Comparative analysis of the chloroplast genomes of eight Piper species and insights into the utilization of structural variation in phylogenetic analysis

Abstract

With more than 2000 species, Piper is regarded as having high medicinal, cosmetic, and edible value. There also remain some taxonomic and evolutionary uncertainties about the genus. This study performed chloroplast genome sequencing of eight poorly studied Piper species and a comparative analysis with black pepper (Piper nigrum). All examined species were highly similar in gene content, with 79 protein-coding genes, 24 tRNAs, and four rRNAs. They also harbored significant structural differences: The number of SSRs ranged from 63 to 87, over 10,000 SNPs were detected, and over 1,000 indels were found. The spatial distribution of structural differences was uneven, with the IR and LSC being relatively more conserved and the SSC region highly variable. Such structural variations of the chloroplast genome can help in evaluating the phylogenetic relationships between species, deciding some hard-to-distinguish evolutionary relationships, or eliminating improper markers. The SSC region may be evolving at high speed, and some species showed a high degree of sequence variation in the SSC region, which seriously affected marker sequence detection. Conversely, CDS sequences tended to lack variation, and some CDSs can serve as ideal markers for phylogenetic reconstruction. All told, this study provides an effective strategy for selecting chloroplast markers, analyzing difficult-to-distinguish phylogenetic relationships and avoiding the taxonomic errors caused by high degree of sequence variations.

Keywords: DNA barcode; chloroplast genome; high degree of sequence variations; phylogenetic relationships; piper.

FIGURE 1

Gene map of Piper chloroplast genome. Genes belonging to different functional categories are color-coded. Genes inside the circle are transcribed in a clockwise direction and those located on the outside are transcribed in a counter-clockwise direction. LSC, large-single-copy; SSC, small-single-copy; IR, inverted repeat.

FIGURE 2

The distribution of repeat sequences in Piper chloroplast genomes. (A) The proportion of repeat sets in the three regions (IR, LSC, and SSC). (B) The proportion of repeat sets in introns, intergenic regions, and CDS. (C) The number of repeat sets in different genes.

FIGURE 3

The SSRs in Piper chloroplast genomes. (A) The number of SSRs in different Piper species. (B) The proportion of SSRs in the three regions (IR, LSC, and SSC). (C) The number of different types of SSRs (mononucleotides, dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides). (D) The number of each SSR.

FIGURE 4

The SNP and indel in Piper chloroplast genomes. (A) The (A1) shows the proportion of SNP in the three regions (IR, LSC, and SSC). The (A2) shows the average number of nucleic acids per SNP in the three regions (IR, LSC, and SSC). (B) The proportion of indels in the three regions (IR, LSC, SSC). (C) The heat map of the correlations between species that constructed by the average length of the nucleic acid sequence in which an SNP appears. Each value was normalized by dividing the maximum value. A higher value indicates a greater correlation between the two species. (D) The heat map of correlations between species that constructed by the number of indels. Each value was normalized by dividing the maximum value. A higher value indicates a lower correlation between the two species.

FIGURE 5

The mean Pi values between species.

FIGURE 6

Sliding window analysis of the whole chloroplast genome of Piper species. X-axis shows the position; Y-axis, nucleotide diversity of each window. The coding genes that can be used as barcodes were screened out and their locations are highlighted in blue boxes. (A) The distribution of Pi values in IRa region; (B) The distribution of Pi values in IRb region; (C) The distribution of Pi values in LSC region; (D) The distribution of Pi values in SSC region; (E) The distribution of Pi values in SSC region of the species set with 5 species; (F) The distribution of Pi values in SSC region of the species set with 4 species.

FIGURE 7

Comparison of boundaries among IR, LSC, and SSC regions of nine Piper species. Genes above lines are transcribed forward and those below the lines are transcribed reversely.

FIGURE 8

The ENc-GC3 plot of different Piper species. The solid line represents the expected curve of positions of genes when the codon usage was only determined by the GC3s composition. The red dots highlighted the screened markers. (A) P. nigrum; (B) P. boehmeriifolium; (C) P. austrosinese; (D) P. mutabile; (E) P. bonii; (F) P. betle; (G) P. retrofractum; (H) P. hainanense; (I) P. umbellatum.

FIGURE 9

The phylogenetic trees. (A) The phylogenetic tree of Piper species [A tree constructed from screened markers. The other trees from the sequence and SNPs of IR and LSC also showed similar results (Supplementary Figure S3)]. (B) The conflicting phylogenetic relationships that constructed by unsuitable markers from SSC region [The trees from SNPs and sequence of SSC region also showed similar result (Supplementary Figure S3)].