Recent molecular and karyologic studies have significantly modified delimitation of Lilium. However, despite the importance of pollen evolution in the genus comprehensive studies with electron microscopy and evaluation of pollen evolution are lacking. Therefore, we studied pollen morphology in a sample of 65 individuals from 37 taxa covering all the sections distributed in the world, using scanning electron microscopy. Our collection of 49 individuals from 21 taxa covering all five sections in China was also included in the database. We found pollen tetrads in L. bakerianum. Based on present and previous studies, our results suggest that pollen from L. formosanum should be classified as a new type, Formosanum. Combined with morphological and molecular evidence, pollen sculpture patterns appear to reflect phylogenetic relationships and are useful for species or subsection delimitation. Based on a comprehensive survey and correlation with potential functional implications, we propose the following hypothesis: evolution of an exine sculpture shows pollen type trends from Martagon → Callose → Concolor → Formosanum. The evolutionary trend regarding pollen sculpture and size could be related to selective pressure to adapt to environmental conditions. Pollen size and shape showed a significantly positive correlation with annual precipitation, and smaller pollen grains appear to adapt better in habitats with extreme conditions. Evolution trends in exine sculpture do not appear to be definitively correlated with pollen size and shape.
Citation: Du Y-p, Wei C, Wang Z-x, Li S, He H-b, Jia G-x (2014) Lilium spp. pollen in China (Liliaceae): Taxonomic and Phylogenetic Implications and Pollen Evolution Related to Environmental Conditions. PLoS ONE 9(1): e87841. https://doi.org/10.1371/journal.pone.0087841
Editor: Paul Adam, University of New South Wales, Australia
Received: July 10, 2013; Accepted: January 1, 2014; Published: January 31, 2014
Copyright: © 2014 Du et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by Fundamental Research Funds for the Central Universities (Project No.BLYJ201207) (http://www.dost.moe.edu.cn/dostmoe/), the project from the National Natural Science Foundation of China (Project No. 31071819) (http://www.nsfc.gov.cn/Portal0/default152.htm), and Special Fund for Forestry Scientific Research in the Public Interest (Project No. 201204609) (http://www.forestry.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Approximately 110 to 115 Lilium species are distributed in the cold and temperate regions of the Northern Hemisphere , , particularly in East Asia, the Himalayas and Hengduan Mountains, North America and Europe. A total of 55 species occur in China . De Jong  and Patterson and Givnish  consider southwest China and the Himalayas to be the center of origin of this genus.
Classification of this genus has been historically complicated. Several classifications for Lilium have been proposed based on morphological characters. Detailed studies have been performed for East Asia, European and North American Lilium species –. Based on 13 morphological characters and two germination types, Comber divided this genus into the following seven sections: Martagon Rchb., Pseudolirium Endl. which is limited to North America, Liriotypus Asch. and Graeb. which is distributed across Europe and the Caucasus, Archelirion Baker, Sinomartagon Comber, Leucolirion Wilson, and Daurolirion Comber, representing the most widely accepted taxonomical divisions . Wang and Tang recognized sect. Lophophorum (Bur. et Franch.) Wang et Tang out of sect. Sinomartagon Comber and included campaniform-flowered species . Liang  and Haw  modified sect. Lophophorum to accommodate the Nomocharis-like Lilium species in sect. Sinomartagon Comber. Chinese species were divided into five sections: Martagon, Archelirion, Sinomartagon, Leucolirion and Lophophorum.
Recently, molecular phylogenetic analyses and chromosome techniques have improved the understanding of several groups within the genus and modified the phylogenetic position of Comber’s classification, such as placement of sect. Daurolirion Comber in sect. Sinomartagon, L. henryi in subsect. Leucolirion 6b, modification of sect. Lophophorum and relationship confirmation in sect. Liriotypus –, –. Preliminary research found that sect. Sinomartagon, which mainly occurred in China, was complicated and polyphyletic. As indicated by Patterson and Givnish , intercontinental dispersal details of the genus Lilium are not yet clear. The division of subsect. Sinomartagon 5c and sect. Lophophorum are controversial and will require further research. Nishikawa et al. ,  suggested that L. henryi be classified into subsect. Leucolirion 6a and that it showed similar morphological features with L. rosthornii, thereby demonstrating that the phylogenetic position of L. rosthornii needs further study.
There are few relevant studies regarding pollen morphology which defines the taxonomic and reflect the evolution of the genus. According to the description within Lilium by Baranova  based on the number, shape and arrangement of columellae that form the muri, there are three morphological types of pollen: (1) Martagon (muri formed by rectangular columellae); (2) Callose (muri formed by rounded columellae); and (3) Concolor (muri formed by separated rounded and polygonal columellae). Previous studies found that most Lilium species have single pollen grains. However, pollen tetrads were found in L. sempervivoideum H. Lév. and L. amoenum E. H. Wilson ex Sealy, and the size and sculptural elements confirmed the taxa as two subspecies in L. sempervivoideum . The pollen morphology of L. lophophorum (Bur. et Franch.) Franch., L. henrici Franch., L. souliei (Franch.) Sealy and L. nanum Klotz. et Garcke supported placement in Lilium, which differs from Nomocharis in apture and sculptural elements, and showed an evolutionary aperture trend from monocolpate to porate . Muratović et al.  showed that two related European species, L. bosniacum and L. carniolicum, share similar pollen morphology. In addition, pollen morphology of some Chinese species under scanning electron microscopy (SEM), including 9 species described by Li and Qin , 10 species and 3 cultivars by Zhang et al. , and 12 species and 6 cultivars by Wu et al. , could provide taxonomic implications within Lilium:pollen has not only the commonness of genus, but also the specificity of single species. Interspecific pollen size and morphological characteristics has some difference, which has a certain reference value for the classification of Lilium. For example, there are differences in pollen size among L. cernnum, L. lanciflium and L. pumilum. Also there are significant differences in pollen ornamentation and morphology between L. leucanthum and its variety L. leucanthum var. centifolium from Qinling Mountains. Results obtained by Wang et al.  and Liu  indicate that pollen size parameters of L. pumilum and L. concolor from different provenances showed different degrees of variation. Determining whether this is a universal phenomenon in other species or if