University of Isfahan Journal of Plant Biological Sciences 3041-9603 14 4 2023 02 20 Investigating Specific Diversity of Aquatic Plant Ceratophyllum L. in Iran Using Morphological Characters and ISSR and SRAP Molecular Markers Investigating Specific Diversity of Aquatic Plant Ceratophyllum L. in Iran Using Morphological Characters and ISSR and SRAP Molecular Markers 71 86 28055 10.22108/ijpb.2023.139564.1339 FA Hanieh Bahadori Department of Plant and Animal Biology, Faculty of Biological Scienceand Technology, University of Isfahan, Isfahan, Iran Saeed Afsharzadeh Department of Plant and Animal Biology, Faculty of Biological Scienceand Technology, University of Isfahan, Isfahan, Iran Shabnam Abbasi Department of Biology Education, Farhangian University, P.O. Box 14665-889, Tehran, Iran Journal Article 2023 10 23 The genus Ceratophyllum L. is a submerged aquatic plant of the Ceratophyllaceae family, with six species recognized worldwide. So far, two species, C. demersum and C. submersum, have been reported for the flora of Iran. Considering the importance of this plant from an environmental and ecological point of view, its taxonomic and molecular studies have been carried out in this study to investigate the species diversity of this genus. Following the identification of species by assessing morphological features in individuals from different populations, 7 ISSR primers and 3 pairs of SRAP primers were used to confirm the identifications and study the genetic diversity of distinct populations. Molecular investigations showed the number of 5 genetic groups, whose intra-population diversity (52%) was higher than inter-population diversity (48%) with P<0.05. Furthermore, the data revealed that the populations of this plant in Iran have relatively the same genetic pool. Based on this, it can be concluded that several elements, including the connectedness of aquatic ecosystems, particularly in the north of the country, as well as the high adaptability of this submerged plant with aquatic habitats, can be effective in producing such conditions. Recent droughts have increased the sexual reproduction of this plant, and as a result, the species diversity within the populations has increased. Introduction Aquatic plants (macrophytes) are important components of freshwater ecosystems that affect the functioning and diverse services of the ecosystem. Aquatic plants grow along the edge of the lake and are both a protective and nourishing component of the ecosystem for healthy lakes. Ceratophyllum has about 2 to 30 species that have global distribution. The determination of genetic relationships of plant materials is important in terms of choosing the right parental combination for producing suitable populations, designing effective breeding programs, responding to long-term selection and reducing genetic vulnerability. The study of genetic relationships is a prerequisite for the improvement of agricultural plants and also a prerequisite for the preservation of genetic resources. Considering that in the Ceratophyllum, the separation of species is done by intermediate morphological characteristics, the study of the genetic diversity of different populations of this species in Iran using molecular markers seems necessary. In addition, considering the recent droughts in the country and the destruction of many aquatic habitats, the study of this aquatic plant can provide useful knowledge for the protection of the country's environment. Therefore, in this study, some of the important morphological traits of the samples, the relationships of the species of this genus, as well as their genetic diversity were investigated using molecular markers. Materials and Methods In this study, 50 individuals and 29 populations of two species, C. submersum and C. demersum, were examined. The modified CTAB method was used to extract DNA from 50 individuals from 29 studied aquatic plant populations. Seven ISSR primers and three SRAP primer pairs were selected. For data analysis and statistical analysis of populations, individuals were scored based on the presence of band (1) and the absence of band (0). Using NTSYSpc software and the Jaccard and Nei similarity coefficient, cluster analysis was performed and two dendrograms were obtained. GenAlex software was used for principal component analysis and STRUCTURE software was used to examine the genetic structure of populations. Results and Discussion The morphological studies of this plant showed an herbaceous perennial state. The stem of this plant has multiple nodes and internodes, and the stem mostly branches in the upper 1/3. In the C. demersum species, they have 1 or 2 divisions and in the C. submersum species, they have 3 or 4 dichotomy divisions. The shape of the leaves is thin, long, pointed, and fork-like, which are stringy or linear. In both dendrograms, the samples from the north of the country and the central region of the country (Isfahan) are almost separated, but a kind of genetic connection can be seen between the samples from the north of the country and the center of the country. Through humans and also through water birds, they show a kind of genetic connection. Regarding the C. demersum and C. submersum species that exist in Iran, it seems that the C. demersum species has a wider genetic treasure in the aquatic habitats of the country and the species C. submersum has been found only in a region in the north of the country (Izbaran, Amirkalaye, and Bandar Anzali) in our sampling. Therefore, it seems that C. demersum is ecologically more compatible with various environmental factors and the C. submersum species exists only in limited ecological water niches in the country due to the lack of suitable living conditions. The results of the principal component analysis were in agreement with the cluster analysis and showed some kind of connection and continuity between populations. The results of AMOVA showed 52% genetic diversity within populations and 48% genetic diversity between populations. The results of the Mantel test indicated that there is no relationship between the genetic distance and the geographical distance (R= 0.019 and P = 0.320), which is due to the fact that in the principal component analysis, we saw the lack of separation of the populations. The molecular analysis of Cerarophyllum intraspecies relationships through STRUCTURE analysis showed 5 genetic groups and demonstrated the relationship between individuals of different populations, whose gene pool was relatively similar. Conclusion According to analyses done, it can be said that the gene pool of Ceratophyllum population in Iran is somewhat distorted and there is a relationship and genetic similarity between different populations and only some populations in the north of the country are more uniform. As was stated, various factors can be effective in creating such conditions. A high intra-population genetic diversity has been shown in the populations of this study. The genus Ceratophyllum L. is a submerged aquatic plant of the Ceratophyllaceae family, with six species recognized worldwide. So far, two species, C. demersum and C. submersum, have been reported for the flora of Iran. Considering the importance of this plant from an environmental and ecological point of view, its taxonomic and molecular studies have been carried out in this study to investigate the species diversity of this genus. Following the identification of species by assessing morphological features in individuals from different populations, 7 ISSR primers and 3 pairs of SRAP primers were used to confirm the identifications and study the genetic diversity of distinct populations. Molecular investigations showed the number of 5 genetic groups, whose intra-population diversity (52%) was higher than inter-population diversity (48%) with P<0.05. Furthermore, the data revealed that the populations of this plant in Iran have relatively the same genetic pool. Based on this, it can be concluded that several elements, including the connectedness of aquatic ecosystems, particularly in the north of the country, as well as the high adaptability of this submerged plant with aquatic habitats, can be effective in producing such conditions. Recent droughts have increased the sexual reproduction of this plant, and as a result, the species diversity within the populations has increased. Introduction Aquatic plants (macrophytes) are important components of freshwater ecosystems that affect the functioning and diverse services of the ecosystem. Aquatic plants grow along the edge of the lake and are both a protective and nourishing component of the ecosystem for healthy lakes. Ceratophyllum has about 2 to 30 species that have global distribution. The determination of genetic relationships of plant materials is important in terms of choosing the right parental combination for producing suitable populations, designing effective breeding programs, responding to long-term selection and reducing genetic vulnerability. The study of genetic relationships is a prerequisite for the improvement of agricultural plants and also a prerequisite for the preservation of genetic resources. Considering that in the Ceratophyllum, the separation of species is done by intermediate morphological characteristics, the study of the genetic diversity of different populations of this species in Iran using molecular markers seems necessary. In addition, considering the recent droughts in the country and the destruction of many aquatic habitats, the study of this aquatic plant can provide useful knowledge for the protection of the country's environment. Therefore, in this study, some of the important morphological traits of the samples, the relationships of the species of this genus, as well as their genetic diversity were investigated using molecular markers. Materials and Methods In this study, 50 individuals and 29 populations of two species, C. submersum and C. demersum, were examined. The modified CTAB method was used to extract DNA from 50 individuals from 29 studied aquatic plant populations. Seven ISSR primers and three SRAP primer pairs were selected. For data analysis and statistical analysis of populations, individuals were scored based on the presence of band (1) and the absence of band (0). Using NTSYSpc software and the Jaccard and Nei similarity coefficient, cluster analysis was performed and two dendrograms were obtained. GenAlex software was used for principal component analysis and STRUCTURE software was used to examine the genetic structure of populations. Results and Discussion The morphological studies of this plant showed an herbaceous perennial state. The stem of this plant has multiple nodes and internodes, and the stem mostly branches in the upper 1/3. In the C. demersum species, they have 1 or 2 divisions and in the C. submersum species, they have 3 or 4 dichotomy divisions. The shape of the leaves is thin, long, pointed, and fork-like, which are stringy or linear. In both dendrograms, the samples from the north of the country and the central region of the country (Isfahan) are almost separated, but a kind of genetic connection can be seen between the samples from the north of the country and the center of the country. Through humans and also through water birds, they show a kind of genetic connection. Regarding the C. demersum and C. submersum species that exist in Iran, it seems that the C. demersum species has a wider genetic treasure in the aquatic habitats of the country and the species C. submersum has been found only in a region in the north of the country (Izbaran, Amirkalaye, and Bandar Anzali) in our sampling. Therefore, it seems that C. demersum is ecologically more compatible with various environmental factors and the C. submersum species exists only in limited ecological water niches in the country due to the lack of suitable living conditions. The results of the principal component analysis were in agreement with the cluster analysis and showed some kind of connection and continuity between populations. The results of AMOVA showed 52% genetic diversity within populations and 48% genetic diversity between populations. The results of the Mantel test indicated that there is no relationship between the genetic distance and the geographical distance (R= 0.019 and P = 0.320), which is due to the fact that in the principal component analysis, we saw the lack of separation of the populations. The molecular analysis of Cerarophyllum intraspecies relationships through STRUCTURE analysis showed 5 genetic groups and demonstrated the relationship between individuals of different populations, whose gene pool was relatively similar. Conclusion According to analyses done, it can be said that the gene pool of Ceratophyllum population in Iran is somewhat distorted and there is a relationship and genetic similarity between different populations and only some populations in the north of the country are more uniform. As was stated, various factors can be effective in creating such conditions. A high intra-population genetic diversity has been shown in the populations of this study.

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