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PHYLOGENETIC CHANGES IN CHLOROPLAST GENOMES

Zotov V.S.1, Punina N.V.1, Dorokhov D.B.1, Schaad N.W.2, Ignatov A.N.*1
1 *

Centre Bioengineering, RAS, Moscow, Russia; 2 FDWSRU, USDA-ARS, MD, USA Corresponding author: e-mail: ignatov@biengi.ac.ru

Key words:

evolution, chloroplast, adaptation, phenotype, computer analysis

SUMMARY Motivation: Evolution of green plants cross a few major adaptation boundaries: land plants vs. algae, seed plants (Embryophyta) vs. others, and monocotyledons (Liliopsida) vs. dicots (eudicotyledons). Evolution of chloroplasts symbiotic bacteria inside eukaryote cells, is different from evolution of nuclear genome in both rate and driving forces of phylogenetic modifications. Several distinct approaches were applied to evaluate an impact of evolutionary leaps on chloroplast genome content and composition in aim to connect structural changes to adaptive advantages of progressive phylogenetic lineages in green plants. Results: A comparison of nucleotide and amino acid similarity for conservative genes, gene content, intron presence, amino acid and codon usage, frequency and strand asymmetry of oligonucleotides, and average distance between mononucleotides was made for 51 complete chloroplast genomes of 53 published to the date. The hypothesis of quantum leaps in chloroplast genome organization was tested by comparison of different parameters with consensus evolutionary distances obtained by nucleotide and amino acid similarity for conservative ribosomal rRNA and protein genes. The result demonstrates that transaction from algae to land plants, origin of seed plants and monocotyledons was attributed by significant changes in codon usage, frequency of asymmetric oligonucleotides and average similar nucleotide distance. Availability: http://www.bionet.nsc.ru/bgrs2006/.

INTRODUCTION Green plant chloroplast is a symbiotic prokaryote acquired by ancestor of green plants (Buetow, 1976), closely related to Cyanobacteria (fam. Nostocaceae). Evolution of chloroplasts and other parasitic and symbiotic bacteria have many striking similarities (Eremeeva et al., 2005), including massive loss of genes and reduction of GC content. Generally, changes of chloroplast genome correlate to evolution of nuclear one, with some exception (Chu et al., 2004). Unfortunately, methods of comparison applied for chloroplast genes rarely considered conventional bioinformatics approaches for analysis of genome composition developed for bacterial genome (Wolf et al., 2001). Driving forces of bacterial evolution are different from ones in complex eukaryote genome. Gene gain/loss and changes in DNA composition in bacteria substitute more complex regulatory mechanisms in the host nuclear genome. Evolution of green plants cross a few major adaptation boundaries: land plants vs. algae, seed plants (Embryophyta) vs. others, and monocotyledons (Liliopsida) vs. dicotyledons (eudicotyledons), and those macro-evolutionary events can be traced in organization of such symbiotic organism as chloroplast.

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METHODS

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We compared 51 published complete chloroplast genome using nucleotide and amino acid similarity for gene content, amino acid and codon usage, frequency and strand asymmetry of oligonucleotides, and average distance between similar nucleotides (Zotov, unpublished). Genetic distances obtained for different criteria ways were compared to consensus matrixes for conservative ribosomal 16S-23S-5S rRNA and ribosomal proteincoding genes using Mantels test (Manly, 1985). Genetic distance matrixes for the used genes were calculated using Tamura-Nei Model for nucleotides and Poisson Model for amino acid sequences (Nei, Kumar, 2000). Significant differences between pooled phylogenetic close by the ribosomal genes genomes were considered as evidence of novel mechanism of adaptation of chloroplast genome to beneficial properties of host organism. Tree major evolutionary events were tested: conquest of land by algae, origin of seed plants, and separation of monocotyledons. A phylogenetic tree was built by Wards algorithm for Pearsons distance (Fig. 1) to illustrate the significant leaps in asymmetry of frequent octamer repeats, and principal component analysis was used to illustrate codon usage differences (Fig. 2). Both analyses were made in STATISTICA 6.0 (StatSoft, USA).

Figure 1. Wards tree based on Pearsons distance between asymmetry of frequent octamer repeats in 40 complete chloroplast genomes. Arrow indicate a major junction between algaes, dicotyledons and monocotyledons.

RESULTS Summarized results of comparison are showed in Table 1. Evolutionary independent gene loss was observed at different branches of the phylogenetic tree of chloroplasts. The major evolutionary event in plant life transition from algaes to land plants was attributed by significant changes in strand asymmetry of frequent oligonucleotides (Fig. 1), when appearance of seed plants by codon usage (Fig. 2). The most significant changes were found in chloroplast of monocotyledons amino acid and codon usage, strand asymmetry of oligonucleotides and average distance between similar mononucleotides were remarkably distinct in this highly progressive but genetically narrow group.

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Figure 2. Principal Component Analysis of codon usage in 51 chloroplast genome. A algaes, P parasitic plants, C conifers; D dicotyledons; M monocotyledons. The first and the second factors explained 54 % of total variation.

Table 1. Significant differences in matrixes of genetic distances between pooled chloroplast genomes Parameter Pooled comparison* Difference p-value Gene content 1 N na** 2 N 3 N Amino acid usage 1 N na 2 N 3 Y < 0.01 Codon usage 1 N na 2 Y < 0.01 3 Y < 0.001 Frequency of oligonucleotides 1 N na 2 N 3 N Strand asymmetry of oligonucleotides 1 Y < 0.01 2 N na 3 Y < 0.001 Average distance between similar 1 N na mononucleotides 2 N 3 Y < 0.001 * Pooled comparison: 1, Algae vs. land plants; 2, Seed plants and conifers vs. others; 3, monocotyledons vs. others; ** na, not applicable.

CONCLUSIONS The hypothesis of quantum leaps in chloroplast genome organization was supported by several parameters of the complete genome sequences. The obtained results demonstrate that evolution of chloroplasts led to significant and rapid changes in usage of codons and

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frequency of oligonucleotides that can be indicative for more strict control of chloroplast gene expression from nuclear genes for better adaptive reaction of the whole plant.

ACKNOWLEDGEMENTS The work was supported in part by the grant 3431 of the International Science and Technology Center.

REFERENCES
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