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Previous analysis of charophyte green algal (CGA) genomes and transcriptomes for specific protein families revealed that numerous land plant characteristics had already evolved in CGA. In this study, we have sequenced and assembled the transcriptome of Zygnema circumcarinatum UTEX 1559, and combined its predicted protein sequences with those of 13 additional species [five embryophytes (Emb), eight charophytes (Cha), and two chlorophytes (Chl) as the outgroup] for a comprehensive comparative genomics analysis. In total 25,485 orthologous gene clusters (OGCs, equivalent to protein families) of the 14 species were classified into nine OGC groups. For example, the Cha+Emb group contains 4,174 OGCs found in both Cha and Emb but not Chl species, representing protein families that have evolved in the common ancestor of Cha and Emb. Different OGC groups were subjected to a Gene Ontology (GO) enrichment analysis with the Chl+Cha+Emb group (including 5,031 OGCs found in Chl and Cha and Emb) as the control. Interestingly, nine of the 20 top enriched GO terms in the Cha+Emb group are cell wall-related, such as biological processes involving celluloses, pectins, lignins, and xyloglucans. Furthermore, three glycosyltransferase families (GT2, 8, 43) were selected for in-depth phylogenetic analyses, which confirmed their presence in UTEX 1559. More importantly, of different CGA groups, only Zygnematophyceae has land plant cellulose synthase (CesA) orthologs, while other charophyte CesAs form a CGA-specific CesA-like (Csl) subfamily (likely also carries cellulose synthesis activity). Quantitative real-time-PCR experiments were performed on selected GT family genes in UTEX 1559. After osmotic stress treatment, significantly elevated expression was found for GT2 family genes ZcCesA, ZcCslC and ZcCslA-like (possibly mannan and xyloglucan synthases, respectively), as well as for GT8 family genes (possibly pectin synthases). All these suggest that the UTEX 1559 cell wall polysaccharide synthesis-related genes respond to osmotic stress in a manner that is similar to land plants.

A phylogenomic analysis of 42 complete plastid genomes (plastomes), including 16 that were newly sequenced, was conducted. Plastomes were sampled from 19 subtribes of Pooideae, to investigate relationships within and between Chloroplast Group 1 (Aveneae) and Group 2 (Poeae) species. Two data partitions: complete plastomes, and a combined plastome and rare genomic change (RGC) data matrix, were analyzed. Overall, 156 non-ambiguous RGC were identified, of which homology was inferred for 38 RGC. Among the 38 RGC identified, six were synapomorphic among the Group 1 subtribes: Aveninae, Agrostidinae, and Anthoxanthinae, (Phalaridinae + Torreyochloinae), and 27 were synapomorphic among the Group 2 subtribes: Loliinae, (Ammochloinae + Parapholiinae + Dactylidinae), Parapholiinae, Dactylidinae, Poinae, and Coleanthinae. Four RGC were determined to be homoplasious in Groups 1 and 2. Two other RGC originated through intrastrand deletion events. The remaining RGC events likely originated through recombination given their size and lack of sequence evidence for other types of mutations. This study also determined that relationships between taxa, even those only weakly supported in previous studies, could be inferred with strong support when utilizing complete plastomes.

This project examines the relationships within the genus Zea using complete plastid genomes (plastomes). While Zea mays has been well studied, congeneric species have yet to be as thoroughly examined. For this study four complete plastomes and a fifth nearly complete plastome were sequenced in the five species (Zea diploperennis, Zea perennis, Zea luxurians, Zea nicaraguensis, and Zea mays subsp. huehuetenangensis) by Sanger or next-generation methods. An analysis of the microstructural changes, such as inversions, insertion or deletion mutations (indels) and determination of their frequencies were performed for the complete plastomes. It was determined that 193 indels and 15 inversions occurred across the examined plastomes of Zea. Tandem repeat indels were the most common type of microstructural change observed. Divergence times were estimated using a noncorrelated relaxed clock method. Divergence dates for specific nodes relative to Zea were calculated to fall between 38,000 years before present (YBP) for the subspecies included in this study and 23,000 YBP for section Luxuriantes included in this study. The stem lineage of all Zea species was calculated to have diverged at 176,000 YBP. The calculated mutation rates for the genus fell within the range of 1.7E-8 to 3.5E-8 microstructural changes per site per year. These rates of change are not uniform, despite the close relationships of taxa in this study. Phylogenomic analyses using full plastome alignments were also conducted to compare tree topologies from different types of mutations. In most cases, the previous work examining Zea mitochondrial and nuclear data was confirmed.

The complete chloroplast and mitochondrial genomes of Charophyta have shed new light on land plant terrestrialization. Here, we report the organellar genomes of the Zygnema circumcarinatum strain UTEX 1559, and a comparative genomics investigation of 33 plastomes and 18 mitogenomes of Chlorophyta, Charophyta (including UTEX 1559 and its conspecific relative SAG 698-1a), and Embryophyta. Gene presence/absence was determined across these plastomes and mitogenomes. A comparison between the plastomes of UTEX 1559 (157 548 bp) and SAG 698-1a (165 372 bp) revealed very similar gene contents, but substantial genome rearrangements. Surprisingly, the two plastomes share only 85.69% nucleotide sequence identity. The UTEX 1559 mitogenome size is 215 954 bp, the largest among all sequenced Charophyta. Interestingly, this large mitogenome contains a 50 kb region without homology to any other organellar genomes, which is flanked by two 86 bp direct repeats and contains 15 ORFs. These ORFs have significant homology to proteins from bacteria and plants with functions such as primase, RNA polymerase, and DNA polymerase. We conclude that (i) the previously published SAG 698-1a plastome is probably from a different Zygnema species, and (ii) the 50 kb region in the UTEX 1559 mitogenome might be recently acquired as a mobile element.