Bell Karen L, Fowler Julie, Burgess Kevin S, Dobbs Emily K, Gruenewald David, Lawley Brice, Morozumi Connor, Brosi Berry J
Department of Environmental Sciences, Emory University, 400 Dowman Drive, Atlanta, Georgia 30322 USA.
School of Biological Sciences, The University of Western Australia, Perth, Western Australia 6008, Australia.
Appl Plant Sci. 2017 Jun 12;5(6). doi: 10.3732/apps.1600124. eCollection 2017 Jun.
To study pollination networks in a changing environment, we need accurate, high-throughput methods. Previous studies have shown that more highly resolved networks can be constructed by studying pollen loads taken from bees, relative to field observations. DNA metabarcoding potentially allows for faster and finer-scale taxonomic resolution of pollen compared to traditional approaches (e.g., light microscopy), but has not been applied to pollination networks.
We sampled pollen from 38 bee species collected in Florida from sites differing in forest management. We isolated DNA from pollen mixtures and sequenced and ITS2 gene regions from all mixtures in a single run on the Illumina MiSeq platform. We identified species from sequence data using comprehensive and ITS2 databases.
We successfully built a proof-of-concept quantitative pollination network using pollen metabarcoding.
Our work underscores that pollen metabarcoding is not quantitative but that quantitative networks can be constructed based on the number of interacting individuals. Due to the frequency of contamination and false positive reads, isolation and PCR negative controls should be used in every reaction. DNA metabarcoding has advantages in efficiency and resolution over microscopic identification of pollen, and we expect that it will have broad utility for future studies of plant-pollinator interactions.
为了研究变化环境中的传粉网络，我们需要准确、高通量的方法。先前的研究表明，相对于实地观察，通过研究从蜜蜂身上采集的花粉负载可以构建分辨率更高的网络。与传统方法（如光学显微镜）相比，DNA代谢条形码技术可能能够更快、更精细地对花粉进行分类解析，但尚未应用于传粉网络。
我们从佛罗里达州不同森林管理地点采集的38种蜜蜂中采集花粉。我们从花粉混合物中分离出DNA，并在Illumina MiSeq平台上对所有混合物的 和ITS2基因区域进行单次测序。我们使用综合的 和ITS2数据库从序列数据中鉴定物种。
我们成功地利用花粉代谢条形码技术构建了一个概念验证性的定量传粉网络。
我们的工作强调，花粉代谢条形码技术本身并非定量技术，但可以基于相互作用个体的数量构建定量网络。由于污染和假阳性读数的频率，每个反应都应使用分离和PCR阴性对照。DNA代谢条形码技术在效率和分辨率方面优于花粉的显微镜鉴定，我们预计它将在未来植物-传粉者相互作用的研究中具有广泛的用途。
