Nurah M. Alzamel^*

Department of Biology, College of Science and Humanities, Shaqra University, Shaqra 11961, Saudi Arabia

*Corresponding author’s emails: nurahalzamel@gmail.com; nalzamel@su.edu.sa

Received: 06 October 2024 / Accepted: 06 May 2025 / Published Online: 24 May 2025

Abstract

Irrigation with saline water significantly reduces maize cultivation and productivity worldwide. Therefore, it is essential to investigate sustainable approaches to mitigate diverse effects of irrigation with saline water on various physiological and agronomical characteristics of maize, such as foliar application of selenium and proline. Two experiments, consequently, were carried out in the experimental farm of the College of Sciences and Humanities, Shaqra University, Al-Quwayiyah Governorate, Kingdom of Saudi Arabia during the two successive autumn seasons of 2022 and 2023. Three saltwater irrigation treatments were separately applied using a randomized complete block design with a split-plot arrangement with three replications. In each one of these, three rates of selenium were distributed in the main plots, whereas three rates of proline were applied in the sub-plots. Selenium and proline applications, individually or combined, significantly reduced the effects of saltwater irrigation stress on physiological and agronomic traits of maize. Obviously, optimal results were achieved with selenium at 10 mg L^-1 and proline at 10 mM, effectively mitigating the adverse effects of saltwater irrigation on the physiological and agronomic traits of maize. At a moderate salinity level of 2000 mg L^-1 NaCl, the application of 5, and 10 mM proline improved grain yield per plant by 7.76 and 5.54 %, as well as 17.62 & 7.99%; grain yield per hectare by 7.18 and 7.72 %, along with 19.11 & 8.33 %; and stover yield per hectare by 8.56 and 6.87 %, as well as 16.38 and 9.05 %, in both seasons, respectively under the application of 5, and 10 mg L^-1 selenium, respectively, as compared to severe salinity level of 3000 mg L^-1 NaCl. This in turn showed that exogenous selenium and proline application, individually or combined, were effective strategies in mitigating the harmful effects of salinity on physiological and agronomic traits of maize.

Keywords: Grain yield, Maize, Proline, Saline irrigation stress, Selenium

Asim Sultan¹, Muhammad Kashif Saleemi¹, Muhammad Saqib², Shafia Tehseen Gul^1*

¹Department of Pathology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan

²Department of Clinical Medicine and Surgery, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan

*Corresponding author’s email: drshafia66@yahoo.com; dr.shafia.gul@uaf.edu.pk

Received: 27 January 2025 / Accepted: 06 April 2025 / Published Online: 04 May 2025

Abstract

In the present study, Clostridium perfringens (C. perfringens) was isolated from clinically suspected cases of necrotic enteritis. For this purpose, the intestinal samples collected, after enrichment in thioglycolate broth were inoculated on perfringens-specific media (tryptose sulphite cycloserine agar) in an anaerobic jar having a gas pack. After incubation of 24hrs at 37°C, characteristic black colonies on TSC agar were used for confirmation by gram staining. Molecular confirmation was carried out with species-specific 16 s rRNA primer and DNA was amplified further for different toxin genes i.e. cpa, cpb, etx, iap, cpe, and net B for toxinotyping of C. perfringens. These isolates were further used to induce necrotic enteritis in poultry birds and for this purpose experimental trials were carried out to compare the ameliorative effects of antimicrobial alternatives i.e. Tibetan tea extract, black tea extract. and they were compared with commercial available AGPs. Data was analyzed through analysis of variance (ANOVA) through SAS University Edition. Results of the experimental trial demonstrated that there is a significant improvement in physiological performance and immunity of the birds supplemented with tea extracts and exposed to the NE challenge. The current study finding conclude that the both Tibetan and Black tea extracts can be used separately or in combination for the control of necrotic enteritis in poultry. Tea phenolics have proved their potential as an alternative to antimicrobials and to address antimicrobial resistance in poultry.

Keywords: Necrotic enteritis, Herbal tea extract, Immunity, Physical parameters

SM Azizul Karim Hussaini¹, Farhan Ibne Siddique², Mokammel Hossain Tito⁸, Abdullah Al Mamun¹, Md Arifuzzaman³, Afzal Haq Asif⁴, Shahzad Khan⁵, Muhammad Shahzad Chohan⁵, Samar Sindi⁶, Talha Yusuf ⁷, Md. Siddiqur Rahman¹*

¹Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Bangladesh

²Armed Forces Medical College, Dhaka Cantonment, Dhaka, Bangladesh

³Department of Civil & Environmental Engineering, King Faisal University, Saudi Arabia

⁴Department of Pharmacy Practice, College of Clinical Pharmacy, King Faisal University, Saudi Arabia

⁵Department of Biomedical Sciences, College of Clinical Pharmacy, King Faisal University, Saudi Arabia

⁶Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Saudi Arabia

⁷Department of Computer Sciences, FCIT, King Abdulaziz University, Saudi Arabia

⁸Department of Medicine, Gazipur Agricultural University, Bangladesh

*Corresponding author’s email: siddique.medicine@bau.edu.bd

Received: 23 January 2025 / Accepted: 15 April 2025 / Published Online: 04 May 2025

Abstract

Brucellosis is a highly contagious zoonotic disease caused by the bacterium Brucella spp. that distresses mutually animals and humans, especially in underdeveloped countries with poor control programs. In adult cattle, the disease affects mainly the reproductive organs, thus causing major losses in production and reproduction, such as abortion and reduced fertility. This study involves the collection of 460 blood samples from dairy farms, which were analysed for brucellosis infection using the Rose Bengal Test (RBT). Additionally, data on the animals’ history, including placenta (retained), repeat breeding, their age, abortion, and lastly calving, were also recorded. To address the problem of class imbalance between the positive and negative classes, a technique, known as Synthetic Minority Over-sampling Technique (SMOTE) was applied in the research work. A total of five algorithms were used in this paper among them multilayer perceptron (MLP) and weekadeeplearning4j showed the best results for the prediction of brucellosis having 93.59% and 93.94% accuracy, respectively. Besides, risk factors are ranked based on their importance as ordered as retained placenta > repeat breeding > calving > abortion > age, and three association rules are made to understand the correlation of the factors for occurring the disease. By applying this study, early diagnosis of the disease could be possible to mitigate the economic losses.

Keywords: Brucellosis, Zoonotic disease, Dairy cattle, Machine learning, Risk factors, Public health

Qurat ul Ain¹, Muhammad Adil Rasheed^1*, Imran Tariq², Muhammad Ovais Omer¹, Muhammad Yasir Zahoor³

¹Department of Pharmacology and Toxicology, Faculty of Biosciences, University of Veterinary and Animal Sciences, Lahore, Pakistan

²Punjab University College of Pharmacy, University of the Punjab, Lahore, 54000, Pakistan

³Institute of Biochemistry and Biotechnology, Faculty of Biosciences, University of Veterinary and Animal Sciences, Lahore, Pakistan

*Corresponding author’s email: dr_aadil@uvas.edu.pk

Received: 06 December 2024 / Accepted: 11 March 2025 / Published Online: 25 March 2025

Abstract

Lipid-polymer hybrid nanoparticles (LPHNPs) are innovative composite structures featuring a core-shell design, with a polymeric nanoparticle core surrounded by PEGylated lipid layers. This architecture combines liposome circulation with nanoparticle strength, offering a promising solution for multidrug-resistant cancers. The current study aimed to develop an integrated platform that leverages these advantageous characteristics. To achieve this, Tamoxifen-loaded nanoparticles were formulated using emulsion solvent evaporation technique with PLGA poly (lactic-co-glycolic) acid polymer, while Doxycycline-containing liposomes were developed through a film hydration method. These two components were then combined to fabricate LPHNPs. A range of physicochemical and structural analyses, including dynamic light scattering (DLS), laser diffraction analysis (LDA), and scanning electron microscopy (SEM), were performed. Cellular cytotoxicity was quantitatively assessed using the MTT assay, and biocompatibility was evaluated through in vivo acute toxicity tests on female albino mice. The average hydrodynamic sizes of Tamoxifen and Doxycycline formulations were 198.80 ± 2.10 nm and 100.5 ± 1.29 nm, respectively. After coating Tamoxifen nanoparticles with Doxycycline liposomes, resultant LPHNPs exhibited a diameter of 200.4 ± 2.51 nm and a zeta potential of 4.45 ± 2.51 mV. Notably, LPHNPs demonstrated a significant increase in cytotoxicity (p < 0.001) and showed reduced in vivo toxicity compared to free drugs, with no discernible toxicity observed in histopathology of vital organs, confirming their safety and efficacy.

Keywords: Cytotoxicity, Tamoxifen, Doxycycline, Nanoparticles, Cancer

Wardah Wardah¹, Ridwan Ridwan^2*, Marwan Setiawan¹, Riska Ayu Purnamasari¹, Asep Sadili¹, Siti Sundari¹, Emma Sri Kuncari¹, Dini Ariani³, Parwa Oryzanti¹, Gusmaini², Dibyo Pranowo², Handi Supriadi², Tintin Febrianti⁴

¹ Research Center for Ecology and Ethnobiology, National Research and Innovation Agency, Indonesia

² Research Center for Estate Crops, National Research and Innovation Agency, Indonesia

³ Research Center for Food Technology and Processing, National Research and Innovation Agency, Indonesia

⁴ Faculty of Agriculture, Garut University, Indonesia

*Corresponding author’s email: ridw009@brin.go.id

Received: 17 July 2024 / Accepted:28 February 2025 / Published Online: 18 March 2025

Abstract

Tacca (Tacca leontopetaloides (L.) Kuntze) is a tuberous plant commonly growing in coastal areas, both in open and shaded areas. This study aimed to identify the agronomical characteristics and metabolite content of Tacca growing under various levels of natural shading. The research was conducted in the southern coastal area of Garut Regency (West of Java-Indonesia) by observing the plant growth and production, and metabolite content of Tacca tubers under conditions of natural full sunlight (0% shade/FL), moderate shade (40% shade/MS), and heavy shade (75% shades/HS) in three different locations as replications. Three mature plants (±7 months old) in each shade level and replication were randomly selected as observed samples. The result presented that Tacca’s growth increased with the increasing shading levels. Nonetheless, tuber production showed the highest value under MS, which increased by 76.64% and 35.20% compared to FL and HS, respectively. Fourteen metabolites were detected in Tacca tuber grown in MS and FL, while only eight metabolites were detected in HS. The highest concentration of the metabolites obtained in MS were 8 metabolites (57%) and followed by FL and HS with 3 (21%) and 1 metabolite (7%), respectively. Moderately shaded areas have great potential to be used as cultivation land to produce high-quality Tacca tubers.

Keywords: Coastal area, Light intensity, Metabolites, Shading

Muhammad Shahid Khan¹, Tariq Hussain², Saeed Ahmad Buzdar³, Muhammad Raza Hameed⁴, Muhammad Taslim Ghori⁵, Aliza Maheen⁶, Nadeem Ali⁷, Beenish Imtiaz⁸, Muhammad Rafi Qamar⁵, Ahmed Saleh Summan⁹, Arooj Ali^3*, Rashid Iqbal¹⁰, Riaz Hussain^11*

¹Department of Physics, University of Okara, Pakistan

²Department of Basic Sciences, College of Veterinary and Animal Science, Jhang, Pakistan

³Institute of Physics, Faculty of Physical and Mathematical Sciences, The Islamia University, Bahawalpur 63100, Pakistan ⁴Department of Pathobiology, Faculty of Veterinary Sciences, Bahauddin Zakariya University Multan Pakistan ⁵Department of Clinical Medicine and Surgery, Faculty of Veterinary & Animal Sciences, The Islamia University of Bahawalpur, 63100, Pakistan

⁶Department of Zoology, The Islamia University of Bahawalpur, 63100, Pakistan

⁷Centre of Excellence in Environmental Studies, King Abdulaziz University, Jeddah, Saudi Arabia

⁸Department of Animal Nutrition and Nutritional Disease, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Türkiye

⁹Faculty of Environmental Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

¹⁰Department of Life Sciences, Western Caspian University, Baku, Azerbaijan

¹¹Department of Pathology, Faculty of Veterinary & Animal Sciences, The Islamia University of Bahawalpur, 63100, Pakistan

*Corresponding authors’ emails: aroojali4088@gmail.com; dr.riaz.hussain@iub.edu.pk

Received: 28 September 2024 / Accepted: 22 January 2025 / Published Online: 06 March 2025

Abstract

Vibrating sample magnetometry (VSM), transmission electron microscopy (TEM), and X-ray diffraction (XRD) were used to characterize iron oxide nanoparticles (IONPs) synthesized via co-precipitation. TEM showed that the synthesized NPs had an average size of 13.8 nm and exhibited a saturation magnetization of 50.26 emu/gm, while XRD confirmed their crystallinity. This study assessed the toxicological impacts of IONPs on hematology, serum biochemistry, genotoxicity, and the histoarchitecture ailments in different visceral organs (liver, kidneys, spleen, brain, and heart of rabbits (Oryctolagus cuniculus). A total of 16 healthy adult rabbits without infections were randomly assigned to two groups (A and B). Group A was the control whereas group B received 0.5 mg/kg bw of IONP dose once for 10 days via marginal ear vein. Visceral tissues and blood were obtained from each rabbit on days 5 and 10 of trial. The results showed a significant reduction in antioxidant enzymes and an increase in oxidative stress. The hematological profile indicated lower values of red blood cell counts, hematocrit, lymphocyte, and monocyte while significantly higher values of total white blood cell counts and neutrophil (%) in IONPs-treated rabbits. Serum biomarkers of the liver, kidneys, and heart exhibited escalated concentrations in IONPs-treated rabbits. Histopathological examination revealed notable tissue alterations like necrosis of hepatocyte, congestion, and bile duct hyperplasia in the liver, neuronal degeneration, necrosis, and microgliosis in the brain, tubular degeneration, necrosis, and congestion in the kidneys and disorganization of cardiac myofibers and edema in cardiac tissue. A significantly increased DNA damage was assessed in multiple visceral organs of treated rabbits. In conclusion, our findings demonstrate that exposure to IONPs induces toxic effects in multiple visceral organs including the kidneys, liver, spleen, heart, and brain in rabbits.

Keywords: Magnetic Oxide nanoparticles (MNPs), VSM, ROS production, Hemato-biochemistry, DNA Damage, Histopathology

Ummul Buneen Zafar^1,2, Iqrar Ahmad Rana^1,2*, Sultan Habib Ullah Khan^1,2, Rana Muhammad Atif ^1,3

¹Center of Agricultural Biotechnology and Biochemistry, University of Agriculture, Faisalabad, Pakistan

²Center for Advanced Studies in Agriculture and Food Security, University of Agriculture, Faisalabad, Pakistan

³Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan

*Corresponding author’s email: iqrar_rana@uaf.edu.pk

Received: 30 July 2024 / Accepted: 05 February 2025 / Published Online: 06 March 2025

Abstract

The Shisham (Dalbergia sissoo) population is threatened by the fungal pathogens causing dieback disease. Research over the past decade has shown the involvement of multiple fungal pathogens in causing the dieback disease. Here we reported the RAPD-PCR-based genetic diversity in three potential fungal pathogens including Botrydiplodia theobromae, Fusarium solani, and F. oxysporum isolated from diseased plants across the Punjab province in Pakistan. Twenty fungal isolates of three major fungal species were isolated from dieback-infected trees from eight districts in Punjab Pakistan to investigate the genetic diversity with a set of fifteen RAPD markers of OPB, OPK, and OPL series.  Consequently, fifteen markers produced 166 loci with an average of 55.33 loci per population. Of these, 76.31% loci were polymorphic highlighting the presence of abundant genetic diversity in pathogens. Moreover, OPK-06 primer showed a higher PIC value of 0.416 along with higher heterozygosity contents of 0.49. However, population structure analysis of each fungal strain grouped similar and dissimilar ones based on their amplification into the same and different clusters respectively. The isolates of B. theobromae of the Ayub Agriculture Research Institute (AARI) and Attock district showed higher genetic distance (0.6812) and were found in different clusters. Similarly, isolates of F. solani from UAF formed a different group which further highlighted its degree of polymorphism. Moreover, the UAF isolate displayed a maximum of 0.6432 genetic distance from the AARI isolate. Conversely, the isolates of F. oxysporum were grouped into two main groups highlighting the limited genetic diversity. Further, isolates from UAF also exhibited a maximum of 0.7372 genetic distance from AARI. Consistent results of UAF and AARI isolates of F. oxysporum and F. solani are suitable grounds for further genetic studies.

Keywords: Fusarium oxysporum, RAPD, Botrydiplodia theobromae, Fusarium solani, Dieback, Pathogens

Muharib Alruwaili^1*†, Sonia Younas ^2,3†, Muhammad Umer Khan^4*, Hammad Saleem⁵, Yasir Alruwaili^1,6, Abualgasim Elgaili Abdalla¹, Bi Bi Zainab Mazhari⁷, Khalid Abosalif¹, Hasan Ejaz¹

¹Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Sakaka 72388, Saudi Arabia

²Centre for Immunology and Infection (C2i), Hong Kong Science and Technology Park, Hong Kong, SAR China

³HKU-Pasteur Research Pole, School of Public Health, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China

⁴Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore, 54590, Pakistan

⁵Institute of Pharmaceutical Sciences (IPS), University of Veterinary & Animal Sciences (UVAS), Lahore, Pakistan

⁶Sustainable Development Research and Innovation Center, Deanship of Graduate Studies and Scientific Research, Jouf University, Sakaka 72388, Saudi Arabia

⁷Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Qurayyat 75911, Saudi Arabia

^† These authors contributed equally

*Corresponding authors’ emails: mfalrwaili@ju.edu.sa; muhammad.umer4@mlt.uol.edu.pk

Received: 30 November 2024 / Accepted: 27 January 2025 / Published Online: 06 March 2025

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen, especially affecting individuals with weakened immune systems. Laccase enzymes are pivotal in its pathogenicity, making them promising targets for therapeutic intervention. This study aims to identify and characterize potent laccase inhibitors against C. neoformans using advanced in-silico analysis. The laccase protein (UniProt ID: Q55P57) was retrieved via AlphaFold and validated with ProCheck. Pharmacophore-based virtual screening (PBVS) identified 19 potential inhibitors, which were docked using CB-Dock2. The top six compound’s pharmacokinetic properties were assessed using SwissADME, PKCSM, and StopTox. Bioactivity was predicted via SwissTargetPrediction. Density Functional Theory (DFT) calculations were conducted using Gauss view 5.0.8. The validated 3D structure of the target protein Q55P57 demonstrated high quality, with 86.5% of residues in favored regions. The molecular docking revealed that L-11 exhibited the highest binding affinity (-13.2 kcal/mol), forming crucial interactions within the active site. L-11 displayed favorable physicochemical properties, including high lipophilicity and good Caco2 permeability, positioning it as a strong candidate for therapeutic development. Toxicity predictions indicated non-toxicity for acute inhalation and oral exposure, while bioactivity analysis highlighted its broad target interactions. DFT analysis demonstrated L-11’s enhanced reactivity due to its high dipole moment and low HOMO-LUMO energy gap. The identification of L-11  (8-[4-[9,9-Dimethyl-7-(2,3,4,5,6,7,8,9,10-nonahydroxypyren-1-yl)fluoren-2-yl]phenyl]pyrene-1,2,3,4,5,6,7,9,10-nonol) as a potent inhibitor of C. neoformans laccase represents a novel approach to antifungal drug discovery, marking a significant step to combat fungal infections and a way forward to perform in-vitro and in-vivo studies and ultimately its clinical application.

Keywords: C. neoformans, Ellagic acid, Laccase inhibitor, Molecular docking, ADMET, Antifungal

Mohei EL-Din Solliman¹*, Heba Allah A. Mohasseb¹

¹Plant Biotechnology Department, College of Agricultural and Food Sciences, King Faisal University, Al-Ahsa 31982, Saudi Arabia.

*Corresponding author’s email: msolliman@kfu.edu.sa

Received: 01 December 2024 / Accepted: 23 January 2025 / Published Online: 22 February 2025

Abstract

Dioecious plants, like Date palm (Phoenix dactylifera) and Jojoba (Simmondsia chinensis), are a type of plant that do not have sexual identification in the same way that animals or humans do. Sexual identification in plants typically involves determining the sex of individual plants within a dioecious species. Date palms and jojoba are dioecious, with male and female trees, and their sexual identification is important in agriculture for proper pollination, but until now it doesn’t involve methods like RPA (Recombinase Polymerase Amplification) or CRISPR. We successfully developed a CRISPR/Cas12a-based fluorescence test for the sexual identification of date palm and jojoba as models for dioecious, with RPA and CRISPR-Cas12a, which could be used as a simple, accurate, and fast detection method in agricultural fields due to its advantages of high-sensitivity. This investigation uses our proprietary technology to detect sub-kilobase non-repetitive genomic loci, CRISPR-FISH. In this study, we designed 16 different sgRNAs using full-length Human SRY as the substitute target in date palm and jojoba, given the extremely high homology between Human SRY and Date Palm SRY gene. These sgRNAs were labeled them 3 Atto565 dyes per sgRNA to visualize them at a single-cell level. Fluorescence in situ hybridization (FISH) has been employed as a potent and practical method for directly identifying certain DNA segments inside the genome. In this method, plant chromosomes are physically mapped using ribosomal DNA genes (45S and 5S rDNA) as markers to examine genomic organization. The initial result for SRY staining in a substitute cell line, the Human A549 cell, a male cell line containing a single copy of SRY was promising albeit with high background staining. Prominent dots (red) in the nuclei region were observed, but with high background staining in both nuclei and cytosol. This refined technique is perfect for quickly localizing tiny DNA segments and single-copy genes. Additionally, PRINS is a quicker and less expensive option than FISH. Furthermore, the successful application of the assay for Sexual identification of some Dioecious plant samples highlighted its potential for rapid and accurate sex detection in agricultural settings. In summary, this RPA-CRISPR/Cas12a diagnostic method offers a potentially valuable technological solution and management for Dioecious plant sex-determination at very early stage.

Keywords: Dioecious plants, Date palm; Jojoba; female; male; sex determination; FISH; CRISPR/Cas12a, RPA-CRISPR/Cas12a

Iqra Shah^1,2*, Muhammad Qasim Hayat^1*, Umme Laila¹, Saleem Haider¹, Kaleem Ullah³, Beenish Anjum¹, Madeeha Khan¹, Alvina Gul¹, Summya Malik¹

¹Plant Systematics and Evolution Laboratory, Department of Agricultural Sciences and Technology, Atta ur Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan

²Department of Plant Sciences, University of California, Davis, 95616, CA, USA

³Department of Biotechnology, Faculty of Science and Technology, University of Central Punjab, Lahore, Pakistan

*Corresponding author’s email: m.qasim@asab.nust.edu.pk; ishah.phdabs15asab@student.nust.edu.pk

Received: 21 July 2024 / Accepted: 27 January 2025 / Published Online: 22 February 2025

Abstract

Cyperaceae, commonly known as a sedge family, is widely distributed and is cosmopolitan, it is the third largest among the monocots. It has great ecological and ethnobotanical value but the taxonomic classification of the family at both morphological and molecular level is not much studied. The species resemble a lot and hence are very difficult to differentiate without proper identification. For the taxonomic implication of nut (one of the important parts of inflorescence) in plant identification, sixteen different sedge species were collected from various parts of the country. They were analyzed under stereo and scanning electron microscope to understand the differences among nut characters which help to differentiate among species of the same family. The three most important characteristics considered were Nut shape, size and color. All the species showed variation enabling identification easily. The surface of nut was also observed under the scanning electron microscope and showed great variation. Principal Component Analysis (PCA) was performed to check the impact of characters on the grouping of the species. Based on the results of this study, it can be said that nuts play a very important role in the identification and classification of the plant on a morphological and micromorphological basis.

Keywords: Sedges, Nut, Scanning Electron Microscopy, Stereomicroscopy, Micromorphology, PCA, UPGMA