First Record of Comparative Plastid Genome Analysis and Phylogenetic Relationships among Corylopsis Siebold & Zucc. (Hamamelidaceae).

Corylopsis Siebold & Zucc. (Hamamelidaceae) is widely used as a horticultural plant and comprises approximately 25 species in East Asia. Molecular research is essential to distinguish Corylopsis species, which are morphologically similar. Molecular research has been conducted using a small number of genes but not in Corylopsis. Plastid genomes of Corylopsis species (Corylopsis gotoana, Corylopsis pauciflora, and Corylopsis sinensis) were sequenced using next-generation sequencing techniques. Repeats and nucleotide diversity that could be used as DNA markers were also investigated. A phylogenetic investigation was carried out using 79 protein-coding genes to infer the evolutionary relationships within the genus Corylopsis. By including new plastomes, the overall plastid genome structure of Corylopsis was similar. Simple sequence repeats of 73-106 SSRs were identified in the protein-coding genes of the plastid genomes, and 33-40 long repeat sequences were identified in the plastomes. The Pi value of the rpl33_rps18 region, an intergenic spacer, was the highest. Phylogenetic analysis demonstrated that Corylopsis is a monophyletic group and Loropetalum is closely related to Corylopsis. C. pauciflora, C. gotoana, and C. spicata formed a clade distributed in Japan, whereas C. sinensis, C. glandulifera, and C. velutina formed a clade that was distributed in China.

Comparative transcriptome analysis of leaf, stem, and root tissues of Semiliquidambar cathayensis reveals candidate genes involved in terpenoid biosynthesis.

BACKGROUND: Semiliquidambar cathayensis is a traditional medicinal plant and endemic species in China. Its roots, branches, leaves, bark, and nectar are known to have therapeutic effects against rheumatoid arthritis, lumbar muscle strain, and several other diseases. However, limited knowledge regarding the molecular properties of S. cathayensis highlights the need for further research in order to elucidate the underlying pathways governing the synthesis of its active ingredients and regulation of its accumulation processes. METHODS: We conducted transcriptome sequencing of the leaf, stem and root epidermises, and stem and root xylems of S. cathayensis with three biological replicates. Moreover, candidate genes involved in terpenoid biosynthesis, such as IDI, FPPS, DXR, SQS, GPPS, and HMGR were selected for quantitative real-time PCR analysis. RESULTS: We identified 88,582 unigenes. Among which, 36,144 unigenes were annotated to the nr protein database, 21,981 to the Gene Ontology database, 11,565 to the Clusters of Orthologous Groups database, 24,209 to the Pfam database, 21,685 to the SWISS-PROT database, and 12,753 to the Kyoto Encyclopedia of Genes and Genomes (KEGG), with 5072 unigenes common to all six databases. Of those annotated using the KEGG database, 187 unigenes were related to the terpenoid metabolism pathway, and expression analysis of the related genes indicated that the mevalonate and methylerythritol 4-phosphate pathways play different roles in terpenoid biosynthesis in different tissues of S. cathayensis. CONCLUSIONS: These findings greatly expand gene resources of S. cathayensis and provide basic data for the study of the biosynthetic pathways and molecular mechanisms of terpenoids.

Mining and characterization of novel EST-SSR markers of Parrotia subaequalis (Hamamelidaceae) from the first Illumina-based transcriptome datasets.

Parrotia subaequalis is an endangered Tertiary relict tree from eastern China. Despite its important ecological and horticultural value, no transcriptomic data and limited molecular markers are currently available in this species. In this study, we first performed high-throughput transcriptome sequencing of two individuals representing the northernmost (TX) and southernmost (SJD) population of P. subaequalis on the Illumina HiSeq 2500 platform. We gathered a total of 69,135 unigenes for P. subaequalis (TX) and 84,009 unigenes for P. subaequalis (SJD). From two unigenes datasets, 497 candidate polymorphic novel expressed sequence tag-simple sequence repeats (EST-SSRs) were identified using CandiSSR. Among these repeats, di-nucleotide repeats were the most abundant repeat type (62.78%) followed by tri-, tetra- and hexa-nucleotide repeats. We then randomly selected 54 primer pairs for polymorphism validation, of which 27 (50%) were successfully amplified and showed polymorphisms in 96 individuals from six natural populations of P. subaequalis. The average number of alleles per locus and the polymorphism information content values were 3.70 and 0.343; the average observed and expected heterozygosity were 0.378 and 0.394. A relatively high level of genetic diversity (HT = 0.393) and genetic differentiation level (FST = 0.171) were surveyed, indicating P. subaequalis maintained high levels of species diversity in the long-term evolutionary history. Additionally, a high level of cross-transferability (92.59%) was displayed in five congeneric Hamamelidaceae species. Therefore, these new transcriptomic data and novel polymorphic EST-SSR markers will pinpoint genetic resources and facilitate future studies on population genetics and molecular breeding of P. subaequalis and other Hamamelidaceae species.

Identification and development of microsatellite (SSRs) makers of Exbucklandia (HAMAMELIDACEAE) by high-throughput sequencing.

Hamamelidaceae (Saxifragales, previously Rosales) comprises approximately six subfamily, 30 genera and 140 species, most of which are Tertiary relicts. Exbucklandia is the only genus of the subfamily Exbucklandioideae, Hamelidaceae, containing only 2-4 species. Of them, the species E. longipetala H. T. Chang is endemic to China and listed as endangered in The Biodiversity Red List of China: Higher Plant, yet some taxonomists put forward that E. longipetala should be merged into E. tonkinensis (Lecomte) H. T. Chang. Currently, there was nearly no phylogeographic studies on this genus possibly due to the deficiency of efficient molecular markers. In this study, we sequenced the genome of E. tonkinensis based on high throughput sequencing technology, and obtained approximately 6 G raw data, which was further de novo assembled into 303,481 contigs. Based on them, 15,326 SSRs were identified from 13,596 contigs, and primers were successfully designed for 10,660 SSRs. A total of 139 paired primers were synthesized, 106 of them were successfully amplified in six Exbucklandia individuals with expected PCR product size, and 24 demonstrated to be polymorphic among three Exbucklandia populations. Accordingly, the expected and observed heterozygosity were between 0.097-0.717 and 0.098-0.583. Based on these efforts, future researches on genetic diversity and population structure of Exbucklandia can be performed to understand its phylogenetic origins and phylogeographic pattern.

Genetic diversity and ex situ conservation of Loropetalum subcordatum, an endangered species endemic to China.

BACKGROUND: Loropetalum subcordatum is an endangered species endemic to China that is characterized by narrow distribution, small population size, and delayed fertilization. However, the genetic diversity of the entire extant natural and ex situ populations has not been assessed to date. In this study, we evaluated the genetic diversity and structure of six natural populations and a single ex situ population (the only known ex situ population of L. subcordatum) using sequence-related amplified polymorphism data. RESULTS: In total, 553 reliable DNA bands, of which 359 (63.28%) were polymorphic, were amplified by polymerase chain reaction with combinations of 15 primers. Low average gene diversity within populations and high genetic differentiation were detected in L. subcordatum. A Mantel test demonstrated that there was a positive correlation between genetic and geographic distances, indicating that significant genetic divergence was likely the result of geographic isolation among natural populations. Furthermore, based on genetic structure patterns, populations of L. subcordatum were divided into three clusters. Group 1 was composed of specimens from Libo, Guizhou Province (GZ) and Huanjiang, Guangxi Zhuang Autonomous Region (GX). Group 2 was composed of Mt. Wuguishan, Guangdong Province (GD). Group 3 was composed of three populations in Hong Kong Special Administrative Region. Additionally, clonal reproduction probably existed in GD population. According to the genetic information analysis and field survey, the ex situ population did not match its source population (GD) in terms of genetics, and its habitat was different from the original natural habitat. We observed that a few individual GD seeds were needed to improve ZS ex situ in the future. CONCLUSIONS: Compared to previous SRAP-based studies of endangered plants, L. subcordatum had extremely low average gene diversity within populations and high genetic differentiation among populations. At present, the unique ex situ population has not been successful due to non-representative samples being taken, a smaller population size, and man-made changes in habitat. Potential strategies are suggested to improve the conservation of this species.

Novel microsatellite markers for Distylium lepidotum (Hamamelidaceae) endemic to the Ogasawara Islands.

BACKGROUND: Distylium lepidotum is a small tree endemic to the Ogasawara Islands located in the northwestern Pacific Ocean. This species is a sole food for an endemic locust, Boninoxya anijimensis. Here, we developed microsatellite markers to investigate genetic diversity and genetic structure and to avoid a genetic disturbance after transplantation to restore the Ogasawara Islands ecosystem. RESULTS: Microsatellite markers with perfect dinucleotide repeats were developed using the next-generation sequencing Illumina MiSeq Desktop Sequencer. Thirty-two primer pairs were characterized in two D. lepidotum populations on Chichijima and Hahajima Islands of the Ogasawara Islands. The number of alleles for the markers ranged from three to 23 per locus in the two populations. Expected heterozygosity per locus in each population ranged from 0.156 to 0.940 and 0.368 to 0.845, respectively. CONCLUSIONS: These microsatellite markers will be useful for future population genetics studies of D. lepidotum and provide a basis for conservation management of the Ogasawara Islands.

Genetic variation and genetic structure of the endangered species Sinowilsonia henryi Hemsi. (Hamamelidaceae) revealed by amplified fragment length polymorphism (AFLP) markers.

Comprehensive research of genetic variation is crucial in designing conservation strategies for endangered and threatened species. Sinowilsonia henryi Hemsi. is a tertiary relic with a limited geographical distribution in the central and western areas of China. It is endangered because of climate change and habitat fragmentation over the last thousands of years. In this study, amplified fragment length polymorphism markers were utilized to estimate genetic diversity and genetic structure in and among S. henryi. In this study, Nei's genetic diversity and Shannon's information index were found to be 0.192 and 0.325 respectively, indicating a moderate-to-high genetic diversity in species. According to analysis of molecular variation results, 32% of the genetic variation was shown to be partitioned among populations, demonstrating a relatively high genetic divergence; this was supported by principal coordinate analysis and unweighted pair-group method with arithmetic average analysis. Moreover, the Mantel test showed that there was no significant correlation between genetic and geographical distances. The above results can be explained by the effects of habitat fragmentation, history traits, and gene drift. Based on the results, several implications were indicated and suggestions proposed for preservation strategies for this species.

A comparative study on genetic effects of artificial and natural habitat fragmentation on Loropetalum chinense (Hamamelidaceae) in Southeast China.

Elucidating the demographic and landscape features that determine the genetic effects of habitat fragmentation has become fundamental to research in conservation and evolutionary biology. Land-bridge islands provide ideal study areas for investigating the genetic effects of habitat fragmentation at different temporal and spatial scales. In this context, we compared patterns of nuclear microsatellite variation between insular populations of a shrub of evergreen broad-leaved forest, Loropetalum chinense, from the artificially created Thousand-Island Lake (TIL) and the Holocene-dated Zhoushan Archipelago of Southeast China. Populations from the TIL region harboured higher levels of genetic diversity than those from the Zhoushan Archipelago, but these differences were not significant. There was no correlation between genetic diversity and most island features, excepting a negative effect of mainland-island distance on allelic richness and expected heterozygosity in the Zhoushan Archipelago. In general, levels of gene flow among island populations were moderate to high, and tests of alternative models of population history strongly favoured a gene flow-drift model over a pure drift model in each region. In sum, our results showed no obvious genetic effects of habitat fragmentation due to recent (artificial) or past (natural) island formation. Rather, they highlight the importance of gene flow (most likely via seed) in maintaining genetic variation and preventing inter-population differentiation in the face of habitat 'insularization' at different temporal and spatial scales.

Genetic variability and population structure of Disanthus cercidifolius subsp. longipes (Hamamelidaceae) based on AFLP analysis.

Disanthus cercidifolius subsp. longipes is an endangered species in China. Genetic diversity and structure analysis of this species was investigated using amplified fragments length polymorphism (AFLP) fingerprinting. Nei's gene diversity ranged from 0.1290 to 0.1394. The AMOVA indicated that 75.06% of variation was distributed within populations, while the between-group component 5.04% was smaller than the between populations-within-group component 19.90%. Significant genetic differentiation was detected between populations. Genetic and geographical distances were not correlated. PCA and genetic structure analysis showed that populations from East China were together with those of the Nanling Range. These patterns of genetic diversity and levels of genetic variation may be the result of D. c. subsp. longipes restricted to several isolated habitats and "excess flowers production, but little fruit set". It is necessary to protect all existing populations of D. c. subsp. longipes in order to preserve as much genetic variation as possible.

Evolution and biogeographic diversification of the witch-hazel genus (Hamamelis L., Hamamelidaceae) in the Northern Hemisphere.

The evolution of the eastern Asian and eastern North American disjunction of the witch-hazel genus Hamamelis L. (Hamamelidaceae) was examined through phylogenetic and biogeographic analyses. Phylogenetic relationships of all Hamamelis species were reconstructed using parsimony and Bayesian analyses of sequence data from six plastid (trnL-F, psaA-ycf3, rps16, matK, atpB-rbcL, and psbA-trnH) and two nuclear (ITS and ETS) DNA regions. The phylogeny was then used to infer the biogeographic origin and subsequent diversification using both event-based (DIVA) and maximum likelihood (LAGRANGE) methods incorporating fossil data. The times of divergence within Hamamelis were estimated with the Bayesian approach using the program BEAST. A very low level of molecular variation was detected in both the plastid and the nuclear DNA regions within Hamamelis. The combined analyses resulted in a phylogeny of the genus with higher resolution and support values. Hamamelis was supported to be monophyletic with H. mollis from eastern China diverged first in the genus. All North American species formed a clade and was sister to the eastern Asian H. japonica. Within the North American clade, H. mexicana was sister to H. vernalis, and the recently described species H. ovalis was found to be closely related to the widespread species H. virginiana. The stem age of Hamamelis was estimated to be at the Eocene (51.2 mya, with 95% HDP: 49.0-54.6 mya), and the crown age of the genus was dated to be at the late Miocene (9.7 mya, with 95% HDP: 3.6-18.1 mya, or 10.6 mya, with 95% HDP: 4.2-19.6 mya). The disjunction between the eastern Asian and the eastern North American species was dated to be 7.1 mya (95% HDP: 3.1-13.6 mya) or 7.7 mya (95% HDP: 3.4-13.6 mya). Biogeographic analyses incorporating fossils resulted in more equally possible solutions at the stem lineage of Hamamelis than those including extant species only. Eastern Asia is inferred to be the most-likely area for the origin of Hamamelis. The current disjunction was due to the extinction in western North America and Europe from Eocene to late Miocene, and later migration from eastern Asia into North America. The Bering land bridge was hypothesized to have played an important role in the evolution of this disjunction. The current species diversity of the genus was the result of relatively recent diversification events during the late Miocene rather than long accumulation of lineages from the early Tertiary.

Resolving an ancient, rapid radiation in Saxifragales.

Despite the prior use of approximately 9000 bp, deep-level relationships within the angiosperm clade, Saxifragales remain enigmatic, due to an ancient, rapid radiation (89.5 to 110 Ma based on the fossil record). To resolve these deep relationships, we constructed several new data sets: (1) 16 genes representing the three genomic compartments within plant cells (2 nuclear, 10 plastid, 4 mitochondrial; aligned, analyzed length = 21,460 bp) for 28 taxa; (2) the entire plastid inverted repeat (IR; 26,625 bp) for 17 taxa; (3) "total evidence" (50,845 bp) for both 17 and 28 taxa (the latter missing the IR). Bayesian and ML methods yielded identical topologies across partitions with most clades receiving high posterior probability (pp = 1.0) and bootstrap (95% to 100%) values, suggesting that with sufficient data, rapid radiations can be resolved. In contrast, parsimony analyses of different partitions yielded conflicting topologies, particularly with respect to the placement of Paeoniaceae, a clade characterized by a long branch. In agreement with published simulations, the addition of characters increased bootstrap support for the putatively erroneous placement of Paeoniaceae. Although having far fewer parsimony-informative sites, slowly evolving plastid genes provided higher resolution and support for deep-level relationships than rapidly evolving plastid genes, yielding a topology close to the Bayesian and ML total evidence tree. The plastid IR region may be an ideal source of slowly evolving genes for resolution of deep-level angiosperm divergences that date to 90 My or more. Rapidly evolving genes provided support for tip relationships not recovered with slowly evolving genes, indicating some complementarity. Age estimates using penalized likelihood with and without age constraints for the 28-taxon, total evidence data set are comparable to fossil dates, whereas estimates based on the 17-taxon data are much older than implied by the fossil record. Hence, sufficient taxon density, and not simply numerous base pairs, is important in reliably estimating ages. Age estimates indicate that the early diversification of Saxifragales occurred rapidly, over a time span as short as 6 million years. Between 25,000 and 50,000 bp were needed to resolve this radiation with high support values. Extrapolating from Saxifragales, a similar number of base pairs may be needed to resolve the many other deep-level radiations of comparable age in angiosperms.