Identification of AP2/ERF family genes in the moso bamboo genome

The conserved AP2/ERF domains of Arabidopsis AP2/ERF protein sequences were originally applied as seed sequences to search the National Center for Gene Research (http://www.ncgr.ac.cn/bamboo) [32] and the NCBI database (www.ncbi.nlm.nih.gov). The search was based on a BLASTP search with an expected value of 100. The identified AP2/ERF genes were used as queries to reconfirm the multiple databases to ensure that no additional related genes were missing from the database. All sequences that met the requirements were analyzed to eliminate genes that did not contain the known conserved domains and motifs using the Pfam database (http://pfam.janelia.org/) [43], the SMART database (http://smart.embl-heidelberg.de/) [44] and the Conserved Domain Database of the NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [45]. Bioinformatic analysis of AP2/ERF genes was performed using ExPASy (http://www.expasy.ch/tools/pi_tool.html) to determine the number of amino acids of the open reading frame (ORF), molecular weight (MW), isoelectric point (pI) and length of the open reading frame (length) for each gene. Subcellular localization was predicted using Softberry (http://linux1.softberry.com/). The AP2/ERF genes in Arabidopsis were searched using the Arabidopsis Information Resource TAIR (http://www.arabidopsis.org/), and the AP2/ERF genes in rice were obtained from the MSU database (http://rice.plantbiology.msu.edu/) and the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/index.shtml).

Phylogenetic, motif recognition and gene structure analyses

The multiple alignment analysis was performed with ClustalX 1.83 software [46], and phylogenetic trees were generated by the neighbor-joining (NJ) method and bootstrap analysis (1000 replicates) [47] and displayed using MEGA 6.0 software [48]. The conserved motifs were analyzed using MEME version 4.9.1 [49, 50]. Gene structure was investigated using the Gene Structure Display Server websites (http://gsds.cbi.pku.edu.cn/).

Evolutionary patterns and divergence of the AP2/ERF gene family in rice and moso bamboo analysis

Pairwise alignment of AP2/ERF gene encoding sequences of the orthologous and paralogous pairs was performed using ClustalX 1.83 software. The occurrence of duplication events and divergence of homologous genes, as well as selective pressure on duplicated genes, were estimated by calculating synonymous (Ks) and non-synonymous substitutions (Ka) per site between the duplicated gene-pairs using DnaSP version 5.10.1 [51, 52]. The Ks rate was considered as the proxy for time to estimate the dates of the gene-pair duplication events, and the dates of the duplication events were further deduced using the formula T = Ks/2λ, assuming that clock-like rates (λ) of rice and moso bamboo were 6.5 × 10^–9 substitutions/synonymous site/year [32, 53, 54].

Analysis of the putative promoter regions of the DREB gene subfamily in moso bamboo

The 2000-bp upstream sequences of the transcriptional start site of PeDREBs were chosen to identify the cis-elements in the putative promoter regions. The PLACE website (http://www.dna.affrc.go.jp/PLACE/) [55] was applied to identify the putative cis-regulatory elements along the promoter sequences.

Plant treatment and qRT-PCR analysis

Moso bamboo seeds were collected from Guilin, Guang Xi Province, China. The seeds from individual plants were germinated on sterile filter papers in culture dishes, while keeping the filter papers moist and in darkness at 25°C. The seedlings were transferred to plastic pots containing vermiculite and grown in an illuminated incubator with 16/8 h of light/dark at 25/18°C and humidity of 80%, and watered with Hoagland nutrient solution every week. Plants were cultivated for three months.

For drought-stress treatments, a 20% PEG-6000 solution (and for salinity treatments, a 250 mM NaCl solution) was poured over the culture medium vermiculite. For low-temperature stress treatments, plants were transferred to an illuminated incubator at 4°C with other culture conditions unchanged. Plant leaves and roots were collected for analysis at 0, 0.5, 1, 3, 6, 12 and 24-h time points. Plant roots were rapidly washed with distilled water (4°C incubated water for low-temperature treatment) after treatment at each time-point, frozen immediately in liquid nitrogen and stored at—80°C prior to RNA extraction.

The total RNA was extracted using TRIZOL reagent (Invitrogen, Germany) based on the manufacturer’s instructions, and extensively pre-treated using RNase-free DNase I (Promega, Madison, WI, USA) to digest any genomic DNA. RNA quality was characterized initially on a 1% agarose gel with Tris–acetate–EDTA (TEA) buffer and NanoDrop 8000 spectrophotometer (Thermo Scientific) and then the integrity of RNA samples was further evaluated using an Agilent 2100 Bioanalyzer (USA). For first-strand cDNA synthesis, 2 μg of total RNA in a 20 μl reaction volume treated with DNase was transcribed using M-MLV reverse transcriptase in accordance with the manufacturer’s protocols (Promega).

Due to the small number of members characterized in the DREB subfamily, we analyzed the expression patterns of 24 DREB genes (excluding PH01000343G0780, PH01003107G0070 and PH01003928G0080, because of lack of specific primers) by using real-time quantitative PCR (qRT-PCR) reactions to survey their functions in response to abiotic stress. The qRT-PCR reactions were carried out with a LightCycler480H System (Roche) using SYBRH Premix EX TaqTM kit (Roche). The 20-μl reaction system contained 0.4 μl (10 mM) of each primer, 2 μl (20 ng) of cDNA, 10 μl of SYBR Green I Master and then ddH2O was added to make up the final volume, following the manufacturer’s instructions. Amplification reactions were performed as follows: 95°C for 10 s, 60°C for 10 s and 72°C for 20 s. All reactions were performed in triplicate, both technical and biological. The primers, listed in S1 Table, were chosen using Primer 3.0 software (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi). Tonoplast intrinsic protein 41 gene (TIP41) [56] was used as the reference gene. The data of relative expression levels were calculated using the comparative ΔΔ^CT method, and presented in clusters using fold-change values transformed to log₂ format and clustered by Multiple Array Viewer using the average method linkage method with Pearson’s correlation distance metric [57].

Detection of AP2/ERF transcription factors

We identified a total of 121 putative AP2/ERF genes, predicted to include one or two complete or incomplete AP2/ERF domains. The ORF and gene lengths, MW, pI and subcellular localization of these putative genes were analyzed, and results are listed in S2 Table. Gene length ranged from 309 (PH01000069G0560) to 1926 bp (PH01001338G0140), MW from 11.15 (PH01000069G0560) to 64.71 kDa (PH01001338G0140) and pI from 4.45 (PH01002652G0240) to 11.07 (PH01002393G0230). Subcellular localization prediction indicated that 119 genes were located in the nucleus, with score ranging from 4.40 (PH01000084G1170) to 9.95 (PH01001938G0330); however, two genes (PH01003811G0100 and PH01000443G0450) may have been located in the chloroplast. Five of the 121 genes (PH01000303G0100, PH01000028G1600, PH01002652G0240, PH01004233G0070 and PH01195697G0010) were excluded from further analysis due to having a very small domain and resulting in an unacceptable phylogenetic tree. Therefore, the remaining 116 genes were used for phylogenetic analysis, which had corresponding locus IDs in the database National Center for Gene Research.

Phylogenetic analysis of the AP2/ERF family

To determine the phylogenetic relationships between the genes in the moso bamboo AP2/ERF family, multiple alignment analyses were executed using amino acid sequences of the AP2/ERF domain. In total, 17 consensus residues, 4G, 12G, 18I, 31L, 32G, 34T, 41A, 42A, 44A, 45Y, 55D, 57A, 58A, 63G, 66A, 72N and 73F were more than 85% conserved among the 116 proteins in the PeAP2/ERF family (S1 Fig). The alignment indicated that 80 of these proteins were in the ERF subfamily, 28 were in the AP2 subfamily, seven were in the RAV subfamily and there was one soloist. Because 80 proteins possessed one AP2/ERF domain and shared high homology, they belonged to the ERF subfamily (S1 Fig). Three proteins, PH01000320G0460, PH01003811G0100 and PH01004434G0040, had C-terminal regions of the AP2/ERF domain that contained very low homology to the consensus sequence (S1 Fig)—these three proteins belonged to group VI–L. There were 17 proteins that included two AP2/ERF domains and these were in the AP2 subfamily; whereas, 11 proteins possessed one AP2/ERF domain that shared high homology with AP2 proteins, and so were classified in the AP2 subfamily (S1 Fig). Seven proteins each contained one AP2/ERF domain and one B3 domain and hence were classified into the RAV subfamily (S1 Fig).

Based on above observations, an unrooted phylogenetic tree with 116 PeAP2/ERF domain sequences was constructed (Fig 1A), and an additional tree was generated using the domain sequences of 135 AtAP2/ERF, 147 OsAP2/ERF and 116 PeAP2/ERF proteins (S2 Fig). These trees distinguished the AP2 subfamily; the RAV subfamily; groups I–VI, VI–L and VII–X; and a soloist. The reliability of the clustering was supported by the method described in Nakano et al. [7]. The numbers and members of groups I–X are shown in Fig 2 and Table 1. The classification of subgroups was analyzed by the presence and position of introns and the motifs within the AP2/ERF proteins, as described later. Several striking points were found in the PeAP2/ERF family: (1) no members belonged to groups Ia, IIc, Vb, IXa and Xc; (2) there were more members in groups IIId, VIIa, VIIIa and Xb than that from Arabidopsis and rice, whereas there were fewer in other groups; (3) group VIIb existed in moso bamboo and rice, but groups IIIa and Xb-L were specific to Arabidopsis; and (4) there were no members in groups XI—XIV, and these groups may be specific to rice (see Table 1 and Fig 2).

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Fig 1. Unrooted phylogenetic tree and exons/intronsof moso bambooAP2/ERF family genes.

(A) The phylogenetic tree was generatedusing the AP2/ERF domain amino acid sequences by the Clustal 1.83 software with the NJ method.Groups I–IV are CBF/DREB subfamily proteins, and groups V–X are ERF subfamily proteins. (B) The distribution of exons/introns within AP2/ERF genes. The green boxes represent exons, black lines linked two exons represent introns and the number over lines represents the splicing phases.

https://doi.org/10.1371/journal.pone.0126657.g001

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Fig 2. Comparison of group/subgroup size between moso bamboo, Arabidopsis and rice AP2/ERF families.

The subclassification was by Nakano et al. (2006) [7].

https://doi.org/10.1371/journal.pone.0126657.g002

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Table 1. The ERF subfamily genes in moso bamboo.

https://doi.org/10.1371/journal.pone.0126657.t001

Gene structure and conserved motifs analysis of the AP2/ERF gene family

To further understanding of the structural diversity of moso bamboo AP2/ERF genes, we analyzed exon/intron organization within PeAP2/ERF family members. AP2 subfamily members had 1–9 introns (excluding PH01004181G0030), the soloist gene had four introns, and most members of group VII had one or two introns, whereas the other subfamily and groups seldom had introns (Fig 1B). To identify the potential motifs in the PeAP2/ERF family, full-length amino acid sequences of PeAP2/ERF genes and the homologous genes of Arabidopsis and rice were analyzed, and we listed the homologous clades with any PeAP2/ERF genes (see Fig 3 and S3 Fig). The motifs in different subfamilies or subgroups were different, including numbers and conserved sequences. For example, motif analysis identified CMPeII-4, CMPeIII-8, CMPeIII-12 and CMPeVII-3 motifs as LWSY motifs in groups IIb, IIId, IIIc and VIIa, respectively. The ERF-associated amphiphilic repression (EAR) motif of motifs CMPeIII-5 and CMPeVIII-3 in groups IIId, IIIc and VIIIa, was identified as a conserved sequence, LxLxLxLPP, in the C-terminal regions. CMPeVI-4, CMPeVII-6 and CMPeIX-4, identified as putative phosphorylation site sequences, were conserved in groups VI, VIIa and IXb. A unique motif, CMPeX-3, containing a characteristic consensus sequence, Cx₂Cx₂Cx_2~4C, was conserved in the N-terminal region of members in group Xb. CMPeA-6 existed in PH01001341G0150, PH01002603G0280, Os12g03290 and Os11g03540 of AP2 subfamily proteins; and CMPeR-8 only existed in the C-terminal region of PH01000015G1290, PH01000281G0890 and PH00101568G0220 of RAV subfamily proteins. Of soloists (S3 Fig), PH01001018G0090 and Os02g29550 genes had higher homology than AT4G13040 (S3 Fig and S3 Table).

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Fig 3. Gene motifs within each group of the ERF subfamily in moso bamboo, Arabidopsis and rice.

The clades of the phylogenetic tree detected within moso bamboo, Arabidopsis and rice ERF subfamilies. The conserved motifs were identified in the proteins of every group respectively: groups I (A), II (B), III (C), IV(D), V (E), VI (F), VI–L (G), VII (H), VIII (I), IX (J) and X (K). Each colored box below the tree represents the conserved motifs. The CMPeI-1 to VI-1, VI-L-1, and VII-1 to X-1 represent the AP2/ERF domain in every group.

https://doi.org/10.1371/journal.pone.0126657.g003

Characteristics of each group in the PeAP2/ERF family

The characteristics of each group in the PeAP2/ERF family in association with AtAP2/ERF and OsAP2/ERF families were reported by Nakano et al. [7]. The AP2 and RAV subfamilies were characterized and the 11 groups within the ERF subfamily were further analyzed as follows. Group I only contained two genes in subgroup Ib, which had high homology with Os05g49700 and AT1G64380 genes in subgroup Ib; and there were no subgroup Ia genes in moso bamboo (Fig 3A). Group II was divided into two subgroups, IIa and IIb, and lacked subgroup IIc (Fig 3B). The motif analysis identified CMPeII-4 as a LWSY motif in the C-terminal region—similar to CMII-3 in subgroup IIb as described by Nakano et al. [7]. Os04g55520 and Os06g07030 genes in subgroup IIa and PH01002393G0230 gene were clustered into one clade, indicating that PH01002393G0230 belonged in subgroup IIa. Group III consisted of four subgroups, IIIb, IIIc, IIId and IIIe, with no subgroup IIIa (Fig 3C). The putative subgroup IIIc proteins, PH01000131G1240 and PH01001480G0400, contained three consensus motifs, CMPeIII-4, -9 and -12. The CMPeIII-4 and -9 motifs were similar to the CMIII-3 motif’s region I and region II on both sides of the AP2/ERF domain [7]; the CMPeIII-12 motif has also been identified as a LWSY motif. Despite the PH01000017G0950 protein containing motifs different to the former two proteins, these three proteins had no introns and were grouped into a single clade (Fig 1), so the PH01000017G0950 protein was also assigned to subgroup IIIc. The CMPeIII-5 (EAR motif) and -8 motifs were similar to CMIII-6 and CMIII-7, which were contained in subgroup IIId—so proteins containing CMPeIII-5 and -8 motifs were assigned to subgroup IIId and, further based on phylogeny, 10 proteins were assigned to subgroup IIId. PH01003475G0200 and PH01000124G0270 were grouped into a single clade and shared the same motifs, and so were assigned to subgroup IIIe—using a similar method, PH01001205G0030 was assigned to subgroup IIIb. Group IV was divided into two subgroups, IVa and IVb (Fig 3D). The CMPeIV-2 and -4 motifs were identified as similar to CMIV-1 and -2, which were conserved throughout the N-terminal region outside the AP2/ERF domain in subgroup IVa of Arabidopsis and rice; while PH01000098G1180 and PH01003928G0080 possessed the CMPeIV-2 and -4 motifs, and so were assigned to subgroup IVa. The phylogenetic tree and the other motif analysis indicated that the remaining proteins were from subgroup IVb. Followed the classification method of Nakano et al. [7], groups I–IV belonged to the CBF/DREB subfamily as described by Sakuma et al. [5], which was also consistent with the present study. In total, 27 proteins belonged to the CBF/DREB subfamily.

The ERF subfamily genes were divided into two subfamilies: CBF/DREB and ERF [7]. The genes of groups V, VI, VI–L and VII–X were ERF subfamily genes. Group V contained only one protein (Fig 3E), PH01000791G0820, sharing two motifs, CMPeV-2 and CMPeV-3, identified as similar to CMV-1 and CMV-2 which have been described in subgroup Va [7], so the PH01000791G0820 gene was assigned to subgroup Va; there were no subgroup Vb genes in moso bamboo. Group VI consisted of proteins that shared two conserved motifs, CMPeVI-3 and -5, in the N-terminal region (Fig 3F), and analysis indicated that they were similar to CMVI-1 and CMVI-2 [7]; the six genes in group VI had no introns (Fig 1B). PH01000320G0460, PH01003811G0100 and PH01004434G0040 shared a motif, CMPeVI-L-2, in the N-terminal region outside the AP2/ERF domain, similar to CMPeVI-3, and so these three genes were assigned to subgroups VI–L (Fig 3G). Group VII was divided into two subgroups: VIIa and VIIb (Fig 3H). There were 17 proteins that contained four motifs, CMPeVII-2, -3 and -4 were identified as similar to CMVII-1, -5 and -6, respectively, and were in subgroup VIIa of OsERF genes. Among the 17 proteins, 14 had one or two introns (Fig 1B), a phenomenon also described in Arabidopsis and rice [7]. Based on these analyses, the 17 proteins were assigned to subgroup VIIa. PH01001704G0270 had high homology to Os09g11460 protein, and so was assigned to subgroup VIIb. Group VIII was divided into two subgroups: VIIIa and VIIIb (Fig 3I). The putative proteins of subgroup VIIIa had two conserved motifs, CMPeVIII-3 and -4, identified as similar to CMVIII-1 and -2 in subgroup VIIIa. The protein, PH01000069G0560, contained no motif outside the AP2/ERF domain and possessed two introns (Fig 1B), and so was assigned to subgroup VIIIb. Group IX consisted of two subgroups, IXb and IXc, with no IXa (Fig 3J). PH01000171G0880 and PH01002571G0300 genes contained two motifs, CMPeIX-4 and -5, the CMPeIX-4 motif sequence was homologous to CMIX-5 and -6 motifs; the CMPeIX-5 motif sequence was homologous to CMIX-2; the CMIX-2, -5 and -6 motifs were conserved in subgroup IXb; and so the two genes were assigned to subgroup IXb. The CMPeIX-2 and -3 motifs were conserved in PH01001229G0220, PH01000085G0010 and PH01001938G0330 genes, according to Arabidopsis and rice genes—these three genes were assigned to subgroup IXc. Group X consisted of subgroups Xa and Xb (Fig 3X). Five proteins, PH01001490G0850, PH01000493G0150, PH01000559G0630, PH01000173G0700 and PH01000437G0390, contained two conserved motifs, CMPeX-3 and -4. The CMPeX-3 motif, in the N-terminal region, was homologous to the CMX-2 motif in subgroup Xb proteins in Arabidopsis and rice, and so these five proteins were assigned to subgroup Xb. Analyses of phylogenetics and the presence of the CMPeX-2 motif indicated that PH01000004G0570, PH01001201G0350 and PH01002648G0300 genes belonged to subgroup Xa.

Evolutionary patterns and divergence of the AP2/ERF gene family in moso bamboo and rice

In comparative genomics, the phylogeny-based and bidirectional best-hit methods are popular strategies to identify possible paralogous or orthologous genes. Using these two strategies, we found 77 putative paralogous pairs in the moso bamboo genome and 86 orthologous pairs between OsAP2/ERF and PeAP2/ERF—all gene-pairs are listed in S4 Table. In order to evaluate the divergence time between rice and moso bamboo, we used a relative Ks measure as a proxy for time—the frequency distributions of the relative Ks values obtained from duplicated orthologous and paralogous gene-pairs in the rice and moso bamboo genomes are shown in Fig 4C and 4A. The relative Ks distribution peaks around 0.2 in moso bamboo suggest a large-scale event around 15 million years ago (MYA). A recent report found that bamboo underwent whole-genome duplication 7–12 MYA [32], according to analyses of clustered gene families and gene collinearity and, when compared with the result of present study, shows that AP2/ERF family genes underwent a longer large-scale event. Similarly, the relative Ks distribution peaks at 0.2–0.3 for the duplicated orthologous gene-pairs between rice and moso bamboo indicate division within the two groups of AP2/ERF genes at 15–23 MYA. A previous study estimated that the divergence time of rice and moso bamboo was 7–15 MYA [32] and, when compared with our study, reveals that the AP2/ERF family underwent gene evolution before separation of the two progenitors. We also obtained a Ka/Ks ratio of 0.3–0.5 from the duplicated paralogous gene-pairs in the moso bamboo genome (Fig 4D); however, the duplicated orthologous gene-pairs between the rice and moso bamboo genomes gave Ka/Ks of 0.3–0.4 (Fig 4B), suggesting purifying selection for the moso bamboo genome, as well as between the rice and moso bamboo genomes.

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Fig 4. Ks and Ka/Ksvalue distributions of the AP2/ERF genes in the genomes of rice and moso bamboo viewed through the frequency distribution of relative Ks and Ka/Ks modes.

Distribution of Ks and Ka/Ks values were obtained from orthologous gene-pairs (A and B) between the moso bamboo and rice genomes, and paralogous gene-pairs (C and D) in the moso bamboo genome. Ka/Ks = 1 indicates neutral selection, Ka/Ks > 1 indicates positive selection and Ka/Ks < 1 indicates purifying selection [79, 80].

https://doi.org/10.1371/journal.pone.0126657.g004

Analysis of the putative promoter regions of the DREB gene subfamily

Cis-regulatory elements play very important roles in determining the tissue-specific or stress-responsive expression patterns of genes [57], and multi-stimulus responsive genes are closely correlated with cis-regulatory elements in the promoter regions [58, 59]. Cis-elements located upstream of genes within 2000 bp have decisive effects on binding to target genes. To further understand transcriptional regulation and the potential functions of DREB subfamily genes in moso bamboo, 2000-bp putative promoter regions were used to identify putative stress-responsive cis-regulatory elements [60, 61]. Numerous abiotic stress cis-elements—S000176 and S000415 for drought stress, S000453 for salt stress, S00030 for heat stress, S000407 for cold stress and S000457 for wound stress—were found widely in the promoter regions of DREBs in moso bamboo, and are listed in S5 Table. This clearly showed that DREB subfamily transcription factors might respond to abiotic stress and have potential functions in enhancing abiotic stress resistance. For instance, PH01000343G0830 possessed up to 24 drought-stress elements (S000415), PH01001205G0030 had up to 34 cold-stress elements (S000407), and 24 drought-stress elements (S000415) and 34 cold-stress elements (S000407) were identified in PH01002279G0250. Further research on the function of the DREB subfamily genes in moso bamboo will be important in furthering understanding of the stress tolerance mechanism in moso bamboo.

Expression profiles of PeDREBs under drought, cold and salt stresses

To understand the expression profiles of the PeDREB transcription factors under drought, cold and salt stresses, 24 PeDREBs were analyzed by qRT-PCR. Clustering divided them into six clades: named clades I–VI (Fig 5). Clade I had two members, one belonged to IIId, the other to IIIe, and they had up-regulated expression under cold stress and down-regulated under drought and salt stresses in leaves, but were up-regulated under these three treatments in roots. Clade II had two IIb, three IIId, one IIIc and one IVb genes, and the expression levels of these seven genes showed negative responses in leaves, but strongly positive responses in roots. Clade III included one IIa, one IIIc, two IIId and one IIIe genes, the transcripts of PH01000131G1240 initially increased and then dropped at 12 h under salt treatment in leaves, and had continuous high expression levels under drought stress in roots; PH01003475G0200 had continuous high expression levels under drought stress in roots, and the expression levels under cold and salt stresses in roots initially increased and finally dropped. PH01000668G0350 and PH01001480G0400 were involved in drought and cold stress responses in leaves, they were up-regulated under the three treatments in roots; expression levels of PH01002293G0230 initially decreased and then increased under drought stress in leaves, and almost up-regulated under the three treatments in roots. Clade IV, containing one Ib, two IIId and one IVa genes, genes in clade IV were down-regulated under drought stress in leaves and salt stress in roots, had low expression levels under cold stress in leaves, was up-regulated under salt stress in leaves (excluding PH01000242G1390), and was also up-regulated under drought conditions in roots. Clade V had two IIId and one IVb genes, the expression levels of which were down-regulated or little-changed under these three stresses both in leaves and roots. Clade VI consisted of one Ib and two IVb genes, which were up-regulated in leaves and slightly down-regulated in roots under the three stresses.

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Fig 5. Heat map of the real-time quantitative PCR (qRT-PCR) analysis results of PeDREB genes in leaves and roots under drought, low-temperature and high salinity treatments, with three biological and technical replicates.

The expression levels of genes are presented using fold-change values transformed to log₂ format compared to controls. The log₂ (fold-change values) and the color scale are shown at the right of the heat map.

https://doi.org/10.1371/journal.pone.0126657.g005