Integrative taxonomy in a rapid speciation group associated with mating system transition: A case study in the Primula cicutariifolia complex.

Accurate species delimitation is the key to biodiversity conservation and is fundamental to most branches of biology. However, species delimitation remains challenging in those evolutionary radiations associated with mating system transition from outcrossing to self-fertilization, which have frequently occurred in angiosperms and are usually accompanied by rapid speciation. Here, using the Primula cicutariifolia complex as a case, we integrated molecular, morphological and reproductive isolation evidence to test and verify whether its outcrossing (distylous) and selfing (homostylous) populations have developed into independent evolutionary lineages. Phylogenetic trees based on whole plastomes and SNPs of the nuclear genome both indicated that the distylous and homostylous populations grouped into two different clades. Multispecies coalescent, gene flow and genetic structure analyses all supported such two clades as two different genetic entities. In morphology, as expected changes in selfing syndrome, homostylous populations have significantly fewer umbel layers and smaller flower and leaf sizes compared to distylous populations, and the variation range of some floral traits, such as corolla diameter and umbel layers, show obvious discontinuity. Furthermore, hand-pollinated hybridization between the two clades produced almost no seeds, indicating that well post-pollination reproductive isolation has been established between them. Therefore, the distylous and homostylous populations in this studied complex are two independent evolutionary lineages, and thus these distylous populations should be treated as a distinct species, here named Primula qiandaoensis W. Zhang & J.W. Shao sp. nov.. Our empirical study of the P. cicutariifolia complex highlights the importance of applying multiple lines of evidence, in particular genomic data, to delimit species in pervasive evolutionary plant radiations associated with mating system transition.

Simplified Genomic Data Revealing the Decline of Aleuritopteris grevilleoides Population Accompanied by the Uplift of Dry-Hot Valley in Yunnan, China.

Understanding the evolutionary history of endangered species is crucial for identifying the main reasons for species endangerment in the past and predicting the changing trends and evolutionary directions of their future distribution. In order to study the impact of environmental changes caused by deep valley incision after the uplift of the Qinghai-Tibet Plateau on endangered species, we collected 23 samples belonging to four populations of Aleuritopteris grevilleoides, an endangered fern endemic to the dry-hot valleys (DHV) of Yunnan. Single-nucleotide variation sites (SNPs) were obtained by the genotyping-by-sequencing (GBS) method, and approximately 8085 SNP loci were identified. Through the reconstruction and analysis of genetic diversity, population structure, population dynamics, evolution time, and ancestral geographical distribution, combined with geological historical events such as the formation of dry-hot valleys, this study explores the formation history, current situation, reasons for endangerment and scientifically sound measures for the protection of A. grevilleoides. In our study, A. grevilleoides had low genetic diversity (Obs_Het = 0.16, Exp_Het = 0.32, Pi = 0.33) and a high inbreeding coefficient (Fis = 0.45). The differentiation events were 0.18 Mya, 0.16 Mya, and 0.11 Mya in the A. grevilleoides and may have been related to the formation of terraces within the dry-hot valleys. The history of population dynamics results shows that the diversion of the river resulted in a small amount of gene flow between the two clades, accompanied by a rapid increase in the population at 0.8 Mya. After that, the effective population sizes of A. grevilleoides began to contract continuously due to topographic changes resulting from the continuous expansion of dry-hot valleys. In conclusion, we found that the environmental changes caused by geological events might be the main reason for the changing population size of A. grevilleoides.

Over 30 Years of Misidentification: A New Nothospecies Lycoris × jinzheniae (Amaryllidaceae) in Eastern China, Based on Molecular, Morphological, and Karyotypic Evidence.

Based on the complete chloroplast genome, morphology, and karyotype evidence, we identified a new nothospecies, Lycoris × jinzheniae S.Y. Zhang, P.C. Zhang & J.W. Shao, in eastern China. This new nothospecies has been inappropriately named Lycoris × albiflora in the previous literature for more than 30 years. However, the new nothospecies resulted from the hybridization of L. sprengeri and L. chinensis and had the following characteristics: the karyotype was 2n = 19 = 3V + 16I, the leaves emerged in the spring, the ratio of filament to corolla length was approximately 1.2, tepals were slightly undulated and curved, and it was distributed throughout eastern China. These characteristics are quite different from those of L. × albiflora; thus, in this study, we named it and provided a detailed morphological description and diagnosis.

 Polygonatumpraecox (Asparagaceae), a new species from mid-eastern China revealed by morphological and molecular evidence.

A new species, Polygonatumpraecox Y.F.Hu & J.W.Shao (Asparagaceae), is described and illustrated. This species is similar to P.cyrtonema, P.odoratum and P.caulialatum, but can be distinguished from P.cyrtonema by its racemose inflorescence, cylindrical hairless filaments and apex without a retrorse spur; from P.odoratum by its stout moniliform rhizome, straight stem and longer (1.7-2.2 cm long) floral tube; and from P.caulialatum by its upper part straight stem, yellowish-green corolla, lobes excurved and earlier flowering. The complete chloroplast genome of this new species is 155,115-155,256 bp in length. Phylogenetic analysis revealed that P.praecox is not genetically related to the above three morphological similar species, but is closely related to the two European species (P.multiforum and P.latifolium). This species is relatively common in mid-eastern China and has previously been confused with P.cyrtonema. As its wild resources have decreased in recent years due to over-exploitation for medicinal or edible purposes, we classify it as Near Threatened (NT) according to the IUCN Red List Criteria.

Lycorisinsularis (Amaryllidaceae), a new species from eastern China revealed by morphological and molecular evidence.

Lycorisinsularis S.Y.Zhang & J.W.Shao, a new fertile diploid species from coastal provinces in eastern China is described. This new species is most similar to L.sprengeri in morphology and has been misidentified as the latter for a long time. However, it can be distinguished from the latter by the relatively longer perianth tube (1.5‒2.5 cm vs. less than 1.3 cm), a characteristic that was overlooked before. Phylogenetic analysis, based on complete plastid genome, showed that L.insularis is not genetically related to L.sprengeri in the genus. The former was a sister group of L.sanguinea, while the latter was closely related to L.longituba and L.chinensis and they were respectively located on different clades that were separated at the base of the phylogenetic tree. The chromosome number of L.insularis is 2n = 22. At present, as the new species is relatively widely distributed and the wild population can normally reproduce by seeds, we evaluate it as LC (Least Concern) according to criteria of the IUCN Red List.

Measuring Vehicle Profile Size: Lidar-Based System and K-Frame-Based Methodology.

At present, light curtain is a widely-used method to measure the vehicle profile size. However, it is sensitive to temperature, humidity, dust and other weather factors. In this paper, a lidar-based system with a K-frame-based algorithm is proposed for measuring vehicle profile size. The system is composed of left lidar, right lidar, front lidar, control box and industry controlling computer. Within the system, a K-frame-based methodology is investigated, which include several probable algorithm combinations. Three groups of experiments are conducted. An optimal algorithm combination, A16, is determined through the first group experiments. In the second group experiments, various types of vehicles are chosen to verify the generality and repeatability of the proposed system and methodology. The third group experiments are implemented to compare with vision-based methods and other lidar-based methods. The experimental results show that the proposed K-frame-based methodology is far more accurate than the comparative methods.

Lycoris wulingensis, a dwarf new species of Amaryllidaceae from Hunan, China.

Lycoris wulingensis S.Y. Zhang, a new species from Hunan Province (central South China), is described and illustrated. This new species is a fertile diploid plant and its karyotype is 2n = 22. It is most similar to L. × haywardii in morphology, but the latter is a hybrid species and distributed in East China and the plant is much larger. Amongst the original species, L. wulingensis is similar to L. radiata, but differs from it in its flowers being rose-red (vs. red) and stamens and tepals are nearly the same length (vs. stamens significantly longer than tepals).

Pollen micromorphology of eastern Chinese Polygonatum and its role in taxonomy by using scanning electron microscopy.

Although considerable progress has recently been made in the taxonomic study of Asparagaceae, but the Polygonatum remains one of the most poorly investigated genus. Therefore, the present study was designed with the aim, to document the pollen micromorphology of closely related Polygonatum taxa and its taxonomic significance. Pollen micro-morphological features were observed using scanning electron microscopy-SEM). The pollen grains are monad, navicular, monocolpate, and radially symmetrical. A significant variation was observed in the exine sculpturing. Moreover, most of the Polygonatum taxa have perforate pollen while some of them were mixed with psilate to perforate, perforate to microreticulate, sometimes scabrate, gemmate with baculate. Hence, this study documented the pollen morphology of Polygonatum taxa and strengthens the taxonomic identification of the genus Polygonatum based on pollen characters, which helps and can be used as an additional tool for the correct identification and discrimination of the species of Polygonatum at generic and species level.

Foliar epidermal micromorphology and its taxonomic significance in Polygonatum (Asparagaceae) using scanning electron microscopy.

In the current investigation, 16 samples of six species of the genus Polygonatum were collected from different geographical regions in China. Leaf micromorphological traits were studied in detail using scanning electron microscopy. About five-leaf samples were used for each species collected from different geographical regions. A significant variation was found in the foliar epidermal traits such as stomata types and their distribution, epidermal cell shape, anticlinal wall pattern, and various types of trichomes. Polygonal epidermal cells were observed in P. cyrtonema, P. odoratum, P. sibiricum, and P. filipes, rectangular in P. zanlanscianense and P. odoratum and irregular shape in P. filipes and P. zanlanscianense. Straight anticlinal wall pattern was observed in most of the species, but straight to little undulate was observed in P. cyrtonema, P. filipes, and P. zanlanscianense. Similarly, various shape stomata that are, oval, oblong, and elliptical and types that is, paracytic, anomocytic, and actinocytic have been observed. Based on the foliar traits, the P. odoratum and P. sibiricum were found closely related to one another. On the other hand, P. involucratum and P. zanlanscianense shows a strong association. However, P. filipes showed very less similarity with the other five species. Hence this study adds useful information that may be helpful in the further taxonomic classification of the taxa at subspecies level and was found significant in the correct identification and discrimination of the closely related taxa of the genus Polygonatum.

Comparison Among Five Eucalyptus Species Based on Their Leaf Contents of Some Primary and Secondary Metabolites.

BACKGROUND: Eucalyptus belongs to the Myrtaceae family. It is the most planted hardwood forest crop worldwide, representing a global renewable resource of fiber, pharmaceuticals and energy. OBJECTIVE: To compare the five species, E. maidenii, E. robusta, E. citriodora, E. tereticornis and E. camaldulensis, seeking for the richest source of nutrients and pharmaceuticals. METHODOLOGY: Eucalyptus samples were subjected to some chemical determinations for both primary and secondary metabolites to verify their nutritional and pharmaceutical importance related to different extracts. GC-MS analysis was applied to detect the presence of some individual phenolic constituents in their leaves. RESULTS: E. robusta recorded the maximum contents of carbohydrates (40.07%) and protein (31.91%). While E. camaldulensis contained the highest contents of total phenolic compounds (46.56 mg/g), tannins (40.01 mg/g) and antioxidant activities assayed by the phosphomolybednum method (57.60 mg/g), followed by E. citridora. However, E. tereticornis exhibited the highest reducing power ability (151.23 mg/g). The GC-MS highlighted 20 phenolic constituents and antioxidants which varied in their abundance in Eucalyptus leaves, 8 individual phenolics (hydroquinone, hesperitin, pyrogallol, resorcinol, protocatechuic acid, naringenin, chlorogenic acid and catechin) were maximally recorded with E. camaldulensis and secondly, with E. citridora in case of at least 5 components. Nevertheless, gallic and quinic acids were more abundant in the leaves of E. tereticornis, which may explain its high corresponding reducing powers. CONCLUSION: Acetone-water combination has enhanced phenolics extraction from Eucalyptus tissues. This is the first report aiming to compare between the aforementioned Eucalyptus species highlighting either their nutritional or medicinal importance.

Intraspecific variation of self-incompatibility in the distylous plant Primula merrilliana.

Heteromorphic self-incompatibility can prevent self- and intramorph fertilization while favouring intermorph mating and the maintenance of morph-ratio stability in heterostylous populations. However, variation in the expression of self-incompatibility intraspecies has seldom been assessed. Through hand pollinations and microsatellite markers, the variation in the expression of self-incompatibility and genetic diversity were studied in distylous plant Primula merrilliana. We discovered that the strength of self-incompatibility varied extensively among individuals and populations, from pronounced to weaker self-incompatibility in distylous populations, all the way to strong self-compatibility in homostylous populations. Each distylous population included self-incompatible (SI), partly self-compatible (PSC) and self-compatible (SC) individuals, with the index of self-compatibility (ISC) ranging from 0.07 to 0.68 across populations. Self-compatible populations (ISC > 0.25) were not genetically clustered but were more closely related to populations with high SI and SC individuals were mixed with SI individuals within populations. The ISC and the proportions of SC and PSC individuals were higher in peripheral than in central populations, but no decrease of genetic diversity and no deviations of floral morph ratio from isoplethy were detected in peripheral populations. Additionally, the expression of self-incompatibility was stronger in long-styled flowers than in short-styled flowers. The variation in the strength of self-incompatibility documented in P. merrilliana cautions against the estimation of ISC from a few individuals or populations in distylous species and provides a more complex and nuanced understanding of the role of self-incompatibility in heterostyly.

Molecular phylogeography of East Asian Boea clarkeana (Gesneriaceae) in relation to habitat restriction.

Subfamily Cyrtandroideae (Gesneriaceae) comprises a broadly distributed group of rocky-slope herbs, with China being the center of its distributional range. The normal growth of many species within the family is particularly dependent on special habitats. Due to the paucity of molecular studies, very little is known regarding East Asian herb phylogeographic pattern. Here, we investigate the molecular phylogeography of Boea clarkeana Hemsl., a unique resurrection herb endemic to China, focusing on geographically restrictive effects of habitat distribution on evolutionary history. Variation in three chloroplast DNA (cpDNA) intergenic spacers (psbA-trnH, rps12-rpl20, and trnL-trnF), the ribosomal internal transcribed spacer (ITS) and simple sequence repeats in expressed sequence tags (EST-SSRs) was investigated across 18 populations to assess genetic diversity, genetic structure and historical dynamics. Genetic diversity was low within populations (cpDNA, hS = 0.03, πS×10(3) = 0.17; ITS, hS = 0.16, πS×10(3) = 0.43) but high for species (cpDNA, hT = 0.82, πT×10(3) = 3.12; ITS, hT = 0.88, πT×10(3) = 6.39); 76 alleles were detected in this highly inbred species (FIS = 0.22), with a significantly low average of 1.34 alleles per locus. No cpDNA or ITS haplotypes were shared between regions. Based on cpDNA results, the Mt. Huangshan-Tianmu and Mt. Qinling-Daba haplotypes are ancestral; these two regions represent potential refugia. Although no evidence of significant retreat-migration phenomena during glacial cycles was detected, interglacial range expansion from northern Mt. Qinling-Daba was identified (121,457 yr BP). Rapid agricultural growth caused bottlenecks in many populations, especially on Mt. Huang-Tianmu. Habitat restriction and fragmentation, weak seed and pollen dispersal abilities, and long-term isolation caused by human-induced or environmental changes are considered the main causes of extinction of several populations and low genetic diversity within populations and regions. These analyses clarify the effects of habitat restriction on B. clarkeana, representing an evolutionary reference for similar gesneriads, and enrich our understanding of the molecular phylogeography of East Asian rocky-slope herbs.

Characterization of the transcriptome and EST-SSR development in Boea clarkeana, a desiccation-tolerant plant endemic to China.

BACKGROUND: Desiccation-tolerant (DT) plants can recover full metabolic competence upon rehydration after losing most of their cellular water (>95%) for extended periods of time. Functional genomic approaches such as transcriptome sequencing can help us understand how DT plants survive and respond to dehydration, which has great significance for plant biology and improving the drought tolerance of crops. Boea clarkeana Hemsl. (Gesneriaceae) is a DT dicotyledonous herb. Its genomic sequences characteristics remain unknown. Based on transcriptomic analyses, polymorphic EST-SSR (simple sequence repeats in expressed sequence tags) molecular primers can be designed, which will greatly facilitate further investigations of the population genetics and demographic histories of DT plants. METHODS: In the present study, we used the platform Illumina HiSeq™2000 and de novo assembly technology to obtain leaf transcriptomes of B. clarkeana and conducted a BLASTX alignment of the sequencing data and protein databases for sequence classification and annotation. Then, based on the sequence information, the EST-SSR markers were developed, and the functional annotation of ESTs containing polymorphic SSRs were obtained through BLASTX. RESULTS: A total of 91,449 unigenes were generated from the leaf cDNA library of B. clarkeana. Based on a sequence similarity search with a known protein database, 72,087 unigenes were annotated. Among the annotated unigenes, a total of 71,170 unigenes showed significant similarity to the known proteins of 463 popular model species in the Nr database, and 59,962 unigenes and 32,336 unigenes were assigned to Gene Ontology (GO) classifications and Cluster of Orthologous Groups (COG), respectively. In addition, 44,924 unigenes were mapped in 128 KEGG pathways. Furthermore, a total of 7,610 unigenes with 8,563 microsatellites were found. Seventy-four primer pairs were selected from 436 primer pairs designed for polymorphism validation. SSRs with higher polymorphism rates were concentrated on dinucleotides, pentanucleotides and hexanucleotides. Finally, 17 pairs with stable, highly polymorphic loci were selected for polymorphism screening. There was a total of 65 alleles, with 2-6 alleles at each locus. Primarily due to the unique biological characteristics of plants, the HE (0-0.196), HO (0.082-0.14) and PIC (0-0.155) per locus were very low. The functional annotation distribution centered on ESTs containing di- and tri-nucleotide SSRs, and the ESTs containing primers BC2, BC4 and BC12 were annotated to vegetative dehydration/desiccation pathways. DISCUSSION: This work is the first genetic study of B. clarkeana as a new plant resource of DT genes. A substantial number of transcriptome sequences were generated in this study. These sequences are valuable resources for gene annotation and discovery as well as molecular marker development. These sequences could also provide a valuable basis for future molecular studies of B. clarkeana.

[Isolation and analysis of polymorphic microsatellite loci in Dendrobium huoshanense].

OBJECTIVE: To investigate the genetic diversity and structure of Dendrobium huoshanense, a (CT)n enriched microsatellite library was constructed using a magnetic beads enrichment procedure. METHOD: The 3'-biotinylated oligonucleotide probe was used to hybridize with the digested D. huoshanense genomic DNA fragments whose both ends were ligated with adaptors. The hybridized complex was then combined with the streptavidin-coated magnetic beads. The captured microsatellite fragments were eluted, collected and cloned into pMD19-T vector. The recombinant plasmids were transformed into Escherichia coli DH5alpha competent cells. The clones that yielded two or more bands contained microsatellite fractions. Positive clones were screened and sequenced. Thirty pairs of primers were designed and synthesized. Polymorphism at each locus was determined using 24 individuals from a natural population from Huoshan county town in Anhui province. RESULT: Twelve polymorphic microsatellite loci from the microsatellite-enriched genomic library were newly developed across 24 D. huoshanense individuals. In total, 65 alleles were identified, and the number of alleles per locus ranged from 2 to 8. The mean observed and expected heterozygosities were 0.500 and 0.638, respectively. Two loci significantly deviated from Hardy-Weinberg equilibrium (P<0.05), which could be due to the presence of null alleles. Furthermore, three of twelve loci showed significant linkage disequilibrium (P<0.05). CONCLUSION: These results suggest that the identified polymorphic microsatellite markers will be useful in population genetic studies of D. huoshanense.

Effects of population size on reproductive success of the endangered and endemic species Primula merrilliana.

The reproductive ability related to the population size of the endangered and endemic species Primula merrilliana Schltr. was investigated. In 26 populations observed, only four contain more than 500 flowering individuals, whereas most of them (53.8%) consist of less than 100 flowering individuals. Though the ratio of pin and thrum plants keeps its balance at 1:1 for all populations, the frequency of pin and thrum flowers was significantly biased in most small populations. Population size strongly affected reproductive success; plants in small populations produced significantly fewer flowers and fruits per plant and fewer seeds per fruit, and therefore fewer seeds per plant. The floral density was another important factor that influenced the reproductive success of P. merrilliana, because four main reproductive success parameters (fruits per plant, seeds per fruit, seeds per plant, and the proportion of flowers setting fruit) were all positively correlated with floral density. The size of plants and the number of leaves per plant (measure of habitat quality) were not influenced by the variation of population size, suggesting that the reduced fecundity in small populations may not be a consequence of lower habitat quality. Inbreeding depression and pollen limitation as a result of less attractiveness in small populations are therefore likely explanations for these patterns.