PEER-REVIEWED ARTICLE bioresources.com Yigit et al. (2023). “Clonal variation in black pine,” BioResources 18(3), 4850-4865. 4850 Clonal Variation Based on Some Morphological and Micromorphological Characteristics in the Boyabat (Sinop/Turkey) Black Pine (Pinus nigra subsp. pallasiana (Lamb.) Holmboe) Seed Orchard Nurcan Yigit,a Ayşe Öztürk,a Hakan Sevik,b Halil Barış Özel,c,* Fathi Elmabruk Ramadan Kshkush,a and Berkant Işık c Seed orchards with high hereditary qualities and the improvement studies used are of great importance. This study was carried out on individuals in a Boyabat grafted black pine seed orchard, Sinop. The morphological and micromorphological measurements of the characteristics were performed on needle samples taken from individuals, and the genetic diversity was determined on a clonal basis. According to the analysis of variance applied to the data obtained from the measurements and the morphological and micromorphological characters of the clones, it was determined that there was a significant difference among the clones at the P<0.001 confidence level. In this context, according to Duncan’s Range test, the creation of a large number of groups is an indicator of it. The highest heritability rates were obtained in needle diameter, stipule diameter, number of the dorsal stoma, and needle length characteristics. DOI: 10.15376/biores.18.3.4850-4865 Keywords: Black pine; Boyabat; Seed orchard; Morphology; Clones Contact information: a: Kastamonu University, Faculty of Forestry, Department of Forest Engineering, Kuzeykent, Kastamonu-TURKEY; b: Kastamonu University, Faculty of Engineering and Architecture, Department of Environmental Engineering, Kuzeykent, Kastamonu-TURKEY; c: Bartın University, Faculty of Forestry, Bartın-TURKEY; *Corresponding author: halilbarisozel@gmail.com INTRODUCTION Climate types, quite different from each other, prevail across Turkey, and accordingly, there is a wide variety of species in forested areas (Atalay and Efe 2015). However, they enable us to study domestic and foreign species together in afforestation studies. Thus, the opportunities to establish healthy forest stands are increasing in terms of both quality and quantity. In afforestation studies, it is necessary to select appropriate species of the appropriate origin, to follow the principles of improvement studies, and to pay attention to afforestation techniques. For this purpose, the selection of seed sources with high hereditary qualities and the improvement studies used are of great importance (Wu et al. 2015; Kaviriri et al. 2020; Weng et al. 2020). Improved seeds can increase wood production by up to 40% (Üçler and Turna 2005; Yahyaoğlu and Ölmez 2005). Seeds of a certain origin ensure afforestation and provide economic and ecological benefits (Wu et al. 2015). The most important purpose of tree breeding is to promote ecological and economic benefits (Dyjakon 2019). PEER-REVIEWED ARTICLE bioresources.com Yigit et al. (2023). “Clonal variation in black pine,” BioResources 18(3), 4850-4865. 4851 Black pine (Pinus nigra Arnold.) is the most important species which can be spread to the steppe regions in Türkiye. Away from its native areas Black pine is planted for its timber production purposes (Topacoglu et al. 2016). Black pine, which is one of the dominant species in the forest assets of Türkiye, is a primary forest tree species that has a very wide distribution area starting from South Europe up to Türkiye. It can be argued that black pine is a typical south European forest tree species that is ecologically and economically important in the abovementioned distribution area (Gülsoy and Cinar 2019). Depending on changing environmental conditions, it is almost impossible to predict what kinds of threats forests will face in the future. Ensuring the continuity of the genetic diversity of species will make the presence of individuals carrying genes that will be needed in the upcoming years possible (Sevik et al. 2013). It is preferable for improvement studies that the genetic diversity of the selected populations is as high as possible. It is easier to find proper improvement materials, and the chance of success is higher with populations having a broad genetic base (Velioğlu et al. 2002; Lindgren et al. 2008). High intraspecific genetic diversity is a guarantee of adaptation to changing environmental conditions. Genetic diversity determines the adaptation potential of a species and is an important part of ecosystem stability. Therefore, the conservation of genetic diversity is essential to maintain adaptability. Genetic diversity is a raw material that will be shaped in improvement studies and through which results can be obtained accordingly. High genetic diversity evenly increases the chance of genetic staff to choose genotypes and populations appropriate for their study objectives (Şevik 2012). Seed orchards are one of the most important seed sources in terms of creating a connection between present and future, such as gene conservation areas, and future forest plantations (Bilir and Temiraga 2012). Seed orchards are areas where seeds are produced in large quantities to obtain the highest genetic gain as cheaply and fast as possible (Wu et al. 2015). According to another definition, they are plantations that are operated for the frequent, abundant, and easy harvesting of forest tree seeds, with use of selected clones or fertilizers, and where pollen flow from isolated or external sources can be blocked or reduced (Zobel and Talbert 1984). They are also shown as the most appropriate way to put the genetic gain obtained from tree breeding studies into practice (Tulukçu et al. 2002). Genetic diversity can be determined by physiological and morphological characteristics or molecular markers (Suangtho et al. 1999). To date, genetic variation studies have been generally initiated based on morphological characteristics, and after obtaining sufficient data, detailed information has been reached through isoenzyme and DNA studies. However, morphological characteristics were mainly examined in the studies carried out, and the number of studies carried out based on anatomical characteristics has remained quite limited (Donnelly et al. 2016). Studies on needle sizes and anatomical characteristics of conifers showed significant differences between and within populations (Bobowicz and Korczyk 1994; Urbaniak et al. 2003; Androsiuk and Urbaniak 2006). Many studies have been carried out on clonal seed orchards in terms of the morphological characteristics of seeds and cones (Deligöz and Gezer 2005; Çılgın et al. 2007; Hauke- Kowalska et al. 2019; Kaviriri et al. 2020; Weng et al. 2020). Variation among clones and within clones is an important factor in terms of the seed production (Prescher et al. 2007). Many studies have been carried out on the variations of the productivity of forest tree species (Kang and Lindgren 1998; Benowicz and El- Kassaby 1999; Kang 2001; Bilir et al. 2002; Sengün and Semerci 2002; Bilir et al. 2004; PEER-REVIEWED ARTICLE bioresources.com Yigit et al. (2023). “Clonal variation in black pine,” BioResources 18(3), 4850-4865. 4852 Lindgren et al. 2009). The study was carried out on grafted black pine individuals in the Boyabat seed orchard, Sinop. The Boyabat Pinus nigra seed orchard, Sinop, was established by taking seed stands of Kastamonu origin. Clones were planted by grafting to seedlings at a distance of 8 × 8 m. The seed orchard is 10.9 Ha in size and located at an altitude of 450 m. This study determined the structuring of adaptive genetic diversity on a clonal basis by analyzing the morphological and micromorphological characteristics of needle samples taken from individuals. For this purpose, the characters measured were analyzed using the SPSS package program, and it was attempted to determine the structuring of adaptive genetic diversity on a clonal basis. EXPERIMENTAL The study was carried out in the Boyabat black pine seed orchard, Sinop. The black pine seed orchard affiliated to Sinop Provincial Directorate was planted by the Forest Trees and Seed Improvement Institute in 1995 with 30 clones. From the administrative aspect, the seed orchard is within the boundaries of Kastamonu Regional Directorate of Forestry, Boyabat Forest Management Directorate, Bürnük Forest Sub-district Directorate, and has a size of 10.1 ha (Fig. 1). Within the scope of the study, genetic variation attributes in the seed orchards were determined with the help of some morphological and anatomical characteristics. Morphological characteristics have been used to determine genetic variations in many studies carried out to date (Sengün and Semerci 2002; Sevik et al. 2013). However, the number of studies in which anatomical characteristics have been used to determine genetic variations is very limited. However, it is known that all phenotypic characters, including anatomical characters, are formed under the mutual interaction of genetic structure and environmental conditions (Yayla et al. 2022; Kuzmina et al. 2023; Cobanoglu et al. 2023). The most dominant factors affecting phenotypic characters are climatic (Koç 2022; Dogan et al. 2023) and edaphic (Cetin et al. 2022; Key et al. 2022) factors. It can be accepted that in seed orchards where climatic and edaphic factors are relatively homogeneous, anatomical characters, like other phenotypic characters, are shaped largely depending on the genetic structure, since they are established in a limited and nearly flat area. In fact, it is accepted that anatomical characters are less affected by environmental conditions and therefore reflect genetic structure more clearly (Yigit 2016; Yigit et al. 2021). Therefore, anatomical characters are important instruments that can be used especially in genetic variation studies, but the number of studies on this subject is negligible. PEER-REVIEWED ARTICLE bioresources.com Yigit et al. (2023). “Clonal variation in black pine,” BioResources 18(3), 4850-4865. 4853 Fig. 1. Location of the Boyabat black pine seed orchard on the map Within the scope of the study, needle samples were collected in December, except for the vegetation season. Needle samples were collected from a total of 90 trees, three ramets from each of 30 clones, and last year's needles from the same direction, labeled, airtight packed, and brought to the laboratory. Morphological characteristics, such as needle length (NL) (cm), needle width (NW) (mm), needle diameter (ND) (mm), stipule diameter (SD) (mm), number of the dorsal stoma (NDS), number of dorsal stoma channels (NDSC), number of the ventral stoma (NVS), and number of ventral stoma channels (NVSC), were determined on the needle PEER-REVIEWED ARTICLE bioresources.com Yigit et al. (2023). “Clonal variation in black pine,” BioResources 18(3), 4850-4865. 4854 samples collected. Furthermore, ash determination (A) was performed as follows: an empty crucible and cover were dried for 15 min on a heater or in an indirectly heated furnace at approximately 600 °C. The porcelain crucibles were allowed to wait in the desiccator for 45 min and weighed with a precision of 0.1 mg. The sample was put in the crucible. The cover removed and the crucible and its contents were put in an indirectly heated furnace and burned until all carbon was removed. Initially, temperature was gradually increased to avoid volatilization. The crucible was allowed to wait for 3 h at 575 ± 25 °C in the furnace. At the end of this period, the sample in the crucible should be completely bleached, or the particles should have been lost. Ash was calculated as follows, Ash% = (A*100)/B where A is the ash weight, and B is the complete dry sample weight (g). Anatomical characteristics, such as needle wet weight (WW), the weight values of needles taken when they were fresh (g), needle dry weight (DW), and oven-dried weights of samples (at a precision of 0.001 g), were determined. The data obtained were analyzed using the SPSS 20.0 statistical package program. The analysis of variance was applied to clones in terms of the characters measured, and Duncan’s test was applied to the data in case of statistically significant (P ≤ 0.05) differences as a result of the analysis. As a result of Duncan’s test, new homogeneous groups were determined (Kalıpsız 1994; Ercan 1995). Broad sense heritability values were estimated both on individual tree basis (H1) and clone mean basis (H2) as the ratio of total genetic variance (s2c) to total phenotypic variance (s2c + s2E) for H1 and to (s2c + s2E/n) for H2 (n = graft number). Cloning effect variance biases heritability values, but the magnitude is negligible and can be ignored. In the present study, heritability components were estimated as E= error mean square and 2c = (clone mean square-error mean square/ number of grafts per clone). This formula has been used in different articles (Sevik and Topacoglu 2015). RESULTS AND DISCUSSION As a result of the measurements and calculations performed on the samples taken in the Boyabat black pine clone orchard, A, WW, DW, NL, NW, ND, SD, NDS, NDSC, NVS, and NVSC values were determined. The study was carried out on 141 trees of 30 different clones, and the analysis of variance was applied to the values obtained as a result of the calculations. The results are presented in Table 1. Significant differences were found in terms of all the morphological characteristics studied at a confidence level of 99.9% both among the clones and within the clones. Duncan’s test was applied for each anatomical characteristics to determine how the clones were grouped within themselves, and the test results are presented in Table 2. According to Duncan’s test results of the A character, clone 81 (1.839) formed a class alone, and clone 87 had the highest value (1.361) and was included only in the last homogeneous group according to the ash content of the needle. According to the wet weight values of the needle, clone 95 with the lowest value (1.833) was included only in the first homogeneous group, and clone 82 with the highest value (2.11) was included only in the last homogeneous group. According to the results of Duncan’s test applied to the dry weight values of the needle, Clone 95 was included only in the first homogeneous group PEER-REVIEWED ARTICLE bioresources.com Yigit et al. (2023). “Clonal variation in black pine,” BioResources 18(3), 4850-4865. 4855 (0.817) according to the dry weight values of the needle and that clones 1 and 82 (2.003 and 2.087) were included only in the fifth and last homogeneous group (Table 2). In terms of the NL morphological character, the clones formed 16 different classes among themselves. Clone 85 was included in the first homogeneous group with a needle length value of 10.5 cm and clone 93 was included in the last homogeneous group with a needle length value of 12.6 cm. In terms of the ND morphological character, it is observed that three different groups were formed. Clone 88 was included in the first homogeneous group, clone 93 was included in the second homogeneous group, and all other clones were in the 1st class. In terms of the NW character, the test revealed that 15 different groups were formed. When the groups were examined, clone 85 was included in the 1st homogenous group, clones 81 and 85 were included in the 2nd homogenous group, and clone 8 was included in the last homogeneous group alone. In terms of the SD morphological character, clone 85 formed a homogeneous group alone, and clone 81 formed the 2nd homogenous group. Clone 93 was included in the last homogenous group (Table 2). The clones were grouped in 11 different groups in terms of the NDS character according to Duncan’s test results of micromorphological characteristics. Clone 98 formed the first and single group and clones 95 and 97 formed the second group together. Clone 89 formed the last class alone. When Duncan’s test results were examined in terms of the NDSC character, 7 groups were formed. Clone 82 formed the last class alone and clones 85 and 89 formed a class together. When Duncan’s test results were examined in terms of the NVS micromorphological character, clones 86, 93, 88, 96, and 10 formed a class by themselves. Clone 8 formed the last class alone. When the results of Duncan’s test applied to micromorphological characteristics were examined in terms of the NVSC character, clones 7, 93, and 99 formed a class by themselves and clone 8 formed the last class. Correlation analysis was performed to determine the relationship between the elements, and the results are presented in Table 3. The studied relationship that was not measured in the correlation analysis was related to the linear part of the relationship between the variables. PEER-REVIEWED ARTICLE bioresources.com Yigit et al. (2023). “Clonal variation in black pine,” BioResources 18(3), 4850-4865. 4856 Table 1. Analysis of Variance Applied to All Characteristics Characters Sum of Squares Degree of Freedom Means Square F-Value Significant A Between groups 59.454 29 2.05 6.462 .000* Within groups 47.591 150 0.317 Total 107.045 179 WW Between groups 41.932 29 1.446 3.113 .000* Within groups 69.682 150 0.465 Total 111.614 179 DW Between groups 17.533 29 0.605 2.577 .000* Within groups 35.188 150 0.235 Total 52.721 179 NL Between groups 573.725 29 19.784 8.463 .000* Within groups 4488.475 1920 2.338 Total 5062.2 1949 NW Between groups 68.01 29 2.345 5.486 .000* Within groups 820.713 1920 0.427 Total 888.723 1949 ND Between groups 15.938 29 0.55 20.027 .000* Within groups 52.688 1920 0.027 Total 68.626 1949 SD Between groups 27.168 29 0.937 10.036 .000* Within groups 179.229 1920 0.093 Total 206.397 1949 NDS Between groups 35876.015 29 1237.104 9.192 .000* Within groups 19650.167 146 134.59 Total 55526.182 175 NDSC Between groups 184.917 29 6.376 3.604 .000* Within groups 258.333 146 1.769 Total 443.25 175 NVS Between groups 44654.432 29 1539.808 4.395 .000* Within groups 51149 146 350.336 Total 95803.432 175 NVSC Between groups 216.045 29 7.45 4.467 .000* Within groups 243.5 146 1.668 Total 459.545 175 P<0.05 (95% confidence level); P<0.01 (99% confidence level); P<0.001 (99.9% confidence level) ns: Not significant PEER-REVIEWED ARTICLE bioresources.com Yigit et al. (2023). “Clonal variation in black pine,” BioResources 18(3), 4850-4865. 4857 Table 2. Duncan’s Test Results of Ash Content in All Characteristics Clone No A WW DW NL NW ND SD NDS NDSC NVS NVSC 1 2.929gh 2.926defghı 2.087e 10.872 abcdef 1.4020 a 1.7849 cdef 2.09 bcdefgh 119.33ıjk 10defg 137ghı 11.67efgh 2 2.602efgh 2.693cdefg 1.694cde 11.375 efghıjkl 1.3847 a 1.7899 cdef 2.03 bcde 81.67abc 8abc 112.67abcdefg 9.67abc 3 1.966abcdef 3.068efghı 1.257abcd 11.733 hıjklmn 1.4325 a 1.7769 cde 2.08 bcdefg 90.67abcde 10defg 115abcdefgh 11.67efgh 4 2.361cdefgh 2.215bcd 1.549bcde 11.944 klmno 1.5104 a 1.9331 lmn 2.27 jkl 110ghıj 9.33bcdefg 140hı 11.67efgh 5 1.794abcdef 3.546hıj 1.326abcd 10.635 ab 1.4245 a 1.8715 hıjklm 2.13 efghı 102.67defgh 10defg 125bcdefghı 12.33gh 6 1.819abcdef 3.130efghı 1.235abcd 10.831 abcde 1.4869 a 1.8836 ıjklm 2.11 efghı 102.33defgh 9.67cdefg 116.67abcdefgh 10.67abcdefg 7 1.520abc 3.361fghıj 1.145abc 11.263 cdefghıj 1.4657 a 1.8589 ghıjk 2.16 efghıj 99.33defgh 9bcdef 100.33ab 9a 8 2.654fgh 3.573hıj 1.906de 12.205 nop 1.5717 ab 1.9843 no 2.27 jkl 125jk 10.33efg 148.33ı 13.33gh 9 1.657abcde 2.460bcde 1.224abcd 10.720 abcd 1.3606 a 1.7526 bc 1.98 bcd 86.5abcd 9.5bcdefg 100.5ab 10.5abcdef 10 2.069abcdefg 3.685ıj 1.547bcde 11.548 ghıjklm 1.5542 ab 1.9377 mn 2.23 ıjk 97.25cdefgh 9bcdef 97a 9.75abcd 81 1.839abcdef 1.126a 1.328abcd 10.975 abcdefg 1.4128 a 1.7081 b 1.95 b 107.33fghı 10defg 132.33defghı 11.67efgh 82 3.175h 2.116bc 2.003e 11.295 cdefghıj 1.4140 a 1.8035 cdefgh 2.11 efghı 112.5hıj 11g 134defghı 11.5defg 83 1.315ab 2.626cdef 0.961ab 11.771 ıjklmn 1.5367 ab 1.9224 klmn 2.21 hıjk 90.67abcde 9.67cdefg 108abcd 10.67abcdefg 84 1.880abcdef 2.956defghı 1.443abcde 12.089l mnop 1.4433 a 1.896jklm 2.14 efghı 94abcdefg 10defg 102ab 12fgh 85 1.678abcde 3.186efghı 1.294abcd 10.463 a 1.3713 a 1.5581a 1.81 a 119ıjk 10.67fg 136.67fghı 11.67efgh 86 2.353cdefgh 2.786cdefgh 1.681cde 11.636 hıjklmn 1.4487 a 1.7656bcd 2.06 bcdef 91.67abcdef 8.33abcd 92.67a 10.33abcdef 87 1.361ab 4.015j 1.058abc 11.334 defghıjk 1.4892 a 1.8502ghıj 2.14 efghı 97bcdefgh 10defg 116.33abcdegh 11.33cdefg 88 1.640abcde 3.086efghı 1.129abc 11.093 bcdefgh 2.562 c 1.8302defghıj 2.31 kl 81.33abc 8abc 94.67a 9.33ab 89 2.187bcdefg 2.866cdefgh 1.719cde 11.597 ghıjklmn 1.4148 a 1.8333defghıj 2.16 efghıj 132k 10.67fg 135.33efghı 11.67efgh 90 1.730abcdef 2.665cdef 1.161abc 12.458 op 1.4709 a 1.8976jklm 2.16 efghıj 101.33defgh 8.67abcde 130cdefghı 11.67efgh 91 1.827abcdef 2.673cdef 1.193abc 10.683 abc 1.4229 a 1.8639hıjk 2.07 bcdefg 81.33abc 8.33abcd 101.33ab 10.33abcdef 92 2.188bcdefg 3.095efghı 1.670cde 11.643 hıjklmn 1.4307 a 1.7953cdefg 2.10 defgh 105.33efghı 9.33bcdefg 118.33abcdefgh 10.33abcdef 93 2.061abcdefg 3.193efghı 1.550bcde 12.611 p 1.7853 b 2.0127o 2.36l kl 88abcd 9bcdef 93a 9a 94 2.057abcdefg 3.056efghı 1.529bcde 11.151 bcdefghı 1.4747 a 1.8349efghıj 2.29 kl 105.33efghı 9bcdef 128.33cdefghı 11bcdefg 95 1.134a 1.833b 0.817a 11.480 fghıjkl 1.3482 a 1.7092b 1.98 bcd 80.67cbefghı 9.33bcdefg 104abc 10abcde 96 2.516defgh 2.790cdefgh 1.593bcde 11.879 jklmno 1.5681 ab 1.9225klmn 2.20 ghıjk 81.33abc 7.67ab 96a 9.67abc 97 1.304ab 3.466ghıj 0.936ab 12.152 mnop 1.4705 a 1.8672hıjkl 2.19 fghıjk 80.67ab 7a 110.33abcdef 9.67abc 98 1.672abcde 2.835cdefgh 1.239abcd 12.047l mnop 1.368 a 1.7868cdef 1.97 bc 78.67a 8abc 101ab 9.33ab 99 2.4312cdefgh 2.235bcd 1.727cde 11.573 ghıjklmn 1.4475 a 1.8673hıjkl 2.22 hıjk 82.33abc 7a 110abcde 9a 100 1.600abcd 2.620cdef 1.215abc 11.852 jklmno 1.3783 a 1.8257defghı 2.07 bcdefg 94.67abcdefg 8.33abcd 110.67abcdefg 10.33abcdef PEER-REVIEWED ARTICLE bioresources.com Yigit et al. (2023). “Clonal variation in black pine,” BioResources 18(3), 4850-4865. 4858 The correlation coefficient calculated as a result of the correlation analysis was indicated with r and took values between –1 and +1. The fact that the coefficient was close to +1 indicates that there was a good correlation between the two variables, and the fact that it was close to -1 indicates that there was a good but inverse correlation, in other words, one of the variables increased while the other one decreased. Upon evaluating the results in this respect, it is observed that the level of relationship between some elements was high. Table 3. Correlation Analysis Results NW ND SD A WW DW H A% NDS NDSC NVS NVSC NL -.105 .544** .447** -.007 .001 .043 .061 -.113 .063 -.057 .014 .038 NW 1 -.144 -.062 .079 .066 .101 .160 .044 .128 .130 .157 .108 ND 1 .682** .350** .006 .013 .049 -.005 -.016 .047 -.090 .092 SD 1 .334** -.045 -.013 .045 .030 .029 -.070 .016 -.010 A 1 -.198 -.127 -.134 .002 .050 -.007 -.134 -.056 WW 1 .950** .895** -.163 .212 .046 .239* .189 DW 1 .915** -.196 .311** .104 .289* .242* H 1 -.221 .361** .152 .334** .273* A% 1 -.212 -.113 -.061 -.075 NDS 1 .743** .716** .634** NDSC 1 .433** .634** NVS 1 .800** There was a positive correlation in general with respect to the measured characteristics. When the results were examined in this context, the relationship between some elements was observed to be quite high. For example, the correlation coefficients between WW and DW (0.950), NVS and NVSC (0.800) were quite high. Similarly, the correlation coefficient calculated between A and WW (-0.198) was negative but very strong. Very strong relationships were observed among many characteristics. Analysis of variance, variance components and heritability estimates for studied characteristics are shown in Table 4. Table 4. Analysis of Variance, Variance Components and Heritability Estimates for Studied Characteristics Characteristics Between Populations Within Populations s2c s2E H1 2 H2 A 2.05 0.317 0.347 0.317 0.52 0.87 WW 1.446 0.465 0.196 0.465 0.30 0.72 DW 0.605 0.235 0.074 0.235 0.24 0.65 NL 19.784 2.338 3.489 2.338 0.60 0.90 NW 2.345 0.427 0.384 0.427 0.47 0.84 ND 0.55 0.027 0.105 0.027 0.80 0.96 SD 0.937 0.093 0.169 0.093 0.65 0.92 NDS 1237.104 134.59 220.503 134.59 0.62 0.91 NDSC 6.376 1.769 0.921 1.769 0.34 0.76 NVS 1539.808 350.336 237.894 350.336 0.40 0.80 NVSC 7.45 1.668 1.156 1.668 0.41 0.81 PEER-REVIEWED ARTICLE bioresources.com Yigit et al. (2023). “Clonal variation in black pine,” BioResources 18(3), 4850-4865. 4859 When the table values were examined, it was seen that the characters with the highest heritability were ND, SD, NDS, and NL. According to the table values, the H2 value was 0.90 and above in terms of these characters. The images of dorsal and ventral stoma channels and stomas taken on the needles during the measurements performed to determine micromorphological characteristics are presented in Fig. 2. Fig. 2. The images of dorsal and ventral stoma channels The results of the analysis of variance show that there were statistical differences at a confidence level of at least 95%, in terms of all the micromorphometric characteristics examined in the Boyabat clonal seed orchard. There were statistically significant differences among the clones in terms of all the characteristics studied. According to the results of Duncan’s test, it can be said that the creation of a large number of groups is an indicator of it. It is possible to interpret the results in a way that the genetic diversity in the seed orchard is sufficient. Most studies of clonal variations of seed orchards are morphological, physiological, and phenological, and the determination of anatomical characteristics brings a different dimension to such studies. Afforestation studies conducted on thousands of hectares of land are performed with the seeds obtained from seed orchards. The degree of success of the outcomes of afforestation studies emerges after many years. Genetic differences among clones in seed orchards should be clearly revealed by determining anatomical characteristics to observe successful outcomes in a shorter time. Genetic variation studies were initiated based on morphological characteristics. As well as any phenotypic characteristic, plant metabolism is shaped by the mutual interaction between genetic structure (Özel et al. 2022; Tandogan et al. 2023; Kurz et al. 2023) and environmental conditions (Varol et al. 2022; Ghoma et al. 2022; Cetin et al. 2023). Therefore, genetic variation studies have been carried out on many species based on morphological characteristics. Topaçoğlu (2013), Güney et al. (2014), and Sevik (2012) determined genetic variations based on morphological characteristics in Pinus nigra, Pinus brutia, and Abies, respectively. In addition to these, there are many studies aimed at determining genetic variations among clones in seed orchards (Cilgin et al. 2007; Buğday 2008). Morphological and anatomical characteristics are a result of the interaction between genetics and the environment (Yigit et al. 2018a) and are shaped by the effects of genetic factors (Sevik et al. 2017; Yigit et al. 2018b) and environmental factors (Yigit et al. 2016; Turkyilmaz et al. 2020). This can be explained by the effect of microenvironment conditions on the clones. Microenvironment conditions affect morphological characteristics significantly (Cetin et al. 2018; Yucedag et al. 2019). This is a seed orchard. Therefore, the genetic structure is thought to be the same on a clone basis. However, it is thought that the differences arise from environmental conditions. Therefore, micro environmental conditions are quite effective on clones. PEER-REVIEWED ARTICLE bioresources.com Yigit et al. (2023). “Clonal variation in black pine,” BioResources 18(3), 4850-4865. 4860 In this study, the clones differed significantly at a confidence level of 99.9% in terms of all characteristics and formed a large number of homogeneous groups, according to Duncan’s test results. This can be interpreted based on a hypothesis that the genetic diversity in the seed orchard is high. Genetic diversity is desired to be high, especially in the population. In the studies carried out, it was determined that intrapopulation genetic diversity in many species was higher than interpopulation genetic diversity. In the studies carried out, it was determined that the ratio of interpopulation variation was 9% in Pinus contorta (Wheeler and Guries 1982), 6% in Pinus nigra (Velioğlu et al. 2002), 6.1% in Pinus strobus (Rajora et al. 1998), 1.5% in Abies sachalinensis (El-Kassaby et al. 1992), 2.6% in Abies mariesii (Suyama et al. 1992), 4.8% in Abies cephalonica (Fady and Conkle 1993), and 13.3% in Abies alba (Vendramin et al. 1999). Considering that the interclone variation was quite high in this study, it can be said that the results obtained in the literature are generally compatible with the results of the present study. There are many genetic variation studies that have been carried out using morphological characteristics to date. However, both the number of studies, in which genetic variation was determined using anatomical characteristics, and the number of the characteristics used are quite limited. Matziris (1993) and Lamhamedi et al. (2000) determined the variation using needle resin channels and electron microscope images, respectively. The number of studies in which wood density was used to determine genetic variation is quite high (Hernandez and Adams 1991; Zhang and Morgenstern 1995; Hylen 1997; Chave et al. 2006). The anatomical characteristics of wood have been generally analyzed to investigate whether the fiber source is suitable for papermaking in the paper industry (Ay and Şahin 1996; İstek et al. 2009). Seed orchards continue to make significant contributions to improved seed production until the genetic values of clones in Turkey are completed. The protection and maintenance of these orchards should be sustained meticulously. Along with the establishment of seed orchards, it is aimed to meet all seed needs that will arise in the future from seed orchards. Furthermore, it has been aimed to provide resources for seed orchard genetic studies and to protect populations that are of good quality and under the danger of extinction. CONCLUSIONS 1. This study determined the genetic diversity by needle morphological and micromorphological characteristics in the Boyabat black pine clonal seed orchard. There were statistically significant differences at a confidence level of 99.9% among the clones in terms of all the studied characteristics. 2. There were differences among the clones in terms of revealing the hereditary value of the clonal seed orchard in this respect and most of the characteristics studied. One of the largest deficiencies of seed orchards is the narrowing of the gene pool. 3. There were statistical differences at a confidence level of at least 95% according to the results of the analysis of variance in terms of most of the characteristics examined in the seed orchard. 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