Research Article |
Corresponding author: Mohamed N. Al-Yahya’ei ( mohamed.alyahyaei@moheri.gov.om ) Corresponding author: Abdullah M. Al-Sadi ( alsadi@squ.edu.om ) Academic editor: Alfredo Vizzini
© 2024 Shah Hussain, Moza Al-Kharousi, Dua’a Al-Maqbali, Arwa A. Al-Owaisi, Rethinasamy Velazhahan, Mohamed N. Al-Yahya’ei, Abdullah M. Al-Sadi.
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Citation:
Hussain S, Al-Kharousi M, Al-Maqbali D, Al-Owaisi AA, Velazhahan R, Al-Yahya’ei MN, Al-Sadi AM (2024) Two new species of Hymenagaricus (Agaricales, Agaricaceae) from Oman, based on morphology and molecular phylogeny. MycoKeys 105: 1-19. https://doi.org/10.3897/mycokeys.105.113591
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Hymenagaricus has small to medium-sized mushrooms and the cap surface with squamulose pellicles, consisting of hymeniform or pseudoparenchymatous cells and yellowish-brown basidiospores. The species of Hymenagaricus are very similar to those of Xanthagaricus and it is extremely difficult to differentiate the species of both genera in the field. However, phylogenetically, both the genera are clearly distinct. In this study, we describe two new species of Hymenagaricus, i.e. H. wadijarzeezicus and H. parvulus from the southern part of Oman. Species descriptions are based on a combination of morphological characteristics of basidiomata and phylogenetic analyses of three gene regions: internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), the large subunit of nuclear ribosomal DNA (28S) and translation elongation factor one alpha (EF-1α). Full descriptions, micrographs and illustration of anatomical features, basidiomata photos and phylogenetic analyses results of the new taxa are provided. Morphological comparisons of new taxa with similar species and a key to species included in the phylogenetic analyses are also provided.
Dhofar, diversity, taxonomy, termite mounds, two new taxa
Three species previously in Agaricus subgenus Conioagaricus, Agaricus hymenopileus, A. alphitochrous and A. nigrovinosus, were placed in a new and separate genus called Hymenagaricus Heinem. by Heinemann in 1981. This taxonomic change was likely due to the distinct features observed in the cap of these species during different stages of their development. At the young stage, the pilei are entirely covered with a pellicle, but as they mature, the pellicle is disrupted, leaving a single large squamulose pellicle at the centre of the pileus. The squamules are composed of hymeniform or pseudoparenchymatous cells, which set these species apart from others within the genus Agaricus (
Species of Hymenagaricus are saprotrophic in nature and are mostly distributed in the Palaeotropical Regions. Members of this genus are recognised by the squamulose pellicle on the pileus surface that mostly consists of hymeniform cells or pseudoparenchymatous tissues, yellow to yellowish-brown basidiospores and the absence of both pleurocystidia and clamp connections (
Phylogenetically, species of Hymenagaricus are intermixed with the monotypic genus Heinemannomyces Watling (
Four species of Agaricaceae, namely Agaricus arabiensis S. Hussain & Al-Sadi, Micropsalliota ventricocystidiata Al-Sadi & S. Hussain, Xanthagaricus appendiculatus Al-Sadi & S. Hussain and X. omanicus Al‐Kharousi, Al‐Sadi & S. Hussain have recently been described from Dhofar Region, Oman (
During the years 2022–23, macrofungal exploration missions were conducted in the Dhofar Region, in which we collected ten (10) collections of Hymenagaricus. Morphological characterisation and multigene (ITS, 28S, EF-1α) phylogenetic analyses revealed that the 10 collections represent two new species, which are described in this study.
The specimens were collected in the Dhofar Region, located in the south of the Sultanate of Oman. The region experiences a monsoon-influenced climate with a distinct wet season known as the Khareef, which occurs from June to early September (
In the current study, mushroom specimens were collected from three localities (Wadi Naheez, Wadi Jahaneen, Wadi Jarzeez) of the Dhofar Region, in the months of August–September 2022 to 2023. The specimens were photographed in the field and field characteristics such as the shape, colour, size and smell of basidiomata were noted. The samples were dried using a fruit dehydrator with temperature adjusted at 45 °C (
For microscopic study, handmade sections were made from lamellae, cap and stipe surfaces and annulus. Thin small sections were initially mounted in 5% aqueous potassium hydroxide (KOH) (w/v) and then re-hydrated in 1% aqueous Cong red (w/v) for a more obvious appearance. Microscopic features such as the size, shape and colour of basidiospores, basidia, cheilocystidia, pellicle structure, veil and annulus morphology were studied under a compound microscope (ECLIPSE Ni-U, Nikon Co., Ltd., Japan). For size measurements of these structures, Piximetre (http://ach.log.free.fr/Piximetre/) was used. For the morphological terminology,
Genomic DNA was extracted from dried specimens using X-AMP DNA reagent kit (Dubuque, Iowa, USA), following the manufacturer’s protocol. A volume of 200 µl X-AMP DNA reagent was taken in an Eppendorf tube containing the sample (approximately 5–15 mg of gills) and incubated for 15 minutes at 70 °C. After cooling, 2 µl solution from the sample was used as a DNA template directly for polymerase chain reaction (PCR) without any further treatment. We amplified three gene regions, including the internal transcribed spacer (ITS), the large subunit of nuc rDNA (28S) and the translation elongation factor 1 alpha (EF-1α) gene. The primer combinations were: ITS1F and ITS4 for ITS (
Consensus sequences were created from the forward and reverse primer reads of the newly-generated ITS, 28S and EF-1α sequences using BioEdit v.7.0.9.0 (
Species | Origin | Voucher number | GenBank accession | Reference | |
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ITS | 28S | ||||
Agaricus campestris | Spain | LAPAG370 | KM657927 | KP739803 | Parra et al. (2016) |
Heinemannomyces splendidissimus | China | Q. Zhao 2591 | KY039571 | KY039576 |
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Hei. splendidissimus | China | Z.W. Ge2540 | KY039570 | KY039575 |
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Hei. splendidissimus | China | GDGM46633 | MF621038 | MF621039 |
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Hei. splendidissimus | China | GDGM46634 | – | MF621040 |
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Hei. splendidissimus | Thailand | ecv3586 | HM488760 | HM488769 |
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Hei. sp. | Thailand | ZRL185 | KT951346 | KT951527 |
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Hymenagaricus ardosiaecolor | Togo | LAPAF9 | JF727840 | – |
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H. ardosiaecolor | Tanzania | Z4 | KM360160 | – |
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H. cf. kivuensis | Burundi | BR6089 | KM982454 | – |
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H. wadijarzeezicus | Oman | JRZ2-22-015 | OR613000 | OR613018 | This study |
H. wadijarzeezicus | Oman | JRZ2-22-013 | OR612999 | OR613019 | This study |
H. wadijarzeezicus | Oman | NHZ-22-019 | OR612997 | OR613020 | This study |
H. wadijarzeezicus | Oman | JHN-22-019 | OR612998 | OR613021 | This study |
H. wadijarzeezicus | Oman | JRZ-22-005 | OR612996 | OR613022 | This study |
H. pakistanicus | Pakistan | FAK196 | OP082405 | – |
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H. pakistanicus | Pakistan | FAK195 | OP082404 | – |
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H. parvulus | Oman | JRZ-22-004 | OR612994 | OR613017 | This study |
H. parvulus | Oman | JRZ2-22-002 | OR612995 | – | This study |
H. siamensis | Thailand | SDBR-CMUWP038 | OP837533 | OP836600 |
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H. siamensis | Thailand | SDBR-CMUNK1508 | OP836301 | OP836385 |
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H. saisamornae | Thailand | ZRL3103 | KM982450 | KM982452 |
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H. saisamornae | Thailand | SDBRCMUNKNW0474 | MW349605 | MW349603 |
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H. saisamornae | Thailand | SDBR-CMUNK0369 | MW345912 | MW345917 |
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H. saisamornae | Thailand | SDBR-CMUNK0567 | MW349602 | MW349604 |
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H. saisamornae | Thailand | LD2012186 | KM982451 | KM982453 |
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H. sp. | Pakistan | LAH35329 | OQ998344 | – |
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H. sp. | Thailand | CA833 | JF727858 | – |
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In this study, 19 new sequences (7 ITS, 6 28S and 6 EF-1α) were generated from our collections of Hymenagaricus. There were no EF-1α sequences of the genus available in GenBank; therefore, only combined ITS-28S sequences were used in the final data matrix. The final ITS-28S dataset was comprised of 1516 characters including 1272 constant sites, 141 informative sites and 103 uninformative sites. The topology of trees revealed similar patterns in both ML and BI methods; therefore, the phylogeny inferred from ML analysis is presented here with values from both BT and PPs in Fig.
Maximum Likelihood phylogeny of Hymenagaricus and Heinemannomyces, based on combined ITS-28S sequence data, with Agaricus campestris as the outgroup taxon. Values above the node represent ML bootstrap percentages and BI posterior probabilities; the new species are represented in bold fonts.
The new species Hymenagaricus wadijarzeezicus can be differentiated from other species of the genus by its unique whitish woolly veil, covering both the cap and the stipe surfaces.
The specific epithet ‘wadijarzeezicus’ refers to the valley Jarzeez in the south of Oman, where the holotype was found.
Basidiomata small to medium-sized. Pileus 30–80 mm in diam., at the young stage, broadly ovoid to parabolic, covered completely by a smooth, pale brownish pellicle; at mature stage, pulvinate to convex, pellicle disrupting except at the centre where it is retained as one large, smooth, brownish squamule, surface is woolly, covered with whitish, strigose to villose or floccose veil towards the margin; margin appendiculate with long, whitish, fibrils of veil. Context dark pinkish on cutting, 3–5 mm thick at the pileus centre. Lamellae free, pale pinkish at young stage, at mature stage greyish-pink to brownish, ventricose, up to 3 mm wide, densely crowded, with 1–3 series of lamellulae. Stipe 30–60 × 5–10 mm, equal, with a slightly bulbous base, with root-like rhizoid structure at the base, annulus floccose, concolorous to veil; stem covered with floccose veil below the annulus, smooth above the annulus, context pinkish on cutting, fistulose. Smell pleasant. Taste not recovered.
Basidiospores (6.5)7.0–8.0(8.5) × (4.0)4.5–5.5(6.0) µm, average size 7.5 × 5.0 µm, Q = 1.4–1.6, av. Q = 1.5; ellipsoid to broadly ellipsoid, yellowish to dark brown, smooth, thick-walled, apiculus visible, germ-pore not observed. Basidia 20–25 × 7–9 µm, on average 22.5 × 8.0 µm, clavate to cylindrical, smooth, hyaline in KOH, mostly tetrasporic, rarely bisporic. Cheilocystidia 16–23 × 7–9 µm, on average 19.5 × 8.0 µm, ellipsoid to subclavate, smooth, thin-walled, hyaline in KOH. Pleurocystidia absent. Lamellar trama regular, with 4–6.6 µm diam., cylindrical to inflated, thin-walled, hyaline hyphae. Subhymenium consisted of subglobse to irregular cells, measuring 12–18 µm diam. Pellicle is a hymeniform, consisting of chains of two or three elements, measuring 13–17 × 10–16 µm each element, globose to subglobose or ovoid, hyaline, or pale yellowish, smooth, thin-walled, these chains of elements attached to inflated hyphae with encrusted walls. Pileus veil is a cutis to ixocutis, consisting of elongated or cylindrical elements, easily detached, hyaline, thin-walled, each element measuring 13–45 × 6–9 µm. Annulus is an intricate trichoderm, composed of hyaline hyphae, 6–8 µm diam., cylindrical, constituted by short elements, constricted at septa, easily disarticulated. Stipe veil similar to pileus veil. Clamp connections absent in all tissues.
Basidiomata of Hymenagaricus wadijarzeezicus A–C holotype collection (JRZ2-22-013) D, E NHZ-22-019 F young fruiting bodies where the cap is entirely covered by pellicle represented by arrows (JRZ2-22-015) G context changed into pinkish on cutting, the arrow represents the root-like rhizoid (JRZ2-22-015). Scale bars: 20 mm.
Occurring in July to early September, as saprotrophic, solitary or scattered in small groups, on or near the termite mounds, under the trees of Anogeissus dhofarica. Currently only known from southern Oman.
Sultanate of Oman: Dhofar, Salalah, Wadi Naheez, on termite mounds, under the trees of Anogeissus dhofarica, 07 August 2022, S. Hussain, A. Al-Owaisi, Al-Yahya’ei & Al-Sadi, NHZ-22-019 (Mawarid-NHZ-22-019), GenBank accession: ITS = OR612997, 28S = OR613020, EF-1α = OR729602; Wadi Jarzeez, under the trees of Anogeissus dhofarica, 08 August 2022, S. Hussain, A. Al-Owaisi, Al-Yahya’ei & Al-Sadi, JRZ-22-005 (Mawarid-JRZ-22-005), GenBank accession: ITS = OR612996, 28S = OR613022, EF-1α = OR729603; Wadi Jaheen, under the trees of Anogeissus dhofarica, 10 August 2022, S. Hussain, A. Al-Owaisi, Al-Yahya’ei & Al-Sadi, JHN-22-019 (Mawarid-JHN-22-019), GenBank accession: ITS = OR612998, 28S = OR613021, EF-1α = OR729600; Wadi Jarzeez, on termite mounds, under the trees of Anogeissus dhofarica, 11 August 2022, S. Hussain, A. Al-Owaisi & Al-Yahya’ei, JRZ2-22-015 (Mawarid-JRZ2-22-015), GenBank accession: ITS = OR613000, 28S = OR613018, EF-1α = OR729601; Wadi Gogob, on termite mounds, under the trees of Anogeissus dhofarica, 22 August 2023, S. Hussain & Al-Yahya’ei, GOB-23-008 (Mawarid-GOB-23-008); Sahalanawt, on termite mounds, 27 August 2023, S. Hussain & Muhammad Salim, Sahalanawt-23-001 (Mawarid-Sahalanawt-23-001); Tetam, on termite mounds, 30 August 2023, S. Hussain & Amer Qattan, Tetam-23-001 (Mawarid-Tetam-23-001).
The new species Hymenagaricus wadijarzeezicus with medium-sized basidiomata, can be distinguished from the known species of the genus by its remarkable woolly cap and stipe surfaces. In Hymenagaricus, there are four species with a cap diameter of 50 mm or above, which are: Hymenagaricus cf. kivuensis, H. mlimaniensis Mwanga & Tibuhwa, H. ardosiaecolor and H. alphitochrous (Berk & Broome) Heinem. Hymenagaricus wadijarzeezicus is the 5th species with a cap diameter above 50 mm. None of these species has a woolly basidiomata surface, except Hymenagaricus wadijarzeezicus.
In ML phylogeny, the most similar species to the new species H. wadijarzeezicus is H. saisamornae. Hymenagaricus saisamornae is a recently described species from Thailand, with substantially smaller basidiomata (10–25 mm cap diam.), pileus surface covered with minute brownish squamules, stipe smooth to finely whitish squamulose and smaller basidiospores (5.5–7.0 × 4.0–4.5 µm;
The new species Hymenagaricus parvulus can be differentiated from other species of the genus by its small-sized, creamy basidiomata, umbonate pileus covered with appressed pellicle.
The specific epithet ‘parvulus’ refers to the small-sized basidiomata of the new species.
Basidiomata small-sized. Pileus 15–25 mm in diam., at young stage globose to parabolic, surface floccose squamulose, squamules light pinkish to creamy, with appressed pellicle at the centre, margin appendiculate; at mature stage cap convex to hemispherical with the broadly umbonate disc, with appressed, pale brownish pellicle at the disc, surface finely floccose squamulose, squamules pale creamy to light greyish, margins striate, just exceeding the lamellae; context membranous, pinkish on cutting. Lamellae free, pale pinkish to brownish, ventricose, sparsely crowded, with 1–2 series of lamellulae. Stipe 25–35 × 2–5 mm, equal, annulus cortinate, concolorous to squamules; stem surface creamy, covered with finely floccose squamules below the annulus, smooth above the annulus, context pinkish on cutting, fistulose. Smell pleasant. Taste not recorded.
Basidiospores 5.0–6.5 × 4.0–4.5 µm, average size 6.0 × 4.2 µm, Q = 1.3–1.5, av. Q = 1.4; ellipsoid to broadly ellipsoid, yellowish to dark brown, smooth, thick-walled, apiculus visible, germ-pore not observed. Basidia 16.5–22.5 × 6.5–8.5 µm, on average 19.0 × 7.5 µm, clavate to cylindrical, smooth, hyaline in KOH, tetrasporic. Cheilocystidia 19–25 × 9–11 µm, on average 22 × 10 µm, clavate to broadly clavate, often turning to one side, with multiseptate base, smooth, thin-walled, hyaline in KOH. Pleurocystidia absent. Subhymenium consisting of cylindrical to elongated cells, measuring 6–9 µm diam. Pellicle is a hymeniform, consisting of chains of several elements, each element measuring 14–22 × 12–17 µm, globose to subglobose or ovoid, hyaline or pale yellowish, smooth, thin-walled; these chains of elements attached to inflated hyphae with encrusted walls. Veil is a cutis to ixocutis, consisting of elongated or cylindrical elements, not easily detached, hyaline, thin-walled, with terminal element fusiform with papillate end, each element measuring 15–18 × 5–7 µm. Annulus is an intricate trichoderm, composed of hyaline hyphae, 4–7 µm diam., cylindrical, constituted by short elements, constricted at septa and easily disarticulated. Clamp connections absent in all tissues.
Fruiting body formation occurs in early August to early September, saprotrophic, scattered in small groups, found on termite mounds. Currently only known from southern Oman.
Sultanate of Oman: Dhofar, Salalah, Wadi Jarzeez, on termite mounds, under the trees of Anogeissus dhofarica, 11 August 2022, S. Hussain, A. Al-Owaisi, Al-Yahya’ei & Al-Sadi, JRZ2-22-002 (Mawarid-NHZ-22-002), GenBank accession: ITS = OR612995.
Hymenagaricus parvulus is a small, cream-coloured species, differentiated from other species of the genus by its whitish to pale pinkish floccose squamules on pileus and stipe surfaces with a broadly umbonate centre. Hymenagaricus parvulus shares basidiomata size and basidiospores morphology with H. pakistanicus. However, H. pakistanicus can be differentiated from the new species by its caesptiose fruiting habit, pileus with pinkish to brownish squamulose pellicle, consisting of pseudoparenchymatous cells (
Species of Hymenagaricus and Xanthagaricus are morphologically very similar and it is extremely difficult to differentiate the species of these genera in the field. However, in most species of Xanthagaricus, the cap surface is covered with small, brownish to purplish scales. These scales are concentrated at the pileus centre, while a large central, undisrupted scale at the cap centre has been observed in the most species of Hymenagaricus. Phylogenetically, both the genera are clearly distinct.
Phylogenetically, the species of Hymenagaricus are closely related to the monotypic genus Heinemannomyces. Morphologically, both these genera are clearly distinct. Species of Hymenagaricus have a squamulose cap surface and these squamules consist of hymeniform or pseudoparenchymatous cells and yellowish-brown basidiospores. The monotypic genus with single species Heinemannomyces splendidissimus has a brownish to greyish-red pileus, covered with a finely woolly veil and greyish to dark bluish basidiospores (
In our phylogenetic analyses, the specimens representing Heinemannomyces formed a basal group. Species of Hymenagaricus were recovered in three groups. One group consisted of Hymenagaricus wadijarzeezicus, the new species, H. saisamornae and H. cf. kivuensis. In this group, Hymenagaricus saisamornae with small-sized basidiomata intermix with H. cf. kivuensis and H. wadijarzeezicus both with medium-sized basidiomata. Similarly, another group consisting of Hymenagaricus pakistanicus, H. parvulus the new species and unnamed species H. sp. (LAH35329). All these taxa, including the unnamed species, have a small fruiting body. The third group consists of Hymenagaricus ardosiaecolor (medium-sized basidiomata) and H. siamensis, the small-sized species.
Both basidiomata size and pellicle structure are species delimitation characters in the genus Hymenagaricus. Based on our analyses, we can predict that these characters could be used in the future for infrageneric classification of the genus.
The two new species, Hymenagaricus wadijarzeezicus and H. parvulus, were collected in the Dhofar Region, located in the southern part of Oman. Hymenagaricus wadijarzeezicus is medium-sized and H. parvulus is a small-sized species. Both are widespread in the Region, under the trees of Anogeissus dhofarica. It is interesting to note that both collections of H. parvulus (JRZ-22-002, JRZ2-22-004) and several collections of H. wadijarzeezicus (NHZ-22-019, JRZ2-22-013, JRZ2-22-015, GOB-23-008, Sahalanawt-23-001, Tetam-23-001) were found on termite mounds. However, we did not find any study reporting the association of Hymenagaricus with termites. However, secotioid fungal genus Podaxis Desv. in the family Agaricaceae has an apparent relationship with termites (
Several species of Agaricaceae were recently reported from the Dhofar Region (
A taxonomic key to the species of Hymenagaricus included in our phylogenetic analyses is presented below. This key is based on cap diameter (small-sized with cap less than 40 mm in diam. and medium-sized with cap ranging from 50–100 mm in diam.) and pellicle structure either hymeniform or pseudoparenchymatous cells.
1 | Basidiomata small-sized, pileus diam. below 40 mm | 2 |
– | Basidiomata medium-sized, pileus diam. up to 100 mm | 5 |
2 | Fruiting bodies solitary or gregarious | 3 |
– | Fruiting bodies appear in cluster (casepitose), pileus 20-30 mm diam., pellicle consisted of pseudoparenchymatous cells | Hymenagaricus pakistanicus |
3 | Pileus whitish or pinkish-brown, pellicle consisted of hymeniform cells | 4 |
– | Pileus brownish, pellicle consisted of pseudoparenchymatous cells | H. siamensis |
4 | Pileus 15–25 mm diam., whitish to creamy, pellicle smooth, finely appressed | H. parvulus |
– | Pileus 10–15 mm diam., pinkish to brownish, squamulose pellicle | H. saisamornae |
5 | Pileus covered with brownish squamules | 6 |
– | Pileus and stipe surfaces covered with whitish woolly fibrils | H. wadijarzeezicus |
6 | Pileus diam. 40–60 mm, basidiospores 5.8–6.5 × 3.9–4.5 µm | H. ardosiaecolor |
– | Pileus diam. up to 100 mm, hymeniform squamules | H. cf. kivuensis |
We would like to thank Bader Al Quyudhi, Shamsa Al Balushi and Maryam Al Hinai for their invaluable help in the sampling trip to the Dhofar Region, which was essential to the success of our research. We would also like to thank Amer Qattan and Mohammed Al Jahwari for their guidance and support throughout the project.
The authors have declared that no competing interests exist.
No ethical statement was reported.
This research was funded by the Agriculture and Fisheries Development Fund, Sultanate of Oman (grant number 104/1/1).
Shah Hussain and Moza Al-Kharousi: conceptualisation, writing – original draft and review and editing, data curation, formal analysis, investigation, methodology and visualisation. Dua'a Al-Maqbali and Arwa A. Al-Owaisi: sampling, data curation and investigation. Mohamed N. Al-Yahya'ei and Abdullah M. Al-Sadi: project administration, resources, supervision , writing - review and editing. Rethinasamy Velazhahan: writing - review and editing, formal analysis.
Shah Hussain https://orcid.org/0000-0002-5772-7206
Rethinasamy Velazhahan https://orcid.org/0000-0002-9263-4371
Mohamed N. Al-Yahya’ei https://orcid.org/0000-0002-9516-5339
Abdullah M. Al-Sadi https://orcid.org/0000-0002-3419-8268
All of the data that support the findings of this study are available in the main text.