The significance of recent diversification in the Northern Hemisphere in shaping the modern global flora revealed from the herbaceous tribe of Rubieae (Rubiaceae).

The global herbaceous flora is probably shaped by both ancient and/or recent diversification, companied with the impacts from geographic differences between the Northern and Southern Hemispheres. Therefore, its biogeographic pattern with respect to temporal and spatial divergence is far from full understanding. Tribe Rubieae, the largest herbaceous tribe in the woody-dominant Rubiaceae, provides an excellent opportunity for studying the macroevolution of worldwide colonization. Here, we aim to reconstruct the evolutionary history of Rubieae with regard to climate fluctuation and geological history in the Cenozoic. A total of 204 samples of Rubieae representing all the distribution areas of the tribe were used to infer its phylogenetic and biogeographic histories based on two nrDNA and six cpDNA regions. The ancestral area of Rubieae was reconstructed using a time-calibrated phylogeny in RASP and diversification rates were inferred using Bayesian analysis of macroevolutionary mixtures (BAMM). Our results show Rubieae probably originated in European region during the middle Oligocene, with the two subtribes separating at 26.8 million years ago (Ma). All the genera in Rubieae formed separate clades between 24.79 and 6.23 Ma. The ancestral area of the subtribe Rubiinae was the Madrean-Tethyan plant belt and the North Atlantic land bridge (NALB) provided passage between North America and Europe for Rubiinae. The subtribe Galiinae clade originated in Europe/central Asia during the late Oligocene. Two diversification shifts were detected within Rubieae in the late Neogene. Most extant Rubieae species diverged recently during the Neogene within clades that generally were established during the late Paleogene. The tribe shows complex migration/dispersal patterns within the North Hemisphere combined with multiple recent dispersals into Southern Hemisphere. Our results highlighted the important role of recent biogeographic diversification in the Northern Hemisphere in shaping the modern global herbaceous flora during the latest and rapid worldwide expansion in the Neogene.

The complete plastid genome of Kelloggia chinensis Franch. (Rubiaceae), an endemic species from East Asia.

Kelloggia chinensis Franch. is an herbal plant species endemic to East Asia. Its complete plastid genome sequence is 155, 665 bp in length, with a large single-copy (LSC) region of 85, 788 bp, a small single-copy (SSC) region of 16, 977 bp, and a pair of inverted repeat regions (IRs) of 26, 450 bp. The whole plastid genome contains 132 genes, including 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The overall GC content of K. chinensis plastid genome is 37.1%. K. chinesis is evolutionarily close to tribe Rubieae according to the Maximum likelihood phylogenetic analysis based on 12 taxa.

Seed Dormancy and Soil Seed Bank of the Two Alpine Primula Species in the Hengduan Mountains of Southwest China.

The timing of germination has long been recognized as a key seedling survival strategy for plants in highly variable alpine environments. Seed dormancy and germination mechanisms are important factors that determining the timing of germination. To gain an understanding of how these mechanisms help to synchronize the germination event to the beginning of the growing season in two of the most popular Primula species (P. secundiflora and P. sikkimensis) in the Hengduan Mountains, Southwest China, we explored their seed dormancy and germination characteristics in the laboratory and their soil seed bank type in the field. Germination was first tested using fresh seeds at two alternating temperatures (15/5 and 25/15°C) and five constant temperatures (5, 10, 15, 20, and 25°C) in light and dark, and again after dry after-ripening at room temperature for 6 months. Germination tests were also conducted at a range of temperatures (5-30, 25/15, and 15/5°C) in light and dark for seeds dry cold stored at 4°C for 4 years, after which they were incubated under the above-mentioned incubation conditions after different periods (4 and 8 weeks) of cold stratification. Base temperatures (T b) and thermal times for 50% germination (θ 50) were calculated. Seeds were buried at the collection site to test persistence in the soil for 5 years. Dry storage improved germination significantly, as compared with fresh seeds, suggesting after-ripening released physiological dormancy (PD); however, it was not sufficient to break dormancy. Cold stratification released PD completely after dry storage, increasing final germination, and widening the temperature range from medium to both high and low; moreover, the T b and θ 50 for germination decreased. Fresh seeds had a light requirement for germination, facilitating formation of a persistent soil seed bank. Although the requirement reduced during treatments for dormancy release or at lower alternating temperatures (15/5°C), a high proportion of viable seeds did not germinate even after 5 years of burial, showing that the seeds of these two species could cycle back to dormancy if the conditions were unfavorable during spring. In this study, fresh seeds of the two Primula species exhibited type 3 non-deep physiological dormancy and required light for germination. After dormancy release, they had a low thermal requirement for germination control, as well as rapid seed germination in spring and at/near the soil surface from the soil seed bank. Such dormancy and germination mechanisms reflect a germination strategy of these two Primula species, adapted to the same alpine environments.

Cold stratification, temperature, light, GA3, and KNO3 effects on seed germination of Primula beesiana from Yunnan, China.

Primula beesiana Forr. is an attractive wildflower endemically distributed in the wet habitats of subalpine/alpine regions of southwestern China. This study is an attempt to understand how this plant adapts to wet habitats and high altitudes. Specifically, we examined the effects of cold stratification, light, GA3, KNO3, and temperature on P. beesiana seed germination. KNO3 and GA3 increased germination percentage and germination rate compared to control treatments at 15/5 and 25/15 °C. Untreated seeds germinated well (> 80%) at higher temperatures (20, 25 and 28 °C), whereas at lower (5, 10 and 15 °C) and extremely high temperatures (30 and 32 °C) germination decreased significantly. However, after cold stratification (4-16 weeks), the germination percentage of P. beesiana seeds at low temperatures (5-15 °C) and the germination rate at high temperatures (30 °C) increased significantly, suggesting that P. beesiana has type 3 non-deep physiological dormancy. The base temperature and thermal time for germination decreased in seeds that were cold stratified for 16 weeks. Cold-stratified seeds incubated at fluctuating temperatures (especially at 15/5 °C) had significantly high germination percentages and germination rates in light, but not in dark, compared to the corresponding constant temperature (10 °C). Seeds had a strict light requirement at all temperatures, even after experiencing cold stratification; however, the combinations of cold stratification and fluctuating temperature increased germination when seeds were transferred from dark to light. Such dormancy/germination responses to light and temperature are likely mechanisms that ensure germination occurs only in spring and at/near the soil surface, thus avoiding seedling death by freezing, inundation and/or germination deep in the soil.

Molecular phylogeny of Galium L. of the tribe Rubieae (Rubiaceae) - Emphasis on Chinese species and recognition of a new genus Pseudogalium.

Galium L. is the largest genus in the tribe Rubieae, with about 667 species distributed worldwide. Previous researches mainly focused on species from the Americas and Europe. In the present paper, we greatly increased the number of samples examined from eastern Asia (especially China), representing the most comprehensive sampling of Galium to date. A total of 194 species and variations (subspecies) of Galium were sampled to determine phylogenetic relationships, using two nuclear and five chloroplast markers. Our data are largely consistent with all previous phylogenetic results and confirmed that Galium is non-monophyletic, as are most of its sections. Most members of Galium, including the Chinese taxa, fall into three large clades mixed with other genera from the Galium s.l. group; the exception being the distinct Galium paradoxum Maxim., the first diverged lineage in the Galium s.l. group, which was treated as a new genus (Pseudogalium L.-E. Yang, Z.-L. Nie & H. Sun, gen. nov.). The Galium s.s is a well-supported clade comprised entirely of Galium species, usually with six or more leaves per whorl, mostly from the Old World. Samples from G. maximowiczii (Kom.) Pobed, G. sect. Depauperata and sect. Aparinoides, together with a few from Asperula sect. Glabella and Microphysa (Schrenk ex Fisch. & C.A. Mey.) Pobed., form the second clade. The third clade comprises taxa purely from Galium that usually have four leaves per whorl, from both the New and Old World. Our results also indicated that the monotypic genus Microphysa should be retained and clarified phylogenetic relationships of some specific confused taxa from China. Unlike prior inferences, the combination of opposite leaves associated with two stipules is proposed as the ancestral characteristic of the Galium s.l. group and even the tribe. In addition, the shapes of different corolla and inflorescence types are important for distinguishing some taxa within Rubieae.

Host specificity of two pollinating seed-consuming fly species is not related to soil moisture of host plant in the high Himalayas.

Studying the drivers of host specificity can contribute to our understanding of the origin and evolution of obligate pollination mutualisms. The preference-performance hypothesis predicts that host plant choice of female insects is related mainly to the performance of their offspring. Soil moisture is thought to be particularly important for the survival of larvae and pupae that inhabit soil. In the high Himalayas, Rheum nobile and R. alexandrae differ in their distribution in terms of soil moisture; that is, R. nobile typically occurs in scree with well-drained soils, R. alexandrae in wetlands. The two plant species are pollinated by their respective mutualistic seed-consuming flies, Bradysia sp1. and Bradysia sp2. We investigated whether soil moisture is important for regulating host specificity by comparing pupation and adult emergence of the two fly species using field and laboratory experiments. Laboratory experiments revealed soil moisture did have significant effects on larval and pupal performances in both fly species, but the two fly species had similar optimal soil moisture requirements for pupation and adult emergence. Moreover, a field reciprocal transfer experiment showed that there was no significant difference in adult emergence for both fly species between their native and non-native habitats. Nevertheless, Bradysia sp1., associated with R. nobile, was more tolerant to drought stress, while Bradysia sp2., associated with R. alexandrae, was more tolerant to flooding stress. These results indicate that soil moisture is unlikely to play a determining role in regulating host specificity of the two fly species. However, their pupation and adult emergence in response to extremely wet or dry soils are habitat-specific.

Overlapping Leaves Covering Flowers in the Alpine Species Eriophyton wallichii (Lamiaceae): Key Driving Factors and Their Potential Impact on Pollination.

Extrafloral structures are supposed to have evolved to protect flowers from harsh physical environments but might have effects on pollination. Overlapping leaves cover flowers in Eriophyton wallichii, an alpine perennial endemic to the Himalaya-Hengduan Mountains. In previous study, it has showed that these extrafloral leaves can protect interior flowers from temperature fluctuations caused by drastic solar radiation fluctuations, but these leaves may also protect interior flowers from rain wash and UVB damage, and we do not know which one is the main function. In this study, we investigated whether rain and UVB protection are the main functions of overlapping leaves covering flowers and their potential impact on pollination. We first measured the intensities of UVB radiation in open air, beneath leaves and corollas, and then examined pollen susceptibility to different intensities of UVB and rain in the laboratory to estimate whether corollas per se protect interior pollen from UVB and rain damage. We also carried out pollination treatments and observed pollinator visitation of flowers with and without leaves in the field to assess whether the overlapping leaves covering flowers impair pollinator attraction. Our results showed that (1) water and strong UVB significantly decreased pollen germinability, but corollas per se could protect pollen from UVB and rain damage; (2) no autonomous self-pollination and apomixis occurred, and pollinators were essential for the reproduction of E. wallichii; however, flower coverage by overlapping leaves did not limit pollination. We suggested that rain and UVB protection was not the main function of overlapping leaves covered flowers, given that this protection can be provided by corollas per se. Alternatively, this extrafloral structure in E. wallichii may have evolved in response to extreme high temperatures associated with the strong solar radiation fluctuations. This indicates that, even in alpine plants, extreme high temperature may affect the evolution of plant extrafloral structures.

Oviposition by mutualistic seed-consuming pollinators reduces fruit abortion in a recently discovered pollination mutualism.

A prerequisite for the evolutionary stability of pollinating seed-consuming mutualisms is that each partner benefits from the association. However, few studies of such mutualism have considered the benefit gained by the pollinators. Here, we determined how the pollinating seed-predators ensure the provisioning of their offspring in the recently discovered mutualism between Rheum nobile and Bradysia flies. The correlation between flower fate and fly oviposition was examined. Floral traits and patterns of variation in fruit abortion and fly oviposition were investigated to determine whether female flies exhibit preferences for particular flowers when laying eggs. Indole-3-acetic acid (IAA) was quantified to determine whether female flies manipulate host physiology. Flowers that flies oviposited on had a significantly lower probability of fruit abortion compared with intact flowers. Females did not exhibit oviposition preference for any of the floral traits examined. There was no significant correlation between fruit abortion and fly oviposition in terms of either flower position or timing of flowering. IAA concentrations in oviposited flowers were significantly higher than in intact flowers. Our results suggest that oviposition by the mutualistic seed-consuming pollinator Bradysia sp., greatly reduces the probability of fruit abortion of its host, R. nobile; this may be attributed to the manipulation of host physiology through regulating IAA levels.

A new pollinating seed-consuming mutualism between Rheum nobile and a fly fungus gnat, Bradysia sp., involving pollinator attraction by a specific floral compound.

Pollinating seed-consuming mutualisms are regarded as exemplary models for studying coevolution, but they are extremely rare. In these systems, olfactory cues have been thought to play an important role in facilitating encounters between partners. We present a new pollinating seed-consuming mutualism from the high Himalayas between the endemic herb, Rheum nobile, and a fly fungus gnat, Bradysia sp. Seed production resulting from pollination by Bradysia flies and seed consumption by their larvae were measured to determine the outcome of this interaction. Floral scent analyses and behavioural tests were conducted to investigate the role of olfactory cues in pollinator attraction. Rheum nobile is self-compatible, but it depends mainly on Bradysia sp. females for pollination. Seed production resulting from pollination by adult flies is substantially higher than subsequent seed consumption by their larvae. Behavioural tests showed that an unusual floral compound, 2-methyl butyric acid methyl ester, emitted by plants only during anthesis, was attractive to female flies. Our results indicate that the R. nobile-Bradysia sp. interaction represents a new pollinating seed-consuming mutualism, and that a single unusual compound is the specific signal in the floral scent of R. nobile that plays a key role in attracting its pollinator.

Grey leaves in an alpine plant: a cryptic colouration to avoid attack?

Cryptic colouration is a common predation-avoidance strategy in animals that is postulated to occur in plants, but few experimental studies have rigorously tested this hypothesis. We investigated the colouration of Corydalis benecincta, an alpine plant with remarkably dimorphic leaf colours (grey and green), based on a cost-benefit analysis. First we tested the premise that herbivores (Parnassius butterflies) cannot distinguish grey leaves from a scree background by spectrographic measurements and by estimating discriminability between leaves and scree using a butterfly colour vision model. Then we estimated the potential costs of inconspicuousness by comparing the photosynthetic performance and visual attractiveness to flower visitors of the two colour morphs. Finally, we examined the potential benefits of inconspicuousness by comparing damage, survivorship and female reproductive success. It is difficult for herbivores to distinguish grey-coloured morphs against the background. This grey colour originates in a combination of anthocyanins and chlorophylls. The two colour morphs had similar photosynthetic performance, visual attractiveness and female reproductive success. However, grey morphs had significantly lower herbivore damage and higher survivorship. Grey leaves benefit C. benecincta by reducing herbivory with low investment in anthocyanin synthesis, and little cost on photosynthesis and mating opportunity. This cryptic colouration may have evolved through selection pressure imposed by visually foraging herbivores.