The complete chloroplast genome of Microcycas calocoma (Miq.) A. DC. (Zamiaceae, Cycadales) and evolution in Cycadales

Abstract

Cycadales is an extant group of seed plants occurring in subtropical and tropical regions comprising putatively three families and 10 genera. At least one complete plastid genome sequence has been reported for all of the 10 genera except Microcycas, making it an ideal plant group to conduct comprehensive plastome comparisons at the genus level. This article reports for the first time the plastid genome of Microcycas calocoma. The plastid genome has a length of 165,688 bp with 134 annotated genes including 86 protein-coding genes, 47 non-coding RNA genes (39 tRNA and eight rRNA) and one pseudogene. Using global sequence variation analysis, the results showed that all cycad genomes share highly similar genomic profiles indicating significant slow evolution and little variation. However, identity matrices coinciding with the inverted repeat regions showed fewer similarities indicating that higher polymorphic events occur at those sites. Conserved non-coding regions also appear to be more divergent whereas variations in the exons were less discernible indicating that the latter comprises more conserved sequences. Phylogenetic analysis using 81 concatenated protein-coding genes of chloroplast (cp) genomes, obtained using maximum likelihood and Bayesian inference with high support values (>70% ML and = 1.0 BPP), confirms that Microcycas is closest to Zamia and forms a monophyletic clade with Ceratozamia and Stangeria. While Stangeria joined the Neotropical cycads Ceratozamia, Zamia and Microcyas, Bowenia grouped with the Southern Hemisphere cycads Encephalartos, Lepidozamia and Macrozamia. All Cycas species formed a distinct clade separated from the other genera. Dioon, on the other hand, was outlying from the rest of Zamiaceae encompassing two major clades-the Southern Hemisphere cycads and the Neotropical cycads. Analysis of the whole cp genomes in phylogeny also supports that the previously recognized family-Stangeriaceae-which contained Bowenia and Stangeria, is not monophyletic. Thus, the cp genome topology obtained in our study is congruent with other molecular phylogenies recognizing only a two-family classification (Cycadaceae and Zamiaceae) within extant Cycadales.

Keywords: Chloroplast genomes; Cycadales; Cycads; Evolution; Microcycas; Phylogeny; Plastids; Zamiaceae; cpDNA.

Figure 1. Microcycas calocoma annotated circular genome.

Gene map of Microcycas calocoma whole chloroplast genome. Innermost circle indicates partition of large single copy (LSC) region, small single copy (SSC) region and two regions of inverted repeats (IR_A and IR_B) which are also highlighted by the thick lines found in the outermost circle. Genes within the outermost circle are transcribed in clockwise direction whereas genes on the outside are transcribed in counter clockwise direction. Colors denote the different functions of genes.

Figure 2. Sequence identity plots of various genera in Cycadales using mVISTA.

Sequence variations among 10 genera in Cycadales using C. debaoensis as base reference. Complete chloroplast genomes generated by mVISTA. Regions in pink indicate conserved non-coding sequences, purple are conserved exons, while white-colored regions identify more variable sites. The Y-axis represents percent identity ranging from 50% to 100%. IR junctions are indicated in parentheses to show LSC, SSC and IR regions.

Figure 3. cpDNA tree obtained using ML and BI.

Maximum likelihood (ML) tree and Bayesian inference (BI) tree of concatenated data matrix of 81 protein-coding genes from complete chloroplast genome sequences of 10 genera of Cycadales available in GenBank. Ginkgo biloba was used as outgroup. Bootstrap support values are given at the nodes annotated as ML/BI. Branches with bootstrap values >75 for ML and Bayesian posterior probabilities >0.95 for BI were considered as highly supported.