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Genetic Diversity Analysis of cpDNA in Turkish Abies Taxa Türk Göknar Taksonlarında Genetik Çeşitlilik Analizleri

Aim of study: Five Abies taxa naturally distributed in Turkey. Abies nordmanniana has three subspecies and A. cilica has two subspecies. In this study, we aimed to show phylogenetic relationships both in Turkish taxa and in other Abies taxa from around the world based on cpDNA regions, trnR-trnN and rps18-rpl20 regions. Material and methods: Following CTAB-based DNA isolation method, the relevant fir DNA regions were amplified and sequenced. Phylogenetic trees were constructed using maximum likelihood method with 1000 bootstrap replicates. Main results: It was difficult to make distinctions among the Turkish Abies taxa based on the sequenced DNA regions. Based on rps18-rpl20 phylogenetic tree, some members of Abies cilicica subsp. isaurica , A. nordmanniana subsp. equi-trojani and A. cilicica subsp. cilicica were in the same clade with A. spectabilis and A. densa; However, some members of A. nordmanniana subsp. bornmuelleriana , A. cilicica subsp . isaurica and A. nordmanniana subsp. equi-trojani were placed in a clade with A. alba placed near Turkey and A. amabilis known from North America. Highlights: This study provides new insights into the distribution of cpDNA variation in Abies species in Turkey and the genetic variation between firs in Turkey and the rest of the world.


Introduction
Firs (Abies Mill.) are coniferous trees in the Pinaceae family which includes 51 species that are native to the Northern Hemisphere. They distribute naturally in the temperate and boreal regions of the Northern Hemisphere and are mainly found in the mountainous regions of North America, Central America, Europe, North Africa and Asia (Himalaya, South China, and Taiwan) (Li, 1975). The genus is restrained to the mountainous areas in the subtropical and temperate latitudes of the Northern Hemisphere (Farjon, 1990).
Fir tree species are found in many forest areas of Turkey. Five taxa, which are placed in two fir tree species (Abies nordmanniana and Abies cilicica) are naturally distributed in Turkey.
Three of these five taxa are endemic. These taxa are Abies nordmanniana subsp. nordmanniana (distributed in the North of Kızılırmak in East Black Sea region), A. nordmanniana subsp. bornmuelleriana (distributed from the West of Kızılırmak to Uludağ in the east Blacksea region), Abies nordmanniana subsp. equitrojani (distributed between the Çanakkale, Balıkesir and Bursa), Abies cilicica subsp. isaurica (distributed in the middle and West Taurus Mountains in the South Anatolia region) and A. cilicica subsp. cilicica (distributed in the East Taurus Mountains in the South Anatolia region). However, A. nordmanniana subsp. bornmuelleriana and A. nordmanniana subsp. equi-trojani cannot be morphologically distinguished. Therefore, these two taxa were combined under the name of ''Kazdagi Fir'' in ''Turkey Plant List (Vascular Plants)'' (Güner et al., 2000;Güner et al., 2012).
Molecular genetic methods are widely used to analyze the conserved regions of the genome (Ateş, 2011). Molecular phylogenetic analysis using DNA sequence data enables molecular botanists to better define various taxonomic categories. Nuclear (nDNA), chloroplast (cpDNA) and mitochondrial (mtDNA) DNA sequences have been utilized to evaluate phylogenetic relationships in plants (Suyama et al., 2000;Xiang et al., 2004;Tozkar et al., 2009;Semerikova et al., 2011;Aguirre-Planter et al., 2012). However, some cpDNA regions are more informative and useful than other DNA regions for phylogenetic reconstructions (Liang, 1997). In addition, non-coding DNA regions might be more significant because of their rapid evolution than coding DNA regions. This property of non-coding DNA regions causes more variable characters and these DNA regions more useful for better construction and resolution of the phylogenetic tree (Wang et al., 1999).
Although there are many studies on the morphological classification of fir tree species (Kormutak et al., 2004;Hansen et al., 2005;Ziegenhagen et al., 2005;Xiang et al., 2009), a small number of molecular phylogenetic studies in the literature have examined Abies taxa in Turkey. These studies focused on the isozyme variation in a limited number of Anatolian fir populations (Şimşek, 1992;Gülbaba et al., 1996). To date, chloroplast matK, some trn regions, and nuclear ITS DNA regions have been examined to determine the molecular differentiation of Turkish fir tree populations (Ateş, 2011;Özdemir Değirmenci, 2011;Tayanç et al., 2013). Analyses of chloroplast rbcl and mitochondrial nad5-4 regions were limited to Mediterranean firs (in the South Anatolia region) which were compared with other fir species from around the world (Kormutak et al., 2004;Ziegenhagen et al., 2005). In addition, microsatellite markers were also evaluated to uncover the genetic diversity of the fir populations in Turkey (Hansen et al., 2005;Kaya et al., 2008;Hrivnák et al., 2017;Tayanç et al., 2013).
In this study, we aim to show phylogenetic relationships in Turkish fir taxa based on two non-coding cpDNA markers, i.e. trnR-trnN and rps18-rpl20. In addition, we used other fir species from previous phylogenetic studies to show taxonomic and phylogenetic position of the Turkish taxa in large phylogeny.

Plant Material
Samples of fir taxa distributed in Turkey were investigated to determine their phylogenetic relationships (Güner et al., 2012). The morphological characteristics of Kastamonu Uni., Orman Fakültesi Dergisi, 2021, 21(1): 41-54 Celik Altunoglu et al. Kastamonu Univ., Journal of Forestry Faculty 43 taxa were used to identify different members of the genus Abies. Studied specimens were identified using Flora of Turkey (Davis, 1965). The geographical locations of sampled Turkish Abies taxa are shown in Table 1.

DNA Isolation, PCR Amplification and Sequence Analysis
A modified cetyl trimethylammonium bromide (CTAB)-based method was used to isolate DNA from fresh needles (Saghai-Maroof et al., 1984). RNA from the extracted DNA solution was removed by RNAse enzyme (2 mg/mL) at 37 °C for 30 min. Concentration and purity of DNA were calculated using MultiscanGo Spectrophotometer (Thermo Scientific, USA). In addition, the DNA quality was checked by 2 % agarose gel and stored at -80 °C until use.
PCR reactions included 1.5 mM MgCl2, 1.25 U Taq DNA polymerase enzyme, 5 μL 10x PCR buffer, 5μL 2 mM dNTP, 25 pmol/μL of each gene's specific forward and reverse primer, 100 ng of template DNA and distilled water to achieve a final volume of 50 μL. After testing for amplification efficiency, trnR-trnN and rps18-rpl20 cpDNA regions were amplified by PCR.
Reaction conditions for the trnR-trnN region were as follows: initial denaturation at 95 °C for 5 min and denaturation at 95 °C for 1 min, annealing at 61.4 °C for 1 min and extension at 72 °C for 1 min which was repeating 35 times, then final extension at 72 °C for 7 min. After the initial denaturation step at 95 °C for 5 min, the following three steps were repeated for 35 times for the rps18-rpl20 region: denaturation at 95 °C for 1 min, then annealing at 50.1 °C for 1 min and extension at 72 °C for 1 min, then final extension step was at 72 °C for 7 min. The primer sequences used were as follows: trnR-trnN-F 5'GCCTGTAGCTCAGAGGATTA3', trnR-trnN-R 5'TCCTCAGTAGCTCAGTGGTA 3', and rps18-rpl20F 5'AGTCGATTTATTAGTGAGCA3', rps18-rpl20R 5'CTTCGTCGTTTGTGGATTAC 3' (Wang et al., 1999;Suyama et al., 2000). Amplification efficiency was checked with agarose gel electrophoresis and visualized by Kastamonu Uni., Orman Fakültesi Dergisi, 2021, 21( Forward and reverse sequence reads were compared for the most accurate DNA sequence in the studied DNA regions. PCR product purification and DNA sequencing were performed by Refgen Biotechnology (Ankara University, Teknokent, Ankara). ABI 310 Genetic Analyzer User' s Manual was used for sequence analysis. Sequencing was performed using the Big Dye Cycle Sequencing Kit (Applied Biosystems) with a ABI 310 Genetic Analyzer (PE Applied Biosystem, USA) automatic sequencer with forward and reverse primers of the relevant cpDNA regions.

Phylogenetic Tree Analysis
Finch TV software (Geospiza Inc.) was used to visualize DNA sequence results. SnapGene software (from GSL Biotech; available at snapgene.com) was used for contig forward and reverse sequences. Sequences obtained from trnR-trnN and rps18-rpl20 sub-regions and sequences retrieved from NCBI were combined and analyzed to understand genetic diversity among Abies taxa. A phylogenetic tree was constructed by the maximum likelihood method with bootstrap analysis for 1000 replicates and JTT (Jones-Taylor-Thornton) substitution model after multiple sequence alignments by ClustalW using the MEGA 6 program (Tamura et al., 2013). For alignment analysis, ClustalW was used according to following parameters: pairwise alignment gap opening = 15, gap extension = 6.6 and multiple alignment gap-opening = 15, gap extension =6.7, delay divergent sequences = 30% and transition weight = 0.5. Alignments were controlled and visually examined. Gaps in the aligned sequence data were considered as missing data. Pairwise distances between Abies taxa distributed in Turkey and pairwise distances between Turkish Abies members and Abies members from around the world were compared. Phylogenetic trees were displayed using interactive Tree Of Life software (iTOL) (http://itol.embl.de) (Letunic & Bork, 2016). Turkish fir taxa were marked with ▼.
Keteleeria davidiana (JN935765.1) and Picea abies (AJ001025.1) were used as outgroups in the constructed phylogenetic trees (Semerikova & Semerikov, 2014). The total trnR-trnN sequences of 14 fir species and total rps18-rpl20 sequences of 21 fir species were obtained from the NCBI database and used to evaluate phylogenetic relationships between fir species in Turkey and those from around the world (Table 2). Table 2. trnR-trnN and rps18-rpl20 sequences retrieved from NCBI.

trnR-trnN Sequences
rps18-rpl20 Sequences Species Name Accession Numbers Species Name Accession Numbers

Results and Discussion
Turkish Abies taxa were classified in previous studies but there were inconsistences in the taxonomy because of the variable and complex morphological features of fir species. The trnR-trnN sequence analysis showed that, the total length of this region was 823 bp and GC content was 45.5 %. Computed pairwise (p) distances between the studied samples ranged between 0.000 and 0.077 (Supplementary Table 1). All Turkish fir taxa were in the same clade of the phylogenetic tree ( Figure 1). Considering that the phylogenetic tree was constructed by the maximum likelihood method and low p distance values, it is difficult to make distinctions among Turkish Abies taxa members based on this cpDNA region. In the Türkiye Bitkileri Listesi (Damarlı Bitkiler (Güner et al. 2012), A. nordmanniana subsp. bornmuelleriana and A. nordmanniana subsp. equi-trojani were combined. Our findings support this taxonomic treatment based on the low p distance values compared with p distance values among the other Turkish fir taxa (Supplementary Table 1).
In addition, Abies taxa in Turkey were compared with other fir species from around the world to elucidate their phylogenetic relationship (Figure 2 The rps18-rpl20 regions of cpDNA were compared between Turkish fir taxa and other fir species from around the world (Figure 3). The total length of this region was 513 bp and its GC content was 33.6%. According to the phylogenetic tree, Abies taxa members were closer to each other in the clade and low p values (varied between 0.000 and 0.063) promoted this aggregation (Supplementary Table 3). Two main clusters appeared when Turkish fir taxa and other fir species from around the world were compared (Figure 4). The p distance values were varied between 0.000 and 0.599 (Supplementary Table 4   A genetic study of eight classified Mediterranean and one North American fir species shown that North American species (A. concolor) had the most divergent haplotypes (Parducci & Szmidt, 1999). According to a study conducted by Kormutak et al. (2004) Mediterranean firs including closely related species differed from both Asian and North American firs based on PCR-RFLP analysis of eight genes from cpDNA. In this study, Kormutak et al. (2004) reported that Mediterranean fir species has the lowest level polymorphism. In another study, Asian, North American and Mediterranean fir species were phylogenetically classified based on trnL and trnF region sequences. The Mediterranean fir species (European) A. alba and A. nordmanniana differed from other fir species based on their tandem repeat types (Isoda et al., 2000). In addition, the sequence analysis of the trnL region of Turkish fir taxa revealed the existence of a single clade, which includes Turkish-European fir species. In the same study, Turkish fir species were aggregated and separated from Asian-American fir species in a phylogenetic tree based on their trnF sequence analysis (Özdemir Değirmenci, 2011). A similar aggregation of Turkish fir species was also observed in our phylogenetic tree based on the sequences of the trnR-trnN regions. Another important finding was made by comparing the matK1 region sequence of Turkish fir species. All Turkish fir taxa and A. numidica (Algerian fir) that grow in the Mediterranean phytogeographic region were found in the same clade, whereas other fir species from around the world dispersed into various phylogenetic clades (Ateş, 2011). In this study, A. nordmanniana subsp. nordmanniana was observed in a group with A. numidica based on the sequences of rps18-rpl20. A possible explanation for this might be their similar ecological requirements. Another important finding was that A. cephalonica (Greek Fir) which is closely related to A. nordmanniana (distributed in Northern Turkey) (Fady et al., 1992) was the closest species to the Turkish fir taxa with low distance rates (0.008) based on the ITS region sequence data (Tayanç et al., 2013). However, studied DNA regions in these last three studies were conserved and no differences were observed among Turkish fir taxa members (Ateş, 2011;Özdemir Değirmenci, 2011;Tayanç et al., 2013). Therefore, these results are in line with those of the previous studies. Xiang et al. (2009) analyzed internal transcribed spacer (ITS) region for 31 Abies species to examine phylogenic classification of the genus. Small and large sub-repeats were identified for Pinaceae and Abies, respectively. Four major groups, (1) Western North American monotypic section, (2) Eastern Asian-North American lineage, (3) Western North America group including species from Mexico and Eastern Asia and (4) Eurasian lineage group, were determined based on ITS regions. These results are consistent with data obtained from our study, although relationship among the four major clades remained unconvincingly resolved. This combination of findings supports the conceptual premise that Turkish fir members are in same phylogenetic clade when cpDNA or nDNA regions are used to construct a phylogenetic tree. However, fir taxa in Turkey phylogenetically differ from firs distributed in the Mediterranean and European regions and were commonly found in a distinct clade according to their cpDNA regions. Interestingly, our study shows that based on rps18-rpl20 cpDNA, some members in Turkish fir taxa are in the same clade as A. amabilis located in the Pacific Northwest of North America.

Conclusion
Previous studies on fir species in Turkey have focused on their morphological differences and there are limited data on their molecular phylogenetic relationships. The current study provides new insights into the distribution of cpDNA variation for Abies taxa in Turkey and examines the genetic variation between Turkish fir species and other fir species distributed around the world. Based on two cpDNA markers, Turkish Abies taxa are not separated from one another. This may stem from recent divergence among the taxa and indicate their common ancestor. Therefore, further studies may shed lighter on the classification of Abies by comparing the maternally inherited