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

Phylogenetic study of Lythraceae family in Iran, focusing on the genera Lythrum, Ammannia, and Rotala | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Phylogenetic study of Lythraceae family in Iran, focusing on the genera Lythrum, Ammannia, and Rotala Rana Mahmoodi, Marzieh Beygom Faghir, Robabeh Shahi Shavvon This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4612594/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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 trn H -psb A, 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 Lythrum species (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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction 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 rbc L 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 atp B- rbc L, and trn K- mat K, along with previously sequenced data from trn L- trn F, rbc L, psb A- ycf 3, 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. Material and Methods 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 trn H- psb A 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/ trn H- psb A 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 psb A- trn H and nuclear ribosomal ITS markers for our molecular investigations. The psb A- trn H intergenic spacer region was amplified using primers trn Hf-05 and psb A3- 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 psb A- trn H 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 trn H- psb A 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 ). Results 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 trn H- psb A 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, trn H- psb A, and combined datasets. Bootstrap values and posterior probabilities are displayed along the branches in Figs. 1 – 3 . Cladistic analysis of trn H- psb A 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 ). Discussion 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 trn H- psb A 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 g labrous 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). Conclusion 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 trn H -psb A, 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 trn H -psb A 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. Declarations 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. References APG IV (2016) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Bot J Linn 181:1–20. https://doi.org/10.1111/boj.12385 Conti E, Litt A, Wilson PG, et al (1997) Interfamilial relationships in Myrtales: Molecular phylogeny and patterns of morphological evolution. Syst Bot 22:629. https://doi.org/10.2307/2419432 Cronquist A (1982) An integrated system of classification of flowering plants. Columbia University Press, New York, USA Dahlgren R, Thorne RF (1984) The order Myrtales: circumscription, variation, and relationships. Ann Missouri Bot Gard 71:633. https://doi.org/10.2307/2399158 Davis PH (1988) Flora of Turkey and the East Aegean Islands. Edinburgh University Press, Edinburgh Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19:11–15 Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution (NY) 39:783. https://doi.org/10.2307/2408678 Graham S. A. (2007) Lythraceae. In: Kubitzki K (ed) Flowering Plants Eudicots. The families and genera of vascular plants. Springer, Berlin Graham SA (1985) A revision of Ammannia (Lythraceae) in the Western Hemisphere. J Arnold Arbor 66:395–420. https://doi.org/10.5962/bhl.part.13185 Graham SA, Cavalcanti TB (2001) New Chromosome counts in the Lythraceae and a review of chromosome numbers in the family. Syst Bot 26:445–458 Graham SA, Hall J, Sytsma K, Shi S (2005) Phylogenetic analysis of the Lythraceae based on four gene regions and morphology. Int J Plant Sci 166:995–1017. https://doi.org/10.1086/432631 Gu C, Ma L, Wu Z, et al (2019) Comparative analyses of chloroplast genomes from 22 Lythraceae species: inferences for phylogenetic relationships and genome evolution within Myrtales. BMC Plant Biol 19:281. https://doi.org/10.1186/s12870-019-1870-3 Haining HN, Graham S, Gilbert MG (2007) Lythraceae. In: Wu ZY (ed) Flora of China. Science Publishers, Beijing, China Huang Y, Shi S (2002) Phylogenetics of Lythraceae sensu lato: A preliminary analysis based on chloroplast rbcL gene, psaA-ycf3 spacer and nuclear rDNA internal transcribed spacer (ITS). Int J Plant Sci 163:215–225. https://doi.org/10.1086/338392 Inglis PW, Cavalcanti TB, Facco MG, et al (2023) A comprehensive genus-level phylogeny and biogeographical history of the Lythraceae based on whole plastome sequences. Ann Bot 132:293–318. https://doi.org/10.1093/aob/mcad091 Johnson LAS, Briggs BG (1984) Myrtales and Myrtaceae-A phylogenetic analysis. Ann Missouri Bot Gard 71:700. https://doi.org/10.2307/2399159 Johnson PN, Brooke PA (1989) Wetland plants in New Zealand. DSIR Publishing, Wellington. Koehne E (1903) Lythraceae. In: Engler A (ed) Das Pflanzenreich IV. 216, Heft 17, W. Engelmann, Germany. Koehne E (1881) Lythraceae monographice describuntur. Botanische Jahrbucher fur Systematik, Pfl anzengeschichte und Pfl anzengeographie 1:142–157 Mahmoodi R, Faghir MB, Parsapanah S (2022a) Palynological study of the family Lythraceae J.St.‐Hil. in Iran; with special emphasis on the genera Ammannia , Lythrum , and Rotala . Feddes Repert 133:289–304. https://doi.org/10.1002/fedr.202100052 Mahmoodi R, Faghir MB, Shahi Shavvon R (2022b) Foliar micromorphology of the family Lythraceae in Iran with special emphasis on the genera Lythrum , Ammannia , and Rotala . Rostaniha 23:161–179 Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: in Proceedings of the Gateway Computing Environments Workshop (GCE). New Orleans, pp 1–8 Morris JA (2007) A molecular Phylogeny of Lythrum (Lythraceae): Preliminary analyses based on the atpB-rbcL Intergenic Spacer and ITS. A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Naqinezhad A, Naseri Larijani N (2017) Ammannia coccinea (Lythraceae), a new record for the Flora Iranica area. Phytol Balc (Sofia) 23:35–38 Page DM (2001) TreeView (Win32). Retrieved from http://taxonomy.zoology.gla.ac.uk/rod Polatschek A, Rechinger KH (1968) Lythraceae. In: Rechinger KH (ed) Flora Iranica. Akad." Druck-und Verlagsanstalt, Graz POWO (2024) Plants of the World Online. In: Published on the Internet; http://www.plantsoftheworldonline.org/. Retrieved 24 May 2024. Rambaut A, Suchard MA, Xie D, Drummond AJ (2014) Tracer v1.6. http://beast.bio.ed.ac.uk/Tracer Sang T, Crawford DJ, Stuessy TF (1997) Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). Am J Bot 84:1120–1136. https://doi.org/10.2307/2446155 Shi S, Huang Y, Tan F, et al (2000) Phylogenetic analysis of the Sonneratiaceae and its relationship to Lythraceae based on ITS sequences of nrDNA. J Plant Res 113:253–258. https://doi.org/10.1007/PL00013926 Swofford DL (2002) PAUP*, Phylogenetic Analysis Using Parsimony, version 4.0b10 Sytsma KJ, Litt A, Zjhra ML, et al (2004) Clades, clocks, and continents: Historical and biogeographical analysis of Myrtaceae, Vochysiaceae, and relatives in the Southern hemisphere. Int J Plant Sci 165: S85–S105. https://doi.org/10.1086/421066 Takhtajan A (1987) System of Magnoliophyta. Academy of Sciences USSR, Leningrad Tate JA, Simpson BB (2003) Paraphyly of Tarasa (Malvaceae) and diverse origins of the polyploid species. Syst Bot 28:723–737 Webb CJ, Sykes WR, Garnock-Jones PJ (1988) Flora of New Zealand. Naturalised Pteridophytes, Gymnosperms, Dicotyledons. Botany Division DSIR, Christchurch Webb DA (1967) Lythraceae. In: Tutin TG (ed) Flora Europaea. Cambridge University Press, Cambridge, pp 300–303 Yadav S, Kumar A, Dora J, Kumar A (2013) Essential perspectives of Lawsonia inermis . Journal of Pharmaceutical and Chemical Sciences 2:888–896 Yousef Naanaie S (2010) Lythraceae. In: Assadi M, Maassoumi A. A., Babakhanlou P, Mozaffarian V (eds) Flora of Iran. Research Institute of Forests and Rangelands, Tehran Additional Declarations No competing interests reported. Supplementary Files Appendix.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4612594","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322020746,"identity":"f0ff2a19-122b-4b38-bc81-1674bb36d8c8","order_by":0,"name":"Rana Mahmoodi","email":"","orcid":"","institution":"University of Guilan","correspondingAuthor":false,"prefix":"","firstName":"Rana","middleName":"","lastName":"Mahmoodi","suffix":""},{"id":322020747,"identity":"5ffb9fc2-35e6-4cd2-836d-781553308626","order_by":1,"name":"Marzieh Beygom Faghir","email":"","orcid":"","institution":"University of Guilan","correspondingAuthor":false,"prefix":"","firstName":"Marzieh","middleName":"Beygom","lastName":"Faghir","suffix":""},{"id":322020748,"identity":"c3559324-bb22-47c8-9dcc-391b3aa17405","order_by":2,"name":"Robabeh Shahi Shavvon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYHACNgbGBgk5KEeCeC3GJGthSGwg2lXmDezPHvzcYZG+4UYC44cfDBb5BLXIHOAxN+w9I5EL1MIs2cMgYUnQOgkGHjYJ3jawFgZpIN+AoC0SDOzPJP+2SaQbAG35TaQWBjNpoC0JQC1sRNrCzGMmLdsmYTjzzMM2yx4DYrSwtz+TfNtWJ893PPnwjR8VdYS1MDBDaYUDwNhhIEIDAsg3kKJ6FIyCUTAKRhQAAJSzMAHF16K6AAAAAElFTkSuQmCC","orcid":"","institution":"Yasouj University","correspondingAuthor":true,"prefix":"","firstName":"Robabeh","middleName":"Shahi","lastName":"Shavvon","suffix":""}],"badges":[],"createdAt":"2024-06-20 14:54:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4612594/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4612594/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60179759,"identity":"6216a50f-a85d-419c-a770-76386365ed85","added_by":"auto","created_at":"2024-07-12 17:07:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":45249,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree inferred from \u003cem\u003etrn\u003c/em\u003eH-\u003cem\u003epsb\u003c/em\u003eA data of Lythraceae species. Bootstrap support from the maximum parsimony and the maximum likelihood analyses higher than 50% and posterior probability of Bayesian inference higher than 0.5 are indicated above branches, respectively.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4612594/v1/3a8568f594e3ccbdf023b8b3.jpg"},{"id":60180557,"identity":"9ab578e7-3973-4c69-b0f8-710f4f020bbe","added_by":"auto","created_at":"2024-07-12 17:23:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56876,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree inferred from ITS data of Lythraceae species. Bootstrap support from the maximum parsimony and the maximum likelihood analyses higher than 50% and posterior probability of Bayesian inference higher than 0.5 are indicated above branches, respectively.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4612594/v1/3eb1205e4e02ad9d1a56e324.jpg"},{"id":60180242,"identity":"ff89271f-31b9-4a3e-954a-9b9fb0f35510","added_by":"auto","created_at":"2024-07-12 17:15:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63909,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree inferred from combined data of Lythraceae species. Bootstrap support from the maximum parsimony and the maximum likelihood analyses higher than 50% and posterior probability of Bayesian inference higher than 0.5 are indicated above branches, respectively.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4612594/v1/0d29b6ab248d79284159358c.jpg"},{"id":60179760,"identity":"2afebf13-fe24-46cb-a600-8d80a7c198c7","added_by":"auto","created_at":"2024-07-12 17:07:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":96668,"visible":true,"origin":"","legend":"\u003cp\u003eCharacter reconstruction (including leaf epicuticular wax ornamentation, number of pseudocolpi, pseudocolpi membrane and exine sculptures, inflorescence type, and leaf arrangement) on the ML tree topology.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4612594/v1/aa1e9c5dc8d2a17abbe2ba9e.jpg"},{"id":62511960,"identity":"099c79ca-3b90-462b-8ac3-dadb945efe6f","added_by":"auto","created_at":"2024-08-15 07:01:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":864990,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4612594/v1/d08f6067-17be-47af-97a4-b7c1b2144182.pdf"},{"id":60179758,"identity":"f88a5795-eec1-43a5-b4d5-2c74cd2fe81d","added_by":"auto","created_at":"2024-07-12 17:07:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15327,"visible":true,"origin":"","legend":"","description":"","filename":"Appendix.docx","url":"https://assets-eu.researchsquare.com/files/rs-4612594/v1/fa64bb1d949b5f66977e0ea5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phylogenetic study of Lythraceae family in Iran, focusing on the genera Lythrum, Ammannia, and Rotala","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe family Lythraceae, with approximately 28 genera and 700 species (POWO \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), belongs to the order Myrtales, within Eudicots, in the Malvids clade, forming a monophyletic group with Geraniales (APG IV \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Phylogenetic, morphological, and embryological evidence suggests a close relationship between Lythraceae and Onagraceae (Dahlgren and Thorne \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Johnson and Briggs \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Molecular analyses also support the affinity between these two families (Conti et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Sytsma et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; APG IV \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Fossil evidence indicates that the Lythraceae family originated in the late Cretaceous to early Paleocene period (Graham et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Nevertheless, molecular evidence suggests that the divergence of this family from Myrtales occurred earlier (Sytsma et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). 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. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). 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 \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1903\u003c/span\u003e; Cronquist \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Dahlgren and Thorne \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Johnson and Briggs \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Takhtajan \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). According to Inglis et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the main centers of Lythraceae species richness are the mountains in western and southern Mexico, as well as the Espinha\u0026ccedil;o mountain range and central plateau within the Cerrado biome of eastern Brazil.\u003c/p\u003e \u003cp\u003eFurthermore, the greatest genus-level diversity was identified in central and eastern Brazil, the southeastern USA, and mountainous regions of Colombia (Inglis et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe 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 (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1881\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1903\u003c/span\u003e), 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 \u0026amp; Thorne (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1984\u003c/span\u003e) suggested that the genera of these three families be considered as subfamilies of Lythraceae. Johnson \u0026amp; Briggs (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1984\u003c/span\u003e) 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.\u003c/p\u003e \u003cp\u003eConti et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) evaluated chloroplast \u003cem\u003erbc\u003c/em\u003eL gene sequences for several genera of the order Myrtales, including \u003cem\u003eTrapa\u003c/em\u003e, 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. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and Huang \u0026amp; Shi (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), as well as Graham et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Additionally, Morris (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) conducted analyses on molecular data from two chloroplast regions, specifically \u003cem\u003eatp\u003c/em\u003eB-\u003cem\u003erbc\u003c/em\u003eL, and \u003cem\u003etrn\u003c/em\u003eK-\u003cem\u003emat\u003c/em\u003eK, along with previously sequenced data from \u003cem\u003etrn\u003c/em\u003eL-\u003cem\u003etrn\u003c/em\u003eF, \u003cem\u003erbc\u003c/em\u003eL, \u003cem\u003epsb\u003c/em\u003eA-\u003cem\u003eycf\u003c/em\u003e3, 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.\u003c/p\u003e \u003cp\u003eIn the study by Gu et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), 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.\u003c/p\u003e \u003cp\u003e(Polatschek and Rechinger \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1968\u003c/span\u003e) introduced the genera and species of this family in the Flora Iranica, listing five genera: \u003cem\u003eLawsonia\u003c/em\u003e L., \u003cem\u003eLythrum\u003c/em\u003e L., \u003cem\u003eAmmannia\u003c/em\u003e L., \u003cem\u003eRotala\u003c/em\u003e L., and \u003cem\u003ePeplis\u003c/em\u003e L. \u003cem\u003eLawsonia\u003c/em\u003e, \u003cem\u003eRotala\u003c/em\u003e, and \u003cem\u003ePeplis\u003c/em\u003e were each represented by one species, while \u003cem\u003eLythrum\u003c/em\u003e and \u003cem\u003eAmmannia\u003c/em\u003e encompass nine and four species, respectively. Diversity center of the \u003cem\u003eAmmannia\u003c/em\u003e, \u003cem\u003eLythrum\u003c/em\u003e, and \u003cem\u003eRotala\u003c/em\u003e is in the Old-World (Graham, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In the flora of Iran, four genera of this family are mentioned, \u003cem\u003eLawsonia\u003c/em\u003e, \u003cem\u003eLythrum\u003c/em\u003e, \u003cem\u003eAmmannia\u003c/em\u003e, and \u003cem\u003eRotala\u003c/em\u003e. \u003cem\u003eLawsonia\u003c/em\u003e has one species, \u003cem\u003eLa. inermis\u003c/em\u003e L., cultivated in the western and southern regions of Iran. \u003cem\u003eLythrum\u003c/em\u003e and \u003cem\u003eAmmannia\u003c/em\u003e include seven and four species, respectively, scattered in different parts of Iran (Yousef Naanaie \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). A new species of \u003cem\u003eAmmannia\u003c/em\u003e, named \u003cem\u003eA. coccinea\u003c/em\u003e Rottb., was reported from Guilan Province by Naqinezhad \u0026amp; Naseri Larijani (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Only one species, \u003cem\u003eR\u003c/em\u003e. \u003cem\u003eindica\u003c/em\u003e (Willd.) Koehne, is reported from northern Iran in the \u003cem\u003eRotala\u003c/em\u003e genus. Additionally, two species, \u003cem\u003eRotala densiflora\u003c/em\u003e (Roth) Koehne and \u003cem\u003ePeplis hyrcanica\u003c/em\u003e Sosn., which were listed in the Flora Iranica by Polatschek \u0026amp; Rechinger (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1968\u003c/span\u003e), 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 (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), there is no evidence to support the presence of these species in natural environments within Iran.\u003c/p\u003e \u003cp\u003eThe 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 \u003cem\u003eLythrum\u003c/em\u003e, \u003cem\u003eAmmannia\u003c/em\u003e, and \u003cem\u003eRotala\u003c/em\u003e 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.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Sampling\u003c/h2\u003e \u003cp\u003eIn this study, 13 species of the Lythraceae family growing in Iran, along with the species \u003cem\u003eL. portula\u003c/em\u003e 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\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The collected specimens were carefully examined for taxonomic identification. The Flora Iranica (Polatschek and Rechinger \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1968\u003c/span\u003e), the Flora of Iran (Yousef Naanaie \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and the Flora of Turkey (Davis \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) were the primary sources for species identification. The names of the species were matched with the online site POWO (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) (Plants of the World Online) at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://powo.science.kew.org/\u003c/span\u003e\u003cspan address=\"https://powo.science.kew.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The contradictions observed concerning the scientific names of some species are presented in the \u003cspan refid=\"Sec12\" class=\"InternalRef\"\u003eappendix\u003c/span\u003e. Additionally, sequence information from previous studies involving ten taxa for the \u003cem\u003etrn\u003c/em\u003eH-\u003cem\u003epsb\u003c/em\u003eA region and 21 taxa for the nrDNA ITS region was utilized from the GenBank for comparative analysis of Iranian species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As outgroups, \u003cem\u003eLudwigia grandiflora\u003c/em\u003e (Michx.) Greuter \u0026amp; Burdet and \u003cem\u003eLu. peruviana\u003c/em\u003e (L.) H. Hara were employed.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePlant species examined in this research, along with their corresponding herbarium detail and GenBank accession numbers.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDNA/Sequence source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGenBank accession no.\u003c/p\u003e \u003cp\u003enrDNA ITS/\u003cem\u003etrn\u003c/em\u003eH-\u003cem\u003epsb\u003c/em\u003eA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. verticillata\u003c/em\u003e (Ard.) Lam.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLorestan: Khorramabad, Cham-Divan, 1000 m, Veise Karami, 24111 (TUH).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. auriculata\u003c/em\u003e Willd.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLorestan: Khorramabad, Cham-Divan, 1000 m, Veise Karami, 24108 (TUH).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMH808727.1/ -\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. baccifera\u003c/em\u003e L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGilan: Rudbar, Rahimabad, Reshterud Village, 211 m, Ashouri, 1396, 8554 (GUH).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.\u0026nbsp;coccinea\u003c/em\u003e Rottb.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMG237340.1/ DQ006199.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA. multiflora\u003c/em\u003e Roxb.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGilan: Someh Sara, Hendekhaleh District, Nokhaleh Akbari Village, 21 m, Ashouri, 1396, 8553 (GUH).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMF599386.1/ -\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDuabanga grandiflora\u003c/em\u003e (Roxb. ex DC.) Walp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAF163695.1/ KR532980.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLawsonia inermis\u003c/em\u003e L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKF850586.1/ GQ435226.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLudwigia peruviana\u003c/em\u003e (L.) H. Hara\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKX168366.1/ GU135322.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLu. grandiflora\u003c/em\u003e (Michx.) Greuter \u0026amp; Burdet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKX168323.1/ KC996841.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLythrum silenoides\u003c/em\u003e Boiss. \u0026amp; No\u0026euml;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLorestan: Khorramabad, Cham-e Divan, elevation 1000 meters, Veise Karami, 1998, 24113 and 1-24113 (TUH).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. virgatum\u003c/em\u003e L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWest Azerbaijan: between Salmas and Qushchi, Khan Takhti, 1400 m, Ghahreman and Mozaffarian, 1372, 17444 (TUH).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMG975397.1/ -\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. salicaria\u003c/em\u003e L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMK895653.1/ KC584962.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. junceum\u003c/em\u003e Banks \u0026amp; Sol.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKohgiluyeh and Boyer-Ahmad: Dogonbadan, Belgheis spring, 1400 m, Ghahreman, Attar, and Sheikhi, 1375, 20328 (TUH).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMG975401.1/ -\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. hyssopifolia\u003c/em\u003e L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGilan: Langarud, Chamkhaleh, 25 m, Naqinezhad, 1378, 21459 (TUH).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMG975399.1/ -\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. thymifolia\u003c/em\u003e L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGilan: Bandar Anzali, elevation 20 m, Mozaffarian and Maassoumi, 6901 (TUH).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMG975400.1/ -\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. portula\u003c/em\u003e (L.) D. A. Webb.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMorocco: T\u0026eacute;touan, 9 kilometers southwest of Souk-e-Araba-Ayacha, 120 m, Podlech, 1365, 43652 (MSB).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMG237615.1/ -\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. thesioides\u003c/em\u003e M. Bieb.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFars: 6 kilometers south of Jahrom, between Hood and the village of Kooreh, 800 m, Assadi and Akhani, 61867 (TARI).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMH245820.1/ -\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. tribracteatum\u003c/em\u003e Salzm. ex Spreng.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFars: 30 kilometers south of Jahrom, near the village of Harm, at an approximate elevation of 800 m, Assadi and Akhani, 61852 (TARI).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMG975398.1/ -\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePunica granatum\u003c/em\u003e L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAY035761.1/ GQ435338.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRotala indica\u003c/em\u003e (Willd.) Koehne\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGilan: Siahkal, Khararud District, Salash Village, 55 m, Ashouri, 1396, 8555 (GUH).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR. rotundifolia\u003c/em\u003e (Buch.-Ham. ex Roxb.) Koehne\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMH071602.1/ GU135292.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSonneratia\u0026nbsp;alba\u003c/em\u003e Sm.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAF163701.1/ MH583031.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTrapa natans\u003c/em\u003e L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenBank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKX098577.1/ KP280472.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and Sanger sequencing\u003c/h2\u003e \u003cp\u003eThe total DNA was extracted from fresh and dried leaf tissues using the 2 x CTAB procedure developed by Doyle \u0026amp; Doyle (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) and the DNeasy Plant Mini Kit (Qiagen Valencia, CA, USA) following the instructions specified by the manufacturer. We opted for plastid \u003cem\u003epsb\u003c/em\u003eA-\u003cem\u003etrn\u003c/em\u003eH and nuclear ribosomal ITS markers for our molecular investigations. The \u003cem\u003epsb\u003c/em\u003eA-\u003cem\u003etrn\u003c/em\u003eH intergenic spacer region was amplified using primers \u003cem\u003etrn\u003c/em\u003eHf-05 and \u003cem\u003epsb\u003c/em\u003eA3- f, developed by (Tate and Simpson \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and Sang et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), respectively. PCR reactions were conducted in a 25 \u0026micro;L volume, comprising 9.5 \u0026micro;L deionized water, 12.5 \u0026micro;L of 2x Taq DNA polymerase master mix RED (Amplicon, Cat No. 180301), 1 \u0026micro;L of each primer (10 pmol/\u0026micro;L), and 1 \u0026micro;L of template DNA (20 ng/\u0026micro;L). For the \u003cem\u003epsb\u003c/em\u003eA-\u003cem\u003etrn\u003c/em\u003eH intergenic spacer region, PCR conditions consisted of an initial denaturation step at 93\u0026deg;C for 5 minutes, followed by 35 cycles of denaturation at 94\u0026deg;C for 45 seconds, annealing at 48\u0026ndash;56\u0026deg;C for 45 seconds, extension at 72\u0026deg;C for 90 seconds, and a final extension at 72\u0026deg;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\u0026rsquo; protocols, using the same primers as employed for PCR amplification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analyses\u003c/h2\u003e \u003cp\u003eSequences were aligned using MAFFT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngphylogeny.fr\u003c/span\u003e\u003cspan address=\"https://ngphylogeny.fr\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), 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 \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), Bayesian inference was conducted using MrBayes on the CIPRES (Miller et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) gateway, and maximum likelihood analysis was carried out on \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://iqtree.cibiv.univie.ac.at\u003c/span\u003e\u003cspan address=\"http://iqtree.cibiv.univie.ac.at\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 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 \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). The rescaled consistency index (RC), consistency index (CI), and retention index (RI) were calculated using PAUP* 4.b10 software (Swofford \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) to assess tree quality. To determine the appropriate evolutionary model for the data, the web server \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://iqtree.cibiv.univie.ac.at/\u003c/span\u003e\u003cspan address=\"http://iqtree.cibiv.univie.ac.at/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e was utilized, and based on the Akaike information criterion (AIC), the TVM\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;I model was selected for cpDNA \u003cem\u003etrn\u003c/em\u003eH-\u003cem\u003epsb\u003c/em\u003eA data, the GTR\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;G4 model for nrDNA ITS and combined data.\u003c/p\u003e \u003cp\u003eBayesian analyses were conducted with the following configurations: Four Markov chain Monte Carlo heuristic searches, each comprising 10\u0026nbsp;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. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). 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 \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe remaining trees were utilized to create a consensus tree representing the majority agreement (50% threshold) and viewed using TreeView v. 1.6.1 (Page \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003ePhylogenetic 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 \u003cem\u003etrn\u003c/em\u003eH-\u003cem\u003epsb\u003c/em\u003eA 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, \u003cem\u003etrn\u003c/em\u003eH-\u003cem\u003epsb\u003c/em\u003eA, and combined datasets. Bootstrap values and posterior probabilities are displayed along the branches in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCladistic analysis of\u003c/b\u003e \u003cb\u003etrn\u003c/b\u003e\u003cb\u003eH-\u003c/b\u003e\u003cb\u003epsb\u003c/b\u003e\u003cb\u003eA intergenic spacer data\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOn the maximum likelihood tree, the main node A is placed with high support (MP BS\u0026thinsp;=\u0026thinsp;100; ML BS\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;1). \u003cem\u003eLythrum\u003c/em\u003e species are in the lower, distinct clades, forming a non-monophyletic group. However, these clades lack strong support. The two species \u003cem\u003eL. virgatum\u003c/em\u003e L. and \u003cem\u003eL. salicaria\u003c/em\u003e L. diverged first from the other species in the group (MP BS\u0026thinsp;=\u0026thinsp;80; ML BS\u0026thinsp;=\u0026thinsp;78; PP\u0026thinsp;=\u0026thinsp;0.87). Following this, the remaining species are observed on branches A1 (above) and A2 (below). A2, which holds the species \u003cem\u003eL. thesioides\u003c/em\u003e M. Bieb. and \u003cem\u003eL. silenoides\u003c/em\u003e Boiss. \u0026amp; No\u0026euml; (MP BS\u0026thinsp;=\u0026thinsp;78; ML BS\u0026thinsp;=\u0026thinsp;87; PP\u0026thinsp;=\u0026thinsp;0.93), while A1, a branch with low support, contains \u003cem\u003eRotala\u003c/em\u003e and \u003cem\u003eAmmannia\u003c/em\u003e species, species \u003cem\u003eS. alba\u003c/em\u003e Sm., \u003cem\u003eT. natans\u003c/em\u003e L., \u003cem\u003eP. granatum\u003c/em\u003e L., and \u003cem\u003eLa. inermis\u003c/em\u003e. Additionally, the genera \u003cem\u003eAmmannia\u003c/em\u003e form a monophyletic group (MP BS\u0026thinsp;=\u0026thinsp;81; ML BS\u0026thinsp;=\u0026thinsp;85; PP\u0026thinsp;=\u0026thinsp;0.97) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCladistic analysis of nrDNA ITS data\u003c/h2\u003e \u003cp\u003eOn the maximum likelihood tree based on nrDNA ITS data, the Lythraceae family is monophyletic (node A) with high support (MP BS\u0026thinsp;=\u0026thinsp;100; ML BS\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;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 \u003cem\u003eLythrum\u003c/em\u003e species in the \"Lythrum\" clade, among which three species, \u003cem\u003eL. hyssopifolia\u003c/em\u003e L., \u003cem\u003eL. junceum\u003c/em\u003e Banks \u0026amp; Sol., and \u003cem\u003eL. thymifolia\u003c/em\u003e L., along with two species, \u003cem\u003eL. virgatum\u003c/em\u003e and \u003cem\u003eL. salicaria\u003c/em\u003e, are placed as monophyletic groups with high support (MP BS\u0026thinsp;=\u0026thinsp;97; ML BS\u0026thinsp;=\u0026thinsp;96; PP\u0026thinsp;=\u0026thinsp;1). The sub-clade A2, includes \u003cem\u003eAmmannia\u003c/em\u003e species the \"Ammannia\" clade with high support (MP BS= -; ML BS\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;1), along with \u003cem\u003eLa. inermis\u003c/em\u003e, \u003cem\u003eD. grandiflora\u003c/em\u003e (Roxb. ex DC.) Walp., \u003cem\u003eS. alba\u003c/em\u003e, and \u003cem\u003eT. natans\u003c/em\u003e. The sub-clade A3, also with weak support (MP BS= -; ML BS\u0026thinsp;=\u0026thinsp;43; PP= -), comprises the \"Rotala\" clade (\u003cem\u003eR. indica\u003c/em\u003e, \u003cem\u003eR. rotundifolia\u003c/em\u003e (Buch.-Ham. ex Roxb.) Koehne) (MP BS\u0026thinsp;=\u0026thinsp;100; ML BS\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;1), and \u003cem\u003eP. granatum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCladistic analysis of combined data\u003c/h2\u003e \u003cp\u003eOn the maximum likelihood phylogenetic tree based on combined data, the main node A is observed with high support (MP BS\u0026thinsp;=\u0026thinsp;100; ML BS\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;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, \u003cem\u003eR. indica\u003c/em\u003e and \u003cem\u003eR. rotundifolia\u003c/em\u003e, forming a monophyletic group (MP BS\u0026thinsp;=\u0026thinsp;100; ML BS\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;1), along with \u003cem\u003eP. granatum\u003c/em\u003e on a separate branch. Additionally, three species, \u003cem\u003eT. natans\u003c/em\u003e, \u003cem\u003eS. alba\u003c/em\u003e (in a small subgroup), and \u003cem\u003eD. grandiflora\u003c/em\u003e (on a separate branch), are situated in this clade (MP BS= -; ML BS\u0026thinsp;=\u0026thinsp;99; PP\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e \u003cp\u003eA1b carries the species of \u003cem\u003eAmmannia\u003c/em\u003e in the \"Ammannia\" clade (MP BS= -; ML BS\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;1), along with \u003cem\u003eLa. inermis\u003c/em\u003e (MP BS\u0026thinsp;=\u0026thinsp;100; ML BS\u0026thinsp;=\u0026thinsp;73; PP\u0026thinsp;=\u0026thinsp;0.93). The species of \u003cem\u003eLythrum\u003c/em\u003e are placed in subgroup A2 or the \"Lythrum\" clade (MP BS\u0026thinsp;=\u0026thinsp;98; ML BS\u0026thinsp;=\u0026thinsp;95; PP\u0026thinsp;=\u0026thinsp;1). Among them, two species, \u003cem\u003eL. silenoides\u003c/em\u003e and \u003cem\u003eL. thesioides\u003c/em\u003e, are grouped in subgroup A2b (MP BS\u0026thinsp;=\u0026thinsp;97; ML BS\u0026thinsp;=\u0026thinsp;97; PP\u0026thinsp;=\u0026thinsp;0.93), while three species, \u003cem\u003eL. hyssopifolia\u003c/em\u003e, \u003cem\u003eL. junceum\u003c/em\u003e, and \u003cem\u003eL. thymifolia\u003c/em\u003e, are placed in subgroup A2a (MP BS\u0026thinsp;=\u0026thinsp;92; ML BS\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;1). Finally, two species, \u003cem\u003eL. virgatum\u003c/em\u003e and \u003cem\u003eL. salicaria\u003c/em\u003e, are clustered together (MP BS\u0026thinsp;=\u0026thinsp;99; ML BS\u0026thinsp;=\u0026thinsp;100; PP\u0026thinsp;=\u0026thinsp;1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe 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, \u003cem\u003eLythrum, Ammannia\u003c/em\u003e and \u003cem\u003eRotala\u003c/em\u003e 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. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Morris \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The species of the genus \u003cem\u003eLythrum\u003c/em\u003e showed a monophyletic relationship (except for the cpDNA \u003cem\u003etrn\u003c/em\u003eH-\u003cem\u003epsb\u003c/em\u003eA data). The genus is identified by its alternate upper leaves, spike-like inflorescence, and multi-flowered or solitary cymes (Yousef Naanaie \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In all trees resulting from this study, the species \u003cem\u003eL. virgatum\u003c/em\u003e and \u003cem\u003eL. salicaria\u003c/em\u003e were consistently grouped in one clade. These two species also formed a monophyletic group in the study by Morris (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Koehne (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1903\u003c/span\u003e) classified these two species under the subgenus \u003cem\u003eSalicaria\u003c/em\u003e 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\u0026thinsp;=\u0026thinsp;5, 10). The basic chromosome number is reported to be n\u0026thinsp;=\u0026thinsp;15 in \u003cem\u003eL. virgatum\u003c/em\u003e and n\u0026thinsp;=\u0026thinsp;15, 25, and 30 in \u003cem\u003eL. salicaria\u003c/em\u003e (Graham and Cavalcanti \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). \u003cem\u003eL. virgatum\u003c/em\u003e and \u003cem\u003eL. salicaria\u003c/em\u003e 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 \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). 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. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003eb\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cem\u003eL. thesioides\u003c/em\u003e and \u003cem\u003eL. silenoides\u003c/em\u003e were also united in chloroplast and combined trees. They are annual species with 2 to 8 stamens (Yousef Naanaie \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), 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.\u003c/p\u003e \u003cp\u003eThe three species, \u003cem\u003eL. hyssopifolia\u003c/em\u003e, \u003cem\u003eL. thymifolia\u003c/em\u003e, and \u003cem\u003eL. junceum\u003c/em\u003e, 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 \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1968\u003c/span\u003e; Yousef Naanaie \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), 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. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003eb\u003c/span\u003e). Among them, \u003cem\u003eL. hyssopifolia\u003c/em\u003e and \u003cem\u003eL. junceum\u003c/em\u003e 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 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1903\u003c/span\u003e) placed these two species close to the Eurasian species \u003cem\u003eLythrum\u003c/em\u003e by having one or two flowers in the axis, a thick nectar ring in the ovary and relatively long style.\u003c/p\u003e \u003cp\u003e \u003cem\u003eL. hyssopifolia\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;10) is an annual herb with an erect ascending stem, homomorphic flowers, 2\u0026ndash;6 stamens, capsule shorter than hypanthium (Koehne \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1903\u003c/span\u003e; Webb \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Yousef Naanaie \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), It is widespread in seasonally moist habitats in southern parts of Eurasia and has become invasive in many parts of the world. \u003cem\u003eL. junceum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;5) is a biennial and perennial plant characterized by a decumbent stem, tristylous flowers, 12 stamens, and a corolla tube length of 5\u0026ndash;6 mm. It is distributed in southern Europe and Central Asia (Polatschek and Rechinger \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1968\u003c/span\u003e; Morris \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). While, \u003cem\u003eL. thymifolia\u003c/em\u003e is an annual plant with homomorphic flowers, 2\u0026ndash;6 stamens, and a short corolla length; distinguished from \u003cem\u003eL. hyssopifolia\u003c/em\u003e by its hypanthium and corolla length (Polatschek and Rechinger \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1968\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on the present findings, \u003cem\u003eL. portula\u003c/em\u003e (L.) D. A. Webb. appeared in an independent clade, and sister group relationship with other species (\u003cem\u003eL. salicaria\u003c/em\u003e, \u003cem\u003eL. virgatum\u003c/em\u003e, \u003cem\u003eL. thymifolia\u003c/em\u003e, \u003cem\u003eL. Junceum\u003c/em\u003e, \u003cem\u003eL. hyssopifolia\u003c/em\u003e, and \u003cem\u003eL. tribracteatum\u003c/em\u003e) of \u003cem\u003eLythrum\u003c/em\u003e 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. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Johnson and Brooke \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe species of \u003cem\u003eAmmannia\u003c/em\u003e demonstrated monophyletic groups on all trees. Three species of \u003cem\u003eA. auriculata\u003c/em\u003e Willd., \u003cem\u003eA. coccinea\u003c/em\u003e, and \u003cem\u003eA. multiflora\u003c/em\u003e Roxb. in cpDNA trees and four species of \u003cem\u003eA. auriculata\u003c/em\u003e with \u003cem\u003eA. baccifera\u003c/em\u003e L. and \u003cem\u003eA. multiflora\u003c/em\u003e with \u003cem\u003eA. coccinea\u003c/em\u003e in combined trees united in small groups.\u003c/p\u003e \u003cp\u003eAccording to Graham's classification (1985), species of \u003cem\u003eAmmannia\u003c/em\u003e were primarily divided into two subgenera, \u003cem\u003eAmmannia\u003c/em\u003e and \u003cem\u003eCryptotheca\u003c/em\u003e (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 \u003cem\u003eA. baccifera\u003c/em\u003e were placed in the section \u003cem\u003eAmmannia\u003c/em\u003e (having style approximately 5.0 mm or less), while \u003cem\u003eA. auriculata\u003c/em\u003e and \u003cem\u003eA. coccinea\u003c/em\u003e were placed in the \u003cem\u003eEustylia\u003c/em\u003e section (having style approximately 3.0-5.1 mm or less). Additionally, in the Flora Iranica (Polatschek and Rechinger \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1968\u003c/span\u003e) and Flora of Iran (Yousef Naanaie \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), based on style length and inflorescence type, \u003cem\u003eA. auriculata\u003c/em\u003e was classified with \u003cem\u003eA. multiflora\u003c/em\u003e (due to style length of 1.5-3.0 mm), and \u003cem\u003eA. baccifera\u003c/em\u003e with \u003cem\u003eA. verticillata\u003c/em\u003e (Ard.) Lam. (with shorter style, 0.25\u0026ndash;0.5 mm). However, in the current study \u003cem\u003eA. baccifera\u003c/em\u003e grouped with \u003cem\u003eA. auriculata\u003c/em\u003e and \u003cem\u003eA. multiflora\u003c/em\u003e with \u003cem\u003eA. coccinea\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) formed monophyletic groups. This indicates that the phylogenetic reconstruction of \u003cem\u003eAmmannia\u003c/em\u003e 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 \u003cem\u003eA. baccifera\u003c/em\u003e and \u003cem\u003eA. auriculata\u003c/em\u003e 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. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Yousef Naanaie \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). They exhibit similarities in leaf micro-morphological features (such as stomatal size and inner stomata rim shape) (Mahmoodi et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e) 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., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Furthermore, the close affinity between the two species, \u003cem\u003eA. multiflora\u003c/em\u003e and \u003cem\u003eA. coccinea\u003c/em\u003e, is supported by their morphological similarities. Both are herbaceous, annual species, with opposite, linear or lanceolate leaves, cyme inflorescence, and 4 stamens.\u003c/p\u003e \u003cp\u003eThe phylogenetic tree reconstructed in the present study also indicates the sister relationship between \u003cem\u003eLa. inermis\u003c/em\u003e and the species of \u003cem\u003eAmmannia\u003c/em\u003e. \u003cem\u003eLa. inermis\u003c/em\u003e is \u003cem\u003eg\u003c/em\u003elabrous shrub or small tree 2\u0026ndash;6 m tall native of North Africa and southwest Asia (Yadav et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). It is cultivated and occurs naturally in western and southern Iran. Species of \u003cem\u003eRotala\u003c/em\u003e, including \u003cem\u003eR. indica\u003c/em\u003e and \u003cem\u003eR. rotundifolia\u003c/em\u003e, 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. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Previous molecular studies (Graham et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) also support this grouping.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCharacter evolution in Lythraceae in Iran\u003c/h2\u003e \u003cp\u003eThe 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These morphological features are taxonomically informative and used for genus classification (Polatschek and Rechinger \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1968\u003c/span\u003e; Yousef Naanaie \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). 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 \u003cem\u003eAmmannia\u003c/em\u003e and \u003cem\u003eRotala\u003c/em\u003e. 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 \u003cem\u003eLythrum\u003c/em\u003e. Among these, \u003cem\u003eRotala\u003c/em\u003e 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).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, a comprehensive phylogeny of the family Lythraceae, focusing on the three genera \u003cem\u003eLythrum\u003c/em\u003e, \u003cem\u003eAmmannia\u003c/em\u003e, and \u003cem\u003eRotala\u003c/em\u003e, was presented for the first time. Phylogenetic analyses using cpDNA \u003cem\u003etrn\u003c/em\u003eH\u003cem\u003e-psb\u003c/em\u003eA, nrDNA ITS, and combined data of maximum parsimony, Bayesian inference, and maximum likelihood revealed monophyly of the three genera \u003cem\u003eLythrum\u003c/em\u003e, \u003cem\u003eAmmannia\u003c/em\u003e, and \u003cem\u003eRotala\u003c/em\u003e (except for \u003cem\u003etrn\u003c/em\u003eH\u003cem\u003e-psb\u003c/em\u003eA 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 \u003cem\u003eL. thesioides\u003c/em\u003e with \u003cem\u003eL. silenoides\u003c/em\u003e; \u003cem\u003eL. salicaria\u003c/em\u003e with \u003cem\u003eL. virgatum\u003c/em\u003e; and the three species \u003cem\u003eL. thymifolia\u003c/em\u003e, \u003cem\u003eL. junceum\u003c/em\u003e, and \u003cem\u003eL. hyssopifolia\u003c/em\u003e 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 \u003cem\u003eA. coccinea\u003c/em\u003e and \u003cem\u003eA. multiflora\u003c/em\u003e, as well as between \u003cem\u003eA. baccifera\u003c/em\u003e and \u003cem\u003eA. auriculata\u003c/em\u003e, 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.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors do not have any conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eR. 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.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe 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.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during the current study are available in the GenBank repository.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAPG IV (2016) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Bot J Linn 181:1\u0026ndash;20. https://doi.org/10.1111/boj.12385\u003c/li\u003e\n\u003cli\u003eConti E, Litt A, Wilson PG, et al (1997) Interfamilial relationships in Myrtales: Molecular phylogeny and patterns of morphological evolution. Syst Bot 22:629. https://doi.org/10.2307/2419432\u003c/li\u003e\n\u003cli\u003eCronquist A (1982) An integrated system of classification of flowering plants. 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A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy\u003c/li\u003e\n\u003cli\u003eNaqinezhad A, Naseri Larijani N (2017) \u003cem\u003eAmmannia coccinea\u003c/em\u003e (Lythraceae), a new record for the Flora Iranica area. Phytol Balc (Sofia) 23:35\u0026ndash;38\u003c/li\u003e\n\u003cli\u003ePage DM (2001) TreeView (Win32). Retrieved from http://taxonomy.zoology.gla.ac.uk/rod\u003c/li\u003e\n\u003cli\u003ePolatschek A, Rechinger KH (1968) Lythraceae. In: Rechinger KH (ed) Flora Iranica. Akad.\u0026quot; Druck-und Verlagsanstalt, Graz\u003c/li\u003e\n\u003cli\u003ePOWO (2024) Plants of the World Online. In: Published on the Internet; http://www.plantsoftheworldonline.org/. 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Int J Plant Sci 165: S85\u0026ndash;S105. https://doi.org/10.1086/421066\u003c/li\u003e\n\u003cli\u003eTakhtajan A (1987) System of Magnoliophyta. Academy of Sciences USSR, Leningrad\u003c/li\u003e\n\u003cli\u003eTate JA, Simpson BB (2003) Paraphyly of \u003cem\u003eTarasa\u003c/em\u003e (Malvaceae) and diverse origins of the polyploid species. Syst Bot 28:723\u0026ndash;737\u003c/li\u003e\n\u003cli\u003eWebb CJ, Sykes WR, Garnock-Jones PJ (1988) Flora of New Zealand. Naturalised Pteridophytes, Gymnosperms, Dicotyledons. Botany Division DSIR, Christchurch\u003c/li\u003e\n\u003cli\u003eWebb DA (1967) Lythraceae. In: Tutin TG (ed) Flora Europaea. Cambridge University Press, Cambridge, pp 300\u0026ndash;303\u003c/li\u003e\n\u003cli\u003eYadav S, Kumar A, Dora J, Kumar A (2013) Essential perspectives of \u003cem\u003eLawsonia inermis\u003c/em\u003e. Journal of Pharmaceutical and Chemical Sciences 2:888\u0026ndash;896\u003c/li\u003e\n\u003cli\u003eYousef Naanaie S (2010) Lythraceae. In: Assadi M, Maassoumi A. A., Babakhanlou P, Mozaffarian V (eds) Flora of Iran. Research Institute of Forests and Rangelands, Tehran\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lythraceae, Monophyly, Phylogenetic relationship, Taxonomy","lastPublishedDoi":"10.21203/rs.3.rs-4612594/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4612594/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe 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 \u003cem\u003etrn\u003c/em\u003eH\u003cem\u003e-psb\u003c/em\u003eA, 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 \u003cem\u003eLythrum, Ammannia\u003c/em\u003e, and \u003cem\u003eRotala \u003c/em\u003ein 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 \u003cem\u003eLythrum \u003c/em\u003especies on the lower branches and the derivation of \u003cem\u003eAmmannia\u003c/em\u003e species on the higher clades and \u003cem\u003eRotala \u003c/em\u003especies on the terminal clades. The results of the phylogenetic study revealed the close relationship of some \u003cem\u003eLythrum\u003c/em\u003especies (such as \u003cem\u003eL. thesioides\u003c/em\u003e M. Bieb. with \u003cem\u003eL. silenoides\u003c/em\u003e Boiss. \u0026amp; Noë and three species \u003cem\u003eL. thymifolia \u003c/em\u003eL\u003cem\u003e.\u003c/em\u003e, \u003cem\u003eL. junceum \u003c/em\u003eBanks \u0026amp; Sol., and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003ehyssopifolia \u003c/em\u003eL.) and \u003cem\u003eAmmannia\u003c/em\u003e (\u003cem\u003eA. coccinea\u003c/em\u003e Rottb. with \u003cem\u003eA. multiflora \u003c/em\u003eRoxb., and \u003cem\u003eA. baccifera\u003c/em\u003e L\u003cem\u003e. \u003c/em\u003ewith \u003cem\u003eA. auriculata \u003c/em\u003eWilld\u003cem\u003e.\u003c/em\u003e), indicating the need for taxonomic revision.\u003c/p\u003e","manuscriptTitle":"Phylogenetic study of Lythraceae family in Iran, focusing on the genera Lythrum, Ammannia, and Rotala","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-12 17:07:35","doi":"10.21203/rs.3.rs-4612594/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"38674378-3ea0-4007-9a4f-321a2a7537a5","owner":[],"postedDate":"July 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-15T06:53:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-12 17:07:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4612594","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4612594","identity":"rs-4612594","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}