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. 2019 Mar 28;11(2):plz019. doi: 10.1093/aobpla/plz019

Comparative transcriptomics sheds light on differential adaptation and species diversification between two Melastoma species and their F1 hybrid

Wei Lun Ng 1,2, Wei Wu 1, Peishan Zou 1, Renchao Zhou 1,✉
PMCID: PMC6481908  PMID: 31037213

Variation in gene expression promotes adaptive divergence. In this study, we performed comparative transcriptomics on Melastoma candidum, M. sanguineum and their F1 hybrid to investigate the role of gene expression in plant species diversification and hybridization. Differentially expressed genes were mostly found between the two parental species, and were related to adaptive traits that limit the species to their habitats. In the F1 hybrid, although the expression levels of most genes were similar to either parent, a small number of genes had expression levels exceeding both parents, possibly explaining its vigour in certain morphological and adaptive traits.

Keywords: Adaptive divergence, comparative transcriptomics, Melastoma, transgressive segregation, trichome

Abstract

Variation in gene expression has been shown to promote adaptive divergence, and can lead to speciation. The plant genus Melastoma, thought to have diversified through adaptive radiation, provides an excellent model for the study of gene expressional changes during adaptive differentiation and following interspecific hybridization. In this study, we performed RNA-seq on M. candidum, M. sanguineum and their F1 hybrid, to investigate the role of gene expression in species diversification within the genus. Reference transcriptomes were assembled using combined data from both parental species, resulting in 50 519 and 48 120 transcripts for the leaf and flower petal, after removing redundancy. Differential expression analysis uncovered 3793 and 2116 differentially expressed (DE) transcripts, most of which are between M. candidum and M. sanguineum. Differential expression was observed for genes related to light responses, as well as genes that regulate the development of leaf trichomes, a trait that among others is thought to protect plants against sunlight, suggesting the differential adaptation of the species to sunlight intensity. The analysis of positively selected genes between the two species also revealed possible differential adaptation to other abiotic stresses such as drought and temperature. In the hybrid, almost all possible modes of expression were observed at the DE transcripts, although at most transcripts, the expression levels were similar to that of either parent instead of being intermediate. A small number of transgressively expressed transcripts that matched genes known to promote plant growth and adaptation to stresses in new environments were also found, possibly explaining the vigour observed in the hybrid. The findings in this study provided insights into the role of gene expression in the diversification of Melastoma, which we believe is an important example for more cross-taxa comparisons in the future.

Introduction

In speciation, the formation of new lineages can be driven by two basic evolutionary processes—lineage divergence and lineage fusion. Lineage divergence can happen through differential adaptation or genetic drift, with the former expected to proceed more rapidly when the effective population size is large (Jensen and Bachtrog 2011). Lineage fusion through hybridization, on the other hand, can provide raw material for adaptation to various habitats by creating new allelic combinations and phenotypes (Arnold et al. 2012). When the two drivers of speciation take effect on a single group of taxa, rapid species radiation can occur, as seen in the cichlid fishes of Africa (Meier et al. 2017) and the kiwifruit (Liu et al. 2017).

While the majority of studies on lineage divergence has focused on sequence or structural variation (e.g. Turner et al. 2005; Schluter and Conte 2009; Hendrick et al. 2016; Lindtke et al. 2017), variation in gene expression has also been shown to be relevant in speciation (e.g. Wolf et al. 2010). Its interaction with the environment can promote adaptive divergence—when a population colonizes a new environment, the regulation of gene expression becomes critical in ensuring the persistence of a population. In time, this also promotes genetic divergence in adaptive traits, eventually causing reproductive isolation between populations (Pavey et al. 2010)—and this can have profound impacts on the diversification of species. A similar impact can be attained following lineage fusion: in hybridization, a first-generation (F1) hybrid receives half of its genetic material from each of its parents. The resulting gene expressional patterns may or may not be additive of that observed in the parental taxa (Birchler et al. 2003). Transcriptomic shock, usually presented in the form of the complete suppression of transcripts from one parent and widespread up- or down-regulation of gene expression (Hegarty 2011; Barreto et al. 2015), can result in novel phenotypes or even transgressive phenotypes that exceed that of the parents (i.e. transgressive segregation; deVicente and Tanksley 1993; Rieseberg et al. 1999; Rieseberg et al. 2003). These attributes, given the right environment and intrinsic compatibility, may then lead to rapid establishment of the hybrid population into an independent evolutionary lineage (Abbott et al. 2013).

The shrub genus Melastoma (Melastomataceae) provides an excellent model to study the gene expressional changes during adaptive differentiation and following interspecific hybridization. Melastoma is thought to have diversified through adaptive radiation (Renner and Meyer 2001), with many species within the genus having evolved to fit into different ecological niches, such as occupying lowlands vs. montane elevations and open vs. shady environments (Wong 2016). At the same time, their recent divergence (i.e. 1 million years ago; Renner and Meyer 2001) and shared life history characteristics, such as partially overlapping geographic distribution and flowering periods (Chen 1984), as well as shared pollinators (Gross 1993; Luo et al. 2008), allow hybridization to happen easily between the co-occurring species. Two species, M. candidum and M. sanguineum, are sympatrically distributed in southern China and hybridize extensively (Liu et al. 2014). Despite the largely shared geographical distribution, they have different habitat preferences: in the wild, M. candidum prefers exposed environments such as open fields, grasslands and roadsides, while M. sanguineum prefers shady environments and is often found at the edges of forests. This adaptation to different sunlight intensities seems to be related to the presence and absence of trichomes (hair-like structures) on the surface of their mature leaves (Fig. 1). Of their various functions, trichomes are thought to play a role in protection of the leaves from sunlight (Wagner et al. 2004; Hauser 2014), and thus may hold the key to adaptive divergence between the two Melastoma species. Their F1 hybrid, on the other hand, displays trichome length intermediate between its parents, while having more vigorous growth (e.g. bigger leaves and flowers, faster growth) and seem capable of crossing over adaptation barriers (e.g. sunlight exposure) that limit the parental species to their habitats (W. L. Ng and R. Zhou, pers. obs.). In fact, during a survey at the Diaoluo Mountain in Hainan, China, we observed hybrid individuals that flourished in constant-shaded habitats and at higher altitudes compared to the parents. Such displays of both intermediate and transgressive phenotypes in the hybrids demonstrate the possible role of gene expressional regulation following hybridization.

Figure 1.

Figure 1.

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Leaf morphology (top) and trichome structure observed under ×10 magnification (bottom) for M. candidum, M. sanguineum and their F1 hybrid. Differences in the length and density of trichome present on the leaf surface partly contribute to the difference in visible coloration of the leaves.

Earlier studies that use transcriptomic data to compare gene expression in plants have mostly looked at gene expressional changes within a single species subjected to different treatments (e.g. Carvajal et al. 2018) or following hybridization of different varieties/ecotypes of the same species (e.g. Shen et al. 2012; Bell et al. 2013; He et al. 2013). Few have actually looked at differences between divergent species, presumably due to the complexity of comparing large-scale gene expression across species (Wolf et al. 2010; Kristiansson et al. 2013; Roux et al. 2015; Shimizu-Inatsugi et al. 2016). Our study therefore aimed to look further to compare gene expression between two plant species, and the expressional changes following hybridization, using M. candidum, M. sanguineum and their F1 hybrid, as a model system. Such comparisons can elucidate the molecular mechanisms that allow for differential adaptation of the taxa to differing habitats, which could be a major underlying cause for species radiation within the genus Melastoma.

Materials and Methods

Sampling and RNA extraction

Two individuals (biological replicates) each of M. candidum, M. sanguineum and their F1 hybrid (‘hybrid’) were grown in the greenhouse at Sun Yat-sen University, Guangzhou, China, under a white shade cloth with a low level of shade (i.e. 20 %), during which all individuals showed normal growth. The individuals were initially collected from Qiongzhong, Hainan, China. The F1 hybrid status of the hybrid individuals was confirmed by sequencing six nuclear loci used in our previous studies on Melastoma hybrid identification (Dai et al. 2012; Zhou et al. 2017; Zou et al. 2017). Samples of two tissue types, i.e. leaf and flower petal (‘petal’), were harvested at 0900 h in the morning of 30 June 2017, when the petals exhibited complete opening, snap-frozen in liquid N2 and stored at −80 °C until RNA extraction. Total RNA was extracted using the RNAprep Pure Plant Kit (polysaccharides and polyphenolics-rich; TIANGEN Biotech Co. Ltd, Beijing, China). Voucher specimens of the samples used in this study (NG201801–NG201806) were deposited in the Herbarium of Sun Yat-sen University (SYS).

RNA-seq and data preprocessing

Upon RNA integrity checking and quantification (integrity of RNA samples were measured with an Agilent 2100 Bioanalyzer and RIN (RNA Integrity Number) of >7 is required), the total RNA samples were subjected to library construction (insert size ~450 bp, strand-specific using the dUTP protocol; Parkhomchuk et al. 2009) and Illumina HiSeq sequencing (150 bp paired-end). The quality of the obtained raw sequencing reads was first examined using FastQC (Babraham Bioinformatics; www.bioinformatics.babraham.ac.uk/projects/fastqc/) ver. 0.11.3. Then, a series of quality filtering steps were conducted on the raw reads, including the removal of adapter sequences, removal of reads containing >5 % of N bases, trimming of bases with Phred Q score <20 (i.e. error rate ≥1 %) and removal of sequences with average Phred Q score <20. Only paired-end reads with length ≥50 bp were kept. All filtering steps were conducted using BBDuk embedded in BBTools (jgi.doe.gov/data-and-tools/bbtools) ver. 37.76. The quality-filtered reads were considered high-quality (‘HQ’) and were used for the subsequent assembly and mapping steps.

De novo assembly and redundancy reduction

The Trinity (Grabherr et al. 2011; Haas et al. 2013) pipeline for genome-guided de novo assembly was used to assemble transcripts from the RNA-seq data. In order to conduct analysis on differential expression among M. candidum, M. sanguineum and the hybrid, a common set of reference transcripts had to be generated. The common set of reference transcripts were obtained by generating a ‘master’ transcriptome assembled from reads from both parental species. All four HQ data sets of M. candidum and M. sanguineum (two replicates per species, each tissue type treated separately) were first combined and normalized using the Trinity script insilico_read_normalization.pl [max_cov = 50, pairs_together, SS_lib_type = RF]. Then, the normalized reads were aligned to a M. candidum draft genome (W. Wu et al., unpubl. data) using the STAR aligner (Dobin et al. 2013) ver. 2.5.3, prior to de novo assembly using Trinity ver. 2.5.1.

To reduce redundancy, the tr2aacds pipeline of the EvidentialGene package (Gilbert 2016) ver. 2017.03.10 was used to process all the Trinity-assembled transcripts. Leveraging the functions of CD-HIT (Fu et al. 2012), Exonerate (Slater and Birney 2005) and NCBI’s BLAST, the pipeline trims down the set of assembled transcripts and categorizes the transcripts into ‘okay’, ‘okay-alternate’ and ‘drop’ sets, based on coding potential. The ‘okay’ sets of transcripts were regarded as optimal and hence as the representative transcript sets, i.e. the transcriptomes.

For comparison of transcriptomic properties across M. candidum, M. sanguineum and the hybrid, the transcriptomes of each tissue type of M. candidum, M. sanguineum and the hybrid were also separately assembled. To do this, RNA-seq data of both replicates of a single tissue type from each of M. candidum, M. sanguineum and the hybrid were combined, normalized, assembled and trimmed down, as described above. For convenience, the two types of transcriptomes assembled in this study, i.e. the transcriptomes assembled from combined M. candidum and M. sanguineum RNA-seq data, and the transcriptomes assembled from RNA-seq data of M. candidum, M. sanguineum or the hybrid samples, shall hereinafter be known as the ‘master transcriptomes’ and the ‘taxon-specific transcriptomes’, respectively.

Quality assessment of assembled transcriptomes

To assess the quality of the transcriptomes, TransRate (Smith-Unna et al. 2016) ver. 1.0.3 was used to conduct a reference-free quality assessment of the assembled transcriptomes. It generates basic metrics that describe a transcript set, and maps the HQ reads back to the transcriptomes to estimate various metrics from the alignment.

The transcriptomes were compared to a set of 956 core plant genes using the Benchmarking Universal Single-Copy Ortholog (BUSCO) assessment tool (Simao et al. 2015) ver. 3.0.2 to assess the completeness of the transcriptome. Contigs were categorized as ‘complete, single copy’, ‘complete, duplicated copy’, ‘fragmented’ or ‘missing’, depending on the length of the aligned sequence. The BUSCO analysis [lineage = plantae, mode = transcriptome, e-value ≤ 1e-3] was run on the Cyverse Discovery Environment (de.cyverse.org; accessed March 2018).

The number of full-length coding transcripts present in the transcriptomes were also used as an assessment of assembly quality. This was done by comparing the transcriptomes to the Viridiplantae protein database of UniProtKB/Swiss-Prot using BLASTx [e-value ≤ 1e-20, max_target_seqs = 1]. The number of transcripts estimated to be full-length (>90 %) or nearly full-length (>70 %) were counted using the Trinity script analyze_blastPlus_topHit_coverage.pl.

Differential expression and functional enrichment

To quantify the expression abundance of each transcript, the Trinity script align_and_estimate_abundance.pl was used, employing Kallisto (Bray et al. 2016) to generate normalized abundance estimates across transcripts of the master transcriptomes. EdgeR (Robinson et al. 2010) was subsequently used to identify differentially expressed (DE) transcripts (with Trinity script run_DE_analysis.pl), with those satisfying at the same time a false discovery rate (FDR) of ≤0.001 and a log2 fold-change (logFC) of ≥2 (as suggested by Vijay et al. 2013) considered as DE. Heatmaps displaying the relative expression of DE transcripts and expression profile clustering were generated using the Trinity scripts analyze_diff_expr.pl and define_clusters_by_cutting_tree.pl [Ptree = 60], respectively. Then, DE transcripts were extracted and queried against the Arabidopsis thaliana protein database using KOBAS ver. 3.0 (Xie et al. 2011) to identify enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) terms [e-value ≤ 1e-5]. The definitions of GO terms were referenced from AmiGO 2 (amigo.geneontology.org).

Identification of DE genes related to trichome development

The presence of trichomes on the leaves is thought to influence differential adaptation of plant species to sunlight. Among M. candidum, M. sanguineum and the hybrid, leaf trichome length is greatest in M. candidum, followed by the hybrid (intermediate length), and M. sanguineum (shortest, but often absent). To determine the molecular factors behind the two extreme leaf trichome phenotypes (presence and absence), we looked at DE transcripts between M. candidum and M. sanguineum in the leaf that could be related to trichome development. First, A. thaliana TAIR10 proteins (ver. 20101214) with functional descriptions containing the keyword ‘trichome’ were extracted from the TAIR10 functional description list (ver. 20170331). Differentially expressed transcripts between M. candidum and M. sanguineum identified from the leaf master transcriptome were then queried against these trichome-related protein sequences using BLASTx [e-value ≤ 1e-3], and significant matches (best-hit only, bit score ≥ 50, % identity ≥ 30; as suggested by Pearson 2013) were recorded. The same procedures were conducted on DE transcripts between M. candidum and M. sanguineum in the petal as control, to determine if the trichome-related DE genes found in the leaf were tissue-specific.

Characterization of transgressive segregation in the F1 hybrids

To further dissect the phenomenon of transgressive phenotypes following hybridization, DE transcripts identified from the leaf and petal master transcriptomes that were found to be significantly up- and down-regulated in the hybrid in comparison to both parental species were isolated. These transcripts were then queried against the A. thaliana TAIR10 proteins (ver. 20101214) using BLASTx [e-value ≤ 1e-3], and significant matches (best-hit only, bit score ≥ 50, % identity ≥ 30) were then cross-referenced to the TAIR10 functional description list (ver. 20170331) and recorded.

Identification of positively selected genes between M. candidum and M. sanguineum

To identify genes under positive selection between M. candidum and M. sanguineum, orthologous transcripts were first identified from leaf and petal taxon-specific transcriptomes of both species using the bidirectional-best-hits method. A custom script incorporating MAFFT (for sequence alignment; Katoh and Standley 2013), PAL2NAL (for conversion of protein sequence alignment into codon alignments; Suyama et al. 2006) and KaKs_Calculator ver. 2.0 (for calculation of Ka and Ks values; Wang et al. 2010) was then used to estimate the non-synonymous (Ka) and synonymous (Ks) substitution rates between transcript pairs. Pairs with Ka/Ks ratios >1 and P ≤ 0.05 (Fisher’s exact test) were considered as positively selected, and were queried against the A. thaliana TAIR10 proteins (ver. 20101214; www.arabidopsis.org/, accessed 4 June 2018) using BLASTx [e-value ≤ 1e-3]. Significant matches (best-hit only, bit score ≥ 50, % identity ≥ 30) were then cross-referenced to the TAIR10 functional description list (ver. 20170331; www.arabidopsis.org/, accessed 4 June 2018) and recorded.

Results

De novo assembly of transcriptomes from RNA-seq data

Of the 50.0–62.7 million raw read pairs generated from Illumina sequencing of the cDNA libraries of M. candidum, M. sanguineum and the hybrid, 46.4–59.1 million read pairs survived quality filtering [see Supporting Information—Table S1]. Trinity normalization retained 5.6–7.3 % and 8.2–13.1 % HQ reads at a maximum coverage of 50 for the assembly of the master and taxon-specific transcriptomes, respectively. Trinity generated 209 516 and 196 293 transcripts for the leaf and petal master transcriptomes, respectively, which were subsequently reduced to 50 519 and 48 120 by EvidentialGene. The EvidentialGene-reduced transcriptomes were regarded as the optimal, and hence as references for subsequent DE analyses. Together with the outcome of other assessments, the high mapping rates (>80 %) of HQ reads to the transcriptomes assembled in this study signified that they were of good quality. Details on the characterization and various assessments of the master and taxon-specific transcriptomes are listed in Table 1 and Supporting Information—Table S2, respectively.

Table 1.

Statistics based on the master transcriptomes. Mc = M. candidum; Ms = M. sanguineum; Hy = F1 hybrid; rep = replicate. aBased on EvidentialGene ‘okay’ transcripts. bFiltered reads to EvidentialGene ‘okay’ transcripts.

Leaf Flower petal
No. of read pairs after normalization (% of total) 15 389 729 (7.3 %) 11 362 570 (5.6 %)
No. of contigs (Trinity output) 209 516 196 293
No. of contigs (EvidentialGene output, ‘okay’ transcripts) 50 519 48 120
Smallest contiga (bp) 201 201
Largest contiga (bp) 16 762 16 810
Total lengtha (bp) 65 471 851 63 130 782
Mean lengtha (bp) 1295.99 1311.95
% GCa 48.1 48.1
N50a (bp) 1864 1885
Mapping rateb (%)
 Mc (rep 1; rep 2) 88.3; 88.4 88.8; 89.5
 Ms (rep 1; rep 2) 85.3; 84.6 89.7; 90.3
 Hy (rep 1; rep 2) 88.4; 80.9 87.5; 89.6
BUSCO assessmenta
 Complete (%) 92.8 92.6
  Single-copy (%) 75.1 76.8
  Duplicated (%) 17.7 15.8
 Fragmented (%) 2.8 2.9
 Missing (%) 4.4 4.5
Transcript length assessmenta
 No. of transcripts >90 % 5404 5224
 No. of transcripts >70 % 7558 7372
No. of DE transcripts (logFC ≥ 2, FDR ≤ 0.001) 3973 2116
KOBAS annotation (Mc-Ms-Hy)
 No. of transcripts annotated (% of total) 34 640 (68.6 %) 33579 (69.8 %)
 No. of DE transcripts annotated (% of total) 3165 (79.7 %) 1545 (73.0 %)
  No. of KEGG terms (corrected P ≤ 0.05) 2 3
  No. of GO terms (corrected P ≤ 0.05) 92 61
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GC=the nucleotides Guanine and Cytosine.

Overall differential gene expression

To examine gene expressional differences, data from two biological replicates of each of M. candidum, M. sanguineum and the hybrid were mapped to the master transcriptomes for abundance estimation. Satisfying logFC ≥ 2 and FDR ≤ 0.001, EdgeR estimated a total of 3973 and 2116 DE transcripts in three-way comparisons (M. candidum–M. sanguineum–hybrid) for the leaf and petal transcriptomes, respectively (Table 1). In both tissue types, differential expression was highest between the parental species (M. candidum–M. sanguineum). The hybrid had less DE transcripts with M. candidum (hybrid–M. candidum) compared to with M. sanguineum (hybrid–M. sanguineum) in the leaf, and had similar numbers of DE transcripts with both parental species (hybrid–M. candidum and hybrid–M. sanguineum) in the petal (Table 2). Heatmaps that summarize the relative expression levels at all DE transcripts in the leaf and petal are shown in Fig. 2. Clustering the transcripts with similar patterns of expression across the samples resulted in four clusters each for the leaf and petal (Fig. 3). As seen from Figs 2 and 3, the expression levels of the hybrid at the majority of DE transcripts were similar to either M. candidum or M. sanguineum, rather than intermediate.

Table 2.

Number of differentially expressed transcripts found in the leaf and flower petal master transcriptomes. Lower diagonal = leaf master transcriptome; upper diagonal = flower petal master transcriptome. Based on logFC ≥ 2, FDR ≤ 0.001.

M. candidum M. sanguineum F1 hybrid
M. candidum – 1886 418
M. sanguineum 3243 – 407
F1 hybrid 572 2025 –
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Figure 2.

Figure 2.

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Heatmaps of relative expression levels of DE transcripts in the (A) leaf and (B) flower petal. (Mc = M. candidum; Ms = M. sanguineum; Hy = F1 hybrid; rep = replicate).

Figure 3.

Figure 3.

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Clustering of transcripts with similar expression profiles, at 60 % height of the dendrograms of heatmaps in Fig. 2, for the (A) leaf and (B) flower petal. The number above each graph shows the number of transcripts that fall under the same cluster (Mc = M. candidum; Ms = M. sanguineum; Hy = F1 hybrid; rep = replicate).

Functional enrichment analysis of DE transcripts

Using KOBAS, enrichment of KEGG and GO terms was conducted to explore the biological functions of the DE transcripts. In the three-way comparisons (M. candidum–M. sanguineum–hybrid), 79.7 % (3165 of 3973) and 73.0 % (1545 of 2116) of the leaf and petal DE transcripts were functionally annotated. Significant correlations were found for two and three KEGG terms, and 92 and 61 GO terms, for the leaf and petal, respectively (Table 1; see Supporting Information—Table S3). In the leaf, the enriched KEGG terms were ‘biosynthesis of secondary metabolites’ and ‘stilbenoid, diarylheptanoid and gingerol biosynthesis’; in the petal they were ‘biosynthesis of secondary metabolites’, ‘phenylpropanoid biosynthesis’ and ‘stilbenoid, diarylheptanoid and gingerol biosynthesis’. For the leaf, most GO terms fell under ‘biological process’ (63), followed by ‘cellular component’ (20) and ‘molecular function’ (9); for the petal, most GO terms fell under ‘biological process’ (41), followed by ‘molecular function’ (13) and ‘cellular component’ (7).

Between M. candidum and M. sanguineum, 78.9 % (2560 of 3243) and 74.1 % (1398 of 1886) of the leaf and petal DE transcripts were annotated, respectively. In the leaf, up-regulated transcripts in M. candidum were significantly related to two KEGG terms (‘biosynthesis of secondary metabolites’ and ‘flavonoid biosynthesis’) and 79 GO terms, while up-regulated transcripts in M. sanguineum were related to two KEGG terms (‘ribosome’ and ‘thiamine metabolism’) and 55 GO terms. In the petal, up-regulated transcripts in M. candidum were not significantly related to any KEGG or GO terms, while up-regulated transcripts in M. sanguineum were related to six KEGG terms (‘biosynthesis of secondary metabolites’, ‘phenylpropanoid biosynthesis’, ‘stilbenoid, diarylheptanoid and gingerol biosynthesis’, ‘flavonoid biosynthesis’, ‘phenylalanine metabolism’ and ‘limonene and pinene degradation’) and 82 GO terms [see Supporting Information—Table S4].

Genes related to trichome development

Trichomes on the leaf surface are thought to be a morphological characteristic that plays a role in differential adaptation between M. candidum and M. sanguineum. Searching through the list of descriptions for A. thaliana functional genes, 122 genes were found to be related to trichome development. BLASTx of leaf DE transcripts between M. candidum and M. sanguineum revealed 159 transcripts significantly matching to 44 trichome-related A. thaliana genes [see Supporting Information—Table S5a], while 108 petal DE transcripts between M. candidum and M. sanguineum significantly matched 31 trichome-related A. thaliana genes [see Supporting Information—Table S5b]. Twenty and seven of these trichome-related genes were tissue-specific in the leaf and petal, respectively.

Transgressive segregation in hybrids

Transcripts that were significantly up- or down-regulated in the leaf and petal of the hybrid compared to M. candidum and M. sanguineum were extracted and annotated. In the leaf, 11 and 20 transcripts were found to be significantly up- and down-regulated, respectively, while in the petal, only one transcript was found to be significantly up-regulated but no transcripts were found to be significantly down-regulated, in the hybrid. BLASTx correlated seven and 14 transgressively segregated transcripts in the leaf of the hybrid to the same numbers of genes [see Supporting Information—Table S6] in the list of A. thaliana functional genes. No significant match was found for the up-regulated transcript in the petal.

Genes under positive selection

To examine genes possibly under positive selection between M. candidum and M. sanguineum, taxon-specific transcriptomes assembled for both species [see Supporting Information—Table S2] were subjected to reciprocal BLAST, retaining only best hits that occurred in both directions for downstream Ka and Ks estimation. In total, 23 602 and 22 338 transcript pairs (M. candidum–M. sanguineum) were retained for the leaf and petal, respectively. Filtering for transcript pairs with Ka/Ks ratio >1 and P ≤ 0.05, 32 and 31 transcripts were obtained for the leaf and petal, respectively. BLASTx correlated 24 and 26 transcripts from the leaf and petal to the same numbers of genes [see Supporting Information—Table S7] in the list of A. thaliana functional genes.

Discussion

Having diversified through adaptive radiation (during which new species with very close genetic relationships are formed in a short period of time), frequent hybridization between many Melastoma species in nature has been observed, and some recognized ‘species’ have even been shown to be of hybrid origin (Dai et al. 2012; Liu et al. 2014; Zhou et al. 2017; Zou et al. 2017), highlighting the importance of hybridization in the evolution of Melastoma. Using two commonly found species in South China, M. candidum and M. sanguineum, as well as their F1 hybrid, this study aimed to characterize gene expressional patterns following hybridization. We also looked at the possible role of gene regulation in the differentiation between the two parental species, as well as between the parental species and their hybrid. Such insights may provide glimpses into possible mechanisms of diversification within Melastoma, especially since key morphological traits likely linked to their adaptive divergence are few, and reproductive isolation between many member species appears to be weak, illustrated by the many known cases of interspecific hybridization. In addition, we also identified positively selected genes to explore the role of natural selection in the evolution of Melastoma species.

Assembly of master transcriptomes for cross-species comparison

Most studies on comparative transcriptomics of plants to date compared patterns of gene expression across treatments (e.g. Carvajal et al. 2018) or across ecotypes (e.g. Shen et al. 2012; Bell et al. 2013; He et al. 2013) of a single species. Examples of studies that conducted cross-species comparisons in plants using high-throughput sequencing data are relatively rare, and these either used the transcriptome/genome of one of the species in the comparison (e.g. Zhang et al. 2013) or of a closely related species (e.g. Guo et al. 2015), combined the genomes of the species in the comparison (e.g. Zhu et al. 2017), or isolated only the orthologous transcripts (e.g. Davidson et al. 2012; Roberts and Roalson 2017), as reference. While an unpublished draft genome of M. candidum (one of the target parental species) was available during this study, using it as a reference for mapping and estimation of expression abundance in M. candidum, M. sanguineum and the hybrid would introduce bias when these estimates are directly compared. Therefore, we explored the method of a ‘master’ reference transcriptome assembled using RNA-seq data from the two parental species to allow for cross-taxa comparison of gene expression. This was possible given the low sequence divergence between M. candidum and M. sanguineum (<2 %, data not shown; high cross-species mapping rates of >85 %, see Supporting Information—Table S2). Similar methods of using combined data from more than one closely related taxa for de novo assembly have been employed in comparative studies of birds (Wolf et al. 2010) and fish (Goetz et al. 2010). In this study, the draft genome of M. candidum was merely used as a substrate for grouping overlapping reads into clusters that will then be separately fed into Trinity for de novo transcriptome assembly (i.e. genome-guided assembly; Haas et al. 2013). Using this method, fewer, but more contiguous transcripts were assembled than without the reference genome (data not shown), generally suggesting a better assembly.

Differential adaptation of M. candidum and M. sanguineum

In the functional analysis of DE transcripts between the two parental species, up-regulated transcripts in the leaf of M. candidum were enriched with functions associated with light-related responses (i.e. ‘response to light stimulus’, ‘response to radiation’, ‘response to light intensity’, ‘response to high light intensity’, ‘phototropism’ and ‘photoprotection’). This may explain the better adaptation of M. candidum to high sunlight intensity compared to M. sanguineum, reflected in their habitat preferences in the wild, whereby the former prefers habitats exposed to strong sunlight while the latter prefers shady environments. We note, however, that up-regulated genes do not necessarily confer better performance or better adaptation, and the true implications could only be established through future functional studies. Interestingly, response to karrikin, a group of plant growth regulators found in the smoke of burning plant material (Flematti et al. 2015), was found to also be up-regulated in the leaf of M. candidum. While usually associated with plants that grow immediately after bushfires or wildfires, sensitivity to karrikin has been associated with enhanced light responses in A. thaliana seedlings (Nelson et al. 2010; Flematti et al. 2015), possibly contributing to better response to sunlight in M. candidum. On the other hand, in M. sanguineum, up-regulated transcripts were enriched with functions related to the chloroplast, likely a characteristic of plants adapted to low-light conditions for more efficient light uptake (Leong and Anderson 1984).

The analysis on positively selected genes between M. candidum and M. sanguineum also identified some interesting genes related to the differential adaptation between M. candidum and M. sanguineum. MYB transcription factors are well-known regulators of phenylpropanoid metabolism in plants, which play roles in growth and development as well as in defence against biotic and abiotic stresses (Liu et al. 2015). The positively selected MYB20 and MYB55 have been associated with adaptive response to drought stress in Arabidopsis (Gao et al. 2014) and tolerance to high temperatures in rice (El-kereamy et al. 2012), respectively. The correlation between these genes and habitat preferences of both species are apparent—in the wild, M. candidum is found mostly on level ground while M. sanguineum prefers to grow on slopes (W. L. Ng and R. Zhou, pers. obs.); and when along an elevation, M. candidum grows at lower altitudes (<120 m) compared to M. sanguineum (<400 m; Chen 1983). In addition, several genes associated with the regulation of auxin were found to be under positive selection. Auxin affects plant growth and orientation in response to, among others, sunlight (Weijers et al. 2018), and this again seems to correlate with the adaptation of M. candidum and M. sanguineum to different sunlight conditions.

In this study, transcriptomic data from the petal can be considered as a control when making comparisons between M. candidum and M. sanguineum, since morphological differences observed in their flowers seem to confer little adaptive advantage. In fact, none of the DE transcripts found in the petals of M. candidum and M. sanguineum suggested any direct association with differential adaptation between M. candidum and M. sanguineum, such as responses to light, drought or temperature—the main factors that, we believe, contribute to differential adaptation between the two species. Noteworthy, however, is the up-regulation of transcripts related to cellular components found in the petal of M. sanguineum, which could explain the larger flowers of M. sanguineum compared to M. candidum.

Trichome development in M. candidum and M. sanguineum

One of the main morphological differences in the leaves of the two species is in the presence (M. candidum) and absence (M. sanguineum) of trichomes on the surface of their mature leaves. Usually as defences against biotic attacks and abiotic stressors, trichomes are thought to play a major role in the protection from sunlight by increasing reflectance and reducing the heat load (Hauser 2014). This could be true given our observations in the field, as well as in our greenhouse experiments, in which the leaves of M. candidum tend to turn yellow and wilt when placed in the shade, while the leaves of M. sanguineum tend to turn red and appear scotched when placed under constant sunlight. We cannot, however, discount the importance of other adaptive roles that trichomes may play in M. candidum and M. sanguineum, such as protection against herbivory (Hauser 2014), given that in the wild the former (with longer trichomes) often occurs at open areas while the latter (with little/no trichomes) prefers shaded environments.

Nonetheless, it was expected that many genes with functions related to trichome development are differentially expressed between M. candidum and M. sanguineum. These included members of a transcriptional network known to positively regulate trichome development, i.e. the MYB, basic helix-loop-helix (bHLH) and WD-40 repeat (WDR) proteins. In Arabidopsis, these proteins come together to form a trimeric complex that activates the expression of GLABROUS2, which induces trichome formation (Zhao et al. 2008). GLABROUS2 was also found in this study to be up-regulated in M. candidum. The involvement of several other up-regulated gene products in M. candidum in the positive regulation of trichome development, e.g. the C2H2 zinc finger protein, zinc finger protein 6 (ZFP6) and the ENHANCER OF GLABRA 3 (EGL3), is also well documented (Schellmann and Hulskamp 2005; Zhou et al. 2013; Pattanaik et al. 2014; Yan et al. 2014). Interestingly, the expression levels of these transcripts in the hybrid were mostly in between those of M. candidum and M. sanguineum (Fig. 4), likely a cause for the intermediate trichome length observed in the hybrid (Fig. 1).

Figure 4.

Figure 4.

Open in a new tab

Expression levels of several genes related to trichome development in M. candidum (blue), M. sanguineum (red), and the F1 hybrid (grey).

A considerable number of proteins with trichome-related functions were also up-regulated in the glabrous leaves of M. sanguineum; M. sanguineum plantlets initially produce leaves with trichomes (1–2 mm in length) sparsely distributed on the upper surface, but lose them as the plants mature. Under the microscope, trichome ‘roots’ can still be observed below the epidermis of mature M. sanguineum leaves (Fig. 1), and the density of these structures remains almost constant throughout the growth phase of the leaf (R. Zhou, unpubl. data). These point to the possibility that trichomes actually continue to differentiate and form throughout a leaf’s development in M. sanguineum, although the part above the epidermis does not form, or forms and quickly aborts, leaving only the ‘root’ and a small bump on the surface intact. The detection of gene products in this study known to be expressed in trichomes proved that the trichomes (or part of) in mature M. sanguineum leaves are still intact and viable. One of the genes found up-regulated in M. sanguineum is GLABRA2, which positively regulates trichome initiation. However, ubiquitous expression of this gene was found to negatively affect subsequent trichome formation and development in Arabidopsis, resulting in glabrous leaves with aborted trichomes that appear stubby (Rerie et al. 1994; Srinivas 2004), like that observed in M. sanguineum. This resemblance hints at the possible role of GLABRA2 in causing the ‘absence’ of trichomes on the leaves of M. sanguineum.

Patterns of gene expression in the F1 hybrid

The clustering of DE transcripts based on expression levels among M. candidum, M. sanguineum and the hybrid provided a general idea of the patterns of expression across them. Almost all possible modes of expression were observed in the hybrid—expression levels similar to either M. candidum or M. sanguineum (i.e. parental expression dominance), expression levels intermediate between M. candidum and M. sanguineum (i.e. additive expression), as well as expression levels exceeding both M. candidum and M. sanguineum (i.e. transgressive expression). Among the DE transcripts found in this study, parental level gene expression dominance was the most common in the hybrid, demonstrating that the mode of expression in plants following hybridization may not always be mostly additive (cf. Swanson-Wagner et al. 2006; Wu et al. 2016; Taliercio et al. 2017). In fact, various mechanisms, such as genomic imprinting (Wolf et al. 2014) and regulatory divergence between parental alleles (Combes et al. 2015), are known to alter gene expression in the hybrids in relation to the parents.

Transgressive segregation following hybridization

The hybrid between M. candidum and M. sanguineum is widespread in South China, to a point where it was once even recognized as a species (Chen 1983) until a recent molecular study (Liu et al. 2014) confirmed its hybrid status. Aside from intermediate trichome structures on the leaf, branch and hypanthium, the hybrid displays obvious vigour in several other morphological characteristics—faster growth, as well as larger leaf and flower (diameter of ~6 cm in M. candidum, ~7.5 cm in M. sanguineum and ~9 cm in the hybrid)—in comparison to both of its parents (W. L. Ng and R. Zhou, pers. obs.). The functional information of the transgressively expressed transcripts in the hybrid suggested some molecular mechanisms behind its vigour, as several up-regulated gene products are thought to be associated with enhanced growth in plants. For example, the overexpression of SAUR proteins has been shown to promote plant growth in Arabidopsis (Li et al. 2015) possibly through inducing cell elongation and growth (van Mourik et al. 2017), and the overexpression of tonoplastic intrinsic protein has been associated with cell enlargement and accelerated cell division in Arabidopsis and tobacco (Ludevid et al. 1992; Okubo-Kurihara et al. 2008).

Aside from morphological changes, the hybrid between M. candidum and M. sanguineum has also been observed to be able to grow under various conditions, even those not preferred by either parent, for example across sunlight intensities, terrains and temperatures (W. L. Ng and R. Zhou, pers. obs.). In the hybrid, we found an up-regulated gene product associated with adaptation to stress in new environmental conditions—the plant ABC transporter, which helps infer tolerance to many kinds of stresses by transporting chemicals critical for adaptation to terrestrial habitats (Hwang et al. 2016). To afford such a regulation of adaptability would be an advantageous and a critical property that could have led to the rapid adaptive speciation within Melastoma in only the past 1 million years (Renner and Meyer 2001), and transgressive segregation following hybridization could have been the force that initially drove the generation of this diversity (Kagawa and Takimoto 2018).

Data

Illumina sequencing data from this study have been deposited in the NCBI SRA database (accession number PRJNA526807). Transcriptomes assembled in this study and the custom scripts used for analysis in this study have been deposited in Figshare (available via the link https://doi.org/10.6084/m9.figshare.7824845.v1).

Sources of Funding

This study was supported by grants from the National Natural Science Foundation of China (31670210 and 31811530297), Guangdong Natural Science Foundation (2015A030302011), Science and Technology Program of Guangzhou (201707010090) and Chang Hungta Science Foundation of Sun Yat-sen University.

Contributions by the Authors

W.L.N. and R.Z. conceived and designed the study, W.L.N. and P.Z. performed the experiments, W.L.N. and W.W. performed the analyses, W.L.N. wrote the manuscript. All authors revised the manuscript.

Conflict of Interest

None declared.

Supporting Information

The following additional information is available in the online version of this article—

Table S1. Sequence data used in this study.

Table S2. Statistics based on the taxon-specific transcriptomes.

Table S3. Functional annotation of differentially expressed (DE) transcripts among Melastoma candidum, M. sanguineum and their F1 hybrid.

Table S4. Functional annotation of differentially expressed (DE) transcripts between Melastoma candidum and M. sanguineum.

Table S5. Annotation of trichome-related differentially expressed (DE) transcripts between Melastoma sanguineum and M. candidum.

Table S6. Annotation of transgressively segregated transcripts in the F1 hybrid leaf tissue.

Table S7. Annotation of putative genes under positive selection between Melastoma candidum and M. sanguineum.

Supplementary Tables
Click here for additional data file. (652.6KB, pdf)

Acknowledgements

The authors thank the anonymous reviewers for suggestions to improve this manuscript, and B. Haas at the Broad Institute for support on the use of the Trinity package.

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