Grass plastomes reveal unexpected paraphyly with endemic species of Micrairoideae from India and new haplotype markers in Arundinoideae

PREMISE OF THE STUDY: We investigated the little-studied Arundinoideae/Micrairoideae clade of grasses with an innovative plastome phylogenomic approach. This method gives robust results for taxa of uncertain phylogenetic placement. Arundinoideae comprise ∼45 species, although historically was much larger. Arundinoideae is notable for the widely invasive Phragmites australis. Micrairoideae comprise nine genera and ∼200 species. Some are threatened with extinction, including Hubbardia, some Isachne spp., and Limnopoa. Two micrairoid genera, Eriachne and Pheidochloa, exhibit C4 photosynthesis in this otherwise C3 subfamily and represent an independent origin of the C4 pathway among grasses. METHODS: Five new plastomes were sequenced with next-generation sequencing-by-synthesis methods. Plastomes were assembled by de novo methods and phylogenetically analyzed with eight other recently published arundinoid or micrairoid plastomes and 11 outgroup species. Stable carbon isotope ratios were determined for micrairoid and arundinoid species to investigate ambiguities in the proxy evidence for C4 photosynthesis. KEY RESULTS: Phylogenomic analyses showed strong support for ingroup nodes in the Arundinoideae/Micrairoideae subtree, including a paraphyletic clade of Hubbardieae with Isachneae. Anatomical, biochemical, and positively selected sites data are ambiguous with regard to the photosynthetic pathways in Micrairoideae. Species of Hubbardia, Isachne, and Limnopoa were definitively shown by δ13C measurements to be C3 and Eriachne to be C4. CONCLUSIONS: Our plastome phylogenomic analyses for Micrairoideae are the first phylogenetic results to indicate paraphyly between Isachneae and Hubbardieae. The definitive δ13C data for four genera of Micrairoideae indicates the breadth of variation possible in the proxy evidence for photosynthetic pathways of both C3 and C4 taxa.