Anales de Biología 42: 105-114, 2020 ARTICLE http://dx.doi.org/10.6018/analesbio.42.12. Morpho-anatomical variation and their phylogenetic impli- cations in native and exotic species of Pinus L. growing in the Indian Himalayas Lav Singh1,4, Pooja Dixit1, Ravi Prakash Srivastava1, Shivaraman Pandey2, Praveen Chandra Verma3 & Gauri Saxena1 1 Department of Botany, University of Lucknow, Lucknow, India. 2 Plant Diversity, Systematics and Herbarium Division, CSIR-National Botanical Research Institute, Lucknow, India. 3 Plant Molecular Biology and Genetic Engineering Division, CSIR-National Botanical Research Institute, Lucknow, India. 4 Department of Botany, R. D. and D. J. College, Munger University, Munger 811-201, India. Resumen Correspondence Variaciones morfoanatómicas y sus implicaciones filogenéticas en G. Saxena especies nativas y exóticas de Pinus L. del Himalaya indio E-mail: [email protected] Se realizó un estudio anatomico completo y detallada de agujas en Received: 15 March 2020 diez especies de Pinus utilizando nueve rasgos morfológicos y Accepted: 23 June 2020 anatómicos. Los datos pueden usarse como una herramienta para Published on-line: 7 July 2020 la identificación y clasificación de Pinus hasta el nivel de género y especie. También encontramos que la similitud y las diferencias en los rasgos anatómicos de la hoja respaldaban la filogenia molecu- lar de Pinus realizada por varios investigadores. Palabras clave: Pinus; Acícula; Fascículo; Haz vascular; Phylogenia. Abstract Comprehensive and detailed anatomy of needles in ten species of Pinus using four morphological traits and nine anatomical traits was carried out. The data can be used as a tool for identification and classification of Pinus upto genus and species level. We also found that similarity and differences in leaf anatomical traits sup- ported the molecular phylogeny of genus Pinus conducted by se- veral researchers. Key words: Pinus; Needles; Fascicles; Vascular bundle; Phylogeny. Introduction tropical regions of Central America and Asia. They may form forest or co-exist with other trees Order Pinales represents an outstanding group of (Farjon 1984 & 2005, Gaussen et al. 1993). They gymnosperms and is omnipresent in terrestrial have received much attention in past few years habitat. These monoecious woody plants usually because they form a major component of many grow naturally or have been introduced in both temperate forests, and not only have ecological the hemispheres, mainly in the Northern hemi- implications but are also of economical signifi- sphere sometimes occurring in subtropical and cance as a source of timber, pulp and paper, nuts, 106 L. Singh et al. Anales de Biología 42, 2020 seeds, resins, construction materials, and other by- within a specific range. The data generated was products (Richardson & Rundel 1998). further used to draw evolutionary relationship Genus Pinus L. is a large group of different among these ten exotic and indigenous species of pine species in Indian Himalayas with high genus Pinus namely Pinus merkusii Jungh. & de medicinal value and has played a significant role Vriese, Pinus kesya (Basionym of Pinus insularis in maintaining health (Sharma et al. 2018). Genus Endl. ), Pinus taeda L., Pinus elliottii Engelm., Pinus is the largest genus in the family of conifer- Pinus echinata Miller, Pinus thunbergii Parl., ous trees with broad climate adaptability. Genus Pinus patula Schiede ex Schlectendahl et Pinus has 110 species (Gernandt et al. 2005) and Chamisso, Pinus greggii Engelm, Pinus wallichi- is usually divided into two subgenera, Strobus (D. ana A.B. Jackson and Pinus roxburghii Sargent. Don) Lemmon (soft pines) and Pinus L. (hard pines), which are further divided into sections and Materials and methods subsections (Little & Critchfield 1969, Gernandt et al. 2005). The taxonomic history of the genus Sample collection was reviewed by Price et al. (1998), that included morphology, anatomy, crossability, cytology, sec- Altogether 150 observation by analysing 30 plant samples (3 trees for each species and five needles ondary metabolites, DNA and protein compa- per tree), comprising 10 pine species (Table 1) risons. The morphological traits that distinguish were collected using random block design (RBD) various species from each other include characters in September, 2016, from a cultivated population like the length and width of needles, the number in the region of Ranikhet (located at 357 km NSE of needles per fascicle, arrangement and orienta- of New Delhi (Fig. 1); 29°39’52.2"N, 79°28’ tion of needles (pendulous or erect), and anatomi- 40.9"E; altitude 1,727 m.a.s.l.). The site is charac- cal characteristics like epidermal cells, number terized by an average temperature of 14.4 °C, av- and position of resin ducts, and number of vascu- erage rainfall (about 1,575 mm of annual precipi- lar bundles in the needle (Gernandt et al. 2005). tation) and low soil fertility. A voucher specimen It has been observed that morphological and of all the species selected for study was deposited anatomical traits of needles as well as wood are in the herbarium of the National Botanical Re- strongly controlled by environmental conditions search Institute (NBRI), Lucknow (India) and like temperature, light availability and moisture identified. content in the habitat (Abrams & Kubiske 1990, Dixit et al. 2016). These morphological as well as Methodology for morpho-anatomical studies anatomical differences could provide new infor- Tree height was measured using stick method and mation that can be used to establish phylogenetic other macroscopic and microscopic analysis were relationship among various species (Ghimire et performed at the laboratory. Needle length was al. 2014). Although the needle structure of the measured (Table 1) using a measuring scale, common conifers like that of genus Pinus is com- paratively well studied and known, a comprehen- whereas other anatomical characteristics were ob- sive treatment of the comparative histological served (Table 2, Fig. 2) under the microscope organization of the needles is still lacking. The (Nikon Eclipse 80i). For morphological studies, main focus of present work was to perform a parameters like a number of needles per fascicle detailed anatomical study of needles from native and needle length were taken. The other morpho- Indian and cultivated species of genus Pinus to logical characters taken into account included the make a detailed histological comparison of height of the plant and bark color. For anatomical selected Pinus species. In our study, we found a studies, fresh plant needles were collected from high level of morphological variability among Pinus species under study. The microtome sec- native and exotic species as well as within their tioning (using Radical Di-cast Microtome, RMT- population particularly needle traits like needle 20A) and processing of needles was done accord- length that shows a higher degree of variation. ing to the procedure given by Federica & Ruzin Same is also true for plant height and bark colour. (2000). Fresh needles were fixed in a formalin- Further, the individual has a specific norm of acetic acid solution (50 ml 95% ethanol, 5 ml reactions to environmental factors and has a glacial acetic acid, 10 ml 37% formaldehyde and capacity for certain morphological modifications 35 ml distilled water) and kept for 24 hours Anales de Biología 42, 2020 Pinus in the Indian Himalayas 107 Height No of needle Size of needle Sample code Pinus species (in meters) Bark color per fascicles (in cm) PM Pinus thunbergii* 27-39 Greyish brown 2 10.66±3.3 PR Pinus roxburghii* 45-54 Dark grey 3 29.97±6.8 PW Pinus wallichiana* 15-46 Greyish brown 5 15.49±4.8 PM Pinus merkusii* 14-18 Grey to brown 2 21.59±4.0 PK Pinus kesya* 30-46 Light brown 3 18.54±1.2 PTd Pinus taeda 28-34 Reddish brown 3 14.98±2.7 PE Pinus echinata 25-37 Reddish 2 35.56±2.5 PG Pinus gregii 12-15 Grey 3 13.2±4.3 PP Pinus patula 12-19 Reddish brown 3 20.06±4.3 PEl Pinus elliottii 18-30 Greyish brown 2 21.08±3.3 *Pine species native to India Tabla 1. Especies de Pinus seleccionadas en los Himalayas noroccidentales y análisis de sus rasgos morfológicos. Table 1. Selected species of Pinus in nortwestern Himalayas and morphological trait analysis. Figura 1. Mapa que muestra el área de donde provienen las especies de Pinus que han sido recolectados durante el presente estudio (Uttarakhand, India). Figure 1. Map showing area from where the species of Pinus have been collected during present study (Uttarakhand, India). 108 L. Singh et al. Anales de Biología 42, 2020 Needle Pinus Pinus Pinus Pinus Pinus characteristics roxburghii* wallichiana* merkusii* kesya* thunbergii* 1. NT (µm) 771.93±9.6a 438.60±2.38h 664.34±3.46b 495.74±9.98f 501.36±2.80f 2. NW (µm) 1174.88±1.99b 755.76±6.51i 1265.81±16.19a 869.80±2.96g 1267.32±3.40a 3. C+ET (µm) 10.32±0.36f 12.13±0.57e 18.90±0.54a 10.42±0.73f 12.18±0.80e 4. ECW (µm) 11.68±0.57c 12.71±0.37c 15.24±0.64b 17.01±0.49a 12.77±1.13c 5. ENCT (µm) 24.40±0.69a 17.79±0.69bc 22.96±1.46a 17.94±1.91b.c 18.40±1.00bc 6. ENCW (µm) 45.59±2.45b 35.37±0.78f 41.77±1.32b 29.65±1.38g 38.15±1.82e 7. VBT (µm) 446.55±3.98a 239.81±2.37h 363.29±2.15d 240.62±0.73g,h 301.34±3.97d 8. VBW (µm) 681.61±3.98b 252.06±4.49j 701.05±3.26a 401.50±1.30g 288.77±2.69i 9. RCD (µm) 61.50±2.57a 40.57±1.27e 57.85±1.11b 41.62±2.16e 40.34±1.00e Needle Pinus Pinus Pinus Pinus Pinus characteristics taeda echinata greggii patula elliottii 1.