BACKGROUND
tropical intertidal zones are extreme environments characterized by high salinity, drought, hypoxia and high ultraviolet  radiation, which severely limit plant growth, development and reproduction  <cit> .
as the dominant forest community and ecosystem in the coasts, mangrove plants struggle and survive in these environments with remarkable morphological and physiological characteristic, for example, exposed breathing roots, support roots and buttresses, salt-excreting leaves, and viviparous water-dispersed propagules  <cit> .
understanding the genetic basis underlying those adaptive traits at the genomic level could provide important clues to the molecular mechanisms of stress resistance in marine halophytes.
mangroves are constituent plants of approximately  <dig> species from  <dig> genera belonging to  <dig> families  <cit> .
studies based on fossils and phylogenetic analysis have suggested that these biogeographically and taxonomically diverse genera are of independent origins in different geologic epochs  <cit> .
however, the divergent time and the species radiation within some genera are still unclear and are of great interest to many botanists.
comparative analysis among mangrove species and model terrestrial plants using rna-sequencing has revealed that specific sequence divergence and transcriptional regulation play major roles in response to salt stress in many mangrove species, such as aegiceras corniculatum  <cit> , bruguiera gymnorhiza  <cit>  and sonneratia alba  <cit> .
however, the model plants used in these studies, such as arabidopsis thaliana and populus tomentosa, are distantly related to mangroves, which makes it difficult to distinguish adaptive processes from those caused by phylogenetic effects, thus reducing the resolution of selection signals  <cit> .
among all true mangrove genera, acanthus is the only genus that includes both mangrove and terrestrial species; therefore, it is an ideal system to investigate the adaptive evolution of mangrove plants to stressful intertidal environments while minimizing phylogenetic influences.
this genus consists of three representative mangrove species and approximately  <dig> terrestrial species  <cit> .
the three mangrove species, which are also called holly mangroves, are found in the intertidal zones from india to the west pacific and tropical australia.
unlike other terrestrial species of acanthus, which are distributed in the mediterranean basin, a. leucostachyus is restrict to south to southeast asia and grows under rain forest canopies at the edges of streams 600– <dig> m above the sea level  <cit> .
similarities in geographic distribution and ecological requirements of a. leucostachyus and the congeneric mangrove species suggested a. leucostachyus is a suitable outgroup for illustrating the evolutionary history of the mangrove species in acanthus.
nguyen et al.
<cit>  identified  <dig> genes that are involved in response to salt stress from  <dig> expressed sequence tags  of a. ebracteatus.
however, these studies only listed the annotations of these stress-response genes.
the role of natural selection in adaptive evolution of mangrove species has not been characterized.
in the current study, we performed rna-sequencing to assess the mangrove species a. ilicifolius and its terrestrial congener a. leucostachyus, to provide insights into the evolutionary process underlying the adaptation of mangroves to intertidal zones.
we asked the following specific questions: 1) what is the origination pattern of the mangrove acanthus taxa and when mangrove species diverged from their terrestrial relatives within this genus?
2) are there any marked differences in gene ontology  classification among the mangrove transcriptome profiles?
finally, 3) which genes have experienced adaptive evolution and contributed to the adaptation of a. ilicifolius to stressful intertidal habitats?
RESULTS
transcriptome sequencing and de novo assembly
a total of  <dig>  million 90-bp and  <dig>  million 100-bp paired-end reads were sequenced for a. ilicifolius and a. leucostachyus, respectively .
the raw data were deposited in the ncbi sequence read archive  with the accession number srp <dig>  to minimize sequencing errors, we individually trimmed each read to its longest contiguous segment, using quality scores of higher than  <dig> for all remaining segments.
after the trimming processes, reads with length of less than 50 bp were removed from each dataset.
a total of  <dig>  and  <dig>  million high-quality reads for a. ilicifolius and a. leucostachyus, respectively, were used for a further de novo assembly.
this assembly was performed with trinity software  <cit> , and generated  <dig>  and  <dig>  contigs for the two species, respectively.
of them,  <dig>  and  <dig>  similar contigs were clustered into  <dig> and  <dig> clusters by tgicl  <cit>  using a default threshold of  <dig>  and cd-hit  <cit>  using a global identity threshold of  <dig> .
a total of  <dig>  and  <dig>  contigs were remained in the two datasets, respectively.table  <dig> sequencing and assembly statistics for the transcriptome data of acanthus ilicifolius and a. leucostachyus.
no.
is short for number
a. ilicifolius
a. leucostachyus
a. ilicifolius
a. leucostachyus
to enhance the reliabilities of the assembly results, we calculated the coverage and the average depth for each contig and removed  <dig> and  <dig> contigs with less than 75 % sites with at least 2× depth from the two data sets, respectively.
at last,  <dig>  and  <dig>  unigenes with average depths of 32× and 31× were retained for a. ilicifolius and a. leucostachyus, respectively.
the average lengths of the remained contigs were  <dig> and 913 bp and n <dig> values were  <dig> and 1580 bp, respectively, suggesting that the transcriptomes for both species were of good quality.
the discrepancy in n <dig> values between the two species may be due to the difference of the genomic complexity.
the length distribution of these contigs is shown in additional file  <dig>
phylogenetic analysis and the divergent time estimation
in this study, we estimated the synonymous substitution rate  of the orthologs between each pair of the two acanthus species and avicennia marina .
the results showed that the peak of ks distribution was  <dig>  between a. ilicifolius and a. leucostachyus, while it was  <dig>  and  <dig>  between av.
marina and a. ilicifolius and a. leucostachyus, respectively .
this unusual high ks value between a. ilicifolius and its terrestrial congener suggests long-term divergence, albeit within the same genus.
our estimation indicated that a. ilicifolius diverged with a. leucostachyus at approximately  <dig>  million years ago  with a 95 % credibility interval  of  <dig>  to  <dig>  my, while the split time of acanthus and avicennia was dated at  <dig>  mya .
the estimated divergence time between acanthaceae s.l.
and pedaliaceae was approximately  <dig>  mya .fig.
<dig> genetic divergence and the divergent time among acanthus ilicifolius, a. leucostachyus and avicennia marina.
a synonymous substitution rates  distribution of the orthologs between each pair of acanthus ilicifolius, a. leucostachyus and av.
marina.
the numbers in parentheses after the species name indicate the number of orthologs used for ks distribution plotting.
b divergent time of a. ilicifolius, a. leucostachyus and av.
marina, as well as the two outgroups sesamum indicum and mimulus guttatus.
the scale bar is  <dig> million years.
the value and purple bar at each node indicate the estimated divergent time  with a 95 % credibility interval
to infer the origin of the mangrove species in acanthus, we reconstructed the phylogenetic tree for the three mangrove species, a. ilicifolius, a. ebracteatus and a. volubilis, and their terrestrial relatives, a. leucostachyus and two european species a. mollis and a. montanus, based on the combined sequences of four nuclear segments, including three nuclear genes  and the nrits region, and the three chloroplast fragments .
maximum likelihood analysis revealed that acanthus fell into two clades with high bootstrap support ; three mangrove species formed one clade, and the three terrestrial species formed the other .
within the mangrove clade, a. ebracteatus formed a single clades with a bs value of 80 %.
compared to a. volubilis, a. ilicifolius and a. ebracteatus was close to each other with strong support .
within the terrestrial clade, a. mollis is sister to a. montanus with 100 % bs.fig.
<dig> phylogenetic tree of six species of acanthus.
the branches of mangroves species were marked in red.
numbers at each node indicate maximum likelihood bootstrap values
furthermore, we calculated the ks for the combined sequences of the three nuclear genes and the kimura two-parameter distances for the nrits and chloroplast fragments .
for the three nuclear genes, the average ks between a. ilicifolius and a. leucostachyus was  <dig> .
based on the divergence time of the two species of  <dig>  mya, the synonymous substitution rate within acanthus was estimated to be  <dig> e- <dig> per synonymous site per year.
according to this synonymous substitution rate, the splitting time between the mangrove clade and the two european terrestrial species was estimated at  <dig>  mya.
within the mangrove clade, the divergence time between a. ilicifolius and a. ebracteatus  was estimated to be  <dig>  mya, while it was estimated to be  <dig>  mya between a. volubilis and the clade of a. ilicifolius and a. ebracteatus .
comparatively, the divergence of a. leucostachyus and the clade of a. montanus and a. mollis was  <dig> , corresponding to  <dig>  mya.
similarly, for nrits and the combined chloroplast fragments, the mean of the kimura two-parameter distances between the mangrove and terrestrial clades were  <dig>  and  <dig> , which are one order of magnitude higher than those among the three mangrove taxa , respectively.
functional annotation
to access the go classification for each gene, the full transcriptome sequences of a. ilicifolius and a. leucostachyus were annotated with the swiss-prot database in agbase  <cit>  with a cutoff e-value of 1e- <dig>  a total of  <dig>   and  <dig>   genes, respectively, were successfully annotated with known go terms.
the go distributions for the two species are shown in fig.
<dig>  in general, the go classifications did not show significant differences between the transcriptome profiles of a. ilicifolius and a. leucostachyus.
in the cellular component category, cell and organelle component-related functions were predominant.
a total of  <dig>   and  <dig>   genes were assigned to cell and  <dig>   and  <dig>   to cell part for the two species, respectively.
there were  <dig>  and  <dig>  genes in a. ilicifolius and  <dig>  and  <dig>  genes in a. leucostachyus annotated with organelle and organelle part.
in molecular function category, binding and catalytic activity were the most enriched, comprising  <dig>   and  <dig>   genes in a. ilicifolius and  <dig>   and  <dig>   genes in a. leucostachyus, respectively.
in the last category, biological process,  <dig>  ,  <dig>   and  <dig>   genes were annotated to three major go terms in a. ilicifolius, including cellular and metabolic process and biological regulation, while  <dig>  ,  <dig>   and  <dig>   genes were assigned to these three terms in a. leucostachyus.
it should be noted that  <dig>   and  <dig>   genes were assigned to the go term response to stimulus, which also comprised a large proportion of the biological process category for both species.
these results indicate that functions with enriched annotation may be quite fundamental and essential for plants.fig.
<dig> gene ontology  distributions for acanthus ilicifolius  and a. leucostachyus .
annotation results were mapped to categories in the second level of go terms, respectively.
go terms that contain less than 1 % of total genes were excluded from this graph.
*, p-value < <dig> .
**, p-value <  <dig>
several go terms significantly differed between a. ilicifolius and a. leucostachyus.
compared with a. leucostachyus, a lower percentage of genes in the cellular component category were annotated with extracellular region and extracellular region part , and a higher percentage to symplast  for a. ilicifolius.
in the molecular function category, genes related to molecular transducer activity were more abundant in a. ilicifolius compared with a. leucostachyus , but those involved in structural molecular activity were less prominent .
in the biological process category, more genes were assigned to multicellular organismal and development processes  as well as cellular component organization  in a. ilicifolius than in a. leucostachyus.
identification of genes under positive selection in a. ilicifolius
we calculated and plotted the non-synonymous to synonymous substitution ratio  for the  <dig> pairs of orthologs between the two acanthus species, a. ilicifolius and a. leucostachyus, as shown in fig.
<dig>  only  <dig> pairs were identified with a ka/ks ratio that was significantly larger than  <dig>  indicating signals of positive selection in these two species .
the functional descriptions of the  <dig> pairs are listed in additional file  <dig>  of them, four were found to be involved in the response to biotic and/or abiotic stimuli .fig.
<dig> ka/ks distribution of  <dig> pairs of orthologs between acanthus ilicifolius and ac.
leucostachyus.
the solid line marks ka/ks =  <dig>  whereas the red dots mark the genes with ka/ks ratio significantly larger than 1
to identify candidate genes under positive selection along the branch of a. ilicifolius, we further tested the  <dig> pairs of orthologs among four species, a. ilicifolius, a. leucostachyus, av.
marina and s. indicum, using the improved branch-site likelihood method in the codeml module of paml .
a total of  <dig> genes were identified as positively selected genes  in a. ilicifolius.
go annotations revealed that  <dig>  and  <dig>  of the  <dig> genes were assigned to metabolic and cellular process, respectively .
of the  <dig> genes,  <dig> sequences were assigned to  <dig> kyoto encyclopedia of genes and genomes  pathways   <cit> .
detailed annotations were performed on these genes and the results are listed in additional file  <dig>  interestingly,  <dig> genes were annotated with functions involving stimulus responses and reproduction, and  <dig> were found to be directly involved in response to abiotic stresses .
these genes fell into three functional groups.
the first group consisted of four genes involved in salt-stress resistance.
one gene of interest, ail_c57143_g1_i <dig>  catalyzes the transfer of an amino group from ornithine to the precursor of proline, l-glutamate 5-semialdehyde, which plays a key role in the proline synthesis pathway.
twelve genes with annotations of response to heat and uv stress were assigned to the second groups.
two genes, ail_c48553_g1_i <dig> and ail_c56385_g1_i <dig>  were found to be involved in the last step of the synthesis of glutathione, which is an important antioxidant that protects important cellular components from damage caused by reactive oxygen species  in plants.
the third groups included seven genes involved in seed germination and embryo development.fig.
<dig> positively selected and expanded key genes in stress resistance pathways of acanthus ilicifolius.
genes, products and pathways for responding to salt-stress and for heat and ultraviolet-stress were marked in blue and orange shading, respectively
we further calculated the fragments per kilobase of exon per million fragments mapped   <cit>  for the  <dig> genes in a. ilicifolius and a. leucostachyus .
we found that  <dig> of the  <dig> genes were expressed at low levels and that their expression levels did not differ between the two species.
in contrast, three genes, ail_c56385_g1_i <dig>  ail_c48553_g1_i <dig> and ail_c52163_g1_i <dig>  were expressed at significantly higher levels in a. ilicifolius than in a. leucostachyus , and two gene, ail_c53207_g1_i <dig> and ail_c51326_g2_i <dig>  showed decreased expression in a. ilicifolius than in a. leucostachyus .
discussion
high divergence between the mangrove and terrestrial species within acanthus
most genera comprising mangrove species contain exclusively mangroves  <cit> , and each genus is considered to be a monophyletic group.
acanthus is the only genus consisting of both true mangroves and terrestrial plants; thus, it is a good system for dissecting the adaptive evolution of mangroves.
in this study, we reconstructed the phylogenetic tree for the genus acanthus, including three mangrove species and three terrestrial relatives, and av.
marina was used as the outgroup.
the topological structure revealed that the mangrove and terrestrial species of acanthus fell into different clades, with the peak of ks distribution of  <dig>  .
the pairwise kimura two-parameter distances of the six acanthus species also revealed that the divergence between the mangrove and terrestrial species of acanthus is one order of magnitude higher than that among the three mangrove taxa .
the splitting time of a. ilicifolius and its terrestrial relative a. leucostachyus was estimated to be  <dig>  mya , while  <dig>  mya between the mangrove clade and the two european species.
these estimations suggest that the mangrove acanthus species entered the intertidal environment in early to middle miocene .
the transgression that occurred from  <dig>  to  <dig>  mya caused dramatic environmental changes and the loss of tropical forests, which may be the major reason for new lineages entering the intertidal environment  <cit> .
in contrast with the high divergence of the mangrove clade and their terrestrial relatives, the three mangrove acanthus species had quite low interspecific divergence, especially between a. ilicifolius and a. ebracteatus.
the average ks between a. volubilis, and the clade of a. ilicifolius and a. ebracteatus was  <dig> , suggesting that the divergence time between these two clades was  <dig>  mya according to the assumed synonymous substitution rate of  <dig> e- <dig> per synonymous site per year.
the two sibling species, a. ilicifolius and a. ebracteatus, show very close but distinct relationship in the phylogenetic trees.
this result is in agreement with the taxonomy based on morphological characteristics, including the open inflorescences, white or purple flowers, absent bracteoles and stem without axillary spines of a. ebracteatus  <cit> , although these two species are considered to be the same species by some authors  <cit> .
in this study, the divergence time between a. ilicifolius and a. ebracteatus was estimated as  <dig>  mya, based on the three nuclear genes.
such recent speciation has also been observed in several mangrove genera, such as sonneratia  <cit> .
however, unlike other mangrove species, a. ilicifolius and a. ebracteatus have nearly identical distributions, suggesting that the primary force driving the divergence of these two species cannot be simply attributed to geographic isolation but that there is a high probability of ecological or adaptive differentiation.
however, this hypothesis requires a further testing in the future.
absence of genetic components specific to the mangrove transcriptomes
resistance to extreme abiotic stress in the intertidal zone is a complex process involving numerous genes that underlie relevant the morphological and metabolic characteristics across the transcriptome.
in the current study, we compared the go classifications between the transcriptomes of a. ilicifolius and its terrestrial relative, a. leucostachyus.
we did not observe any noteworthy differences in go classifications between these two species, except for a small number of go terms.
most of the genes were assigned to fundamental functions, such as cellular and organelle structure, binding and catalytic activity, metabolic process and biological regulation, in agreement with a previous report of the mangrove species s. alba  <cit> .
however, these results are inconsistent with reports of two other mangrove species, r. mangle and heritiera littoralis, which have been suggested to possess mangrove-specific genomic characteristics  <cit> .
the discrepancy between our results and previous study may due to the species specificity and the differences in the tissue types for rna sequencing.
in acanthus, we found that the go classifications between the mangrove and terrestrial species were similar, suggesting few genetic components specific to the mangrove transcriptomes.
positively selected genes in a. ilicifolius
in the past decade, more and more studies have suggested that the differential adaptation of populations is the primary force driving population divergence and speciation .
identifying and characterizing the pattern of selective pressures across a genome, such as sequence divergence, may provide insights into the processes and mechanisms of speciation .
in this study, although no genetic components specific to the mangrove transcriptomes were identified, we identified signals of natural selection in a few genes.
according to the ka/ks ratios,  <dig> of the  <dig> pairs of orthologs between two acanthus species were found to be under positive selection .
of them, one ortholog  was annotated as tip1; <dig>  a gene that is involved in response to salt stress.
the expression of this gene in nacl stress-sensitive yeast mutant strains has been shown to highly increase the resistance to nacl treatment  <cit> .
the ortholog ail_c53470_g4_i <dig> was found to be a homolog of gf <dig> omega, which is considered to be involved in rescuing defects in dna-damage and dna-replication checkpoints and may play an important role in the oxidative stress response  <cit> .
another two orthologs, ail_c48391_g1_i <dig> and ail_c52685_g1_i <dig>  function in response to attack by microbes  <cit> .
furthermore, to investigate the genetic bases of the adaptive traits of a. ilicifolius, we employed the improved branch-site likelihood method and identified  <dig> candidate psgs out of the  <dig> orthologous genes in a. ilicifolius.
in contrast with its terrestrial relative a. leucostachyus, a. ilicifolius is faced with three major abiotic stresses in the intertidal environment, high salinity, high temperatures and ultraviolet radiation, and periodic inundation.
the annotations indicated that a total of  <dig> psgs were involved in the responses to these three stress conditions .
four genes that function in response to salt stress were assigned to the first group .
of them, ail_c57143_g1_i <dig>  annotated as ornithine aminotransferase, may play a key role in responding to salt stress in a. ilicifolius.
the product of this gene transfers the amine group from ornithine to glutamate, a precursor of proline  <cit> .
proline is known to be the major osmolyte involved in response to salt stress in a. ilicifolius, as well as many other mangroves and terrestrial plants .
another gene, ail_cl2465contig <dig>  is homologous to the gene encoding ubiquitin-specific protease  <dig>  of arabidopsis thaliana, which could increase salt tolerance by positively regulating plasma membrane na/h antiport activity under salt stress condition  <cit> .
the second group consisted of  <dig> genes related to scavenging and the repairing of damage due to oxidative and superoxide stress and uv radiation under high temperature and light conditions .
in the intertidal zone, high temperatures and ultraviolet radiation may result in the dramatic accumulation of ros, leading to severe damage of dna and cell structures  <cit> .
two of the ten genes, ail_c48553_g1_i <dig> and ail_c56385_g1_i <dig>  are involved in the last step of the synthesis of glutathione, which is an important antioxidant in plants that protects vital cellular components from damage caused by ros  <cit> .
another gene ail_c51326_g2_i <dig> showed homology to the gene encoding the chloroplastic drought-induced stress protein of  <dig> kd  of solanum tuberosum.
cdsp <dig> is a thioredoxin that participates in defense against oxidative damage in photosynthetic membranes .
seven genes involved in seed germination and embryo development were assigned into the third group.
zhang et al.
<cit>  have revealed that short-term tide inundation of <15 h per day has no impact on the time of seed germination; however, long-term inundation of ≥15 h per day delays seed germination and decreases the germination rate in a. ilicifolius.
genes ail_cl5647contig <dig> is homologous to briz <dig> of arabidopsis thaliana.
briz <dig> is involved in forming a heteromeric e <dig> ligase complex, which is required for seed germination and post-germination growth  <cit> .
another three genes ail_cl3016contig <dig>  ail_cl3870contig <dig> and ail_c54714_g1_i <dig> were found to play important role in embryo development .
thus, these genes may greatly improve the reproductive success of a. ilicifolius in harsh coastal environments.
these three groups of resistance related psgs are crucial to the survival and adaptation of a. ilicifolius to extreme habitats.
adaptation to the differential environments might be the primary force driving species diversification in acanthus.
the fpkm values of the most of the  <dig> genes were low for both a. ilicifolius and a. leucostachyus, suggesting that there was no apparent increase in the expression of these genes in response to salt stress in a. ilicifolius under natural conditions .
in contrast to terrestrial glycophytes, mangrove trees are more adaptive to moderate levels of salt than to freshwater environments  <cit> .
transcription analysis of ceriops tagal has also revealed that most salt-induced genes are involved in long-term strategies in response to short-term high-salinity stress  <cit> .
a total of three genes were more highly expressed in a. ilicifolius than in a. leucostachyus.
two of the three were homologous to the gene encoding gamma-hydroxybutyrate dehydrogenase , which functions in oxidative stress tolerance in arabidopsis thaliana .
in contrast with a. leucostachyus, which was collected under rainforest canopies, a. ilicifolius was sampled from frontal thickets on the stream edges of recently accreting estuarine banks of zhujiang river.
direct sunlight may cause the higher expression of these two oxidative stress-related genes in a. ilicifolius.
the third gene, ail_c52163_g1_i <dig>  is homologous to the gene encoding chloroplast stem-loop binding protein of 41 kda, which is involved in oxidative stress tolerance and chloroplast rna metabolism in seedlings.
in contrast, the expression of ail_c51326_g2_i <dig>  the product of which is cdsp <dig>  was two-fold higher in a. leucostachyus compared with a. ilicifolius.
this result is in agreement with parida  <cit> , who has reported that polypeptides with a molecular weight of 32 kda are decreased in b. parviflora as a result of salt stress.
however, in this study, only one individual was collected for each species with no biological replicates; therefore, the comparison of expression levels between the two species are not that reliable and only represent preliminary findings that may be expanded upon in a further investigation based on more samples.
CONCLUSIONS
acanthus is the only genus consisting of both mangrove and terrestrial species.
in this study, we characterized the transcriptomes of one mangrove species in acanthus, a. ilicifolius, and its terrestrial relative a. leucostachyus.
this comparative transcriptome analysis has greatly enriched the current knowledge of the origin of the mangrove species in acanthus and their adaptive evolution to abiotic stresses in the intertidal environment.
phylogenetic analysis indicated that the initial entry of the mangrove species in acanthus into the intertidal environment might have occurred during the transgression in the early to middle miocene.
however, our study found that positive selection plays an important role in the process of adaption to harsh intertidal zones.

 tropical intertidal zones are extreme environments characterized by high salinity, drought, hypoxia and high ultraviolet radiation, which severely limit plant growth, development and reproduction. as the dominant forest community and ecosystem in the coasts, mangrove plants struggle and survive in these environments with remarkable morphological and physiological characteristic, for example, exposed breathing roots, support roots and buttresses, salt-excreting leaves, and viviparous water-dispersed propagules. understanding the genetic basis underlying those adaptive traits at the genomic level could provide important clues to the molecular mechanisms of stress resistance in marine halophytes. mangroves are constituent plants of approximately species from genera belonging to families. studies based on fossils and phylogenetic analysis have suggested that these biogeographically and taxonomically diverse genera are of independent origins in different geologic epochs. however, the divergent time and the species radiation within some genera are still unclear and are of great interest to many botanists. comparative analysis among mangrove species and model terrestrial plants using rna-sequencing has revealed that specific sequence divergence and transcriptional regulation play major roles in response to salt stress in many mangrove species, such as aegiceras corniculatum, bruguiera gymnorhiza and sonneratia alba. however, the model plants used in these studies, such as arabidopsis thaliana and populus tomentosa, are distantly related to mangroves, which makes it difficult to distinguish adaptive processes from those caused by phylogenetic effects, thus reducing the resolution of selection signals. among all true mangrove genera, acanthus is the only genus that includes both mangrove and terrestrial species; therefore, it is an ideal system to investigate the adaptive evolution of mangrove plants to stressful intertidal environments while minimizing phylogenetic influences. this genus consists of three representative mangrove species and approximately terrestrial species. the three mangrove species, which are also called holly mangroves, are found in the intertidal zones from india to the west pacific and tropical australia. unlike other terrestrial species of acanthus, which are distributed in the mediterranean basin, a. leucostachyus is restrict to south to southeast asia and grows under rain forest canopies at the edges of streams 600– m above the sea level. similarities in geographic distribution and ecological requirements of a. leucostachyus and the congeneric mangrove species suggested a. leucostachyus is a suitable outgroup for illustrating the evolutionary history of the mangrove species in acanthus. nguyen et al. identified genes that are involved in response to salt stress from expressed sequence tags of a. ebracteatus. however, these studies only listed the annotations of these stress-response genes. the role of natural selection in adaptive evolution of mangrove species has not been characterized. in the current study, we performed rna-sequencing to assess the mangrove species a. ilicifolius and its terrestrial congener a. leucostachyus, to provide insights into the evolutionary process underlying the adaptation of mangroves to intertidal zones. we asked the following specific questions: 1) what is the origination pattern of the mangrove acanthus taxa and when mangrove species diverged from their terrestrial relatives within this genus? 2) are there any marked differences in gene ontology classification among the mangrove transcriptome profiles? finally, 3) which genes have experienced adaptive evolution and contributed to the adaptation of a. ilicifolius to stressful intertidal habitats?  transcriptome sequencing and de novo assembly a total of million 90-bp and million 100-bp paired-end reads were sequenced for a. ilicifolius and a. leucostachyus, respectively. the raw data were deposited in the ncbi sequence read archive with the accession number srp to minimize sequencing errors, we individually trimmed each read to its longest contiguous segment, using quality scores of higher than for all remaining segments. after the trimming processes, reads with length of less than 50 bp were removed from each dataset. a total of and million high-quality reads for a. ilicifolius and a. leucostachyus, respectively, were used for a further de novo assembly. this assembly was performed with trinity software, and generated and contigs for the two species, respectively. of them, and similar contigs were clustered into and clusters by tgicl using a default threshold of and cd-hit using a global identity threshold of. a total of and contigs were remained in the two datasets, respectively.table sequencing and assembly statistics for the transcriptome data of acanthus ilicifolius and a. leucostachyus. no. is short for number a. ilicifolius a. leucostachyus a. ilicifolius a. leucostachyus to enhance the reliabilities of the assembly results, we calculated the coverage and the average depth for each contig and removed and contigs with less than 75 % sites with at least 2× depth from the two data sets, respectively. at last, and unigenes with average depths of 32× and 31× were retained for a. ilicifolius and a. leucostachyus, respectively. the average lengths of the remained contigs were and 913 bp and n values were and 1580 bp, respectively, suggesting that the transcriptomes for both species were of good quality. the discrepancy in n values between the two species may be due to the difference of the genomic complexity. the length distribution of these contigs is shown in additional file phylogenetic analysis and the divergent time estimation in this study, we estimated the synonymous substitution rate of the orthologs between each pair of the two acanthus species and avicennia marina. the results showed that the peak of ks distribution was between a. ilicifolius and a. leucostachyus, while it was and between av. marina and a. ilicifolius and a. leucostachyus, respectively. this unusual high ks value between a. ilicifolius and its terrestrial congener suggests long-term divergence, albeit within the same genus. our estimation indicated that a. ilicifolius diverged with a. leucostachyus at approximately million years ago with a 95 % credibility interval of to my, while the split time of acanthus and avicennia was dated at mya. the estimated divergence time between acanthaceae s.l. and pedaliaceae was approximately mya.fig. genetic divergence and the divergent time among acanthus ilicifolius, a. leucostachyus and avicennia marina. a synonymous substitution rates distribution of the orthologs between each pair of acanthus ilicifolius, a. leucostachyus and av. marina. the numbers in parentheses after the species name indicate the number of orthologs used for ks distribution plotting. b divergent time of a. ilicifolius, a. leucostachyus and av. marina, as well as the two outgroups sesamum indicum and mimulus guttatus. the scale bar is million years. the value and purple bar at each node indicate the estimated divergent time with a 95 % credibility interval to infer the origin of the mangrove species in acanthus, we reconstructed the phylogenetic tree for the three mangrove species, a. ilicifolius, a. ebracteatus and a. volubilis, and their terrestrial relatives, a. leucostachyus and two european species a. mollis and a. montanus, based on the combined sequences of four nuclear segments, including three nuclear genes and the nrits region, and the three chloroplast fragments. maximum likelihood analysis revealed that acanthus fell into two clades with high bootstrap support; three mangrove species formed one clade, and the three terrestrial species formed the other. within the mangrove clade, a. ebracteatus formed a single clades with a bs value of 80 %. compared to a. volubilis, a. ilicifolius and a. ebracteatus was close to each other with strong support. within the terrestrial clade, a. mollis is sister to a. montanus with 100 % bs.fig. phylogenetic tree of six species of acanthus. the branches of mangroves species were marked in red. numbers at each node indicate maximum likelihood bootstrap values furthermore, we calculated the ks for the combined sequences of the three nuclear genes and the kimura two-parameter distances for the nrits and chloroplast fragments. for the three nuclear genes, the average ks between a. ilicifolius and a. leucostachyus was. based on the divergence time of the two species of mya, the synonymous substitution rate within acanthus was estimated to be e- per synonymous site per year. according to this synonymous substitution rate, the splitting time between the mangrove clade and the two european terrestrial species was estimated at mya. within the mangrove clade, the divergence time between a. ilicifolius and a. ebracteatus was estimated to be mya, while it was estimated to be mya between a. volubilis and the clade of a. ilicifolius and a. ebracteatus. comparatively, the divergence of a. leucostachyus and the clade of a. montanus and a. mollis was, corresponding to mya. similarly, for nrits and the combined chloroplast fragments, the mean of the kimura two-parameter distances between the mangrove and terrestrial clades were and, which are one order of magnitude higher than those among the three mangrove taxa, respectively. functional annotation to access the go classification for each gene, the full transcriptome sequences of a. ilicifolius and a. leucostachyus were annotated with the swiss-prot database in agbase with a cutoff e-value of 1e- a total of and genes, respectively, were successfully annotated with known go terms. the go distributions for the two species are shown in fig.  in general, the go classifications did not show significant differences between the transcriptome profiles of a. ilicifolius and a. leucostachyus. in the cellular component category, cell and organelle component-related functions were predominant. a total of and genes were assigned to cell and and to cell part for the two species, respectively. there were and genes in a. ilicifolius and and genes in a. leucostachyus annotated with organelle and organelle part. in molecular function category, binding and catalytic activity were the most enriched, comprising and genes in a. ilicifolius and and genes in a. leucostachyus, respectively. in the last category, biological process,, and genes were annotated to three major go terms in a. ilicifolius, including cellular and metabolic process and biological regulation, while, and genes were assigned to these three terms in a. leucostachyus. it should be noted that and genes were assigned to the go term response to stimulus, which also comprised a large proportion of the biological process category for both species. these results indicate that functions with enriched annotation may be quite fundamental and essential for plants.fig. gene ontology distributions for acanthus ilicifolius and a. leucostachyus. annotation results were mapped to categories in the second level of go terms, respectively. go terms that contain less than 1 % of total genes were excluded from this graph. *, p-value <. **, p-value <  several go terms significantly differed between a. ilicifolius and a. leucostachyus. compared with a. leucostachyus, a lower percentage of genes in the cellular component category were annotated with extracellular region and extracellular region part, and a higher percentage to symplast for a. ilicifolius. in the molecular function category, genes related to molecular transducer activity were more abundant in a. ilicifolius compared with a. leucostachyus, but those involved in structural molecular activity were less prominent. in the biological process category, more genes were assigned to multicellular organismal and development processes as well as cellular component organization in a. ilicifolius than in a. leucostachyus. identification of genes under positive selection in a. ilicifolius we calculated and plotted the non-synonymous to synonymous substitution ratio for the pairs of orthologs between the two acanthus species, a. ilicifolius and a. leucostachyus, as shown in fig.  only pairs were identified with a ka/ks ratio that was significantly larger than indicating signals of positive selection in these two species. the functional descriptions of the pairs are listed in additional file of them, four were found to be involved in the response to biotic and/or abiotic stimuli.fig. ka/ks distribution of pairs of orthologs between acanthus ilicifolius and ac. leucostachyus. the solid line marks ka/ks =  whereas the red dots mark the genes with ka/ks ratio significantly larger than 1 to identify candidate genes under positive selection along the branch of a. ilicifolius, we further tested the pairs of orthologs among four species, a. ilicifolius, a. leucostachyus, av. marina and s. indicum, using the improved branch-site likelihood method in the codeml module of paml. a total of genes were identified as positively selected genes in a. ilicifolius. go annotations revealed that and of the genes were assigned to metabolic and cellular process, respectively. of the genes, sequences were assigned to kyoto encyclopedia of genes and genomes pathways. detailed annotations were performed on these genes and the results are listed in additional file interestingly, genes were annotated with functions involving stimulus responses and reproduction, and were found to be directly involved in response to abiotic stresses. these genes fell into three functional groups. the first group consisted of four genes involved in salt-stress resistance. one gene of interest, ail_c57143_g1_i catalyzes the transfer of an amino group from ornithine to the precursor of proline, l-glutamate 5-semialdehyde, which plays a key role in the proline synthesis pathway. twelve genes with annotations of response to heat and uv stress were assigned to the second groups. two genes, ail_c48553_g1_i and ail_c56385_g1_i were found to be involved in the last step of the synthesis of glutathione, which is an important antioxidant that protects important cellular components from damage caused by reactive oxygen species in plants. the third groups included seven genes involved in seed germination and embryo development.fig. positively selected and expanded key genes in stress resistance pathways of acanthus ilicifolius. genes, products and pathways for responding to salt-stress and for heat and ultraviolet-stress were marked in blue and orange shading, respectively we further calculated the fragments per kilobase of exon per million fragments mapped for the genes in a. ilicifolius and a. leucostachyus. we found that of the genes were expressed at low levels and that their expression levels did not differ between the two species. in contrast, three genes, ail_c56385_g1_i ail_c48553_g1_i and ail_c52163_g1_i were expressed at significantly higher levels in a. ilicifolius than in a. leucostachyus, and two gene, ail_c53207_g1_i and ail_c51326_g2_i showed decreased expression in a. ilicifolius than in a. leucostachyus. discussion high divergence between the mangrove and terrestrial species within acanthus most genera comprising mangrove species contain exclusively mangroves, and each genus is considered to be a monophyletic group. acanthus is the only genus consisting of both true mangroves and terrestrial plants; thus, it is a good system for dissecting the adaptive evolution of mangroves. in this study, we reconstructed the phylogenetic tree for the genus acanthus, including three mangrove species and three terrestrial relatives, and av. marina was used as the outgroup. the topological structure revealed that the mangrove and terrestrial species of acanthus fell into different clades, with the peak of ks distribution of. the pairwise kimura two-parameter distances of the six acanthus species also revealed that the divergence between the mangrove and terrestrial species of acanthus is one order of magnitude higher than that among the three mangrove taxa. the splitting time of a. ilicifolius and its terrestrial relative a. leucostachyus was estimated to be mya, while mya between the mangrove clade and the two european species. these estimations suggest that the mangrove acanthus species entered the intertidal environment in early to middle miocene. the transgression that occurred from to mya caused dramatic environmental changes and the loss of tropical forests, which may be the major reason for new lineages entering the intertidal environment. in contrast with the high divergence of the mangrove clade and their terrestrial relatives, the three mangrove acanthus species had quite low interspecific divergence, especially between a. ilicifolius and a. ebracteatus. the average ks between a. volubilis, and the clade of a. ilicifolius and a. ebracteatus was, suggesting that the divergence time between these two clades was mya according to the assumed synonymous substitution rate of e- per synonymous site per year. the two sibling species, a. ilicifolius and a. ebracteatus, show very close but distinct relationship in the phylogenetic trees. this result is in agreement with the taxonomy based on morphological characteristics, including the open inflorescences, white or purple flowers, absent bracteoles and stem without axillary spines of a. ebracteatus, although these two species are considered to be the same species by some authors. in this study, the divergence time between a. ilicifolius and a. ebracteatus was estimated as mya, based on the three nuclear genes. such recent speciation has also been observed in several mangrove genera, such as sonneratia. however, unlike other mangrove species, a. ilicifolius and a. ebracteatus have nearly identical distributions, suggesting that the primary force driving the divergence of these two species cannot be simply attributed to geographic isolation but that there is a high probability of ecological or adaptive differentiation. however, this hypothesis requires a further testing in the future. absence of genetic components specific to the mangrove transcriptomes resistance to extreme abiotic stress in the intertidal zone is a complex process involving numerous genes that underlie relevant the morphological and metabolic characteristics across the transcriptome. in the current study, we compared the go classifications between the transcriptomes of a. ilicifolius and its terrestrial relative, a. leucostachyus. we did not observe any noteworthy differences in go classifications between these two species, except for a small number of go terms. most of the genes were assigned to fundamental functions, such as cellular and organelle structure, binding and catalytic activity, metabolic process and biological regulation, in agreement with a previous report of the mangrove species s. alba. however, these results are inconsistent with reports of two other mangrove species, r. mangle and heritiera littoralis, which have been suggested to possess mangrove-specific genomic characteristics. the discrepancy between our results and previous study may due to the species specificity and the differences in the tissue types for rna sequencing. in acanthus, we found that the go classifications between the mangrove and terrestrial species were similar, suggesting few genetic components specific to the mangrove transcriptomes. positively selected genes in a. ilicifolius in the past decade, more and more studies have suggested that the differential adaptation of populations is the primary force driving population divergence and speciation. identifying and characterizing the pattern of selective pressures across a genome, such as sequence divergence, may provide insights into the processes and mechanisms of speciation. in this study, although no genetic components specific to the mangrove transcriptomes were identified, we identified signals of natural selection in a few genes. according to the ka/ks ratios, of the pairs of orthologs between two acanthus species were found to be under positive selection. of them, one ortholog was annotated as tip1; a gene that is involved in response to salt stress. the expression of this gene in nacl stress-sensitive yeast mutant strains has been shown to highly increase the resistance to nacl treatment. the ortholog ail_c53470_g4_i was found to be a homolog of gf omega, which is considered to be involved in rescuing defects in dna-damage and dna-replication checkpoints and may play an important role in the oxidative stress response. another two orthologs, ail_c48391_g1_i and ail_c52685_g1_i function in response to attack by microbes. furthermore, to investigate the genetic bases of the adaptive traits of a. ilicifolius, we employed the improved branch-site likelihood method and identified candidate psgs out of the orthologous genes in a. ilicifolius. in contrast with its terrestrial relative a. leucostachyus, a. ilicifolius is faced with three major abiotic stresses in the intertidal environment, high salinity, high temperatures and ultraviolet radiation, and periodic inundation. the annotations indicated that a total of psgs were involved in the responses to these three stress conditions. four genes that function in response to salt stress were assigned to the first group. of them, ail_c57143_g1_i annotated as ornithine aminotransferase, may play a key role in responding to salt stress in a. ilicifolius. the product of this gene transfers the amine group from ornithine to glutamate, a precursor of proline. proline is known to be the major osmolyte involved in response to salt stress in a. ilicifolius, as well as many other mangroves and terrestrial plants. another gene, ail_cl2465contig is homologous to the gene encoding ubiquitin-specific protease of arabidopsis thaliana, which could increase salt tolerance by positively regulating plasma membrane na/h antiport activity under salt stress condition. the second group consisted of genes related to scavenging and the repairing of damage due to oxidative and superoxide stress and uv radiation under high temperature and light conditions. in the intertidal zone, high temperatures and ultraviolet radiation may result in the dramatic accumulation of ros, leading to severe damage of dna and cell structures. two of the ten genes, ail_c48553_g1_i and ail_c56385_g1_i are involved in the last step of the synthesis of glutathione, which is an important antioxidant in plants that protects vital cellular components from damage caused by ros. another gene ail_c51326_g2_i showed homology to the gene encoding the chloroplastic drought-induced stress protein of kd of solanum tuberosum. cdsp is a thioredoxin that participates in defense against oxidative damage in photosynthetic membranes. seven genes involved in seed germination and embryo development were assigned into the third group. zhang et al. have revealed that short-term tide inundation of <15 h per day has no impact on the time of seed germination; however, long-term inundation of ≥15 h per day delays seed germination and decreases the germination rate in a. ilicifolius. genes ail_cl5647contig is homologous to briz of arabidopsis thaliana. briz is involved in forming a heteromeric e ligase complex, which is required for seed germination and post-germination growth. another three genes ail_cl3016contig ail_cl3870contig and ail_c54714_g1_i were found to play important role in embryo development. thus, these genes may greatly improve the reproductive success of a. ilicifolius in harsh coastal environments. these three groups of resistance related psgs are crucial to the survival and adaptation of a. ilicifolius to extreme habitats. adaptation to the differential environments might be the primary force driving species diversification in acanthus. the fpkm values of the most of the genes were low for both a. ilicifolius and a. leucostachyus, suggesting that there was no apparent increase in the expression of these genes in response to salt stress in a. ilicifolius under natural conditions. in contrast to terrestrial glycophytes, mangrove trees are more adaptive to moderate levels of salt than to freshwater environments. transcription analysis of ceriops tagal has also revealed that most salt-induced genes are involved in long-term strategies in response to short-term high-salinity stress. a total of three genes were more highly expressed in a. ilicifolius than in a. leucostachyus. two of the three were homologous to the gene encoding gamma-hydroxybutyrate dehydrogenase, which functions in oxidative stress tolerance in arabidopsis thaliana. in contrast with a. leucostachyus, which was collected under rainforest canopies, a. ilicifolius was sampled from frontal thickets on the stream edges of recently accreting estuarine banks of zhujiang river. direct sunlight may cause the higher expression of these two oxidative stress-related genes in a. ilicifolius. the third gene, ail_c52163_g1_i is homologous to the gene encoding chloroplast stem-loop binding protein of 41 kda, which is involved in oxidative stress tolerance and chloroplast rna metabolism in seedlings. in contrast, the expression of ail_c51326_g2_i the product of which is cdsp was two-fold higher in a. leucostachyus compared with a. ilicifolius. this result is in agreement with parida, who has reported that polypeptides with a molecular weight of 32 kda are decreased in b. parviflora as a result of salt stress. however, in this study, only one individual was collected for each species with no biological replicates; therefore, the comparison of expression levels between the two species are not that reliable and only represent preliminary findings that may be expanded upon in a further investigation based on more samples.  acanthus is the only genus consisting of both mangrove and terrestrial species. in this study, we characterized the transcriptomes of one mangrove species in acanthus, a. ilicifolius, and its terrestrial relative a. leucostachyus. this comparative transcriptome analysis has greatly enriched the current knowledge of the origin of the mangrove species in acanthus and their adaptive evolution to abiotic stresses in the intertidal environment. phylogenetic analysis indicated that the initial entry of the mangrove species in acanthus into the intertidal environment might have occurred during the transgression in the early to middle miocene. however, our study found that positive selection plays an important role in the process of adaption to harsh intertidal zones. 
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. 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. 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. we obtained and contigs with n values of and 1557 bp for a. ilicifolius and a. leucostachyus, respectively. based on orthologs, a. ilicifolius and a. leucostachyus were found to be highly divergent, with a peak of synonymous substitution rate distribution of and an estimated divergence time of approximately million years ago. gene ontology 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. twenty-three of the positively selected genes were found to be involved in salt, heat and ultraviolet stress tolerance, seed germination and embryo development under periodic inundation. 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. a total of genes in a. ilicifolius were identified with signals of positive selection. 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. however, little is known regarding the two respects of mangrove species in acanthus. 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.
 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.  we obtained and contigs with n values of 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 orthologs, a. ilicifolius and a. leucostachyus were found to be highly divergent, with a peak of synonymous substitution rate distribution of and an estimated divergence time of approximately million years ago. 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 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 genes in a. ilicifolius were identified with signals of positive selection. twenty-three of the positively selected genes 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.  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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords abiotic stressadaptationmangrovescomparative transcriptomeissue-copyright-statement© the author 2015 