Introduction of three cryptic lineages of invasive Nymphoides cristata in the southeastern United States

Abstract

Crested floating heart [ Nymphoides cristata (Roxb.) Kuntze] is an invasive aquatic plant in the southeastern United States. For clonal plants like N. cristata , clonal diversity may influence response to control tactics and/or evolutionary potential. However, little is known about the diversity of introduced N. cristata . In this study, we used genotyping-by-sequencing to quantify N. cristata diversity in the southeastern U.S. and determine how that diversity is distributed across the invaded range. Our results show that at least three distinct genetic lineages of N. cristata are present in the southeastern U.S. Geographic distribution of the lineages varied, with one widespread lineage identified across several states and others only found in a single waterbody. There is also evidence of extensive asexual reproduction, with invaded waterbodies often host to a single genetic lineage. The genetic diversity reported in this study likely results from multiple introductions of N. cristata to the southeastern U.S. and should be considered by managers when assessing control tactics such as screening for biocontrol agents or herbicide testing. The extent and distribution of genetic diversity should also be considered by researchers studying the potential for invasive spread of N. cristata within the U.S. or hybridization with native Nymphoides species.

Full text

This is a “preproof” accepted article for Invasive Plant Science and Management. This version
may be subject to change in the production process, and does not include access to
supplementary material. DOI: 10.1017/inp.2024.28
Short title: Diversity of floating heart
Introduction of three cryptic lineages of invasive Nymphoides cristata in the southeastern
United States
Zachary J. Kuzniar1, Nathan E. Harms2, Sarah M. Ward3, and Ryan A. Thum4
1 Graduate student (ORCID 0009-4211-6304), Department of Pant Science and Plant Pathology,
Montana State University, Bozeman, MT, USA; 2 Research Biologist, US Army Engineer
Research and Development Center, Lewisville, TX, USA; 3Affiliate Professor, Department of
Land Resources and Environmental Sciences, Montana State University, Bozeman, MT, USA;
4Associate Professor, Department of Plant Science and Plant Pathology, Montana State
University, Bozeman, MT, USA.
Author for correspondence: Zachary J. Kuzniar. Email: zachary.kuzniar@student.montana.edu
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

Abstract
Crested floating heart [Nymphoides cristata (Roxb.) Kuntze] is an invasive aquatic plant in the
southeastern United States. For clonal plants like N. cristata, clonal diversity may influence
response to control tactics and/or evolutionary potential. However, little is known about the
diversity of introduced N. cristata. In this study, we used genotyping-by-sequencing to quantify
N. cristata diversity in the southeastern U.S. and determine how that diversity is distributed
across the invaded range. Our results show that at least three distinct genetic lineages of N.
cristata are present in the southeastern U.S. Geographic distribution of the lineages varied, with
one widespread lineage identified across several states and others only found in a single
waterbody. There is also evidence of extensive asexual reproduction, with invaded waterbodies
often host to a single genetic lineage. The genetic diversity reported in this study likely results
from multiple introductions of N. cristata to the southeastern U.S. and should be considered by
managers when assessing control tactics such as screening for biocontrol agents or herbicide
testing. The extent and distribution of genetic diversity should also be considered by researchers
studying the potential for invasive spread of N. cristata within the U.S. or hybridization with
native Nymphoides species.
Key words: aquatic plant, biological invasions, clonal reproduction, genotyping-by-sequencing
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

Management Implications
The identification of three distinct Nymphoides cristata (crested floating heart) lineages
here – and a fourth interspecific hybrid lineage (N. cristata × N. aquatica) reported elsewhere –
has implications for the management of this invasive species. Studies of control techniques,
spread, and impacts of N. cristata should explicitly consider the genetic diversity identified in
this study. For example, herbicides are currently the most common option for controlling N.
cristata, including submersed applications of diquat, endothall, and florpyrauxifen-benzyl, foliar
applications of endothall, imazamox, and imazapyr, and foliar combinations of flumioxazin and
glyphosate. However, herbicide testing has not explicitly considered whether N. cristata lineages
differ in their responses. As differences in herbicide response has been found among distinct
clonal genotypes of other aquatic plants (e.g., 2,4-D and fluridone in Myriophyllum spp.;
fluridone in Hydrilla verticillata), we posit that herbicide trials for N. cristata should include
representatives of the four distinct lineages identified thus far. Similarly, N. cristata has been
identified as a candidate for biological control. However, there are no biocontrol agents currently
in operation. The identification of distinct lineages in the U.S. suggests at least three independent
introductions. Genetic survey of N. cristata in its native range could help inform the search for
biological control agents, especially if introductions can be traced to distinct geographic origins.
Further, any biological control agents identified should be tested on the distinct genetic lineages
identified in the U.S., as biocontrol efficacy can vary at the subspecific level. Finally, it is
possible that the distinct lineages could have distinct environmental preferences or tolerances
that could be important for predicting their spread across the landscape.
Introduction
Genetic variation can influence plant invasions and management (i.e., Barrett 1992;
Bossdorf et al. 2005; Sakai et al. 2001; Ward et al. 2008). Additionally, genetic variation can
facilitate adaptation to new environments encountered after introductions, and during range
expansion (e.g., Lee 2002; Prentis 2008). For example, distinct genetic populations or lineages of
invasive plants may vary in their response to herbicides (e.g., Kay 1992; Netherland and Willey
2017; Chorak and Thum 2020; Williams et al. 2020; Kurniadie et al. 2021). Similarly, genetic
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

lineages can differ in the effect of biological control agents (e.g., Sobhian et al. 2003; Bruckart et
al. 2004; Williams et al. 2014; Blossey et al. 2018).
The amount and distribution of genetic variation in invasive plants will be primarily
influenced by the number of introductions, the genetic structure and diversity of source
populations, and the extent of admixture, sexual reproduction, and asexual reproduction in the
introduced range. At one extreme, introduction of a single genet followed by exclusively asexual
reproduction will result in essentially no genetic diversity in the introduced range (e.g.,
Hollingsworth and Bailey 2000; Le Roux et al. 2007; Loomis and Fishman 2009; Zhang et al.
2010). At the other extreme, multiple introductions from genetically distinct source populations,
followed by admixture and sexual reproduction, can generate greater and novel genetic diversity
in introduced populations (Facon et al. 2008; Kolbe et al. 2004; Lavergne & Molofsky 2007).
Population genetic and genomic descriptions of invasive species can help determine the number
of distinct lineages present in the introduced range, and provide insight into the extent or
potential for admixture of distinct lineages which in turn can inform studies on herbicidal and
biological control development.
Crested floating heart [Nymphoides cristata (Roxb.) Kuntze] is a floating-leaved aquatic
plant native to Asia that has spread across the southeastern United States. The first introduction
of N. cristata appears to have occurred in south Florida circa 1996 when plants escaped from
cultivation for the aquatic garden trade (Burks 2002). Since then, N. cristata has been observed
in Louisiana (2012), Mississippi (2016), North Carolina (2017), South Carolina (2006), and
Texas (2014), and is considered an invasive/noxious species in several of the inhabited states
(Thayer and Pfingsten 2024). The scattered distribution of N. cristata populations throughout the
southeastern U.S. may reflect multiple introductions (Burks 2002), but no genetic analysis has
been conducted to date to test whether introduced N. cristata consists of one or more distinct
lineages.
Nymphoides cristata is capable of both asexual and sexual reproduction, although the
prevailing mode of reproduction remains largely unknown. In the native range, N. cristata
reproduces sexually through a gynodioecious breeding system derived from heterostyly, where
female plants with reduced, sterile stamens, rely on the bisexual-morph plants for pollen (Nair
1973). In addition, bisexual plants from the native range have shown self-compatibility in an
experimental setting (Nair 1973). However, asexual reproduction via vegetative propagation is
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

likely responsible for the majority of biomass (Nair 1973; Sculthorpe 1967), a strategy common
in the Nymphoides genus (Gettys et al. 2017; Sivarajan and Joseph 1993). Similarly, N. cristata
is thought to reproduce primarily through vegetative means in the introduced range
(fragmentation, daughter plants, tubers, and rhizomes) (Burks 2002; Willey and Langeland 2011).
Spread and range expansion of the species is likely facilitated via fragmentation caused by
contact with boat motors, wave action, and mechanical harvesting (Burks 2002; Willey et al.
2014). It is also capable of producing seeds (Burks 2002; Gettys et al. 2017), and has hybridized
with a native species, big floating heart [Nymphoides aquatica (J.F. Gmel.) Kuntze] (Harms et al.
2021), but the extent of sexual reproduction in U.S. populations remains unknown.
In this study, we conducted a population genomic survey of introduced N. cristata in the
southern U.S. to determine how much genetic diversity is in the introduced range. For example,
if the N. cristata invasion results from asexual spread of a single genetic clone, then we would
expect to find no population genomic variation in our survey (barring somatic mutation and
genotyping error). In contrast, identification of multiple distinct genetic lineages would suggest
multiple introductions from different sources, and the potential for intermediate genotypes if
distinct lineages have hybridized.
Materials and Methods
Sample collection:
Samples were obtained from a previous study on N. cristata (see Harms et al. 2021),
using preserved DNA samples and dried plant tissues. Briefly, Harms et al. (2021) collected 1-13
plants per waterbody, depending on the size of the infestation, with small populations minimally
sampled (i.e., only a few plants). Within waterbodies, plants were sampled 3-5 meters apart to
avoid repeatedly collecting the same plant. The final data set consisted of 62 samples (i.e., leaves
from N. cristata plants) from 12 different waterbodies throughout the southeast United States,
including lakes, ponds, roadside ditches, and canals (Table 1). We acknowledge that our
sampling strategy limits out inference regarding diversity within waterbodies. However, the main
focus of this study was to evaluate evidence for one versus multiple genetic lineages in the U.S.,
and given the likelihood of local clonal reproduction, we prioritized the number of waterbodies
examined over the number of individuals per waterbody.
DNA sequencing:
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

We used next-generation, genotyping-by-sequencing (GBS) to generate a single
nucleotide polymorphism (SNP) data set. Prior to sequencing, DNA was extracted from plant
tissues using the Qiagen DNeasyPlant Kit (Qiagen Corporation, 27220 Turnberry Lane, Suite
200, Valencia, CA 91355) following the standard plant protocol. A Qubit fluorometer
(ThermoFisher Scientific, 168 Third Ave., Waltham, MA, 02451) was used to confirm genomic
DNA content was high enough for sequencing (60ng total gDNA), then all extracts were sent to
the University of Minnesota Genomic Center for library assembly and sequencing. The
sequencing library was prepared for double digest restriction-site associated DNA sequencing
(ddRAD; Peterson et al. 2012) using the restriction enzyme pair PstI and MspI and size-selected
for 101bp fragments using the PippinHT system. The library was sequenced on an Illumina
NovaSeq 6000 (Illumina, Inc., 5200 Illumina Way, San Diego, CA, 92122) system targeting
approximately 2.5M single-end reads per sample.
Bioinformatics and filtering:
Following sequencing, we processed the raw sequence data using a bioinformatics
pipeline to produce a SNP data set for downstream diversity analyses. First, the reads were
demultiplexed by barcode and adapters were trimmed using gbstrim, a custom script designed to
pre-process GBS data generated by UMGC (Garbe 2022). The demultiplexed reads were then
passed to Stacks v2.55 (Catchen et al. 2013), where the process_radtags module removed low-
quality reads (i.e., “Phred” score < 25). Next, the core de novo pipeline was executed in Stacks
with the minimum stack depth (m), mismatch distance between loci within an individual (M),
and number of mismatches between loci in the catalog (n) parameters set at 5, 4, 4, respectively.
These were selected following a parameter optimization procedure similar to that outlined in
Paris et al. (2017). After catalog creation, SNP identification and genotyping were also
performed in Stacks, retaining only one biallelic SNP per RAD-locus. The resulting variant-
calling files were exported for further filtering and analysis. All bioinformatic work in this study
was performed on the Montana State University Tempest computing cluster.
To ensure only high-quality variants were included in downstream analysis, the genetic
data were filtered with the R package vcfR v1.13.0 (Knaus and Grunwald 2017). To reduce the
amount of missing data, we excluded loci that were absent in >25% of the individuals and
removed any individuals with >75% missing genotype calls. We also removed loci with unusual
read depth (under 10th percentile or over 90th percentile) and set the minimum minor allele
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

frequency to 0.05. After filtering, the final genetic data set contained 62 N. cristata samples
genotyped at 2,242 SNPs.
Data analysis:
To avoid violating any model assumptions associated with the clonality of N. cristata, we
used a model-free approach to explore the amount and distribution of genetic diversity. A
Euclidean genetic distance matrix was generated in adegenet v2.1.7 (Jombart and Ahmed 2011)
for use in Principal Components Analysis (PCA). The PCA was completed in the ade4 package
(Dray and Dufour 2007) to visualize genetic variation among samples and determine whether
distinct genetic groups of N. cristata exist. We also generated a genetic dissimilarity matrix using
poppr v.2.9.3 (Kamvar et al. 2014) to summarize the actual number of SNP differences between
individuals. The distribution of N. cristata across the invaded range was mapped with ggplot2
(Wickham 2016). All data analyses were conducted in R version 4.2.1 (R Core Team 2021).
Results and Discussion
Our survey of N. cristata diversity identified three distinct genetic groups (Figure 1). We
refer to those groups as genetic lineages, collections of closely related individuals distinguished
by the genomic variants (i.e., SNPs) inherited from a common ancestor. Genetic variation was
found both within- and between lineages, although the amount of between-lineage variation was
far greater than within. Within lineages, individuals averaged approximately 35 SNP differences,
while individuals compared across lineages differed by an average of 1028 – 1514 differences
(Table 2).
The CFH-2 lineage was the most common and widespread; it was found in Florida,
Louisiana, and Texas, and occurred in 10 of the 12 waterbodies sampled overall (Figure 2). In
contrast, CFH-3 was only found in one waterbody (Lake Marion, SC), and CFH-1 was only
found in two waterbodies, both in southeast Florida (Figure 2).
The differences in relative abundance among lineages may be due to introduction
dynamics and/or ecological differences among lineages. N. cristata was likely brought to the U.S.
for trade as an aquatic garden ornamental and remains available for purchase from vendors in the
industry. Although it is now illegal to possess, import, or distribute N. cristata in several
southern states (FL, SC, NC, TX), the ornamental industry is a likely candidate for initial
introduction(s) and subsequent range expansion. The relative abundance of the CFH-2 lineage
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

may indicate that it was a preferential/popular lineage in the industry, or that it was the first to
escape cultivation. Indeed, N. cristata is clonally propagated for sale in the industry, although it
was not possible to include commercially available ornamental samples in this study due to time
and funding constraints. In addition, CFH-2 may be more vigorous and widely adapted in the
introduced range than others, facilitating its spread across the southern states while CFH-1 and
CFH-3 remained isolated. Further investigation would be necessary to determine whether CFH-2
presents a greater management challenge. We recognize that the number of waterbodies sampled
and number of individuals sampled within a waterbody was limited and there may be more
diversity in the introduced range than we detected.
Burks (2002) suggested multiple introductions, possibly escaped from ornamental water
gardens, based on the scattered distribution of N. cristata across southern Florida. Indeed, we
identified two distinct lineages in Florida: one that was restricted to southeastern Florida, and a
second lineage that is widespread across Florida and the U.S. Gulf Coast (Figure 2). These may
represent two independent introductions in Florida followed by range expansion of CFH-2. It is
also possible that CFH-2 has been repeatedly introduced across the Gulf Coast states, with its
widespread distribution representing numerous independent introductions. Further, we identified
a third unique lineage found only in South Carolina (CFH-3), which could represent another
introduction.
We cannot rule out with certainty the alternative hypotheses of a single introduction from
a genetically variable source, or accumulation of new mutations following introduction.
However, we would have expected more within-waterbody lineage diversity if N. cristata was
introduced from a genetically variable source (although we recognize that within waterbody
sample sizes were low). Similarly, we find clonal evolution post-introduction unlikely because of
the relatively large number of allelic differences (1028 - 1514) separating the three distinct
lineages combined with the relatively recent introduction (~1996). Plant invasions resulting from
multiple introductions are common (Dlugosch and Parker 2008), particularly for ornamental
species like N. cristata where the plant trade increases the likelihood of repeated introductions
(Dehnen-Schmutz et al. 2007). Sampling efforts in the native range (i.e., southeastern Asia),
along with additional sampling in the U.S., could help clarify the number and location(s) of
sources of N. cristata introduction. In addition, N. cristata has been identified as a good
candidate for biological control, although natural enemies/potential agents have yet to be
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

described (Harms and Nachtrieb 2019). Identification of source populations in the native range
might also yield natural enemies of this plant that could be tested as potential biocontrol agents
(Bossdorf et al. 2005; Gaskin et al. 2011).
The introduction of three distinct lineages provides opportunities for genetic admixture
and the generation of novel genetic variants in the introduced range (e.g., Facon et al. 2008;
Kolbe et al. 2004; Lavergne & Molofsky 2007). Although interspecific hybrids between N.
cristata and native N. aquatica have been identified in the Santee Cooper Reservoir system in
South Carolina (Harms et al. 2021), we did not find any evidence for sexual reproduction among
the three lineages, as evidenced by the lack of genetically intermediate individuals (Figure 1). In
addition, the average pairwise genetic distances between individuals were approximately 97%
greater when comparing between lineages versus within lineages. It is possible that these
lineages have sexually reproduced with one another, but that we did not sample them.
Alternatively, it is possible that the distinct lineages are capable of sexual reproduction but have
not had sufficient opportunity, yet. The three lineages were largely allopatric, but there was some
overlap between two of them in south Florida. Finally, it is possible that the different lineages
have some reproductive barriers (e.g., pre or post mating, pre or post zygotic) that limit sexual
reproduction between them. Additional study of reproductive potential among the introduced
lineages is warranted.
While we cannot rule out some degree of sexual reproduction within lineages, we
hypothesize that the low genetic variation observed within each of the lineages primarily reflects
sequencing/genotyping errors, and that the N. cristata lineages identified here have primarily
reproduced asexually throughout the southeastern United States. Although individuals within
each lineage were not genetically identical, per se, clonal genotypes are not expected to have
identical genotypes across thousands of SNPs generated by ddRAD due to
sequencing/genotyping error and somatic mutations (da Cuhna et al. 2021; Reynes et al. 2021).
The interpretation of the low within lineage variation as clonal reproduction is consistent with
previous field observations and reports of reproductive biology of N. cristata that hypothesize
prolific vegetative propagation and spread in the invaded range (Burks 2002; Harms and
Nachtrieb 2019; Willey and Langeland 2011). Inbreeding could also account for the within-
lineage variation we observed. Nymphoides cristata has been proven to be self-compatible
through artificial pollination of bisexual plants in an experimental setting (Nair 1973). However,
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

heterostyly provides a morphological based incompatibility system in N. cristata. Although
incomplete, this system is thought to promote reproduction between the female and bisexual
plant morphs (i.e., dioecism) in the native range (Nair 1973). Further sampling and detailed
description of flower morphology in the introduced range could help decipher the reproductive
capabilities of the introduced populations. Finally, we acknowledge that the low sample sizes
within waterbodies preclude an understanding of the relative extent of sexual versus asexual
reproduction in any single population of N. cristata.
Acknowledgements
The authors would like to thank Greg Chorak, Del Hannay, and Ashley Wolfe for providing
thoughtful comments to improve the manuscript. We thank Cassidy Kempf, Michael Coulon,
Tim Bister, Jacob Green, Casey Moorer, John Riser, and Carl Bussells for assistance with sample
collections.
Funding: This work was supported by the U.S. Army Engineer Research and Development
Center Aquatic Plant Control Research Program Cooperative Agreement W912HZ-18-2-0010,
and the Montana Agricultural Experiment Station (Project MONB00249).
Competing Interests: The authors declare none.
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

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https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

Table 1. Waterbodies where Nymphoides cristata were collected for use in this study, including
genetic lineage assignments (Lineage ID) and number of individuals (N) collected from each
waterbody.
Waterbody
Latitude
Longitude
County
State
Lineage ID
N
Lake Fairview
28.6005
-81.4128
Orange
FL
CFH-2
1
JW Corbett WMA
26.8579
-80.4165
Palm Beach
FL
CFH-2
2
Roadside canal
26.6548
-80.1747
Palm Beach
FL
CFH-1, CFH-2
2, 6
Flying Cow ditch
26.6348
-80.3001
Palm Beach
FL
CFH-1
6
Business pond
26.0057
-80.3018
Broward
FL
CFH-2
2
Flat Lake
30.2817
-90.8182
Ascension
LA
CFH-2
13
Private pond
30.4043
-91.1576
Avoyelles
LA
CFH-2
1
Lake Marion
33.5372
-80.428
Berkeley
SC
CFH-3
7
Caddo Lake
32.7195
-94.1198
Harrison
TX
CFH-2
10
Lake Conroe
30.5643
-95.6358
Montgomery
TX
CFH-2
3
Houston Arboretum
29.7618
-95.4498
Harris
TX
CFH-2
7
Roadside ditch
30.4399
-94.7201
Hardin
TX
CFH-2
2
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

Table 2. Summary of individual-based genetic distance within and between lineages. Reported
values along the diagonal are the absolute number of SNP differences between individuals within
a lineage. Between lineage values represent the average number of SNP differences observed
across lineages.
CFH-1
CFH-2
CFH-3
CFH-1
30
-
-
CFH-2
1514
31
-
CFH-3
1099
1028
41
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

Figure 1. Principle component analysis of 62 Nymphoides cristata samples. Principle
components 1 and 2 account for 96.1% of cumulative variation. Colored ellipses (non-statistical)
were added a posteriori to denote putative genetic lineages identified by ordination.
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press

Figure 2. Distribution of Nymphoides cristata lineages across the introduced range. Points
represent waterbodies where samples were collected and colors denote the genetic lineage(s)
present, assigned according to the PCA analysis in Figure 1. *Note there are two overlapping
points representing the roadside canal in south Florida where CFH-1 and CFH-2 co-occurred.
https://doi.org/10.1017/inp.2024.28 Published online by Cambridge University Press