Jawad Ali Shah

Background/Objectives: The current research work aimed to evaluate the cryptic walnut genotypes of the Hazara region in Pakistan by using DNA barcoding and phylogenetic analysis. Methods: Based on morphological traits such as nut size, nut shape, and the number of leaflets, five genotypes were chosen and samples were collected for the current study. For molecular analysis, gDNA was isolated from the fresh leaves, and the five most effective angiosperm-specific markers, ITS2, rbcLa, rbcLc, rpoC1, and UBE3, were utilized. Based on amplification, sequencing, and identification success rates, ITS2 and UBE3 were recorded as the most efficient markers followed by rbcLa, rbcLc, and rpoC1. Results: During phylogenetic analysis, the query genotype-1 based on ITS2 and genotype-2 based on UBE3 clustered with (KF454101.1-Juglans regia) and (KC870919.1-J. regia) with bootstraps of 56 and 100, respectively. Genotype-3 based on rbcla clustered in a major clade with J. regia L., cultivars (MN397935.1 J. regia ‘Vina’) and (MN397934.1-J. regia ‘Serr’), (MN397933.1 J. regia ‘Pedro’), (MN397932.1 J. regia ‘Lara’), (MN397931.1 J. regia ‘Howard’), and (MN397930.1 J. regia ‘Hartley’) with bootstrap of 100. Meanwhile, genotype-4 and genotype-5 based on rbclc and rpoC1 clustered with (MN397935.1 J. regia ‘Vina’) and (MN397934.1 J. regia ‘Serr’), across the database sequences. To clarify the taxonomic status of cryptic walnut genotypes, it is necessary to combine diverse DNA barcodes. The results of ITS2 and UBE3, followed by rbcL barcoding markers, are promising taxonomic tools for cryptic walnut genotypes in Pakistan. Conclusions: It has been determined that the genotypes of walnuts in the study area are both J. regia L. and its cultivars and that the accuracy of discrimination regarding the genus Juglans L. is greater than 90%. The reported DNA barcodes are recommended for the correct identification and genetic evaluation of Juglans taxa and its population.

The management of understory vegetation and anthropogenic nitrogen (N) deposition has significantly resulted in a nutrient imbalance in forest ecosystems. However, the effects of canopy nitrogen addition and understory vegetation removal on N transformation processes (mineralization, nitrification, ammonification, and leaching) along with seasonal variations (spring, summer, autumn, and winter) remain unclear in subtropical forests. To fill this research gap, a field manipulation experiment was conducted with four treatments, including: (i) CK, control; (ii) CN, canopy nitrogen addition (25 kg N ha⁻¹ year⁻¹); (iii) UR, understory vegetation removal; and (iv) CN+UR, canopy nitrogen addition plus understory vegetation removal. The results revealed that CN increased net mineralization and nitrification by 294 mg N m⁻² month⁻¹ in the spring and 126 mg N m⁻² month⁻¹ in the winter, respectively. UR increased N mineralization and nitrification rates by 618 mg N m⁻² month⁻¹ in the summer. In addition, CN effectively reduced N leaching in the spring, winter, and autumn, while UR increased it in the spring and winter. UR increased annual nitrification rates by 93.4%, 90.3%, and 38.9% in the winter, spring, and summer, respectively. Additionally, both net N ammonification and annual nitrification rates responded positively to phosphorus availability during the autumn. Overall, UR potentially boosted nitrification rates in the summer and ammonification in the spring and winter, while CN reduced N leaching in the spring, winter, and autumn. Future research should integrate canopy nitrogen addition, understory vegetation removal, and phosphorus availability to address the global N deposition challenges in forest ecosystems.