Floral adaptations of two lilies: implications for the evolution and pollination ecology of huge trumpet-shaped flowers.

PREMISE: Evolutionary transitions among floral morphologies, many of which provide evidence for adaptation to novel pollinators, are common. Some trumpet-shaped flowers are among the largest flowers in angiosperms, occurring in different lineages. Our goal was to investigate the role of pollinators in the evolution of these flowers using Lilium. METHODS: We investigated floral traits and pollinators of L. primulinum var. ochraceum and L. brownii var. viridulum and reviewed reports of visitors to huge trumpet-shaped flowers. Using a published phylogeny of Lilium, we reconstructed ancestral floral morphological states in Lilium to elucidate the origins of trumpet-shaped lilies. RESULTS: Both lilies are largely self-incompatible and show floral syndromes indicative of hawkmoth pollination. The short trumpet-shaped lily can be pollinated by short-tongued (<40 mm) but not long-tongued hawkmoths (>65 mm), while the huge trumpet-shaped lily can be pollinated by both. A literature review including 22 species of trumpet-shaped flowers suggests that their pollinator guilds commonly include both short- and long-tongued moths. A phylogenetic reconstruction indicates that trumpet-shaped lilies possibly have multiple origins from tepal-reflexed ancestors, at least six of which have evolved huge flowers (>50 mm). CONCLUSIONS: Adaptation to short-tongued hawkmoths may have initiated the evolution of trumpet-shaped lilies. Huge trumpet-shaped lilies may have evolved as a response to selection by long-tongued hawkmoths, without excluding the short-tongued ones. This evolutionary pathway leads to a functionally more generalized pollination system instead of an increasingly specialized one and is not necessarily associated with pollinator shifts.

Molecular phylogenetics and historical biogeography of Sorbus sensu stricto (Rosaceae).

Explaining how plants from eastern Asia migrated to other Northern Hemisphere regions is still challenging. The genus Sorbus sensu stricto (including c. 88 species) is considered as a good example to illuminate this scenario, due to the wide distribution in the temperate zone and high diversity in the Himalayas and Hengduan Mountains. Based on four nuclear markers (LEAFY-2, GBSSI-1, SBEI and WD) and one chloroplast marker (rps16-trnK), we reconstructed the phylogenetic relationship of Sorbus using 54 taxa (60% of the genus representing all subgenera, sections, or series and geographical areas in the previous classifications), and estimated divergence time and historical biogeography of the genus. Phylogenetic analyses supported that the subgenera Sorbus and Albo-carmesinae, as defined by traditional morphological characters (such as fruit color), are suitable. However, five clades defined by phylogenetic analysis of the genus are not consistent with traditionally defined sections or series. Molecular dating and biogeographic reconstruction showed that the age of the most recent common ancestor was estimated at 41mya (95% HPD: 49-35mya) in eastern Asia. Four dispersal events are assumed to explain the wide distribution of Sorbus in the temperate zone and diversification in the edges of Qinghai-Tibet Plateau (QTP). Species dispersed from eastern Asia to the Tianshan Mountains, North America and Europe during the Oligocene and Miocene period. We found that polyploidization occurred multiple times in the subgenus Albo-carmesinae, in the Tianshan Mountains, Himalayas, and H-D Mountains. Finally, we suggest that apomixis, polyploidization, and hybridization may have combined with the multistage uplifting of Himalayas and H-D Mountains (and the subsequent increases in geologic, ecological and climatic heterogeneity) to drive the striking species diversity of Sorbus in this region.

Revisiting the evolutionary events in Allium subgenus Cyathophora (Amaryllidaceae): Insights into the effect of the Hengduan Mountains Region (HMR) uplift and Quaternary climatic fluctuations to the environmental changes in the Qinghai-Tibet Plateau.

The respective roles that the Hengduan Mountains Region (HMR) uplift around 4-3 Ma and Quaternary climatic oscillations played in causing the environmental changes in the Qinghai-Tibet Plateau (QTP) remain unknown. Here, we reconstruct the evolutionary history of two varieties of Allium cyathophorum and A. spicatum of subgenus Cyathophora, restricted to the HMR and the western QTP, respectively. Forty-five populations were surveyed for chloroplast and nuclear sequence variation to evaluate phylogenetic relationships, dates of divergence and ancestral area/inflorescence reconstructions. In addition, analyses were conducted on discernable micromorphologies, cytotypes and seed size variation. Our results indicated that two varieties of A. cyathophorum are separate species, i.e. A. farreri and A. cyathophorum, and the initial split of Cyathophora was triggered by the HMR uplift around 4-3 Ma. Subsequently, A. spicatum originated through the strengthened aridification in the western QTP induced vicariance of the ancestral populations in the HMR during the early Pleistocene. A self-sustaining allotetraploid species from A. farreri and A. cyathophorum was established during an interglacial period of penultimate glaciation of the QTP. Seed size variation also supports these by the colonization-competition tradeoff among small and large seeds. Our findings appear to suggest that the HMR uplift could have strengthened the development of the Asian monsoon regimes in this region and aridification in the western QTP, while the Quaternary climatic oscillations spurred the allopatric species' range shifts and created new open microhabitat for the alloploid species.

Morphological and ecological divergence of Lilium and Nomocharis within the Hengduan Mountains and Qinghai-Tibetan Plateau may result from habitat specialization and hybridization.

BACKGROUND: Several previous studies have shown that some morphologically distinctive, small genera of vascular plants that are endemic to the Qinghai-Tibetan Plateau and adjacent Hengduan Mountains appear to have unexpected and complex phylogenetic relationships with their putative sisters, which are typically more widespread and more species rich. In particular, the endemic genera may form one or more poorly resolved paraphyletic clades within the sister group despite distinctive morphology. Plausible explanations for this evolutionary and biogeographic pattern include extreme habitat specialization and hybridization. One genus consistent with this pattern is Nomocharis Franchet. Nomocharis comprises 7-15 species bearing showy-flowers that are endemic to the H-D Mountains. Nomocharis has long been treated as sister to Lilium L., which is comprised of more than 120 species distributed throughout the temperate Northern Hemisphere. Although Nomocharis appears morphologically distinctive, recent molecular studies have shown that it is nested within Lilium, from which is exhibits very little sequence divergence. In this study, we have used a dated molecular phylogenetic framework to gain insight into the timing of morphological and ecological divergence in Lilium-Nomocharis and to preliminarily explore possible hybridization events. We accomplished our objectives using dated phylogenies reconstructed from nuclear internal transcribed spacers (ITS) and six chloroplast markers. RESULTS: Our phylogenetic reconstruction revealed several Lilium species nested within a clade of Nomocharis, which evolved ca. 12 million years ago and is itself nested within the rest of Lilium. Flat/open and horizon oriented flowers are ancestral in Nomocharis. Species of Lilium nested within Nomocharis diverged from Nomocharis ca. 6.5 million years ago. These Lilium evolved recurved and campanifolium flowers as well as the nodding habit by at least 3.5 million years ago. Nomocharis and the nested Lilium species had relatively low elevation ancestors (<1000 m) and underwent diversification into new, higher elevational habitats 3.5 and 5.5 million years ago, respectively. Our phylogeny reveals signatures of hybridization including incongruence between the plastid and nuclear gene trees, geographic clustering of the maternal (i.e., plastid) lineages, and divergence ages of the nuclear gene trees consistent with speciation and secondary contact, respectively. CONCLUSIONS: The timing of speciation and ecological and morphological evolutionary events in Nomocharis are temporally consistent with uplift in the Qinghai-Tibetan Plateau and of the Hengduan Mountains 7 and 3-4 million years ago, respectively. Thus, we speculate that the mountain building may have provided new habitats that led to specialization of morphological and ecological features in Nomocharis and the nested Lilium along ecological gradients. Additionally, we suspect that the mountain building may have led to secondary contact events that enabled hybridization in Lilium-Nomocharis. Both the habitat specialization and hybridization have probably played a role in generating the striking morphological differences between Lilium and Nomocharis.

Evolutionary events in Lilium (including Nomocharis, Liliaceae) are temporally correlated with orogenies of the Q-T plateau and the Hengduan Mountains.

The Hengduan Mountains (H-D Mountains) in China flank the eastern edge of the Qinghai-Tibet Plateau (Q-T Plateau) and are a center of great temperate plant diversity. The geological history and complex topography of these mountains may have prompted the in situ evolution of many diverse and narrowly endemic species. Despite the importance of the H-D Mountains to biodiversity, many uncertainties remain regarding the timing and tempo of their uplift. One hypothesis is that the Q-T Plateau underwent a final, rapid phase of uplift 8-7 million years ago (Mya) and that the H-D Mountains orogeny was a separate event occurring 4-3 Mya. To evaluate this hypothesis, we performed phylogenetic, biogeographic, divergence time dating, and diversification rate analyses of the horticulturally important genus Lilium, including Nomocharis. The Lilium-Nomocharis complex is distributed throughout the temperate Northern Hemisphere but is most diverse within the H-D Mountains and Q-T Plateau. Our matK and ITS phylogenies support previous studies showing that Nomocharis is nested within Lilium. However, we detected incongruence between the two gene trees which may result from hybridization. Dating analyses performed using the ITS dataset showed that the evolution of major lineages within Lilium-Nomocharis may be temporally coincident with Q-T Plateau uplift occurring 8-7 Mya and H-D Mountains uplift approximately 4-3 Mya. Our analyses of diversification times and rates among Lilium-Nomocharis clades are less conclusive. However, these do suggest high extinction rates among H-D Mountains lineages.

Phylogeny of the Diploid Species of Rubus (Rosaceae).

Rubus L. (Rosaceae, Rosoideae) contains around 700 species distributed on all continents except Antarctica, with the highest species diversity in temperate to subtropical regions of the northern hemisphere. The taxonomy of Rubus is challenging due to the frequency of polyploidy, hybridization and apomixis. Previous studies mostly sampled sparsely and used limited DNA sequence data. The evolutionary relationships between infrageneric taxa, therefore, remain to be further clarified. In the present study, genotyping by sequencing (GBS) reduced-representation genome sequencing data from 186 accessions representing 65 species, 1 subspecies and 17 varieties of Rubus, with emphasis on diploid species, were used to infer a phylogeny using maximum likelihood and maximum parsimony methods. The major results were as follows: (1) we confirmed or reconfirmed the polyphyly or paraphyly of some traditionally circumscribed subgenera, sections and subsections; (2) 19 well-supported clades, which differed from one another on molecular, morphological and geographical grounds, were identified for the species sampled; (3) characteristics such as plants with dense bristles or not, leaves leathery or papyraceous, number of carpels, instead of inflorescences paniculate or not, aggregate fruits and leaves abaxially tomentose or not, may be of some use in classifying taxa whose drupelets are united into a thimble-shaped aggregate fruit that falls in its entirety from the dry receptacle; and (4) a preliminary classification scheme of diploid species of Rubus is proposed based on our results combined with those from previous phylogenetic analyses.

Chromosome diversity and evolution in tribe Lilieae (Liliaceae) with emphasis on Chinese species.

In this paper, karyotype data of the tribe Lilieae in China were analyzed and been superimposed onto a phylogenetic framework constructed by the internal transcribed spacer to investigate the karyotype evolution. Ten parameters for analyzing karyotype asymmetry were assessed and karyotypic idiogram of five genera of Lilieae were illustrated. The results showed that, the relationship of genera in Lilieae that inferred from Maximum Parsimony criteria and Bayesian Inference were congruent with previous studies, which focused on higher level of Liliales. The karyotype showed distinctive among genera, mainly expressed on the location and amount of secondary constrictions and intercalary satellites: the genus Notholirion have neither of them, and the genera Cardiocrinum and Fritillaria have the secondary constriction alone; the genera Lilium and Nomocharis showed both features, and the distribute pattern of the intercalary satellites showed similarity among related clades. The asymmetry that assessed by several methods indicated that the evolution trend of Lilieae did not follow a single direction, but different in each genus. On the sectional level of the genus Lilium (including Nomocharis) the karyotype evolution included three major periods. Combining the chromosomal structure variations and karyotype asymmetry, the chromosome diversity and evolution in Lilieae were quite clear in the light of molecular inference.

Chromosome-level de novo genome assembly and whole-genome resequencing of the threatened species Acanthochlamys bracteata (Velloziaceae) provide insights into alpine plant divergence in a biodiversity hotspot.

The Hengduan Mountains region is an important hotspot of alpine plant diversity and endemism. Acanthochlamys bracteata is a species of a threatened monotypic genus endemic to the Hengduan Mountains. In this study, we present a high-quality, chromosome-level reference genome for A. bracteata, constructed using long reads, short reads and Hi-C technology. We characterized its genetic diversity, population structure, demographic history and gene flow by resequencing individuals collected across its distribution. Comparative genomics analyses based on sequence information from single-copy orthologous genes revealed that A. bracteata and Dioscorea rotundata diverged ~104.5 million years ago. Whole-genome resequencing based on population genetic analysis revealed that the division of the 14 populations into 10 distinct clusters reflected geographical divergence, and three separate high levels of gene flow occurred sequentially between isolated populations of the Hengduan Mountains, a finding which is consistent with the turnover between ice ages and interglacial periods. Our findings indicate that Quaternary climatic changes played an important role in shaping the genetic structure and demographic trajectories of A. bracteata, and provide critical insights into the genetic status and evolutionary history of this poorly understood species, and possibly other alpine plants with a similar distribution. This study demonstrates the usefulness of population genomics for evaluating the effects of past climatic changes and identifying conservation units for the conservation and management of threatened species. Our high-quality genome represents a valuable resource for future studies of the underlying molecular mechanisms of adaptive evolution and provides insight for further comparative genomic analysis with other Velloziaceae species.

The first complete chloroplast genome of the fern genus Polystichum (Dryopteridaceae).

The first chloroplast genome of the fern genus Polystichum Roth (Dryopteridaceae) is reported here. Polystichum deltodon (Baker) Diels belongs to subgenus Haplopolystichum (Polystichum; Dryopteridaceae), many species of which are endangered or critically endangered species. The complete chloroplast genome of P. deltodon was determined for the first time in this work, which is revealed a circle quadripartite structure of 154,143 bp in length comprising a large single-copy region (LSC) of 86,990 bp, a small single-copy region (SSC) of 21,593 bp and a pair of inverted regions (IRs) of 22,780 bp, respectively. Based on the reported chloroplast genomes of Dryopteridaceae, phylogenetic analyses suggested that P. deltodon was located nearly to the genus Crytomium, which is in agreement with previous systematic research.

Species Boundaries and Parapatric Speciation in the Complex of Alpine Shrubs, Rosa sericea (Rosaceae), Based on Population Genetics and Ecological Tolerances.

Discerning species boundaries among closely related taxa is fundamental to studying evolution and biodiversity. However, species boundaries can be difficult to access in plants because ongoing divergence and speciation may leave an evolutionary footprint similar to introgression, which occurs frequently among species and genera. In this study, we sought to determine species boundaries between two closely related alpine shrubs, Rosa sericea and Rosa omeiensis, using population genetics, environmental data and ecological niche modeling, and morphological traits. We analyzed populations of R. sericea and R. omeiensis using genetic markers comprising a fragment of the single-copy nuclear gene, LEAFY, micro-satellites (EST-SSR), and plastid DNA sequences. The DNA sequence data suggested clusters of populations consistent with geography but not with previously proposed species boundaries based on morphology. Nevertheless, we found that the ecological niches of the previously proposed species only partially overlap. Thus, we suspect that these species are in the process of parapatric speciation; that is, differentiating along an ecological gradient, so that they exhibit differing morphology. Morphology has previously been the basis of recognizing the species R. sericea and R. omeiensis, which are the most widely distributed species within a broader R. sericea complex that includes several other narrow endemics. Here, we recognize R. sericea and R. omeiensis as independent species based on morphological and ecological data under the unified species concept, which emphasizes that these data types are of equal value to DNA for determining species boundaries and refining taxonomic treatments. While the DNA data did not delimit species within the R. sericea complex, we expect to develop and utilize new, robust DNA tools for understanding speciation within this group in future studies.

[Relationship between the melanocortin receptor 1 (MC1R) gene and the coat color phenotype in cattle].

The MC1R gene is related to coat color in cattle and to lactoprotein content in milk. Polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing were used to analyze the MC1R of China Holstein Black-white Cattle, China Holstein Red-white Cattle, Luxi Yellow Cattle and Bohai Black Cattle. Three alleles (ED, E+ and e) were identified. Most China Holstein Black-white Cattle had ED and E+ alleles (ED = 0.12, E+ = 0.80). The same was true for Bohai Black Cattle (ED = 0.52, E+ = 0.47). On the other hand, most China Holstein Red-white Cattle and Luxi Yellow Cattle had the e allele (e/e = 0.95). E+/e genotype was present in China Holstein Red-white Cattle and Luxi Yellow Cattle. We reasoned that ED and E+ lad to the synthesis of the black pigment. Furthermore, we found an important SNP (Single Nucleotide Polymorphism) at 725 position of the MC1R coding region.

The demographic response of a deciduous shrub (the Indigofera bungeana complex, Fabaceae) to the Pleistocene climate changes in East Asia.

East Asia harbors the highest level of floristic diversity among the world's temperate regions. Despite the increase in phylogeographic studies of temperate plants in East Asia, far less attention has been paid to widely distributed deciduous shrubs that widespread across several floral regions. We sequenced two chloroplast DNA (cpDNA) fragments (ndhJ-trnF and trnD-trnT) and one nuclear DNA (Pgk1) of 472 individuals from 51 populations of such a group, the Indigofera bungeana complex. We used population genetic data as well as ecological niche modelling to examine the evolutionary history and glacial refugia during the Last Glacial Maximum (LGM) of this group. We recovered 133 cpDNA and 68 nuclear haplotypes. The star-phylogeny of the recovered cpDNA and nuclear haplotypes and demographic analyses suggested distinct range expansion of I. bungeana complex have occurred during the early and middle Pleistocene. The climate change of the LGM might have affected little on the distribution of this complex based on the niche modelling. However, these climate changes and geographic isolation probably resulted in fixtures of the private haplotypes and genetic differentiations between regions. Our results suggested that this arid-tolerant species complex may have different responses to the Quaternary climate changes with those climate-sensitive species.

Cloning and expression of goat interleukin-18 gene.

We isolated and sequenced a 480 bp cDNA encoding mature goat interleukin-18 (gIL-18) from alveolar macrophages and splenocytes activated with LPS by RT-PCR. The gIL-18 gene was cloned into pET32a (+) vectors and sequenced. Nucleotide sequence of gIL-18 shares high homology with cattle. Fusional expression with pET32a (+) of gIL-18 of about 38kD was obtained by SDS-PAGE analysis after induction by IPTG in the E. Coli BL21 expression system. The recombinant protein can induce IFN-gamma production in PBMC. The IL-18 mRNA was constitutively detected in goat alveolar macrophages with or without LPS, While, enhanced expression was detected in splenocytes and liver cells if treated by LPS, and can be weakly detected in Peripheral blood mononuclear cells (PBMCs) treated by activators. Significant deference of IL-18 mRNA level may reflect the capacity to produce mature IL-18 in such tissues.

Pleistocene glaciations, demographic expansion and subsequent isolation promoted morphological heterogeneity: A phylogeographic study of the alpine Rosa sericea complex (Rosaceae).

While most temperate plants probably underwent glacial constriction to refugia and interglacial expansion, another type of interglacial refugia might have existed to maintain alpine plants during warm periods. To test this hypothesis, we applied phylogeographic methods to 763 individuals (62 populations) which belong to 7 taxonomically difficult species of the Rosa sericea complex distributed in alpine regions of the temperate and subtropical zones in eastern Asia. We used three chloroplast (cp) DNA fragments (trnL-trnF, ndhF-rpl32 and ndhJ-trnF) approximately 3,100 bp and nuclear microsatellite (nSSR) on eight sites to determine whether cold tolerant plants experienced expansion during the Pleistocene. The neutral test and mismatch distribution analysis (MDA) indicated that whole populations and major lineages of the Qinghai-Tibet Plateau (QTP) underwent expansion during the middle to late Pleistocene. Environmental niche modeling (ENM) indicates more suitable habitats during the Last Glacial Maximum (LGM) than at present. We concluded that the demographic history of R. sericea, which diverged in the middle Pleistocene, was mostly affected by climatic oscillations instead of by geographical barriers. The low genetic divergence, as well as the weak phylogenetic structure in the R. sericea complex both support treating this complex as a single taxon.

[ShRNA against NSP4 gene inhibits the proliferation of bovine rotavirus in vitro].

Based on the NSP4 sequence of bovine rotavirus (BRV), the shRNA was designed and synthesized, and a shRNA recombinant lenti-virus vector RNAi-H1-89 was constructed. The recombinant RNAi-H1-89 Lenti-virus was packaged by transfecting the 293T cell with the recombinant vector RNAi-H1-89 and two helper plasmids using lipofectamine, and then used to infect MA104 cells. The MA104 cells were further infected with BRV strain G6 24h post-infection, with the LacZ shRNA recombinant lenti-virus as control. Thirty-six hours later, the CPE of the infected cells was observed under microscope, shRNA of NSP4 gene inhibited CPE in MA104 cell; the shRNA against NSP4 gene also inhibited NSP4 gene expression by RT-PCR, The virus titer in the cell culture supernatant was significant lower compared with the control group. The above results showed that RNAi-H1-89 against NSP4 gene could specifically silence NSP4 gene expression, and inhibit the proliferation of BRV.

[Expression, purification of recombinant Luxi yellow cattle IFN-alpha fusion protein and its antiviral activities].

Interferon a gene was cloned from genomic DNA of Chinese Luxi yellow cattle by PCR, and the PCR product was inserted into vector pET32a( + ) to make a recombinant plasmid pET32a( + )/BoIFN-alpha. The expression of BoIFN-alpha in Escherichia coli was induced by addition of IPTG. Sequence analysis showed that the Chinese Luxi yellow cattle IFN-alpha gene is composed of 498 nucleotides, encoding a mature polypeptide of 166 amino acids. Compared with other BoIFN-alpha subtypes, it shares the highest identity of 97.6% to the C-subtype. SDS-PAGE results showed that recombinant proteins were expressed in inclusion bodies in Escherichia coli with molecular weight of 40 kD and the recombinant proteins accounted for 26.7% of the whole proteins.The expressed product was purified by affinity chromatography with immobilized nickel chelating NTA (Ni-NTA) and its antiviral activities were tested on MDBK/VSV cell system. Its antiviral activities were 5 x 10(5) u/mg on MDBK/VSV cell system. The results showed that the expression plasmid was successfully constructed and BoIFN-alpha C2 protein was expressed in Escherichia coli. Moreover the purification had good effects on antiviral activities.