Genetic diversity is key to survival, adaptation, and evolution in response to abiotic and biotic stresses. Adapting to changing environmental conditions is especially important in the Mediterranean region, which is characterized by heterogeneous environmental conditions. Many species including Pinus species face a loss of adaptation ability owing to potential natural and artificial events like climate change, human activities, and fires (Richardson et al., 2007; StClair & Howe, 2011). Intraspecific variability is essential to modulate adaptive responses to changing local conditions, and forest tree species (particularly Mediterranean pines, except for P. pinea) usually harbor extremely large intraspecific genetic diversity (Heuertz et al., 2010; Fady, 2012).

Two conifers play a key role in Mediterranean coastal forests (Fady et al., 2003; Boydak, 2004). Aleppo pine (Pinus halepensis Mill.) and Brutia pine, also known as Calabrian or Brutian pine (Pinus brutia Ten.) cover together more than 8 million hectares (Daskalakou & Thanos, 2010). The natural distribution of Aleppo pine is in southern Europe (Spain, Italy, and Greece) and northern Africa (Morocco, Algeria, and Tunisia). Brutia pine is predominantly distributed in Türkiye, but some Brutia pine populations are also found in Crete, Cyprus, Syria, and Lebanon (Mauri et al., 2016). Aleppo and Brutia pines are vicariant species; the former is mainly found in the coastal areas of the western Mediterranean region while the latter is native to the eastern Mediterranean region, both of which play important roles in local economies (Fady et al., 2003; Boydak, 2004). The evolutionary and ecological characteristics of Aleppo and Brutia pines are closely related (Tozkar et al., 2009). These pines live in a Mediterranean-type climate, which is characterized by warm, dry summers and cool, mild winter conditions, and thrive in a complex range of ecological habitats from sea level up to 2,000 meters (Fady et al., 2003; Boydak, 2004; Daskalakou & Thanos, 2010). Both species are known to adapt to the Mediterranean climate. Natural and artificial hybridization between these two species has been described in many studies. Viable seeds were only obtained when Aleppo pine was used as the pollen donor and Brutia pine was used as the maternal parent (Schiller et al., 1986; Korol et al., 1995; Bucci et al., 1998). Despite their closeness, the two pines are recognized as separate species occupying different geographical ranges and bioclimates (Fady et al., 2003). Both pines can be identified using morphological, palynological, biochemical traits and DNA markers (Bucci et al., 1998; Korol et al., 2002a; Tozkar et al., 2009).

Characterizing the genetic resources of these two pines is essential to predict their adaptive potential, thereby implementing adequate strategies for the conservation, management, and improvement of these species. Aleppo and Brutia pines, separately or together were previously analyzed for their background levels of diversity, using morphological (Panetsos et al., 1997; Isik & Isik, 1999; Isik et al., 1999), biochemical traits (Korol et al., 2002a,b; Kaya et al., 2006) and DNA markers (Bucci et al., 1998; Kandemir et al., 2004; İçgen et al., 2006; Lise et al., 2007; Grivet et al., 2011; Kurt et al., 2012; Ruiz Daniels et al., 2018; Olsson et al., 2021). The results of several studies have suggested that both species are distinct and that Brutia pine is more diverse than Aleppo pine (Bucci et al., 1998; Kurt et al., 2012).

However, researchers have rarely analyzed these two pines simultaneously using the same markers. To date, only one study (Bucci et al., 1998) has used chloroplast simple sequence repeats (cpSSR) markers to analyze the genetic diversity of these two closely related species (Aleppo pine and Brutia pine). The latter study was limited by the number of populations analyzed within each species, limiting the inferences. The cpSSR primers developed by Vendramin et al. (1996) from the Pinus thunbergii Parl. chloroplast genome have been used in many pine species (Bucci et al., 1998; Robledo-Arnuncio et al., 2005; Grivet et al., 2009; Heurtz et al., 2010; Kurt et al., 2012). cpSSR markers are very useful tools for determining genetic diversity and phylogeographic analysis of closely related species due to their high polymorphism levels and easy optimization for related species (Bucci et al., 1998; Kurt et al., 2012).

Here we analyzed a comprehensive set of Aleppo and Brutia pine populations across the Mediterranean Basin using common chloroplast microsatellite markers. The main goal of this study was to characterize the genetic diversity of these two key Mediterranean pines to gain insights into the evolutionary history of the Aleppo-Brutia species complex.

The studies with the same molecular markers, especially with paternally inherited chloroplast microsatellites, are limited for both Aleppo and Brutia pine and have been rarely studied (Olsson et al., 2021). According to our literature knowledge, halepensis-complex pine species (Turkish red, Aleppo, and eldarica pines) were analyzed only in the study of Bucci et al. (1998) using cpSSR markers. Our study is based on the first extensive sampling of two species through their natural distribution range across Mediterranean Basin countries (Fig. 1 and Table 1). This study had almost three-fold population numbers (20 vs 57) and more than five-fold (247 vs 1338) individual numbers (Table 1) more than Bucci et al. (1998). Therefore, our study provides comprehensive data contributing to Aleppo and Brutia pine genetic diversity, phylogeographic structure, conservation actions, and phylogenetic relationships.

In this study, we selected ten primer pairs for preliminary analysis, and the five most polymorphic primers were used for all analyses. We found a high number of haplotypes for only five cpSSR loci in two species (Table 1). The eldarica pine population (Khorasan-Razavi) alone had four haplotypes. Bucci et al. (1998) reported 27 alleles for the same SSR markers in the studied Aleppo and Brutia pine populations. The higher number of alleles (51) detected in the present study could be explained by various reasons such as sampling comprehensive populations, analyzing a large number of individuals, the inclusion of ecologically marginal populations (Fig. 1 and Table 1), and effective population size of the eastern Mediterranean tree species (suggested for Mediterranean conifers by Fady (2005) and for Aleppo pine by Grivet et al. (2009)). We found 63 and 150 haplotypes, and 33 and 44 alleles for Aleppo and Brutia pine, respectively. Twenty-six out of 51 alleles were common for both species. Of the 204 haplotypes, only nine were shared by the two species. As previously reported by different studies (Bucci et al., 1998; Kurt et al., 2012), similar to our study, the Brutia pine populations analyzed had higher levels of genetic diversity compared to those of Aleppo pine populations. Olsson et al. (2021), combining molecular and fossil information, suggested that at the beginning of the Pleistocene (~ 2 million years), P. brutia may have expanded in the eastern Mediterranean, while P. halepensis populations went through bottlenecks. The suggestion by Olsson et al. (2021) clarifies our results.

The haplotypic and allelic variations observed in this study are consistent with the results of cpSSR analysis in other pine species, P. pinaster (25 alleles and 108 haplotypes for six cpSSR loci, Ribeiro et al., 2001), P. sylvestris (29 alleles and 139 haplotypes for six cpSSR loci, Robledo-Arnuncio et al., 2005), P. uncinata (62 alleles and 174 haplotypes for ten cpSSR loci, Dzialuk et al., 2009) and P. cembra (22 alleles and 41 haplotypes for six cpSSR loci, Hohn et al., 2005). The allelic and haplotypic differentiation of cpSSR markers depends on the number of loci, populations, individuals, and polymorphism levels of the related loci and species (Robledo-Arnuncio et al., 2005; Kurt et al., 2012). Moreover, the species-specific alleles reported by Bucci et al. (1998) were 21 for Aleppo and one for Brutia pine, compared to 7 and 18 respectively in the present study (Table S2). There were three population-specific alleles for eldarica pine (Khorasan-Razavi). These results suggest that the five cpSSR analyzed are optimal for species delimitation in Brutia pine, but more loci should be included in the analyses in Aleppo pine.

The allele size variants and allele range between the minimum and maximum in this study (Table S1) were also more diverse than those reported by Bucci et al. (1998). All the studied loci in the Brutia pine populations showed 1 to 5-bp mutational steps (Fig. S1). Those steps were seen from 1 to 7-bp only in a few loci of the Aleppo and eldarica pine populations. However, Kurt et al. (2012) reported the 1 and 2-bp gap in six cpSSR loci (four of them are the same as in this study) of Brutia pine populations. Also, Robledo-Arnuncio et al. (2005) detected the 2-bp gap in the Pt71936 region, and they reported that size variants were due to a 5-bp deletion in the microsatellite flanking region according to sequence analysis. The chloroplast microsatellites in this study are mononucleotide-repeats (Vendramin et al., 1996) like the aforementioned studies (Bucci et al., 1998; Robledo-Arnuncio et al., 2005; Kurt et al., 2012). Therefore, individuals carrying all mutational steps in this study should be sequenced to reveal the source of mutations.

The analysis of molecular variance showed that population-level differentiation was reduced for both species, while most of the variation (83% and 73% for Aleppo and Brutia pine, respectively) was found among individuals within populations (Table 2). Therefore, tree improvement programs and conservation studies need to consider mainly individuals within a population. Although Western Aleppo pine populations have lower genetic variation than their eastern counterparts, they still maintain historical demography. This situation can be explained as the Western Aleppo pine populations have potentially adapted gene pools to their environment. For this reason, Western Aleppo pine populations should be conserved more effectively than their eastern counterparts. The Eastern Aleppo pine populations might be preferable for studies on tree improvement and especially the investigation of adaptive traits. In addition, Brutia pine and Aleppo pine populations of the eastern Mediterranean are important to constitute a reservoir of genetic diversity for improvement and conservation programs. Conservation of both pine forests is not only crucial for related species but is also very vital for the biodiversity of all living organisms related to forests and individual trees. Conservation and sustainable use of both pine forests by ex-situ and in-situ conservation efforts and protected areas are one of the most important steps to prevent biodiversity loss. The global temperature rises and increase in human activities will probably affect Mediterranean forests harsher than other ecosystems, especially at the lower altitudinal distribution of both pine species (StClair & Howe, 2011). Therefore, conservation and protection strategy studies should be increased at the local, regional and international levels to maintain sustainable biodiversity levels for the two pine forests and related species (Climent et al., 2021).

Aleppo pine populations showed clear geographic differentiation, while the Brutia pine populations did not show a clear geographic pattern like Aleppo pine; however, geographically close populations and/or populations from their continual distribution range were genetically closer than fragmented and/or ecologically marginal populations (Figs. 1 and 3; Table 1). As expected, the eldarica pine population was genetically closer to the Turkish red pine populations (Fig. 3). The same results for eldarica pine were reported by Bucci et al. (1998). Some populations of Brutia pine (Amasya-Amasya, Amasya-Bafra, K. Maras-K.Maras and Balikesir-Ayvacik) clustered with the Tabarka population of Aleppo pine from Tunisia (Fig. 3). These populations have been sharing a haplotype (H3), which is a common second haplotype for Aleppo pine. This phenomenon could be explained by unidirectional mating (Aleppo pine as pollen donor and Brutia pine as a female parent not reciprocally) between two species reported by different researchers using morphological traits (Panetsos et al., 1997), allozymes (Korol et al., 1995, 2002a) and DNA markers (Bucci et al., 1998; Tozkar et al., 2009). Kremer et al. (2012) reported that airborne pollens from forest trees have the potential to be transported in considerable amounts over hundreds to thousands of kilometers based on the results of aerobiological studies. The evolutionary history (derived from a common ancestor) and recent colonization of both species (in the last 10,000 years) in the Mediterranean Basin could be also explained by the aforementioned phenomenon (Conkle et al., 1988; Bucci et al., 1998; Tozkar et al., 2009).

The present study indicated that Aleppo pine populations were divided into two different groups: one included the populations from Spain (except Tivissa), and the other included Turkish, Greek, and Italian populations (Fig. 3). The structure analysis results also supported the dendrogram (Figs. S2 and S3). Our findings showed similarity with Mediterranean conifers (Fady, 2005) and Aleppo pine populations (Grivet et al., 2009). The clear east-west differentiation of Aleppo pine populations was previously described by Schiller et al. (1986) as an Eastern Mediterranean race and Western Mediterranean race by their isozymes which was also confirmed by our results. Bucci et al. (1998) reported two main groups for Aleppo pine populations: a central Mediterranean group (Italian and Spanish populations) and a southern Mediterranean group (Greek and Algerian populations). The different results observed in the study of Bucci et al. (1998) could be related to sampling from individual trees in Aleppo pine populations and a small number of populations. The genetic diversity patterns from east to west and the high variation levels in the eastern population could be attributed to various reasons: (i) climatic conditions at the beginning of the Quaternary in Europe and the Mediterranean Basin (Tzedakis et al., 2002; Petit et al., 2005); (ii) demographic bottlenecks and founder effects with a small population size of Aleppo pine in the western basin (Morgante et al., 1998; Grivet et al., 2009; Ruiz Daniels et al., 2018; Olsson et al., 2021); (iii) limited gene flow with ancestor populations and accumulation of new mutations (Petit et al., 2005); (iv) the effect of different selection pressures at various environments (Kremer et al., 2002; Kurt et al., 2012); and (v) the more and larger refugia of eastern Mediterranean and to be a possible genetic diversity center for the two pine species (Fady, 2005; Grivet et al., 2009; Fady & Conord, 2010), either alone and/or by combinations of those.

As a result, this study adds new information about cpSSR variation of an extensive sampling of Aleppo and Brutia pine populations across the Mediterranean Basin. The Brutia pine populations are much more diverse than the Aleppo pine populations and have no clear geographical differentiation in Türkiye. Aleppo pine populations show a clinal east-west variation across the Mediterranean Basin. Eastern populations are more diverse than western ones and the Eastern Mediterranean Basin could be considered the genetic diversity center of both pine species.