Genetic Diversity of Pinus species in New York: a base line study for fungal endophytes assemblage analysis

Ravishankar Narayana


Endophytes are microorganisms inhabiting plant organs that at some time in their life, can colonize internal plant tissues without causing apparent harm to the host.They produce a wide range of compounds useful for plants for their growth, protection to adverse environmental conditions, and herbivory. The endophytic population varies among plants, species, climatic conditions and host locality. Resolving the importance of locality and host identity is important for understanding fundamental aspects of endophyte population.

Chloroplast intergenic trnH-psbA spacer has recently become a popular tool in plant molecular phylogenetic studies at low taxonomic level and is the region suitable for DNA barcoding studies. The main objective of this study is to verify the genetic identity of pine species at different location in New York before beginning analysis of their resident endophytes.

Materials and Methods

Needles from one red pine and white pine were obtained from Fordham University’s Rose Hill campus in Bronx, NY and Central Park in Manhattan. DNA was extracted from all four samples.

Primers were used to amplify the trnH-psbA (Fig. 2) barcoding region (Armenise et al. 2012). The PCR products were sequenced, and a BLAST search was performed to identify the trees.


Each of the four samples barcoding loci was successfully amplified using standard primer pair and PCR protocol (Fig. 1). We obtained trnH-psbA DNA sequences from every single individual analyzed. BLAST searches performed on GenBank displayed multiple congeneric hits. Highest hits (100% identity) of the trnH-psbA sequence of white pine (Rose Hill campus) was with Pinus strobus, a eastern white pine belong to subgenus Strobus and native species of eastern North America. Whereas the sequence of white pine (Central Park) displayed 100% identity with a Himalayan white pine (Pinus wallichiana), a native species of Himalayan region. On the other hand, the trnH-psbA sequences of red pine (Rose Hill and Central Park) displayed 100% identity with a black pine (Pinus nigra) sequence, a native species of southern and eastern Mediterranean Europe, but not with Pinus resinosa.


The present study detected considerably genetic differentiation between samples located in the Rose Hill and Central Park. The present barcoding results demonstrate that pine samples from Rose Hill campus of Fordham University and Central Park, Manhattan are different species, even though they look morphologically similar to white and red pines. The study of genetic diversity will helps in discrimination and identification of species which are morphologically similar but genetically different (cryptic species). Ideally, future study would include pine species identification from a geographic region along with endophyte population analysis in urban-rural gradient.


Armenise L, Simeone M. C, Piredda R and Schirone, B (2012). Validation of DNA barcoding as an efficient tool for taxon identification and detection of species diversity in Italian conifers. Eur J Forest Res. 131:1337–1353.

Burton Verne Barnes and Warren Herbert Wagner (2004). Michigan Trees: A Guide to the Trees of the Great Lakes Region. University of Michigan Press. pp. 81. ISBN 0-472-08921-8.

Chareprasert S, Piapukiew J, Thienhirun S, Whalley A. J. S. and Sihanonth P (2006). “Endophytic fungi of teak leaves Tectona grandis L. and rain tree leaves Samanea saman Merr,” World Journal of Microbiology and Biotechnology. 22(5):.481–486.

Das A and Varma A (2009). “Symbiosis: the art of living,” in Symbiotic Fungi Principles and Practice, Eds., pp. 1–28, Springer, Berlin, Germany.

Farjon, A. 1984, 2nd edition 2005. Pines. E. J. Brill, Leiden. ISBN 90-04-13916-8.

Hebert PDN, Ratnasingham S, deWaard JR (2003) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B 270:S96–S99.

Petrini, O (1991). Fungal endophytes of tree leaves. In :Microbial ecology of leaves (edn) by Andrews J.H. and Hiran S.S.Springer Verlag. NewYork. USA.

Taghavi S, Garafola C, Monchy S et al., (2009). Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees. Applied and Environmental Microbiology 75 (3): 748–57.

Von Cra¨utlein M, Korpelainen H, Pietila¨inen M, Rikkinen J (2011). DNA barcoding: a tool for improved taxon identification and detection of species diversity. Biodiv. Conserv. 20:373–380.


Understanding how fungal endophyte communities differ in abundance, diversity, taxonomic composition, and host affinity over the geographic ranges of their hosts is key to understanding the ecology and evolutionary context of endophyte–pine associations. This study was undertaken to investigate the samples being characterize to make certain that any observed differences along Urban-Rural gradient were due to locality and not due to miss identification of pine species. The choloplast intergenic trnH-psbA spacer gene was used to characterize the pine samples. Results show that the white pine tree specimens located on the Rose Hill campus of Fordham University and Central Park are Pinus strobus and Pinus wallichiana respectively, even though both appeared to be morphologically white pine (Pinus strobus). Sequence analysis performed on samples which morphologically identified as red pines on the Rose Hill campus of Fordham University and in Central park were identified to be Pinus nigra (black pine). Our finding emphasis the importance of the genetic identification of samples utilized in any studies across an Urban-Rural gradient.


Figure 1-PCR results from amplification using primers for regions of the genes trnH-psbA.

Figure 2-The gene structure of trnH-psbA in Pinus

Figure 3-White pine (Central Park)

Figure 4-White pine (Rose Hill)

Full Paper


I would like to thank Catharina Grubaugh, and Kate Reid for their guidance and patience through all stages of this project. I would also like to thank Dr. Berish Rubin for his support and guidance throughout this project.

This document was last modified 05/07/2015.
This site is powered by the versatile Zope platform.
This is a project of the Biology Department of Fordham University Home