A novel approach to study the morphology and chemistry of pollen in a phylogenetic context, applied to the halophytic taxon Nitraria L.(Nitrariaceae)

Abstract

Nitraria is a halophytic taxon (i.e., adapted to saline environments) that belongs to the plant family Nitrariaceae and is distributed from the Mediterranean, across Asia into the south-eastern tip of Australia. This taxon is thought to have originated in Asia during the Paleogene (66–23 Ma), alongside the proto-Paratethys epicontinental sea. The evolutionary history of Nitraria might hold important clues on the links between climatic and biotic evolution but limited taxonomic documentation of this taxon has thus far hindered this line of research. Here we investigate if the pollen morphology and the chemical composition of the pollen wall are informative of the evolutionary history of Nitraria and could explain if origination along the proto-Paratethys and dispersal to the Tibetan Plateau was simultaneous or a secondary process. To answer these questions, we applied a novel approach consisting of a combination of Fourier Transform Infrared spectroscopy (FTIR), to determine the chemical composition of the pollen wall, and pollen morphological analyses using Light Microscopy (LM) and Scanning Electron Microscopy (SEM). We analysed our data using ordinations (principal components analysis and non-metric multidimensional scaling), and directly mapped it on the Nitrariaceae phylogeny to produce a phylomorphospace and a phylochemospace. Our LM, SEM and FTIR analyses show clear morphological and chemical differences between the sister groups Peganum and Nitraria. Differences in the morphological and chemical characteristics of highland species (Nitraria schoberi, N. sphaerocarpa, N. sibirica and N. tangutorum) and lowland species (Nitraria billardierei and N. retusa) are very subtle, with phylogenetic history appearing to be a more important control on Nitraria pollen than local environmental conditions. Our approach shows a compelling consistency between the chemical and morphological characteristics of the eight studied Nitrariaceae species, and these traits are in agreement with the phylogenetic tree. Taken together, this demonstrates how novel methods for studying fossil pollen can facilitate the evolutionary investigation of living and extinct taxa, and the environments they represent.

Chemical variations in Quercus pollen as a tool for taxonomic identification: implications for long-term ecological and biogeographical research

“…Recent research has shown that spectroscopic methods such as FTIR are effective at differentiating pollen species between distantly related families and/or genera using their chemical composition (Gottardini et al, 2007;Dell'Anna et al, 2009;Zimmermann, 2010;Zimmermann, Bağcıoğlu, Sandt, & Kohler, 2015;Zimmermann, Tafintseva, Bağcıoğlu, Berdahl, & Kohler, 2016;Julier et al, 2016;Zimmermann et al, 2017;Woutersen et al, 2018;Jardine et al, 2019;Zimmermann & Kohler, 2014). Our results build on these previous studies to reveal the potential for chemical variations in pollen to distinguish sub-generic variation between species belonging to three different Quercus sections in pollen samples from 297 individuals from Portugal.…”

“…It has been shown that multivariate-regression methods (e.g. partial least squares; PLS) perform better with preprocessed spectra, as shown by classification of FTIR spectral pollen data in other studies (Zimmermann & Kohler, 2013;Woutersen et al, 2018). For further analyses, the spectra were constrained between 700 and 1800 cm -1 .…”

“…those species belonging to different genera). In another study, Woutersen et al (2018) report ~95% recall on largely congeneric species in the Nitrariaceae family using single grain FTIR, but also note that lack of environmental variability (pollen from one individual per species) could have led to an overestimation of classification success. In contrast, Zimmermann et al (2017) achieve ~100% accuracy on species identification and 75% accuracy on identification of origin using hierarchical PLSR on pollen from three species of Poaceae (Festuca ovina, Anthoxanthum odoratum, Poa alpina) of different genera and origins (Sweden, Norway, Finland) grown under controlled conditions (45 individual per species).…”

“…For example, Zimmermann (2010) classified pollen from 11 species at comparable accuracy to conventional light-microscopy methods, whilst later work by Zimmermann & Kohler (2014) and Depciuch, Kasprzyk, Drzymała, & Parlinska-Wojtan (2018) expanded on the differences in pollen composition by analysing over 300 different species and identified lipids (specifically triglycerides) as being major sources of variation in the pollen spectra of congeneric species across several diverse genera (Quercus, Iris, Pinus, Betula, etc). Differences between the sporopolleninspectral regions of the pollen of 15 morphologically-similar Pinales species have also recently been observed, whilst two independent studies have demonstrated that the pollen spectra of pollen of both Poaceae and Nitrariaceae can be used to generate sub-family level differentiation using FTIR (Julier et al, 2016;Woutersen et al, 2018;Zimmermann et al, 2017;P. Jardine et al, 2019).…”

“…One potential solution lies in the chemical analysis of pollen and spores. Evidence is mounting that analysis of pollen grains using Fourier Transform Infrared Spectroscopy (FTIR) may provide a new tool for differentiating between pollen types extracted from sediment sequences (Julier et al, 2016;Woutersen et al, 2018;Jardine, Gosling, Lomax, Julier, & Fraser, 2019), since pollen-grain chemistry may itself show biogeographic patterns related to phylogeny and environmental conditions (Bağcıoğlu, Kohler, Seifert, Kneipp, & Zimmermann, 2017;Zimmermann et al, 2017). FTIR is a nondestructive method which is used to infer the chemical composition of a sample based on the fact that different molecular-functional groups have different wavelength-specific absorbances of infrared radiation (Gottardini, Rossi, Cristofolini, & Benedetti, 2007;Ivleva, Niessner, & Panne, 2005;Pappas, Tarantilis, Harizanis, & Polissiou, 2003;Parodi, Dickerson, & Cloud, 2013;Schulte, Lingott, Panne, & Kneipp, 2008;Zimmermann, 2010;Zimmermann & Kohler, 2014;.…”