Wuxia Guo

De Novo Assembly of Coding Sequences of the Mangrove Palm (Nypa fruticans) Using RNA-Seq and Discovery of Whole-Genome Duplications in the Ancestor of Palms

Nypa fruticans (Arecaceae) is the only monocot species of true mangroves. This species represents the earliest mangrove fossil recorded. How N. fruticans adapts to the harsh and unstable intertidal zone is an interesting question. However, the 60 gene segments deposited in NCBI are insufficient for solving this question. In this study, we sequenced, assembled and annotated the transcriptome of N. fruticans using next-generation sequencing technology. A total of 19,918,800 clean paired-end reads were de novo assembled into 45,368 unigenes with a N50 length of 1,096 bp. A total of 41.35% unigenes were functionally annotated using Blast2GO. Many genes annotated to “response to stress” and 15 putative positively selected genes were identified. Simple sequence repeats were identified and compared with other palms. The divergence time between N. fruticans and other palms was estimated at 75 million years ago using the genomic data, which is consistent with the fossil record. After calculating the synonymous substitution rate between paralogs, we found that two whole-genome duplication events were shared by N. fruticans and other palms. These duplication events provided a large amount of raw material for the more than 2,000 later speciation events in Arecaceae. This study provides a high quality resource for further functional and evolutionary studies of N. fruticans and palms in general.

Pronounced genetic differentiation and recent secondary contact in the mangrove tree Lumnitzera racemosa revealed by population genomic analyses.

Systematically investigating the impacts of Pleistocene sea-level fluctuations on mangrove plants may provide a better understanding of their demographic history and useful information for their conservation. Therefore, we conducted population genomic analyses of 88 nuclear genes to explore the population dynamics of a mangrove tree Lumnitzera racemosa across the Indo-West Pacific region. Our results revealed pronounced genetic differentiation in this species between the populations from the Indian Ocean and the Pacific Ocean, which may be attributable to the long-term isolation between the western and eastern coasts of the Malay Peninsula during sea-level drops in the Pleistocene glacial periods. The mixing of haplotypes from the two highly divergent groups was identified in a Cambodian population at almost all 88 nuclear genes, suggesting genetic admixture of the two lineages at the boundary region. Similar genetic admixture was also found in other populations from Southeast Asia based on the Bayesian clustering analysis of six nuclear genes, which suggests extensive and recent secondary contact of the two divergent lineages in Southeast Asia. Computer simulations indicated substantial migration from the Indian Ocean towards the South China Sea, which likely results in the genetic admixture in Southeast Asia.

Development and characterization of microsatellite loci for smooth cordgrass, Spartina alterniflora (Poaceae).

Premise of the study: Spartina alterniflora is one of the nine most notoriously invasive plants in China. Microsatellite markers were developed for this species to investigate its invasiveness and genetic diversity. Methods and Results: Fifteen polymorphic and seven monomorphic simple sequence repeat (SSR) markers derived from expressed sequence tags (ESTs) were identified and screened in 60 samples of S. alterniflora. The number of alleles per polymorphic locus ranged from two to eight, with an average of 3.8 alleles per polymorphic locus. The expected heterozygosity and observed heterozygosity based on seven disomic loci ranged from 0.27 to 0.46 and 0.21 to 0.51, respectively. The average Shannon index ranged from 0.26 to 0.94 in eight nondisomic loci. Conclusions: The SSR markers described here may be useful for further investigation of population genetics and invasion dynamics of S. alterniflora.

Transcriptome analyses provide insights into the phylogeny and adaptive evolution of the mangrove fern genus Acrostichum.

The mangrove fern genus Acrostichum grows in the extremely unstable marine intertidal zone under harsh conditions, such as high salt concentrations, tidal rhythms and long-term climate changes. To explore the phylogenetic relationships and molecular mechanisms underlying adaptations in this genus, we sequenced the transcriptomes of two species of Acrostichum, A. aureum and A. speciosum, as well as a species in the sister genus, Ceratopteris thalictroides. We obtained 47,517, 36,420 and 60,823 unigenes for the three ferns, of which 24.39–45.63% were annotated using public databases. The estimated divergence time revealed that Acrostichum adapted to the coastal region during the late Cretaceous, whereas the two mangrove ferns from the Indo West-Pacific (IWP) area diverged more recently. Two methods (the modified branch-site model and the Kh method) were used to identify several positively selected genes, which may contribute to differential adaptation of the two Acrostichum species to different light and salt conditions. Our study provides abundant transcriptome data and new insights into the evolution and adaptations of mangrove ferns in the inhospitable intertidal zone.

Comparative Analysis of Transcriptomes in Rhizophoraceae Provides Insights into the Origin and Adaptive Evolution of Mangrove Plants in Intertidal Environments

Mangroves are woody plants that grow at the interface between land and sea in tropical and subtropical latitudes, where they exist in conditions of high salinity, extreme tides, strong winds, high temperatures, and muddy, anaerobic soils. Rhizophoraceae is a key mangrove family, with highly developed morphological and physiological adaptations to extreme conditions. It is an ideal system for the study of the origin and adaptive evolution of mangrove plants. In this study, we characterized and comprehensively compared the transcriptomes of four mangrove species, from all four mangrove genera, as well as their closest terrestrial relative in Rhizophoraceae, using RNA-Seq. We obtained 41,936–48,845 unigenes with N50 values of 982–1,185 bp and 61.42–69.48% annotated for the five species in Rhizophoraceae. Orthology annotations of Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Clusters of Orthologous Groups revealed overall similarities in the transcriptome profiles among the five species, whereas enrichment analysis identified remarkable genomic characteristics that are conserved across the four mangrove species but differ from their terrestrial relative. Based on 1,816 identified orthologs, phylogeny analysis and divergence time estimation revealed a single origin for mangrove species in Rhizophoraceae, which diverged from the terrestrial lineage ~56.4 million years ago (Mya), suggesting that the transgression during the Paleocene–Eocene Thermal Maximum may have been responsible for the entry of the mangrove lineage of Rhizophoraceae into intertidal environments. Evidence showed that the ancestor of Rhizophoraceae may have experienced a whole genome duplication event ~74.6 Mya, which may have increased the adaptability and survival chances of Rhizophoraceae during and following the Cretaceous–Tertiary extinction. The analysis of positive selection identified 10 positively selected genes from the ancestor branch of Rhizophoraceae mangroves, which were mainly associated with stress response, embryo development, and regulation of gene expression. Positive selection of these genes may be crucial for increasing the capability of stress tolerance (i.e., defense against salt and oxidative stress) and development of adaptive traits (i.e., vivipary) of Rhizophoraceae mangroves, and thus plays an important role in their adaptation to the stressful intertidal environments.

Genetic diversity, population structure, and genetic relatedness of native and non-native populations of Spartina alterniflora (Poaceae, Chloridoideae)

Spartina alterniflora Losiel. is a highly invasive species found on the Chinese coast. To characterize the evolutionary mechanisms underlying the success of S. alterniflora in China, we examined the distribution and structure of genetic variation among three native populations at their source locations and five non-native populations in China, at both the chloroplast and nuclear microsatellite loci. Both cpDNA and microsatellite data revealed comparable genetic diversity and population differentiation between the native and introduced regions, which is consistent with the deliberate historical planting of heterogeneous founding groups in China. Bayesian clustering analysis showed that the best two clusters in the introduced region correspond to populations from the Atlantic coast and Gulf coast, respectively, instead of the three ecotypes proposed by a previous study. Investigating genetic composition of individuals suggested that most of the non-native plants might be of Atlantic origin, while some individuals in the Hong Kong population could be genetic admixtures of Atlantic and Gulf origin. This study indicated varied genetic components among populations in China, which imply different sources for the present Chinese populations.

Identification and characterization of evolutionarily conserved alternative splicing events in a mangrove genus Sonneratia

Alternative splicing (AS), which produces multiple mRNA transcripts from a single gene, plays crucial roles in plant growth, development and environmental stress responses. Functional significances of conserved AS events among congeneric species have not been well characterized. In this study, we performed transcriptome sequencing to characterize AS events in four common species of Sonneratia, a mangrove genus excellently adaptive to intertidal zones. 7,248 to 12,623 AS events were identified in approximately 25% to 35% expressed genes in the roots of the four species. The frequency of AS events in Sonneratia was associated with genomic features, including gene expression level and intron/exon number and length. Among the four species, 1,355 evolutionarily conserved AS (ECAS) events were identified from 1,170 genes. Compared with non-ECAS events, ECAS events are of shorter length and less possibility to introduce premature stop codons (PTCs) and frameshifts. Functional annotations of the genes containing ECAS events showed that four of the 26 enriched Gene Ontology (GO) terms are involved in proton transport, signal transduction and carbon metabolism, and 60 genes from another three GO terms are implicated in responses to osmotic, oxidative and heat stresses, which may contribute to the adaptation of Sonneratia species to harsh intertidal environments.

Chloroplast phylogeography of a widely distributed mangrove species, Excoecaria agallocha , in the Indo-West Pacific region

Excoecaria agallocha is one of the predominant mangrove species in the Indo-West Pacific (IWP) region with an extensive range of distribution. To infer the current geographical patterns of genetic variation and provide new insights on the historical population dynamics of mangrove species in the IWP region, we sampled E. agallocha across its distribution range and investigated the phylogeography of this species using four chloroplast DNA (cpDNA) fragments. Our results showed that E. agallocha possessed a high degree of species-level genetic diversity, while the average genetic diversity within populations was much lower. The presence of population genetic structure was supported by the estimates of genetic differentiation and the analysis of molecular variance (AMOVA). Of the ten haplotypes identified, no haplotypes were shared between the East Indian Ocean (EIO), the West Pacific Ocean (WPO), and the North Australian (NA) regions. Genealogy analysis, haplotype distribution patterns, and the principal coordinate analysis (PCoA) consistently suggested the existence of three haplotype groups distributed in distinct geographical locations. The genealogical breaks observed and further analysis of geographic/genetic barriers indicated that both land barriers and oceanic currents may have played important roles in the divergence and demography of E. agallocha.

Comparative transcriptome analysis of the invasive weed Mikania micrantha with its native congeners provides insights into genetic basis underlying successful invasion

BackgroundMikania micrantha H.B.K. (Asteraceae) is one of the world’s most invasive weeds which has been rapidly expanding in tropical Asia, including China, while its close relative M. cordata, the only Mikania species native to China, shows no harm to the local ecosystems. These two species are very similar in morphology but differ remarkably in several ecological and physiological traits, representing an ideal system for comparative analysis to investigate the genetic basis underlying invasion success. In this study, we performed RNA-sequencing on the invader M. micrantha and its native congener M. cordata in China, to unravel the genetic basis underlying the strong invasiveness of M. micrantha. For a more robust comparison, another non-invasive congener M. cordifolia was also sequenced and compared.ResultsA total of 52,179, 55,835, and 52,983 unigenes were obtained for M. micrantha, M. cordata, and M. cordifolia, respectively. Phylogenetic analyses and divergence time dating revealed a relatively recent split between M. micrantha and M. cordata, i.e., approximately 4.81 million years ago (MYA), after their divergence with M. cordifolia (8.70 MYA). Gene ontology classifications, pathway assignments and differential expression analysis revealed higher representation or significant up-regulation of genes associated with photosynthesis, energy metabolism, protein modification and stress response in M. micrantha than in M. cordata or M. cordifolia. Analysis of accelerated evolution and positive selection also suggested the importance of these related genes and processes to the adaptability and invasiveness of M. micrantha. Particularly, most (77 out of 112, i.e. 68.75%) positively selected genes found in M. micrantha could be classified into four groups, i.e., energy acquisition and utilization (10 genes), growth and reproduction (13 genes), protection and repair (34 genes), and signal transduction and expression regulation (20 genes), which may have contributed to the high adaptability of M. micrantha to various new environments and the capability to occupy a wider niche, reflected in its high invasiveness.ConclusionsWe characterized the transcriptomes of the invasive species M. micrantha and its non-invasive congeners, M. cordata and M. cordifolia. A comparison of their transcriptomes provided insights into the genetic basis of the high invasiveness of M. micrantha.

Development of polymorphic EST-SSR markers in Itea chinensis (Iteaceae) and cross-amplification in related species

Premise of the Study We isolated and characterized 16 expressed sequence tag–simple sequence repeat (EST‐SSR) markers in Itea chinensis (Iteaceae), a common evergreen broadleaf tree, for future studies on the genetic diversity and spatial genetic structure of the species. Methods and Results Based on transcriptome data of I. chinensis, a total of 36 primer pairs were initially designed and tested. Of these, 16 were successfully amplified and showed clear polymorphism. For these markers, the number of alleles per locus varied from two to 15. The observed and expected heterozygosity ranged from 0 to 0.600 and 0.072 to 0.554, respectively. Furthermore, all loci were successfully cross‐amplified in two congeneric species, I. oblonga and I. yangchunensis. Conclusions The EST‐SSR markers described here can be used to study the genetic diversity and phylogeographic patterns of I. chinensis and other related species in Itea.