Correlational analysis of halotolerant palynomorphs peculiarities by integrating morphological and morphometric characterization using scanning electron microscopy

Abstract

Pollen analysis of halophytes is crucial for their accurate identification and taxonomic classification within respective families. This study aimed to highlight the systematic pollen analysis of 15 dominant halotolerant species from twelve families collected in the Kalabagh Salt Range, Pakistan. Light microscopic (LM) and scanning electron microscopic (SEM) techniques were used to examine the pollen’s discriminative traits that support the phenetic grouping and morphometric differentiation among halotolerant taxa. Pollen exine sculpture varied from punctate, verrucate, microechinate, perforate, to reticulate. Pollen shape also exhibited variance ranging from oblate-spheroidal (4 spp.), prolate (3 spp.), prolate-spheroidal (3 spp.), subprolate (3 spp.), Oblate (1 sp.) to spheroidal (1 sp.). The high pollen viability value of Euphorbia caducifolia (92.75%) describes its ecological adaptation to a saline environment. Principal component (PCA) and cluster analysis were used to analyze pollen morphometric data, revealing strong affinities among the studied halotolerant. Morpho pollinic traits, combined with advanced systematic tools, are crucial for identifying lineages within morphogroup taxa and may contribute to the taxonomic position of halotolerant plant species.

Introduction

Halotolerant plants are highly versatile, diverse, and worthy scientific models with a high level of salt tolerance resulting from various morphological, biochemical, and physiological traits, such as salt secretion, osmotic adjustment, reactive oxygen species detoxification, ion compartmentalization, and other mechanisms that need further exploration^1,2. The optimal salinity for the halophytes’ growth ranges from 50 to 250 mM NaCl³. Halophytes have adaptive mechanisms that enable them to withstand high levels of salinity and to complete their entire life cycles⁴. Halotolerant taxa exhibit a remarkable diversity of form and functions⁵. Halophytes are the basic and integral component of the saline ecosystem. The reconstructed vegetation dynamics of the past can be utilized to infer associated environmental conditions. Therefore, halophytes are considered the best indicators of climatic and ecological changes, both spatially and temporally^6,7.

Halophytism evolved several times throughout the plant kingdom. All plant kingdoms contain members that have adaptations for saline habitats and are mainly distributed in semi-arid and arid inlands along the temperate zones^3,8. According to Lombardi⁹, 6000 species of marine and terrestrial plants, and 2% of angiosperms are classified as halophytes. The complexity of the halophytic group is also linked to the multitude of ecosystems. The small number of halophytic floras in the world supports the fact that salt tolerance appeared later in evolution, and it is not a basic trait in terrestrial plants¹⁰. The potential of halophytes in the form of land cover cannot be denied, as they function as sand dune binders and prevent soil erosion¹¹.

Palynology, as a conserved character, has high genetic stability and is more stable than external macrographical morphology, because it is scarcely affected by changes in environmental conditions. Pollen’s characteristics are of potential significance in plant taxonomy and are utilized to infer genetic relationships between inter-specific taxonomic levels or among ecological groups¹². Palynology helps to understand the phylogenetic relationship of plants and assemble new pollen traits from extant vegetation¹³. Pollen symmetry, polarity, exine sculpture, and type and number of apertures are significant traits phylogenetically¹⁴. The inaperturate pollen is considered a derivative trait as it is closely linked with the evolution of exine structure¹⁵.

The pollen’s morphological features are often used as additional diagnostic characters in taxonomy¹⁶. For example, Fagonia and Zygophyllum belong to Zygophyllaceae, grow in similar saline habitats, and exhibit overlapping vegetative characters. The medium-sized, isopolar, subprolate shape with tectate exine pollen of Fagonia species serves as a reliable diagnostic characters to differentiate them from Zygophyllum species, which show small size, prolate-spheroidal pollen with microreticulate-foveolate exine, despite similar ecological adaptations¹⁷. Numerous studies have exhibited pollen observations as effective evidence for the identification and classification of plant species at both specific and generic levels^18–20. Recent advances in microscopic technologies facilitate the resolution of taxonomic issues. Light and scanning electron microscopy help in the evaluation of the taxonomic relevance of taxa by unveiling the pollen’s ultra-sculpture²¹. Scanning electron microscopy (SEM) provides qualitative ultrastructural insights into the specimen morphology²².

Halotolerant taxa are included in several groups of plants that need pollen characterization for identification and discrimination in a taxonomic hierarchy²³. A rich diversity of halophytes in Pakistan represents 19% of its flora, with 410 species reported²². Ashfaq et al²⁴. conducted a study on the palynological characteristics of 12 Convolvulaceae taxa from Pakistan’s arid regions and confirmed the significance of pollen morphology in species characterization and identification. Khan et al²⁵. declared the Aizoaceae as an eurypalynous family. Elkordy and Faried²⁶ studied pollen morphological and numerical analysis of 06 Tamarix species of Egypt. They exhibited radially symmetrical, small, and isopolar pollen and identified two major clades of morphological traits. Woutersen et al²⁷. examined the 8 halophytic pollen taxa of family Nitrariaceae and analyzed chemical and morphological traits of Peganum species using SEM, LM, and Fourier Transform Infrared spectroscopy (FTIR).

Pollen characters of three varieties of Fagonia Taxa were studied by Taia et al²⁸. in Libya and exhibited tricolporate, prolate, and subprolate shape pollen. They declared genus Fagonia as the most difficult genus in its species circumscription. Euphorbiaceae exhibit succulents to large desert trees, and Euphorbia is one of the largest and widespread genera in tropical, subtropical, and temperate zones. The taxonomic significance of qualitative and quantitative palynological traits of tropical Euphorbia species was observed by Shah et al²⁹., who declared it a cosmopolitan genus. Nguyen et al³⁰. analyzed pollen morphology of Cleomaceae species of Vietnam using LM and SEM to understand their phylogenetic relationship and significance in taxonomic delimitation. They reported monad, tricolporate, and prolate-shaped pollen in Cleome species. Baser et al³¹. provided solutions to taxonomical problems of Polygonaceae species using palynological information in Turkey and reported 3-colpate, pantoporate, prolate pollen with reticulate and microechinate-perforate exine sculpture. Palynological studies are significant for group taxonomy and support conservation and reproductive research³². Pollens’ studies of any particular geographic region unravel the intricate relationships between location, climatic variability, and pollen characteristics³³.

This study aimed to contribute to the knowledge of the pollen flora of halotolerant plant species that inhabit the Kalabagh Salt Range. The characterization of 15 halotolerant palynomorphs and their viability utilizing advanced microscopic techniques was analyzed for the correct identification and delimitation of taxa at species and genus levels. Multivariate analysis, such as principal component and cluster analysis, was performed to identify the affinities among the studied taxa. It is the first novel halotolerant palynomorphs study from the Kalabagh Salt range, Punjab, Pakistan, that was conducted to understand the relationship between pollen traits and environmental stresses and to compile the comparative palynological database of halotolerant plants of the Kalabagh Salt range that can aid as a reference system for evolutionary, ecology, and conservation studies.

Materials and methods

Plant collection and sampling site

A total of 15 wild and dominant halotolerant species belonging to 12 genera were collected from the Kalabagh region of the Salt Range, Punjab, Pakistan (Fig. 1). The Kalabagh Salt Range is located on the western bank of the Indus River at 32° 57’ 57.60” N and 71° 33’ 10.80” E. in Mianwali District, Punjab, Pakistan. Kalabagh Salt Range is famous for its red hills and traditional handicrafts. QGIS ver. 3.34³⁴ was used to create a map of the Kalabagh Salt Range and plant collection sites. Sterile forceps were used to take fresh stamens from 6 specimens of each plant collected during the flowering season of the year 2024. The samples were collected from public land in the Salt Range with the permission of the central regional plant protection and biodiversity stations. Salt-tolerant plants were pressed, dried, and preserved. Catalogue of Life³⁵ was accessed to identify the collected specimens. Dr. Muhammad Zafar (Professor of ISL Herbarium) confirmed the identification of plant specimens. The World Flora Online was accessed to validate and authenticate the botanical names of collected specimens³⁶. Plants were mounted on Herbarium sheets that provide detailed information about each taxon, including their botanical name, local name, family, altitude, and collected site. Plants were assigned a voucher number and deposited (Deposition number: HALO-ISL/9931/24) at the Herbarium of Pakistan (ISL) in Quaid-i-Azam University, Islamabad. A complete taxa list, their full voucher data, and reproductive morphological characters are given in Table 1.

Fig. 1.

Map of Kalabagh Salt Range, Punjab, Pakistan, created using QGIS ver. 3.34 (https://qgis.org/).

Table 1.

Halotolerant species sampling and their reproductive morphological characters.

Taxon	Status	Voucher specimen number	Habit	Life cycle	Flowering period	Inflorescence type	Flower type	Sepals	Petals	Collector name	Altitude (m)
Zaleya pentandra (L.) C.Jeffrey	Wild	ISL.240,082	Herb	Perennial	Mar-Dec	Solitary, axillary	Hermaphrodite, actinomorphic	Oblong ovate, pale green	Oval, white	Moona Nazish	219
Malcolmia africana (L.) R.Br.	Wild	ISL.240,009	Herb	Annual	Mar-July	Raceme	Cruciform, actinomorphic	Ovate or oblong, purple	Oblanceolate, pink	Moona Nazish	181
Cleome viscosa L.	Wild	ISL.240,067	Herb	Annual	June-Oct	Racemose	Solitary, axillary, asymmetrical	Oblong-lanceolate, green	Oblong-obovate, yellow	Moona Nazish	235
Cressa cretica L.	Wild	ISL.240,093	Shrub	Perennial	Mar-Oct	Solitary	Sub-sessile, actinomorphic	Obovate, pinkish white	Oblong-ovate, white	Moona Nazish, Asif Kamal	233
Citrullus colocynthis (L.) Schrad.	Wild	ISL.240,088	Herb	Perennial	April-July	Solitary	Solitary monoecious, actinomorphic	Lanceolate, pale yellowish green	Ovate-acute, yellowish green	Moona Nazish	232
Euphorbia caducifolia Haines	Wild	ISL.240,011	Shrub	Perennial	April-Aug	Cyathium	Pseudanthium, actinomorphic	Turbinate, green	Globose, yellow	Moona Nazish, Asif Kamal	251
Euphorbia granulata Forssk.	Wild	ISL.240,037	Herb	Perennial	May-Oct	Cyathia axillary	Cyathia, actinomorphic	Absent	Globose, yellowish white	Moona Nazish, Asif Kamal	225
Peganum harmala L.	Wild	ISL.240,056	Herb	Perennial	Mar-Sept	Solitary	Hermaphrodite, actinomorphic	Linear, green	Oblong-elliptic, yellowish white	Moona Nazish	185
Plantago lanceolata L.	Wild	ISL.240,075	Herb	Perennial	Mar-June	Spicate	Capitate, actinomorphic	Obovate, brown	Narrowly ovate, brownish white	Moona Nazish, Asif Kamal	224
Polygonum effusum Meisn.	Wild	ISL.240,063	Herb	Annual	June-Sep	Axillary, cymes	Spikes, actinomorphic	Tepals	5 tepals (obovate), Pink	Moona Nazish	235
Polygonum maritimum L.	Wild	ISL.240,081	Subshrub	Perennial	July-Oct	Solitary/cluster, axillary	Inconspicuous, spikes, actinomorphic	Tepals	5 tepals (oblong elliptic), Whitish-pink	Moona Nazish	186
Salvadora oleoides Decne.	Wild	ISL.240,015	Tree	Perennial	Mar-June	Axillary panicles	Sessile, actinomorphic	Obtuse, pale green	Spathulate, Pale green	Moona Nazish, Asif Kamal	234
Tamarix salina Dyer	Wild	ISL.240,018	Shrub	Perennial	Apr-July	Raceme, paniculate	Regular, sub-sessile, actinomorphic	Obtuse, Pink	Obovate, violet	Moona Nazish	251
Tamarix smyrnensis Bunge	Wild	ISL.240,045	Shrub	Perennial	Mar-Aug	Raceme, panicle	Hermaphrodite, zygomorphic	Ovate, green	Elliptic, Pink	Moona Nazish	219
Fagonia indica Burm.f.	Wild	ISL.240,042	Shrub	Annual	Mar-Sep	Solitary	Hermaphrodite, actinomorphic	Ovate, green	Spathulate, pinkish-purple	Moona Nazish	237

Light microscopic analysis of pollens of halotolerant species

Several flowers were collected randomly from each plant. Pollen measurements were based on fully developed 30 pollen grains per individual plant specimen, as recommended by Qiu et al³⁷.. Fresh anthers were taken for light microscopy. Anthers were placed in a test tube and crushed in 70% alcohol using a glass rod and filtered. The alcohol was decanted, and the remaining sediment was covered with glacial acetic acid and acetolysis mixture and centrifuged. The acetolysis mixture was prepared by mixing one part of concentrated sulphuric acid and nine parts of acetic anhydride. The tube containing the mixture was placed in a hot water bath to obtain brownish black pollen grains. Then, the test tube was cooled, and pollen samples were gently transferred from the test tube to glass slides³⁸.

The Wodehouse technique was followed to stain the pollen using glycerin gel mixed with 1% safranin³⁹. The safranin was prepared using the method of Ali et al⁴⁰.. A cover slip was placed on glass slides and sealed with paraffin wax. Five slides were prepared per specimen to serve as biological replicates, ensuring measurement consistency. The prepared slides were observed under a Meiji Techno, Iruma-gun Saitama, Japan, biological microscope (model no. MX5300H). Qualitative data were observed, and quantitative data were measured using 40x magnification. Measurements of each plant species, including pollen size, colpus length, exine thickness, colpus width, and polar and equatorial diameters, were obtained from duplicate measurements of each pollen grain to minimize technical errors. Pollen shape was determined based on the P/E ratio. Halbritter et al⁴¹. classification was followed to identify the different pollen types. Pollens micrographs were taken using a Light microscope (Leica Dialux, Mannheim, Germany; Model 1000) at different resolutions. The measured pollen characters are given in Table 2.

Table 2.

Descriptive statistics of SOC stocks before sowing and after harvesting for soybean and wheat.

Family/plant name	Pollen type	Pollen shape	Colpi	Polar diameter (µm) min (mean ± std error) Max	Equatorial diameter (µm) min (mean ± std error) Max	P/E ratio	Colpi length (µm) min (mean ± std error) Max	Colpi width (µm) min (mean ± std error) Max	Exine thickness (µm) min (mean ± std error) Max	Pollen viability (%)	Exine sculpturing
Aizoaceae Zaleya pentandra (L.) C.Jeffrey	Tricolpate	Subprolate	+	12.25(13.35 ± 0.5)15.25	12.25(13.5 ± 0.54) 15.5	0.98	3.75(4.85 ± 0.37) 6.0	3.5(4.65 ± 0.35) 5.5	0.5(0.6 ± 0.06) 0.75	89.14	Punctate
Brassicaceae Malcolmia africana (L.) R.Br.	Tricolpate	Prolate	+	15.25(17.75 ± 1.05)21.0	16.5(18.9 ± 0.81) 21.5	0.93	4.0(4.35 ± 0.15) 4.75	3.0(3.3 ± 0.14) 3.75	2.25(2.6 ± 0.15) 3.0	87.64	Reticulate
Cleomaceae Cleome viscosa L.	Tricolporate	Prolate	+	13.0(17.3 ± 1.51) 21.50	23.75(27.0 ± 1.23) 29.75	0.64	5.75(6.10 ± 0.12) 6.50	4.75(5.10 ± 0.15) 5.50	1.50(1.85 ± 0.12) 2.25	87.68	Reticulate-rugulate
Convolvulaceae Cressa cretica L.	Tricolpate	Prolate	+	19.5(22.85 ± 1.36) 25.75	23.0(30.4 ± 2.63) 36.25	0.75	4.5(5.1 ± 0.20) 5.5	3.25(3.85 ± 0.23) 4.5	2.75(3.0 ± 0.07) 3.25	82.69	Tectate-perforate
Cucurbitaceae Citrullus colocynthis (L.) Schrad.	Tricolpate	Oblate- spheroidal	+	19.75(23.15 ± 1.39)28.0	18.0(21.60 ± 1.41) 25.25	1.07	3.00(3.25 ± 0.11) 3.50	3.25(3.55 ± 0.09) 3.75	2.75(3.10 ± 0.12) 3.50	88.15	Reticulate
Euphorbiaceae Euphorbia caducifolia Haines	Tricolporate	Oblate- spheroidal	+	13.50(16.55 ± 1.52)24.1	13.55(17.75 ± 1.33) 21.55	0.93	1.75(2.05 ± 0.11) 3.14	2.15(3.62 ± 0.12) 3.20	1.25(2.15 ± 0.25) 2.0	92.75	Reticulate
Euphorbia granulata Forssk.	Tricolporate	Oblate- spheroidal	+	14.25(17.25 ± 0.88) 20.45	15.35(17.75 ± 0.71) 20.35	0.97	1.41(1.85 ± 0.21) 2.35	3.45(3.60 ± 0.19) 3.8	1.35(1.55 ± 0.25) 2.35	91.45	Reticulate
Nitrariaceae Peganum harmala L.	Tricolporate	Oblate-spheroidal	+	19.75(20.10 ± 0.18)20.75	17.25(19.25 ± 0.57) 20.50	1.04	3.25(4.85 ± 0.45) 6.0	2.75(3.70 ± 0.36) 4.50	1.25(1.55 ± 0.14) 2.0	82.81	Reticulate
Plantaginaceae Plantago lanceolata L.	Tricolporate	Spheroidal	+	17.75(21.95 ± 1.39)25.50	23.50(26.95 ± 1.17) 30.25	0.81	1.86(2.83 ± 0.25) 4.30	2.25(2.70 ± 0.18) 3.75	1.25(1.45 ± 0.09) 1.75	86.17	Verrucate
Polygonaceae Polygonum effusum Meisn.	Tricolporate	Subprolate	+	13.5(17.75 ± 0.7) 16.5	14.5(14.65 ± 0.5) 13.75	1.21	4.20(5.55 ± 0.30) 5.25	4.25(3.09 ± 0.39) 6.35	3.0(3.5 ± 0.25) 4.0	89.25	Microechinate, Perforate
Polygonum maritimum L.	Tricolporate	Subprolate	+	13.45(16.5 ± 0.29) 15.5	11.5(13.25 ± 0.8) 13.0	1.24	6.25(5.0 ± 0.35) 6.0	3.55(7.5 ± 6.0) 7.55	1.65(2.25 ± 0.11) 3.35	90.45	Psilate
Salvadoraceae Salvadora oleoides Decne.	Tricolpate	Prolate-spheroidal	+	17.25(19.95 ± 1.05)23.0	20.75(22.85 ± 0.60) 24.0	0.87	4.25(4.9 ± 0.26) 5.75	3.0(10.55 ± 7.36) 40.0	1.25(1.9 ± 0.26) 2.75	86.86	Micro-reticulate
Tamaricaceae Tamarix salina Dyer	Tricolpate	Prolate-spheroidal	+	11.0(19.55 ± 0.91)15.25	10.0(19.05 ± 1.12) 17.0	1.02	3.40(5.35 ± 0.23) 4.35	3.50(4.25 ± 0.12) 4.50	1.35(1.5 ± 0.05) 1.25	85.67	Reticulate
Tamarix smyrnensis Bunge	Tricolpate	Prolate-spheroidal	+	17.0(18.55 ± 0.12) 16.75	14.25(17.05 ± 0.85) 20.05	1.08	3.75(4.7 ± 0.35) 3.75	3.45(5.5 ± 0.49)6.05	1.5(1.75 ± 0.17) 1.75	82.70	Reticulate
Zygophyllaceae Fagonia indica Burm.f.	Tricolporate	Oblate	+	10.75(13.4 ± 1.08)15.50	22.25(25.3 ± 0.90) 27.75	0.52	6.25(7.3 ± 0.33) 8.25	9.0(9.45 ± 0.14) 9.75	1.0(1.45 ± 0.16) 2.0	87.67	Reticulate

SEM analysis of halotolerant species pollens

Pollen samples were examined using a scanning electron microscope (SEM) at the Central Resource Laboratory, University of Peshawar. Prior to SEM analysis, pollen grains were subjected to the standard acetolysis procedure²¹ to remove cytoplasmic content and enhance exine visibility. The treated pollen grains were then mounted on polished aluminum stubs (12.5 mm diameter) using conductive adhesive tape. The stubs were sputter-coated with a 25 nm layer of gold-palladium using a JEOL JSM-5910 SEM (Peabody, USA) to enhance conductivity and image resolution. SEM observations were carried out at an accelerating voltage of 15 kV and a working distance of 10 to 15 mm, with magnifications ranging from 3,000 to 10,000X to capture detailed pollen morphology, including exine ornamentation and apertural features. Morphometric measurements, such as pollen diameter, polar axis, equatorial axis, and aperture dimensions, were taken from twenty independent pollen grains per species to ensure statistical reliability and account for natural intraspecific variation. The terminology and classification of pollen features followed the guidelines of Hesse et al⁴².. All SEM micrographs were acquired under uniform brightness and contrast settings to allow consistent comparison among species.

Statistical analysis

The mean, maximum, minimum, and standard deviation values of the examined pollen characters were determined using GNU PSPP (ver 1.6.2). The pollen grains’ polar and equatorial diameters, exine thickness, colpus lengths, and widths were described quantitatively. The P/E ratio was calculated for each taxon using the formula P/E ratio = P/E × 100, based on the same pollen polar to equatorial diameter. P represents the polar axis, and E represents the equatorial axis of the same pollen grain.

Pollen viability shows the pollen longevity and dispersion ability. Identification of viable pollen is crucial for conservators and breeders and provides reasonable indications of pollen’s ability to grow and germinate⁴³. Pollen’s viability was determined through their staining capacity by adding 2 drops of 1% acetocarmine. Five slides of each plant species were prepared and examined under an optical microscope (Zeiss microscope 3014, Germany) at 10× magnification. Inviable or aborted pollen grains were stained yellow, while viable pollen grains were stained pink⁴⁴. Pollen viability was calculated using the formula, Pollen viability = F/(F + S) ×100, where F represents the viable pollen grains and S represents the inviable pollen grains.

Interrelationships of the evaluated characters were analyzed using Principal Component Analysis (PCA). XLSTAT (ver. 2018) used pollen metrical variables, including P/E ratio, exine thickness, polar axis, equatorial axis, colpus length, colpus width, and pollen viability for variance evaluation. The Euclidean distance matrix of the standardized data was subjected to cluster analysis by an unweighted pair group method with arithmetic mean (UPGMA). OriginPro 2021 was used to make a cluster heatmap. Statistical differences were evaluated to exhibit the patterns of linkage among halophytes based on pollen data.

Results

Fifteen halotolerant species (Zaleya pentandra, Malcolmia africana, Cleome viscosa, Cressa cretica, Citrullus colocynthis, Euphorbia caducifolia, Euphorbia granulata, Peganum harmala, Plantago lanceolata, Polygonum effusum, Polygonum maritimum, Salvadora oleoides, Tamarix salina, Tamarix smyrnensis, and Fagonia indica) belonging to twelve families (Aizoaceae, Brassicaceae, Cleomaceae, Convolvulaceae, Cucurbitaceae, Euphorbiaceae, Nitrariaceae, Plantaginaceae, Polygonaceae, Salvadoraceae, Tamaricaceae, and Zygophyllaceae) were analyzed for pollens morphological traits using light and scanning electron microscopic techniques. These families and their representative species were selected due to their dominance in the Kalabagh Salt range. A detailed account of qualitative (pollen type, shape, and exine sculpture) and quantitative (polar and equatorial diameters, colpi length and width, exine thickness, P/E ratio, and pollen viability) palyno-morphological traits of the studied halotolerant species is given in Table 2. Figures 2, 3, 4, and 5 represent pollen’s light and scanning electron micrographs. Figures 6 and 7 illustrate the annotation plot and Sankey diagram of pollen traits of halotolerant species, which exhibit variations among pollen size, shape, aperture, and exine sculpture that can be used as potential taxonomic attributes in the identification and delimitation of similar plant group species at the family and genus level. Figure 8 represents the rows and columns of the data matrix in a cluster heatmap that are ordered according to the clustering output. Different colors exhibit pollen’s morphometric variables for easy recognition that include polar and equatorial diameters, colpi length and width, exine thickness, P/E ratio, and viability values.

Fig. 2.

LM pollens photomicrographs of halotolerant species at 100X magnification (a) Zaleya pentandra, (b) Malcolmia africana, (c) Cleome viscosa, (d) Cressa cretica, (e) Citrullus colocynthis, (f) Euphorbia caducifolia, (g) Euphorbia granulata, (h) Peganum harmala, (i) Plantago lanceolata, (j) Polygonum effusum, (k) Polygonum maritimum, (l) Salvadora oleoides, (m) Tamarix salina, (n) Tamarix smyrnensis, (o) Fagonia indica.

Fig. 3.

Scanning electron microscopic pollens micrographs of halotolerant species. Zaleya pentandra (1,2), Malcolmia africana (3), Cleome viscosa (4,5), Cressa cretica (6,7), Citrullus colocynthis (8,9).

Fig. 4.

Scanning electron microscopic pollens micrographs of halotolerant species. Euphorbia caducifolia (10,11), Euphorbia granulata (12,13), Peganum harmala (14,15), Plantago lanceolata (16,17), Polygonum effusum (18).

Fig. 5.

Scanning electron microscopic pollens micrographs of halotolerant species. Polygonum maritimum (19), Salvadora oleoides (20), Tamarix salina (21,22), Tamarix smyrnensis (23), Fagonia indica (24).

Fig. 6.

Annotation graph showing variations among P/E ratio and pollens viability of the studied species.

Fig. 7.

Sankey diagram exhibiting comparative pollen morphological traits of halotolerant species.

Fig. 8.

Cluster heatmap of pollen’s morphometric variables of halotolerant taxa.

Pollens’ description of Aizoaceae halotolerant taxa

Family Aizoaceae’s pollens are typically stenopalynous⁴⁵. The subprolate-shaped tricolpate pollens were observed in Zaleya species. The polar and equatorial diameters were 13.35 μm and 13.5 μm, respectively. The exine thickness of 0.6 μm has been noted for selected salt-tolerant species. Punctate exine sculpture was observed. The mean length and width of colpi were 4.85 μm and 4.65 μm. The polar to equatorial ratio was 0.98 μm. Z. pentandra pollens showed 89.14% viability.

Pollens’ description of Brassicaceae halotolerant taxa

Pollen’s morphology has provided a systematic relationship among different genera of Brassicaceae⁴⁶. The pollens of the examined halotolerant species were monad and tricolpate. The prolate-shaped pollen with reticulate exine ornamentation was observed. The equatorial diameter (18.9 μm) was the maximum as compared to the polar diameter (17.75 μm). The exine thickness was 2.6 μm. The mean length and width of colpi were 4.35 μm and 3.3 μm, respectively. A high pollen viability value (87.64%) was shown by the examined species in the Salt Range. In the annotation plot of pollen traits (Fig. 6), the Brassicaceae species Malcolmia africana grouped with intermediate polar and equatorial diameters, showing close resemblance to other mid-sized pollen taxa such as Euphorbia caducifolia and Euphorbia granulata. This visualization highlights that Brassicaceae pollen was not extreme in size but exhibited high viability, distinguishing it from species with either very large or very small pollen grains.

Pollens’ description of Cleomaceae halotolerant taxa

Tricolporate and monad pollens were observed in Cleome viscosa. The pollen shape was prolate with reticulate-rugulate exine sculpture. The equatorial diameter of pollens was found to be maximum (27.0 μm) as compared to the polar diameter (17.3 μm). The P/E ratio was 0.64. Pollen exhibited an exine thickness of 1.85 μm. Colpi were greater in length (6.10 μm) than in width (5.10 μm). Pollens showed an 87.68% viability rate for the Salt range. In the pollen trait plot, C. viscosa appears distinct from most species due to its larger equatorial diameter and relatively small polar axis, reflected in its low P/E ratio. Its placement close to Fagonia indica demonstrates how the plot allows comparison across families, showing convergent pollen dimensions in unrelated taxa (Fig. 6).

Pollens’ description of Convolvulaceae halotolerant taxa

Cressa cretica is a thermo-cosmopolitan halophyte that usually grows in muddy or sandy saline habitats^47,48. C. cretica, with other succulent plants, characterizes thermophilous vegetation that colonizes abandoned fields in the Salt range after farming on saline soil. The prolate-shaped tricolpate with tectate-perforate exine sculpture was observed in C. cretica pollens. The equatorial diameter (30.4 μm) was greater than the polar diameter (22.85 μm). Pollens showed an exine thickness of 3.0 μm. The polar-to-equatorial ratio was 0.75 for the selected salt-tolerant species. The length of colpi (5.1 μm) was greater than the width (3.85 μm). Pollens exhibited 82.69% viability. In the annotation plot, C. cretica is positioned at the far right with the largest polar and equatorial diameters among all studied species (Fig. 6). This separation emphasizes its morphological distinctness and explains its isolation into its own clade in cluster analysis. The graph also shows that the largest pollen size and low viability than many other taxa, illustrating how the visualization integrates multiple traits (size, shape, viability, and P/E ratio) to compare halotolerant species.

Pollens’ description of Cucurbitaceae halotolerant taxa

Citrullus colocynthis is a perennial plant that grows in warmer climatic conditions, such as on sub-desert soils and sandy loams⁴⁹. The monad, oblate-spheroidal, and tricolpate pollens were found in C. colocynthis. Reticulate exine sculpture was examined with 3.10 μm of exine thickness. The polar and equatorial diameters were 23.15 μm and 21.6 μm. Colpi width was observed to be slightly greater than length, which is 3.55 μm and 3.25 μm, respectively. The noted P/E ratio was 1.07, and pollen viability was 88.15%. In the Sankey diagram of pollen traits (Fig. 7), C. colocynthis is clearly grouped within the tricolpate aperture type, linked to its oblate-spheroidal shape and reticulate exine sculpture. This visualization highlights the structural pathway from aperture to shape to surface ornamentation, showing how the species fits into broader trait categories and how its combination of traits differentiates it from other halotolerant taxa.

Pollens’ description of Euphorbiaceae halotolerant taxa

Euphorbiaceae is a palynologically heterogeneous family⁵⁰. Tricolporate pollens were observed in Euphorbia caducifolia and Euphorbia granulata. Oblate-spheroidal pollen with reticulate exine sculpture were examined. Similar pollen traits exhibit interspecific relations and specify the position of plants of the same group in the same family. Both halotolerant species exhibited similar equatorial diameters (17.75 μm) but different polar diameters, which were 16.55 μm in E. caducifolia and 17.25 μm in E. granulata. E. granulata exhibited a greater P/E ratio of 0.97. Exine thickness varies from 1.55 μm (E. granulata) to 2.15 μm (C. caducifolia). The results revealed the maximum colpi length and width in pollen of C. caducifolia (2.05 μm and 3.62 μm) as compared to E. granulata. E. caducifolia showed high pollen viability (92.75%). In the Sankey diagram, both Euphorbia species are mapped to the tricolporate aperture type, associated with an oblate-spheroidal shape and reticulate exine. Their placement in the same structural flow of the diagram reinforces the close interspecific relationship inferred from their similar pollen morphology, while still reflecting subtle differences in size and exine thickness.

Pollens’ description of Nitrariaceae halotolerant taxa

Peganum is present in semiarid to arid regions of Central Asia⁵¹. The monad, tricolporate, and oblate-spheroidal pollen were exhibited by Peganum harmala. Reticulate exine sculpture has been observed. The polar diameter (20.1 μm) was slightly greater than the equatorial diameter (19.25 μm). Exine thickness was 1.55 μm. The colpi length (4.85 μm) was greater than the colpi width (3.7 μm). The noted polar-to-equatorial ratio was 1.04. The value of pollen viability was 82.81%. In the cluster heatmap of morphometric traits, P. harmala grouped closely with species sharing oblate-spheroidal shape and reticulate exine, confirming its phenetic similarity to other salt-tolerant taxa with comparable pollen dimensions. This visualization allows us to see how P. harmala bridges families with similar pollen morphology despite taxonomic distance (Fig. 8).

Pollens’ description of Plantaginaceae halotolerant taxa

The family Plantaginaceae is stenopalynous in nature⁵². Tricolporate and spheroidal pollens with verrucate exine ornamentation were examined in Plantago lanceolata. The equatorial diameter was 26.95 μm while the polar diameter was 21.95 μm. Exine thickness of 1.45 μm was measured. The P/E ratio was 0.81, whereas the value of pollen viability was 86.17%. The pore length and width were 5.35 μm and 5.15 μm in the examined Plantago species. In the heatmap, P. lanceolata was clearly separated from the rest due to its unique verrucate exine and spheroidal shape (Fig. 8). This distinct clustering highlights the diagnostic value of its pollen traits and underlines its divergence from other halotolerant families.

Pollens’ description of Polygonaceae halotolerant taxa

Polygonum genus has a polyphyletic origin and eurypalynous in nature⁵³. Tricolporate pollens having subprolate shapes were found in both investigated Polygonum species. Microechinate, perforate exine ornamentation was observed in P. effusum, while psilate was observed in P. maritimum. P. effusum exhibited greater polar and equatorial diameters (17.75 μm and 14.65 μm) as compared to P. maritimum (16.5 μm and 13.25 μm). Exine thickness in P. effusum (3.5 μm) was also found greater than in P. maritimum (2.25 μm). A slight variation was observed in the P/E ratio of P. effusum (1.21) and P. maritimum (1.24). P. effusum showed greater colpi length (5.55 μm) and P. maritimum showed greater colpi width (7.5 μm). Pollen viability was maximum in P. effusum (90.45%). The cluster heatmap placed both Polygonum species close to each other, but a subtle distance between them reflected their differences in exine ornamentation (microechinate-perforate and psilate) and exine thickness. This graphical grouping confirmed interspecific diversity within the same genus.

Pollens’ description of Salvadoraceae halotolerant taxa

The tricolpate and prolate-spheroidal pollen were studied in S. oleoides. The pollen with micro-reticulate exine sculpture and 1.9 μm of exine thickness were examined. The polar and equatorial diameters were 19.95 μm and 22.85 μm. A polar-to-equatorial ratio of 0.87 was noted for the halotolerant S. oleoides. The colpi width (10.55 μm) was greater than the colpi length (4.9 μm). Pollens exhibited a high viability value (86.86%). In the heatmap, S. oleoides clustered tightly with other tricolpate and prolate-spheroidal taxa, underscoring the strong consistency of its pollen traits with the general Salvadoraceae profile.

Pollens’ description of Tamaricaceae halotolerant taxa

Tamarix is the most complex genus taxonomically among angiosperms that has reticulate evolution⁵⁴. The subprolate-shaped pollens with reticulate exine were examined in both studied Tamarix species. Tricolpolate pollens with polar (19.55 μm) and equatorial diameter (19.05 μm) were examined in T. salina, while polar (18.55 μm) and equatorial diameter (17.05 μm) were noted for T. smyrnensis. Both salt-tolerant species show slight variation in exine thickness. The P/E ratio ranges from 1.02 (T. salina) to 1.08 (T. smyrnensis). The length of colpi was greater in T. salina (5.35 μm) than T. smyrnensis (4.7 μm). The slight difference in pollen viability values was noticed in T. salina (85.67%) and T. smyrnensis (82.70%). The heatmap revealed that the two Tamarix species formed a close sub-cluster, reflecting their shared tricolpate, subprolate, and reticulate traits (Fig. 8). However, their slight morphometric differences (polar/equatorial diameters and colpi dimensions) were sufficient to maintain a measurable distinction between them.

Pollens’ description of Zygophyllaceae halotolerant taxa

Fagonia indica primarily has a distribution in salt-affected lands⁵⁵. It is a woody shrub that can grow up to 60 cm in height. It is greenish gray with basal branches. Tricolporate pollens with reticulate exine were observed in F. indica. The polar and equatorial diameters were 13.4 μm and 25.3 μm, respectively. The P/E ratio was 0.52, which determines the oblate shape of pollen. The colpi width (9.45 μm) was greater than the colpi length (7.3 μm). The measured exine thickness was 1.45 μm. Fagonia pollen exhibited high viability (87.67%). In the cluster heatmap, F. indica occupied a position among tricolporate species with reticulate exine, showing strong grouping with taxa of similar morphology. This placement visually reinforced its phenotypic alignment within Zygophyllaceae and its broader similarity with other halotolerant plant families.

Principal component analysis (PCA)

Principal Component Analysis (PCA) is a statistical approach used to assess a set of factors and highlight the variations among them, reducing dimensionality while providing insights into taxonomic relationships within taxa (Fig. 9). For this analysis, variables were selected based on their biological relevance and variability among species, including polar and equatorial diameters, colpus length and width, pollen viability, and the P/E ratio of pollen grains from 15 halotolerant species. Before PCA, the data were standardized using z-score normalization to ensure that variables with different units and scales contributed equally to the analysis. The standardized dataset allowed accurate comparison and minimized bias due to scale differences. The overall variance explained by PC-1 was 35.8%, with equatorial diameter, colpus length, and width identified as the most significant contributors. On the positive side of the first axis, Fagonia indica, Salvadora oleoides, and Cleome viscosa were positioned, while Tamarix salina, Peganum harmala, and Cressa cretica occupied the negative side. PC-2 accounted for 30.1% of the variance, with pollen viability, polar diameter, P/E ratio, and exine thickness being the most influential variables. Positive and negative sides of PC-2 included species such as Zaleya pentandra, Polygonum maritimum, Euphorbia granulata, and Euphorbia caducifolia, versus Polygonum effusum, Malcolmia africana, and Citrullus colocynthis, respectively. The analysis revealed that species on the positive side of PC-1 generally exhibited larger colpi length and width and greater equatorial diameters, whereas species on the negative side showed smaller polar diameters and thinner exine. This approach ensures that the PCA results are robust, reproducible, and accurately reflect interspecific variations in pollen morphology.

Fig. 9.

Principal component analysis (PCA) of the halotolerant taxa exhibiting pollens morphometrical features.

Cluster analysis

Cluster analysis (UPGMA) was employed to investigate the phenetic relationship of reported halotolerant taxa. Variables for the analysis were carefully selected based on their biological relevance and contribution to interspecific variability, including quantitative traits such as polar and equatorial diameters, pollen size, shape, and exine thickness, as well as qualitative traits like aperture type and exine ornamentation patterns. Prior to clustering, all quantitative data were standardized using z-score normalization to ensure that differences in measurement scales did not bias the Euclidean distance calculations. Morphological traits of pollen from 15 halotolerant species were used to perform cluster analysis, which revealed strong affinities among plants of the complex group, halophytes. The halotolerant species were categorized into two main clusters using a dendrogram (Fig. 10). Cluster I contained eight species, while cluster II contained seven species, reflecting contrasts and similarities in pollen morphological traits. A high degree of similarity was observed between C. viscosa and F. indica in cluster I as compared to P. effusum and P. maritimum. In cluster II, T. salina and T. smyrnensis exhibited great similarity as compared to P. lanceolata. Similarly, M. africana and C. colocynthis were placed in the same clade due to a lower Euclidean distance between their morphological pollen traits, while C. cretica was placed in a separate clade. The clusters were primarily defined based on variation in quantitative pollen traits, such as size (polar axis and equatorial diameter), shape, and exine thickness, as well as qualitative traits like aperture type and ornamentation patterns. The strong similarity between C. viscosa and F. indica can be attributed to their nearly identical pollen sizes, aperture (tricolporate), and exine sculpture, while species such as P. effusum and P. maritimum differed in both size range and surface ornamentation, placing them further apart in the analysis. The dendrogram provides significant insight into the taxonomic relationships of halotolerant species by reflecting morphological similarities based on phenetic palyno-taxonomical traits.

Fig. 10.

Cluster dendrogram of halotolerant species based on pollens morphological characters.

Discussion

Halotolerant taxa contribute to the biodiversity and primary productivity of the saline habitats^56,57. This study provides a detailed account of the pollen’s morphological traits of 15 wild halotolerant plants belonging to Aizoaceae, Brassicaceae, Cleomaceae, Convolvulaceae, Cucurbitaceae, Euphorbiaceae, Nitrariaceae, Plantaginaceae, Polygonaceae, Salvadoraceae, and Tamaricaceae families. Morpho pollinic traits study of a variety of taxa belonging to different families could be of immense aid in plant systematics^58–60. Palynological characterization aids in exploring important aspects of systematic study that predict the evolution of problematic and closely related taxa⁶¹.

Salt ranges constitute a habitat of high conservation value and potential ecological interest⁶². The Kalabagh Salt Range of North Punjab is a remote area surrounded by mountains and naturally enriched with halophytic floral diversity. Increasing salinization in the Salt Ranges is a major threat to food security in Pakistan. Plants with high salt tolerance are one of the probable options for the economic use of saline soil. In arid regions of Pakistan, saline lands are abundant due to low precipitation and high evaporation, which brings up most of the water via capillary rise from the soil surface, resulting in the salt accumulation on the soil’s surface^63,64.

Pollen characterization using advanced microscopic techniques is a key method for taxonomic verification, as distinct traits act as diagnostic markers for taxa identification²². High resolution imaging plays a critical role in decoding the evolutionary details contained within the pollen’s microscopic morphology⁶⁵. The halotolerant Zaleya pentandra exhibited tricolpate and subprolate pollens in this study. The palyno-morphological features of 9 plants from 6 genera of the family Aizoaceae were studied by El-Naga et al⁶⁶., who reported subprolate and tricolporate pollen types in Zaleya pentandra, which is consistent with our findings. The prolate-shaped 3-zonocolpate pollens with acute ends, punctate-scabrate and spinulate exine sculpturing in Zaleya pentandra were examined by Osman and Hassan⁶⁷ in Egypt and Sudan. The studies on southern African Aizoaceae plants have suggested a relationship between their members and arid environmental conditions⁶⁸.

The highly dominant salt-tolerant plant of the family Brassicaceae, Malcolmia africana, was collected from the Salt range of Kalabagh, Pakistan. Brassicaceae is a stenopalynous family that has tricolpate pollen with reticulate surface ornamentation⁶⁹. Tricolpate pollen with reticulate exine in the genus Malcolmia were reported by Noor et al⁷⁰., similar to our study. Exine sculpture plays a significant role in the family Brassicaceae by showing variations among genera within the same tribe and among species within the genera⁷¹. Umber et al⁴⁶. reported that Eruca vesicaria, a glycophyte of the family Brassicaceae, exhibited small, oblate-spheroidal pollen with psilate exine. High viability (87.64%) was exhibited by M. africana pollen, which is an indispensable factor and one of the prerequisites that show successful fertilization in the respective environment⁷².

Cleomaceae include mostly herbaceous plants, widely distributed in subtropical and tropical regions⁷³. Cooke⁷⁴ placed Cleome in the family Capparidaceae, but now this genus is taxonomically separated and treated as a separate family, i.e., Cleomaceae⁷⁵. Perveen and Qaiser⁷⁶ also reported tricolporate and trilobed pollen with regulate-reticulate exine in C. viscosa. Aparadh and Karadge⁷⁷ observed a similar pollen type. Exine ornamentation has diagnostic importance in the Cleome genus and supports the species classification⁷⁸. Mir et al⁷⁹. studied prolate shape tricolporate pollens in C. viscosa, like our results. Nguyen et al³⁰. examined pollen characters of two glycophytes, Cleome houtteana and Cleome gynandra, and observed echinate and striate-foveolate exine. The reticulate exine is an adaptive feature that provides mechanical stability and enhanced viability under salt stress in halophytes.

Convolvulaceae is one of the dominant families in arid regions where the weather remains unpredictable and vegetation is sparse⁸⁰. Ashfaq et al²⁴. observed the palyno-morphological characters of family Convolvulaceae taxa and described their eurypalynous nature. Our results indicated prolate-shaped pollens with tectate-perforate exine sculpture in halotolerant C. cretica. Abu-Serag et al⁸¹. also reported monad and medium-sized pollen in C. cretica, consistent with our findings. Al-Anbari et al⁸². studied pollen of Cuscuta sp., a non-halotolerant plant of the family Convolvulaceae, and reported tricolporate pollen having microreticulate exine sculpture.

The morphometric pollen characters provide additional information to resolve conflicts based on macro-morphology³⁷. C. colocynthis exhibited tricolpate, oblate-spheroidal pollens with reticulate exine in this study. Jarret et al⁸³. examined Citrullus spp. pollen and revealed their tricolpate, prolate, and reticulate exine ornamentation in all species, which confirmed our findings. Perveen and Qaiser⁸⁴ investigated 10 pollen taxa belonging to 7 genera of Cucurbitaceae using SEM and LM, and observed tricolporate, oblate-spheroidal to prolate or subprolate pollen with a reticulate exine in C. colocynthis. The large, tricolporate, prolate pollen was observed in the glycophyte Citrullus lanatus by Sadia et al⁸⁵.. The medium-sized pollen with reticulate columellae in C. colocynthis was examined by Alhomaidi⁸⁶ to highlight the potential of morpho pollinic traits in plant systematics.

Euphorbia is the 2nd largest genus of flowering plants, including xerophytic and succulent plants characterized by diagnostic morphological synapomorphy and cyathium inflorescence⁸⁷. Both studied halotolerant species exhibited tricolporate, oblate-spheroidal pollens with reticulate exine. Tangsariya and Sharma⁸⁸ also reported tricolporate apertures in Euphorbia spp. Reticulate exine sculpture in Euphorbia spp. was also observed by Effiom et al⁸⁹.. Kürşat⁹⁰ examined 27 Euphorbia pollen taxa and reported tricolporate and tricolpate pollen with reticulate exine sculpture and oblate-spheroidal shape pollens in 20 out of 27 species. Odabaşı et al⁹¹. observed perforate exine ornamentation in non-saline Turkish E. helioscopia and E. oblongata. Alyas et al⁹². reported oblate-spheroidal shape tricolporate pollens with reticulate exine ornamentation in salt-tolerant E. indica and E. serpens.

P. harmala is a perennial herbaceous plant native to the Mediterranean region and Central Asia⁹³. Semerdjieva and Yankova-Tsvetkova⁹⁴ studied P. harmala pollen in Bulgaria, highlighting its distinctive size and reticulate exine sculpture, consistent with our results. In our study, P. harmala exhibited tricolporate, oblate-spheroidal pollen with reticulate exine sculpture. Fazal et al⁹⁵. and Koyuncu et al⁹⁶. also observed tricolporate pollens with striate-rugulate and reticulate exine in P. harmala. Pollen characterization of the genus Peganum was examined by Woutersen et al²⁷., who reported monad, isopolar, and tricolporate pollens.

Many researchers used pore number as a diagnostic trait for different families^16,23,97. In this study, the halotolerant species P. lanceolata exhibited spheroidal tricolporate pollen having verrucate exine. P. lanceolata showed a large equatorial diameter as compared to the polar diameter. Al-Quran⁹⁸ reported polypantoporate pollens in 11 species of Plantaginaceae distributed in Jordan. Klimko et al⁹⁹. also examined pantoporate pollens in 9 Plantago species native to Poland, similar to our study. Plantaginaceae members exhibit little variation in exine type. The verrucate exine ornamentation and large equatorial axis, as compared to the polar axis, were also observed by Habib et al¹⁰⁰.. Butt et al¹⁰¹. examined pollen of P. major, a non-halotolerant species, and reported pantoporate pollen with scabrate exine.

Polygonum is the largest genus of Polygonaceae, comprising roughly 300 species all over the world¹⁰². P. maritimum is a glycohalophyte because salt avoidance is its salt protection mechanism¹⁰³. P. maritimum has white homostylous flowers. Mahmoudi et al¹⁰⁴. studied the taxonomy and evolution of the Polygonum genus and reported tricolporate and prolate to spheroidal pollen in P. maritimum, similar to our findings. Shiha et al¹⁰⁵. and Coşkunçelebi et al¹⁰⁶. also reported that Polygonum taxa are characterized by prolate pollen with microechinate exine sculpture. Polygonum molle is a non-halotolerant species that exhibited tricolpate pollen with micro-reticulate exine as compared to our studied halotolerant species¹⁰⁷.

Salvadoraceae is represented by 1 genus with 3 species, i.e., S. oleoides, S. alii, and S. persica in Pakistan¹⁰⁸. The members of this family are well adapted to arid environments and help in the reclamation of saline soil and sand dune habitats in arid ecosystems^109,110. S. oleoides is a facultative halophyte that can be found in arid and dry regions¹¹¹. In the Kalabagh Salt Range, S. oleoides was found dominant in the present study. Perveen and Qaiser¹¹² studied the pollen flora of the family Salvadoraceae and reported prolate-spheroidal pollens with reticulate tectum. The conservation of S. oleoides is needed because this plant species is vulnerable regionally and under threat due to its low seed viability and germination rate¹¹³.

The genus Tamarix comprises shrubs and trees that are recretohalophytes and grow mainly in saline regions. Tamarix plants can tolerate extreme abiotic stress and are utilized to repair the soil structure, sculpt landscapes in saline soils, and enhance ecological environments¹¹⁴. Both studied Tamarix species exhibited tricolpate, prolate-spheroidal shape pollens with reticulate exine ornamentation. Qaiser and Perveen¹¹⁵ studied Tamarix pollen morphology and exhibited sub-prolate pollen with reticulate exine in T. salina, like our findings.

Zygophyllaceae is a xerophytic, heterogeneous, and eurypalynous family⁹¹. Fagonia indica is a spiny small shrub present in dry regions and deserts of Asia¹¹⁶. Fagonia taxonomy is complex due to its high morphological plasticity, which reflects its adaptations to environmental conditions¹¹⁷. The prolate pollen with psilate exine in Fagonia species was examined by Ahmad et al¹¹⁸. using LM, contrary to our findings. Sultana et al¹¹⁹. examined tricolpate and prolate-spheroidal pollen in xerophytic Fagonia bruguieri, which has adaptations to cope with water stress. Ahmad et al¹²⁰. revealed an 80.48% pollen viability for F. indica during their study on sub-tropical flora of Pakistan. In the current study, F. indica pollens exhibited 87% viability, indicating their ability to mature and fertilize in the saline environment.

Pollen’s fertility is a quantitative feature that provides valuable information about the regeneration of plant species through sexual reproduction, to ensure the species’ survival in a region¹²¹. Pollen with high viability can make saline agriculture more productive and efficient, and more suitable for breeding programs and commercial purposes. The fertility of any plant can be determined by phylogenetic relatedness, as selective pressures can explain efficient fertilization based on common metabolic pathways and similar floral morphologies¹²². All the studied halotolerant species exhibited high pollen viability, indicating their successful reproductive potential in the Salt Range and their tolerance towards environmental stresses.

PCA analysis of halotolerant species belonging to different families exhibits little variation in pollen traits such as exine thickness, viability, and polar and equatorial diameters. Both axes (PC1 and PC2) showed 65.9% of the variance. Qualitative data is not sufficient to delimit taxa; therefore, quantitative data was also incorporated. PCA outcomes can be interpreted easily by examining spectral features and score plots in corresponding loadings¹²³. PCA is useful for describing the relationships between environmental variables and taxonomic values¹²⁴. PCA is useful for morphological traits used in taxonomic studies for the delimitation of similar morphogroups in the flora of a specific region¹²⁵.

Salt tolerance in angiosperms has evolved in different lineages. The tolerance ability of salinity also increases tolerance to other environmental stresses¹²⁶. The ecological balance of arid ecosystems is dependent on halophytes; however, over-exploitation, climate change effects, and habitat degradation are primary threats to halotolerant taxa. In situ conservation initiatives are needed for the long-term preservation of endangered halotolerant species, which must be implemented using a multidisciplinary and holistic approach.

Conclusion

The potential of advanced microscopic techniques for pollen’s morphological peculiarities to efficiently identify and discriminate halotolerant species belonging to different families is elucidated in this study. This is the first comprehensive pollen morphological analysis of halotolerant species of Kalabagh Salt Range, Punjab, Pakistan. Reproductive morphological traits were compared, and palynomorphs’ shape, type, size, exine, aperture, and viability emerged as the most informative traits to assess affinities and differentiation among halotolerant plant species. It is concluded from the broad pollen descriptions that halotolerant species exhibit a strong association between pollen viability and traits. This type of affinity has potential applications in environmental monitoring and ecological studies. Principal component and cluster analysis results provide insights into the classification of halotolerant species, grouping them into two major groups based on pollen traits. This study highlights the significance of quantitative data together with pollen features that allow the identification and confirmation of species affinities at various levels. Despite the promising features of halotolerant species, further investigations integrating palynomorphology with molecular and phylogenetic analyses are required to strengthen the systematic studies of these taxa and phylogenetic resolution. Although this study focuses on fifteen halotolerant species from a single geographic region, which may not fully capture the broad diversity of halophytes in other saline environments. Additionally, the analysis focused primarily on pollen ultrastructure, but this could be used as a baseline to relate their functional and ecological performance in natural habitats. While the findings provide valuable insights for taxonomic classification, their practical application in large-scale ecological restoration, conservation, or breeding programs remains limited without further field validation and complementary physiological studies.

Author contributions

MN. Original draft preparation, conceptualization, design the work, resources, methodology, supervision, project administration, data interpretation, approved data presentation, softwares, and field survey. MZ. formal analysis, draft reviewing, validation. MHS. Visualization, presentation of submitted work, draft reviewing, investigation. MM. Formal analysis, methodology, validation. RM. Formal analysis, draft reviewing and editing. FB. Visualization, investigation, draft reviewing, data collection. AK. Methodology, contribution to design work, data collection, draft reviewing and editing, data interpretation, field survey, statistical analysis. MAB. Draft reviewing, resources, funding acquisition, formal analysis. MTN. Visualization, investigation, draft reviewing, editing, presentation of the submitted work. All authors agree with the final version of the manuscript.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.