Morphological and molecular genetic variations of some taxa of genus Acacia and their taxonomic significance1

A resemble in Acacia s.l. spp. morphology causes a great confusion about its identification. So, the current study's objectives are using morphological and molecular traits for accurate identification of Acacia s.l. taxa, assess the discrimination of the newly proposed genera of Vachellia and Senegalia and detect the genetic variations between studied taxa. The morphological characters of 24 taxa of genus Acacia s.l. were investigated with emphasis on seeds. DNA fingerprint and the genetic relationships among the studied taxa were investigated by using 10 (RAPD) and 10 (ISSR) markers that showed a relatively equal polymorphism (about 15%). RAPD marker generated a total of 395 fragments of which 64 bands were polymorphic, while ISSR primers generated a total of 314 fragments, 46 of them were polymorphic. The generated dendrogram that based on 893 traits divided the studied taxa of Acacia s.l. into two major groups; Acacia s.s. group and Vachellia group that containing Senegalia taxa. The complementary use of morphological and molecular data gave an accurate characterization and clear the genetic variation between studied taxa of Acacia.


Introduction
Various phylogenetic studies demonstrated that Acacia s.l. is not monophyletic, it is currently broadly accepted to be partitioned into at least five genera Viz. Acacia s.s., Vachellia, Senegalia, Acaciella and Mariosousa [1]. The term Acacia s. s. was retained for the bulk of Australian taxa, with Vachellia and Senegalia being used in Asia and Africa [2]. Morphological characteristics were considered significant, but not enough for the characterization of Acacia s.l. [3]. Hence, in this study, we investigated the position of Acacia s.l. spp. in the new conventional arrangement, based on data generated from both morphological and molecular information from two molecular markers.
Chaudhary and Al-Jowaid [4] studied the morphological characteristics of 18 species of the genus Acacia s.l. in Saudi Arabia. They used several vegetative and floral characteristics to build a key to define the genus. Further, Migahid [5] described the morphology of 14 species belonging to genus Acacia s.l. In addition, Khan et al [6] investigated the morphological and anatomical characteristics of eight Acacia s.l. species found in the Wadi Fatimah in Makkah Al-Mukarramah region. Al-Gohary and Mohamed [7] studied the morphological variations between 12 taxa of genus Acacia s.l. Their results completed the construction of an indented identification key at the specific and infra-specific levels.
Few authors have investigated the seed morphology of Acacia s. l. [8]. The seed surface sculpture patterns of nine species, one subspecies and two varieties of Acacia s.l. of Saudi Arabia were described [9]. The seed coat's morphological and anatomical characteristics have been used to distinguish 11 Acacia s.l. species in Saudi Arabia [10] using Scanning Electron Microscopy (SEM) [11] and Light Microscopy (LM) [7].
Molecular markers have been used to identify species and populations. Because genetic markers are unaffected by environmental factors, they are useful in the conservation of plant germplasm [12,13]. Many authors have revealed the valuable data obtained from combining morphological characteristics and molecular markers for evaluating genetic diversity among different varieties of Acacia s.l. as well as the role of genetic markers in supporting the taxonomic results for the differentiation of studied varieties [14,15]. Abdel-Hamid [16] demonstrated that Random Amplified Polymorphic DNA (RAPD) was a useful tool for the identification and characterization of plants at the level of subspecies. Mutharaian et al [17] used RAPD markers to determine genetic distance and similarity among seven Acacia s.l. spp., which showed 326 species-specific markers with 55.82% polymorphism to clarify the close relationships among the seven studied species.
RAPD and Inter-Simple Sequence Repeats (ISSR) have been extensively used for plant classification owing to their simplicity, rapidity in detecting DNA polymorphism and high efficiency for analysis of genetic diversity [18][19][20][21][22][23]. RAPD and ISSR markers were used to study the genetic variability between five species of Acacia, where the RAPD marker resulted in 58 bands, with 51 unique bands and seven polymorphic bands, and the ISSR marker showed 75 bands of which 42 were unique and 31 polymorphic between studied taxa. RAPD separated Acacia ehrenbergiana from other investigated species, while ISSR separated both Acacia etbaica and Acacia tortilis from other species [24].
The identities of Acacia s.l. spp. in Saudi Arabia have caused great confusion for field workers in this country. Therefore, the goal of this research was to use morphological characteristics, with emphasis on seed traits for the accurate identification and systematic classification of 24 taxa belonging to the genus Acacia s.l. grown within Kingdom of Saudi Arabia and to assess the discrimination of the newly proposed genera of Vachellia and Senegalia, further objective was to detect genetic diversity via RAPD and ISSR markers that could provide molecular characterization, preservation of germplasm of these important taxa, and investigate the genetic relationships among them.

Materials and methods
In the present study, 24 taxa of Acacia s.l. were collected from five localities in KSA representing eighteen species, four subspecies and two varieties. The collected plants were identified and authenticated according to [5,25,26] (Table 1).

Morphological traits
The macromorphological characters of the whole plant viz. habit, leaf, flowers, inflorescence, pod and seeds were extracted directly from the fresh specimens of the available taxa. Twenty four mature seeds were dried, cleaned, examined by Stereomicroscope and photographed by Digital Camera 7.2 mp; six characters of seed were studied viz. colour, shape, texture, areola shape, hilum position, hilum shape. For SEM investigation, the seeds were dried and coated with golden particles and fixed to the specimen holder of Scanning Electron Microscope (SEM, Inspect S, version 3.1.2) maintained at accelerating potential voltage of 20-30 K.V. and photographed at different magnifications. six characters of seed were viz. sculpture surface, cell shape, anticlinal wall shape, anticlinal wall elevation, periclinal wall elevation and periclinal wall vesititure. The descriptive terminology of seed surface sculpture that is used in the present study was based on the glossary of [27][28][29][30].

Molecular markers
RAPD according to [31] and Inter Simple Sequence Repeats (ISSR) according to [32] markers were used in this study.

DNA isolation
DNA isolation from plant tissues was carried out using DNeasy plant Mini Kit (Dneasy plant mini handbook 2016, Qiagen Inc., USA).
ISSR-PCR reactions were done by using 10 primers with sequences indicated in (Table 2). Reaction conditions and mixtures (25 µl total volumes) consisted of dNTPs, MgCl 2 , 10X buffer, Primer, Taq DNA Polymerase, BSA (bovine serum albumin) and Template DNA were optimized. Amplification was carried out in a (Veriti ® Thermal Cycler, Life Technologies, Applied Biosystem, NY, USA) programmed for 36 cycles as follows: 94°C/5 min (1 cycle); 94°C/30 sec., 44°C/90 sec. 72°C/1.5 min (36 cycles); 72°C/20 min (1 cycle) and 4°C (infinitive). For both RAPD and ISSR PCR amplification was done twice for each sample and a particular primer for giving two amplification replicates in order to saturate polymorphism within samples and ensure reproducibility of the data.

Data analysis
Unweighted Pair-Group Method using Arithmetic Averages with SAHN function due to [33] was followed to evaluate the variation of morphological characters states among the submitted taxa, each taxa was considered as operational taxonomic unit (OTU) and characters states were analyzed as binary features. In addition, Table 2. DNA monomorphic and polymorphic bands and polymorphism ratio for the studied taxa generated by RAPD and ISSR marker and their sequences. the banding patterns generated by RAPD-PCR and ISSR-PCR markers were compared to detect the differences and similarity between samples under study. Clear and distinct bands were scored as "1" for presence and "0" for absence of bands. The configuring of groups was depended on the Jaccard's coefficient similarity values. All statistics were performed by software NTSys-PC version 2.02 by Rohlf [34].

Morphological characteristics
The most informative and diagnostic morphological characters were; plant habit that varied between shrub in Acacia coriacea, A. cuthbertsonii, A. ehrenbergiana and A. ligulata or tree in 19 taxa. The plant length whether small in six studied taxa, medium in another six taxa or large in the remaining. The plant texture glabrous in 9 taxa, whether pubescent in one taxa A. tortilis subsp. raddiana or spiny in 13 taxa (Table 3)  Length: Small (0), Medium (1), Large (2) 3.
Seed shape ovate in 7 taxa, rounded in 7 taxa, quadrate in one taxa A. gerrardii var. gerrardii, elliptic in 7 taxa, Kidney-shaped in one taxa A. senegal.

Molecular markers
The RAPD analysis of the 24 taxa of genus Acacia s.l. with the different random 10-mer primers generated a total of 395 fragments of which 64 bands were polymorphic. The molecular size of amplified fragments ranged from 122.09 bp to 2268.10 bp. The lowest number of amplified fragments was (34) bands by primer U10, While the highest amplified fragment was (47) bands by primer D18. The monomorphic bands were not detected in all samples with all primers. The total polymorphism ratio was 15.80%. The highest ratio of polymorphism generated by primer U18 (30.43%), while the primer V15 produced the lowest polymorphism ratio (2.78%). The total number of species-specific bands scored across the 24 taxa of genus Acacia s.l. by RAPD marker are 64.
The highest number of species-specific bands generated from primer U18 was 14, while the lowest number of species-specific bands was 1 generated from primer V15. Analysis of the 24 taxa of genus Acacia with the ten ISSR primers generated a total of 314 fragments, 47 of them were polymorphic. The molecular sizes of bands varied from 198.42 bp to 1489.14 bp. The number of PCR products ranged from 26 to 37 bands. There were no monomorphic bands detected with all primers and all samples. The total polymorphism ratio was 15.41%. Forty seven molecular markers were detected as species-specific bands. The highest ratio of polymorphism generated by primer 844 (31.03%), while the primer IT1 produced the lowest polymorphism (8.57%). The total number of species-specific markers scored by  L. A. johnwoodii. M. A. ligulata. N. A. nilotica subsp. indica. O. A. nilotica subsp. indica. P. A. nilotica subsp. tomentosa. Q. A.  origena. R. A. pruinocarpa. S. A. salicina. T. A. sclerosperma. U. A. senegal. V. A. tortilis subsp. radiana. W. A. tortilis subsp. totrilis. X. A. victoria.
ISSR is 47. The highest number of species-specific markers generated by primer 844 was 9, while the lowest number of species-specific markers was 3 generated from primer IT1, SAS1 and Terry ( Table 2, Figures 3  and 4).

Genetic relationships by RAPD and ISSR markers
The genetic similarities among the twenty four taxa of genus Acacia s. l. based on Jaccard's coefficient appeared that, the highest pairwise similarity indices (0.55) resulted from RAPD marker was between A. cuthbertsonii and A. sclerosperma, also between A. nilotica subsp. tomentosa and A. nilotica subsp. indica. While the lowest similarity indices (0.10) were noticed between A. ehrenbergiana and A. origena (Appendix A). The highest pairwise similarity indices (0.62) resulted from ISSR were between A. nilotica subsp. tomentosa and A. nilotica subsp. indica. While the lowest similarity indices (0.14) were observed between A. coriacea and A. gerrardii.var. iraquensis (Appendix B). The highest pairwise genetic similarity among the 24 taxa of genus Acacia resulted from both RAPD and ISSR markers was (0.58)

Phenetic analysis of both morphological and molecular characteristics
The phenetic analysis of both morphological and molecular attributes generated a dendrogram ( Figure 5) that clarifies the splitting of genus Acacia s.l. into two main series; Series I included 16 taxa, A. gerrardii var. najdensis was segregated early at 0.2 taxonomic distance as a distinct identity and the remaining taxa distributed into three groups; the first one included five taxa where A. asak was segregated early at a taxonomic distance 0.2. A. ehrenbergiana and A. johnwoodii formed sister taxa at a taxonomic distance 0.35. A. farnesiana and A. senegal were grouped together at a taxonomic distance 0.22. The second group divided into two subgroups; one comprised A. etbaica and A. origena at taxonomic distance less than 0.3, the second subgroup contained four taxa where A. gerrardii var. gerrardii and A. gerrardii var. iraquensis the last segregated taxa at 0.32 taxonomic distance. A. tortilis subsp. tortilis and A. tortilis subsp. raddiana were grouped together in the present study. The third group contained two sister subgroups the first was A. nilotica subsp. indica and A. pruinocarpa at taxonomic distance 0.25. While the second was another accession of A. nilotica subsp. indica and A. nilotica subsp. tomentosa at taxonomic distance less than 0.5.
Series II included 8 taxa that distributed into two groups; the first one included four taxa where A. coriacea was segregated early followed by A. ampliceps at a taxonomic distance about 0.2, while A. ligulata and A. salcina formed sister taxa at a taxonomic distance less than 0.3. The second group contained two sister groups one of them comprised A. cuthberstonii and A. sclerosperma at a taxonomic distance less than 0.3. While the second contained A. cyclops and A. victoriae at a taxonomic distance about 0.2.

Discussion
Considering a legitimate concern for clearness, all species talked about here are alluded to by their Acacia s.l. names and after resolution of the interrelationships among the studied species, the name of Acacia s.s., Vachellia and Senegalia are imposed. The present study included 24 taxa of the former broadly circumscribed genus Acacia s.l. that occur in Kingdom of Saudi Arabia, either naturally or as major intruders. Genus Acacia s.l. should be treated as comprising at least five genera, this new classification is based on results from many morphological, genetic studies [2]. The obtained results confirmed the segregation of Acacia s.l into Senegalia and Vachellia [1], where the generated dendrogram that based on the morphological and molecular attributes was divided into two main series one of them contained the taxa belonging to genus Vachellia, two species of Senegalia were nested within this group while the second group contained the taxa that belonging to Acacia s.s. Although the majority of Acacia s.s. spp. form a single series, the placement of A. pruniocarpa outside the main Acacia s.s. series and migrates to the Vachellia series represents a novel finding, indicating the close relationship among some taxa of Acacia s.s. and Vachellia that may be resolved by use of more morphological characters and genetic markers.
From the morphological results we can realize the benefits of using Light Microscope (LM) and SEM as diagnostic characteristics for separation and distinguishing between studied taxa of Acacia s.l. and the importance of seed description as a valuable taxonomic character.
The analysis of the twenty four taxa of Acacia s.l. with both markers RAPD and ISSR generated 709 bands, 111 from them are polymorphic bands (species-specific bands). This indicates that these markers can be used to characterize taxa used in this study [16,[35][36][37][38]. However, RAPD marker detects 64 polymorphic bands that are higher than polymorphic bands scored for ISSR (47) between different taxa of Acacia s. l.. This may be due to the ratio of coding and non-coding sequences within the genome and differences in genome composition of different taxa used in this study [39,40]. The combined data of both markers give more balanced results for genetic variations among studied taxa [24].
The results of RAPD and ISSR polymorphism showed differences between the same taxa of A. nilotica subsp. indica, one was collected from Al-Qassim and another one collected from Al-Riyadh. The dendrogram generated by both RAPD and ISSR markers data separated them from each other, although they have the same morphological characters. This is indicating the power of these markers to distinguish between closely related samples. The explanation of the molecular genetic variability between studied taxa is dependent on the sexual reproduction, in which low levels of genetic variation as often related with sexual propagation, in addition the differences of the geographical distributions of these taxa and the effect of abiotic factors of the environment; these agreed with [24,[41][42][43][44][45]. Also, Yadav et al. [46] demonstrated that climatic and geographical conditions have a great impact on genetic diversity of wild population of A. nilotica and also the high transferability of genetic resources from other related species of A. nilotica.
From the generated phenogram using UPGMA cluster method that was based on combined pooled data of RAPD, ISSR and morphological traits, we noticed that A. asak separated in a distinct entity while A. ehrenbergiana and A. johnwoodii grouped together, this is in agreement with [7], while Alaklabi [47] separated A. ehrenbergiana away from A. johnwoodii based on molecular characters investigated with nrDNA-ITS. So, the combined results of morphological and molecular attributes in this study concomitant together and solve this problems.
Acacia farnesiana and A. senegal were grouped together according to this study although, Al-Watban et al. [48] concluded that A. farnesiana was not closely related with A. senegal based on only morphological characteristics indicating that the reliance on only one tool for resolving the relationships among the studied taxa is not enough. Also. A. cyclops and A. victoriae grouped together this is not in agreement with [24] where they separate A. victoriae under distinct group based on two molecular markers examined with ITS and ETS, this may be due to the difference in the discrimination power of the markers that were used in both studies leading to the confirmation of that the more tools used, the more clearer the relationship.
The relationship between some taxa of Acacia s.l. of this study is with agreement with many previous studies. A. etbaica and A. origena found in one group this is in agreement with [49], A. gerrardii var. gerrardii and A. gerrardii var. iraquensis grouped together, this is in according with [50,51]. A. nilotica subsp. nilotica and A. nilotica subsp. tomentosa are in one clade [24,52] that demonstrated this result. A. tortilis subsp. tortilis and A. tortilis subsp. raddiana were grouped together in the present study, this is in according with [11] and [53]. A. coriacea was segregated early followed by A. ampliceps, while A. ligulata and A. salcina formed a sister taxa this agree with [54]. A. cuthberstonii and A. sclerosperma grouped together this agree with [55].

Conclusion
From this study, we can concluded that the complementary use of morphological and molecular data could enable accurate identification of the studied taxa of Acacia s.l., in addition, the results of RAPD and ISSR markers considered as powerful techniques for molecular characterization, detecting genetic polymorphisms that can discriminate between studied taxa and giving a clear view about the genetic diversity among them. The combined data gained from morphological and molecular traits succeeded for distinguishing and separated studied taxa into two major groups; Acacia s.s. group and Vachellia group that containing Senegalia taxa and give a valuable results that can be used for future taxonomical and genetic studies on genus Acacia s.l. so the segregation of Acacia s.l into Senegalia and Vachellia is confirmed.