1. Introduction
Ammannia belongs to the annual herbs of the Lythraceae, containing 25 species, mainly distributed in humid areas such as marshes, waters, or paddy fields worldwide [
1]. The genus comprises upright and solid stems extending approximately 150 cm high, flowers have four petals, and seeds are inverted pyramidal; one side is round, and the other is irregularly concave [
2]. This genus is the most common broad-leaved weed in rice fields and is a successful competitor for nutrition and space during the rice growth period [
3,
4,
5]. The most common
Ammannia weeds in China’s paddy fields are
A. arenaria and
A. multiflora [
5,
6,
7]. These two weeds have very a strong ability to adapt to the environment and can grow under water in the early stage of rice planting and complete vegetative growth and reproduce, provided the soil is maintained moist in the later stage. Although
A. arenaria and
A. multiflora seeds are very small, their quantity is very large, making them the most problematic in rice fields [
5,
6].
Managing
A. arenaria and
A. multiflora in paddy fields is a considerable challenge in crop protection. Because the seedbanks of this genus are very large [
5,
6], the use of pre-emergence herbicides to control it is necessary [
8]. The sulfonylureas herbicide, bensulfuron-methyl, which belongs to the acetolactate synthase (ALS) inhibitors [
9], was most frequently used to control
A. arenaria and
A. multiflora [
10,
11,
12]. However, such herbicides acting on a single target-site are prone to develop resistance [
13,
14]. Unfortunately, bensulfuron-methyl resistance has been reported in
A. arenaria [
15,
16] and
A. multiflora [
17,
18]. The failure of weed control at pre-emergence inevitably strengthens post-emergence control. The harmfulness of the two weeds is different. Generally, adult
A. arenaria is taller than rice and requires more space and nutrients, whereas adult
A. multiflora is smaller [
19]; therefore, the focus of management should be inclined. However, before flowering,
A. arenaria and
A. multiflora are difficult to distinguish. Presently, chloroplast (cp) genomes sequencing and identifying their genetic lines contribute to distinguishing two
Ammannia weeds, establishing a basis for scientific management. From a different perspective,
A. arenaria and
A. multiflora are important medicinal plants [
20], which is helpful for the treatment of many diseases, including otitis media [
21] and thyroid nodules [
22]. Chloroplast genome information also facilitates the utilization of
Ammannia resources and exploitation of allied species.
Chloroplasts (cp) are organelles in photosynthetic plants or algae that perform photosynthesis [
23]. Chloroplasts contain genetic material, and their genomes are highly conserved owing to the lack of recombination, haploidy, and uniparental inheritance. Fundamentally, they can provide rich evolutionary information [
24,
25,
26]. In addition, the cp genome is small and easy to obtain completely compared to the nuclear genome; therefore, it has unique research value in phylogeny, species identification, and population genetics [
27]. Because of these characteristic properties, determining and analyzing ribosome organization in the cp system has become an important mechanism for addressing plant phylogeny and assessing biodiversity. Generally, the cp genome has a typical quadripartite structure [
28,
29,
30] and its circular structure is organized into large single-copy (LSC) and small single-copy (SSC) regions, separated by inverted repeats (IRs), which are a pair of sequences with opposite orientations, named IRa and IRb [
24,
31,
32]. Sequences between IRa and IRb regions can generate triggered flip-flop recombination, stabilizing single-copy regions [
33]. The cp genome is particularly useful for studies characterizing the phylogeny and history of most plant lineages in the context of reticular-type evolution (hybridization) and polyploidy [
34,
35,
36]. With the advancement of the cp genome-sequencing technology and in-depth understanding of the cp genome by researchers, the genetic relationship of multiple genera, such as
Camellia,
Taxodium, and
Pterocarpus, have been uncovered [
27,
33,
37]. To date, information about the composition, structure, and differences between species, as well as the evolutionary relationships of
Ammannia species based on cp genome is still limited.
This study aimed to explore a complete analysis and comparison of germination conditions, field morphology, herbicides sensitivity, and cp genomes of
Ammannia species,
A. arenaria and
A. multifloras (
Supplementary Figure S1) collected in paddy fields for the first time, and provide knowledge for the identification of the two morphologically similar plants. Therefore, this study also provides a theoretical basis for the regeneration of diversity and resource utilization of this genus.
4. Discussion
Ammannia species,
A. arenaria and
A. multiflora, are the most common broad-leaved weeds in paddy fields in China. Although farmers use various methods for weed management, situations may still exist where
Ammannia species are uncontrollable (
Supplementary Figure S2). The conditions required for seed germination of the two
Ammannia species were similar (
Figure 1). However,
A. multiflora seeds can germinate at 15 °C. This should be taken seriously when planting early rice. Therefore, the results can provide a theoretical basis for predicting the occurrence of two weeds under different environmental conditions. Based on our investigation, the height and maximum lateral distance of
A. arenaria were higher than
A. multiflora (
Table 2), indicating that
A. arenaria has a considerable advantage in competing for resources with rice. Therefore, identifying and managing
A. arenaria in the early stages is particularly important. Simultaneously, we should also be alert to the risk of future damage to rice from plants closely related to
Ammannia species.
We selected three populations for each species to avoid the impact of herbicide use history on the study of sensitivity differences between
A. arenaria and
A. multiflora. The synthetic auxin herbicides, florpyrauxifen-benzyl [
44] and MCPA-Na [
45], had the best control effect on the two
Ammannia species; however, the traditional acetolactate synthase inhibitor, pyrazosulfuron-ethyl [
46], and the new 4-hydroxyphenylpyruvate dioxygenase inhibitor, pyraquinate [
47], were ineffective in managing them. This study can serve as a basis for herbicide selection. Accurately identifying and managing
Ammannia species can also help reduce herbicide costs and environmental pollution.
Many plant cp genome sequences have been determined following the first reported cp genome sequence of tobacco [
48]. Presently, there are no studies on the evolutionary relationships of
Ammannia. The present study found that the cp genomes of
A. arenaria and
A. multiflora, 158,401 and 157,900 bp (
Figure 3), were relatively larger than those of common plants, such as
Echinochloa and
Oryza, and smaller than those of
Cyperus species in paddy fields [
49]. The typical circular tetramerous structure of the cp genome is conserved in plants, and the length of each quadripartite structure of the cp genome in the same genus is generally similar [
37,
50]. The cp genome of
A. arenaria and
A. multiflora also revealed these features, with similar LSC, SSC, and IR lengths (
Figure 3;
Table 3).
Simple sequence repeats, or microsatellites, are tandem repeats comprising 1–6 nucleotide repeat units that are widely distributed in plant cp genomes [
51,
52]. As valuable molecular genetic markers, SSRs are widely used in plant genotyping and population genetics [
53,
54,
55,
56]. These repeats promote intermolecular recombination and enrich the diversity of cp genomes in the population [
57]. This study showed that the cp genome of
A. arenaria had one more SSR than that of
A. multiflora, including one SSR with an encoding function. Thus, differential SSRs can be used as important molecular markers in the two species. Additionally, long repeats are special DNA sequences that are repeated in the genome in various forms and usually occupy a large proportion of the genome [
58]. Repeated segments also have important molecular significance in the study of plant evolution [
59]. The cp genome of
A. arenaria had 28 more LRs than that of the cp genome of
A. multiflora (
Table 4). The repeat sequences detected in this study are important biological information resources for
Ammannia, and are of considerable significance for the identification of
Ammannia species and the study of genetic diversity and population structure.
Chloroplast genome genes are highly conserved in plants [
24,
25,
26]. As a result, 86 and 85 protein-coding genes were identified in
A. arenaria and
A. multiflora, respectively. Although the genes were not completely consistent, the categories of genes were similar, mainly belonging to the categories of photosynthesis and self-replication (
Table 5), further verifying the conservation of protein-encoding genes in chloroplasts [
27,
50,
60]. The difference in the number of protein-coding genes between the two
Ammannia species is caused by one gene,
infA, which exists only in the cp genome of
A. arenaria (
Figure 3;
Table 5). The
infA gene is a ribosomal protein L23 operon component and is transcribed into polycistronic mRNA [
61]. The
infA gene is considered to be the most mobile chloroplast gene in plants so far [
62], which may have caused the difference between
A. arenaria and
A. multiflora in evolution. The
infA gene in
A. arenaria had an initiation codon, unlike without an initiation codon in tobacco [
48]. Additionally, this different gene can be used to distinguish between the morphologically-similar
A. arenaria and
A. multiflora. Except for protein-coding genes, noncoding RNAs are conservative in the two
Ammannia species, similar to other plants of the same genus [
27,
60].
Expansion and contraction of the cp genome is a common phenomenon in plants [
24], which occurs mainly at the IR/SC junction [
63]. Although highly conserved, IR expansion and contraction are directly related to cp genome rearrangement and variation in size, which is also a major determining factor in plant genome evolution [
27,
33,
37]. This study showed that the IR expansion and contraction of the cp genome were highly conserved between
A. arenaria and
A. multiflora. All boundary genes or genes that cross two regions are consistent in the two
Ammannia species, including the length of these genes away from the nearest boundary. There was a difference in the length of only one gene,
trnH, between
A. arenaria and
A. multiflora, which was 74 and 75 bp, respectively (
Figure 5). This revealed that the expansion and contraction in the IR and SC regions did not result in large changes to the junction boundaries in
Ammannia.
Genome data are valuable for addressing species definitions, as they can be used to establish organelle-based “barcodes” for certain species, which can be used to reveal phylogenetic relationships [
64]. Chloroplast genome sequences are essential for plant species identification, phylogenetic relationships, and the determination of plant taxonomic status. With the continuous discovery of plant cp genome information, the genetic evolutionary relationships of some Lythraceae plants have been successfully elucidated in the form of phylogenetic trees [
65,
66,
67]. However, the phylogenetic relationships of
Ammannia have not yet been studied. In the present study, the two cp genomes of
Ammannia, model plant of dicotyledon (
A. thaliana), common dicotyledonous weeds in rice field, Lythraceae plants, and Onagraceae plants were used to perform phylogenetic analysis. The analysis showed that the morphologically-similar
Ammannia species,
A. arenaria and
A. multiflora, were close phylogenetically (
Figure 6). Thirteen Lythraceae plants, including
A. arenaria and
A. multiflora, are more closely related, with support values of 100%, while Lythraceae and Onagraceae have a sister relationship, which is consistent with previous research results [
66]. However, the genetic relationship between
Ammannia species and another two dicotyledonous weeds in rice fields,
E. prostrata and
P. lapathifolia, was distant. Although analysis of the complete cp genome may not be sufficient to adequately resolve all phylogenetic relationships [
68,
69,
70], it still provides a viable way to clarify species relationships.
Single nucleotide polymorphisms are important indicators of evolutionary differences between plants of the same genus, with the advantage of low cost by high-throughput techniques [
71]. These direct molecular markers evidently show the exact nature and location of allelic variations [
72]. Therefore, SNPs have recently attracted increasing attention [
33,
53]. Considering the cp genome of
A. multiflora as a reference, 47 SNPs in the intergenic region and 20 SNPs in the CDS region were identified in
A. arenaria (
Table 6), showing the difference between the two species. This is one of the important molecular foundations for the differentiation of two species. The nine nonsynonymous SNPs may result in the differences in protein function. These SNPs can be important differential nucleotide databases to distinguish the two species. Generally, SNPs occur at a higher frequency in variable, less conserved genes [
72]. The present study identified nine nonsynonymous SNPs across six encoding genes (
Supplementary Table S5), accounting for only approximately 7% of all genes in the cp genome of
A. arenaria. This is because the nonsynonymous rate is typically slower owing to the purifying selection acting on the gene [
73].