Glacial vicariance and oceanic circulation shape population structure of the coastal legume Derris trifoliata in the Indo-West Pacific.

PREMISE: The phylogeography of coastal plant species is shaped by contemporary and historical biogeographic processes. In this study, we aim to decipher the phylogeography of Derris trifoliata, a woody legume of relatively recent origin and wide distribution, in coastal areas in the Indo-West Pacific (IWP) region. METHODS: Genetic diversity and population structure were assessed by analyzing six nuclear and three chloroplast DNA sequences from 30 populations across the species' range. Phylogeography was inferred by estimating gene flow, divergence time, historical population size changes, and historical habitat suitability using paleoclimatic niche modeling. RESULTS: High genetic diversity was observed at the species level. The populations of three oceanic regions included in this study (i.e., Indian Ocean, South China Sea, and Pacific Ocean) formed distinct clades and likely diverged during the late Pleistocene. Potential barriers to gene flow were identified, including the Sunda and Sahul shelves, geographic distance, and current patterns of oceanic circulation. Analysis of changes in population size supported the bottleneck model, which was strengthened by estimates of habitat suitability across paleoclimatic conditions. CONCLUSIONS: The once widespread distribution of D. trifoliata was fragmented by changes in climatic suitability and biogeographic barriers that arose following sea-level changes during the Pleistocene. In addition, contemporary patterns of oceanic circulation and geographic distance between populations appear to maintain genetic differentiation across its distribution in the IWP.

Phylogeographic pattern of a cryptoviviparous mangrove, Aegiceras corniculatum, in the Indo-West Pacific, provides insights for conservation actions.

MAIN CONCLUSION: This study identified the historical geoclimatic factors which caused low genetic diversity and strong phylogeographic structure in a cryptoviviparous mangrove. The phylogeographic pattern was used to suggest conservation actions. Phylogeographic studies are used to understand the spatial distribution and evolution of genetic diversity, and have major conservation implications, especially for threatened taxa like the mangroves. This study aimed to assess the phylogeographic pattern of Aegiceras corniculatum, a cryptoviviparous mangrove, across its distribution range in the Indo-West Pacific (IWP) region. We genotyped 398 samples, collected from 37 populations, at four chloroplast DNA (cpDNA) loci, and identified the influence of historical processes on the contemporary population structure of the species. Low genetic diversity at the population level was observed. The evolutionary relationship between 12 cpDNA haplotypes suggested a strong phylogeographic structure, which was further validated by the clustering algorithms and proportioning of maximum variation among hierarchical population groups. The magnitude and direction of historical gene flow indicated that the species attained its wide distribution from its likely ancestral area of the Malay Archipelago. The divergence time estimates of the haplotypes indicated that the geoclimatic changes during the Pleistocene, especially the glacial sea-level changes and emergence of landmasses, hindered genetic exchange and created genetic differentiation between the phylogenetic groups. The species overwintered the last glacial maxima in multiple refugia in the IWP, as identified by the environmental niche modelling. Overall, our findings indicated that ancient glacial vicariance had influenced the present genetic composition of A. corniculatum, which was maintained by the current demographic features of this region. We discussed how these findings can be used to prioritize areas for conservation actions, restore disturbed habitats and prevent further genetic erosion.

Going with the flow: analysis of population structure reveals high gene flow shaping invasion pattern and inducing range expansion of Mikania micrantha in Asia.

BACKGROUND AND AIMS: Mikania micrantha, a climbing perennial weed of the family Asteraceae, is native to Latin America and is highly invasive in the tropical belt of Asia, Oceania and Australia. This study was framed to investigate the population structure of M. micrantha at a large spatial scale in Asia and to identify how introduction history, evolutionary forces and landscape features influenced the genetic pattern of the species in this region. METHODS: We assessed the genetic diversity and structure of 1052 individuals from 46 populations for 12 microsatellite loci. The spatial pattern of genetic variation was investigated by estimating the relationship between genetic distance and geographical, climatic and landscape resistances hypothesized to influence gene flow between populations. KEY RESULTS: We found high genetic diversity of M. micrantha in this region, as compared with the genetic diversity parameters of other invasive species. Spatial and non-spatial clustering algorithms identified the presence of multiple genetic clusters and admixture between populations. Most of the populations showed heterozygote deficiency, primarily due to inbreeding, and the founder populations showed evidence of a genetic bottleneck. Persistent gene flow throughout the invasive range caused low genetic differentiation among populations and provided beneficial genetic variation to the marginal populations in a heterogeneous environment. Environmental suitability was found to buffer the detrimental effects of inbreeding at the leading edge of range expansion. Both linear and non-linear regression models demonstrated a weak relationship between genetic distance and geographical distance, as well as bioclimatic variables and environmental resistance surfaces. CONCLUSIONS: These findings provide evidence that extensive gene flow and admixture between populations have influenced the current genetic pattern of M. micrantha in this region. High gene flow across the invaded landscape may facilitate adaptation, establishment and long-term persistence of the population, thereby indicating the range expansion ability of the species.

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

BACKGROUND: Mikania 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. RESULTS: A 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. CONCLUSIONS: We 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.

Transcriptome analysis of the Holly mangrove Acanthus ilicifolius and its terrestrial relative, Acanthus leucostachyus, provides insights into adaptation to intertidal zones.

BACKGROUND: Acanthus is a unique genus consisting of both true mangrove and terrestrial species; thus, it represents an ideal system for studying the origin and adaptive evolution of mangrove plants to intertidal environments. However, little is known regarding the two respects of mangrove species in Acanthus. In this study, we sequenced the transcriptomes of the pooled roots and leaves tissues for a mangrove species, Acanthus ilicifolius, and its terrestrial congener, A. leucostachyus, to illustrate the origin of the mangrove species in this genus and their adaptive evolution to harsh habitats. RESULTS: We obtained 73,039 and 69,580 contigs with N50 values of 741 and 1557 bp for A. ilicifolius and A. leucostachyus, respectively. Phylogenetic analyses based on four nuclear segments and three chloroplast fragments revealed that mangroves and terrestrial species in Acanthus fell into different clades, indicating a single origin of the mangrove species in Acanthus. Based on 6634 orthologs, A. ilicifolius and A. leucostachyus were found to be highly divergent, with a peak of synonymous substitution rate (Ks) distribution of 0.145 and an estimated divergence time of approximately 16.8 million years ago (MYA). The transgression in the Early to Middle Miocene may be the major reason for the entry of the mangrove lineage of Acanthus into intertidal environments. Gene ontology (GO) classifications of the full transcriptomes did not show any apparent differences between A. ilicifolius and A. leucostachyus, suggesting the absence of gene components specific to the mangrove transcriptomes. A total of 99 genes in A. ilicifolius were identified with signals of positive selection. Twenty-three of the 99 positively selected genes (PSGs) were found to be involved in salt, heat and ultraviolet stress tolerance, seed germination and embryo development under periodic inundation. These stress-tolerance related PSGs may be crucial for the adaptation of the mangrove species in this genus to stressful marine environments and may contribute to speciation in Acanthus. CONCLUSIONS: We characterized the transcriptomes of one mangrove species of Acanthus, A. ilicifolius, and its terrestrial relative, A. leucostachyus, and provided insights into the origin of the mangrove Acanthus species and their adaptive evolution to abiotic stresses in intertidal environments.

DnFCA Isoforms Cooperatively Regulate Temperature-Related Flowering in Dendrobium nobile.

Timely flowering is a determinative trait for many economically valuable species in the Dendrobium genus of the Orchidaceae family, some of which are used for ornamental and medicinal purposes. D. nobile, a representative species of nobile-type Dendrobium, normally flowers in spring after exposure to sufficient low temperatures in winter. However, flowering can be stopped or disrupted by the untimely application of high temperatures. Little is known about the regulation and the mechanisms behind this switch. In this study, we report two isoforms from the KFK09_017173 locus of the D. nobile genome, named DnFCAγ and DnFCAß, respectively, that cooperatively regulate flowering in D. nobile. These two isoforms are generated by alternative 3' polyadenylation of DnFCA (FLOWERING CONTROL LOCUS C in D. nobile) pre-mRNA and contain a distinct 3'-terminus. Both can partially rescue late flowering in the Arabidopsis fca-1 mutant, while in wild-type Arabidopsis, they tend to delay the flowering time. When introduced into the detached axillary buds or young seedlings of D. nobile, both were able to induce the transcription of DnAGL19 (AGAMOUS LIKE 19 in D. nobile) in seedlings, whereas only DnFCAγ was able to suppress the transcription of DnAPL1 (AP1-LIKE 1 in D. nobile) in axillary buds. Furthermore, the time-course change of DnFCAγ accumulation was opposite to that of DnAPL1 in axillary buds, which was remarkable under low temperatures and within a short time after the application of high temperatures, supporting the suggestion that the expression of DnAPL1 can be inhibited by a high accumulation of DnFCAγ in floral buds. In leaves, the accumulation of DnFCAß was in accordance with that of DnAGL19 and DnFT (FLOWERING LOCUS T in D. nobile) to a large extent, suggesting the activation of the DnAGL19-DnFT pathway by DnFCAß. Taken together, these results suggest that the DnFCAγ-DnAPL1 pathway in axillary buds and the DnFCAß-DnAGL19 pathway in the leaves cooperatively promote flowering under low temperatures. The long-term and constant, or untimely, application of high temperatures leads to the constitutive suppression of DnAPL1 by a high level of DnFCAγ in axillary buds, which consequently delays floral development.

Contrasting Phylogeographic Patterns in Lumnitzera Mangroves Across the Indo-West Pacific.

Mangroves are ecologically important forest communities in tropical and subtropical coasts, the effective management of which requires understanding of their phylogeographic patterns. However, these patterns often vary among different species, even among ecologically similar taxa or congeneric species. Here, we investigated the levels and patterns of genetic variation within Lumnitzera consisting of two species (L. racemosa and L. littorea) with nearly sympatric ranges across the Indo-West Pacific (IWP) region by sequencing three chloroplast DNA regions (for both species) and genotyping 11 nuclear microsatellite loci (for L. littorea). Consistent with findings in studies on other mangrove species, we found that both L. racemosa and L. littorea showed relatively high genetic variation among populations but low genetic variation within populations. Haplotype network and genetic clustering analyses indicated two well-differentiated clades in both L. racemosa and L. littorea. The relationship between geographic and genetic distances and divergence time estimates of the haplotypes indicated that limited dispersal ability of the propagules, emergence of land barriers during ancient sea-level changes, and contemporary oceanic circulation pattern in the IWP influenced the current population structure of the two species. However, the position of genetic break was found to vary between the two species: in L. racemosa, strong divergence was observed between populations from the Indian Ocean and the Pacific Ocean possibly due to land barrier effect of the Malay Peninsula; in L. littorea, the phylogeographic pattern was created by a more eastward genetic break along the biogeographic barrier identified as the Huxley's line. Overall, our findings strongly supported previous hypothesis of mangrove species divergence and revealed that the two Lumnitzera species have different phylogeographic patterns despite their close genetic relationship and similar current geographic distribution. The findings also provided references for the management of Lumnitzera mangroves, especially for the threatened L. littorea.

Land masses and oceanic currents drive population structure of Heritiera littoralis, a widespread mangrove in the Indo-West Pacific.

Phylogeographic forces driving evolution of sea-dispersed plants are often influenced by regional and species characteristics, although not yet deciphered at a large spatial scale for many taxa like the mangrove species Heritiera littoralis. This study aimed to assess geographic distribution of genetic variation of this widespread mangrove in the Indo-West Pacific region and identify the phylogeographic factors influencing its present-day distribution. Analysis of five chloroplast DNA fragments' sequences from 37 populations revealed low genetic diversity at the population level and strong genetic structure of H. littoralis in this region. The estimated divergence times between the major genetic lineages indicated that glacial level changes during the Pleistocene epoch induced strong genetic differentiation across the Indian and Pacific Oceans. In comparison to the strong genetic break imposed by the Sunda Shelf toward splitting the lineages of the Indian and Pacific Oceans, the genetic differentiation between Indo-Malesia and Australasia was not so prominent. Long-distance dispersal ability of H. littoralis propagules helped the species to attain transoceanic distribution not only across South East Asia and Australia, but also across the Indian Ocean to East Africa. However, oceanic circulation pattern in the South China Sea was found to act as a barrier creating further intraoceanic genetic differentiation. Overall, phylogeographic analysis in this study revealed that glacial vicariance had profound influence on population differentiation in H. littoralis and caused low genetic diversity except for the refugia populations near the equator which might have persisted through glacial maxima. With increasing loss of suitable habitats due to anthropogenic activities, these findings therefore emphasize the urgent need for conservation actions for all populations throughout the distribution range of H. littoralis.

Convergent adaptation of the genomes of woody plants at the land-sea interface.

Sequencing multiple species that share the same ecological niche may be a new frontier for genomic studies. While such studies should shed light on molecular convergence, genomic-level analyses have been unsuccessful, due mainly to the absence of empirical controls. Woody plant species that colonized the global tropical coasts, collectively referred to as mangroves, are ideal for convergence studies. Here, we sequenced the genomes/transcriptomes of 16 species belonging in three major mangrove clades. To detect convergence in a large phylogeny, a CCS+ model is implemented, extending the more limited CCS method (convergence at conservative sites). Using the empirical control for reference, the CCS+ model reduces the noises drastically, thus permitting the identification of 73 convergent genes with P true (probability of true convergence) > 0.9. Products of the convergent genes tend to be on the plasma membrane associated with salinity tolerance. Importantly, convergence is more often manifested at a higher level than at amino-acid (AA) sites. Relative to >50 plant species, mangroves strongly prefer 4 AAs and avoid 5 others across the genome. AA substitutions between mangrove species strongly reflect these tendencies. In conclusion, the selection of taxa, the number of species and, in particular, the empirical control are all crucial for detecting genome-wide convergence. We believe this large study of mangroves is the first successful attempt at detecting genome-wide site convergence.

Characterization of the complete chloroplast genome of Camellia granthamiana (Theaceae), a Vulnerable species endemic to China.

Camellia granthamiana is a wild camellia resource endemic to China and is listed as a Vulnerable species globally. Here, we reported and characterized its complete chloroplast (cp) genome by using Illumina pair-end sequencing data. The total chloroplast genome size was 157,001 bp, including inverted repeats (IRs) of 26,042 bp, separated by a large single copy (LSC) and a small single copy (SSC) of 86,622 and 18,295 bp, respectively. A total of 131 genes, including 36 tRNA, 8 Rrna, and 87 protein-coding genes were identified. Phylogenetic analysis showed that C. granthamiana is sister to C. sinensis with 100% value support.

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.

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.

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.

The origin, diversification and adaptation of a major mangrove clade (Rhizophoreae) revealed by whole-genome sequencing.

Mangroves invade some very marginal habitats for woody plants-at the interface between land and sea. Since mangroves anchor tropical coastal communities globally, their origin, diversification and adaptation are of scientific significance, particularly at a time of global climate change. In this study, a combination of single-molecule long reads and the more conventional short reads are generated from Rhizophora apiculata for the de novo assembly of its genome to a near chromosome level. The longest scaffold, N50 and N90 for the R. apiculata genome, are 13.3 Mb, 5.4 Mb and 1.0 Mb, respectively. Short reads for the genomes and transcriptomes of eight related species are also generated. We find that the ancestor of Rhizophoreae experienced a whole-genome duplication ~70 Myrs ago, which is followed rather quickly by colonization and species diversification. Mangroves exhibit pan-exome modifications of amino acid (AA) usage as well as unusual AA substitutions among closely related species. The usage and substitution of AAs, unique among plants surveyed, is correlated with the rapid evolution of proteins in mangroves. A small subset of these substitutions is associated with mangroves' highly specialized traits (vivipary and red bark) thought to be adaptive in the intertidal habitats. Despite the many adaptive features, mangroves are among the least genetically diverse plants, likely the result of continual habitat turnovers caused by repeated rises and falls of sea level in the geologically recent past. Mangrove genomes thus inform about their past evolutionary success as well as portend a possibly difficult future.

Occurrence and levels of nitrofuran metabolites in sea cucumber from Dalian, China.

The occurrence and levels of nitrofuran metabolites (NFMs) in sea cucumber (SC) from Dalian, China, are reported. Four metabolites including 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM) and 1-aminohydantoin (AHD) in different SC products (fresh, instant and dry salted SCs) were measured. The frequency of occurrence for NFMs in all SC samples was 42.7%. The total NFM concentrations ranged from non-detectable to 64.6 ng g-1, with a mean of 3.59 ng g-1. AOZ and SEM were the dominant congeners, accounting for 40.1% and 59.1% of the total NFMs, respectively. The concentrations and patterns varied among different regions. Higher levels of NFMs were found in the fresh SC products, and the order for the average concentration of ∑4NFM was fresh > dry salted > instant.

De novo assembly of the transcriptome of Aegiceras corniculatum, a mangrove species in the Indo-West Pacific region.

Aegiceras corniculatum (L.) Blanco is one of the most salt tolerant mangrove species and can thrive in 3% salinity at the seaward edge of mangrove forests. Here we sequenced the transcriptome of A. corniculatum used Illumina GA platform to develop its genomic resources for ecological and evolutionary studies. We obtained about 50 million high-quality paired-end reads with 75bp in length. Using the short read assembler Velvet, we yielded 49,437 contigs with the average length of 625bp. A total of 32,744 (66.23%) contigs showed significant similarity to the GenBank non-redundant (NR) protein database. 30,911 and 18,004 of these sequences were assigned to Gene Ontology and eukaryotic orthologous groups of proteins (KOG). A total of 4942 transcripts from our assemblies had significant similarity with KEGG Orthologs and were involved in 144 KEGG pathways, while 9899 unigenes had enzyme commission (EC) numbers. In addition, 9792 transcriptome-derived SSRs were identified from 7342 sequences. With our strict criteria, 4165 candidate SNPs were also identified from 2058 contigs. Some of these SNPs were further validated by Sanger sequencing. Genomic resources generated in this study should be valuable in ecological, evolutionary, and functional genomics studies for this mangrove species.

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.

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.

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.

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.