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Invasive Plant Science and
Management
www.cambridge.org/inp
Research Article
Cite this article: Williams DA, Harms NE,
Knight IA, Grewell BJ, Futrell CJ, and Pratt PD
(2020) High genetic diversity in the clonal
aquatic weed Alternanthera philoxeroides in the
United States. Invasive Plant Sci. Manag 13:
217–225. doi: 10.1017/inp.2020.32
Received: 28 May 2020
Revised: 21 September 2020
Accepted: 13 October 2020
First published online: 4 November 2020
Associate Editor:
Marie Jasieniuk, University of California, Davis
Keywords:
Chloroplast DNA; invasive species; ploidy;
population genetics
Author for correspondence:
Dean A. Williams, Department of Biology, Texas
Christian University, Fort Worth, TX 76129.
(Email: dean.williams@tcu.edu)
© The Author(s), 2020. Published by Cambridge
University Press on behalf of the Weed Science
Society of America.
High genetic diversity in the clonal aquatic weed
Alternanthera philoxeroides in the United States
Dean A. Williams1, Nathan E. Harms2, Ian A. Knight3, Brenda J. Grewell4,
Caryn Joy Futrell5and Paul D. Pratt6
1Professor, Department of Biology, Texas Christian University, Fort Worth, TX, USA; 2Research Biologist, Aquatic
Ecology and Invasive Species Branch, Environmental Laboratory, U.S. Army Engineer Research and
Development Center, Vicksburg, MS, USA; 3Postdoctoral Research Participant, Oak Ridge Institute for Science
and Education, Oak Ridge, TN, USA; 4Research Ecologist, U.S. Department of Agriculture, Agricultural Research
Service, Invasive Species and Pollinator Health Research Unit, Davis, CA, USA; 5Biological Sciences Technician,
U.S. Department of Agriculture, Agricultural Research Service, Invasive Species and Pollinator Health Research
Unit, Davis, CA 95616, USA and 6Research Leader and Entomologist, U.S. Department of Agriculture, Agricultural
Research Service, Invasive Species and Pollinator Health Research Unit, Albany, CA, USA
Abstract
The distribution of genetic diversity in invasive plant populations can have important manage-
ment implications. Alligatorweed [Alternanthera philoxeroides (Mart.) Griseb.] was introduced
into the United States around 1900 and has since spread throughout much of the southern
United States and California. A successful biological control program was initiated in the late
1960s that reduced A. philoxeroides in the southern United States, although control has varied
geographically. The degree to which variation among genotypes may be responsible for varia-
tion in control efficacy has not been well studied due to a lack of genetic data. We sampled 373
plants from 90 sites across the United States and genotyped all samples at three chloroplast
regions to help inform future management efforts. Consistent with clonal spread, there was
high differentiation between sites, yet we found six haplotypes and high haplotype diversity
(mean h=0.48) across states, suggesting this plant has been introduced multiple times.
Two of the haplotypes correspond to previously described biotypes that differ in their suscep-
tibility to herbicides and herbivory. The geographic distribution of the three common haplo-
types varied by latitude and longitude, while the other haplotypes were widespread or localized
to one or a few sites. All the haplotypes we screened are hexaploid (6n=102), which may
enhance biological control. Future studies can use these genetic data to determine whether gen-
otypes differ in their invasiveness or respond differently to control measures. Some states, for
instance, have mainly a single haplotype that may respond more uniformly to a single control
strategy, whereas other states may require a variety of control strategies. These data will also
provide the basis for identifying the source regions in South America, which may lead to the
discovery of new biological control agents more closely matched to particular genotypes.
Introduction
The distribution of genetic diversity in invasive plants can have important management impli-
cations. Different genotypes of the same species and inter- or intraspecific hybrids can require
different control methods due to differing growth potential or resistance to particular agents or
herbicides (Bultemeier et al. 2009; LaRue et al. 2013; Manrique et al. 2008; Michel et al. 2004;
Thum et al. 2012; Williams et al. 2014). Investigations into the distribution of genetic diversity
across the invasive range can potentially shed light on whether management efforts will need to
be tailored to specific regions or whether an overall broad strategy can be employed (Benoit and
Les 2013; Thum et al. 2020). For example, the aquatic invader hydrilla (Hydrilla verticillata L. f.
Royle) is widespread in the United States, but management must be tailored to the infested area,
because there are multiple introduced biotypes with different geographic distributions (Jacono
et al. 2020); introduced biological control agents differ in their effectiveness between biotypes
(Grodowitz et al. 2010); and herbicide-tolerant strains have emerged in some areas, requiring
alternative herbicides for effective control (Netherland and Jones 2015). Understanding and
comparing the distribution of genetic diversity in native and invasive ranges can also shed light
on the number of introductions and evolutionary processes acting during invasion (founder
effects, bottlenecks, etc.) and identify source regions in the native range to facilitate the explo-
ration for biological control agents and the design of appropriate host-specificity plant tests
(Croxton et al. 2011; Gaskin et al. 2011; Ward et al. 2008; Williams et al. 2005,2018).
Alligatorweed [Alternanthera philoxeroides (Mart.) Griseb.] is native to the Parana and
Paraguay River regions in Argentina, Paraguay, Uruguay, and southeastern Brazil (Sosa
et al. 2004). It was introduced into the United States around 1900, most likely through
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contaminated ship ballast, and quickly spread throughout water-
ways in the southern United States and California (Zeiger 1967).
The species has also been introduced to Asia, Europe, Australia,
Central America, the Caribbean, and other regions in South
America. The plant can grow in both terrestrial and aquatic hab-
itats and is a serious threat to waterways, agriculture, and natural
habitats due to its rapid growth rate and ability to outcompete
native species (Buckingham 2002).
Phenotypic plasticity for growth form and sex in A. philoxer-
oides is common and has been documented for aquatic versus ter-
restrial habitats and between different soil types (Geng et al. 2007;
Kay and Haller 1982; Liu et al. 2011). In the United States, two bio-
types have been recognized, one with a broad stem and long, nar-
row leaves (BSA) and one with a narrow stem and short, broad
leaves (NSA), which are differentiated at allozyme loci and are
not a phenotypic response to different environmental conditions
(Kay and Haller 1982; Wain et al. 1984). These biotypes may cor-
respond to two forms described in South America, A. philoxeroides
philoxeroides and A. philoxeroides angustifolia (Sosa et al. 2004,
2008). A study utilizing nuclear inter-simple sequence repeats
(ISSRs) found that populations in the United States have much
higher genetic diversity compared with populations in China
and genetic diversity similar to samples from the plant’s native
range in Argentina (Geng et al. 2016). Geng et al. (2016) suggest
a single clonal lineage was introduced into China, while multiple
lineages were introduced into the United States.
Alternanthera philoxeroides ploidy is difficult to study due to a
large number of small chromosomes, but the presence of aneuploidy
and low pollen viability in a number of samples in the native range
suggests A. philoxeroides exists as a complex of hybrids (Sosa et al.
2008; Telesnicki et al. 2011). In Argentina, the species exists as tetra-
ploid (4n=ca. 68) or hexaploid (6n=ca. 102), while in China, indi-
viduals are hexaploid (6n=102) (Cai et al. 2009; Krug and Sosa 2019;
Sosa et al. 2008). Based on estimates of genome size, A. philoxeroides
may be pentaploid in the United States (Chen et al. 2015). The ploidy
of A. philoxeroides may be important from a control standpoint,
because increased ploidy has been suggested to contribute to invasive-
ness for a number of taxa by increasing adaptive potential or plasticity
to environmental conditions and management actions (Pandit et al.
2014;teBeestetal2012). However, for A. philoxeroides,femalealli-
gator weed flea beetles (Agasicles hygrophila Selman and Vogt,
Coleoptera: Chrysomelidae) prefer to oviposit, and larvae have better
performance on hexaploids than tetraploids in the native range (Krug
and Sosa 2019). Therefore, the role of polyploidy in A. philoxeroides
invasion success and management remains unclear.
Three biological control agents were introduced into the United
States in 1964 to 1971 to control A. philoxeroides (Spencer and
Coulson 1976). These agents, especially A. hygrophila, have signifi-
cantly reduced infestations in the southern United States
(Cofrancesco 1988; Spencer and Coulson 1976). Nevertheless,
there is still widespread variability in control efficacy that may have
a climatic or genetic basis. Because A. hygrophila cannot survive
colder winter temperatures in the northern part of A. philoxeroides
range in the United States, it must migrate annually from overwin-
tering locations in the South (Coulson 1977). This leads to annual
variation in the timing of A. hygrophila activity in more northern
A. philoxeroides populations and contributes to the observed vari-
ability in control in those regions (Harms and Cronin 2020).
Additionally, the two A. philoxeroides biotypes (BSA and NSA)
in the United States appear to differ in their susceptibility to her-
bivory by A. hygrophila (Kay and Haller 1982; Pan et al. 2013) and
they respond differently to some herbicides (Kay 1992). To what
extent observed variability in control may be due to presence of
these different genotypes or others has not been well studied.
Here we report on the largest A. philoxeroides genetic study to
date, conducted across the entire invaded range of the United
States to help inform future management of this species. We char-
acterized the genetic structure of A. philoxeroides in the United
States using chloroplast (cpDNA) markers, to better understand
the invasion history of A. philoxeroides and explore whether this
genetic diversity can be linked to geographic patterns, climate,
morphology, and ploidy, which could have important manage-
ment implications. Chloroplast markers are particularly well suited
for phylogeographic studies, and so these data will also provide the
basis for identifying the specific source regions in South America,
which may lead to the discovery of new biological control agents
more closely matched to particular genotypes (e.g., Cuda et al.
2012; Schaal et al. 2003; Williams et al. 2005,2018). The response
of plants to management such as biological control agents and her-
bicides is expected to be related to nuclear DNA variation rather
than cpDNA haplotypes per se. In clonally reproducing popula-
tions, however, cpDNA variation will be linked to nuclear DNA,
and cpDNA haplotypes may therefore provide a good marker
for distinguishing biotypes, as they are for invasive H. verticillata
in the United States (Madeira et al. 2007). Alternanthera philoxer-
oides produces viable seed in the native range but is believed to
spread only through fragmentation and vegetative growth in the
invasive range (Geng et al. 2007; Sainty et al. 1998; Thayer and
Pfingsten 2020; Ye et al. 2003). Seedlings have never been found
in the invasive range, and although seeds are produced, they do
not appear viable, so cpDNA haplotypes could potentially be useful
as markers to indicate which management efforts will be most
effective for a given area.
We specifically ask: (1) Is cpDNA diversity consistent with high
genetic diversity and the presence of multiple introductions into
the United States? (2) Does genetic diversity in A. philoxeroides
reflect geographic and climatic patterns? (3) Do genotypes corre-
spond to the previously described NSA and BSA biotypes? (4) Do
genotypes correspond to previously described ploidy levels in
A. philoxeroides? These data will provide a better understanding
Management Implications
We found high genetic diversity of Alternanthera philoxeroides
(alligatorweed) across the United States that is structured geographi-
cally. Two of the chloroplast haplotypes (genotypes) we found corre-
spond to two previously described narrow-stem (NSA) and broad-
stem (BSA) biotypes that are reported to respond differently to both
biological control and herbicides. The other genotypes we found may
also differ in their invasiveness (e.g., varying growth rates) or may
respond differently to control measures. The control of these different
haplotypes should be tested with currently available herbicides and
biological control agents. These studies may encourage foreign explo-
ration for additional agents that are better adapted to specific haplo-
types and climates. Managers may need to consider that different
areas will need different management strategies. Some states have
mainly a single invasive haplotype that may respond more uniformly
to a single control strategy, while other states and river basins have a
variety of haplotypes and therefore may require a variety of control
strategies depending on population genetics and specific locality.
The markers we developed in this study could be used to quickly
determine which haplotypes are present in a given area.
218 Williams et al.: A. philoxeroides genetic diversity
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of the genetic variability across the invasive range in the United
States that can be used in future studies to determine whether
genotype differences may be correlated with growth rates and
effectiveness of different control methods.
Materials and Methods
Sampling
Alternanthera philoxeroides tissues (n=375 samples) were col-
lected during 2017 to 2020 at locations in the United States.
Most samples were collected by the authors (66 sites), but in several
cases (33 sites), we received material from collaborators. Sampling
locations were identified through a variety of means, including
chance encounters while driving, directed searching from online
databases, museum herbarium records, or local knowledge.
Plants within sites were chosen for sampling at random, and col-
lections were spaced apart by several meters to avoid sampling the
same plant. At each site, one to eight plants were sampled. The
uppermost two leaf-pairs were removed and placed in a sealable
plastic bag with silica gel desiccant. Once dry, samples were frozen
until they could be processed.
Genetics
We extracted DNA from all samples using the IBI Scientific MINI
Genomic DNA kit (Plants) (Dubuque, IA) as per the manufacturer’s
instructions. We amplified and sequenced three chloroplast (cpDNA)
regions (rpL16,trnS-G,trnL intron, and trnL-F spacer), using primers
reported in Shaw et al. (2005) for 24 samples. Polymerase chain reac-
tions (PCR) (10 μl) contained 10 to 50 ng of DNA, 0.5 μMofeach
primer, and 1X AccuStartTM II PCR SuperMix (2X) (Quanta
Biosciences, Gaithersburg, MD). Reactions were cycled in an ABI
2720 thermal cycler (Thermo Fisher Scientific, Waltham, MA).
The cycling parameters were one cycle at 95 C for 10 min, followed
by 30 cycles of 30 s at 94 C, 30 s at 55 C, 60 s at 72 C, and then a final
extension at 72 C for 5 min. Products were sequenced bidirectionally
with the PCR primers using BrightDye®Terminator Cycle
Sequencing Kit (MCLAB, South San Francisco, CA). The only poly-
morphisms between samples in these cpDNA regions were
differences in the size of mononucleotide repeats. Unique sequences
have been deposited in GenBank (NCBI accession numbers
MW015925–MW015933). We therefore designed primers around
these repeat regions (5 0–30)rpL16F–TGGAATCATAGTGGA
TTGTCAAA, rpL16R–CAATTCATTGGGAAGGATGG; trnS-GF–
AAGTAACAAAGATTCAACGAATTCAA, trnS-GR–AGGCCGT
GGGAATACTCCTT; trnFfF–CTTCTCTCGCATCATCTTCTCA,
trnFfR–GTCCCTCTATCCCCAAAAGC. Each forward primer was
labeled with 6-FAM. All three cpDNA regions were amplified in a
single multiplex reaction. PCRs (10 μl)contained10to50ngof
DNA, 0.1 μM of each primer, and 1X AccuStartTM II PCR
SuperMix (2X). The cycling parameters were one cycle at 95 C for
10 min, followed by 30 cycles of 30 s at 94 C, 30 s at 60 C, 30 s at
72C,andthenafinalextensionat60Cfor30minonanABI
2720 thermal cycler. The resulting multiplexes were diluted 20X with
dH
2
O. For each sample, 1.0 μl of diluted product was loaded in 10 μl
of HIDI formamide with 0.1 μl of LIZ-500 size standard (Thermo
Fisher Scientific) and electrophoresed on an ABI 3130XL Genetic
Analyzer. Genotypes were then scored and binned using
GENEMAPPER v. 5.0 (Thermo Fisher Scientific). Positive controls
for each haplotype were run in all amplification batches to control
for slight size shifts (~ ±0.5 bp) that can occur between runs.
Reamplification of 10% of all samples, including the less common
genotypes, gave identical results.
Leaf Morphology and Plant Architecture
Plants were collected from field locations during 2017 to 2018 and
then clonally propagated three to four times in a common garden
before morphological measurements. Plants from each population
were cultured separately in 20-L plastic buckets with municipal-
delivered tap water supplemented with slow-release Osmocote®
fertilizer (15-9-2; Scotts Miracle-Gro, Marysville, OH). For mor-
phology/plant architecture measurements, plants were recultured
in a standard nutrient solution. Genotyping was conducted using
the original plant material collected from the field.
To assess differences in leaf shape, stem diameter, and stem
branching, plants were grown hydroponically in a temperature-
controlled greenhouse at the U.S. Army Engineer Research and
Development Center, Vicksburg, MS. Six replicate clones from 39
populations (Table 1) were placed into net pots (12.7-mm diameter)
filled with washed expanded clay rocks (8- to 16-mm diameter). Net
pots were placed individually within white 4-L polyethylene food
containers, and charcoal-filtered tap water was added to each con-
tainer. After 1 wk, the water was replaced with 1.5 L of half-strength
Hoagland’s nutrient solution. Six weeks after adding nutrients, mea-
surements were taken on all plants. We collected four leaves from
each plant (n=24 leaves per population), imaged them on a flatbed
scanner, and then measured their dimensions (length and width)
using ImageJ image analysis software (National Institutes of
Health, Bethesda, MD). Leaf shape was calculated as the length-
to-width ratio. Outer stem diameter of each plant was measured
in millimeters at the widest location on the stem. Branching was
measured as the total number of side branches off the main stem.
Ploidy
Shoot cuttings from five haplotypes (Ap 1, 2, 3, 4, and 6) were cul-
tured hydroponically in flasks within an incubator until new roots
emerged for collection. Root tips were excised from actively grow-
ing adventitious roots, fixed for 2 h in 3:1 ethanol: acetic acid, and
stored in 70% ethanol until analysis. Fixed root tips were stained
with Schiff reagent using the Feulgen reaction. Five evaluations
were performed for each of the population samples using meri-
stematic cells from preserved root tip tissue that were “squashed”
between cover slips and microscope slides in 45% acetic acid. Slides
were examined directly under phase-contrast optics at 1,000×(oil
immersion) using a Zeiss Axiostar Plus microscope (Zeiss
Microscopy, Jena, Germany). Images were photographed with
an Infinity 2-1 digital camera (Lumenera, Ottawa, Canada) using
Image-Pro Insight v. 9.0 software (Media Cybernetics, Bethesda,
MD). Composite images, including 10 to 30 images in sequence,
from 6 to 8 cells per squash were then evaluated for chromosome
counts. Ploidy level was assigned based on chromosome numbers.
Statistical Analyses
We merged sampling sites that were within the same river system
or lake to increase sample sizes within sites. This resulted in 90 sites
with 3 to 13 samples (mean 4.1 ±0.18 SE samples per site).
Haplotype diversity (h) and analysis of molecular variance
(AMOVA) were calculated in GenAlEx v. 6.5 (Peakall and
Smouse 2006,2012). Haplotype diversity is the probability that
two randomly selected samples are different haplotypes. A TCS
haplotype network (Clement et al. 2000; Templeton et al. 1992)
Invasive Plant Science and Management 219
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was constructed using POPART (Leigh and Bryant 2015) to visu-
alize similarities among haplotypes.
To explore the spatial distribution of common A. philoxeroides
haplotypes in the United States, we used an information theoretic
approach. We excluded rare haplotypes (Ap2, 4, 5) from this explo-
ration, because they only occurred in a few locations, suggesting
they are recent introductions or post-introduction mutations
and have not yet spread to their potential extent. We extracted cli-
mate data for A. philoxeroides collection locations from the first
five principal components (PCs) of the 35 bioclimatic variables
in the CliMond 1975H data set (Bio36, Bio37, Bio38, Bio39,
Bio40) (Kriticos et al. 2014). The PCs are described in Kriticos
et al. (2014) and explain more than 90% of the variation in the
original CliMond data set. PCs were used to obtain climate infor-
mation for each survey location and as potential explanatory var-
iables for distribution of haplotypes, assessed through model
selection.
We used the Akaike information criterion adjusted for small
sample size (AICc) to select the most informative multinomial
logistic regression model from the full set of candidate models
(Burnham and Anderson 2003). For the full model, haplotype
was the nominal response variable, and latitude, longitude, and
each climate PC were included as covariates. None of the PCs were
strongly correlated with latitude or longitude (i.e., r >0.90), so all
were included in the full model. We used multinomial logistic
regression to test whether haplotype identity was related to lati-
tude, longitude, or any of the five climate PCs. Top models (i.e.,
those with substantial support; Burnham and Anderson 2003)
were those with AICc values within 2 of the model with the lowest
AICc value. AICc weights are also reported, which represent the
relative strength of support that a model is the best given the data
and other candidate models. Spearman rank correlation was used
to test for associations between the relative abundance of haplo-
types and latitude.
The effects of haplotype on leaf shape, stem diameter, and
branching were modeled in R (R Core Team 2013) using general-
ized linear mixed models. Source population was treated as a ran-
dom effects variable. Branching count data were not normally
distributed and thus were modeled using a Poisson error distribu-
tion. Post hoc means separations were conducted using the
Benjamini-Hochberg adjustment for multiple comparisons
(Benjamini and Hochberg 1995). All results displayed are untrans-
formed means with standard error.
Results and Discussion
Genetic Diversity
Six haplotypes were detected across the United States in 375 samples
(Table 2;Figures1and 2). The majority of individuals had haplotypes
Ap1, 3, and 6 (Figure 2). Most sites (74.4% of 90 sites) contained only a
single haplotype, 24.4% contained two haplotypes, and 1.1% had three
haplotypes. There was significant genetic structuring in the United
States, as expected for a clonally spreading plant; AMOVA revealed
that 20% of the variance was found within sites, 53% was found
among sites (PhiPT =0.80), and 27% was found among states
(P =0.001 in all cases). Haplotype diversity of A. philoxeroides within
states was not correlated with the number of sampling sites (ρ
s
=0.09,
P=0.79). Haplotype diversity was lowest in South Carolina (h=0.11)
and highest in Georgia (h=0.79) and averaged 0.48 ±0.06 (SE) across
states (Table 3). The lower Mississippi River and Arkansas River
drainagestogetherhavehighA. philoxeroides diversity, with five of
the six haplotypes present (h=0.64). This high number of haplotypes
and diversity across different statessuggeststhisplantwasintroduced
multiple times. Other invasive clonally spreading aquatic weeds in the
United States have fewer haplotypes. Hydrilla verticillata has three
haplotypes in the United States and was introduced on three separate
occasions (Madeira et al. 2007; Tippery et al. 2020)andhygrophila
[Hygrophila polysperma (Roxb.) T. Anderson] has a single haplotype
Table 1. Alternanthera philoxeroides locations used for morphological leaf
measurements.
Location cpDNAHap
Latitude
°N
Longitude
°W
333 Cove, AR Ap1 35.320 93.214
Aberdeen Lake, MS Ap1 33.825 90.500
Blind River, LA Ap1 30.095 90.779
Choctaw Boat Ramp, LA Ap1 29.850 90.679
Coosa River, AL Ap1 33.055 86.527
Cooter’s Pond, AL Ap1 32.431 86.400
Longbranch, MS Ap1 33.769 90.144
Lake Monroe, FL Ap1 28.835 81.322
Lake Merrisach, AR Ap1 34.033 91.266
Navidad River 1, TX Ap1 29.036 96.563
Navidad River 2, TX Ap1 29.038 96.571
Newnan’s Lake, FL Ap1 29.618 82.253
TennTom Waterway, MS Ap1 33.661 88.487
Lake Waco, TX Ap1 31.610 97.305
Montezuma Slough, CA Ap2 38.097 121.894
Baldwin College, GA Ap3 31.485 83.533
CVS Pond, SC Ap3 32.213 80.701
Lake Marion, SC Ap3 33.535 80.331
Lake Wallace, SC Ap3 34.630 79.680
Suwanee, FL Ap3 30.301 82.932
Valley Park, MS Ap3 32.635 90.863
NorCo, CA Ap4 33.924 117.598
333 Cove, AR Ap6 35.320 93.214
Aberdeen Lake, MS Ap6 33.840 88.508
Anguilla, MS Ap6 33.023 90.848
Boardman Landing, NC Ap6 34.443 78.690
Beard’s Lake, AR Ap6 33.697 93.942
Coal Creek, OK Ap6 35.898 95.398
Coosa River, AL Ap6 33.055 86.527
Ditch, GA Ap6 31.839 81.414
Emerald Valley Lake, AL Ap6 33.759 86.607
Germantown Greenway,
TN
Ap6 35.117 89.820
Lansbrook Lake, OK Ap6 35.561 97.623
Lake Martin, AL Ap6 32.798 85.820
Lake Micosukee, FL Ap6 30.529 83.980
Nickajack, AL Ap6 34.832 87.322
Poverty Point Reservoir,
LA
Ap6 32.530 91.490
Schad ditch, TX Ap6 29.499 98.577
Lake Waccamaw, NC Ap6 34.300 78.552
Table 2. Six chloroplast haplotypes of Alternanthera philoxeroides in the United
States.a
Haplotype rpL16 trnS-G trnFf
Ap1 163 (A
10
) 173 (A
11
) 155 (A
9
/A
11
)
Ap2 163 (A
10
) 172 (A
10
) 155(A
10
/A
10
)
Ap3 163 (A
10
) 172 (A
10
) 156 (A
9
/A
12
)
Ap4 163 (A
10
) 173 (A
11
) 156 (A
9
/A
12
)
Ap5 163 (A
10
) 174 (A
12
) 156 (A
9
/A
12
)
Ap6 164 (A
11
) 172 (A
10
) 157 (A
9
/A
13
)
aHaplotypes are composed of three chloroplast regions. Their respective size in base pairs
and their corresponding mononucleotide repeats are given. The trnFf region encompasses
two separate mononucleotide repeats.
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in the United States and appears to belong to a single clonal lineage
(Mukherjee et al. 2016).
Some A. philoxeroides haplotypes are widespread (Ap1, 3, 6) and
some appear to be relatively localized (Ap2, 4, 5), which is also con-
sistent with a pattern of multiple introductions. California is also rel-
atively distinct from the southeastern United States, although the
two haplotypes in California were also found at one site each in
the southern United States, suggesting they may have been moved
between these areas. Haplotype Ap4 was found in historic invasion
sites in southern California and in one sample from Arkansas, and
Ap5 was found at three sites in Louisiana (Figure 1). Haplotype Ap2
was most common at recently invaded sites in northern California,
suggesting a new introduction rather than dispersal from Ap4 pop-
ulations in southern California, and Ap2 was also found in four
Figure 1. Sampling localities and chloroplast haplotypes for Alternanthera philoxeroides in the United States. The colored pie diagrams indicate the relative abundance of
haplotypes (Ap1–6) in each state, and the red dots are sampling localities.
Figure 2. Haplotype TCS network for Alternanthera philoxeroides. Size of circles is related to relative abundance of each haplotype. Cross hatches on lines indicates one mono-
nucleotide base difference between haplotypes.
Invasive Plant Science and Management 221
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samples from a single site in Georgia (Figure 1). The phylogeo-
graphic structure of A. philoxeroides in the entire native range will
need to be described to better evaluate the introduction history and
determine possible source regions.
Geographic and Climatic Patterns
Latitude, longitude, and Bio40 were influential in explaining varia-
tion in the three most common haplotypes (Ap1, 3, 6) (Table 4;
Figure 3). Three equally plausible models were identified by
AICc (Table 4), and latitude was included in all three. The top
model had only latitude and longitude (AICc weight =0.27) and
had twice the support of the next model, which had latitude and
Bio40 (AICc weight =0.12), and nearly three times the support
of the third-best model, which had latitude only (AICc weight
=0.10). Latitude was common to all three models and was due
to the relative abundance of haplotype Ap1 being negatively cor-
related to latitude (ρ
s
=−0.88, P =0.0008) and haplotype Ap6
being positively correlated with latitude (ρ
s
=0.96, P =0.0005).
Ap3 was widespread from Texas to Florida and did not covary with
latitude. Latitudinal patterns in haplotypes may be related to intro-
duction histories, historical control efforts, or environmental
differences and deserve further study. Longitude may be related
to differences across the southeastern United States in haplotype
composition that may represent different introduction histories.
Bio40, which appeared in one of the models, represents radiation
during the warmest quarter (Bio26) and maximum weekly radia-
tion (Bio21) (Kriticos et al. 2014). Maximum radiation levels
appear to be relatively high in the southeastern United States
but lower in Florida and the East Coast (Kriticos et al. 2014).
Further studies will be necessary to determine whether A. philox-
eroides haplotypes differ in their growth patterns under high and
low radiation levels.
Leaf Morphology and Plant Architecture
Alternanthera philoxeroides haplotypes Ap1 and Ap6 were clearly
distinguished by all three morphological measurements in the
study, with haplotypes Ap2, Ap3, and Ap4 displaying somewhat
intermediate characteristics. The leaves of Ap1, Ap3, and Ap4
plants were more lanceolate in appearance, with significantly
greater length-to-width ratios (3.6 ±0.1, 3.5 ±0.2, and 4.3 ±0.5
cm, respectively) compared with the more ovate leaves of Ap6
(2.7 ±0.1 cm) (Wald X2=30.78; df =4; P <0.001) (Figure 4;
Table 5). Stem diameters of Ap1 were approximately twice as large
as those of Ap6, consistent with descriptions of broad- and narrow-
stemmed biotypes (Wald X2=154.18; df =4; P <0.001).
Branching from the main stem was significantly less frequent in
Ap1 compared with Ap6; however, neither displayed branching
that differed significantly from that of the intermediate haplotypes
(Wald X2=12.60; df =4; P <0.013) (Table 5). Morphologically,
Ap1 and 6 also correspond to descriptions of A. p. angustifolia,
with a more northern distribution in South America, and A. p. phil-
oxeroides, with a more southern distribution (Sosa et al. 2004).
These native ranges are also consistent with Ap1 being more
common in more southern regions and Ap6 being more common
in more northern regions of the United States. Whether Ap1 and 6
actually correspond to the two forms in the native range needs to be
verified by genetically testing samples from the native range.
Ploidy
Invasiveness in plants is associated with both smaller genomes and
high ploidy levels (Pandit et al. 2014; Suda et al. 2015; te Beest et al.
Table 3. Alternanthera philoxeroides genetic diversity in states at chloroplast
loci.a
State LocN N N Hap Ap h
AL 9 41 2 1, 6 0.44
AR 6 34 4 1, 3, 4, 6 0.53
CA 8 37 2 2, 4 0.51
FL 19 79 3 1, 3, 6 0.45
GA 4 16 4 1, 2, 3, 6 0.79
LA 18 66 4 1, 3, 5, 6 0.54
MS 9 32 3 1, 3, 6 0.65
NC 3 11 2 3, 6 0.55
OK 2 8 1 6 0.00
SC 5 19 2 3, 6 0.11
TN 1 7 2 3, 6 0.48
TX 6 25 3 1, 3, 6 0.57
aAbbreviations: LocN, number of sampling sites; N, total sample size; NHap, number of
haplotypes in each state; Ap, numbers correspond to haplotypes; h, haplotype diversity.
Table 4. Top best-fit models explaining variation in Alternanthera philoxeroides
haplotype, based on Akaike information criterion adjusted for small sample size
(AICc) selection.
Response
variable
Candidate
model AICc ΔAICc Likelihood
AICc
weight
Haplotype Latitude,
Longitude
204.3 0 1 0.27
Latitude,
Bio40
205.89 1.59 0.45 0.12
Latitude 206.25 1.95 0.38 0.10
Table 5. Least squares means ±standard error for stem diameter, stem
branching (total number of side branches off of main stem), and leaf shape
(length:width ratio) for Alternanthera philoxeroides haplotypes.a
Haplotype Diameter No. of branches L:W ratio
mm
Ap1 11.06 ±0.32a 6.9 ±0.5b 3.6 ±0.1a
Ap2 8 ±1.19ab 7.2 ±1.7ab 3 ±0.5ab
Ap3 6.94 ±0.49b 8 ±0.7ab 3.5 ±0.2a
Ap4 8.67 ±1.19ab 4.3 ±1.7b 4.3 ±0.5a
Ap6 5.82 ±0.29b 8.3 ±0.4a 2.7 ±0.1b
aDifferent letters indicate significant differences between haplotypes for a given measured
variable.
Figure 3. Dot plot of Alternanthera philoxeroides haplotype distribution in relation to
latitude in the United States. Although all haplotypes are represented here, only
common haplotypes 1, 3, and 6 were used in model selection.
222 Williams et al.: A. philoxeroides genetic diversity
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2012). All chromosome counts from populations with haplotypes
Ap 1, 2, 3, 4, and 6 were 6n=102, indicating the invasive A. phil-
oxeroides cytotype in the United States is hexaploid (Figure 5).
Invasive A. philoxeroides populations in China are also hexaploid,
consistent with invasive populations having higher ploidy levels.
Chen et al. (2015) used flow cytometry to quantify genome size
in four plants from Argentina, two from the United States (NSA
and BSA), and one from China. Alternanthera philoxeroides in
China had a genome size similar to that of a population in
Argentina that is also known to be hexaploid (Sosa et al. 2008).
Another sample from Argentina that was thought to be hexaploid
had a DNA content that was similar to that of known tetraploids in
Argentina, and the U.S. samples had genome sizes that suggested
they were pentaploid. These results suggest that genome size in this
species can vary within a ploidy level. The presence of hexaploids in
the United States and China may be an advantage for control with
A. hygrophila (Krug and Sosa 2019). Whether genome size per se
might also impact biological control agents or is related to invasive-
ness in this species is unknown and deserves further study.
Conclusions
Most studies of A. philoxeroides to date have focused on differences
between plants from the native and invaded range and explored
development and performance of the biocontrol agent or plant
defensive responses rather than biomass response (equivalent to
successful control) of the plants to feeding (Liu et al. 2018;Pan
et al. 2013; Zhang et al. 2019). It will therefore be valuable to deter-
mine how effective the currently available biological control agents
will be on the introduced haplotypes. Our data suggest that haplo-
types Ap1 and 6 correspond to the NSA and BSA biotypes, which
respond differently to both A. hygrophila herbivory and herbicides
(Kay 1992; Kay and Haller 1982). Anecdotal observations in the
native range also suggest that A. p. philoxeroides may be less com-
monly attacked by A. hygrophila than A. p. angustifolia (Sosa et al.
2004). In combination with climatic limitations in some parts of the
invaded range, genetic differences among haplotypes may lead to
reduced control and encourage foreign exploration for additional
agents that are better adapted to specific haplotypes and climates.
Figure 4. Narrow-stem (NSA)/Ap6 (A) and broad-stem (BSA)/Ap1 (B) Alternanthera philoxeroides biotype/haplotype.
Figure 5. Representative composite images of Alternanthera philoxeroides cells with stained chromosomes: (A)haplotype Ap1 (Coosa River,AL); (B) haplotype Ap2(Suisun Marsh,
Montezuma Slough, CA); (C) haplotype Ap3 (Lake Wallace, SC); (D) haplotype Ap4 (Visalia, CA); and (E) haplotype Ap6 (Lake Micosukee, FL).
Invasive Plant Science and Management 223
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If future studies determine that genotypes differ in their inva-
siveness (e.g., varying growth rates) or respond differently to con-
trol measures, then it may be necessary to consider that different
areas will need different management strategies. Florida and South
Carolina, for instance, have mainly a single invasive haplotype, so
plants may respond more uniformly to a single control strategy. In
contrast, the Mississippi River drainage has a variety of haplotypes,
and therefore may require a variety of control strategies depending
on the population genetics and specific locality. The cpDNA gen-
otyping we developed in this study provides a quick method to
determine which haplotypes are present in a given area.
Acknowledgments. We thank the following people for assistance in sample
collection or processing: Jim Cronin, Julie Nachtrieb, Aaron Schad, Lynde
Dodd, Brett Hartis, Keith Thomas, Rachael Klopfenstein, Chris Beals, David
Webb, Curtis Tackett, Diana Rashash, Tim Harris, David Lattuca, Chelsea
Bohaty, Mariah McInnis, Ram Medrano, Al Cofrancesco, Rodrigo Diaz, Kurt
Getsinger, Stacey Springfield, Derek Medina, Mike Pitcairn, and Robin
Carter-Ervin. Daniella Biffi helped with laboratory work. Funding was provided
by the U.S. Army Engineer Research and Development Center, Aquatic Plant
Control Research Program. No conflicts of interest have been declared.
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