Systematic palynology in Korean Piperales with special focus on its exine surface ornamentation and orbicule morphology

The pollen and orbicule morphology of the Korean Piperales (Aristolochia, Asarum, Houttuynia, Piper, and Saururus) were investigated via scanning electron microscopy. Piperales pollen is a monad, its size ranging from very small to large (P = 7.78–51.4 μm, E = 6.68–43.1 μm), and having a mainly circular to sub-circular shape. The aperture type is constant in the genus [inaperturate (Aristolochia), tri to pentaporate (Asarum), and monosulcate (Houttuynia, Piper, and Saururus)]. There are four distinct types of exine ornamentation: Fossulate with perforate, microreticulate with gemmae, microperforate with granula, and microechinate. The orbicules (minute sporopollenin granules) were observed in all studied taxa and thus, may be a possible symplesiomorphic characteristic of Piperales. Further, the observed orbicule surface ornamentation was similar to pollen exine patterns, for example muri, gemmae, or granula. This resemblance between orbicule and pollen exine ornamentation may imply a similar biosynthesis pattern of sporopollenin of pollen exine and orbicules. The phenogram resulting from a cluster analysis using palynological characters was generally consistent with the known molecular phylogeny of Piperales. This initial study will help understand the palynological diversity and provide detailed information of pollen and orbicule characteristics in Piperales.

Exine ornamentation. Four distinct types of exine ornamentation were defined based on the existence of gemmae or granula; perforate or echinate patterns.
All studied taxa within Aristolochia had semitectate exines. The surface ornamentation pattern was fossulate with well-developed muri arranged in an irregular shape. The murus width ranged between 0.29 and 0.80 μm, and the range of perforation diameter and their area was 0.16-0.47 μm and 0.01-0.13 μm 2 , respectively (Table 1). There were no gemmae or granula on the surface; observed in A. contorta (Fig. 2b) and A. manshuriensis (Fig. 2d).

Discussion
Using the palynological characteristics identified through SEM analysis, we determined useful key characteristics for identification and a strong correlation between pollen and orbicule surface ornamentation patterns. This was www.nature.com/scientificreports/ the first study whereby a comprehensive approach using pollen and orbicule morphology covering all genera of Korean Piperales was conducted.
Most previous pollen measurements on the Piperales have been conducted based on acetolysed 21,22,24,29 , air-dried 26 , or fixed/stained pollen grains 28 . In the case of Aristolochia manshuriensis, varying pollen size was reported following different preparation techniques and microscopes; stained pollen using stereo microscope [29-(48. present study). Further, the size and shape of pollen grains could be affected by the different preparation methods 2,3,14,39 . The fixation, staining, and acetolysis methods for pollen measuring have side effects such as distortion of shape and change of size. The striking differences in pollen size and distorted pollen shape were due to harmomegathy, a characteristic infolding of pollen grains to accommodate the decrease in cellular volume due to water loss 40,41 . Thus, we recommend the critical point drying preparation to avoid deformation and distortion of pollen grains.
The pollen aperture types of the Korean Piperales were confirmed by comparing previous palynological studies. It is uncertain whether the aperture of Aristolochia contorta is porate 27 or inaperturate 28 . Our results strongly support the observation of Nakonechnaya and Kalachev 28 because we did not find apertures on the surface of the studied Aristolochia pollen grains. Pollen is usually described as inaperturate or 3-zonocolpate within the Asarum literature [42][43][44] . However, our description of the Asarum corresponds to the combination of Asarum europaeum type 45 and some Chinese Asarum 22 . Moreover, in the case of the Houttuynia, we only observed monosulcate grains, similar to a study by Smith and Stockey 26 , although both monosulcate and trichotomosulcate pollen grains were observed 23 .
The exine surface morphology of pollen grains in the studied taxa was similar to that in other Piperales species [21][22][23][24][26][27][28][29] . The observed variation of exine ornamentation also proved to be a helpful diagnostic characteristic at the inter-/intra generic level. The Aristolochia pollen is fossulate without any gemmae/granula, whereas Saururaceae species do not possess a murus pattern. The Piper pollen is unique, with microechinate elements without any murus patterns and perforation among studied taxa. The Asarum taxa are distinct in two groups based on the gemmae surface patterns (Type II-1 smooth-surfaced vs Type II-2 striate-surfaced). Thus, www.nature.com/scientificreports/ the presence or absence of gemmae/granula, muri, lumen/perforation on their surface can aid in recognizing certain groups of Korean Piperales.
In the case of the Asarum mandshuricum complex, its taxonomic identity remains controversial. A. mandshuricum was recognized as two infra-species, A. mandshuricum var. mandshuricum and A. mandshuricum var. seoulense based on the presence or absence of trichomes on the petiole 46,47 . However, several studies treated the infra-species, seoulense, as synonymous with A. mandshuricum 35,37 . A recent taxonomic study revealed that the A. mandshuricum complex mostly shares common leaf micromorphological characters 38 , which conflicts with our palynological results. In the present study, Asarum mandshuricum complex was distinct in two types (Type II-1 var. mandshuricum vs Type II-2 var. seoulense). Further in-depth integrative taxonomic studies, including molecular phylogeny, micromorphology, and cytology using abundant samples of A. mandshuricum complex, are required to delimitate the taxa to facilitate their identification and classification.  www.nature.com/scientificreports/ Although orbicule characteristics have been studied in various taxonomic groups, the presence of orbicules and their morphological diversity in Piperales has received little attention 15,16 . Orbicules of selected taxa from Piperales in this study, except for Houttuynia cordata, were observed for the first time since the study by Verstraete et al. 16 . The orbicules were identified in all studied taxa, similar to previous research on Piperales [30][31][32] . The presence of orbicules is regarded as a plesiomorphic feature, common in the early-diverging clades with a trend towards orbicule absence in late-branching 9,16 . Moreover, according to a recent review, the orbicules are also observed in the clade, related to the Piperales 48 . Thus, the presence of orbicules could be considered as a possible symplesiomorphic character of Piperales. A further study focusing on the distribution of orbicules in Magnoliids is required to increase the resolution of their evolutionary trends.
The observed orbicule surface ornamentation was similar to the pollen exine patterns with elements such as muri, gemmae, or granula (Table 2). Orbicule size, in addition to the surface pattern, was significantly correlated with quantitative exine ornamentation elements such as the diameter of gemmae/granula and lumen/perforation, muri branching length, and muri width (Fig. 6). This resemblance between orbicules and pollen surface ornamentations was reported in several taxa 9,[12][13][14]16 . Thus, our results offer additional evidence which implies that a similarly patterned biosynthesis of sporopollenin is possible on a pro-orbicule, as well as on a microspore 16 .
Our dendrogram based on cladistic and phenetic analyses, as well as palynological characters is consistent with the previously reported molecular phylogeny, including the monophyletic lineage of both Piperaceae and Saururaceae 49 . Further studies that include various morphological/micromorphological characteristics are required to understand the evolution of the order Piperales better.

Conclusion
Inaperturate, monosulcate, tri to pentaporate and very small to large-sized pollen grains, and constantly existing orbicules are consistent characteristics for Korean Piperales. The four types of exine ornamentation and stable orbicule traits may be of great systematic importance for Piperales. Our results strongly support the developing pollen exine hypothesis, whereby orbicules and pollen surface ornamentations are the result of a similar biosynthesis. Further, exine ornamentation patterns may be useful for defining systematic groups at the intra-or interspecific level in Asarum as they had a great diversity of palynological features. Palynological characteristics, including orbicules, appear to help reconstruct systematic relationships. The present study helps to understand the diversity in Piperales orbicule morphology, and provides information on pollen and orbicules characteristics. Further studies involving more Piperales taxa, including extensive sampling, will contribute to understanding the evolutionary tendencies of pollen and orbicule characteristics.

Materials and methods
Taxon sampling and identification. Mature flowers of 18 accessions (12 species, five genera) were collected from natural habitats to observe the pollen and orbicule morphological features of Korean Piperales (Fig. 1).
All plants described in this paper were collected and used in accordance with the relevant guidelines and regulations. The investigated taxa were neither endangered nor protected. Moreover, all samples were collected with a permit issued from the Korea National Park Service, and voucher specimens were deposited in the Korean Herbarium of Standard Herbal Resources (KIOM) at the Korea Institute of Oriental Medicine, Naju, Korea (see Supplementary Table S1).
Most of the samples were collected from living plants and preserved in FAA solution (40% formalin: 40% glacial acetic acid: 70% ethyl alcohol). To confirm the consistency of palynological characteristics, we compared a minimum of two accessions for each taxon when available.
Microscopic observation. Prior to observing pollen morphology, all dried floral samples were first examined using a stereomicroscope (SM, Olympus SZX16, Olympus, Tokyo, Japan) to select fully mature anthers. The fully matured anthers were prepared using the critical point drying method (CPD; Moon et al. 15,39 ; Song et al. 2,3,14 ) for SEM. Dried anthers were rehydrated overnight in a wetting agent Agepon® (Agepon: distilled water, 1:200) (Agfa Gevaert, Leverkusen, Germany). The rehydrated materials were then dehydrated through an ethanol series (50%, 70%, 90%, 95%, and 100% ethanol) at room temperature for 1 h per ethanol concentration. The dehydrated materials were immersed in liquid carbon dioxide (CO 2 ) for CPD (SPI-13200JE-AB, SPI Supplies, West Chester, USA). The dried materials were then mounted on aluminum stubs with a double-sided adhesive conductive carbon disk (05073-BA, SPI Supplies, West Chester, USA), and the stubs were coated with platinum using an ion-sputtering device (208HR; Cressington Scientific Instruments Ltd., Watford, United Kingdom) for 90 s. Thereafter, the samples were examined using a field emission scanning electron microscope (FE-SEM, S-4700, Hitachi, Tokyo, Japan) at an accelerating voltage of 5-10 kV and an 8-10 mm working distance. Data analysis. The obtained quantitative characteristics were determined using the Digimizer software (Digimizer version 5.4.3, MedCalc Software, Mariakerke, Belgium). Pearson's correlation coefficients were used to estimate relationships among the following quantitative variables: Orbicule diameter, gemmae/granula diameter, lumen/perforation diameter, muri branching length, and muri width. This statistical analysis was conducted using R, version 3.6.3 50 .
For phenetic analysis, 13 pollen and orbicule characteristics (seven qualitative and six quantitative) and their codes were used (Table 3). Cluster analyses were performed calculating UPGMA using NTSYS-PC 2.1 software 51 to visualize the relationship between the different species based on seed characters.