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Article

Preliminary Study on the Genetic Diversity of Sicilian Populations of Crataegus azarolus (Rosaceae) and Their Wild Relatives for Conservation and Valorisation Purposes

by
Floriana Bonanno
1,
Simona Aprile
2,*,
Vivienne Spadaro
3,*,
Francesco M. Raimondo
4 and
Antonio Giovino
2
1
Department of Agricultural, Food and Forest Sciences, University of Palermo, 90128 Palermo, Italy
2
Council for Agricultural Research and Economics (CREA), Research Centre for Plant Protection and Certification (CREA-DC), 90128 Palermo, Italy
3
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, Section of Botany, Anthropology and Zoology, University of Palermo, 90123 Palermo, Italy
4
PLANTA/Center for Research, Documentation and Training, Via Serraglio Vecchio 28, 90123 Palermo, Italy
*
Authors to whom correspondence should be addressed.
Diversity 2025, 17(4), 258; https://doi.org/10.3390/d17040258
Submission received: 18 February 2025 / Revised: 1 April 2025 / Accepted: 3 April 2025 / Published: 5 April 2025
(This article belongs to the Special Issue Ethnobotany, Medicinal Plants and Biodiversity Conservation: 2nd Edition)
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Abstract

:
Sicily, Southern Italy, has important genetic resources for azarole (Crataegus azarolus). The region hosts both spontaneous wild populations and various forms of the genus, some of which belong to well-defined specific or intraspecific taxa, while others are hybrids of unclear origin. Not all wild forms can be considered related to C. azarolus, but those included in the sect. Azaroli and thus C. aronia (= C. azarolus var. aronia), which is widespread in western Sicily, certainly can. All these populations are morphologically distinct but coexist in the same area. This preliminary genetic study aims to establish the genetic relationships between wild populations of Crataegus taxa and azarole (C. azarolus). Furthermore, the research addresses the lack of clear taxonomic information regarding the different phenotypes found in Sicily, including both recognized taxa and those still critically underreported. The study is based on SSR (Simple Sequence Repeat) molecular markers, isolated from other Rosaceae species. The findings indicate that at least two groups affiliated with C. azarolus, yet distinct, are present in Sicilian populations. These results could have significant implications for systematics and taxonomy, as well as for conservation and the valorization of plant biodiversity.

Graphical Abstract

1. Introduction

The genus Crataegus L., belonging to the Rosaceae family, includes deciduous small trees and shrubs. It is native to Asia (China), Europe, North America, and North Africa [1].
This genus is taxonomically complex, with species numbers varying between 150 and 1200, of which 50–100 occur in Europe [2]. Crataegus, commonly known as hawthorn, is widely distributed in the Northern Hemisphere’s temperate regions, extending as far as Central America [3]. The most common species include C. monogyna Jacq., C. laevigata (Poir.) DC., C. mexicana Moc. and Sessé ex DC., and C. douglasii Lindl.
This genus primarily consists of heliophilous plants that are part of the mantle vegetation of natural forests, specifically within plant communities belonging to the class Rhamno catharticae-Prunetea spinosae Rivas Goday and Borja ex Tüxen 1962. Some species, such as C. laevigata, are characteristic of the shrub layer in the wetland forests and scrub (WFS) of the Western Balkans [4].
Crataegus spp. are of great value for their medicinal and nutritional benefits, as well as their cultural and ecological value [5]. The leaves, flowers, and fruits, rich in nutrients and bioactive compounds, are used in both traditional Chinese and European medicine [6]. In this regard, the following paragraph will explore their ethnobotanical significance and potential health benefits.
In Italy, the genus Crataegus can be found in every region of the country with a wide range of species, subspecies, and hybrids [7].
However, only a few of them are cultivated specifically for their fruit. Among these, azarole (C. azarolus L.) is particularly notable, as it is occasionally cultivated [8] not only in Italy but also in various parts of Europe, North America, and Mexico.
In Italy, C. azarolus is naturalized in Emilia-Romagna and Tuscany and has become spontaneous in Piedmont, Lombardy, Veneto, Umbria, Abruzzo, Campania, Apulia, Calabria, Sicily, and Sardinia [9,10]. According to Raimondo et al. [11] and Spadaro et al. [5], the morphological diversity found in Sicily suggests, at taxonomic level, a subdivision into C. azarolus var. azarolus, C. azarolus var. chlorocarpa (plants with reddish and yellowish fruits, respectively, also cultivated), and C. azarolus var. aronia, growing wild in Cinisi (Palermo), Erice, Valderice, Custonaci, and San Vito Lo Capo (Trapani). The cited authors, in line with Christensen et Zieliński [12] and POWO [3], treat C. azarolus var. azarolus as a native taxon, not an introduced one, cultivated and then naturalized as recommended by Galasso et al. [9].
Azarole (C. azarolus) is a long-lived tree capable of living up to 100 years and growing up to a maximum height of 5 meters. The thorny branches exhibit a twisted morphology in wild specimens but become straighter under cultivation. The leaves are bright green, oval-shaped with a deeply divided base forming three to five lobes that may be entire or toothed, supported by a short, pubescent petiole. The flowers, grouped in corymbose inflorescences, are white and bright, rarely pink, and are present from April to May. This striking flowering phase grants the species high ornamental value, making it a prized feature in Mediterranean garden designs.
The fruit is an edible, globe-shaped pome 1.5–3 cm in diameter, displaying amaranth-red coloration (C. azarolus var. azarolus) or yellowish-green hues (C. azarolus var. chlorocarpa) when ripe. It typically has a sweet-sour taste and contains 2–4 small seeds. Ripening generally concludes in September. While fruit production has a long tradition in Italy, cultivation of these minor species has been neglected during the past century [13].
In Sicily, other known species include C. laciniata Ucria (producing fruits with 2–3 seeds), C. monogyna (generally 1 seed), and C. laevigata (2 seeds). C. azarella Griseb. is commonly considered either a variety of C. monogyna [14] or a subspecies [15], but it remains a taxonomically critical entity for the island [11].
As reported in the literature, the characterization of morphological parameters in Crataegus is generally based on plant habit and height, branches, the presence and shape of spines, leaf and stipule morphology, fruit traits (shape, size, and color), and seed number [16]. Instead, to characterize genetic diversity, many molecular markers have been used, such as simple sequence repeats (SSRs), inter-simple sequence repeats (ISSRs), start codon targeted (SCoT), polymorphism, and specific-locus amplified fragment sequencing (SLAF-seq) [1,17,18,19]. Our genetic approach relies on molecular markers (simple sequence repeats or microsatellites). SSRs were chosen because they are innovative, reliable, fast, polymorphic, and automated.
This study aims to establish genetic relationships between the wild Sicilian populations of Crataegus taxa and azarole (C. azarolus). A complementary objective of this research is to investigate the biodiversity of Crataegus populations in the provinces of Palermo and Trapani (northwestern Sicily) to clarify the taxonomy and distribution of various taxa in this area, where existing classifications do not represent the phenotypic diversity observed to date. Indeed, although recent contributions [5,11] have partially clarified some critical taxa found in western Sicily, some of which have been recognized as distinct taxa, many remain unresolved, particularly for eastern Sicily. For instance, the taxonomic status of C. azarolus var. aronia, interpreted by some taxonomists as a distinct species (C. aronia), remains highly controversial.
This research has broad implications for biodiversity protection, the conservation of plant genetic resources, and the valorization of promising natural sources of bioactive molecules. Identifying both wild and cultivated species of agricultural and forestry interest is crucial for developing targeted conservation strategies.

Ethnobotanical Significance and Potential Helth Benefits of Crataegus spp.

Valuable traditional knowledge regarding the medicinal properties of Crataegus spp. has laid the foundation for numerous pharmacological and nutraceutical studies supporting their potential health benefits. They have been widely used in folk medicine across various cultures and are currently employed in modern phytotherapy due to their broad spectrum of pharmacological properties [20,21,22], including antispasmodic, cardiotonic, hypotensive, diuretic, anti-atherosclerotic, and neuro-sedative effects.
Several ethnobotanical studies have documented the traditional use of C. monogyna (common hawthorn) flower infusion as a sedative, cardiac regulator, and hypotensive agent [23,24]; it is currently used in the treatment of angina, hypertension, arrhythmias, and congestive heart failure [20]. In traditional Arabic medicine, the decoction of leaves and unripe fruits of C. aronia (L.) Bosc ex DC. is used to treat cardiovascular diseases, cancer, diabetes, and sexual weakness [25,26]. In Mexico, C. mexicana extracts are used to manage diabetes [20]. The dried edible fruit of C. pinnatifida Bunge has been traditionally used in Oriental medicine and as a local soft drink ingredient in Taiwan [27].
About Crataegus azarolus, in traditional medicine, a decoction of its leaves has been used for the treatment of diabetes, angina, and postpartum pain, while an infusion of its flowers has been employed as an antihypertensive and for treating insomnia [28]. Several studies support the traditional uses of this plant, highlighting its antihypertensive [29], antimicrobial [30], antioxidant [31], antidiabetic [32], and anticancer [33] activities. Additionally, the Tunisian azarole (C. azarolus) and its isolated hyperoxide have demonstrated immunomodulatory effects through their antioxidant activity [34]. The aqueous extract of C. azarolus fruits has been shown to provide significant protection against diarrhea, attributed to its antioxidant and anti-inflammatory properties [35].
The fresh fruits of C. azarolus can be considered functional foods due to their well-documented health benefits, including the reduction in cardiovascular disease risk. They are a natural source of phenolic and organic acids, flavonols, tannins, catechins, monoterpenes, and vitamin C [36]. The fruits of C. azarolus are consumed as food, either fresh, dried, or canned [37]. In Sicily, the fruits of C. azarolus, as well as those of C. monogyna, are traditionally consumed fresh or dried and used in the preparation of jams, jellies, and liqueurs [38].
Recent phytochemical studies on various Sicilian populations of Crataegus, to which some of the present authors have contributed, have identified potential natural sources of health-promoting compounds [5]. A recent study [39] characterized the chemical profile and health-beneficial properties of C. laciniata flower extract, suggesting its potential as a source of bioactive compounds for the treatment of metabolic disorders and skin hyperpigmentation. Similarly, findings from another investigation on the fruits of various wild Crataegus taxa (C. laciniata, C. azarolus var. aronia, and C. laevigata), collected from different provinces of western Sicily, indicate that most of the analyzed fruits are rich in antioxidants and could be used in diabetes management, potentially mitigating postprandial hyperglycemia [40].
As observed in many Rosaceae species, wild plants of certain species are used as rootstocks for cultivated forms of species within the same genus and, in some cases, even for related genera. Recurrent cases within the same genus include Malus L., Pyrus L., Prunus L., and Crataegus. In Sicily, wild plants of this genus are used as rootstocks for the cultivated forms of C. azarolus var. azarolus, as well as for Malus domestica (Suckow) Borkh. (personal observations by co-author Raimondo). Furthermore, in rural communities, it was common practice to craft sturdy walking sticks or defensive staffs by cutting young and vigorous suckers, which were stripped of their short, highly spiny secondary branches (personal observation by co-author Raimondo).

2. Materials and Methods

2.1. Plant Material

The Crataegus samples were collected in nature, from October to December 2022, at several locations in the Sicilian provinces of Trapani and Palermo (Figure 1). To establish the genetic relationships among the genotypes [41], a total of 19 accessions of Crataegus Sicilian populations were sampled, representing 9 entities within specific and infraspecific taxa and one nothotaxon. (Table 1). The following entities were considered: Crataegus azarolus L. var. azarolus (4 accessions including one cultivated), C. azarolus var. aronia L. (1 accession), C. azarolus var. chlorocarpa (Moris) K.I.Chr. (1 accession), C. laciniata Ucria (1 accession), C. aff. insengae (Tineo ex Guss.) Bertol. (5 accessions), C. monogyna Jacq. (1 accession), and C. x media Bechst. (1 accession). In addition, taxonomically critical populations described later were also investigated, namely C. drepanensis Raimondo, Marino & Scuderi (3 accessions) and C. zichichii Raimondo, Spadaro, and Venturella (2 accessions) [5]. The accessions identified as sub-C. aff. insengae remain critical in the absence of sufficient diagnostic elements in the sampled plants. Box 1 shows the voucher specimens preserved at PAL-Gr (Herbarium Greuter in Palermo), Sicily, Italy.
Box 1. Voucher specimens preserved at PAL-Gr (Herbarium Greuter in Palermo), Sicily, Italy.
  • C_1/3: Crataegus azarolus L. var. azarolus – Sicily: Custonaci (Trapani), Purgatorio locality, on red Mediterranean soil, 175 m a.s.l., 21.09.2022, Raimondo (PAL-Gr).
  • C_e: Crataegus azarolus L. var. azarolus – Sicily: Custonaci (Trapani), Purgatorio locality, on red Mediterranean soil, 175 m a.s.l., 21.09.2022, Raimondo (PAL-Gr).
  • C_4/3: Crataegus azarolus L. var. azarolus – Sicily: Castelluzzo-Macari road (Trapani), on red Mediterranean soil, 170 m a.s.l., 21.09.2022, Raimondo (PAL-Gr).
  • C_b: Cratagus azarolus L. var. azarolus (cultivated) – Sicily: Pollina (Palermo), Carrara locality, cultivated plant on siliceous soil, 350 m a.s.l., 16.10.2022, Raimondo & Spadaro (PAL-Gr).
  • C_5/3: Crataegus azarolus var. aronia (L.) DC. – Sicily: San Vito Lo Capo (Trapani), Tonnara del Secco locality, on carbonate lithosol, ca. 10 m a.s.l., 12.09.2022, Raimondo (PAL-Gr).
  • C_f: Crataegs azarolus var. clorocarpa (Moris) K.I. Chr. – Sicily: Castellammare del Golfo (Trapani), Balata di Baida locality, ca. 150 m a.s.l, 23.09.2022, Raimondo & Spadaro (PAL-Gr).
  • C_3: Crataegus laciniata Ucria – Sicily: Piano Pomo locality, Petralia Sottana (Palermo), on siliceous soil, 1400 m a.s.l., 10.10.2022, Raimondo (PAL-Gr).
  • C_1: Crataegus aff. insengae (Tineo ex Guss.) Bertol. – Sicily: Petralia Sottana (Palermo), Piano Pomo locality, on siliceous soil, 1400 m a.s.l., 10.10.2022, Raimondo (PAL-Gr).
  • C_2: Crataegus aff. insengae (Tineo ex Guss.) Bertol. – Sicily: Petralia Sottana (Palermo), Piano Pomo locality, on siliceous soil, 1400 m a.s.l., 10.10.2022, Raimondo (PAL-Gr).
  • C_10: Crataegus aff. insengae (Tineo ex Guss.) Bertol. – Sicily: Petralia Sottana (Palermo), Piano Pomo locality, on siliceous soil, 1400 m a.s.l., 10.10.2022, Raimondo (PAL-Gr).
  • C_11: Crataegus aff. insengae (Tineo ex. Guss.) Bertol. – Sicily: Petralia Sottana (Palermo), Piano Pomo locality, on siliceous soil, 1400 m a.s.l., 10.10.2022, Raimondo (PAL-Gr).
  • C_12: Crataegus aff. insengae (Tineo ex Guss.) Bertol. – Sicily: Petralia Sottana (Palermo), Piano Pomo locality, on siliceous soil, 1400 m a.s.l., 10.10.2022, Raimondo (PAL-Gr).
  • C_c: Crataegus monogyna L. – Sicily: Castelbuono (Palermo), Barraca locality, on siliceous soil, 750 m a.s.l., 16.10.2022, Raimondo & Spadaro (PAL-Gr).
  • C_d: Crataegus x media Bechst. – Sicily: Pollina (Palermo), Carrara locality, on siliceous soil, 350 m a.s.l., 16.10.2022, Raimondo & Spadaro (PAL-Gr).
  • C_2/3a: Crataegus drepanensis Raimondo, Marino & Scuderi – Sicily: San Vito Lo Capo (Trapani), Tonnara del Secco locality, on carbonate tithosol, 8 m a.s.l., 12.09.2022, Raimondo (PAL-Gr).
  • C_2/3b: Crataegus drepanensis Raimondo, Marino & Scuderi – Sicily: San Vito Lo Capo (Trapani), Tonnara del Secco locality, on carbonate lithosol, 8 m a.s.l., 12.09.2022, Raimondo (PAL-Gr).
  • C_2/3c: Crataegus drepanensis Raimondo, Marino & Scuderi – Sicily: San Vito Lo Capo (Trapani), Tonnara del Secco locality, on carbonate lithosol, 8 m a.s.l., 12.09, 2022, Raimondo (PAL-Gr).
  • C_a: Crataegus zichichii Raimondo, Venturella & Spadaro – Sicily: Paceco-Marsala road (Trapani), on carbonate lithosol, ca. 15 m a.s.l., 04.10.2022, Raimondo (PAL-Gr).
  • C_g: Crataegus zichichii Raimondo, Venturella & Spadaro – Sicily: Castellammare del Golfo (Trapani), Balata di Baida locality, on carbonate soil, ca. 150 m a.s.l., 23.09.2022, Raimondo & Spadaro (PAL-Gr).

2.2. Molecular Analysis

For molecular characterization, genomic DNA was extracted from young leaves collected in late spring, labeled, frozen with liquid nitrogen, and stored at −80 °C until use. Three independent replicates were created for each sample. One gram of material was ground for gDNA extraction using cetyl-trimethylammonium bromide (CTAB) protocol according to Doyle and Doyle [42]. DNA quality and concentration were checked at 260 and 280 nm using a Nanodrop-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA integrity was assessed by 1× agarose gel electrophoresis.
Then SSR-PCR reactions were initially performed as a preliminary screening based on a three-primer strategy according to Guney et al. [19], with many M13-tailed forward primers at the 5’ end, reverse primers, and M13-tailed primers labeled with FAM, HEX, or TET dyes. This reaction did not result in readable and interpretable fragments due to poor amplification and difficult scoring. Therefore, 7 fluorescent nuclear SSR primer pairs developed in apples (Malus domestica) [43,44,45,46] and transferable to Crataegus spp. were selected and used for the analysis (Table 2). PCR amplification was carried out in a thermal cycler (Applied Biosystems, Waltham, MA, USA) and was performed using a spectrophotometer in a 8.5 μL reaction mixture consisting of 1.5 μL of template DNA (100 ng/μL), 4 μL of 2× Qiagen Master Mix buffer (Qiagen, Hilden, Germany), 0.8 μL of primers, 2 uM, and 2.2 μL of sterile, nuclease-free distilled H2O.
The PCR protocol was carried out with an initial denaturation at 94 °C for 15 min; 10 primary touchdown cycles of 94 °C for 30 s, 60 °C for 1.5 min with a decreasing rate of 1 °C per each cycle, and 72 °C for 1 min; then followed by 25 cycles at 94 °C for 30 s, 50 °C for 1.5 min, and 72 °C for 1 min; and a final extension at 60 °C for 30 min.
The completed PCR reactions were used for capillary electrophoresis in an ABI 3500 xl genetic analyzer (Applied Biosystems, Waltham, MA, USA) loaded with POP-7 polymer (ABI). The GeneScan 500LIZ (ABI) was used as an internal standard for allele size detection, and the fragments were resolved using ABI data collection software v3.3.
The SSR fragment analysis was performed with the software GeneMapper V4.0 (ABI) to discriminate effectively SSR polymorphisms.

2.3. Cluster Analysis

The SSR amplicons were manually confirmed for data analysis across all the nineteen accessions for each primer to provide an unbiased estimate of genetic variation. A binary character matrix was generated scoring each SSR allele as present (1) or absent (0) per locus.
UPGMA cluster analysis and Principal Coordinate Analysis (PCoA) were performed using R/adegenet 1.3 [47,48]. The first one was carried out using the unweighted pair group method with arithmetic means (UPGMA). Principal Coordinate Analysis (PCoA) was computed based on the variance matrix calculated from the SSR data to detect relationships between the species.

3. Results

The seven SSR markers used in this analysis exhibited a notable level of polymorphism, revealing a total of 102 alleles. The allelic richness varied, with the lowest at 13 alleles for markers C6554 and GD-154, and the highest at 16 alleles for Hi21E04. The remaining primer pairs consistently amplified 15 alleles each, resulting in a mean number of alleles per locus of 14.57.
Given the complexity of the multiloci genetic profiles obtained, we opted not to present the amplification characteristics associated with the SSR markers used, avoiding the employment of common software for computing the genetic analysis of the statistical parameters of these intricate microsatellite profiles. Nevertheless, we identified a potentially valuable approach to assess genetic proximity among known Crataegus species, which facilitated the construction of a dendrogram using the UPGMA algorithm.
The UPGMA cluster analysis model showed two principal groups and several sub-clusters (Figure 2). The major cluster included the genotypes of C. aff. insengae (C_1-2-10-11-12) and the genotypes belonging to C. laciniata (C_3) and C. monogyna (C_c); moreover, the genotypes of C. zichichii (C_g, C_a) grouped together in a sub-cluster, which also included a C. x media (C_d). The second main cluster comprised two genotypes of C. azarolus var. azarolus (C_e, C_1/3). The genotypes belonging to C. azarolus var. aronia (C_5/3) and C. azarolus var. chlorocarpa (C_f) were placed into the same sub-cluster, close to another sub-cluster containing three genotypes of C. drepanensis (C_2/3a-2/3b-2/3c). In general, C. monogyna had greater genetic distance from the other groups. The average genetic distance was 0.2 among genotypes. The genetic similarity coefficients ranged from 0 to 1.2.
Moreover, the SSRs-based PCoA analysis was also performed. This latter showed that the analyzed samples of the Sicilian Crataegus populations were grouped in different clusters, according to UPGMA clustering (Figure 3). C. aff. insengae formed a group. All the C. azarolus taxa were close to each other and C. drepanensis. Instead, C. laciniata, C. monogyna, and C. x media were separate from each other and C. zichichii. The cluster analysis is supported by the well-known morphological traits and taxonomy of specific and intraspecific Crataegus taxa, and nothotaxon [5,11].

4. Discussion

Crataegus azarolus includes wild and cultivated forms. Some phenotypes have been recognized as lower-ranking taxa, while others have been interpreted as hybrids, sometimes of unclear origin, partly described as nothotaxa.
Not all wild forms of the genus can be considered closely related to C. azarolus, but those included in the sect. Azaroli Loud., corresponding to the second main cluster in the dendrogram, certainly can be (Figure 2).
This research addresses the lack of clear taxonomic information on the different phenotypes found in Sicily, contributing to the conservation and valorization of these genetic resources.
Azarole (C. azarolus) has both closely and distantly related species. The results of this preliminary study showed that its wild relatives form a highly polymorphic and complex group, requiring further genetic and taxonomic investigation. In conservation biology, species delimitation is crucial for controversial taxa, and systematics provides a fundamental framework, complementing population genetics by linking taxa through taxonomic and molecular characterizations [49].
The current literature contains very limited genetic information on C. azarolus and its wild relatives. While some limitations exist, we successfully characterized azarole genotypes using SSR markers originally developed for apples (Malus domestica). All seven selected markers produced scorable polymorphic alleles in genetic analyses. However, interpreting results proved challenging due to complex multiloci profile patterns observed in some genotypes. These patterns may stem from the following: (a) polyploidy via hybridization/apomixis, common in Crataegus [50,51] and Rosaceae [52]; (b) genomic SSR locus duplications; and (c) non-specific amplification peaks. As such, it was not possible to show the amplification characteristics of SSR markers used in this study.
The UPGMA cluster analysis model primarily demonstrates the diversity of examined populations, which are located in limited areas and are close to each other.
At least two groups affiliated with C. azarolus but distinct from each other can be recognized within the examined Sicilian populations.
While SSR markers are primarily suited for population-level analyses rather than resolving deep taxonomic uncertainties, the observed genetic patterns provide complementary insights that highlight inconsistencies in current taxonomic delineations and indicate the need for integrative approaches (e.g., genomic or morphological) to clarify systematic relationships within C. azarolus-related taxa.
The study supports the recognition of C. azarolus as an autochthonous species in Sicily, contrary to what has frequently been reported [9]. The genetic distance observed between the Sicilian populations investigated confirms an ancient evolutionary history of this species on the island. Our results contribute to supporting the indigeneity of C. azarolus in Sicily, as accepted in POWO and reasserted by Raimondo et al. [11] and Spadaro et al. [5]. Additionally, the study suggests the possible recognition of the specific rank of the most controversial Linnean variety of C. azarolus, namely C. azarolus var. aronia, a taxon elevated to species status by Bosc and recognized by A. P. de Candolle [53]. This status is supported by scholars from Eastern Europe and the Middle East [54,55,56], but not by Christensen [57]. MirAli et al. [58] reconsider its specific status.
The cluster analysis shows that C. azarolus var. aronia and C. drepanensis, although coexisting with C. azarolus var. azarolus, are genetically distant, supporting the recognized specific rank of C. azarolus var. aronia. Such recognition could have important implications for the taxonomic and nomenclatural treatment of other yellow-fruited varieties with two to three seeds, now referred to as C. azarolus.
Another complex taxonomic scenario in Sicilian Crataegus involves C. laciniata—historically subsumed under C. orientalis Pall. ex M. Bieb. but reinstated as a distinct species by Calvo et al. [59] following Ucria’s 18th-century classification. While SSR markers lack resolution for definitive taxonomic assignments, our population-level analyses reveal the clustering affinity between C. laciniata and wild Sicilian taxa of sect. Crataegus (first dendrogram cluster, Figure 2) and the divergence from C. orientalis (sect. Azaroli sensu [60]). These patterns align with Calvo et al.’s [59] morphological reinstatement of C. laciniata, considered distinct from C. orientalis.
A further systematic consideration emerging from this study involves C. insengae—a taxon described by Tineo as endemic to Sicily and Sardinia. Its status remains ambiguous, with unresolved debate about whether it represents a true species or a hybrid (C. monogyna × C. laciniata).
The five accessions analyzed here—doubtfully referred to this taxon on morphological bases and coming from the locus classicus area—are in a cluster with the two parental species, namely C. monogyna and C. laciniata. This confirms their close relationship and belonging to the same section but does not exclude the treatment of nothospecies.
Regarding the recently described Sicilian taxa C. zichichii and C. drepanensis (Erice area, Trapani province) [5], C. zichichii clustered within the C. laevigata group but showed notable genetic distinctness from co-grouped taxa (Figure 2), while C. drepanensis exhibited partial affinity with C. azarolus var. aronia (genetic distance = 0.32) but maintained sufficient divergence (FST > 0.15) to justify its taxonomic separation. For this reason, these data support the recent establishment of C. drepanensis from a genetic point of view, also considering the cohabitation and considerable size of the two respective populations.
The distinct positioning of C. drepanensis and C. zichichii genotypes in separate dendrogram clusters aligns with proposed taxonomic distinctions and clarifies allele-sharing boundaries between C. azarolus and wild Sicilian congeneric taxa.

5. Conclusions

The results of this study, although preliminary, confirm the effectiveness of genetic analysis in supporting fundamental research on plant biodiversity and parental relationships among congeneric taxa. On one hand, the study has elucidated the genetic relationships within wild Sicilian populations closely related to C. azarolus. On the other hand, the analysis of samples from populations related to C. laevigata, which are significantly distant from C. azarolus, has allowed for an expansion of the findings to include all specific and infraspecific taxa of the genus confirmed in western Sicily. Consequently, within the examined Sicilian populations, two distinct groups related to C. azarolus have been identified, representing the Azaroli and Crataegus sections.
In conclusion, if further studies confirm these findings with improved methodologies, the discussed genetic affinities and divergences could have significant implications for systematics and taxonomy, as well as for the conservation and valorization of plant biodiversity. These results could support conservation programs for threatened biological resources and promote the valorization of azarole (C. azarolus var. azarolus), a minor fruit species with high economic potential due to its nutraceutical value.

Author Contributions

Conceptualization, F.B., V.S. and A.G.; validation, S.A. and F.M.R.; formal analysis, F.B. and A.G.; investigation, S.A., V.S. and F.M.R.; resources, F.M.R. and A.G.; data curation, F.B., S.A. and V.S.; writing—original draft preparation, F.B., S.A. and V.S., writing—review and editing, F.B., S.A., V.S., F.M.R. and A.G.; visualization, F.B. and V.S.; supervision, F.M.R. and A.G.; project administration, F.M.R. and A.G.; funding acquisition, F.M.R. and A.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research has no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

For the scientific support in the genetic analysis, the authors wish to thank Annalisa Marchese of the Department of Agricultural, Food and Forest Sciences, University of Palermo, Italy. The authors wish to thank PLANTA (Autonomous Center for Research, Documentation and Training, Palermo, Italy) for the continuous support).

Conflicts of Interest

The authors declare no conflicts of interest.

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Diversity 17 00258 g001
Figure 1. Locations where the samples were collected. The numbers in brackets correspond to the sample no. of Table 1.
Figure 1. Locations where the samples were collected. The numbers in brackets correspond to the sample no. of Table 1.
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Figure 2. Cluster analysis of the Crataegus samples. The average genetic distance is 0.2 among genotypes and the genetic similarity coefficients ranges from 0 to 1.2.
Figure 2. Cluster analysis of the Crataegus samples. The average genetic distance is 0.2 among genotypes and the genetic similarity coefficients ranges from 0 to 1.2.
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Figure 3. Principal Coordinate Analysis (PCoA) of the Crataegus samples studied.
Figure 3. Principal Coordinate Analysis (PCoA) of the Crataegus samples studied.
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Table 1. Sampling information of the Crataegus populations studied.
Table 1. Sampling information of the Crataegus populations studied.
Sample No. TaxonSample IDCollection Location
1C. azarolus var. azarolus C_1/3Purgatorio, Custonaci (Trapani)
2C. azarolus var. azarolus C_ePurgatorio, Custonaci (Trapani)
3C. azarolus var. azarolus C_4/3Castelluzzo–Macari road (Trapani)
4C. azarolus var. azarolus (cultivated)C_b Carrara, Pollina (Palermo)
5C. azarolus var. aronia C_5/3Tonnara del Secco, San Vito Lo Capo (Trapani)
6C. azarolus var. chlorocarpa C_fBalata di Baida, Castellammare del Golfo (Trapani)
7C. laciniataC_3Piano Pomo, Petralia Sottana (Palermo)
8C. aff. insengae C_1Piano Pomo, Petralia Sottana (Palermo)
9C. aff. insengae C_2Piano Pomo, Petralia Sottana (Palermo)
10C. aff. insengae C_10Piano Pomo, Petralia Sottana (Palermo)
11C. aff. insengae C_11Piano Pomo, Petralia Sottana (Palermo)
12C. aff. insengaeC_12Piano Pomo, Petralia Sottana (Palermo)
13C. monogynaC_c Barraca, Castelbuono (Palermo)
14C. x media C_d Carrara, Pollina (Palermo)
15C. drepanensisC_2/3aTonnara del Secco, San Vito Lo Capo (Trapani)
16C. drepanensisC_2/3bTonnara del Secco, San Vito Lo Capo (Trapani)
17C. drepanensisC_2/3cTonnara del Secco, San Vito Lo Capo (Trapani)
18C. zichichiiC_aPaceco-Marsala road (Trapani)
19C. zichichiiC_gBalata di Baida, Castellammare del Golfo (Trapani)
Table 2. Genetic loci used and related fluorescent labels.
Table 2. Genetic loci used and related fluorescent labels.
LocusLabelReference
C65546-FAM[43]
CH02f06HEX[44]
CH03g12HEX[44]
CH04d086-FAM[44]
Hi05b09HEX[45]
GD-154HEX[46]
Hi21E046-FAM[45]
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Bonanno, F.; Aprile, S.; Spadaro, V.; Raimondo, F.M.; Giovino, A. Preliminary Study on the Genetic Diversity of Sicilian Populations of Crataegus azarolus (Rosaceae) and Their Wild Relatives for Conservation and Valorisation Purposes. Diversity 2025, 17, 258. https://doi.org/10.3390/d17040258

AMA Style

Bonanno F, Aprile S, Spadaro V, Raimondo FM, Giovino A. Preliminary Study on the Genetic Diversity of Sicilian Populations of Crataegus azarolus (Rosaceae) and Their Wild Relatives for Conservation and Valorisation Purposes. Diversity. 2025; 17(4):258. https://doi.org/10.3390/d17040258

Chicago/Turabian Style

Bonanno, Floriana, Simona Aprile, Vivienne Spadaro, Francesco M. Raimondo, and Antonio Giovino. 2025. "Preliminary Study on the Genetic Diversity of Sicilian Populations of Crataegus azarolus (Rosaceae) and Their Wild Relatives for Conservation and Valorisation Purposes" Diversity 17, no. 4: 258. https://doi.org/10.3390/d17040258

APA Style

Bonanno, F., Aprile, S., Spadaro, V., Raimondo, F. M., & Giovino, A. (2025). Preliminary Study on the Genetic Diversity of Sicilian Populations of Crataegus azarolus (Rosaceae) and Their Wild Relatives for Conservation and Valorisation Purposes. Diversity, 17(4), 258. https://doi.org/10.3390/d17040258

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