Evolution of chloroplast matK genes among lower land plants

1. Core eudicots Core eudicots Basal eudicots 90 Basal eudicots Monocots 73 86 Basal angiosperms Lycopodiella Selaginella 84 Gymnosperms Basal angiosperms 75 Basal angiosperms 100 Monocots Gnetophytes Conifers 100 Monocots Cycads 92 0.92 99 Cycads Ginkgo 1.00 100 100 Gymnosperms* Ginkgo Ophioglossum Conifers Adiantum Green algae 99 100 Monocots 0.99 Gnetophytes Gnetophytes 99 96 100 1.00 Ferns and allies 82 rbcL 1.00 1.00 100 Bryophytes Lycophytes and ferns 89 Lycophytes and ferns 0.61 Bryophytes Bryophytes 1.00 (B) (A) 0.52 1.00 1.00 0.56 1.00 1.00 1.00 Genomic DNA Genomic DNA RT-PCR RT-PCR 1.00 Epifagus L O L O L O L O L O L O Selaginella doederleinii 1.00 1.00 M R I1 I 2 M R I1 I2 M M R R I I M M R R I I 1.00 1.00 500bp 500bp 1.00 Intron Free-standing matK pseudogene No intron ycf3 ycf2 atpF petB petD rpl12 ndhA ndhB ndhH rpl16 ycf10 ycf66 rps12 rpoC1 rps16 clpP trnV rrn23 trnT rpl2 rpl6 trnG trnH trnA trnK trnL trnI 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Evolution of chloroplast matK genes among lower land plants Shu-Lan Chuang (莊樹嵐) and Jer-Ming Hu (胡哲明) Institute of Ecology and Evolutionary Biology, National Taiwan University Abstract The introns of chloroplast trnKUUU contain an open reading frame denoted as matK. The putative gene product MatK is the only one having maturase function in chloroplasts. However, only those chloroplasts of land plants and higher green algae such as Characeae have introns in their trnK genes, but not in other green algae examined. Chloroplast matK genes are indispensable since in nonphotosynthetic parasitic plant, Epifagus virginiana, the chloroplast matK is functional even being a free-standing from with dismissed trnK exons. The chloroplasts of Psilotum, moss and liverworts all have trnK5’-matK-trnK3’ structure, but it is found that matK is a pseudogene in hornwort Anthoceros formosae. We found a clear trnK5’-matK-trnK3’ structure in Ophioglossum petiolatum, Lycopodiella cernua and Selaginella doederleinii. RT-PCR results showed matK genes are expressed in Ophioglossum petiolatum and Lycopodiella cernua, but no signal detected in Selaginella doederleinii. So the function and expression of matK are not consistent in lower land plants. Codon usage analysis showed that the use of codons in matK is in congruent with the average use of chloroplast genomes, showing a bias that can be explained by constraints on GC contents. The result of correspondence analysis suggests the codon usage of chloroplast matK has some properties that is correlated with their evolutionary relationship. Lycopodiella cernua and Selaginella doederleinii are placed into different group in matK phylogenetic analysis, but the incongruence is likely due to the disputable sequence alignment, which causes long branch attraction that will affect phylogenetic inference. Nonetheless, the result showed that Pinus, Ginkgo, Cycas from a monophyletic group, which is sistered to angiosperms. Together they form a clade that is sistered to Gnetales. Fig 4. Codon usage analysis. (A, B) Correspondence analysis of codon usage. Major groupings are indicated. (C) Nc-plot shows the bias of matK codon usage is correlated to GC contents. (C) Results (A) (B) Fig. 2. Detection of chloroplast matK expression byRT-PCR. (A) Results from Lycopodiella cernua (L) and Ophioglossum petiolatum(O). On the right showing a PCR of genomic DNA as controls. (B) Results from Selaginella doederienii, and a PCR of genomic DNA is on the right. Chloroplast matKsare expressed in L. cernua and O. petiolatum, but not in S. doederienii.Abbreviations: M (matK), R (rbcL), I and I1 (intergenetic spacer: rbcL/atpB), I2 (trnL intron). (B) Bayesian inference tree (A) Maximum parsimony tree Fig. 6. Phylogenetic analyses of matK showed Gnetales is sister to other seed plants. Discussion Nicotiana tabacum • The matK is present in the chloroplasts of lower land plants, but trnK5’-matK-trnK3’ structure may be lost in ferns due to chloroplast genome rearrangement. • Chloroplast matKs are expressed in Ophioglossum and Lycopodiella, butnot expressed in Selaginella. • Chloroplast matK follows chloroplast average codon usage and the bias is influenced by GC content. • Codon usage of matK does have evolutionary properties. • Phylogenetic analysis of matK showed Gnetales is sister to other seed plants. Atropa belladonna Epifagus virginiana Spinacia oleracea Arabidopsis thaliana Oenothera elata Lotus corniculatus Fig. 3. Dot blot hybridization, indicating that matK is likely present in all of the samples examined. Zea mays Oryza sativa Triticum aestivum Calycanthus floridus Amborella trichopoda Kishino-Hasegawa test Pinus koraiensis Pinus thunbergii Adiatum capillus-veneris A B Psilotum nudum Physcomitrella patens Marchantia polymorpha Anthoceros formosae Chaetosphaeridium globosum Chara vulgaris P<0.05 * Table 1. The results of Kishino-Hasegawa test show that the TreeParsimony is preferred in parsimony criteria, but the TreeBayesian is favored by likelihood criteria, and both the alternative tree topologies are rejected. Fig. 7. Distribution of chloroplast introns. Arrow A indicates the presence of trnK/matK in Chaetosphaeridium + land plant chloroplasts. Arrow B indicates matK being a pseudogene in Anthoceros.