Bayesian analysis—The dataset was analyzed with Bayesian inference using the program MrBayes version 3.2.7a. Sequence data were subjected to a general time reversal model including the estimation of invariant sites and assuming a discrete gamma distribution with six rate categories . The relative fit of various models of nucleotide substitution for the ITS region, chloroplast regions, and combined data set to identify the best model was examined. The best model was selected based on the Akaike Information Criterion . The Markov Chain Monte Carlo sampling, starting from random trees and priors, was run for 1,000,000 generations and every 100th tree was sampled. Four MCMC chains comprising three heated chains and a single cold chain were used in the analyses. Majority rule consensus trees and posterior probabilities for nodes were assembled from all post-burn-in sampled trees. Phylogenetic reconstructions were estimated after a couple of independent runs to confirm that they converged on similar stationary parameter estimates. For the combined data set, plastic nursery plant pot the data for each region were merged and aligned using muscle in MEGA before running on MrBayes.
Maximum Likelihood—For Maximum Likelihood estimation, different regions used different models. For the nuclear region, ITS 4 and ITS 5, sequence alignment was performed using the muscle tool included in the MEGA 10 suite, and the Kimura-2 parameter model was applied. For the chloroplast region involving trnHpsbA primer, alignment was also performed using muscle, but the best model was the Hasegawa-Kishino model. For the combined data, alignments were performed using muscle while the best model was the Tamura 3-parameter model. The bootstrap consensus tree inferred from 1000 replicates in Maximum Likelihood, was taken to represent the evolutionary history of the taxa analyzed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches in the trees generated.After extraction of DNA, poor bands were obtained during PCR for many of the species studied. This could be due to the old nature of the herbarium specimens used. Several primers were tried for the chloroplast region but for many of them, only very few specimens were amplified during PCR. The best result for chloroplast region was obtained from trnH-psbA. When samples were sent for sequencing, most of the results obtained for trnH-psbA were good for one direction, and rarely did we get good results for both directions. To protect the integrity of the results, only directions with clean DNA sequences were used for alignment and analysis. This problem with trnH-psbA that is reported here was also encountered by [2]. A higher number of successful PCR reactions were obtained for the nuclear ITS region than for the chloroplast regions, unlike the result reported by [2]. Sequences obtained for the ITS region ranged from 500 to 550 bp, while those for the chloroplast region were mostly below 500 bp. Although more sequences amplified for ITS, the tree generated for both nuclear and chloroplast regions were not significantly different, so the data were combined.
Clean reads that are suitable for analysis were obtained for the two primer sets used for this study, but more samples were amplified for the nuclear region. To combine the data, only samples that we got sequences for both regions were used. This is to ensure that none of the regions will influence the topology of the tree more than the other. Also, for ease of comparison of clades obtained from the combined dataset to clades from the separate regions.Tree from combined dataset is shown below, Figure 2. The values of the posterior probabilities of the branches obtained are used as measures of branch support. Values below 0.95 are considered to have very low support. All the trees obtained from Bayesian inference and Maximum likelihood show a close relationship between Englerophytum and Synsepalum. Trees from separate analyses are available from corresponding author.Neither Synsepalum nor Englerophytum were not resolved as monophyletic and some of the clades in the polytomy corresponded to the previously recognized genera by Aubréville. Three of the species previously grouped under the genus Afrosersalisia were used in this study namely S. afzelli, S. cerasifera, and S. kassneri. As seen in Figure 2, they belong to the same clade,—clade C. For the Vincentella genus, out of the four species, data were successfully obtained for three species. These include S. muelleri, S. passargei, and S. revoluta. None of the herbaria we loaned specimens from had S. brenanii. Just like the report for Afrosersalisia, the species are in the same clade, -clade A in Figure 2. The case is slightly different for the genus Pachystella. Out of the three species in Pachystella, two species are in the same clade, clade C, separating them from S. subverticillatum, which was found in clade A. For the genus Synsepalum sensu stricto, three species were previously recognized. These include S. dulcificum—the type species for the genus, S. stipulatum, and S. subcordatum. All three species were included in this study. In this group, all the species were in one clade, clade D.
It is important to note, however, that these three species were not the only species found in the clade, other species formed the same clade with them.Two principal issues are addressed in this work. One is the merging of the small genera recognized by [7], while the second issue is the divergent view of some authors on the Synsepalum–Englerophytum complex. The previously recognized small genera Afrosersalisia, Vincentella, Pachystela, Synsepalum, Tulesta. After the revision of Sapotaceae by Pennington in 1991, 19 more species have been added to the genus Synsepalum bringing the total to 35 species currently recognized. Some authors including have called for the separation of Synsepalum from the previous small genera that were combined. As seen in the combined data tree generated, some of the clades corresponded to the previous genera that were recognized by Aubréville. Data analysis using Bayesian inference and Maximum likelihood for ITS grouped all three species of Synsepalum sensu stricto used in this study into one clade. A dichotomous tree having species of Synsepalum in one clade and species of Englerophytum in another clade would have supported the monophyly of Synsepalum, however, the tree obtained from the nuclear, chloroplast region and combined dataset resulted in a phylogenetic separation of subgroups of species belonging to both genera. In the combined tree reported here, clades of Englerophytum are nested within Synsepalum. This consistent nesting of Englerophytum within Synsepalum further suggests that the genera are closely related. Leaf venation, presence or absence of stipules, inflorescence, fusion of sepals, stamens insertion, anther position, and number of ovaries tend to overlap in the previously merged genera. The overlapping morphological character and the molecular evidence seen in tree suggest very strongly that Synsepalum sensu lato should be reversed into small genera.Molecular evidence obtained from this study and all other phylogenetic studies involving the species merged by Pennington and the newly described ones for both Synsepalum and Englerophytum shows that the two genera are not phylogenetically distinct from each other. Thus, the trees obtained in this research do not agree with the circumscription of the genera as defined by Pennington. All the trees obtained using MrBayes for Bayesian inference and MEGA X for Maximum likelihood show that Englerophytum is nested within Synsepalum. It is important to state here that only a few morphological characters were used by [7] to separate Englerophytum from Synsepalum. Synsepalum has an eucamptodromous venation and Englerophytum has a brochidodromous venation and a strong fusion of thefilament into a staminal tube. Although it is taxonomically correct, in some cases, seedling starter pot to use few morphological characters to separate genera, this is not just the case with the EnglerophytumSynsepalum complex. The nesting of Englerophytum within Synsepalum in trees generated for both nuclear chloroplast regions indicates very strongly that the group do not merit distinct generic status.In this study of the phylogenetic relationships of the Synsepalum–Englerophytum complex, neither of the two genera resolved as monophyletic.
Although resolution of relationships among the major clades were only weakly to moderately supported, the clades of Englerophytum are nested within Synsepalum as seen in the combined tree. This shows that the morphological circumscription of the two genera does not align with the molecular evidence. We think that morphological character states that distinguish Synsepalum might be ancestral while the character states that distinguish Englerophytum may be independently derived synapomorphies for each subclade. The polytomy observed in the genus Synsepalum shows that the genus is not a single lineage. Some of the clades in the phylogenetic tree correspond to some of the small genera merged by Pennington. The need to resurrect some of the previously merged genera, like Vincentella, is undeniably evident in the result obtained in this work and that of [2]. The Synsepalum–Englerophytum complex certainly requires some formal taxonomic changes to align with molecular evidence, but such changes cannot be made until a broader sampling of all species currently recognized in the genera is collected for comprehensive morphological as well as molecular studies. The complete genome of Synsepalum has been published. If the complete genome of Englerophytum is published, it would serve as additional information relevant to making definitive and comprehensive conclusions on taxonomic circumscriptions within the Englerophytum–Synsepalum complex.After zinc, boron is the second mostimportant micronutrient deficiency problem in California vineyards. Boron deficiencies are most common in the old flood plains and alluvial fans of the Stanislaus, Merced, San Joaquin, Kings and Kaweah rivers; the Sierra Nevada foothills; and North Coast sites with basaltic soils subject to high rainfall. Vineyard boron deficiencies are mostly associated with soils derived from basaltic and granitic parent material of the Sierra Nevada and North Coast ranges. Low boron is also associated with higher rainfall areas and soils irrigated with snowmelt water originating from the Sierra Nevada. In contrast, boron levels are typically higher and can even be toxic in soils originating from marine sedimentary material, such as in the Central Coast range. Grapevine reproductive tissues are most sensitive to boron deficiency, which results in reduced fruit-set, small “shot berries” that are round to pumpkin-shaped, and flower and fruit cluster necrosis. Boron deficiency can have a drastic effect on fruit quality and yield, even when there are only mild-to-moderate foliar symptoms. At the same time, the over-application of boron can result in plant phytotoxicity. Phytotoxicity begins as a necrosis of the leaf margins that can cause a downward cupping of the young leaves. Most commonly, grape growers have applied boron to the soil by hand or as a direct soil spray, sometimes in combination with an herbicide application . However, such applications must be carefully timed to allow for winter rainfall or irrigation to move the boron into the root zone. Boron applications by foliar spray and drip irrigation are of increased interest for their convenience and the potential for faster vine response. Foliar boron application has been studied in tree crops such as pears, prunes, cherries and almonds, and the application timing was found to A boron-deficient Thompson Seedless cluster in the trial vineyard shows reduced fruit-set, the presence of numerous pumpkin-shaped “shot berries” and necrosis of some branching. Fewer than 10% of the berries are normal size and shape. influence fruit-set and development . However, there is limited research on vine uptake and response to foliar boron and the potential for toxicity. We conducted several studies on the timing of boron foliar-spray applications in an eastern Fresno County vineyard with mild-to-moderate boron deficiency symptoms.The studies were conducted in 1998 and 1999 in an own-rooted, furrow irrigated ‘Thompson Seedless’ grape vineyard on Delhi loamy sand. The vineyard was irrigated with canal water and about 25% supplementation from well water. The extremely low boron content of canal water can contribute to low boron availability in sandy vineyard soils. A preliminary study was conducted in 1998 to determine the influence of foliar sprays on boron concentrations in vegetative and reproductive parts of the vine at bloom. Spray treatment was applied at 2 1/2 weeks pre-bloom on May 6, 1998. The two comparative treat-ments were an untreated control and a foliar boron spray at 1 pound per acre applied as Solubor at 100 gallons per acre . The trial design was a randomized complete block with four-vine plots, replicated 10 times. Vine tissue samples were taken at bloom on May 23, 1998; triple-rinsed with distilled water and oven-dried; and analyzed for boron at the ANR Analytical Laboratory at UC Davis.