# Chloroplast microsatellite diversity of Pinus brutia Ten. and Pinus halepensis Mill. populations across the Mediterranean basin: Inferences of their distributions

Yusuf Kurt, Burcu Cengel, Ercan Velioglu, Santiago C Gonzalez-Martinez, Delphine Grivet, Nuray Kaya

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Yusuf Kurt, Burcu Cengel, Ercan Velioglu, Santiago C Gonzalez-Martinez, Delphine Grivet, et al. Chloroplast microsatellite diversity of Pinus brutia Ten. and Pinus halepensis Mill. populations across the Mediterranean basin: Inferences of their distributions. FOREST SYSTEMS, 2023, 32 (2), pp.e008. 10.5424/fs/202322-19729. hal-04185963

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# Chloroplast microsatellite diversity of *Pinus brutia* Ten. and *Pinus halepensis* Mill. populations across the Mediterranean basin: Inferences of their distributions

● Yusuf KURT¹, ● Burcu CENGEL², ● Ercan VELIOGLU³, ● Santiago C. GONZALEZ-MARTINEZ⁴, ● Delphine GRIVET⁵ and ● Nuray KAYA⁶*

¹ Harran University, Molecular Biology and Genetics Department, Osmanbey Campus, Sanliurfa, Türkiye, ² Forest Tree Seeds and Tree Breeding Research Directorate, Ankara, Türkiye, ³ Poplar and Fast-Growing Forest Trees Research Institute, Kocaeli, Türkiye, ⁴ BIOGECO, INRA, Univ. Bordeaux, 33610, Cestas, France, ⁵ INIA-CIFOR, Dept. Forest Ecology & Genetics, Forest Research Center, 28040 Madrid, Spain., ⁶ Akdeniz University, Biology Department, Campus, Antalya, Türkiye.

*Correspondence should be addressed to Nuray Kaya: nkaya@akdeniz.edu.tr

## Abstract

**Aim of study:** To characterize and compare the genetic resources and gain some insights into the evolutionary history of Aleppo pine (*Pinus halepensis* Mill.) and Brutia pine (*Pinus brutia* Ten.) species which are both distributed across more than 8 million hectares of area in the Mediterranean Basin.

**Area of study:** Fifty-six populations from eight Mediterranean basin countries where *P. halepensis* and *P. brutia* species are located.

**Materials and methods:** We analyzed 1344 seeds belonging to 56 populations using five cpSSR primers (Pt15169, Pt30204, Pt41093, Pt87268, and Pt110048).

**Main results:** The analysis of molecular variance (AMOVA) revealed that the genetic diversity among the Brutia pine populations was slightly higher than that of Aleppo pine (27.06% and 24.27%, respectively). The Aleppo pine populations separately displayed a clear east-west differentiation across the Mediterranean Basin, confirming previous results using other markers. Although the Brutia pine populations showed no spatial genetic pattern, geographically close populations and/or populations from their continual distribution range were genetically closer than the fragmented and/or ecologically marginal populations.

**Research highlights:** The seven Aleppo pine populations from the eastern range (Türkiye, Greece, and Italy) were more than two-fold diverse than the 13 populations from the western range (Spain and Morocco). The eastern range of Aleppo pine and Brutia pine populations had similar levels of genetic diversity parameters. These results suggested that the Eastern Mediterranean Basin is a possible genetic diversity center for the two pine species.

**Additional key words:** halepensis-complex; simple sequence repeats; tree genetics

**Citation:** Kurt, Y; Cengel, B; Velioglu, E; Gonzalez-Martinez, SC; Grivet, D; Kaya, N (2023). Chloroplast microsatellite diversity of *Pinus brutia* Ten. and *Pinus halepensis* Mill. populations across the Mediterranean basin: Inferences of their distributions. Forest Systems, Volume 32, Issue 2, e008. https://doi.org/10.5424/fs/202322-19729

**Supplementary material** (Table S1 and Figs. S1-S2-S3) accompanies the paper on Forest System's website

**Received:** 23 Jul 2022. **Accepted:** 07 Jun 2023.

**Copyright © 2023 CSIC.** This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License.

<table><thead><tr><td>Funding agencies/institutions</td><td>Project / Grant</td></tr></thead><tbody><tr><td>The Scientific and Technical Research Council of Türkiye (TUBITAK)</td><td>1120811</td></tr></tbody></table>

**Competing interests:** The authors have declared that no competing interests exist.
# Introduction

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.

# Material and methods

## Studied populations and DNA extraction

A comprehensive sampling was performed from the natural distribution of *P. halepensis* and *P. brutia* by collecting samples from 56 populations. Twenty-one populations of *P. halepensis* (488 individuals) and 35 populations of *P. brutia* var. *brutia* Ten. (836 individuals), and one population of *P. brutia* var. *eldarica* Medw. (Khorasan-Razavi, 20 individuals) were mostly sampled from the natural distribution range (Fig. 1 and Table 1). Few of the sampled *P. brutia* populations were outside the natural distribution range. Hereinafter, the *P. halepensis* populations and *P. brutia* var. *brutia* Ten. populations are referred to as Aleppo pine and Brutia pine, respectively, while the Khorasan-Razavi (*P. brutia* var. *eldarica* Medw.) population is referred to in the text as Eldarica pine. Previously collected bulked seeds from each population were used for analyses.
<table><caption>Table 1. Geographic information and descriptive genetic parameters of studied populations.</caption><thead><tr><th>No</th><th>Population</th><th>Country</th><th>Altitude (m)</th><th>Latitude</th><th>Longitude</th><th>n</th><th>A</th><th>Ne</th><th>P</th><th>R<sup>h</sup></th><th>H<sub>e</sub></th><th>D<sup>2</sup><sub>sh</sub></th></tr></thead><tbody><tr><td colspan="13" style="text-align: center; font-weight: bold;">Brutia pine</td></tr><tr><td>1.</td><td>Adana-Pos</td><td>Türkiye</td><td>745</td><td>37.541667°</td><td>35.416667°</td><td>24</td><td>8</td><td>4.24</td><td>0</td><td>5.95</td><td>0.80</td><td>0.51</td></tr><tr><td>2.</td><td>Adapazari-Geyve</td><td>Türkiye</td><td>530</td><td>40.386667°</td><td>30.420278°</td><td>24</td><td>5</td><td>3.60</td><td>0</td><td>3.84</td><td>0.75</td><td>0.3</td></tr><tr><td>3.</td><td>Amasya-Amasya</td><td>Türkiye</td><td>430</td><td>40.895278°</td><td>36.355278°</td><td>23</td><td>23</td><td>23.00</td><td>15</td><td>16.00</td><td>1.00</td><td>15.4</td></tr><tr><td>4.</td><td>Amasya-Bafra</td><td>Türkiye</td><td>50</td><td>41.651111°</td><td>35.456111°</td><td>24</td><td>23</td><td>22.15</td><td>16</td><td>15.51</td><td>1.00</td><td>16.24</td></tr><tr><td>5.</td><td>Ankara-Nallihan</td><td>Türkiye</td><td>750</td><td>40.148889°</td><td>30.855556°</td><td>24</td><td>12</td><td>7.02</td><td>0</td><td>8.77</td><td>0.89</td><td>1.1</td></tr><tr><td>6.</td><td>Antalya-Antalya</td><td>Türkiye</td><td>275</td><td>36.995833°</td><td>30.552778°</td><td>24</td><td>12</td><td>10.29</td><td>2</td><td>10.84</td><td>0.94</td><td>0.97</td></tr><tr><td>7.</td><td>Antalya-Alanya</td><td>Türkiye</td><td>350</td><td>36.610000°</td><td>31.965278°</td><td>24</td><td>15</td><td>9.29</td><td>0</td><td>9.21</td><td>0.93</td><td>1.21</td></tr><tr><td>8.</td><td>Antalya-Gundogmus</td><td>Türkiye</td><td>1000</td><td>36.710000°</td><td>32.181944°</td><td>24</td><td>11</td><td>6.86</td><td>0</td><td>8.08</td><td>0.89</td><td>1.25</td></tr><tr><td>9.</td><td>Antalya-Kas</td><td>Türkiye</td><td>1050</td><td>36.408333°</td><td>29.500000°</td><td>24</td><td>5</td><td>1.72</td><td>0</td><td>3.26</td><td>0.44</td><td>0.17</td></tr><tr><td>10.</td><td>Antalya-Kumluca</td><td>Türkiye</td><td>250</td><td>36.433333°</td><td>30.250000°</td><td>24</td><td>12</td><td>6.70</td><td>1</td><td>8.37</td><td>0.89</td><td>0.96</td></tr><tr><td>11.</td><td>Balikesir-Ayvacık</td><td>Türkiye</td><td>300</td><td>39.883333°</td><td>26.416667°</td><td>24</td><td>19</td><td>14.40</td><td>9</td><td>13.18</td><td>0.97</td><td>12.54</td></tr><tr><td>12.</td><td>Bolu-Goynuk</td><td>Türkiye</td><td>750</td><td>40.405000°</td><td>30.659722°</td><td>23</td><td>11</td><td>5.94</td><td>3</td><td>8.06</td><td>0.87</td><td>1.73</td></tr><tr><td>13.</td><td>Bursa-Orhaneli</td><td>Türkiye</td><td>650</td><td>40.011111°</td><td>28.925000°</td><td>24</td><td>7</td><td>3.23</td><td>2</td><td>4.68</td><td>0.73</td><td>0.54</td></tr><tr><td>14.</td><td>Denizli-Acipayam</td><td>Türkiye</td><td>750</td><td>37.181667°</td><td>29.213889°</td><td>23</td><td>5</td><td>2.91</td><td>0</td><td>4.54</td><td>0.68</td><td>0.4</td></tr><tr><td>15.</td><td>Isparta-Sutculer</td><td>Türkiye</td><td>1100</td><td>37.513889°</td><td>30.869444°</td><td>19</td><td>9</td><td>1.45</td><td>1</td><td>2.96</td><td>0.32</td><td>2.19</td></tr><tr><td>16.</td><td>Isparta-Bucak</td><td>Türkiye</td><td>800</td><td>37.500000°</td><td>30.683333°</td><td>21</td><td>10</td><td>6.06</td><td>1</td><td>8.07</td><td>0.88</td><td>1.1</td></tr><tr><td>17.</td><td>Istanbul-Kanlica</td><td>Türkiye</td><td>10</td><td>40.850556°</td><td>29.123333°</td><td>23</td><td>7</td><td>6.81</td><td>2</td><td>7.57</td><td>0.90</td><td>0.39</td></tr><tr><td>18.</td><td>Izmir-Bergama</td><td>Türkiye</td><td>620</td><td>39.235556°</td><td>27.146389°</td><td>20</td><td>9</td><td>2.51</td><td>0</td><td>5.04</td><td>0.63</td><td>0.77</td></tr><tr><td>19.</td><td>Izmir-Izmir</td><td>Türkiye</td><td>150</td><td>38.240556°</td><td>26.600556°</td><td>22</td><td>7</td><td>5.41</td><td>0</td><td>7.35</td><td>0.86</td><td>5.12</td></tr><tr><td>20.</td><td>Kastamonu-Duragan</td><td>Türkiye</td><td>220</td><td>41.419722°</td><td>35.106667°</td><td>24</td><td>14</td><td>3.97</td><td>1</td><td>5.27</td><td>0.78</td><td>1.1</td></tr><tr><td>21.</td><td>K.Maras-Antakya</td><td>Türkiye</td><td>480</td><td>35.900000°</td><td>36.016667°</td><td>24</td><td>9</td><td>7.20</td><td>1</td><td>9.71</td><td>0.90</td><td>0.52</td></tr><tr><td>22.</td><td>K.Maras-K.Maras</td><td>Türkiye</td><td>800</td><td>37.778889°</td><td>36.706944°</td><td>24</td><td>19</td><td>4.57</td><td>3</td><td>6.39</td><td>0.82</td><td>18.21</td></tr><tr><td>23.</td><td>Kutahya-Tavsanlı</td><td>Türkiye</td><td>700</td><td>39.490278°</td><td>29.291389°</td><td>24</td><td>11</td><td>6.26</td><td>1</td><td>7.23</td><td>0.88</td><td>1.86</td></tr><tr><td>24.</td><td>Mersin-Bozyazi</td><td>Türkiye</td><td>350</td><td>36.226389°</td><td>33.105556°</td><td>24</td><td>11</td><td>3.89</td><td>2</td><td>6.43</td><td>0.78</td><td>1.41</td></tr><tr><td>25.</td><td>Mersin-Tarsus</td><td>Türkiye</td><td>1000</td><td>37.091667°</td><td>34.558333°</td><td>24</td><td>15</td><td>14.40</td><td>8</td><td>13.18</td><td>0.97</td><td>1.63</td></tr><tr><td>26.</td><td>Mersin-Gulnar</td><td>Türkiye</td><td>650</td><td>36.241667°</td><td>33.255556°</td><td>24</td><td>11</td><td>5.14</td><td>0</td><td>7.80</td><td>0.84</td><td>1.24</td></tr><tr><td>27.</td><td>Mersin-Silifke</td><td>Türkiye</td><td>100</td><td>36.216667°</td><td>33.716667°</td><td>23</td><td>11</td><td>7.78</td><td>0</td><td>8.49</td><td>0.91</td><td>2.42</td></tr><tr><td>28.</td><td>Mugla-Marmaris</td><td>Türkiye</td><td>60</td><td>37.004722°</td><td>28.328333°</td><td>24</td><td>9</td><td>6.86</td><td>0</td><td>8.08</td><td>0.89</td><td>8.48</td></tr><tr><td>29.</td><td>Mugla-Yilanlı</td><td>Türkiye</td><td>750</td><td>37.288333°</td><td>28.563889°</td><td>24</td><td>8</td><td>10.94</td><td>8</td><td>12.63</td><td>0.96</td><td>0.64</td></tr><tr><td>30.</td><td>S.Urfa-Adiyaman</td><td>Türkiye</td><td>1000</td><td>37.886667°</td><td>37.674722°</td><td>24</td><td>13</td><td>6.22</td><td>0</td><td>8.10</td><td>0.88</td><td>4.47</td></tr><tr><td>31.</td><td>S.Urfa-Sirnak</td><td>Türkiye</td><td>600</td><td>37.483056°</td><td>41.888333°</td><td>24</td><td>12</td><td>8.00</td><td>4</td><td>10.56</td><td>0.91</td><td>1.1</td></tr><tr><td>32.</td><td>Kibris1</td><td>Cyprus</td><td>126</td><td>35.262778°</td><td>33.039167°</td><td>24</td><td>9</td><td>4.36</td><td>4</td><td>6.49</td><td>0.80</td><td>0.59</td></tr><tr><td>33.</td><td>Kibris2</td><td>Cyprus</td><td>157</td><td>35.350833°</td><td>32.982500°</td><td>24</td><td>10</td><td>3.74</td><td>0</td><td>5.74</td><td>0.76</td><td>0.86</td></tr><tr><td>34.</td><td>Mersin-Mut</td><td>Türkiye</td><td>1150</td><td>36.839167°</td><td>33.303333°</td><td>19</td><td>14</td><td>4.88</td><td>7</td><td>8.93</td><td>0.83</td><td>8.17</td></tr><tr><td>35.</td><td>Bursa-MKP</td><td>Türkiye</td><td>400</td><td>39.928611°</td><td>28.625278°</td><td>20</td><td>6</td><td>9.00</td><td>1</td><td>9.06</td><td>0.93</td><td>0.91</td></tr><tr><td colspan="2" style="text-align: center; font-weight: bold;">Mean</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="13" style="text-align: center; font-weight: bold;">Eldarica pine</td></tr><tr><td>36.</td><td>Khorasan-Razavi</td><td>Iran</td><td>1063</td><td></td><td></td><td></td><td>19</td><td>8</td><td>4.31</td><td>5</td><td>7.00</td><td>0.82</td><td>4.07</td></tr><tr><td colspan="13" style="text-align: center; font-weight: bold;">Aleppo pine</td></tr><tr><td>37.</td><td>Mugla-Gokova</td><td>Türkiye</td><td>50</td><td>36.954722°</td><td>29.208333°</td><td>23</td><td>5</td><td>1.76</td><td>1</td><td>2.78</td><td>0.45</td><td>0.42</td></tr><tr><td>38.</td><td>Izmir-Urla</td><td>Türkiye</td><td>50</td><td>38.255556°</td><td>26.709167°</td><td>22</td><td>11</td><td>8.02</td><td>6</td><td>7.76</td><td>0.92</td><td>8.65</td></tr><tr><td>39.</td><td>Cabanellas-Alta</td><td>Spain</td><td>210</td><td>42.248294°</td><td>2.783798°</td><td>27</td><td>9</td><td>3.78</td><td>0</td><td>5.14</td><td>0.76</td><td>0.65</td></tr><tr><td>40.</td><td>Tivissa-Cataluna</td><td>Spain</td><td>400</td><td>41.059193°</td><td>0.760224°</td><td>25</td><td>4</td><td>1.52</td><td>0</td><td>2.05</td><td>0.36</td><td>0.09</td></tr><tr><td>41.</td><td>Zuera-Monegros</td><td>Spain</td><td>575</td><td>41.918800°</td><td>-0.921611°</td><td>25</td><td>6</td><td>2.62</td><td>0</td><td>3.49</td><td>0.64</td><td>0.29</td></tr><tr><td>42.</td><td>Alcantud-Alcarria</td><td>Spain</td><td>950</td><td>40.564133°</td><td>-2.313436°</td><td>27</td><td>5</td><td>2.65</td><td>0</td><td>2.81</td><td>0.65</td><td>0.8</td></tr><tr><td>43.</td><td>Tuejar-Maestrazgo</td><td>Spain</td><td>600</td><td>39.819100°</td><td>-1.159188°</td><td>24</td><td>7</td><td>4.00</td><td>1</td><td>4.68</td><td>0.78</td><td>1.94</td></tr><tr><td>44.</td><td>Tibi-Levante Interior</td><td>Spain</td><td>1010</td><td>38.519440°</td><td>-0.648611°</td><td>26</td><td>4</td><td>2.33</td><td>0</td><td>2.08</td><td>0.59</td><td>0.45</td></tr><tr><td>45.</td><td>Benicassim-Litoral Levantino</td><td>Spain</td><td>430</td><td>40.077655°</td><td>0.025914°</td><td>24</td><td>4</td><td>2.09</td><td>0</td><td>2.17</td><td>0.54</td><td>1.02</td></tr><tr><td>46.</td><td>Villajoyosa-Sudeste</td><td>Spain</td><td>70</td><td>38.496100°</td><td>-0.303656°</td><td>25</td><td>6</td><td>2.99</td><td>1</td><td>3.50</td><td>0.69</td><td>1.21</td></tr><tr><td>47.</td><td>Monovar-Betica Septentrioal</td><td>Spain</td><td>700</td><td>38.385360°</td><td>-0.957389°</td><td>26</td><td>7</td><td>3.22</td><td>1</td><td>3.95</td><td>0.72</td><td>1.82</td></tr><tr><td>48.</td><td>Benamaurel-Betica Meridional</td><td>Spain</td><td>920</td><td>37.702100°</td><td>-2.738858°</td><td>26</td><td>8</td><td>3.25</td><td>1</td><td>4.23</td><td>0.72</td><td>1.12</td></tr><tr><td>49.</td><td>Frigiliana-Sur</td><td>Spain</td><td>570</td><td>36.818198°</td><td>-3.920522°</td><td>23</td><td>6</td><td>2.86</td><td>1</td><td>3.43</td><td>0.68</td><td>1.16</td></tr><tr><td>50.</td><td>Alcotx-Mallorca Menorca</td><td>Spain</td><td>100</td><td>39.971779°</td><td>4.168438°</td><td>25</td><td>6</td><td>3.14</td><td>1</td><td>3.09</td><td>0.71</td><td>0.81</td></tr><tr><td>51.</td><td>Amfilohia</td><td>Greece</td><td>25</td><td>38.883652°</td><td>21.283507°</td><td>28</td><td>12</td><td>8.52</td><td>4</td><td>7.48</td><td>0.92</td><td>6.93</td></tr><tr><td>52.</td><td>Kassandra</td><td>Greece</td><td>25</td><td>40.091078°</td><td>23.881487°</td><td>24</td><td>18</td><td>12.52</td><td>8</td><td>10.18</td><td>0.96</td><td>7.16</td></tr><tr><td>53.</td><td>Litorale Tarantino</td><td>Italy</td><td>10</td><td>40.619829°</td><td>17.116000°</td><td>22</td><td>11</td><td>6.21</td><td>3</td><td>7.26</td><td>0.88</td><td>2.68</td></tr><tr><td>54.</td><td>Gargano Marzini</td><td>Italy</td><td>200</td><td>41.902422°</td><td>15.941800°</td><td>22</td><td>10</td><td>3.56</td><td>1</td><td>6.21</td><td>0.75</td><td>2.4</td></tr><tr><td>55.</td><td>Gargano Monte Pucci</td><td>Italy</td><td>100</td><td>41.547383°</td><td>15.857200°</td><td>14</td><td>6</td><td>2.72</td><td>2</td><td>5.00</td><td>0.68</td><td>1.46</td></tr><tr><td>56.</td><td>Tabarka</td><td>Tunisia</td><td>144</td><td>36.505600°</td><td>9.075704°</td><td>15</td><td>12</td><td>8.33</td><td>6</td><td>9.40</td><td>0.94</td><td>6.93</td></tr><tr><td>57.</td><td>Zaouia Ifrane</td><td>Morocco</td><td>1512</td><td>33.570000°</td><td>-5.140000°</td><td>23</td><td>4</td><td>2.07</td><td>0</td><td>2.46</td><td>0.54</td><td>0.17</td></tr><tr><td colspan="2" style="text-align: center; font-weight: bold;">Western populations mean (Spain, Tunisia and Morocco)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2" style="text-align: center; font-weight: bold;">Eastern populations mean (Türkiye, Greece and Italy)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2" style="text-align: center; font-weight: bold;">Mean</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2" style="text-align: center; font-weight: bold;">Grand mean for all populations</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

n: sample size. A: number of haplotypes. Ne: effective number of haplotypes. P: number of private haplotypes. R<sup>h</sup>: haplotypic richness. H<sub>e</sub>: genetic diversity. D<sup>2</sup><sub>sh</sub>: mean genetic distance among individuals within populations according to stepwise mutation model (Goldstein et al., 1995)
Randomly selected 30-35 seeds from each population
were germinated for DNA extractions. DNA was extract-
ed according to Doyle & Doyle (1990) protocol. The av-
erage number of analyzed samples from each population
was 24, ranging from 19 to 28 individuals per population
(Table 1).

Chloroplast microsatellites and PCR products

For a preliminary analysis, ten primer pairs were selected
from Vendramin et al. (1996) and tested on 16 individuals
from eight geographically distant populations. Based on the
preliminary analysis results, only five primer pairs (Pt15169,
Pt30204, Pt41093, Pt87268, and Pt110048) were used to
analyze all populations, while the others were discarded as
they did not give any product in PCR amplification or were
not polymorphic. PCR amplifications were performed in a
total volume of 10 µL containing 5 ng of template DNA,
2.5 mM MgCl₂, 0.3 mM dNTP mix, 0.5 U Taq polymerase,
1X reaction buffer (Thermo Fisher Scientific), and different
amounts of forward and reverse primers for studied primer
pairs (1 µM for Pt15169, Pt41093 and Pt87268; 2 µM for
Pt110048; and 2.5 µM for Pt30204). PCR reactions were
carried out on an Applied Biosystems 9700 thermal cycler
according to the following conditions: initial denaturation
at 95°C for 5 min, followed by 25 cycles of 1 min at 94°C,
45 s at 55°C, and 1 min at 72°C, and a final extension step
of 8 min at 72°C. The amplified fragments were resolved
on an ABI 310 genetic analyzer (Applied Biosystems). The
size of fragments was determined by GeneScan® Analysis
Software 3.1 (Thermo Fisher Scientific) concerning the
GeneScan-500 LIZ size standard (Applied Biosystems).

Data analysis

The combination of size variants across five chloro-
plast microsatellite regions was defined as haplotypes.
The chloroplast haplotype variation parameters including
the total number of haplotypes within populations, effec-
tive number of haplotypes, number of private haplotypes,
haplotypic richness, and haplotypic Nei's genetic diversi-
ty (Nei, 1987) were calculated using HaplotypeAnalysis
1.05 (Eliades & Eliades, 2009). A median-joining network
including potential median vectors was constructed using
Network 10 with cpSSRs data (Bandelt et al., 1999). The
average genetic distance among individuals within popu-
lations (Goldstein et al., 1995) was estimated according
to the stepwise mutation model, as defined by Vendramin
et al. (1998). Analysis of molecular variance (AMOVA)
was performed using Arlequin 3.1 to estimate the neutral
genetic differentiation of species and populations within
species (Excoffier et al., 2005). The unweighted pair group
method with arithmetic mean (UPGMA) dendrogram of
population pairs was obtained from Nei's (1987) genetic
distance values, which were obtained from haplotype data
using FigTree 1.4.4 Software (Rambaut, 2018).

The spatial and non-spatial genetic mixture analyses
were applied to the haplotypes of populations (Corander
et al., 2003, 2008). The program Structure 2.3.4 (Pritchard
et al., 2000) was used to evaluate the admixture structure
patterns of the populations within each species. The
analysis was performed according to the MCMC algorithm
with 50000 Burn-in periods, 500,000 replications, and 10
iterations. Results of the structure analysis were evaluated
using the web-based program Structure Harvester (Earl
& von Holdt, 2012) and the number of clusters (K) was

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Distribution map of 56 studied populations and their haplotypes in different colors (red dot: Aleppo pine; blue square: Brutia pine (see Table 1 for more information about populations). The haplotypes of 14 western populations (left) and 7 eastern populations (center) of Aleppo pine are presented above. The Brutian pine populations' haplotypes are presented in the right part of the figure. Ind: individuals. Ind1-4: total haplotypes of individuals (from 1 to 4 individuals). The green and yellow areas indicate the natural distribution range of Aleppo and Brutia pine, respectively.</figcaption></figure>
obtained by employing Evanno et al. (2005) and Jakobsson & Rosenberg (2007) computations in Structure Harvester.

# Results

## Chloroplast microsatellite variation

All analyzed primers were polymorphic for Aleppo, Brutia, and Eldarica pines. Overall, 51 size variants were detected from the five primers in the studied species. Almost half of the size variants (24 alleles) were species-specific (private alleles) and each species had private alleles. The total number of size variants for Aleppo pine and Brutia pine were 33 and 42, respectively (Fig. S1 [suppl]). Most private alleles were found in Brutia pine populations (Table S1 [suppl]). The common alleles of primers for each species had distinct size variants. Eldarica pine size variants were found to be closer to the Brutia pine than the Aleppo pine alleles. All the studied primers showed mutational steps among alleles from 1-bp to 5-bp in the Brutia pine populations. Mutational steps of the size variants of alleles were observed in three primers (Pt15169, Pt41093, and Pt87268) for Aleppo pine (Fig. S1).

The detected 51 alleles were combined into 204 different haplotypes. The frequency of the fourteen (H1-H14) haplotypes was greater than 1%, and the total frequency of these haplotypes corresponded to 65.8%. The distribution of haplotypes in the species was 63 and 150 haplotypes in Aleppo and Brutia pines, respectively. The Eldarica pine population had four private haplotypes. The most common haplotypes of each species were found at much lower frequencies

in the other species. Ten haplotypes were shared between Aleppo and Brutia pine populations. The Aleppo pine populations of the eastern and western Mediterranean Basin had different haplotypes, except for two common haplotypes (H2 and H3) (Figs. 1 and 2). The number of haplotypes ranged from 4 to 18, and the effective haplotype number (Ne) was between 1.52 and 12.52 in Aleppo pine populations. In Brutia pine populations, the number (A) of haplotypes ranged from 5 to 23, and Ne was between 1.45 and 23 (Table 1). If all populations were analyzed together, the mean A and the mean Ne were 9.79 and 6.02, respectively (Table 1). Haplotypic richness values ranged from 2.05 (Tivissa) to 10.18 (Kassadra) in Aleppo pine populations. In the Brutia pine populations, the haplotypic richness values ranged from 2.96 (Isparta-Sutculer) to 15.51 (Amasya-Bafra). Nei's gene diversity values (h) showed similarity with the haplotypic richness value of populations. The mean genetic distance among individuals within populations for each species was 2.29 and 3.33 for Aleppo pine and Brutia pine, respectively. If the eastern and western populations of Aleppo pine were considered separately (except for the Tabarka population from Tunisia, which was grouped with Brutia pine populations), the mean genetic distance among individuals within populations changed dramatically (4.24 vs 0.89) (Table 1). The values of genetic diversity parameters were more similar between the Aleppo pine populations of the eastern Mediterranean Basin and Brutia pine populations than the western Aleppo pine populations (Table 1). The Aleppo pine populations from the eastern range (Türkiye, Greece, and Italy) were more than two-fold more diverse than the 13 populations from the western range (Spain and Morocco). When the level of genetic diversity between

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Median-joining network for the 37 most frequent (>5 frequency) cpSSR haplotypes of Aleppo and Brutian pines.</figcaption></figure>
<table><caption>**Table 2.** The results of molecular variance analysis (AMOVA) based on assuming no population structure</caption><thead><tr><th>Source of variation</th><th>df</th><th>Variance components</th><th>Variation (%)</th><th>Fixation indices</th><th>p*</th></tr></thead><tbody><tr><td>Between species</td><td>1</td><td>1.130</td><td>51.98</td><td>F<sub>CT</sub>=0.247</td><td>&lt;0.0001</td></tr><tr><td>Among populations within species</td><td>55</td><td>0.258</td><td>11.87</td><td>F<sub>SC</sub>=0.638</td><td>&lt;0.0001</td></tr><tr><td>Within populations</td><td>1266</td><td>0.786</td><td>36.15</td><td>F<sub>ST</sub>=0.519</td><td>&lt;0.0001</td></tr><tr><td>Total</td><td>1322</td><td>2.174</td><td></td><td></td><td></td></tr></tbody></table>

the eastern range Aleppo pine and Brutia pine populations were compared, the eastern range Aleppo pine populations had slightly higher genetic diversity than Turkish red pine populations (Table 1).

A median-joining network was constructed to understand the relationships between the 37 most common cpSSR haplotypes detected in Aleppo pine and Brutia pine (Fig. 2). The haplotype network showed a minimum number of evolutionary events that separated each haplotype. The network indicated that H1 was centered in Brutia pine haplotypes, while H2 and H3 were centered in the Aleppo pine haplotypes. Although the divergent center is not clear for Brutia pine, H2 might be considered the divergent center of Aleppo pine.

## Phylogeographic comparison of species and populations

The molecular variance analysis based on the stepwise mutation model showed that between species and among populations within species variation based on assuming no population structure were 51.98 and 11.87% of the total variation, respectively (Table 2).

AMOVA results showed that haplotypic differentiation among species was statistically significant. However, the genetic diversity of Brutia pine populations was higher than among Aleppo pine populations (Table 3). The denrogram indicated that the genetic distance between the Aleppo and Brutia pine populations was generally compatible with geographic separation at the species level, except for a few populations. The Amasya-Amasya, Amasya-Bafra, K.Maras-K.Maras and Balikesir-Ayvacik populations of Brutia pine clustered with the Tunisian population (Tabarka) of Aleppo pine. The Eldarica pine population (Khorasan-Razavi) clustered with the Bursa-MKP population of Brutia pine (Fig. 3). Brutia pine populations did not present any clear geographic structure within their natural distribution range. However, the Aleppo pine populations showed clear east-west differentiation across the Mediterranean Basin. The two populations of Aleppo pine from Türkiye were in the same group as those from Greece and Italy. The Aleppo pine populations from the western Mediterranean clustered together, except for the Tivissa population from Spain. The Tivissa population also clustered with the Greek and Turkish populations in the eastern groups (Fig. 3 and Table 1). The eastern Mediterranean

<table><caption>**Table 3.** AMOVA results for Aleppo and Brutia pine populations.</caption><thead><tr><th>Source of variation</th><th>df</th><th>Variance components</th><th>Variation (%)</th><th>Fixation indices</th><th>p*</th></tr></thead><tbody><tr><td colspan="6" style="text-align: center; font-weight: bold;">Aleppo pine populations</td></tr><tr><td>Western and Eastern population groups</td><td>1</td><td>0.137</td><td>15.74</td><td>F<sub>CT</sub>=0.102</td><td>&lt;0.0001</td></tr><tr><td>Among populations within groups</td><td>19</td><td>0.075</td><td>8.63</td><td>F<sub>SC</sub>=0.244</td><td>&lt;0.0001</td></tr><tr><td>Within populations</td><td>474</td><td>2.657</td><td>75.63</td><td>F<sub>ST</sub>=0.157</td><td>&lt;0.0001</td></tr><tr><td>Total</td><td>494</td><td>0.869</td><td></td><td></td><td></td></tr><tr><td colspan="6" style="text-align: center; font-weight: bold;">Brutia pine populations</td></tr><tr><td>Among populations</td><td>34</td><td>0.321</td><td></td><td>F<sub>ST</sub>=0.271</td><td>&lt;0.0001</td></tr><tr><td>Within populations</td><td>776</td><td>0.866</td><td></td><td></td><td></td></tr><tr><td>Total</td><td>810</td><td>1.187</td><td></td><td></td><td></td></tr></tbody></table>

<footnote>*Significance tests were based on 10,000 permutations.</footnote>

<footnote>*Significance tests were based on 10,000 permutations.</footnote>
<figure><img src="image_5.png" /><figcaption>**Figure 3.** UPGMA dendrogram of studied populations based on Nei's genetic distance</figcaption></figure>

group of Aleppo pine is composed of Turkish, Greek, and Italian populations while the western Mediterranean group is composed of Spanish and Moroccan populations.

In the structural analysis, ΔK showed clear peaks for K=2 in both Aleppo and Brutia pine populations, and most of the populations were admixtures of two clusters, while a few populations had specific population structures according to K=2. For Brutia pine, Cluster 1 was reported to be predominantly in Adapazari-Geyve, Amasya-Amasya, Amasya-Bafra, Ankara-Nallıhan, Antalya-Antalya, Antalya-Gündoğmuş, Isparta-Sütçüler, İstanbul-Kanlıca, İzmir-İzmir, Kastamonu-Durağan, Kmaraş-Antakya, Kmaraş-Kmaraş, Mersin-Bozyazi, Mersin-Tarsus and Kibris1 (>90%); Cluster 2 was mainly detected in Balıkesir-Ayvacık, Bolu-Göynük, Denizli-Acipayam, Muğla-Marmaris and S.Urfa-Adiyaman (>90%) (Fig. S2). For Aleppo pine, Cluster 1 was observed to be predominantly in western Mediterranean populations (>90%), while Cluster 2 was generally detected in eastern Mediterranean populations (>90%) (Fig. S3).

## Discussion

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).

Accord- ing 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.

## Authors' contributions

**Conceptualization:** N. Kaya, Y. Kurt

**Data curation:** N. Kaya, Y. Kurt, B. Çengel, E. Velioğlu, D. Grivet, S. C. Gonzalez-Martinez

**Formal analysis:** N. Kaya, Y. Kurt

**Funding acquisition:** N. Kaya

**Investigation:** N. Kaya, Y. Kurt, B. Çengel, E. Velioğlu, D. Grivet, S. C. Gonzalez-Martinez

**Methodology:** N. Kaya, Y. Kurt, B. Çengel, E. Velioğlu, D. Grivet, S. C. Gonzalez-Martinez

**Project administration:** N. Kaya

**Resources:** N. Kaya, Y. Kurt, B. Çengel, E. Velioğlu, D. Grivet, S. C. Gonzalez-Martinez

**Software:** Not applicable

**Supervision:** N. Kaya, Y. Kurt

**Validation:** N. Kaya, Y. Kurt

**Visualization:** N. Kaya, Y. Kurt

**Writing – original draft:** N. Kaya, Y. Kurt

**Writing – review & editing:** N. Kaya, Y. Kurt, B. Çengel, E. Velioğlu, D. Grivet, S. C. Gonzalez-Martinez

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