Insights into Regulation of C2 and C4 Photosynthesis in Amaranthaceae/Chenopodiaceae Using RNA-Seq.

Amaranthaceae (incl. Chenopodiaceae) shows an immense diversity of C4 syndromes. More than 15 independent origins of C4 photosynthesis, and the largest number of C4 species in eudicots signify the importance of this angiosperm lineage in C4 evolution. Here, we conduct RNA-Seq followed by comparative transcriptome analysis of three species from Camphorosmeae representing related clades with different photosynthetic types: Threlkeldia diffusa (C3), Sedobassia sedoides (C2), and Bassia prostrata (C4). Results show that B. prostrata belongs to the NADP-ME type and core genes encoding for C4 cycle are significantly upregulated when compared with Sed. sedoides and T. diffusa. Sedobassia sedoides and B. prostrata share a number of upregulated C4-related genes; however, two C4 transporters (DIT and TPT) are found significantly upregulated only in Sed. sedoides. Combined analysis of transcription factors (TFs) of the closely related lineages (Camphorosmeae and Salsoleae) revealed that no C3-specific TFs are higher in C2 species compared with C4 species; instead, the C2 species show their own set of upregulated TFs. Taken together, our study indicates that the hypothesis of the C2 photosynthesis as a proxy towards C4 photosynthesis is questionable in Sed. sedoides and more in favour of an independent evolutionary stable state.

Biogeography of the xerophytic genus Anabasis L. (Chenopodiaceae).

AIM: Using the extremophile genus Anabasis, which includes c. 28 succulent, xerophytic C4 species, and is widely distributed in arid regions of Northern Africa, Arabia, and Asia, we investigate biogeographical relationships between the Irano-Turanian floristic region (ITfr) and its neighboring regions. We test whether the spread of arid and semi-arid biomes in Eurasia coincides with the biogeography of this drought-adapted genus, and whether the ITfr acted as source area of floristic elements for adjacent regions. LOCATION: Deserts and semi-deserts of Northern Africa, Mediterranean, Arabia, West and Central Asia. METHODS: Four cpDNA markers (rpL16 intron, atpB-rbcL, trnQ-rps16, and ndhF-rpL32 spacers) were sequenced for 58 accessions representing 21 Anabasis species. Phylogenetic relationships and divergence times were inferred using maximum likelihood and a time-calibrated Bayesian approach. To document the extant distribution of Anabasis, material from 23 herbaria was surveyed resulting in 441 well-documented collections used for the coding of eight floristic regions. Using this coded data, ancestral range was estimated using "BioGeoBEARS" under the DEC model. RESULTS: Anabasis originated during the Late Miocene and the ancestral range was probably widespread and disjunct between Western Mediterranean and the Irano-Turanian regions. Diversification started with two divergence events at the Miocene/Pliocene boundary (5.1 and 4.5 mya) leading to Asian clade I with ITfr origin which is sister to a slightly younger Asian clade II, which originated in the Western ITfr, and a Mediterranean/North African clade with an origin in the Western Mediterranean. MAIN CONCLUSIONS: Anabasis did not follow aridification and continuously expanded its distribution area, in fact its probably wide ancestral distribution area seems to have been fragmented during the very Late Miocene and the remnant lineages then expanded into neighboring arid regions. This genus supports the role of the ITfr as source area for xerophytic elements in the Mediterranean and Central Asia.

From cacti to carnivores: Improved phylotranscriptomic sampling and hierarchical homology inference provide further insight into the evolution of Caryophyllales.

PREMISE OF THE STUDY: The Caryophyllales contain ~12,500 species and are known for their cosmopolitan distribution, convergence of trait evolution, and extreme adaptations. Some relationships within the Caryophyllales, like those of many large plant clades, remain unclear, and phylogenetic studies often recover alternative hypotheses. We explore the utility of broad and dense transcriptome sampling across the order for resolving evolutionary relationships in Caryophyllales. METHODS: We generated 84 transcriptomes and combined these with 224 publicly available transcriptomes to perform a phylogenomic analysis of Caryophyllales. To overcome the computational challenge of ortholog detection in such a large data set, we developed an approach for clustering gene families that allowed us to analyze >300 transcriptomes and genomes. We then inferred the species relationships using multiple methods and performed gene-tree conflict analyses. KEY RESULTS: Our phylogenetic analyses resolved many clades with strong support, but also showed significant gene-tree discordance. This discordance is not only a common feature of phylogenomic studies, but also represents an opportunity to understand processes that have structured phylogenies. We also found taxon sampling influences species-tree inference, highlighting the importance of more focused studies with additional taxon sampling. CONCLUSIONS: Transcriptomes are useful both for species-tree inference and for uncovering evolutionary complexity within lineages. Through analyses of gene-tree conflict and multiple methods of species-tree inference, we demonstrate that phylogenomic data can provide unparalleled insight into the evolutionary history of Caryophyllales. We also discuss a method for overcoming computational challenges associated with homolog clustering in large data sets.

De novo Transcriptome Assembly and Comparison of C3, C3-C4, and C4 Species of Tribe Salsoleae (Chenopodiaceae).

C4 photosynthesis is a carbon-concentrating mechanism that evolved independently more than 60 times in a wide range of angiosperm lineages. Among other alterations, the evolution of C4 from ancestral C3 photosynthesis requires changes in the expression of a vast number of genes. Differential gene expression analyses between closely related C3 and C4 species have significantly increased our understanding of C4 functioning and evolution. In Chenopodiaceae, a family that is rich in C4 origins and photosynthetic types, the anatomy, physiology and phylogeny of C4, C2, and C3 species of Salsoleae has been studied in great detail, which facilitated the choice of six samples of five representative species with different photosynthetic types for transcriptome comparisons. mRNA from assimilating organs of each species was sequenced in triplicates, and sequence reads were de novo assembled. These novel genetic resources were then analyzed to provide a better understanding of differential gene expression between C3, C2 and C4 species. All three analyzed C4 species belong to the NADP-ME type as most genes encoding core enzymes of this C4 cycle are highly expressed. The abundance of photorespiratory transcripts is decreased compared to the C3 and C2 species. Like in other C4 lineages of Caryophyllales, our results suggest that PEPC1 is the C4-specific isoform in Salsoleae. Two recently identified transporters from the PHT4 protein family may not only be related to the C4 syndrome, but also active in C2 photosynthesis in Salsoleae. In the two populations of the C2 species S. divaricata transcript abundance of several C4 genes are slightly increased, however, a C4 cycle is not detectable in the carbon isotope values. Most of the core enzymes of photorespiration are highly increased in the C2 species compared to both C3 and C4 species, confirming a successful establishment of the C2 photosynthetic pathway. Furthermore, a function of PEP-CK in C2 photosynthesis appears likely, since PEP-CK gene expression is not only increased in S. divaricata but also in C2 species of other groups.

C3 -C4 intermediates may be of hybrid origin - a reminder.

The currently favoured model of the evolution of C4 photosynthesis relies heavily on the interpretation of the broad phenotypic range of naturally growing C3 -C4 intermediates as proxies for evolutionary intermediate steps. On the other hand, C3 -C4 intermediates had earlier been interpreted as hybrids or hybrid derivates. By first comparing experimentally generated with naturally growing C3 -C4 intermediates, and second summarising either direct or circumstantial evidence for hybridisation in lineages comprising C3 , C4 and C3 -C4 intermediates, we conclude that a possible hybrid origin of C3 -C4 intermediates deserves careful examination. While we acknowledge that the current model of C4 photosynthesis evolution is clearly the best available, C3 -C4 intermediates of hybrid origin, if existing, should not be used for further analysis of this model. However, experimental C3  × C4 hybrids potentially are excellent systems to analyse the genetic differences between C3 and C4 species and, also using segregating progeny, to study the relationship between individual photosynthetic traits and environmental factors.

C3 cotyledons are followed by C4 leaves: intra-individual transcriptome analysis of Salsola soda (Chenopodiaceae).

Some species of Salsoleae (Chenopodiaceae) convert from C3 photosynthesis during the seedling stage to the C4 pathway in adult leaves. This unique developmental transition of photosynthetic pathways offers the exceptional opportunity to follow the development of the derived C4 syndrome from the C3 condition within individual plants, avoiding phylogenetic noise. Here we investigate Salsola soda, a little-studied species from tribe Salsoleae, using an ontogenetic approach. Anatomical sections, carbon isotope (δ13C) values, transcriptome analysis by means of mRNA sequencing, and protein levels of the key C4 enzyme phosphoenolpyruvate carboxylase (PEPC) were examined from seed to adult plant stages. Despite a previous report, our results based on δ13C values, anatomy and transcriptomics clearly indicate a C3 phase during the cotyledon stage. During this stage, the entire transcriptional repertoire of the C4 NADP-malic enzyme type is detected at low levels compared to a significant increase in true leaves. In contrast, abundance of transcripts encoding most of the major photorespiratory enzymes is not significantly decreased in leaves compared to cotyledons. PEPC polypeptide was detected only in leaves, correlating with increased PEPC transcript abundance from the cotyledon to leaf stage.

Evolution of leaf anatomy in arid environments - A case study in southern African Tetraena and Roepera (Zygophyllaceae).

The dry biomes of southern Africa (Desert, Nama Karoo and Succulent Karoo) are home to a rich and diverse xerophytic flora. This flora includes two morphologically diverse clades of Zygophyllaceae, Tetraena and Roepera (Zygophylloideae), which inhabit some of the most arid habitats in the region. Using a plastid phylogeny of Zygophylloideae we assess whether the evolution of putatively adaptive traits (leaf shape, vasculature, mode of water storage and photosynthetic type: C3 versus C4) coincides with the successful colonisation of environments with different drought regimes within southern Africa. Our results show general niche conservatism within arid habitats in Tetraena, but niche shifts from arid to more mesic biomes with longer and/or cooler growing season (Fynbos and Thicket) in Roepera. However, these distinct broad-scale biogeographical patterns are not reflected in leaf anatomy, which seems to vary at more local scales. We observed considerable variability and multiple convergences to similar leaf anatomies in both genera, including shifts between "all cell succulence" leaf types and leaf types with distinct chlorenchyma and hydrenchyma. Our survey of C4 photosynthesis in the Zygophylloideae showed that the C4 pathway is restricted to Tetraena simplex, which also having an annual life history and a widespread distribution, is rather atypical for this group.

When do different C4 leaf anatomies indicate independent C4 origins? Parallel evolution of C4 leaf types in Camphorosmeae (Chenopodiaceae).

Broad-scale phylogenetic studies give first insights in numbers, relationships, and ages of C4 lineages. They are, however, generally limited to a model that treats the evolution of the complex C4 syndrome in different lineages as a directly comparable process. Here, we use a resolved and well-sampled phylogenetic tree of Camphorosmeae, based on three chloroplast and one nuclear marker and on leaf anatomical traits to infer a more detailed picture of C4 leaf-type evolution in this lineage. Our ancestral character state reconstructions allowed two scenarios: (i) Sedobassia is a derived C3/C4 intermediate, implying two independent gains of C4 in Bassia and Camphorosma; or (ii) Sedobassia is a plesiomorphic C3/C4 intermediate, representing a syndrome ancestral to the Bassia/Camphorosma/Sedobassia lineage. In Bassia, a kochioid leaf type (Bassia muricata and/or Bassia prostrata type) is ancestral. At least three independent losses of water-storage tissue occurred, resulting in parallel shifts towards an atriplicoid leaf type. These changes in leaf anatomy are adaptations to different survival strategies in steppic or semi-desert habitats with seasonal rainfall. In contrast, Camphorosma shows a fixed C4 anatomy differing from Bassia types in its continuous Kranz layer, which indeed points to an independent origin of the full C4 syndrome in Camphorosma, either from an independent C3 or from a common C3/C4 intermediate ancestor, perhaps similar to its C3/C4 intermediate sister genus Sedobassia. The enlarged bundle sheath cells of Sedobassia might represent an important early step in C4 evolution in Camphorosmeae.