Species Identification of Pinus Pollen Found in Belukha Glacier, Russian Altai Mountains, Using a Whole-Genome Amplification Method

Fumio Nakazawa,Yoshihisa Suyama,Satoshi Imura,Hideaki Motoyama

doi:10.3390/f9080444

Abstract

Pollen taxa in sediment samples can be identified based on morphology. However, closely related species do not differ substantially in pollen morphology, and accurate identification is generally limited to genera or families. Because many pollen grains in glaciers contain protoplasm, genetic information obtained from pollen grains should enable the identification of plant taxa at the species level. In the present study, species identification of Pinus pollen grains was attempted using whole-genome amplification (WGA). We used pollen grains extracted from surface snow (depth, 1.8–1.9 m) from the Belukha glacier in the summer of 2003. WGA was performed using a single pollen grain. Some regions of the chloroplast genome were amplified by PCR, and the DNA products were sequenced to identify the pollen grain. Pinus includes approximately 111 recognized species in two subgenera, four sections, and 11 subsections. The tree species Pinus sibirica and P. sylvestris are currently found at the periphery of the glacier. We identified the pollen grains from the Belukha glacier to the level of section or subsection to which P. sibirica and P. sylvestris belong. Moreover, we specifically identified two pollen grains as P. sibirica or P. cembra. Fifteen species, including P. sibirica, were candidates for the remaining pollen grain.

Highlights

The natural range of the genus Pinus is confined to the Northern Hemisphere, some species have been introduced to the Southern Hemisphere
Previous pollen DNA barcoding studies have focused on fragments of longer than 300 bp [14], we believe that fragments of around 200 bp may be sufficient to identify pollen at the section or subsection level when the target pollen type is specified and sequence data are available, as demonstrated in the present study
We described an initial attempt to identify pollen grains from a glacier at the species level based on chloroplast DNA sequences

Summary

Introduction

The natural range of the genus Pinus is confined to the Northern Hemisphere, some species have been introduced to the Southern Hemisphere. Pinus pollen grains can reach remote areas, such as mountain glaciers in the Northern Hemisphere, Arctic glaciers, and the Greenland ice sheet, and are found in snow and ice as a predominant pollen type [2,3,4,5,6,7,8,9]. Many pollen grains in glaciers are expected to contain protoplasm, and their maintenance at below 0 ◦ C is favorable for DNA preservation [10]. This property is characteristic of glacial pollen; protoplasm is rarely seen in pollen found in other sediment types, such as peat and lacustrine deposits.

Methods

Results

Conclusion