Background

Polyploidization has long been recognized as an essential force in plant evolution (Raven and Thompson 1964; Leitch and Bennett 1997; Soltis et al. 2014); however, the complex nature of polyploids has restrained research efforts on these plants (Soltis et al. 2009; Dufresne et al. 2014; Baduel et al. 2018). Many questions about polyploid evolution are not yet thoroughly resolved. For example, how did polyploidization occur? How many times has polyploidization occurred in the history of a specific group? Do polyploids interact with closely related taxa; if yes, how? Is a polyploid taxon diverse at the molecular level; if yes, how? An increasing number of studies are attempting to dissect the origins of polyploids and the dynamic nature among and within polyploid taxa by applying the rapidly developing molecular and computational techniques (e.g. Van de Peer et al. 2021; Karbstein et al. 2022; Zhuang et al. 2022). Ludwigia L. section Isnardia (L.) W.L. Wagner & Hoch, a polyploid complex with a moderate number of species and sufficient knowledge from plenty of earlier systematic studies, is an ideal plant group to explore polyploid phylogeny as well as the dynamic nature of polyploids.

Isnardia currently contains 22 taxa, including five diploids, 11 tetraploids, four hexaploids, one octoploid, and one taxon with an unknown ploidy level (Table 1) (Raven 1963; Raven and Tai 1979; Peng 1988, 1989; Peng et al. 2005; Wagner et al. 2007; Arya et al. 2020; Liu et al. 2020). Isnardia are wetland weeds characterized by their creeping or erect stem, 4-merous and haplostemonous flowers, terete or globose capsules, and pluriseriate and free seeds (Wagner et al. 2007). Members of Isnardia were previously classified into three sects. Isnardia, Michelia Ramamoorthy, and Microcarpium Munz (Munz 1944; Raven 1963; Ramamoorthy 1979; Wagner et al. 2007). Early systematic studies have suggested that these three sects. are closely related and, together, compose the Microcarpium complex (Eyde 1977, 1978, 1981; Peng and Tobe 1987; Peng et al. 1988, 2005; Tobe et al. 1988; Peng 1989; Wagner et al. 2007). Based on morphological and anatomic studies, this complex was proposed to be evolutionarily closed to sect. Ludwigia L. (Eyde 1977, 1978, 1981). Recent molecular phylogenies revealed that this complex is a monophyletic group with strong (Liu et al. 2020) or weak (Liu et al. 2017) support, and sect. Ludwigia is sister to the complex (Liu et al. 2017, 2020). However, none of the three sects. in the complex exhibit monophyly (Hung et al. 2009; Liu et al. 2017, 2020), therefore the three sects. in the complex were combined into the sect. Isnardia (Liu et al. 2020). Examining the combination with different data sets will provide valuable information on this taxonomic treatment.

Table 1 Sampled Ludwigia sect. Isnardia taxa, their sample sizes, genome types, ploidy levels, and nucleotide diversities (Tajima’s π) of the studied regions
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Extant Isnardia plants are mainly distributed in North America with two exceptions––L. ovalis Miq. is endemic to East Asia and L. venugopalanii S. Arya, V. Suresh, P. Biju & V.S.A. Kumar occurs in India––and one widely spread taxon, L. palustris (L.) Elliott, which occurrs in North, Central, and South America, Eurasia, and Africa (Raven 1963; Peng et al. 2005; Wagner et al. 2007; Arya et al. 2020). Geographical distribution and phylogeographic analyses indicated that Isnardia plants originated in North America and a few taxa spread to other continents later (Raven 1963; Eyde 1981; Tobe et al. 1988; Liu et al. 2020). The earliest known fossil record of Isnardia indicated that this clade had extended to Europe in Middle Miocene (Friis 1985; Tobe et al. 1988) (ca. 11.63–15.97 million years ago (MYA) referring to the International Commission on Stratigraphy (ICS), https://stratigraphy.org/). That is, Isnardia likely originated before Middle Miocene. However, the results of Hung et al. (2009)’s coalescence analyses suggested a younger the most recent common ancestor (TMRCA) of Isnardia, which was 5.99 ± 0.02 MYA based on chloroplast atpB-rbcL data and 6.59 ± 0.02 MYA based on nuclear ITS data. Additional molecular data, more thorough sampling, and further analyses are warranted to give a better perception of the divergence time of Isnardia.

Origins of Isnardia taxa have been intensively investigated using morphological, anatomic, cytological, and molecular data (Raven 1963; Eyde 1977, 1978, 1981; Raven and Tai 1979; Peng and Tobe 1987; Tobe et al. 1988; Peng 1988, 1989; Peng et al. 2005; Hung et al. 2009; Liu et al. 2017, 2020). Eight ancestral genomes––including genomes A, B (= H), C (= I), D (= G), F1, F2, M1, and M2––have been proposed and assigned to each Isnardia taxon (Table 1) based on cytological and molecular works (Peng 1988, 1989; Peng et al. 2005; Hung et al. 2009; Liu et al. 2020). The reticulate origins of the 16 allopolyploid Isnardia––consisting of 11 tetraploids, four hexaploids, and one octoploid (Table 1)––were also inferred (Liu et al. 2020). Multiple origins are common in polyploid speciation (Soltis and Soltis 1993; Leitch and Bennett 1997; Peng and Chiang 2000; Doyle et al. 2004). To date, however, only two Isnardia––tetraploid L. sphaerocarpa Elliott and hexaploid L. repens J.R. Forst.––have been considered to have multiple origins (Peng 1988; Liu et al. 2020), because of the limitation of small sample sizes for each taxon in the earlier studies. Comprehensively decoding the polyploid Isnardia taxa's origins requires a larger sample size for each taxon.

In addition, higher genetic diversities have been detected in the taxa with higher ploidy levels in several plant groups (e.g. Mallet 2007; García‐Verdugo et al. 2009; Bogačiovienė et al. 2019; Zhang et al. 2019). At the same time, earlier studies have observed plenty of natural hybrids among Isnardia taxa in the field and reported the high capability of interspecific hybridization among Isnardia taxa in breeding experiments at research greenhouses (Raven and Tai 1979; Peng 1988, 1989; Peng et al. 2005). However, these attributes of Isnardia taxa have not been well explored. Hung et al. (2009) measured infraspecific genetic diversities of six Isnardia taxa. Both Liu et al. (2017) and Liu et al. (2020) sampled 20 Isnardia taxa, but they could not approximate interspecific gene flow nor infraspecific genetic diversities due to small sample sizes for each taxon. Moreover, a larger sample size for each taxon will provide a better insight into these characteristics of Isnardia.

In the present study, we intended to meet the following four aims by comprehensively sampling Isnardia. First, we reexamined the reticulate evolution hypotheses implied by earlier studies (Peng 1988, 1989; Peng et al. 2005; Hung et al. 2009; Liu et al. 2020) and investigated multiple origins of polyploid taxa. Second, we reevaluated the estimated ages of Isnardia TMRCA (Hung et al. 2009). Third, we tested the proposition that, as with many other allopolyploid plant groups (Mallet 2007; García‐Verdugo et al. 2009; Bogačiovienė et al. 2019; Zhang et al. 2019), genetic diversities of Isnardia taxa increase with their ploidy levels. Fourth, consistent with earlier field observations and breeding experiments (Raven and Tai 1979; Peng 1988, 1989; Peng et al. 2005), we investigated the interspecific gene flows among Isnardia taxa with different ploidy levels.

Methods

Sampling

We aimed to sample all Isnardia taxa. For each Isnardia taxon, we planned to sample three to ten individuals. Living plants were collected during collecting trips in Alabama, Florida, Massachusetts, Missouri, North Carolina, Oklahoma, South Carolina, Tennessee, Texas, and Virginia in the USA. Leaf tissue for genomic DNA extraction was dried in silica gel immediately after being collected. Vouchers were deposited at the Herbarium of Endemic Species Research Institute (TAIE) (Thiers 2016), Taiwan, for further studies. Samples of the Cuban endemic species, L. stricta (C. Wright ex Griseb.) C. Wright, were obtained from the herbarium vouchers provided by the Herbarium of Missouri Botanical Garden (MO) (Thiers 2016), Saint Louis, Missouri, USA. Unfortunately, samples for the recently described Indian endemic species, L. venugopalanii (Arya et al. 2020), were unavailable. Additional Isnardia taxa from Hung et al. (2009) were also incorporated into this study. In addition, published DNA sequences of some outgroups from other sections in Ludwigia and other genera in Onagraceae were downloaded from the GenBank (Sayers et al. 2019) for the analyses.

Genomic DNA extraction, PCR, cloning, sequencing, and assembly

The genomic DNA of our samples was extracted by optimizing the cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson 1980; Doyle and Doyle 1987) for Isnardia samples. A mixture of 5–10 mg ground leaf tissue, 10 mL 65 °C 3X CTAB isolation buffer, and 40 μL 0.4% ß-mercaptoethanol was incubated at 65 °C for 30 min. After the incubation, 10 mL chloroform-isoamyl alcohol (24:1) was added to the mixture. The solution was gently, thoroughly mixed, and then centrifuged at 6000×g for 10 min at about 20 °C. The supernatant was retained, and the process described in the previous sentence was repeated twice. The final supernatant was mixed well with 10 mL isopropanol, incubated at − 20 °C for 30 min to 24 h, warmed up, and centrifuged at 15,000×g for 10 min at 4 °C. The precipitated DNA was air-dried, dissolved with 500 μL TE buffer, and incubated at 37 °C for 30 min with RNase A. A 500 μL isopropanol was applied to precipitate DNA again. The precipitated DNA was then resuspended with 70% ethanol, centrifuged at 15,000×g for 2–3 min at 4 °C, air-dried, and dissolved in 200 μL TE buffer for the following polymerase chain reactions (PCR).

Both nuclear ITS (ITS1 + 5.8S + ITS2) and chloroplast atpB-rbcL regions were amplified with the universal primers (White et al. 1990; Chiang et al. 1998; Hung et al. 2009) for all Isnardia samples. A 100 μL mixture including 0.5 μL 10U/μL Taq Polymerase (Promega, Madison, Wisconsin, USA), 10 μL 10X PCR buffer, 10 μL 8 mM dNTP, 10 μL 2 pM forward primer, 10 μL 2 pM reverse primer, 10 μL 10 mM MgCl2, 10 μL 2 ng/ μL genomic DNA, and distilled water was applied in each PCR amplification. The genomic DNA in the mixture was initially denatured at 92 °C for 5 min, followed by 31 cycles of 92 °C for 45 s, 53 °C for 75 s, and 72 °C for 90 s, and finally elongated at 72 °C for 10 min. The PCR products were then purified with 1% agarose gel and the Gel/PCR DNA Isolation System (Viogene, Taipei, Taiwan).

In the case where multiple sizes of PCR products were amplified for one individual, TA cloning was applied using the pGEM-T Easy Cloning Vector (Promega, Madison, Wisconsin, USA) to determine variations within the individual.

Subsequently, PCR products and colonies were sequenced on an ABI 3730XL DNA Analyzer (Applied Biosystems, Waltham, Massachusetts, USA) commercially with the universal primers (White et al. 1990; Chiang et al. 1998; Hung et al. 2009). DNA reads were assembled using the De Novo Assemble tool implemented in Geneious Prime 2022.2.1 (Biomatters, Ltd., Auckland, New Zealand). Assembled DNA sequences were deposited at GenBank for further studies.

Sequence alignment, genetic variation, phylogenetic analysis, and network

DNA sequences for ITS and atpB-rbcL regions were aligned with Clustal Omega 1.2.3 (Sievers and Higgins 2014). To understand the infraspecific genetic variations of Isnardia taxa, the nucleotide diversities (Tajima’s π; π) (Tajima 1983) were quantified using MEGA 11 (Tamura et al. 2021) with the Kimura 2-parameter model (Kimura 1980), and 500 replicates were applied to calculate the standard errors. One-way analysis of variance (ANOVA) and student’s t-tests were conducted with SPSS 28.0 (IBM Corp 2021) to test our third hypothesis as well as to assess how sample size affects the genetic diversities of Isnardia.

To reconstruct phylogenetic trees and meet our first goal, we applied Maximum likelihood (ML) and Bayesian inference (BI) algorithms. The nucleotide substitution model for each studied region was obtained using jModelTest 2 (Darriba et al. 2012). The ML trees were generated using RAxML 8.2.11 (Stamatakis 2014) with the best-fitting model for each region, and the branch supports were evaluated using the bootstrapping values (bs) (Felsenstein 1985) with 1000 replicates. Bayesian inference (BI) analyses of phylogeny were conducted using MrBayes 3.2.7 (Huelsenbeck and Ronquist 2001; Ronquist et al. 2012) on the CIPRES Science Gateway 3.3 (Miller et al. 2010) with two independent Markov Chain Monte Carlo (MCMC) runs, 5 × 106 generation Markov chains in each run, and the best-fitting models. Trees were saved every 1000 generations. A 50% majority-rule consensus BI tree and the posterior probabilities (pp) on the branches were yielded by incorporating the last 75% of the saved trees. FigTree 1.4.4 (Rambaut 2018) was applied to depict the ML and BI trees.

To infer the evolutionary relationships within Isnardia, which includes many allopolyploid taxa, a phylogenetic network was conducted. The biparentally inherited nuclear regions are required in the network analyses to take in both maternal and paternal heritages of allopolyploid organisms. Therefore, a subset of the ITS data was applied here. Based on the Isnardia genomes hypothesized by Liu et al. (2020), in the subset, ideally, one sequence would be selected to represent a diploid taxon, two sequences from two different well-supported clades would represent a tetraploid taxon, three sequences from three different well-supported clades would represent a hexaploid, and so on. Here, we randomly selected one sequence to represent a taxon in each well-supported clade from the ITS tree generated above in the present study and made these sequences the subset. The subset was then aligned and analyzed using Clustal Omega 1.2.3 (Sievers and Higgins 2014) and RAxML 8.2.11 (Stamatakis 2014) as described above. The resulting subset ML tree was then converted to a multi-label tree. Subsequently, a phylogenetic network was computed with the multi-label tree algorithm using the Exact Method (Huber et al. 2006) implemented in Dendroscope 3.8.3 (Huson and Scornavacca 2012).

Divergence time estimation

To achieve our second aim, the age of Isnardia TMRCA was estimated using Beast 2.6.7 (Bouckaert et al. 2019) based on the subset ITS data and outgroup sequences from other Ludwigia sections and other genera in Onagraceae (see Additional file 1). Sequences were aligned with Clustal Omega 1.2.3 (Sievers and Higgins 2014), and the best-fitting nucleotide substitution model was determined using jModelTest 2 (Darriba et al. 2012). Applying the birth–death skyline model (BDSKY; Stadler et al. 2013), we ran a Markov Chain length of 4 × 107 generations with the estimated best-fitting nucleotide substitution model, substitution rates of ITS in genus Lopezia under Onagraceae (5.15 × 10–9 substitutions/site/year; O’Kane 1993), and secondary calibration points at the crown node of Onagraceae (71 (88.4–54.3) MYA from Gonçalves et al. (2020) and 46.9 (74.1–2.7) MYA from Zhang et al. (2021)). These two age estimations were both in harmony with those from the fossil records (Grímsson et al. 2011; Lee et al. 2013; Farooqui et al. 2019). Trees were sampled every 1,000 generations. Output log files were checked with Tracer 1.7.1 (Rambaut et al. 2018) to ensure that all Effective Sample Size (ESS) values were greater than 200. The 50% majority-rule consensus trees, pp on the branches, and node heights, or node ages, were produced using TreeAnnotator 2.6.7 (Bouckaert et al. 2019) with a 10% burnin and drawn using FigTree 1.4.4 (Rambaut 2018). Topologies with the uncertainty of the node heights were then generated using DensiTree 2.2.7 (Bouckaert 2010; Bouckaert and Heled 2014).

IMa analyses

To examine the interspecific hybridization among Isnardia taxa as well as to test our fourth hypothesis, we employed the Isolation with Migration coalescent model implemented in IMa2 (Hey and Nielsen 2007) and estimated migration rates in both directions (m0→1 and m1→0) between every two Isnardia taxa. The substitution rates of chloroplast spacers in seed plants (1.01 × 10–9 substitutions/site/year; Graur and Li 2000; Chiang et al. 2006) and nuclear ITS in genus Lopezia (5.15 × 10–9 substitutions/site/year; O’Kane 1993) were adopted to scale all parameters. Since there were multiple changes at some sites in ITS and atpB-rbcL regions, the HKY substitution model (Hasegawa et al. 1985) was applied. For each simulation, 1 × 107 generations with 3 × 106 burn-in were performed. Three independent simulations were conducted for every two taxa to assess the consistency among the results. An average of the three results was reported for every two-taxon pair. The migration rate per gene copy per generation (M) was determined using the formula M = m × u, where u is the substitution rate per year for the studied region. One-way ANOVA and t-tests were applied to investigate the trends in the interspecific gene flow among ploidy levels in Isnardia and test our fourth hypothesis.

Results

Sampling, PCR, sequencing, and assembly

Totally, 111 Isnardia samples––including 70 newly collected samples, 39 from Hung et al. (2009)’s work, and three from the MO herbarium––were processed. An additional six taxa from other Ludwigia sects. and three taxa from other genera in Onagraceae were also included in our analyses. DNA of all newly collected samples and herbarium samples was extracted successfully. Both studied regions were well amplified for most of the newly collected samples. However, after extensive attempts, all PCR failed for the herbarium samples. The herbarium samples were then dismissed from the following analyses. No multiple sizes were found in PCR products, and no polymorphism base was recognized in the assembled sequences. Therefore, cloning was not conducted. The voucher information and GenBank accession numbers of all studied samples are provided in Additional file 1.

Phylogenetic analysis, network, and genetic variation

In total, 192 sequences representing 20 of 22 Isnardia taxa and ten sequences representing nine outgroups were analyzed in the present study, within which 124 Isnardia sequences were newly generated (Table 1; Additional file 1).

Both ML and BI trees were reconstructed for atpB-rbcL and ITS regions. The best-fitting substitution models and descriptive statistics of the two studied regions are shown in Table 2. The alignments and tree files are provided in Additional file 2 and Additional file 3. Since BI trees share most topologies with ML trees, only ML trees are shown here (Figs. 1 & 2). Our ITS tree (Fig. 1) was highly consistent with nuclear trees of Hung et al. (2009) and Liu et al. (2017). However, Isnardia taxa were clustered together with weak support in our ITS tree (pp = 0.66, bs = 44; Additional file 2 & Additional file 3). The well-supported clades revealed in the ITS tree were used to set up the subset ITS data (see network analyses). Adopting the hypothesized genome types of Isnardia inferred by Liu et al. (2020) (Table 1), we identified the A, M1M2, F1F2, and D clades (Fig. 1). For example, the A clade comprised the diploid L. palustris and all other samples which had A in their genome types (Fig. 1). Though the resolutions of our atpB-rbcL tree (Fig. 2) among all other Isnardia were generally low, our tree was largely congruent with the chloroplast phylogenies of Hung et al. (2009) and Liu et al. (2017, 2020), and L. ovalis Miq. was sister to all other Isnardia taxa.

Table 2 The substitution models, characteristics, and maximum parsimony statistics of the regions
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Fig. 1
figure 1

Maximum likelihood tree of Ludwigia sect. Isnardia inferred from the nuclear ITS region. The arrows indicate the crown nodes of Isnardia. Sample numbers (Additional file 1), ploidy levels (Table 1), and genome types (Table 1) are shown right after the taxa. Numbers at nodes show the bootstrapping values (bs) / posterior probabilities (pp) only when the bs at nodes are greater than 50 or pp at nodes are greater than 0.70. The A, M1M2, F1F2, and D clades are indicated. The scale bar denotes the branch length

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Fig. 2
figure 2

Maximum likelihood tree of Ludwigia sect. Isnardia inferred from the chloroplast atpB-rbcL region. The arrow indicates the crown node of Isnardia. Sample numbers (Additional file 1), ploidy levels (Table 1), and genome types (Table 1) are shown right after the taxa. Numbers at nodes show the bootstrapping values (bs) / posterior probabilities (pp) only when the bs at nodes are greater than 50 or pp at nodes are greater than 0.70. The scale bar denotes the branch length

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Moreover, our phylogenetic analyses showed that both tetraploid L. sphaerocarpa (AABB) and hexaploid L. repens (AABBCC) have evolved multiple times, and tetraploid L. arcuata Walter (BBCC) might have multiple origins or a higher infraspecific genetic variation. For L. sphaerocarpa, one evolutionary scenario showed that the paternal and/or maternal donors of L. sphaerocarpa samples 1–3 and 5 were phylogenetically close to L. lanceolata Elliott (AABB) and L. suffruticosa Walter (AABB), two members of the AABB tetraploids (Fig. 1; Table 1; Additional file 1). This evolutionary scenario might fit earlier hypotheses on the homoploid hybridization of L. sphaerocarpa (Peng 1988; Liu et al. 2020) if our atpB-rbcL tree (Fig. 2) had higher resolution and gave more information on the maternal donors of these four L. sphaerocarpa samples. We detected another evolutionary scenario that revealed that the maternal donor of L. sphaerocarpa sample 4 probably carried genome A and was likely closely related to L. lanceolata and L. glandulosa Walter subsp. glandulosa (AABB), which both had an AABB genome type (Fig. 2; Table 1). The paternal donor contributing genome B to this L. sphaerocarpa sample likely also gave rise to L. curtissii Chapm. (BBCCDDF1F1) and L. simpsonii Chapm. (BBCCDD) (Fig. 1; Table 1). The latter evolutionary scenario of L. sphaerocarpa has not been revealed by earlier studies.

For the origins of L. repens, our atpB-rbcL tree (Fig. 2) suggests that the maternal donors were probably tetraploids carrying genome BBCC and closely related to L. arcuata. This result was congruent with earlier studies (Hung et al. 2009; Liu et al. 2020). Moreover, our ITS tree (Fig. 1) indicated that at least two ancestor lineages––likely the maternal donors with genome BBCC––had given birth to L. repens; one gave rise to L. repens sample 4 while another contributed to L. repens samples 1–3 and 5–9. The former maternal donor also played a part in the origination of L. brevipes (Long) Eame (AABBCC). However, no paternal donor of L. repens with genome A was represented in our study.

Our ITS tree shows that L. arcuata samples are clustered in two well-supported groups (Fig. 1). One group consists of L. arcuata samples 2–4, L. brevipes samples 1–6, and L. repens sample 4, while another group includes L. arcuata sample 1 and L. repens samples 1–3 and 5–9. This indicates that L. arcuata probably has multiple origins and/or a relatively high infraspecific nucleotide diversity. In fact, our analyses show that the infraspecific π of L. arcuata was higher than that of most Isnardia (Table 1).

Twenty-nine Isnardia samples were included in the subset ITS data and network analyses. Sample information of the subset is provided in Additional file 1. The phylogenetic network (Fig. 3) concurs with our ITS tree (Fig. 1) completely and indicates the hybrid origins of eight Isnardia taxa. Tetraploids L. lanceolata, L. pilosa Walter, and L. suffruticosa share the same evolutionary histories, and all of them are allopolyploids deriving from two genomes (Fig. 3). One evolutionary signal arose from the lineage sister to the clade/genome A. Another signal is likely from genome B because this signal arose from the lineage sister to L. ravenii C.I Peng, which has B in its genome type. Our ITS and atpB-rbcL trees (Figs. 1, 2) indicate that tetraploid L. sphaerocarpa had evolved at least twice (see previous paragraphs). The first evolutionary scenario, homoploid hybridization (Peng 1988; Liu et al. 2020), could not be shown in the network due to the limitation of our sampling. The second evolutionary scenario is revealed in our network (Fig. 3); that L. sphaerocarpa obtained genome A from the lineage sister to the A clade/genome and obtained its genome B from L. curtissii, which also has genome B. Moreover, two evolutionary signals were detected for the origins of tetraploid L. arcuata and hexaploid L. repens, and these two evolutionary signals were from sister lineages, which both lineages might have genome B (dashed red lines in Fig. 3). This may indicate that both L. arcuata and L. repens have multiple origins. Furthermore, our network implies that hexaploid L. simpsonii stemmed from at least two genomes––D genome from the lineage close to diploid L. microcarpa and B genome from the lineage close to L. curtissii. In addition, two evolutionary signals were mixed to give rise to L. ovalis. However, it was difficult to distinguish M1 and M2 signals in our network. Alignment, the ML tree, and network files are given in Additional file 4.

Fig. 3
figure 3

Evolutionary network of Ludwigia sect. Isnardia inferred from the subset of the ITS region. The arrow indicates the crown node of Isnardia. The A, M1M2, F1F2, and D clades are marked as in Fig. 1. Blue lines show the evolutionary signal from the A genome, red lines from the B genome, dashed red lines likely from the B genome, green lines from the D genome, brown lines from the M1 and M2 genomes

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Furthermore, the results of our genetic variation analyses of Isnardia suggest that the atpB-rbcL region generally has lower infraspecific genetic variations (π = 0.00000–0.01896) compared to the ITS region (π = 0.00000–0.02097). The infraspecific π of each Isnardia taxon are shown in Table 1. Notably, ITS sequences are infraspecific identical in L. microcarpa Michx. and L. spathulata Torr. & A. Gray, and atpB-rbcL sequences are infraspecific identical in L. linifolia Poir. and L. microcarpa. One-way ANOVA analyses showed that the genetic diversities of Isnardia taxa had no significant differences among sample sizes (p-values > 0.05; Additional file 5). In other words, our sampling was appropriate for assessing the genetic diversities in Isnardia. Furthermore, our study detected no relationship between ploidy levels and genetic diversities (Fig. 4). For instance, the π value of the ITS region for diploid L. palustris (AA) was 0.00189 ± 0.00115, but some of the tetraploids with the AABB genome type had lower π values and others had higher π values (Table 1). One-way ANOVA analyses indicated no significant differences in π values of Isnardia taxa among ploidy levels (p-values > 0.05; Fig. 4; Additional file 5). Student’s t-tests also showed no significant differences in π values between diploids and all polyploids (p-values = 0.81 and 0.08 for ITS and atpB-rbcL regions, respectively). That is, our results falsified hypothesis three.

Fig. 4
figure 4

Boxplots showing infraspecific nucleotide diversities (Tajima’s π) of different ploidy levels in Ludwigia sect. Isnardia based on (a) atpB-rbcL and (b) ITS data. Letters above each boxplot indicate pairwise statistical differences between ploidy levels. The complete details for the statistical analysis are shown in Additional file 5. The infraspecific nucleotide diversity of each taxon is available in Table 1

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Divergence time estimation

All (29) Isnardia samples in the subset and nine outgroups were applied to estimate the age of Isnardia TMRCA. Sample information is provided in Additional file 1. The topologies of the resulted chronograms (Fig. 5) are congruent with our ITS tree (Fig. 1). Adopting the secondary calibration points at the crown node of Onagraceae suggested by Zhang et al. (2021), the crown node of genus Ludwigia was approximately 17.2 (20.3–13.2) MYA, the crown node of the North Temperate Haplostemonous (NTH) Ludwigia (= sects. Isnardia + Ludwigia) was 7.6 (10.5–5.3) MYA, and TMRCA of Isnardia was 5.9 (7.7–4.3) MYA (Fig. 5a). When we took Gonçalves et al. (2020)’s results as calibration points instead, the crown node of genus Ludwigia was estimated to be 25.9 (30.5–20.2) MYA, the crown node of the NTH Ludwigia was 11.3 (15.7–7.8) MYA, and TMRCA of Isnardia was 8.9 (11.5–6.5) MYA (Fig. 5b). Alignment and 50% majority-rule consensus tree files are shown in Additional file 6.

Fig. 5
figure 5

Chronograms of Ludwigia sect. Isnardia based on the subset of ITS region with divergence time estimates adopting the substitution rates of ITS (O’Kane 1993) and secondary calibration point at the crown node of Onagraceae following (a) Zhang et al. (2021) or (b) Gonçalves et al. (2020). The arrows indicate the crown nodes of Isnardia. Sample numbers (Additional file 1), ploidy levels (Table 1), and genome types (Table 1) are shown right after the taxa. Stars at nodes indicate the posterior probabilities (pp) only if the pp at the nodes is greater than 0.90. When the pp at the nodes is greater than 0.95, the double star notations are denoted. Numbers at nodes show the median estimated divergence times (MYA) with 95% credible intervals in the brackets. The smears around the nodes display the uncertainty of the estimated divergence times

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IMa analyses

The migration rates per gene copy per generation (M, interspecific gene flow) for every two Isnardia taxa based on ITS and atpB-rbcL regions revealed rampant but low interspecific gene flow in Isnardia, from 9.42 × 10–10 to 3.13 × 10–9, and 2.36 × 10–10 to 6.58 × 10–10, respectively (Fig. 6, Additional file 7). Moreover, our ANOVA and t-tests showed no significant differences in M among ploidy levels based on atpB-rbcL data but found some significant differences in ITS data (Fig. 6, Additional file 8). The M between two ploidy levels––except the M from hexaploids to diploids (group 6 × > 2 ×) and from hexaploids to octoploids (group 6 × > 8 ×) based on ITS data––were compatible with the M between two diploids (group 2 × > 2 ×) (see Fig. 6, Additional file 8). Moreover, Fig. 6b shows that the means of Ms from hexaploids to other ploids (including groups 6 × > 2 × , 6 × > 4 × , 6 × > 6 × , and 6 × > 8 ×) were lower than the means of Ms of other groups. Our data supported hypothesis four and indicated that Ms from hexaploids to other taxa were relatively lower.

Fig. 6
figure 6

Boxplots showing the estimated migration rates (M, migration rates per gene copy per generation) between ploidy levels in Ludwigia sect. Isnardia based on (a) atpB-rbcL and (b) ITS data. Groups on the x axial are coded, showing the direction of the migration rates, e.g., 2 × > 4 × indicates the migration rates from diploids to tetraploids. Letters above each boxplot indicate pairwise statistical differences (p-values < 0.05) between 2 × > 2 × and other groups. The asterisks indicate pairwise statistical differences (p-values < 0.05) between the two groups. The complete details for the statistical analysis are shown in Additional file 8. The estimated migration rates among taxa are available in Additional file 7

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Furthermore, unsymmetrical Ms were detected in most two-taxon pairs (Additional file 7), and ITS data gave more information on the natural dynamic in Isnardia (Fig. 6b, Additional file 8a). Both the M of group 2 × > polyploid were significantly greater than the M of group polyploid > 2 × . In addition, our ITS data indicated that the M of group 4 × > 4 × was considerably higher than the Ms of groups 4 × > 2 × , 4 × > 6 × , and 4 × > 8 × , and hexploids had the same trend but no statistical significance. Diploids, however, showed an opposite trend without statistical significance. The M of group 2 × > 2 × was lower than the Ms of groups 2 × > 4 × and 2 × > 6 × , but higher than the M of group 2 × > 8 × .

Discussions

With an extensive sampling of Isnardia (Table 1; Additional file 1), we met our four aims of this investigation of the polyploid clade’s evolutionary history and dynamic nature.

Evolutionary relationships and polyploid speciation in Isnardia

Incorporating the evolutionary information provided by our phylogenetic trees and network, our data fully support the reticulate evolution and hypotheses as to the genome types of Isnardia taxa indicated by Peng (1989), Peng et al. (1988, 2005), and Liu et al. (2020), despite the generally low resolutions in our atpB-rbcL tree (Fig. 2) and low supports at several basal branches in our ITS tree (Fig. 1). Low resolutions of the chloroplast trees inhibited the understanding of Isnardia evolutionary history in earlier studies (Hung et al. 2009; Liu et al. 2017, 2020) and in ours. Some recent studies on other Ludwigia clade and other plant groups (e.g. Liu et al. 2018; Duvall et al. 2020; Simmonds et al. 2021) have shown that the next-generation sequencing (NGS) data would provide complete plastome sequences and improve the understanding of maternal evolutionary inferences. Further studies with NGS data will be called on to uncover maternal phylogeny in Isnardia thoroughly. Additional nuclear single-copy genes may be needed for the phylogenetic network of Isnardia to comprehend reticulate evolution within this polyploid section (e.g. Díaz-Pérez et al. 2018; Wang et al. 2019; Karbstein et al. 2022).

Moreover, we recognized the A, M1M2, F1F2, and D clades but could not identify the B and C clades (Figs. 1, 2, 3). Very likely, diploids of the latter two clades were extinct (Liu et al. 2020). Interpreting the evolutionary history of polyploid groups without their diploid ancestors is challenging (Holloway et al. 2006; Yuan et al. 2006; Soltis and Soltis 2016). The NGS data and advanced algorithms may be helpful in further evolutionary studies on polyploid groups with extinct diploid progenitors, like Isnardia (e.g. Li et al. 2022; Sancho et al. 2022).

Furthermore, our analyses unveiled the complexity of polyploid speciation in Isnardia, which has not been reported before. Our study revealed that L. repens has evolved at least twice (Figs. 1, 2, 3), which supports the work of Liu et al. (2020). Moreover, based on our data, the multiple origins of L. arcuata are suggested here for the first time (Figs. 1, 2, 3). In addition, our data indicate that L. sphaerocarpa arose in various ways (Figs. 1, 2, 3). As per previous cytological and molecular studies, L. sphaerocarpa has occurred multiple times through the homoploid hybridization between two tetraploid ancestors, which both have an AABB genome type (Peng 1988; Liu et al. 2020). The homoploid hybridization proposition is also supported by our L. sphaerocarpa samples 1–3, and 5 (Figs. 1, 2). Moreover, our L. sphaerocarpa sample 4 uncovers an additional evolutionary scenario showing that genomes A and B were contributed from different lineages (Figs. 1, 2). Unfortunately, the genome types and ploidy levels of maternal and paternal donors of L. sphaerocarpa sample 4 are still unknown because of our limited data. This additional evolutionary scenario not only highlights the complexity of allopolyploidization in L. sphaerocarpa but also offers another route to explore the unidentified or extinct diploid ancestors with genome B. With the application of NGS data, further studies may sample more individuals from polyploid Isnardia taxa with genome B and track back how genome B had contributed to polyploid speciation in Isnardia (e.g. Li et al. 2022; Sancho et al. 2022).

Additionally, we sampled herbarium L. stricta, a Cuban endemic, but obtained no PCR result. However, this was not unexpected and may be attributed to the degraded DNA of herbarium vouchers. Ludwigia stricta is a diploid, morphologically similar to L. linifolia (Peng and Tobe 1987; Peng 1988, 1989), and may be valuable for understanding the reticulate evolution of Isnardia. Some NGS strategies have successfully obtained ample sequences from herbarium samples of various plant groups (e.g. Vatanparast 2018; Couvreur et al. 2019; Vargas et al. 2019) and their use should help obtain evolutionary information from L. stricta and other Isnardia herbarium vouchers.

Divergence time estimation

With a more comprehensive sampling in Isnardia (Table 1 and Additional file 1) and using the secondary calibrations at Onagraceae crown nodes from recent studies (Gonçalves et al. 2020; Zhang et al. 2021), our analyses showed that the ages of Isnardia TMRCA were 5.9 (7.7–4.3) MYA and 8.9 (11.5–6.5) MYA (see Fig. 5), which are in concordance with Hung et al. (2009)’s study (6.59 ± 0.02 MYA), and are younger than the fossil record (ca. 11.63–15.97 MYA, see Friis 1985; Tobe et al. 1988). Many plant and animal study cases have reported incongruences between molecular and fossil dates (Benton and Ayala 2003; Heads 2005). Our case is likely due to the extensive polyploidization in Isnardia. The molecular date estimate based on one paralogue––which is equal to the genome presented in the current study––is theoretically younger than the date of the gene duplication, and all paralogues are requisite to better determine the gene duplication date (Jiao et al. 2011; Ruprecht et al. 2017; Koenen et al. 2021). Unfortunately, as shown in the Results section and Figs. 1 and 3, not all genomes were sequenced for all Isnardia polyploids. This probably led to the finding of a younger age of Isnardia TMRCA by our analyses. Nonetheless, other factors causing inconsistency between the estimated Isnardia TMRCA and fossil records (like inaccurate substitution rate, extinct/unsampled taxa, and unoptimistic algorithm) cannot be ruled out (Linder et al. 2005; Doyle and Egan 2010; Guindon 2020). Obtaining improved divergence time estimates for Isnardia in further studies would not be challenging without all genome/paralogues of single-copy genes, comprehensive taxa sampling, and extensive analyses.

Dynamic nature of Isnardia––infraspecific genetic diversity and interspecific gene flow

Infraspecific genetic diversity reflects the evolutionary history of a taxon, including origin, historical events, life history traits, and geographical distribution pattern. (Avise 2000; Sweigart and Willis 2003; Mallet 2007; Bogačiovienė et al. 2019). As revealed in an earlier study on six Isnardia taxa (Hung et al. 2009), our data also show that the infraspecific nucleotide diversities (π) of Isnardia are generally lower in the chloroplast atpB-rbcL region than in the nuclear ITS region (Table 1). This has been exhibited in many other plant groups and attributed to the chloroplast sequences being more conserved compared to nuclear sequences in plants (e.g. Perdereau et al. 2014; Xu et al. 2015; Li et al. 2018).

Moreover, our data indicate that three Isnardia taxa––L. linifolia, L. microcarpa, and L. spathulata––have infraspecific identical sequences in the atpB-rbcL or ITS regions (Table 1). None or low infraspecific genetic variation may indicate short evolutionary time, reproductive strategies leading to genetic homogeneity (e.g. Bussell 1999; Sweigart and Willis 2003; Duffy et al. 2009), evolutionary historical events resulting in population size reductions (e.g. Pimm et al. 1989; Ellegren and Galtier 2016), or evolutionary constraints (e.g. Futuyma 2010; Baucom 2019) of the taxon. These three Isnardia taxa had arisen at least 0.30 or 0.44 MYA (Fig. 5a or b; Additional file 6), which should be long enough to accumulate some infraspecific genetic variation. None or low genetic variations in these three taxa are unlikely to be ascribable to the short evolutionary time. Furthermore, early systematic studies have reported that L. microcarpa and L. spathulata are facultatively autogamous, while L. linifolia is an outcrossing taxon (Raven and Tai 1979; Peng 1988, 1989; Peng et al. 2005; Wagner et al. 2007). Autogamy might cause none or low infraspecific genetic variation in L. microcarpa and L. spathulata. In contrast, the mating system of L. linifolia contributes little to its infraspecific identical sequence. Furthermore, other plant groups in eastern North America had suffered population shrinkage resulting from climate changes (e.g. Woodruff et al. 1981; Zubakov and Borzenkova 1990; Groot 1991) or habitat destruction (e.g. Vargas-Rodriguez et al. 2015; Mohn et al. 2021; Ony et al. 2021). The three Isnardia taxa may also reduce their population sizes under such environmental disturbances. Additionally, having a restricted distribution is one of the common characteristics of a taxon under evolutionary constraints (Futuyma 2010; Razgour et al. 2019). No genetic variation in L. spathulata, a rare species (Peng et al. 2005), might be credited to the evolutionary constraints of this taxon. However, L. microcarpa and L. linifolia are relatively common (Peng 1988, 1989), and some other causes might apply. Additional sampling and analysis are required to elucidate the taxa without infraspecific genetic variations.

In addition, many earlier studies focusing on infraspecific ploidy taxon or a small polyploid complex with a few taxa unveiled that higher infraspecific genetic diversities were found in taxa with higher ploidy levels (e.g. Mallet 2007; García‐Verdugo et al. 2009; Bogačiovienė et al. 2019; Zhang et al. 2019). We expected to find the same pattern in the massive polyploid complex, Isnardia (hypothesis three). Surprisingly, our data did not support this hypothesis (Table 1; Fig. 4; Additional file 5). This unanticipated result may be attributed to the long history of Isnardia taxa (Fig. 5). Each taxon may undergo different evolutionary events, diluting the effect of ploidy levels on infraspecific genetic diversity (e.g. Riddle et al. 2006; García‐Verdugo et al. 2009). The studied taxa's evolutionary and ecological attributes may also blur this pattern. Apart from the mating systems and evolutionary constraints mentioned in the earlier discussions, other attributes like geographic environments and climate factors were reported to affect infraspecific genetic diversity significantly in polyploid groups (e.g. Zhang et al. 2017; Ahrens et al. 2020; Androsiuk et al. 2021; Tsuruta et al. 2022). Further studies will be required to explain the lack of an effect of ploidy level on infragenetic genetic diversity in Isnardia.

Furthermore, our IMa analyses reveal the prevalent but low interspecific gene flow among Isnardia taxa (Fig. 6, Additional files 7 and 8) and provide a solid genetic basis for the rampant interspecific hybridization in Isnardia reported by earlier field observations and breeding experiments (Raven and Tai 1979; Peng 1988, 1989; Peng et al. 2005). Polyploidization is considered one of the important speciation mechanisms in plants, and prezygotic and/or postzygotic reproductive barriers between ploidy levels are expected. However, an increasing number of studies have reported that polyploidization is extremely complicated (Ramsey and Schemske 1998; Husband and Sabara 2004; Baack et al. 2015) and further suggested that polyploidization in some plant groups could collapse the reproductive barriers among ploidy levels and potentiate interspecific gene flow (e.g. Jørgensen et al. 2011; Bohutínská et al. 2021; Schmickl and Yant 2021). While most studies focused on autopolyploidization-mediated interspecific gene flow and its mechanisms (e.g. Husband and Sabara 2004; Przewieslik-Allen et al. 2021; Schmickl and Yant 2021), only a few reported that allopolyploidization may also break down the reproductive barriers among ploidy levels (e.g. Iqbal et al. 2019). Isnardia is the first plant group with frequent interspecific gene flows among multiple allopolyploids and diploids supported by field observations, greenhouse breeding experiments, and genetic data.

Additionally, our data suggest unsymmetrical interspecific gene flows between every two taxa and two ploidy levels in Isnardia (Fig. 6, Additional files 7 and 8); however, this phenomenon was not detected in earlier Isnardia studies (Peng 1988, 1989; Peng et al. 2005). Previous reports have observed unsymmetrical interspecific gene flows in many plant groups (e.g. Peng and Chiang 2000; Lafon-Placette et al. 2017; Abdelaziz et al. 2021). Both prezygotic and postzygotic reproductive barriers could contribute to this phenomenon (Rahmé et al. 2009; Zhang et al. 2016; Moreira-Hernández and Muchhala 2019). Further studies with genome-wide genetic information and breeding experiments with cytological work may provide more insights into the mechanisms of weakening reproduction barriers among allopolyploids and diploids and asymmetric interspecific gene flow in Isnardia.

Conclusions

In summary, a comprehensive sampling of Ludwigia sect. Isnardia, an allopolyploid complex, was applied to test four hypotheses. First, our phylogenetic trees and network not only support the earlier reticulate evolution hypotheses and proposed genomes but also recognize three multiple-origin taxa––tetraploid L. arcuata, hexaploid L. repens, and tetraploid L. sphaerocarpa. The multiple origins of L. arcuata and a new evolutionary scenario of L. sphaerocarpa are reported in this study for the first time. Second, our age estimates of Isnardia TMRCA (5.9 or 8.9 MYA) align with the earlier estimations but are younger than fossil records. In future studies, sampling all paralogues of the studied DNA regions of all taxa would yield a better TMRCA estimate for a polyploid complex like Isnardia. Our analyses did not support hypothesis three, that higher infraspecific genetic variations would be found in taxa with higher ploidy levels. The long evolutionary history and ecological characteristics may have influenced the infraspecific genetic variation of an Isnardia taxon. In addition, we observed the prevailing and unsymmetrical interspecific gene flows among Isnardia taxa, which suggests that the reproductive barriers among taxa were reduced. This has rarely been reported in an allopolyploid complex and calls for additional research. Taken together, our study provides several new insights into the evolutionary history and dynamic nature of Isnardia and highlights the need for further studies to understand the allopolyploid complex.