Mature seeds of 109 species of Pedicularis representing 13 groups according to Tsoong’s infra-generic classification were investigated using light microscopy (LM) and scanning electron microscopy (SEM). The largest seeds occurred in P. superba (4.82 ± 0.32 mm × 2.18 ± 0.18 mm) and the smallest in P. crenata (0.95 ± 0.16 mm × 0.72 ± 0.08 mm). The individual seed weight ranged from 0.20 to 9.52 mg. There were two types of primary ornamentation, i.e. reticulate and undulate. Reticulate seeds were found in all examined species except for P. pantlingii and P. confluens. The reticulate seeds were divided into three subtypes, i.e. cristate-reticulate, regular-reticulate and membranous-reticulate. Among them, the cristate-reticulate and the undulate primary ornamentation are firstly reported. The inner tangential wall ornamentation was categorized into granulate, ruminate or striate. Seed characters seem to support the transfer of Pedicularis to Orobanchaceae from traditional Scrophulariaceae. Seed length, individual seed weight, primary ornamentation and inner tangential wall ornamentation were considered as potentially useful characters for infra-generic classification. Non-metric multidimensional scaling (NMDS) analyses indicated a clustering of some species in group 1 (Sceptrum) and all species in group 6 (Cyathophora), respectively. However, seed data were not highly congruent with Tsoong’s classification for delimitation of existing groups. Some characters, such as seed shape, primary ornamentation, inner tangential wall ornamentation and epidermal cell shape could be applied to identification of some species in this genus.

Twelve microsatellite markers were developed to determine the genetic diversity and genetic structure of Ypsilandra thibetica, represented by a total of 90 individuals from six natural populations. All twelve microsatellite loci were polymorphic, and the results indicated that a high genetic diversity was present within populations (mean RS = 4.996; mean HE = 0.615), with high levels of genetic structure (mean FST = 0.165; mean FIS = 0.692) among populations. This pattern is likely attributable to consanguineous mating, and this hypothesis is supported by a low relatedness coefficient. Our study suggested that environment factors might restrict gene flow among populations. In addition, physical distances between populations were not related to genetic distances, implying that ancestral populations might have been distributed over a wider area. These results suggest that Y. thibetica should be a high priority for conservation managers.Highlights► Population number declining and habitat shrinking in Ypsilandra thibetica were steadily continuous during the last two decades. ► Twelve SSR marks were developed to determine the genetic diversity and genetic structure. ► The results suggested that the environment factors might restrict gene flow among populations. ► Ypsilandra thibetica should be a high priority for conservation managers.

Charles Darwin was fascinated by the orchid–pollinator interactions, but he did not realize that many orchid species are pollinated by deceit. Cypripedium, a model lineage of nonrewarding orchid flowers, is pollinated primarily by bees. Here we present both an example of floral mimesis of fungus-infected foliage in orchids and an example of flat-footed flies (Agathomyia sp.; Platypezidae) as pollen vectors for angiosperms. Cypripedium fargesii is a nectarless, terrestrial, endangered orchid from southwestern China that requires cross-pollination to produce the maximum number of viable embryos. All insects caught entering or leaving the labellum sac were Agathomyia sp. carrying conidia of Cladosporium sp. on their mouthparts and legs, suggesting mycophagy. Blackish hairy spots on the upper surface of foliage may imitate black mold spots, serving as short-term visual lures. Some odor molecules also associated with Cladosporium cultures were isolated in the floral scent. Mimesis of fungus-infected foliage probably represents an overlooked but important option in angiosperm diversification, because there are three to five more Cypripedium spp. in southwestern China with the same mode of floral presentation and black-spotted hairy leaves.

The flower heliotropism of Anemone rivularis (Ranunculaceae) was investigated on the Yulong Snow Mountain, near Lijiang in the northwest of the Yunnan province of China. We discovered that tepals in this species drive the peduncles to track the sun, and that this flower heliotropism was likely an adaptation for parental environmental effects on reproductive fitness. In brief, A. rivularis flowers retained sun-tracking behavior following removal of pistils and stamens, but lost heliotropic movement, if tepals were removed. Light is the major factor to affect floral heliotropism, the tepal-received light signal was in the blue frequency. Meanwhile, the peduncles were found to bend significantly on the top portion of both control flowers and those lacking pistils and stamens, but instead of keeping a vertical peduncle orientation in flowers with tepals removed. Furthermore, the floral temperature was steadier, and seed sizes and numbers were greater for control flowers than for flowers with tepals removed. Therefore, we conclude that the tepals trigger the flower heliotropism in A. rivularis and play an important role on not only increasing but also keeping optimal thermal condition of flower interior. We further conclude that flower heliotropism enhances the pollen viability and seed production, resulting in higher reproductive success for this alpine species.

Incarvillea mairei (H. Léveillé) Grierson (Bignoniaceae) is an endemic species to the Himalaya-Hengduan Mountains region. Here, we developed 13 microsatellite markers from I. mairei using a modified biotin-streptavidin capture method. Number of alleles per locus (NA) ranged from 3 to 7 with an average of 4.615. The observed (HO) and expected (HE) heterozygosities were from 0.050 to 0.800 and from 0.249 to 0.815, respectively. Additionally, among the 13 identified microsatellite markers, 12 of them were successfully amplified in other three congeneric species, and most of them showed polymorphic. Obtained evidences suggest that these markers provide a useful tool for further study of the population genetic structure and the breeding system in this species or/and infra-generic species.

Floral diversification in the genus Pedicularis (Orobanchaceae) is remarkable among flowering plants. In this genus, floral morphology and pollinator behavior are closely co-adaptive. In the current paper, pollen grains of 23 representative species of Pedicularis mainly from North America, with two species from Japan and two species from China, whose pollination ecology was previously studied, were examined using light microscopy and scanning electron microscopy. Two pollen aperture types and three kinds of exine ornamentation were recognized among these species. In addition, pollen data from previous and the current studies of Pedicularis were integrated and analyzed, together with some pollination characters. There was a significant association between pollen aperture types and corolla types, as well as between pollination syndromes and corolla types. However, there was no association of exine ornamentations with corolla types. The relationships and evolution of this genus were discussed with regards to pollen morphology, corolla types and pollination syndromes.

Pollen morphology of 36 species representing 14 genera within the tribe Rhinantheae in the family Orobanchaceae was studied and illustrated with light microscopy (LM) and scanning electron microscopy (SEM). Five major pollen types were recognized on the basis of exine ornamentation. Within these major types, minor types (subtypes) were distinguished based on exine surface pattern, size, shape, amb form, colpi and colpus membrane. These types and subtypes are as follows: type I. retipilate: subtype Ia. regular retipilate: (1) pollen size < 27 µm, (2) pollen size > 27 µm, subtype Ib. irregular retipilate; type II. verrucate: subtype IIa. macro-verrucate, subtype IIb. verrucate, subtype IIc. sparse verrucate; type III. retirugulate; type IV. granulate; type V. micro-reticulate. A key to pollen morphology of genera studied within the Rhinantheae was made based on pollen morphology from our study and earlier work. Combining with other sources of information on the Rhinantheae, the systematic relationships of this tribe are discussed. Rhinantheae pollen displays considerable variation between genera and species, with taxonomically significant characters at genus and species level. Palynological characteristics provide evidence for interpreting the conflicting views concerning the “Pterygiella Complex”. The evolutionary trend in exine sculpture of Rhinantheae could be proposed, namely that retipilate sculpturing which is the most widespread type is more primitive than the other types (such as foveolate, granulate, regulate, reticulate, retirugulate and verrucate). The pollen data in present study and the view of Hong (1986), as well as the molecular data from Bennett and Mathews (2006) indicated that Asia and related regions were likely to the origin centre of the tribe Rhinantheae.

Primula chungensis is a species with considerable floral and mating-system variation, including distylous (outcrossing), homostylous (selfing) and mixed populations that contain both outcrossing and selfing forms. We isolated 24 microsatellite markers from P. chungensis using Illumina MiSeq sequencing. Polymorphism and genetic diversity were then measured based on a sample of 24 individuals from a natural population in southern Tibet. All loci were polymorphic with the number of alleles per locus ranging from 2 to 4. The observed and expected heterozygosity ranged from 0 to 1 and 0.219 to 0.708, respectively. The microsatellite markers we have identified will serve as valuable tools for the investigation of the population genetic structure and phylogeography of P. chungensis and will inform models of the evolutionary history of mating systems in the species.

The pollen morphology of 86 samples from 84 species of Gaultheria and the closely related genera Chamaedaphne, Craibiodendron, Diplycosia, Eubotrys, Gaylussacia, Leucothoe, Lyonia, Oxydendrum, Pieris, Satyria, and Vaccinium (subfamily Vaccinioideae) was investigated with light and scanning electron microscopy. The tetrahedral tetrads are tri-zonocolporate and range from 22.9 to 51.5 μm in diameter. Exine ornamentation (apocolpia and mesocolpia) of pollen grains is highly variable. Six exine types of apocolpia and five exine types of mesocolpia are distinguished. Viscin threads on the pollen grains of members of Gaultheria are first reported in the study. Colpus length is correlated with both pollen size and colpus maximum width. There is a weak correlation between corolla length and pollen size when the length of the corolla is less than 6 mm. Infrageneric relationships of taxa in Gaultheria are discussed on the basis of pollen data. Pollen morphology is ubiquitously variable at both the species and genus levels. Pollen data support the sister relationships between Satyria and Vaccinium and between Chamaedaphne and Eubotrys of a previously reported molecular phylogenetic analysis. The evolution of pollen characters is traced by character optimization and results discussed. An evolutionary trend of mesocolpium ornamentation within Vaccinioideae from granulate through granulate–rugulate to rugulate is apparent.

Illiciaceae and Schisandraceae, together with other members of Austrobaileyales have been identified as one of the earliest diverging lineages of angiosperms, within the ANITA grade. The specialized Illiciaceae and Schisandraceae comprise a clade defined by apomorphic characters including pollen grains with three or six colpate apertures. In both these families, pollen apertures and exine sculpture were found to be very informative when considered in the context of recent understanding of evolutionary patterns. In the current study, pollen grains of 21 taxa from Illiciaceae and Schisandraceae were investigated. These data, together with palynological data for taxa previously studied, were mapped into recent molecular phylogenetic trees to re-evaluate the existing classification and phylogenetic relationships in the two families. Palynological data were found to be relatively congruent with recent molecular phylogenies, while traditional delimitations of infra-generic taxa were somewhat conflicting and did not reflect phylogeny and evolution. The evolution of pollen morphology in the two families, together with other members of Austrobaileyales, is discussed in comparison with the molecular phylogenies.