Background: For over 50 years, attempts have been made to introgress agronomically useful traits from Erianthus sect. Ripidium (Tripidium) species into sugarcane based on both genera being part of the ‘Saccharum Complex’, an interbreeding group of species believed to be involved in the origins of sugarcane. However, recent low copy number gene studies indicate that Tripidium and Saccharum are more divergent than previously thought. The extent of genus Tripidium has not been fully explored and many species that should be included in Tripidium are still classified as Saccharum. Moreover, Tripidium is currently defined as incertae sedis within the Andropogoneae, though it has been suggested that members of this genus are related to the Germainiinae. Results: Eight newly-sequenced chloroplasts from potential Tripidium species were combined in a phylogenetic study with 46 members of the Panicoideae, including seven Saccharum accessions, two Miscanthidium and three Miscanthus species. A robust chloroplast phylogeny was generated and comparison with a gene locus phylogeny clearly places a monophyletic Tripidium clade outside the bounds of the Saccharinae. A key to the currently identified Tripidium species is presented. Conclusion: For the first time, we have undertaken a large-scale whole plastid study of eight newly assembled Tripidium accessions and a gene locus study of five Tripidium accessions. Our findings show that Tripidium and Saccharum are eight million years divergent, last sharing a common ancestor 12 million years ago. We demonstrate that four species should be removed from Saccharum/Erianthus and included in genus Tripidium. In a genome context, we show that Tripidium evolved from a common ancestor with and extended Germainiinae clade formed from Germainia, Eriochrysis, Apocopis, Pogonatherum and Imperata. We re-define the ‘Saccharum complex’ to a group of genera that can interbreed in the wild and extend the Saccharinae to include Sarga along with Sorghastrum, Microstegium vimineum and Polytrias (but excluding Sorghum). Monophyly of genus Tripidium is confirmed and the genus is expanded to include Tripidium arundinaceum, Tripidium procerum, Tripidium kanashiroi and Tripidium rufipilum. As a consequence, these species are excluded from genus Saccharum. Moreover, we demonstrate that genus Tripidium is distinct from the Germainiinae.
Low copy number gene loci assembled for the project
A tarball (tar.gz) file containing the readme, along with individual assemblies of 5 low copy number gene regions for the following species:
Miscanthidium_junceum Sarga_versicolor
Miscanthus_sacchariflorus Sorghum_propinquum
Miscanthus_sinensis_Andante Andropogon_virginicus
Miscanthus_transmorrisonensis Tripidium_arundinaceum-2
Miscanthus_x_giganteus Sorghum_bicolor-BTx623
Saccharum_hybrid_LCP85-384 Sorghum_arundinaceum
Saccharum_hybrid-SP80-3280 Tripidium_procerum
Saccharum_spontaneum Tripidium_rufipilum
Sarga_timorense Coix_lacryma-jobi (ep2-ex7 and ep2-ex8 only)
The assemblies can be found in individual multi-fasta corresponding to the gene name and exon of the region assembled:
apo1
d8
ep2-ex7
ep2-ex8
rep1
gene-loci.tar.gz
Alignment of low copy number gene loci employed in this study
Concatenated alignment for the five low copy number gene regions employed in the study.
Alignment file is:
low-copy-number-alignment.fas
as well as sequences derived from the paper of Estep et al:
Estep MC, McKain MR, Diaz DV, Zhong J, Hodge JG, Hodkinson TR, et al. Allopolyploidy, diversification, and the Miocene grassland expansion. Proc Natl Acad Sci USA. 2014; 111:15149-54
The Estep et al. datasets can be obtained from the Dryad digital repository:
http://datadryad.org/resource/doi:10.5061/dryad.5kf38
The following data partitions were employed
apo1.fsa
d8.fsa
ep2-ex7.fsa
ep2-ex8.fsa
rep1.fsa
In addition to data from the Estep et al. publication, the Sorghum laxiflorum dataset (lacking rep1) of et al:
Hawkins, J.S., Ramachandran, D., Henderson, A., Freeman, J., Carlise, M., Harris, A. and Willison-Headley, Z., 2015. Phylogenetic reconstruction using four low-copy nuclear loci strongly supports a polyphyletic origin of the genus Sorghum. Annals of botany, 116(2), pp.291-299.
was also emplogyed.
The alignment files include the following additional species data assembled from SRA datasets:
Miscanthidium junceum
Miscanthus_transmorrisonensis
Miscanthus_sacchariflorus
Miscanthus_x_giganteus
Saccharum_hybrid_LCP85-384
Saccharum_hybrid_SP80-3280
Saccharum_spontaneum_RB
Sarga_versicolor
Sarga_timorense
Sorghum_propinquum
Sorguhum_bicolor_BTx623
Sorghum_arundinaceum
Miscanths sinensis cv Andanta and Andropogon virginicus whole genome datasets as well as Tripidium arundinaceum 2, Tripidium procerum and Tripidium rufipilum datasets were sequenced for this study.
The alignment is partitioned as follows
locus partition
apo1 1-674
d8 675-1816
ep2-ex7 1817-2818
ep2-ex8 2819-3640
rep1 3641-4426
low-copy-number-gene-align-phylogeny.tar.gz
Low copy gene locus alignment and phylogeny
Alignment of the five low copy number gene loci analyzed in the paper, along with a maximum likelihood phylogeny derived from the alignment matrix. The alignment matrix is divided into the following gene region partitions: apo1.fsa
d8.fsa
ep2-ex7.fsa
ep2-ex8.fsa
rep1.fsa
The alignment is divided into partitions as follows:
locus partition
apo1 1-674
d8 675-1816
ep2-ex7 1817-2818
ep2-ex8 2819-3640
rep1 3641-4426
Partitions were alanyzed independently to generate the reference phylogeny.
low-copy-number-gene-align-phylogeny.tar.gz
chloroplast-align-phylogeny.tar
Tarball contains the files: README-CP.txt, tripidium-rearranged.fasta and Tripidium-cp-rooted.phy. The tripidium-rearranged.fasta file contains the alignment of whole chloroplasts from 54 species from the Andropogoneae focussing on Tripidium and Saccharum. The alignment is divided into LSC, IRA and SSC partitions. These partitions were further divided into protein-coding gene, RNA-coding gene and non-coding regions. The regions were isolated with the BeforePhylo.pl script and merged into separate partitions which are joined together in this file. The partitions are: p1 = 1-40026
p2 = 40027-41716
p3 = 41717-85819
p4 = 85820-94385
p5 = 94386-99135
p6 = 99136-105301
p7 = 105302-104847
p8 = 104848-123717
Best-fit evolutionary models for each partition were selected using JModelTest2 and the AICc criterion. The best-fit models were as follows: LSC protein coding: TPM1uf+I+Gamma, LSC RNA genes: TVM+Gamma; LSC non-coding: TVM+Gamma; IRA protein coding: TVM+I+Gamma; IRA non-coding: TVM+Gamma; SSC protein coding: TPM1uf+I+Gamma; SSC RNA-gene: TrN+I+Gamma and SSC non-coding: TVM+I+Gamma.
The alignment was analyzed with RAxML using the above partitions and the most likely tree is given in Tripidium-cp-rooted.phy which is the tree topology presented in the BMC Evolutionary Biology paper.