Climatically induced anomalies in tree-ring structure of Abies pindrow (Royal ex D. Don) and Taxus baccata (L.) growing in Hindu-Kush mountainous region of Pakistan

Dendroclimatic potential from Abies pindrow Royal ex D. Don. (fir) and Taxus baccata L. (yew) was investigated by developing four different types of tree-ring width chronologies (by ARSTAN program) in a mixed broadleaf forest of Kalam, KP, Pakistan. Firstly, measured tree-ring width series were cross-dated and identified the best and least (cross-matched with master plot) samples with the most applied Skeleton Plot Model (SPM) method alongside checking the quality through statistical program (COFECHA). Tree-ring attributes (age, growth rate and earlywood and latewood) were also measured with maximum age (677 years) was observed in fir plant and maximum growth rate (6.096 mm year−1) in yew plant with clear demarcations of earlywood and latewood formation. Yew trees were observed to be more sensitive species towards climatic variations (expressed population signal = 0.945, mean sensitivity = 0.321 and signal to noise ratio = 3.214) while fir trees were comparatively less affected by climatic alterations of the study site (expressed population signal = 0.954, mean sensitivity = 0.314 and signal to noise ratio = 2.453). Correlation between tree-ring series of T. baccata with the master series was found to be more significant as compared to A. pindrow after developing series plots of both the species by using package dplR in R. Potential ecological and anatomical explanations for these results were also discussed. Sensitivity of samples showed good climatic signals which are valuable for dendroclimatic growth response investigations leading to reconstruction of past climate as well as possible future climate predictions.


Introduction
Global warming has become one of the main concerns for environmentalists. It has increased the intensity and frequency of climatic extremes . As a consequence, these extreme events especially drought and heat waves lead to degradation of forest ecosystems stability which can contribute to increased risk of desertification. This has been resulted in serious consequences for forests in terms of ecological stability and productivity. According to Inter-governmental Panel of Climate Change (IPCC), a change of climate affects all the aspects of human life (Ahmed et al 2012). Pakistan is highly vulnerable to climate change and ranked as 5th most vulnerable country of the region due to tremendous rise in temperature and Green House Gases (GHGs) emission. It has experienced extreme weather events like drastic temperature fluctuations and heavy rainfall that lead towards heavy floods , Khan et al 2018a, 2018b. Due to these climatic extremes, Hindu-Kush Himalayan region is one of the most susceptible regions. Due to annual resolution and precise dating dendrochronological methods are being widely used to investigate the past climate changes (Waszak et al 2021). Himalayan dry temperate mixed broad leaf forests occur at elevation between 1700-3300 m above sea level. They are located below the sub alpine forests and mix with lower boundaries of moist temperate forests. Climate is with long and cold winters. Due to monsoon penetration, these forests have open canopy as trees are widely spread with poor productivity and open scrub undergrowth (Hussain 2014). Grazing and browsing are common practices by cattle which are damaging the natural balance of species composition. The vegetation includes conifers (deodar, chalgoza, blue pine, spruce, pencil juniper and barmi) broad leaved trees (walnut, white oak, barungi, chan, horse chestnut, ash, maple, poplar) and shrubs (Artemisia, ephedra, jangli badam, astragalus, daphne, phut, unab). These forests receive heavy monsoon rains in summers and heavy snowfall in winters (Rasheet et al 2017).
Wood formation with distinct earlywood and latewood portions in conifers is called Xylogenesis (Cuny and Rathgeber 2016). It comprises of two biological processes, mitotic activity of cambium and differentiation in cambial cells undergoing to expansion. Earlywood is formed when cambium is resumed in spring season with high mitotic activity resulted in high turgor pressure, rapid cell expansion and formation of wide tracheids with thin walls, low density and large lumen area (Micco et al 2019, Zheng et al 2022. Latewood is formed when cambium activity is reduced in late summer resulted in lower cell turgor, a reduction in cell expansion and formation of small tracheids with thick walls, high lignification rate, high density and restricted lumen area (Micco et al 2019). Earlywood and latewood production is regulated by developmental control as well. Plant harmones; Auxin (Indole Acetic Acid) regulate the cell enlargement rate while Cytokinin control the cell division. Besides, carbon metabolism and sugar metabolizing enzymes are also involved in earlywood and latewood transition in conifers (Traversari et al 2022). As natural archives, tree rings provide proxy data for paleo-environmental studies, to analyze ecological issues and reconstructions of past climate (Choi et al 2020).
The dendrochronological method mainly involves the extraction of 'the signal' (common environmental information) and deduction of 'the noise'; the amount of unwanted information (Speer 2010). Tree ring standard protocol mainly comprises of several phases as follows, 1. Selecting the site, (maximum tree growth response to change in environmental factor of interest; mainly temperature and precipitation) after Shah et al (2019) 2. Selecting the trees, should present the best signal to noise ratio (Sullivan et al 2016) 3. Cross-dating the series, before and after the tree ring measurements by comparing and matching the specimens by graphical (Skelton Plot Model) and Statistical (COFECHA) methods (Muhammad et al 2021) 4. Standardizing the time series measurements to remove non-climatic trend and reduce aberrant growth pattern due to disturbances (Park et al 2021) 5. Averaging the standardized individual series into single one that represents the species growth variability (Asad et al 2018 In temperate regions, distinct annual rings are formed in trees by thin divisible tissue enveloping the stem and tree growth is affected by combination of biological and environmental factors . This climate/growth dependency causes synchronicity between trees of one site and other distinct sites. This synchronous growth enables to follow the core principle of dendrochronological and dendroclimatological research 'cross-dating' of trees. Cross-dating is a fundamental principle as it is considered the heart of dendrochronology. It is also described it as the 'art of dendrochronology' (Sesler 2009). It involves tree ring series alignment on the basis of consistent variation in tree ring characteristics. It also facilitates to identify the missing and false rings in series (Brookhouse and Brack 2006). With the help of cross-dating, correlation among the samples, best and least correlated samples with master series to find exact calendar year of ring formation can be determined. In a general way, it is considered that higher elevations with high rainfall, running streams and deep moist soils decrease cross-dating while lower elevations where rainfall is low, sandstone and limestone bed rock under shallow soils steep slopes high ridges and shadow sides of mountains are favorable to cross-dating (Douglass 1941, Muhammad et al 2021. A. Pindrow and T. baccata are more vulnerable to climate changes (Thapa et al 2013, Yousaf et al 2022. These two species in this temperate forest are dominant (Muhammad et al 2021), having rapid growth and according to recent studies these species have potential sensitivity for dendroclimatological studies (Thapa et al 2013, Shaheen et al 2015, Koc et al 2018, Ullah et al 2022. However, little is known on the relationship between climate and growth for these two species in the study site, we explored this correlation with regional temperature and precipitation variability to assess their potential for climate reconstruction. This dendroclimatic analysis of both species will better serve current forest management and conservation efforts when considering future climate scenarios. The objectives of this study were, 1. To determine age/growth of both species. 2. To determine seasonal dynamics (earlywood & latewood) and their relationship with climate. 3. To develop different versions of chronologies and their statistics through COFECHA and ARSTAN. 4. To evaluate climatic signals through statistical analysis of chronologies of both species by using dplr in R.

Study site
Hindu-Kush is a great mountain system of Central Asia extending about 800 km long and 240 km wide. For the present study, Kalam Forest (dry temperate area), located in the high altitude North-Western part of Pakistan in the Hindu-Kush region was selected (figure 1). The site is geographically important since it is located at the junction of three mountainous (Hindu-Kush, Himalaya and Karakoram) areas with alpine peaks varying in elevation from 1900 to 4600 m. The climate of the study area shows great variability as monsoon is dominant during summer while winter nourishes these mountains with snowfall (Hussain 2014, Muhammad et al 2021. According to climate data, the mean monthly maximum temperature observed is 11.9°C for July. The mean monthly minimum temperature observed is −11.9°C for January. The mean max. monthly precipitation observed is 58.4 mm in March while mean min. monthly precipitation is 14.7 mm in June (figures 2(a)-(b)).

Tree-ring sampling
Wood samples from 37 healthy A. pindrow and 13 healthy T. baccata were collected using the Swedish increment borer. Two samples were obtained from each tree to make the cross dating process easy and valuable. The coring technique and samples preparation were carried out following the method given by (Stokes and Smiley 1968). The cores showing good cross matching were measured using Velmex measuring system (TA4021H1).
After skeleton plotting (graphical cross dating method), COFECHA was used to check the measurement accuracy and possible dating error in tree ring series (Holmes et al 1986, Grissino-Mayer 2001. Then cross dated series were subjected to computer software ARSTAN (Cook 1985, Jernej andTom 2018). The climate growth response analysis was performed by using standard chronologies of both species against mean monthly precipitation and temperature for 37 years . The climate data for Kalam forest was obtained from NASA Langley Research Center (LaRC) POWER Project funded through the NASA Earth Science/Applied Science Program (https://power.larc.nasa.gov/).

Results
After successful cross-dating by two methods (visual dating & skeleton plot model; a graphical method) and computer program (COFECHA), three versions of Tree-ring width (TRW) chronologies were developed by another program ARSTAN (autoregressive standardization). COFECHA was used for quality assessment of cross-dating and it added more accuracy in measurements as compared to SPM (Skeleton Plot Model) method as shown in figures 3(a)-(d). The statistical summary is given in tables 2, 3 and 4 respectively.

Crossdating
To crossdate the trees, Skeleton Plot Model was used which is matching of tree ring widths. Skeleton plot is a plot of vertical bars in which bar length is inversely related to ring width. This basic principle of dendrochronology was applied to all the trees either they showed complacent or sensitive growth. The sensitive trees showed best cross matching for example vertical marks were made on graph lines at the desired height by setting a scale; representing the tree ring widths. The longer the bar, narrower is the ring. In case of wide rings, a small-case 'b' is added to skeleton plot. The normal rings were not marked on the graph. After plotting all the rings, master plots were raised and cross matched by dragging right and left the plot (Sheppard 2002). The master chronologies were also raised (figures 3(a)-(d)). The best cross dated and least cross dated trees were recorded (table 1).

Signal strength and standardization
Signal strength usually describes the quality of chronology to reconstruct the variations of past climate history. It depends on sample depth and harmony between individual series combined to a mean chronology. The quality of chronology is affected by sample depth as it is positively correlated with minimum number of sample size to a mean chronology. It is followed to interpret the chronologies with the elimination of age trends and other biological and site related noise from tree ring series. For this purpose, smoothing function was applied to standardize the tree ring series. Commonly dendrochronologists apply negative exponential curve or spline curve method. The resulting chronology was developed with rise of common signals and reflected the climatic variations (figures 4(a)nd 5).

Expressed population signal (EPS) and Rbar
The interval analysis of expressed population signal (EPS) and Rbar with 50 years interval lagged by 25 years was done to determine at what point the number of cores was enough to present a reliable part of the TRW chronologies. The purpose of EPS and Rbar was to investigate the signal strength of chronology and correlations between trees. Autoregressive standardization (ARSTAN) developed four different types of chronologies for both of species named as raw chronology, standard chronology, residual chronology and arstan chronology. The results of these chronologies of both species in terms of mean index, standard deviation, serial correlation, kurtosis coefficient and skewness coefficient are presented (table 3). In raw chronology, the value of mean index for A. pindrow is 1.550 which is less as compared to T. baccata These chronologies provide various statistics of EPS, SNR, Rbar, SSS and eigenvector percentage (table 4, figure 6). The graphical representation of all chronologies is shown in figures 4 and 5. The series plots of both species were developed between the master series and all the samples by using package dplR in R Studio. The correlation between master series and sample series was significant in T. baccata (figure 4(d)) while it was nonsignificant in A. pindrow series plot ( figure 5(d)).

Climate-growth relationship
The annual radial growth of trees was correlated with climatic parameters (precipitation and temperature of Kalam Forest) for the period 1982-2018. The growth response of A. pindrow with precipitation was positively   significant for winter months (previous December, current January and February). The growth also correlated positively with precipitation during current April and August months ( figure 7(a)). Similarly, the growth of T. baccata was found to be positively correlated with precipitation during previous and current summer months. Like A. pindrow, the growth of T. baccata was also positively influenced by current  January to April precipitation ( figure 7(b)). The correlation of temperature with tree growth of A. pindrow was observed to be negative for previous December and current January, May and June. However, temperature correlation during previous October was significantly positive ( figure 8(a)). For T. baccata, temperature was also negatively correlated for most of the current year months and for previous November ( figure 8(b)). The growth of both species was strongly affected by both climatic factors (precipitation, temperature). Precipitation accelerated the radial growth of both trees while temperature hindered the growth of both species.

Seasonal dynamics
Tree ring width and its parts (earlywood & latewood) were measured by using Measure J 2 X (  figure 9. Earlywood of both species is showing variable trends while latewood is usually suppressed and it demarcates the ring growth boundary. The regression analysis was studied between earlywood and latewood of both species with their mean growth rates and it was found to be significant in case of earlywood while R 2 value of latewood correlation indicated that it was non-significant (figures 10 and 11). The ring width was dominated by the earlywood part of the ring as the growing season having more precipitation and favourable growth environment favored more earlywood formation. Figure 12 indicates

Discussion
From the dendrochronological point of view, study site was selected with trees of maximum height and girth after (Ahmed et al 2013). Two conifer species were selected for study with 23 trees of Fir and 16 trees of common Yews. Plants were targeted and two cores from each tree were obtained for cross dating and pattern matching purpose by two methods graphically and statistically after (Fritts 1976). The ring growth pattern on these cores reflected area disturbance by various factors over the period of time. A wide range of environmental factors influence the growth rate of trees (Pumijumnong and Palakit 2021). However, climate is considered as the most prominent factor influencing on year to year growth of trees. Precipitation and temperature are considered the most important factors that show significant correlation with tree ring width of conifers. Earlywood and latewood parts also show significant relationship with climate (Camarero et al 2021, Gricar et al 2021). Four versions of chronologies were developed named as raw, standard, residual and arstan chronology. The variations of tree ring width with respect to statistically developed   chronologies were analyzed for climatic signals that would lead to climate growth response and climatic reconstruction of the past climate of study site (Matisons et al 2015, Roibu et al 2020.
COFECHA analyzed TRW measurements and assessed the quality of measurement with cross dating. It not only properly checks the accuracy of cross-dating the tree rings but also frees TRW measurements from any problem. It gives different parameters for better understanding of cross-dating of tree rings after . In dendrochronological studies, the mean ring width of both species ranged from 1.86-17.71 mm and 2.54-14.58 mm respectively (tables 4, 5). Species having lower mean growth with irregular pattern of rings are considered more valuable as they carry more information about past events (Khan et al 2018a(Khan et al , 2018b. T. baccata wood showed more growth than A. Pindrow as regional climate favored it significantly. Standard deviation of both species ranges as 0.16-0.427 and 0.166-0.821 respectively and found to be greater value of T. baccata. This value is not meaningful for reconstruction although it is used to present some basic information by comparing with other statistical values. However, it is considered an ambiguous statistic for illustration of trees growth variations, as it is largely dependent on autocorrelation and standard deviation of series (Bunn et al 2013). Maximum ring width ranges from 17.71-14.58 mm. This ring width value varies from species to species and between the different sites. Mean sensitivity (MS) usually describes the year to year variations in tree-ring width and is thus considered an estimate of the extent to which the chronology reflects the local climate variations (Fritts 1976, Park et al 2021. It was observed with 0.314 and 0.321 for A. pindrow and T. baccata respectively. MS varies from species to species and even from site to site and it was found to be more meaningful in case of A. pindrow. The correlation with master series for both species was 0.087 and 0.013. However, it does not provide any possible relation to climate reconstruction but it does prove the reliability of chronology (Khan et al 2018a(Khan et al , 2018b. The value of autocorrelation; impact of growth occurred in previous year on growth occurred on current year, was 0.714 and 0.742 respectively. It also varies between different sites and species. We also did interval analysis of expressed population signal (EPS) and Rbar with 50 years interval lagged by 25 years to determine at what point the number of cores was enough to present a reliable part of the TRW chronologies (Cook andKairiukstis 1990, Park et al 2021). The purpose of EPS and Rbar was to investigate signal strength of chronology ad correlations between trees (Fritts 1976, Waszak et al 2021. Autoregressive standardization (ARSTAN) developed four different types of chronologies for both of species named as raw chronology, residual chronology, standard chronology and arstan chronology after (Asad et al 2018, Park et al 2021. ARSTAN program is used in standardization and detrending the series with removal of effects of endogenous stand disturbance by application of robust estimation of mean value. It removes non climatic trends in the given chronology (Zafar et al 2010, Ahmed et al 2012. It also helps in enhancing common signals by autoregressive modeling the index series. The developed chronologies showed distinct variations in both species in the form of trends exhibiting above and below average growth of trees of study site and their dendrochronological potential regarding climatic influences of the past (Esper and Gartner 2001). The climate growth response of both species against climatic parameters was similar. The growth of both species was positively correlated with precipitation and negatively correlated with temperature, similar to the findings of Ullah et al (2022) and Ahmed et al (2010). In dry temperate regions, precipitation becomes the limiting factor for growth of conifer species, and lack of rainfall with increasing temperature results in drought conditions, leading to decreased growth rate of trees (Thapa et al 2013). Our findings also indicate water availability as the most important growth regulating factor for both species. Such results were also summarized by Ahmed et al (2012) and Ali et al (2021). The negative growth response toward temperature rise indicates the vulnerability of both species of Kalam Forest to climatic changes like global warming in future.

Conclusion
This study was conducted to determine the possible growth response of two dominant conifers from the Hindu-Kush mountainous region in Northern Pakistan. The study was aimed to investigate the past climatic changes as well as to understand possible future climate changes for forest management. For this purpose, tree ring width chronology (1740-2019) was developed. Sensitivity of samples showed good climatic signals for dendroclimatological potential that will be helpful in its future applied perspectives. In this study, it is possible to attain an in depth climate variability assessment of conifers. Standardization, used in chronology development to remove age related biasness, sample replication, effects of disturbances and stand dynamics, which although minimized, still remain a significant source of noise. Changing the statistical manipulation through site and sample collection would improve the quality of results and other ecological inferences of the study area. With increases in sample depth, sites and minor adjustments with climate growth relationship development, we can reduce possible biasness and improve the reliability of climate growth response towards reconstruction of past events that would serve as indicators for possible future seasonal changes. This study highlighted the effects of climatic variability upon tree growth of both species, improving our understanding of past climatic changes in dry temperate forests. Furthermore, performing reconstruction of these climatic parameters would be helpful for forest management practices especially in the context of climate change in future.