### Needle phenotype variation among natural populations of Pinus yunnanensis, Pinus kesiya var. langbianensis, and Pinus kesiya

Wei-Ying LI1,Zheng ren Zhang1,Ya xuan Xin2,Fei Wang2,Peiyao Xin2,Jie Gao3

1. 1. Southwest Forestry University；Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences.
2. Southwest Forestry University
3. CAS Key Laboratory of Tropical Forest Ecology, Xishuangbann, Chinese Academy of Sciences
• Received:2022-06-22 Revised:2022-10-28 Published:2022-10-31
• Contact: Jie Gao

Abstract: Aims Pinus kesiya and its sister species P. yunnanensis form savanna and forest communities in tropical and subtropical Southeast Asia and have an overlapping range in southern Yunnan. Introgression and hybridization have occurred in this region, forming a hybrid named P. kesiya var. langbianensis. This complex across a large distribution exhibits abundant climate and genetic variation and significant ecological and economic value. In this paper, we aimed to examine the needle anatomical traits in natural populations to assess phenotypic variation in the species complex to evaluate the association of needle morphological variation with climatic gradients as a reference for adaptation to local environments. Methods We measured eight needle anatomical traits from 31 natural populations, using variance analysis, multiple comparisons, and principal component analysis to understand the needle traits variation between species and populations. We use clustering analysis to investigatethe population structure of the needle morphological traits. We also evaluated the correlation of each trait with latitude using Spearman rank correlations, and we ran a generalized linear modelto identify the best environmental predictors for each trait. Important findings (1) The variation coefficients (CV) of the eight needle traits among the populations ranged from 12.01% to 34.08%. Within species, the CV values of P. kesiya var. langbianensis were highest, followed by P. kesiya and P. yunnanensis. The results showed that the variance components among species and populations were generally higher in the most tested needle traits. The length of stomata guard cell (LSGC), length of the woody thickening layer (WTLL), needle length (NL) and stomatal density (SD) had higher phenotypic differentiation coefficient (Vst), indicating that they were influenced by the phylogenetic conservation and strong environmental selection. (2) Most needle morphological characteristics differed significantly between P. yunnanensis and P. kesiya, while the P. kesiya var. langbianensis showed intermediate values between the two parental pines. Hierarchical clustering analysis showed that the pattern of population variation of needle traits followed the geographical region and genetic components. (3) The NL and SD were positively correlated with latitude and showed a different geographic pattern with pines from the temperate and higher latitude. The key environmental factors affecting the needle traits of the three pine species were mean temperature in the driest quarter, precipitation seasonality, mean diurnal range, precipitation of wettest month, precipitation of driest month. The above studies indicated that the needle phenotype could well explain the geographical substitution distribution pattern of the three pine species. With the average temperature increase in the driest season, the pine populations showed shorter needles, lower stomata density, a more significant woody thickening layer, and guard cells. While with the increase in precipitation, the size of the stomata increases. This reflects the comprehensive adaptation characteristics of tropical pine to high temperatures, high humidity, and a strong monsoon climate to balance the water and heat conditions.