and alternating, whereas P. crispus generally has lighter green or olive leaves with a crispy texture. The width of the leaves was 4-7 mm with a total length of 15-50 mm and around 75% less curly compared to P. crispus. The margins of the leaves were also lacking the serrate quality of P. crispus. Turions typical of P. crispus were not found on the plant, but the apical meristems bore a resemblance of a pseudo turion by being thicker on two sides with leaves coming from each apex.

MATERIALS AND METHODS

On October 3, 2018, an annual macrophyte survey was completed on Franks Tract, the largest waterbody in the Sacramento-San Joaquin Delta. We sampled 100 points that were generated by evenly spacing them over the study area using GIS. We sampled each point using a weighted, doubleheaded, 0.33-m-wide rake, which was dragged for ~3 m along the bottom and then pulled up to the boat for analysis. We recorded each species of submerged macrophyte that was present on the rake. We identified the putative hybrid P. crispus from four sample points.

We extracted total genomic DNA using DNeasy Plant Mini Kits (Qiagen)1 from dried meristem tissue that was preserved in the field with silica gel. We extracted from six putative hybrids from the Frank Tract populations, two putative pure P. crispus individuals from Frank's Tract, and one P. crispus collected in a creek located near Montana State University in Bozeman.

We performed molecular identifications of samples by comparing internal transcribed spacer (ITS) sequences from our samples to those from accessions retrieved from GenBank (Table 1). In particular, for suspected hybrid P. crispus, we predicted to see two unique ITS sequences for each individual that corresponded to sequences from P. crispus and another species.

We amplified ITS using the universal primers ITS4 and ITS 5 (Soltis and Kuzoff 1995). All PGR reactions contained the following: IX GoTaq Hot Start PGR buffer (Promega), 2 mM MgCl2, 2 pmol each primer, 0.2 mM each dNTP, 1 unit of GoTaq Hot Start DNA polymerase (Promega), 2 pl template DNA, and brought to a total volume of 25 pl with molecular biology-grade water. Thermal cycling consisted of the following: one cycle at 94 C for 2 min followed by 25 cycles of 94 C for 1 min, 53 C for 30 sec, 72 C for 1 min, and a final extension at 72 C for 5 min. We visualized 2 pl of PGR products on an agarose gel (^1.5%) to check for size and purity.

We treated PGR products with the enzymes Exonuclease I (New England Biolabs)2 and Antarctic Phosphatase (New England Biolabs)3 to eliminate unincorporated primers and dNTPs before sequencing. PGR products were sent for sequencing to the University of Illinois at Urbana-Champaign's Core Sequencing Facility on an ABI 3730x1 DNA sequencer.

In some cases, direct sequencing of PCR products produced clean and unambiguous sequence. However, in the case of putative hybrids, we found more than one ambiguous base pair, and sequence quality was poor due to insertions and deletions of base pairs (indels). We selected one representative individual to clone the PCR product using the TOPO TA cloning kit (ThermoFisher)4 and sequenced eight positive inserts).

DNA sequences were edited using Sequencher, version 4.2 (Gene Codes Corporation) and aligned using ClustalW, as implemented in MEGA X (Kumar et al. 2018). The final alignment size was 640 bp after trimming. We constructed a neighbor-joining tree in MEGA using a Kimura twoparameter model of DNA sequence evolution with uniform rates among sites, complete deletion of gaps and missing data, and 1,000 bootstrap replicates.

RESULTS AND DISCUSSION

As suspected on the basis of morphology, we found clear evidence for hybridization of P. crispus in Franks Tract in the Sacramento-San Joaquin Delta. Putative hybrids contained two divergent ITS sequences, one of which corresponded to GenBank accessions for P. crispus, and the other ITS sequence corresponded to GenBank accessions for P. berchtoldi (Figure 1) (Les et al. 2009). Although GenBank accessions label the species P. berchtoldi, P. berchtoldi is not recognized as its own species and is widely referred to as P. pusillus. Therefore, we have kept the name P. berchtoldi when referencing the specific GenBank accession, but we refer to the species as P. pusillus throughout the text, as this is the currently accepted official taxonomic name at this time (ITIS 2020). Out of 640 total bp, P. crispus differed from P. pusillus at 36 nucleotide positions. At each of these positions, putative hybrid individuals exhibited clear biparental polymorphisms that corresponded to the two parental ITS sequences (Table 2). Therefore, our data provide strong evidence for hybridization between these two species.

Hybridization in P. crispus has been documented with several species in its native range including crosses with P. praelongus and P. perfoliatus (Kaplan and Fehrer 2013). In its invasive range, hybridization has been documented in New South Wales, Australia, with P. ochreatus (Kaplan et al. 2011). To our knowledge, this is the first documentation of hybridization between P. crispus and P. pusillus.

Generally, hybrid pondweed species have not been recorded to pose management risks. However, hybridization has been widely documented to precede the evolution of invasiveness in many plants (Ellstrand and Schierenbeck 2000). For aquatic plants specifically, hybridization between invasive Eurasian watermilfoil and native northern watermilfoil has concerned managers, as some hybrid genotypes have been found to exhibit faster growth and/ or resistance to some commonly used herbicides (Berger et al. 2012, 2015, LaRue et al. 2012, Thum et al. 2012, Netherland and Willey 2018). There is no indication at this time that this is indeed a viable population, and it may be the case that the single observation of hybridization may be an interim event. It may be of interest to continue to search and track the population with annual surveys and to monitor the increase or potential decline of the hybrid if it is indeed found again.

In addition to P. pusillus, there are five other species in the Potamogetonacae family found in Franks tract including P. richardsonii, P. nodosus, P. illinoensis, Stuckenia pectinata, and S. filiformis (Caudill et al. 2019). It is unclear if P. crispus is capable of hybridizing with these other species. The hybrid did possess pseudo-turions at the apical meristem (Figure 2), which may indicate that it can reproduce both sexually and asexually, as in the case of P. crispus. Asexual reproduction through the formation and sprouting of turions is one of the aspects that makes P. crispus such a successful invasive species. Newman et al. (pers, comm.) found that turions can stay viable for 7 yr in benthic substrate. If the turion-like structures on the hybrid are viable, this means that the hybrid could have the potential for invasive qualities as well. The fact that for P. crispus to hybridize it has to cross-pollinate with other Potamogeton species may make this find somewhat of a rarity given that the plants primary reproductive strategy is largely asexual. The rarity of cross-pollination may be offset by the profuseness of P. crispus in an infestation event, however; P. crispus was found at only 10% of sites and P. bechtoldii was found at 27% of sites. Moreover, both parent species were found at only 3% of sites together. The following year, in October 2019, P. crispus was found at 7% of sites and P. pusillus was found at 13% of sites. The hybrid was not found in 2019, leading us to believe that the high frequency of occurrence of P. pusillus in 2018 led to an increased likelihood of hybridization with P. crispus. Considering that we did not find the hybrid in 2019, it may be possible that the hybrid was incapable of reproduction and likely not of management concern. Despite the lack of management concerns the site where the hybrid was found will continue to be monitored on an annual basis for other macrophyte management goals.

In addition to documenting hybridization between P. crispus and P. pusillus, it is clear from our data that there is at least some genetic diversity even among pure P. crispus in North America. We found three nucleotide positions that exhibited variation among accessions of P. crispus (Table 2). Two P. crispus accessions from North America exhibited heterozygosity at these nucleotide positions (Table 2). The extent of genetic diversity, and any implications for P. crispus ecology or management, are currently unknown. It is not known if treatment methodologies used to control P. crispus would also be successful on the hybrid; however, there is no indication that the hybrid would be more or less resilient to certain herbicides. Given the extent of management of P. crispus in North America, and the recognition that genetic diversity (including hybridization) can impact management outcomes, a geographic survey of genetic diversity for P. crispus is warranted.

SOURCES OF MATERIALS

1DNeasy Plant Mini Kit, Qiagen Corp., 27220 Turnberry Lane, Suite 200, Valencia, CA 91355.

2Exonuclease I, New England Biolabs, 240 County Rd, Ipswich, MA 01938.

"Antarctic phosphatase, New England Biolabs, 240 County Rd, Ipswich, MA 01938.

4TOPO™ TA Cloning™ Kit, Life Technologies Corp., 5791 Van Allen Way Carlsbad, CA 92008.

ACKNOWLEDGEMENTS

We thank Leah Simantel and Emma Rice for assistance with the molecular work. We also would like to thank Patricia Gilbert, Mark Heilman, Scott Shuler, and the California Department of Boating and Waterways for helping find the plant. Support for the project was provided in part by the Montana Agricultural Experiment Station (Project MONB00249).

Footnote

*First author: SePRO Corporation, 11550 North Meridian Street, Suite 600, Carmel, IN 46032-4565. Second Author: Assistant Professor, Department of Plant Science and Plant Pathology, Montana State University, Plant Bioscience Building, PO Box 173150, Bozeman, MT 59717. Corresponding author's E-mail: [email protected]. Received for publication March 31, 2020 and in revised form October 7, 2020.

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