Edited by: Miroslav Obornik, Institute of Parasitology (ASCR), Czechia
Reviewed by: Wei Wu, Zhongkai University of Agriculture and Engineering, China; Isabel Marques, University of Lisbon, Portugal
This article was submitted to Marine and Freshwater Plants, a section of the journal Frontiers in Plant Science
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In many aquatic plant taxa, classification based on morphology has always been difficult. Molecular markers revealed that the complexity in several of these aquatic taxa could be addressed to recurrent hybridization events and cryptic species diversity. The submerged macrophyte genus
Natural hybridization is an important mechanism in plant evolution. Newly formed hybrids can have traits that allow them to colonize new niches that are not occupied by their parental species (
The submerged macrophyte genus
Locations and pond types of the sampled
Molecular studies were performed to clarify the phylogenetic relationships within European
Tetraploid
Furthermore, these molecular studies also found several indications of hybridization events. So far, three groups of hybrids have been detected. The first hybrid lineage has a unique haplotype, the so-called haplotype E, that is unrelated to the
Hybrid zones are secondary contact zones between two species (
Our main goal was to detect different
During the field sampling, we already tried to give a species name to each population, based on their morphological features. We used flower peduncle length as the main discriminative characteristic:
List of sampled populations with the population names (Pop), number of ramets sampled in each population (Ramets), number of multilocus genotypes (MLGs), clonality (R) calculated after clone correction with the formula
Pop | Ramets | MLGs | R | NA | N |
HOBS | HEXP | GIS | Missing values (%) | Salinity (μs/cm) | Flower peduncle length |
P1 | 29 | 29 | 1.00 | 5.23 | 2.59 | 0.61 | 0.54 | −0.08 | 11.8% | 24.4 | >5 cm |
P2 | 12 | 12 | 1.00 | 4.77 | 2.37 | 0.57 | 0.59 | 0.04 | 24.4% | 39.6 | Flowers absent |
P3 | 30 | 30 | 1.00 | 6.58 | 3.13 | 0.65 | 0.65 | 0.00 | 12.6% | 35.2 | Flowers absent |
P4 | 10 | 10 | 1.00 | 2.92 | 2.11 | 0.68 | 0.52 | −0.25 | 9.3% | 31.2 | Flowers absent |
P5 | 25 | 3 | 0.08 | 2.69 | 1.90 | 0.74 | 0.41 | −0.81 | 14.6% | 25.3 | >5 cm |
P6 | 28 | 28 | 1.00 | 5.46 | 2.82 | 0.64 | 0.57 | −0.09 | 21.9% | 57.4 | >5 cm |
P7 | 25 | 17 | 0.67 | 6.00 | 2.95 | 0.74 | 0.59 | −0.24 | 23.4% | 56.4 | Flowers absent |
P8 | 19 | 18 | 0.94 | 5.92 | 3.42 | 0.77 | 0.65 | −0.20 | 8.6% | 12.3 | Flowers absent |
P9 | 17 | 17 | 1.00 | 6.15 | 3.00 | 0.76 | 0.63 | −0.21 | 13.9% | 14.9 | Flowers absent |
D6 | 30 | 29 | 0.97 | 5.50 | 2.87 | 0.63 | 0.55 | −0.17 | 12.7% | 63.8 | >5 cm |
D7 | 29 | 28 | 0.96 | 6.75 | 3.17 | 0.64 | 0.58 | −0.07 | 14.8% | 62.1 | Flowers absent |
T4 | 8 | 8 | 1.00 | 4.00 | 2.60 | 0.71 | 0.58 | −0.19 | 20.1% | 69.4 | >5 cm |
D1 | 16 | 2 | 0.06 | 1.10 | 1.03 | 0.00 | 0.02 | 1.00 | 3.1% | 41.4 | <5 cm |
D2 | 13 | 1 | 0.00 | 1.01 | 1.00 | 0.00 | 0.00 | / | 3.1% | 39.1 | <5 cm |
D3 | 25 | 2 | 0.04 | 1.10 | 1.04 | 0.00 | 0.03 | 1.00 | 3.2% | 34.3 | <5 cm |
D4 | 15 | 2 | 0.07 | 1.10 | 1.05 | 0.00 | 0.03 | 1.00 | 11.3% | 52.8 | <5 cm |
D5 | 21 | 1 | 0.00 | 1.00 | 1.00 | 0.00 | 0.00 | / | 2.9% | 63.8 | <5 cm |
T1 | 26 | 14 | 0.52 | 2.23 | 1.60 | 0.43 | −0.07 | 0.52 | 27.2% | 16 | <5 cm |
T2 | 22 | 6 | 0.24 | 1.60 | 1.46 | 0.24 | −0.22 | 0.24 | 36.5% | 62.1 | <5 cm |
T3 | 22 | 22 | 1.00 | 3.30 | 1.81 | 0.34 | 0.04 | 1.00 | 51.5% | 69.4 | <5 cm |
D1b | 14 | 4 | 0.23 | 1.45 | 1.38 | 0.27 | 0.19 | −0.42 | 30.1% | 41.4 | <5 cm |
T1 | 26 | 26 | 1.00 | 4.67 | 2.69 | 0.40 | 0.53 | 0.25 | 52.1% | 16 | <5 cm |
T2 | 22 | 19 | 0.86 | 3.50 | 1.77 | 0.12 | 0.39 | 0.69 | 32.5% | 62.1 | <5 cm |
T3 | 22 | 13 | 0.57 | 1.60 | 1.20 | 0.15 | 0.12 | −0.22 | 2.7% | 69.4 | <5 cm |
D1b | 14 | 12 | 0.84 | 3.10 | 1.75 | 0.23 | 0.35 | 0.36 | 30,1% | 41.4 | <5 cm |
The locations of the
Plant DNA was extracted from dried leaf tissue using the E.Z.N.A (R) HP Plant DNA Mini Kit Protocols (Omega Bio-Tek, Norcross, GA, United States). To genotype each specimen, we used a set of species-specific nuclear microsatellite markers that were combined in a multiplex reaction as well as a set of four chloroplast DNA markers. We used a QIAGEN multiplex PCR Plus kit to generate DNA-fragments with a PCR reaction in a thermal cycler (MJ research PTC-200 and Bio-Rad My Cycler). These fragments were run on an ABI3730XL sequencer (Macrogen, Seoul, South Korea). We manually scored the results with GeneMarker V2.20 (SoftGenetics LLC®).
Previous work on
Overview of parental
Because previous studies have already shown that morphological identifications of
Usually, hybrids are detected with several microsatellite markers that have diagnostic alleles in both parental species (
First, we examined whether the species name we assigned to a population based on the morphological identification corresponded with the cpDNA identification. If there was a mismatch, we also amplified these populations with the microsatellite set that corresponded with the cpDNA. This allowed us to detect chloroplast capture or strong introgression and corrected for inaccurate morphological identifications.
Secondly, we looked for diagnostic alleles in the markers RUMR4 and RM3, both part of the
Thirdly, we checked the three nuclear markers that were used in both microsatellite sets (RMB5, RMB53, and RMB15). Although these markers are known to amplify in both species, the amplification success in
To visualize the population structure and the differences in microsatellite signature between either pure species or hybrid lineages, we constructed two Principal Component Analyses (PCA). We used R-package Adegenet for this analysis (
Based on the previous species assignment, we considered three different datasets: a pure
To estimate the ploidy of a population, we counted the number of different alleles for each marker. If we detected three or four alleles for at least one locus in a sample, we considered that sample as tetraploid. Because triploids are mostly sterile, we don’t consider the possibility of a triploids in an outcrossing population. Besides, triploids were only rarely detected in
Because most population statistics are developed for diploid populations, there are several restrictions for polyploids and comparisons among different ploidy levels. Above all, there is the problem of calculating observed heterozygosity: diploids have only one state of heterozygosity (e.g., AB) whereas tetraploids have partial and full heterozygotes (e.g., respectively, AABB and ABCD). Secondly, there is the problem of unknown allelic dosages in heterozygotes: if a sample has two different alleles for a single locus, e.g., A and B, it is difficult to deduct whether the true genotype is either AAAB, AABB or ABBB. This uncertainty strongly affects all allele-frequency based calculations (
Parameters measured for each locus.
NA | N |
% miss | HOBS | HEXP | GIS | GST | ρ | DEST | NA | N |
% miss | HOBS | HEXP | GIS | GST | ρ | DEST | |
RMB5° | 10 | 1.8 | 68.8% | 0.21 | 0.48 | 0.72 | 0.25 | 0.06 | 0.53 | 3 | 1.5 | 15.4% | 0.00 | 0.55 | 1.00 | 0.39 | 0.46 | 0.57 |
RM26 | 14 | 4.3 | 3.0% | 0.70 | 0.80 | 0.14 | 0.10 | 0.06 | 0.43 | 6 | 1.5 | 80.8% | 0.50 | 0.80 | 0.01 | 0.34 | 0.35 | 1.00 |
RM12 | 9 | 2.7 | 3.3% | 0.89 | 0.78 | −0.30 | 0.02 | 0.08 | 0.05 | 3 | 2.0 | 33.7% | 1.00 | 0.51 | −0.98 | −0.01 | 0.83 | −0.02 |
RCS9 | 8 | 2.5 | 0.7% | 0.75 | 0.61 | −0.19 | 0.10 | 0.04 | 0.21 | 3 | 1.5 | 67.3% | 1.00 | 0.69 | −1.0 | 0.41 | 0.88 | 0.72 |
RUMR4 | 3 | 1.8 | 5.5% | 0.79 | 0.62 | −0.70 | 0.09 | 0.14 | 0.08 | 6 | 1.3 | 21.2% | 0.26 | 0.60 | −0.15 | 0.64 | 0.65 | 0.64 |
RMB15° | 11 | 2.8 | 1.1% | 0.72 | 0.64 | −0.07 | 0.10 | 0.12 | 0.23 | 6 | 1.3 | 11.5% | 0.06 | 0.60 | 0.75 | 0.59 | 0.64 | 0.63 |
RMB53° | 3 | 1.3 | 93.7% | 0.35 | 0.23 | −0.54 | / | / | / | 2 | 1.0 | 1.9% | 0.00 | 0.04 | 1.00 | 0.04 | 0.25 | 0.01 |
RM3 | 9 | 1.7 | 7.0% | 0.55 | 0.52 | −0.34 | 0.115 | 0.20 | 0.13 | 3 | 1.0 | 36.5% | 0.00 | 0.46 | 1.00 | 0.95 | 0.78 | 0.01 |
RCS5 | 8 | 2.6 | 4.1% | 0.70 | 0.72 | −0.10 | 0.11 | 0.11 | 0.26 | 12 | 1.5 | 76.0% | 1.00 | 0.87 | −0.10 | 0.41 | 0.32 | 0.98 |
RM27 | 7 | 2.0 | 0.7% | 0.99 | 0.52 | −0.90 | −0.02 | 0.01 | −0.04 | 7 | 1.7 | 14.4% | 0.74 | 0.67 | −0.70 | 0.36 | 0.77 | 0.56 |
RCS8 | 8 | 1.9 | 1.5% | 0.62 | 0.50 | −0.22 | 0.13 | 0.09 | 0.16 | 6 | 1.3 | 22.1% | 0.04 | 0.61 | 0.72 | 0.67 | 0.67 | 0.81 |
RC3 | 17 | 3.0 | 4.8% | 0.79 | 0.76 | −0.10 | 0.17 | 0.17 | 0.48 | 7 | 1.4 | 42.3% | 0.50 | 0.77 | −0.58 | 0.58 | 0.50 | 0.89 |
RM22 | 17 | 2.2 | 4.4% | 0.59 | 0.57 | 0.04 | 0.10 | 0.10 | 0.23 | / | / | / | / | / | / | / | / | / |
Total | 11.8 | 2.5 | 10.1% | 0.66 | 0.59 | −0.18 | 0.11 | 0.08 | 0.21 | 5 | 1.4 | 32.5% | 0.40 | 0.50 | −0.13 | 0.48 | 0.45 | 0.54 |
RMV22 | 2 | 1.1 | 3.4% | 0.00 | 0.50 | 1.00 | 0.71 | 0.79 | 0.50 | 4 | 1.6 | 1.3% | 0.29 | 0.46 | 0.19 | 0.63 | 0.61 | 0.43 |
RMV56 | 1 | 1.0 | 0.0% | 0.00 | 0.00 | / | / | / | / | 4 | 1.2 | 41.0% | 0.17 | 0.60 | 0.33 | 0.69 | 0.52 | 0.71 |
RMB5° | 2 | 1.1 | 1.1% | 0.00 | 0.18 | 1.00 | 0.48 | 0.45 | 0.11 | 3 | 1.1 | 1.3% | 0.00 | 0.55 | 1.00 | 0.80 | 0.45 | 0.64 |
RMB53° | 1 | 1.0 | 0.0% | 0.00 | 0.00 | / | / | / | / | 2 | 1.0 | 0.0% | 0.00 | 0.04 | 1.00 | 0.03 | 0.25 | 0.01 |
RMB55 | 1 | 1.0 | 9.1% | 0.00 | 0.00 | / | / | / | / | 13 | 1.6 | 0.0% | 0.37 | 0.71 | 0.15 | 0.59 | 0.72 | 0.62 |
RMB15° | 1 | 1.0 | 0.0% | 0.00 | 0.00 | / | / | / | / | 6 | 1.2 | 0.0% | 0.11 | 0.44 | 0.77 | 0.59 | 0.64 | 0.63 |
RMV10 | 1 | 1.0 | 0.0% | 0.00 | 0.00 | / | / | / | / | 7 | 1.9 | 0.0% | 0.32 | 0.60 | 0.41 | 0.23 | 0.42 | 0.20 |
RMB34 | 2 | 1.0 | 2.3% | 0.00 | 0.00 | 1.00 | 1.00 | 0.97 | 0.60 | 5 | 1.8 | 26.9% | 0.41 | 0.65 | 0.13 | 0.20 | 0.15 | 0.43 |
RMB12 | 1 | 1.0 | 3.4% | 0.00 | 0.48 | / | / | / | / | 7 | 1.7 | 37.2% | 0.06 | 0.80 | 0.87 | 0.53 | 0.34 | 0.86 |
RMV20 | 1 | 1.0 | 13.6% | 0.00 | 0.00 | / | / | / | / | 8 | 1.3 | 3.8% | 0.23 | 0.42 | 0.27 | 0.38 | 0.33 | 0.23 |
RMB16 | 1 | 1.0 | 9.1% | 0.00 | 0.00 | / | / | / | / | 5 | 1.3 | 24.4% | 0.24 | 0.42 | 0.37 | 0.50 | 0.63 | 0.35 |
Total | 1.3 | 1.0 | 5.4% | 0.00 | 0.12 | 1.00 | 0.80 | 0.82 | 0.41 | 6.2 | 1.5 | 23.3% | 0.22 | 0.58 | 0.39 | 0.55 | 0.47 | 0.50 |
Finally, we tested for an association between genetic lineage and habitat type with a two-sided Fisher exact test in R (
Based on the morphological identifications, we considered eight populations as
The three populations with a mismatch between the morphological identification and the cpDNA haplogroup (T1, T2, and T3) were amplified with both sets of microsatellite markers. However, the
A comparable amplification pattern is found in fourteen samples of population D1. This population is identified as
To detect hybrids with the
Next, we considered the three cross-amplifying markers that were included in both marker sets. Markers RMB5 and RMB53 had very bad amplifications in populations with a
Finally, microsatellite amplification success was highly variable between different markers and different populations of assumed hybrids, especially in the
The PCA based on the
A similar clustering pattern could be seen in the PCA based on the
Finally, in the analyses based on the
Microsatellites were also used to detect ploidy levels of these hybrid populations. We found three or four alleles for at least one locus in 40% of the hybrids with a
The five pure
Distribution of the genotypes of pure
Genotype 1 | Genotype 2 | Genotype 3 | Genotype 4 | Genotype 5 | Genotype 6 | |
D1 | 0% | 88% | 12% | 0% | 0% | 0% |
D2 | 0% | 0% | 0% | 0% | 100% | 0% |
D3 | 12% | 0% | 0% | 0% | 88% | 0% |
D4 | 0% | 0% | 0% | 13% | 67% | 20% |
D5 | 0% | 100% | 0% | 0% | 0% | 0% |
Total | 3% | 41% | 2% | 2% | 49% | 3% |
The twelve pure
The three inferred hybrid populations had a genetic diversity that was intermediate to their parental species (mean number of alleles per locus = 5.0,
We also observed strong differences between the two sets of markers in terms of clonality (
In this paper, we used genetic tools to identify different lineages of the genus
We found strong differences in reproductive strategy and habitat preference between
Both
Established hybrid lineages are often found in different habitats than their parental species (
The three hybrid populations can be divided into two different genetic clusters. The first hybrid cluster comprises populations T1 and T2, the second population T3. Each cluster is characterized by a specific group of non-amplifying loci and a set of unique alleles. However, this does not mean that each cluster only groups the interbreeding offspring of a single hybridization event. If a hybrid lineage has a single origin, all hybrid alleles that were not present in the parental individuals would be the result of mutations (
Comparing alleles between hybrids and parental species can reveal information about the origin of these hybrids. The
Several
The
Although the majority of angiosperm hybrid lineages has multiple origins, outcrossing between hybrid lineages with different origins is not ubiquitous in the plant kingdom. Within the macrophyte genus
We could not detect F1 hybrids, nor were they detected in previous molecular studies (
We detected fourteen samples that turned out to be backcrossings between
One limitation in our study is the absence of a good set of cross-amplifying markers that amplified in all samples. In the design of our multiplexes, we tried to overcome this problem by including markers RMB5, RMB15, and RMB53 in both multiplexes, but we did not foresee that only RMB15 could amplify successful in all populations. We also added diagnostic markers RUMR4 and RM3 to the
Flower peduncle length is often considered as the clearest distinguishing characteristic between
The Camargue harbors both
The microsatellite data for both species are available at Dryad (doi:
LB has performed the fieldwork and laboratory work, analyzed the data, and wrote the manuscript. LT helped during the field work and contributed laboratory equipment. LT and BV helped during data-analysis and manuscript writing. All authors contributed to the manuscript and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Patrick Grillas and Marc Thibault (Tour du Valat), Véronique Paliard and Eric Coulet (La Capelière), and Claire Tetrel and Emmanuel Vialet (Domaine de la Palissade) were of great help for finding
The Supplementary Material for this article can be found online at:
Allele frequencies of each microsatellite, calculated for each population.