Phylogenetic study of Lythraceae family in Iran, focusing on the genera Lythrum, Ammannia, and Rotala

doi:10.21203/rs.3.rs-4612594/v1

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Phylogenetic study of Lythraceae family in Iran, focusing on the genera Lythrum, Ammannia, and Rotala

https://doi.org/10.21203/rs.3.rs-4612594/v1

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The family Lythraceae, belonging to the order Myrtales, comprises approximately 28 genera and 700 species of trees, shrubs, and herbs distributed in various regions of the world, especially in tropical and temperate regions. A phylogenetic study based on cpDNA trnH-psbA, nrDNA ITS, and combined sequences datasets were evaluated using maximum parsimony, Bayesian, and maximum likelihood methods. Most of the reconstructed trees revealed the monophyly of the three genera Lythrum, Ammannia, and Rotala in Iran. All trees supported the distinctiveness of species within these three genera. Additionally, the phylogenetic trees of the present study demonstrated the early divergence of Lythrum species on the lower branches and the derivation of Ammannia species on the higher clades and Rotala species on the terminal clades. The results of the phylogenetic study revealed the close relationship of some Lythrumspecies (such as L. thesioides M. Bieb. with L. silenoides Boiss. & Noë and three species L. thymifolia L., L. junceum Banks & Sol., and L. hyssopifolia L.) and Ammannia (A. coccinea Rottb. with A. multiflora Roxb., and A. baccifera L. with A. auriculata Willd.), indicating the need for taxonomic revision.

Lythraceae

Monophyly

Phylogenetic relationship

Taxonomy

The family Lythraceae, with approximately 28 genera and 700 species (POWO 2024), belongs to the order Myrtales, within Eudicots, in the Malvids clade, forming a monophyletic group with Geraniales (APG IV 2016). Phylogenetic, morphological, and embryological evidence suggests a close relationship between Lythraceae and Onagraceae (Dahlgren and Thorne 1984; Johnson and Briggs 1984). Molecular analyses also support the affinity between these two families (Conti et al. 1997; Sytsma et al. 2004; APG IV 2016). Fossil evidence indicates that the Lythraceae family originated in the late Cretaceous to early Paleocene period (Graham et al. 2005). Nevertheless, molecular evidence suggests that the divergence of this family from Myrtales occurred earlier (Sytsma et al. 2004). Nonetheless, molecular studies and fossil records indicate that many members of this family evolved rapidly, dispersed quickly, but then diverged and subsequently became extinct (Graham et al. 2005). This has led to their global distribution as characteristic and diverse genera that include woody trees and shrubs, as well as aquatic herbs. Most genera in this family contain one or two species; some are classified into separate subfamilies, and others are classified into distinct monogeneric families (Koehne 1903; Cronquist 1982; Dahlgren and Thorne 1984; Johnson and Briggs 1984; Takhtajan 1987). According to Inglis et al. (2023), the main centers of Lythraceae species richness are the mountains in western and southern Mexico, as well as the Espinhaço mountain range and central plateau within the Cerrado biome of eastern Brazil.

Furthermore, the greatest genus-level diversity was identified in central and eastern Brazil, the southeastern USA, and mountainous regions of Colombia (Inglis et al. 2023).

The vast habitat diversity and morphological characteristics of flowers have made delineating boundaries between genera within this family challenging. The only monograph of this family, published by Koehne (1881, 1903), includes 22 genera. In this monograph, the monogeneric families Duabangaceae, Punicaceae, and Sonneratiaceae, with inferior and half-inferior ovaries, were classified outside the Lythraceae family with perigynous ovary. Dahlgren & Thorne (1984) suggested that the genera of these three families be considered as subfamilies of Lythraceae. Johnson & Briggs (1984) also conducted cladistic analyses of morphological characteristics within the order Myrtales. The results supported Dahlgren and Thorne's proposal, indicating that Onagraceae and Trapaceae are sister taxa to Lythraceae sensu lato.

Conti et al. (1997) evaluated chloroplast rbcL gene sequences for several genera of the order Myrtales, including Trapa, ten genera from the Onagraceae family, and seven genera from the Lythraceae family. Their analysis indicated sister relationships between the Lythraceae and Onagraceae families. These findings were also supported by molecular studies conducted by Shi et al. (2000) and Huang & Shi (2002), as well as Graham et al. (2005). Additionally, Morris (2007) conducted analyses on molecular data from two chloroplast regions, specifically atpB-rbcL, and trnK-matK, along with previously sequenced data from trnL-trnF, rbcL, psbA-ycf3, and nrDNA ITS, for 29 genera belonging to the family Lythraceae. The findings revealed that multiple convergent evolutions in both morphological characteristics and life history traits have taken place throughout the evolutionary history of this family.

In the study by Gu et al. (2019), the chloroplast genomes of 22 species of the Lythraceae family were compared. A phylogenetic tree was reconstructed using 42 species from the order Myrtales. This study revealed that within the order Myrtales, the families Lythraceae and Onagraceae diverged later compared to the families Melastomataceae and Myrtaceae.

(Polatschek and Rechinger 1968) introduced the genera and species of this family in the Flora Iranica, listing five genera: Lawsonia L., Lythrum L., Ammannia L., Rotala L., and Peplis L. Lawsonia, Rotala, and Peplis were each represented by one species, while Lythrum and Ammannia encompass nine and four species, respectively. Diversity center of the Ammannia, Lythrum, and Rotala is in the Old-World (Graham, 2007). In the flora of Iran, four genera of this family are mentioned, Lawsonia, Lythrum, Ammannia, and Rotala. Lawsonia has one species, La. inermis L., cultivated in the western and southern regions of Iran. Lythrum and Ammannia include seven and four species, respectively, scattered in different parts of Iran (Yousef Naanaie 2010). A new species of Ammannia, named A. coccinea Rottb., was reported from Guilan Province by Naqinezhad & Naseri Larijani (2017). Only one species, R. indica (Willd.) Koehne, is reported from northern Iran in the Rotala genus. Additionally, two species, Rotala densiflora (Roth) Koehne and Peplis hyrcanica Sosn., which were listed in the Flora Iranica by Polatschek & Rechinger (1968), do not have any corresponding herbarium specimens. This absence of specimens suggests that these species may not have been observed or collected in Iran. It is believed that their inclusion in Flora Iranica might have been based on reports from Soviet flora rather than direct evidence from Iranian habitats. Additionally, according to Yousef Naanaie (2010), there is no evidence to support the presence of these species in natural environments within Iran.

The family Lythraceae comprises a group of plants with taxonomic complexities. This generally leads to significant ambiguities in the relationships between genera and species within this family, as well as their taxonomy and phylogenetics. Additionally, as mentioned above, particularly concerning species of the genera Lythrum, Ammannia, and Rotala in Iran, there are many ambiguities and contradictions in the existing literature. Therefore, the main objectives of this study include: 1) conducting a thorough evaluation of the systematic classification of these three genera within the Lythraceae family in Iran, 2) reconstructing the phylogenetic tree and gaining insight into the phylogenetic relationships among these plants, and 3) assessing the evolution of traits based on recent phylogenetic analyses.

Plant Sampling

In this study, 13 species of the Lythraceae family growing in Iran, along with the species L. portula from Morocco were examined. The specimens were collected from the herbarium of the Forests and Rangelands Research Institute (TARI), the herbarium of the University of Tehran (TUH), and the herbarium of the University of Guilan (GUH). Some species were also collected from their habitats (Table 1). The collected specimens were carefully examined for taxonomic identification. The Flora Iranica (Polatschek and Rechinger 1968), the Flora of Iran (Yousef Naanaie 2010), and the Flora of Turkey (Davis 1988) were the primary sources for species identification. The names of the species were matched with the online site POWO (2024) (Plants of the World Online) at https://powo.science.kew.org/. The contradictions observed concerning the scientific names of some species are presented in the appendix. Additionally, sequence information from previous studies involving ten taxa for the trnH-psbA region and 21 taxa for the nrDNA ITS region was utilized from the GenBank for comparative analysis of Iranian species (Table 1). As outgroups, Ludwigia grandiflora (Michx.) Greuter & Burdet and Lu. peruviana (L.) H. Hara were employed.

Table 1

Plant species examined in this research, along with their corresponding herbarium detail and GenBank accession numbers.
Species	DNA/Sequence source	GenBank accession no. nrDNA ITS/trnH-psbA
A. verticillata (Ard.) Lam.	Lorestan: Khorramabad, Cham-Divan, 1000 m, Veise Karami, 24111 (TUH).
A. auriculata Willd.	Lorestan: Khorramabad, Cham-Divan, 1000 m, Veise Karami, 24108 (TUH).
	GenBank	MH808727.1/ -
A. baccifera L.	Gilan: Rudbar, Rahimabad, Reshterud Village, 211 m, Ashouri, 1396, 8554 (GUH).
A. coccinea Rottb.	GenBank	MG237340.1/ DQ006199.1
A. multiflora Roxb.	Gilan: Someh Sara, Hendekhaleh District, Nokhaleh Akbari Village, 21 m, Ashouri, 1396, 8553 (GUH).
	GenBank	MF599386.1/ -
Duabanga grandiflora (Roxb. ex DC.) Walp.	GenBank	AF163695.1/ KR532980.1
Lawsonia inermis L.	GenBank	KF850586.1/ GQ435226.1
Ludwigia peruviana (L.) H. Hara	GenBank	KX168366.1/ GU135322.2
Lu. grandiflora (Michx.) Greuter & Burdet	GenBank	KX168323.1/ KC996841.1
Lythrum silenoides Boiss. & Noë	Lorestan: Khorramabad, Cham-e Divan, elevation 1000 meters, Veise Karami, 1998, 24113 and 1-24113 (TUH).
L. virgatum L.	West Azerbaijan: between Salmas and Qushchi, Khan Takhti, 1400 m, Ghahreman and Mozaffarian, 1372, 17444 (TUH).
	GenBank	MG975397.1/ -
L. salicaria L.	GenBank	MK895653.1/ KC584962.1
L. junceum Banks & Sol.	Kohgiluyeh and Boyer-Ahmad: Dogonbadan, Belgheis spring, 1400 m, Ghahreman, Attar, and Sheikhi, 1375, 20328 (TUH).
	GenBank	MG975401.1/ -
L. hyssopifolia L.	Gilan: Langarud, Chamkhaleh, 25 m, Naqinezhad, 1378, 21459 (TUH).
	GenBank	MG975399.1/ -
L. thymifolia L.	Gilan: Bandar Anzali, elevation 20 m, Mozaffarian and Maassoumi, 6901 (TUH).
	GenBank	MG975400.1/ -
L. portula (L.) D. A. Webb.	Morocco: Tétouan, 9 kilometers southwest of Souk-e-Araba-Ayacha, 120 m, Podlech, 1365, 43652 (MSB).
	GenBank	MG237615.1/ -
L. thesioides M. Bieb.	Fars: 6 kilometers south of Jahrom, between Hood and the village of Kooreh, 800 m, Assadi and Akhani, 61867 (TARI).
	GenBank	MH245820.1/ -
L. tribracteatum Salzm. ex Spreng.	Fars: 30 kilometers south of Jahrom, near the village of Harm, at an approximate elevation of 800 m, Assadi and Akhani, 61852 (TARI).
	GenBank	MG975398.1/ -
Punica granatum L.	GenBank	AY035761.1/ GQ435338.1
Rotala indica (Willd.) Koehne	Gilan: Siahkal, Khararud District, Salash Village, 55 m, Ashouri, 1396, 8555 (GUH).
R. rotundifolia (Buch.-Ham. ex Roxb.) Koehne	GenBank	MH071602.1/ GU135292.2
Sonneratia alba Sm.	GenBank	AF163701.1/ MH583031.1
Trapa natans L.	GenBank	KX098577.1/ KP280472.1

DNA extraction and Sanger sequencing

The total DNA was extracted from fresh and dried leaf tissues using the 2 x CTAB procedure developed by Doyle & Doyle (1987) and the DNeasy Plant Mini Kit (Qiagen Valencia, CA, USA) following the instructions specified by the manufacturer. We opted for plastid psbA-trnH and nuclear ribosomal ITS markers for our molecular investigations. The psbA-trnH intergenic spacer region was amplified using primers trnHf-05 and psbA3- f, developed by (Tate and Simpson 2003) and Sang et al. (1997), respectively. PCR reactions were conducted in a 25 µL volume, comprising 9.5 µL deionized water, 12.5 µL of 2x Taq DNA polymerase master mix RED (Amplicon, Cat No. 180301), 1 µL of each primer (10 pmol/µL), and 1 µL of template DNA (20 ng/µL). For the psbA-trnH intergenic spacer region, PCR conditions consisted of an initial denaturation step at 93°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 48–56°C for 45 seconds, extension at 72°C for 90 seconds, and a final extension at 72°C for 5 minutes. Sequencing was performed on an ABI 3730xl capillary sequencer (Thermo Fisher Scientific, Waltham, USA; Applied Biosystems, USA) through NiaGen (Tehran) company, following the manufacturers’ protocols, using the same primers as employed for PCR amplification.

Phylogenetic analyses

Sequences were aligned using MAFFT (https://ngphylogeny.fr), with subsequent minor manual adjustments. Phylogenetic relationships were analyzed using maximum parsimony (MP), maximum likelihood (ML) methods, and Bayesian inference (BI). Maximum parsimony analysis was performed using PAUP* 4.b10 software (Swofford 2002), Bayesian inference was conducted using MrBayes on the CIPRES (Miller et al. 2010) gateway, and maximum likelihood analysis was carried out on http://iqtree.cibiv.univie.ac.at. In the maximum parsimony method, the heuristic search option was employed, conducting 1,000 replications of random addition sequences with ten trees retained at each step. Tree bisection-reconnection (TBR) branch swapping was utilized, with Mul-Trees enabled and steepest descent disabled. Branch support was evaluated using 1,000 bootstrap replicates, providing bootstrap percentages (bootstrap support, BS), following the same settings as those used for heuristic searches (Felsenstein 1985). The rescaled consistency index (RC), consistency index (CI), and retention index (RI) were calculated using PAUP* 4.b10 software (Swofford 2002) to assess tree quality. To determine the appropriate evolutionary model for the data, the web server http://iqtree.cibiv.univie.ac.at/ was utilized, and based on the Akaike information criterion (AIC), the TVM + F + I model was selected for cpDNA trnH-psbA data, the GTR + F + G4 model for nrDNA ITS and combined data.

Bayesian analyses were conducted with the following configurations: Four Markov chain Monte Carlo heuristic searches, each comprising 10 million generations, with tree sampling every 1,000 generations. We ensured the convergence of parameter estimates and effective sample sizes exceeding 200 for all parameters using Tracer version 1.6 (Rambaut et al. 2014). The initial 25% of trees were discarded as burn-in. Posterior probabilities (PP) were employed to demonstrate node support. The remaining trees were utilized to create a consensus tree representing the majority agreement (50% threshold) and visualized using TreeView v. 1.6.1 (Page 2001).

The remaining trees were utilized to create a consensus tree representing the majority agreement (50% threshold) and viewed using TreeView v. 1.6.1 (Page 2001).

Phylogenetic analyses conducted on the three datasets using maximum parsimony, maximum likelihood methods, and Bayesian inference generally produced congruent tree topologies. Combined data tree enhancing both resolution and support for nodes compared to the individual nrDNA ITS and trnH-psbA trees. Given the absence of discrepancies among analyses, we will exclusively focus on describing and discussing the maximum likelihood tree derived from the nrDNA ITS, trnH-psbA, and combined datasets. Bootstrap values and posterior probabilities are displayed along the branches in Figs. 1–3.

Cladistic analysis of trnH-psbA intergenic spacer data

On the maximum likelihood tree, the main node A is placed with high support (MP BS = 100; ML BS = 100; PP = 1). Lythrum species are in the lower, distinct clades, forming a non-monophyletic group. However, these clades lack strong support. The two species L. virgatum L. and L. salicaria L. diverged first from the other species in the group (MP BS = 80; ML BS = 78; PP = 0.87). Following this, the remaining species are observed on branches A1 (above) and A2 (below). A2, which holds the species L. thesioides M. Bieb. and L. silenoides Boiss. & Noë (MP BS = 78; ML BS = 87; PP = 0.93), while A1, a branch with low support, contains Rotala and Ammannia species, species S. alba Sm., T. natans L., P. granatum L., and La. inermis. Additionally, the genera Ammannia form a monophyletic group (MP BS = 81; ML BS = 85; PP = 0.97) (Fig. 1).

Cladistic analysis of nrDNA ITS data

On the maximum likelihood tree based on nrDNA ITS data, the Lythraceae family is monophyletic (node A) with high support (MP BS = 100; ML BS = 100; PP = 1). The relationship among genera was unresolved, and placed as polytomy in three sub-clades, A1, A2, and A3. Sub-clade A1 includes all the examined Lythrum species in the "Lythrum" clade, among which three species, L. hyssopifolia L., L. junceum Banks & Sol., and L. thymifolia L., along with two species, L. virgatum and L. salicaria, are placed as monophyletic groups with high support (MP BS = 97; ML BS = 96; PP = 1). The sub-clade A2, includes Ammannia species the "Ammannia" clade with high support (MP BS= -; ML BS = 100; PP = 1), along with La. inermis, D. grandiflora (Roxb. ex DC.) Walp., S. alba, and T. natans. The sub-clade A3, also with weak support (MP BS= -; ML BS = 43; PP= -), comprises the "Rotala" clade (R. indica, R. rotundifolia (Buch.-Ham. ex Roxb.) Koehne) (MP BS = 100; ML BS = 100; PP = 1), and P. granatum (Fig. 2).

Cladistic analysis of combined data

On the maximum likelihood phylogenetic tree based on combined data, the main node A is observed with high support (MP BS = 100; ML BS = 100; PP = 1), which includes two clades, A1 and A2. Clade A1 further divides into two sub-clades, A1a and A1b. A1a, or the "Rotala" clade, harbors two species, R. indica and R. rotundifolia, forming a monophyletic group (MP BS = 100; ML BS = 100; PP = 1), along with P. granatum on a separate branch. Additionally, three species, T. natans, S. alba (in a small subgroup), and D. grandiflora (on a separate branch), are situated in this clade (MP BS= -; ML BS = 99; PP = 1).

A1b carries the species of Ammannia in the "Ammannia" clade (MP BS= -; ML BS = 100; PP = 1), along with La. inermis (MP BS = 100; ML BS = 73; PP = 0.93). The species of Lythrum are placed in subgroup A2 or the "Lythrum" clade (MP BS = 98; ML BS = 95; PP = 1). Among them, two species, L. silenoides and L. thesioides, are grouped in subgroup A2b (MP BS = 97; ML BS = 97; PP = 0.93), while three species, L. hyssopifolia, L. junceum, and L. thymifolia, are placed in subgroup A2a (MP BS = 92; ML BS = 100; PP = 1). Finally, two species, L. virgatum and L. salicaria, are clustered together (MP BS = 99; ML BS = 100; PP = 1) (Fig. 3).

The reconstruction of phylogenetic trees based on cpDNA, nrDNA ITS, and combined sequence data using maximum parsimony, Bayesian, and maximum likelihood methods revealed in most cases (except for the cpDNA ML tree) the clear delineation of the monophyly of three genera, Lythrum, Ammannia and Rotala in Iran. In all the resulting trees, the species of these three genera were separated entirely into different groups. The results of this research are supported by previous studies (Graham et al. 2005; Morris 2007). The species of the genus Lythrum showed a monophyletic relationship (except for the cpDNA trnH-psbA data). The genus is identified by its alternate upper leaves, spike-like inflorescence, and multi-flowered or solitary cymes (Yousef Naanaie 2010). In all trees resulting from this study, the species L. virgatum and L. salicaria were consistently grouped in one clade. These two species also formed a monophyletic group in the study by Morris (2007). Koehne (1903) classified these two species under the subgenus Salicaria due to their inflorescence type and heterostyly. Cytogenetic studies have shown that these two species have a higher ploidy level than other species in this genus (n = 5, 10). The basic chromosome number is reported to be n = 15 in L. virgatum and n = 15, 25, and 30 in L. salicaria (Graham and Cavalcanti 2001). L. virgatum and L. salicaria are perennial hers with similar morphological features such as up to 8 axillary flowers, winged-quadrangular stem, sessile leaves, 12 stamens, heterostyle, and capitate stigma (Yousef Naanaie 2010). The association between the two species is strongly supported by the shared features of leaf micromorphology (such as the width of stomata and outer, pristomata, and inner stomata rim type) and palynology (including almost similar colpi width, outline of pollen grains from equatorial and polar views, colpi and pseudocolpi membrane and exine sculpturing) (Mahmoodi et al. 2022a, b)

L. thesioides and L. silenoides were also united in chloroplast and combined trees. They are annual species with 2 to 8 stamens (Yousef Naanaie 2010), showing some affinities in terms of leaf micro morphological (both have glabrous leaves, raised outer periclinal layer, sinuolate-erose inner stomatal rim) and palynological features (such as the pollen II type, colpi, and pseudocolpi membranes micro-verrucate to micro-baculate, colpi longer than pseudocolpi) which is consistent with the results of the phylogeny.

The three species, L. hyssopifolia, L. thymifolia, and L. junceum, formed a monophyletic group on nrDNA ITS and combined trees. These three species have common morphological characteristics (including solitary flowers, hypanthium at least 3 mm long, cylindrical at the fruiting stage) (Polatschek and Rechinger 1968; Yousef Naanaie 2010), which is supported by leaf micro-morphological (such as depressed outer periclinal layer, overlapping-stout pristomata and sinuolate inner stomata rim) and palynological characters (e.g. colpi and pseudocolpi membrane micro-verrucate to micro-baculate) (Mahmoodi et al. 2022a, b). Among them, L. hyssopifolia and L. junceum formed a monophyletic group in the previous phylogenetic study by Morris et al. (2007). The morphological and cytological findings agreed with the molecular data. Koehne (1903) placed these two species close to the Eurasian species Lythrum by having one or two flowers in the axis, a thick nectar ring in the ovary and relatively long style.

L. hyssopifolia (n = 10) is an annual herb with an erect ascending stem, homomorphic flowers, 2–6 stamens, capsule shorter than hypanthium (Koehne 1903; Webb 1967; Yousef Naanaie 2010), It is widespread in seasonally moist habitats in southern parts of Eurasia and has become invasive in many parts of the world. L. junceum (n = 5) is a biennial and perennial plant characterized by a decumbent stem, tristylous flowers, 12 stamens, and a corolla tube length of 5–6 mm. It is distributed in southern Europe and Central Asia (Polatschek and Rechinger 1968; Morris 2007). While, L. thymifolia is an annual plant with homomorphic flowers, 2–6 stamens, and a short corolla length; distinguished from L. hyssopifolia by its hypanthium and corolla length (Polatschek and Rechinger 1968).

Based on the present findings, L. portula (L.) D. A. Webb. appeared in an independent clade, and sister group relationship with other species (L. salicaria, L. virgatum, L. thymifolia, L. Junceum, L. hyssopifolia, and L. tribracteatum) of Lythrum in the tree derived ITS and combined datasets. It is an annual herbaceous plant, hairless, with a sloping creeping stem, roots at the nodes, opposite leaves, short and weak petioles, oval or almost round, full, partly succulent, and rounded apex; native to Europe, and occurs in western Asia, often growing in wet habitats such as marshes (Webb et al. 1988; Johnson and Brooke 1989).

The species of Ammannia demonstrated monophyletic groups on all trees. Three species of A. auriculata Willd., A. coccinea, and A. multiflora Roxb. in cpDNA trees and four species of A. auriculata with A. baccifera L. and A. multiflora with A. coccinea in combined trees united in small groups.

According to Graham's classification (1985), species of Ammannia were primarily divided into two subgenera, Ammannia and Cryptotheca (Blume) Koehne, two sections and four series based on style length, style to carpel length ratio, presence or absence of petal, and petal color. Species such as A. baccifera were placed in the section Ammannia (having style approximately 5.0 mm or less), while A. auriculata and A. coccinea were placed in the Eustylia section (having style approximately 3.0-5.1 mm or less). Additionally, in the Flora Iranica (Polatschek and Rechinger 1968) and Flora of Iran (Yousef Naanaie 2010), based on style length and inflorescence type, A. auriculata was classified with A. multiflora (due to style length of 1.5-3.0 mm), and A. baccifera with A. verticillata (Ard.) Lam. (with shorter style, 0.25–0.5 mm). However, in the current study A. baccifera grouped with A. auriculata and A. multiflora with A. coccinea (Figs. 2 and 3) formed monophyletic groups. This indicates that the phylogenetic reconstruction of Ammannia species in this study does not align with previous classifications based on morphological characteristics and suggests a need for reconsideration in the classification of species within this genus. The grouping of A. baccifera and A. auriculata is further supported by common features in morphology, anatomy, and palynology. These two annual herbaceous species are distinguished by their erect, glabrous stem; opposite, acute leaves; cyme inflorescence, campanulate hypanthium during flowering, four triangular sepals, bracteoles shorter than the floral tube, stamens as long as hypanthium, spherical ovaries, capitate stigma, and spherical capsules (Haining et al. 2007; Yousef Naanaie 2010). They exhibit similarities in leaf micro-morphological features (such as stomatal size and inner stomata rim shape) (Mahmoodi et al., 2022a) and pollen morphology (pollen grain outline from equatorial view, width of colpi and pseudocolpi close to each other as well as the colpi longer than pseudocolpi) (Mahmoodi et al., 2022b). Furthermore, the close affinity between the two species, A. multiflora and A. coccinea, is supported by their morphological similarities. Both are herbaceous, annual species, with opposite, linear or lanceolate leaves, cyme inflorescence, and 4 stamens.

The phylogenetic tree reconstructed in the present study also indicates the sister relationship between La. inermis and the species of Ammannia. La. inermis is glabrous shrub or small tree 2–6 m tall native of North Africa and southwest Asia (Yadav et al. 2013). It is cultivated and occurs naturally in western and southern Iran. Species of Rotala, including R. indica and R. rotundifolia, formed a monophyletic group on all obtained trees (the Rotala clade). This relationship is supported by several morphological features (such as both are annual herbs with creeping stems, opposite, ovoid leaves, four-part flower tube, tetrameric sepals and petals, without epicalyx (Haining et al. 2007). Previous molecular studies (Graham et al. 2005) also support this grouping.

Character evolution in Lythraceae in Iran

The evolutionary trend of some morphological (leaf phyllotaxy, inflorescence type), leaf micro morphological (epicuticular wax type), and palynological features (number of pseudocolpi, colpi and pseudocolpi membrane type, and exine sculpturing type) was traced on the ML tree using combined data sets (Fig. 4). These morphological features are taxonomically informative and used for genus classification (Polatschek and Rechinger 1968; Yousef Naanaie 2010). Opposite upper leaves, solitary flower or in cyme inflorescence, leaf with V to IX epicuticular wax ornamentation types; pollen grains with six pseudocolpi, pseudocolpi membrane either micro-verrucate to micro-baculate or psilate, exine sculpturing striate and rugulate are synapomorphy for the species of Ammannia and Rotala. These traits have evolved from symplesiomorphic traits such as alternate upper leaves, flower arranged in terminal spike-like inflorescences, multi-flowered cymes, or solitary flower; leaf with type I to IV epicuticular wax ornamentation, pollen grains with three pseudocolpi, pseudocolpi membrane micro-verrucate to micro-baculate, exine sculpturing striate in the genus Lythrum. Among these, Rotala species, which have leaf with V and VII sculpturing type; pollen with micro-echinate colpus membrane and psilate pseudocolpi membrane, rugulate exine sculpturing are positioned at the most advanced position on terminal clade (In ML combined tree).

In this study, a comprehensive phylogeny of the family Lythraceae, focusing on the three genera Lythrum, Ammannia, and Rotala, was presented for the first time. Phylogenetic analyses using cpDNA trnH-psbA, nrDNA ITS, and combined data of maximum parsimony, Bayesian inference, and maximum likelihood revealed monophyly of the three genera Lythrum, Ammannia, and Rotala (except for trnH-psbA tree), and species of these three genera were separated entirely into distinct clades (except for ITS tree). Based on the present molecular phylogenetic results, species such as L. thesioides with L. silenoides; L. salicaria with L. virgatum; and the three species L. thymifolia, L. junceum, and L. hyssopifolia formed monophyletic groups and were considered closely related species. Therefore, a revision of the classification of species within these genera is necessary based on the obtained results. In addition, a monophyletic relationship between A. coccinea and A. multiflora, as well as between A. baccifera and A. auriculata, was evident. It was also found that reliance on morphological traits, especially style length, which has been used in previous classifications of species within this genus, is unreliable.

Conflict of interest

The authors do not have any conflicts of interest to declare that are relevant to the content of this article.

Author Contribution

R. Mahmoodi: Data curation. M.B. Faghir: Project conceptualization, formal analysis, manuscript review, and editing. R. Shahi Shavvon: Formal analysis, original draft writing, manuscript review, and editing.

Acknowledgement

We extend our gratitude to all colleagues who supported us in this project, particularly Dr. F. Attar from the herbarium of the University of Tehran (TUH), for supplying the herbarium specimens.

Data Availability

The datasets generated during the current study are available in the GenBank repository.

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No competing interests reported.

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Phylogenetic study of Lythraceae family in Iran, focusing on the genera Lythrum, Ammannia, and Rotala

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Abstract

Figures

Introduction

Material and Methods

Plant Sampling

DNA extraction and Sanger sequencing

Phylogenetic analyses

Results

Cladistic analysis of nrDNA ITS data

Cladistic analysis of combined data

Discussion

Character evolution in Lythraceae in Iran

Conclusion

Declarations

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

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