Discussion
Assessment of Northern Wild Rice via Genotyping-By-Sequencing. Cost-effective sequencing technologies that are capable of generating
robust sets of genome-wide molecular markers, such as GBS, are providing
researchers, especially those working with complex plant genomes and
limited public resources, an avenue for rapid variant detection64–68. In this study, we developed a large
genome-wide SNP dataset, aligned to the NWR reference genome, to assess
the relationship between natural and cultivated populations as well as
to provide a basis for future breeding and conservation studies.
However, we want to emphasize that this GBS approach, which is based onBtg1 and TaqI restriction enzymes, may have introduced a
bias in allele frequencies due to polymorphisms in restriction sites69, which could have led to slightly skewed inferences
regarding the population genetics of NWR.
Relationships within the Zizania Genus. Species of Zizania are endemic to North America 70,71 and split from the
Oryzinae subtribe 20-30 million years ago (MYA)34,72,73. Following this split, the Zizaniinae
subtribe is hypothesized to have experienced a radiation of speciation
across North America and into eastern Asia as individuals made it over
the Bering land bridge, leading to the speciation of Zizania
latifolia 74. Comparison of the Z. palustris and Z. latifolia genomes suggest that the two species split 6-8
MYA 34. Extant North American species split 0.7-1.1
MYA; this split was likely precipitated by increases in the habitat
range of the Zizania progenitor species as climatic conditions
shifted over the last million years. 71,75. Evidence
suggests that Zizania texana , an endangered species living
in a small stretch of the San Marcos River Valley in the southern US, is
a relic, isolated population of the ancestral Z. palustris species 74. However, the evolutionary relationship
between Z. aquatica , a species found from the Great Lakes region
to the east coast of the continental US, and Z. palustris is not
well understood. This is likely due to their overlapping range and
interspecific crossability 74,76. In this study, the
UPGMA, STRUCTURE, and principal coordinate analyses showed only moderate
support for the separation of Z. palustris and Z.
aquatica . Fst values were primarily affected by geographic
distance, and highest within Z. palustris rather than betweenZ. palustris vs Z. aquatica (Figure 4). This may be due to
the limited sample size in this study and more research is needed to
resolve the complex relationship between these Zizania species.
Structure of Northern Wild Rice Populations is Tied to Geography
and a Complex History of Ecosystem Management. Previous genetic
analyses of wild populations of NWR have found limited gene flow between
populations, and a lack of data to support a correlation between
population structure and geographical location5,24,74. In this study, we did not find evidence of
significant gene flow among wild populations of NWR, with populations by
and large clustering according to their lake or river of origin (Figures
2, 3, and S3). This level of differentiation was also evidenced by the
moderate Fst values (0.05-0.15)77 found between
Natural Stand populations (Figure 4). However, the majority of our
analyses, including the Mantel test (Figure S6), suggest a geographic
basis for population structure in NWR. For example, Garfield Lake and
Necktie River, the two closest populations in our study, displayed a
high level of similarity with one another (Figure 2b; Figure 3).
Therefore, it appears that while gene flow is limited in NWR, it likely
occurs between populations in close proximity and more research is
needed to understand the spatial dynamics of NWR populations, whose
aquatic habitats are often discrete and fragmented. While the primary
drivers of gene flow in NWR are not well understood, Lu et al., 2005
found the area and size of a NWR population, along with its degree of
isolation, were major factors affecting the genetic variability and gene
flow among the NWR populations tested. Additionally, recent pollen
travel studies found that most pollen is dispersed within the first 7 m
for Z. palustris 78 and 1.5 m for Z.
texana 79, limiting the likelihood of high levels of
gene flow via wind-pollination in the genus.
The historical management and development of lakes across NWR’s natural
range have likely contributed to the population structure identified in
this study. Efforts to establish new stands of NWR as well as to address
declining population sizes have resulted in reseeding efforts across the
species’ natural range 80,81. For example, Upper Rice
Lake (RRN), which is known to have undergone extensive reseeding efforts
since the 1930s (Dr. Kimball, personal communication ), clustered
primarily with several UMR populations while showing limited overlap
with other RRN populations (Figure 2b; Figure 3). Upper Rice Lake also
showed heavy admixture with a number of lakes in STRUCTURE analyses
(Figure 3). Taken together, these results suggest that human
intervention may have altered the genetic variability and population
structure of the Upper Rice Lake population assessed in this study.
Additionally, Phantom Lake of the SCR watershed displayed heavy
admixture with Cultivated materials. These results were surprising as
Phantom Lake is one of the most geographically distant Natural Stand
populations from cNWR production in this study, and closer populations
displayed little to no admixture with Cultivated materials (Figure 3).
However, Phantom Lake is part of the Crex Meadows State Wildlife Area
and was artificially created in the 1950s, when a series of levee
systems were installed. NWR restoration in this area began in 1991, with
500 lbs (227 kg) of seed sown over the course of three years82. We hypothesize that at least a portion of the seed
utilized in these efforts came from cNWR production, further
highlighting the complexity of population genetic studies in NWR, as
well as the importance of documenting seed sources used in reseeding
efforts. We also suggest that future reseeding efforts should not use
Phantom Lake populations as a seed source based on the recommendations
of the Great Lakes Indian Fish & Wildlife Commission83.
The data presented here for 12 wild populations of NWR likely represents
only a small fraction of the species’ genetic diversity. However, even
with our small sample size, we were able to identify unique genetic
variation within many of the populations in the Natural Stand
collection. This indicates that for conservation efforts, it is
important to consider populations of NWR individually as they may harbor
unique alleles and may be more or less adapted to environmental change.
Further studies, using a broader range and more even distribution of
sampling locations, will increase our knowledge about the population
structure and genetic relationship between wild NWR populations and aid
with decision making for future reseeding and other conservation-based
efforts.
Spatio-Temporal Genetic Diversity Analyses Can Aid in Conservation
Efforts. Comprehensive monitoring of the spatio-temporal genetic
diversity of a species can provide a better understanding of the
evolutionary change a species undergoes over time and help to identify
targets for conservation efforts. Although present-day genetic diversity
studies are available for a wide array of plant species, the majority of
spatio-temporal diversity assessments have focused on large agricultural
commodity crops 84–86. Few studies have focused on
natural populations 87,88 and many, only theoretically89,90. As NWR is an important target for conservation,
monitoring the spatio-temporal diversity of wild populations would
provide impactful data for resource managers and environmental agencies
interested in the health and preservation of NWR populations across the
species natural range.
As a preview of what a more extensive study on the spatio-temporal
genetic diversity of NWR could provide, we evaluated two populations,
Garfield and Shell Lakes, in 2010 and 2018. Comparing these two time
points, we identified a reduction of diversity in samples collected from
Shell Lake in 2018 compared to those collected in 2010, while observing
limited change in the Garfield Lake population (Figure 2d). This may
suggest that Shell Lake has experienced a loss of genetic diversity
during the eight years between collection times, while Garfield Lake has
not. However, more data is needed to confirm this hypothesis. A wide
array of factors could have contributed to this reduction in diversity
in Shell Lake, including the 3 - 5 year boom and bust cycles of NWR91, or environmental conditions favorable for specific
genotypes in the population’s seed bank. It is also possible that
shoreline development and recreation stemming from campgrounds and
resorts on Shell Lake could have impacted the health of its native NWR
population 15.
Cultivated Northern Wild Rice is Distinct from Natural Stand
Populations. Gene flow between domesticated crops and their wild
counterparts can have significant impacts on both natural ecosystems and
agricultural production systems. Genetic contamination, loss of identity
and genetic diversity, and increased weediness are all potential
consequences of gene flow 92. For these reasons, the
extent of gene flow between crops and their wild cohorts has been
evaluated in numerous species and found to be dependent on a variety of
factors including, but not limited to, mating system (i.e. out-crossing
vs selfing), the type and frequency of pollination (i.e. insect vs
wind), the selective (dis)advantage of particular domesticated traits
(i.e. seed shattering resistance reducing seed dispersal), genetic
drift, and genotype × environment interactions 92–95.
Some studies, such as those in soybean (Glycine max ), have
identified limited gene flow, with domesticated and wild samples
separating into monophyletic clades 96,97. Other
studies have identified significant historical gene flow during
domestication, such as Emmer wheat (Triticum dicoccon )98, as well as on-going gene flow between crop-weed
complexes, such as those in cowpea (Vigna unguiculata (L.) Walp)99, pearl millet (Pennisetum
glaucum )100, and species in the Sorghum genus101,102.
Given the out-crossing nature of NWR and that cNWR production occurs
within the centers of origin and diversity of Z. palustris , it is
important to understand the extent of gene flow between cultivated and
wild populations. This study found that Natural Stand and Cultivated
collections are genetically distinct from one another (Figure 2a; Figure
3; Figure S4), indicating minimal gene flow between these two groups and
corroborating the results of previous diversity studies in NWR using
different marker systems 5,24,25. However, based on
the 1st principal coordinate from Figure 2a, we identified more
similarities between the Cultivated collection and Bass, Decker, and
Dahler Lakes than other Natural Stand populations. These lakes are
geographically close to the UMN cNWR paddy complex in Grand Rapids, MN
and could suggest gene flow. However, it is more likely that this is due
to a shared ancestral relationship, as neither STRUCTURE analysis
(Figure 3) nor D -statistics (Table S6) suggest recent gene flow
between the two populations. Importantly, the cultivated germplasm in
use today is all descended from natural stand samples originally
collected from this geographical region within the UMR watershed.
Cultivation and domestication of NWR began in Aitkin, MN and several
small enterprises likely gathered seeds from local populations to build
their germplasm bases 13.
Domestication and Stewardship of Cultivated Northern Wild Rice. As domestication is a process rather than a specific event, species
exhibit varying levels of domestication 103. In
cereals and other major agricultural crops, seed retention and size,
seed dormancy and germination, plant growth habit, and plant size are
domestication traits commonly targeted for selection104. The presence of these common traits across
multiple taxa is known as the domestication syndrome, which
differentiates domesticated species from their wild counterparts. While
many of today’s largest agricultural commodity crops have undergone mass
selection for thousands of years, the advent of new technologies, such
as genomic sequencing, provide today’s plant breeders with new
opportunities for the rapid, targeted domestication of new crops105. Additionally, these technologies afford
researchers the opportunity to study the domestication process in
real-time 106.
To begin exploring the domestication process of cNWR, we evaluated
changes in nucleotide diversity levels and allele frequency
distributions between Natural Stand and cNWR populations using Tajima’s
D, FST , and XP-CLR tests. No significant overlap
was identified between the three tests, suggesting there is limited
evidence for selective sweeps in cNWR. However, two 1-Mb regions on
ZPchr0011 and ZPchr0013 had overlapping top 1% ofFST and XP-CLR scores suggesting there is some
evidence of genetic changes in cNWR compared to the Natural Stands
(Figure 5). A preliminary scan of genes in these two regions identified
5 putative genes whose functions in other species, mainly white rice,
are related to drought and salt stresses as well as abscisic acid (ABA)
signaling. These included a 60S ribosomal protein kinase 32-like gene 107; a CBL-interacting protein kinase
32-like gene 108; a E3 ubiquitin-protein ligase
RZFP34 isoform X2 109,110; and two copies ofras-related protein RABC2a 111 . Unlike
wild populations of NWR, cNWR is grown in man-made irrigated paddies,
which are drained shortly after flowering (Principal Phenological Stage
6)112 to allow for mechanical harvesting of the grain.
Therefore, cNWR experiences conditions similar to upland crops, for
which standing water is not available during the development of fruit,
ripening, and senescence. These results may suggest that stress-related
genes, particularly drought-related genes, were heavily selected for in
cNWR germplasm to adapt to this drastic change in environmental
conditions compared with its natural habitats.
As XP-CLR is more robust than FST for
identifying recent selection events 63, we looked at
the two additional XP-CLR regions that contained the top 1% of the
statistic’s empirical distribution, including a region on ZPchr0005
between 8.5-9.7 Mb and a region on ZPchr0006 between 1.2-1.4 Mb. Within
these regions, we identified a calcium-dependent protein kinase
family protein associated with drought and salt tolerance in white rice113,114; a 2,3-bisphosphoglycerate-independent
phosphoglycerate mutase-like gene involved with chlorophyll synthesis
and photosynthesis in white rice 115; a CTD
nuclear envelope phosphatase 1 homolog associated with seed shattering
resistance in white rice 116; a KH
domain-containing protein SPIN1-like associated with flowering time in
white rice 117; and a pentatricopeptide
repeat-containing protein At1g11900 isoform X1 associated with male
sterility in Petunia 118. Two paralogs ofcytochrome P450 714D1-like were identified on both ZPchr0005 and
ZPchr0006 regions of interest. In white rice, this gene is associated
with seed dormancy and flowering time 119,120. While
not in the scope of our current study, we think these regions merit
further investigation. Given the significance of NWR to a wide range of
stakeholders, it’s important to understand the potential impact of gene
flow from cNWR to wild NWR populations. Therefore, while understanding
the domestication process in cNWR is important for the plant breeding
process, it can also be used to monitor the genetic diversity of natural
stands, allowing for better stewardship of these vital populations.
Domestication indices that account for varying levels of domestication
have been proposed for several species and typically include: the extent
of phenotypic differentiation between the domesticated species and its
wild counterparts; the length of a species’ domestication history;
whether major genetic changes to the domesticated species have been
identified; whether the species has been adapted to agricultural
settings through targeted breeding efforts; and the extent of the
species’ cultivation 121–123. Cultivated NWR is
somewhat phenotypically distinct from wild NWR, mainly in its growth
habit and seed retention characteristics, which have been made possible
through breeding efforts. While the species has a short history of
cultivation, its production has expanded to California, which is outside
the species’ natural range. For these reasons, we suggest that cNWR
should be classified as semi-domesticated.