Genetic Evidence of Stable Northward Extension of Pinus Thunbergii Forests in the Democratic People’s Republic Korea

Japanese black pine with high salt tolerance may be an important constitutive element sustaining terrestrial ecosystem by playing a role of windbreak forests in coastal areas. Korean peninsula would be a notable region in clarifying distribution shift in Pinus species as it has northern distribution limit of Japanese black pine in Asia. Our main object was to verify genetic evidence of stable northward extension of P.thunbergii populations in D. P. R. Korea. We investigated genetic background of 9 populations existing in Korean peninsula using nuclear SSR markers in relation to shifts in climate factors such as temperature and precipitation. Higher genetic diversity in east group (AR=10.7~19.5) and west group (AR=10.3~10.7) compared to north group (AR=6.7~8.8) was found. When number of putative clusters (K) = 2, whole individuals were divided into west group and north-east group, and when K=3, north-east group can be separated into north group and east group. Phylogeographic relationship veried by means of nSSR markers suggest that substantial increment of air temperature in D. P. R. Korea allowed stable anthropogenic transfer of P.thunbergii forests and that articial afforestation may bring rapid establishment of forest ecosystem owing to climate change.


Introduction
Increasing air CO 2 emission, growing global warming resulting in climate change have an in uence on global biome (Lenoir et al. 2008; Keenan et al. 2010; Etterson et al. 2010). In coastal areas, the threat of natural disaster due to climate change and global warming is increasing day by day and windbreak forests formed in these areas might play an important role in damage reduction. Forest may be integral part of the regional biome due to its fundamental position in material circulation and energy ow. Understanding genetic diversity and population dynamics of forest tree species playing an important role in forest composition and functioning in relation to climate change would assist the design of conservation strategy and forest establishment.
The negative impact of climate change on various forest tree species has been widely reported. Freire et al. (2019) assessed the effects of precipitation decrease, air temperature change resulting in frequent forest re on afforestation of Pinus pinea L. and suggested the ways to mitigate the effect of climate change, one of that is to reduce population density. Fettig et al. (2019) reported massive extinction of Pinus ponderosa Dougl. and change of forest composition following air temperature increase accompanied by extreme drought in southern Sierra Nevada, California.
Climate change in different parts may result in change of growth and distribution of forest tree species. Misi et al. (2019) clari ed that the effect of climate on tree's growth could vary between regions based on the assessment of effects of temperature and precipitation on the width of annual ring of Scots pine (Pinus sylvestris L.). Cheaib et al. (2012) assessed potential in uence of air temperature, precipitation and CO 2 concentration on the distribution of Scots pine in France based on several modeling methods and reported upcoming forest contraction. Nevertheless, such researches have not been widely reported in Asia.
The distribution of Japanese black pine is characterized by both natural and arti cial forests (Iwaizumi et al. 2018). Because Japanese black pine forests prevent soil erosion and exhibits tolerance to salty environment, it is suitable for the establishment of wind and wave breaking forest in coastal areas (Konta, 2001;Ogawa 1979, Murai et al. 1992Zhu et al. 2000). That's why there are many arti cial forests as well as natural ones of Japanese black pine in the coastal areas (Iwaizumi et al. 2018).
To establish conservation and management strategy, we assessed genetic background of 9 Japanese black pine populations that has been formed in different period in the context of climate change. Comparing to old natural forests in western and eastern areas, most of the forests in northern part of Korean peninsula are arti cial ones composed of young individuals that aimed to prevent damaging disaster. Some individuals in Thongchon are more than 25 years, even 55~60 years old. In Ongjin, there exists 90 years-old individuals and in Kosan, many individuals are more than 40 years old. Nevertheless, most individuals in the north of Kumya are 8~10 years old planted ones. Despite relatively low genetic diversity indices of Japanese black pine populations in the north of Kumya, genetic diversity indices in all Japanese black pine populations were preserved at high level. Structure analysis showed that when K=2, all Japanese black pine populations were divided into 2 groups-east and north+west ones, and K=3, into 3 groups-east, west and north ones. Investigation on the climate change in northern region showed signi cant air temperature increase but there wasn't a big difference in precipitation during decades.
These results showed that genetic stability of Japanese black pine populations formed to reduce disastrous damage in the north of Kumya has been maintained at a quite level. It is likely for species of genus Pinus distributed in the temperate zone to expand toward north east referring to historical process from refugia to new habitat followed by present and upcoming events. Iwaizumi et al. (2018) reported association between historical expansion of Japanese black pine populations from southwest to northeast and general migration from southern refugia to north after glacial period. Korean peninsula is located in the east of Eurasia and has been reported as putative refugia of various animal and plant species (Ho et al. 2020;Kang et al. 2002). Present air temperature increase could be characterized as reappearance of this historical process. Our results support validity of northward expansion of Japanese black pine populations through recent extensive afforestation and suggest upcoming northward expansion given recent climate change.

Sample collection, DNA extraction
Mature needles were collected from 9 regional population individuals (Fig. 1).
The location information is as indicated in Table 1.
The age scale of sampled individuals was 3-90 years old. Sample sites represent both natural and arti cial distribution of Japanese black pine. We randomly selected trees for sampling within each population keeping at least 30m between sampled trees. The needles were stored at -25℃ before DNA extraction. Total DNA was extracted using cetyl trimethylamonium bromide (CTAB) method (Doyle and Doyle 1987; Rogers and Bendich 1988).

SSR allele genotyping
Genotypes of the sample trees were determined by 7 nuclear SSR markers reported previously (bcpt834: Iwaizumi et al., 2013, bcpt1075, bcpt1549, bcpt1671, bcpt1823, bcpt2532, bcpd119: Iwaizumi et al., 2018 pdms1208). PCR conditions are as follows; an initial denature at 95℃ for 5 min, 35 cycles of denature at 95℃ for 1 min, annealing at optimized temperature for 1min, and chain extension at 72℃ for 1 min, followed by a nal extension for 10 min at 72℃. The size of ampli ed fragments was determined with a DNA sequencer, ABI PRISM 3730 Genetic Analyzer (Applied Biosystems Inc.). The data of mean annual temperature and total annual precipitation in study regions were collected from the State Hydro-Meteorological Administration of DPR Korea.

Association between Japanese black pine range expansion and climate change
To assess the effects of climate on the expansion of distribution of Japanese black pine, we collected annual average air temperature data of 9 sampled sites in 1980-2019. As shown above, 40 years annual air temperature change exhibited increasing trends in spite of its oscillating pattern. In particular, annual air temperature in the north of Kumya exhibited steeper increase than in other regions. In contrast all study regions exhibited slight changes in precipitation and these changes were not signi cant as air temperature. Since 2000, all sampled sites have had higher annual air temperature than that of Ongjin in 1980, and owing to the fact that old Japanese black pine stands exist in Ongjin, it indicates that since 2000, all sampled sites would have suitable climate conditions for the distribution of Japanese black pine.

Discussion
It would be helpful to understand population dynamics, genetic diversity and phylogeographic relationship of regional populations of forest tree species using molecular markers to improve forest conservation and management (Kassa et  Genetic differentiation and relative stability of northern Japanese black pine populations formed as disaster preventive forests suggest that in uence of afforestation on range expansion would be dominant compared to gene ow.
Although annual air temperature increase would enable the cultivation of Japanese black pine in the north of Kumya, it is unlikely to claim annual air temperature rise directly led to expansion of Japanese black pine to the north of Kumya. In study regions, extensive afforestation through cultivation seems to have more in uence than natural migration from south to north. Recently, Japanese black pine is widely cultivated through plantation in northern coastal areas including Kumya and Tanchon. It is no strange that northern regional arti cial forests exhibits lower genetic diversity than that of eastern and western region natural ones.
Different climatic conditions in different regions would lead to different distribution of forest limit. In Korean peninsula, northern limit of overall distribution of Japanese black pine is lower in latitude than that of Japan (Iwaizumi et al. 2018). In China, Japanese black pine has been reported to exist in Dalian, Shandong coastal areas, Wuhan, Nanjing, Shanghai, Hangzhou, which is lower in latitude than that of Korean peninsula (Mao et al. 2019). These differences of forest limit maybe due to the effects of both continental and marine climate on forest in Korean peninsula, whereas China is affected by continental climate, Japanese by seasonal one. Our results support validity of northward extension of Pinus thunbergii Parl. populations through recent extensive afforestation and suggest upcoming northward extension given recent climate change.

Conclusion
The increment of substantial air temperature in study regions, genetic stability and differentiation of northern populations formed recently through afforestation suggests dominant affection of afforestation on expansion of forest distribution. Our results suggest that substantial air temperature increment allowed stable anthropogenic northward extension of Japanese black pine forests.

Declarations
Contributions of the co-authors UH and SS designed the study and conducted the experiments, and HP, KK, TH and IJ analysed the data; UH and SS collected data and wrote the rst draft; and all authors contributed substantially to revisions.

Figure 1
Sample sites of Japanese black pine in Korean peninsula The location information is as indicated in Table 1.
Page 11/12 Annual average air temperature change in 9 sites of Korean peninsula in 1980-2019, The location information is as indicated in Table 1.