Phylogenetic Analysis of Organellar DNA Sequences in the Andropogoneae: Saccharinae
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Theor Appl Genet (1994) 88:933-944 Springer-Verlag 1994 S. M. A1-Janabi M. McClelland C. Petersen B. W. S. Sobral Phylogenetic analysis of organellar DNA sequences in the Andropogoneae: Saccharinae Received: 30 June 1993 / Accepted: 21 December 1993 Abstract To study the phylogenetics of sugarcane (Sac- wheat, rice, barley, and maize, which were used as out- charum officinarum L.) and its relatives we sequenced four groups in phylogenetic analyses. To determine whether loci on cytoplasmic genomes (two chloroplast and two mit- limited intra-complex variability was caused by under sam- ochondrial) and analyzed mitochondrial RFLPs generated pling of taxa, we used seven restriction enzymes to digest using probes for COXI, COXII, COXIII, Cob, 18S+5S, the PCR-amplified rbcL-atpB spacer of an additional 36 26S, ATPase 6, ATPase 9, and ATPase ~ (D'Hont et al. accessions within the Saccharum complex. This analysis 1993). Approximately 650 bp of DNA in the intergenic revealed ten restriction sites (none informative) and eight spacer region between rbcL and atpB and approximately length variants (four informative). The small amount of 150 bp from the chloroplast 16S rDNA through the inter- variation present in the organellar DNAs of this polyptoid genic spacer region tRNA val gene were sequenced. In the complex suggests that either the complex is very young or mitochondrial genome, part of the 18S rRNA gene and ap- that rates of evolution between the Saccharum complex proximately 150 bp from the 18S gene 3' end, through an and outgroup taxa are different. Other phylogenetic infor- intergenic spacer region, to the 5S rRNA gene were se- mation will be required to resolve systematic relationships quenced. No polymorphisms were observed between within the complex. Finally, no variation was observed in maize, sorghum, and 'Saccharum complex' members for commercial sugarcane varieties, implying a world-wide the mitochondrial 18S internal region or for the intergenic cytoplasmic monoculture for this crop. tRNA va~ chloroplast locus. Two polymorphisms (insertion- deletion events, indels) were observed within the 18S-5S Key words Cycle sequencing - PCR - Chloroplast mitochondrial locus, which separated the accessions into Sugarcane Polyploid rbcL. atpB Mitochondria three groups: one containing all of the Erianthus, Eccoilo- Saccharum pus, Imperata, Sorghum, and 1 Miscanthus species; a sec- ond containing Saccharum species, Narenga porphyroc- oma, Sclerostachya fusca, and 1 presumably hybrid Mis- canthus sp. from New Guinea; and a third containing Introduction maize. Eighteen accessions were sequenced for the inter- genic region between rbcL and atpB, which was the most Saccharum L. is part of the Saccharinae subtribe of the An- polymorphic of the regions studied and contained 52 site dropogoneae (Watson et al. 1985, Clayton and Renvoize mutations and 52 indels, across all taxa. Within the Sac- 1986). The Andropogoneae are frequently polyploid and charum complex, at most 7 site mutations and 16 indels many are also of great socio-economic importance (Steb- were informative. The maternal lineage of Erianthus/Ec- bins 1956; Soltis et al. 1992). Very little is known about coilopus was nearly as divergent from the remaining Sac- the speciation and evolution of potyploids even though charum complex members as it was from sorghum, in about 50-70% of all grass species are polyploid (Stebbins agreement with a previous study. Sequences from the rbcL- 1956; Soltis et al. 1992). Furthermore, it is difficult to de- atpB spacer were aligned with GENBANK sequences for limit some polyploid plant species as interspecific boun- daries are unclear because of the possibility of hybridiza- tion and chromosome doubling. These biological phenom- Communicated by A. R. Hallauer ena have helped keep the taxonomy of Saccharum in a state of flux, especially at lower ranks. Mukherjee (1957) coined S. M. A1-Janabi M. McClelland C. Petersen B. W. S. Sobral ([]) California Institute of Biological Research, the term 'Saccharum complex' to refer to members of Na- 11099 North Torrey Pines Road, renga Bor, Sclerostachya (Anderss. ex Hackel) A. Camus, Suite 300, La Jolla CA 92037, USA Erianthus Michx. section Ripidium, and Saccharum that 934 can interbreed and in which species (1) are endemic to the type of Saccharum, and it was a commercial interspecific Bengal-Assam-Sikkim area in India, (2) have been classi- hybrid (S. officinarum x S. spontaneum hybrids). Glasz- fied as Saccharum at one time or another, and (3) have been mann et al. (1990) determined RFLP variation of nuclear directly implicated in the evolution of Saccharum by var- rDNA in some Saccharum species, commercial hybrids, ious authors. More recently, Daniels and Williams (1975) one Erianthus arundinaceus accession, and one Miscan- included Miscanthus Anderss. section Diandra species in thus species. They reported 15 rDNA length variants, some a revision of the complex and extended the geographic of which appeared to be phylogenetically informative, range to include the Indo-Burma-China border region. A though no phylogenies were inferred, possibly because recent taxonomy of the Andropogoneae considers Erian- most accessions studied had more than one rDNA RFLP thus and Narenga to be synonymous with Saccharum, Ec- phenotype and hybridizing fragments varied in intensity. coilopus synonymous with Spodiopogon, and Miscanthus D'Hont et al. (1993) studied organellar RFLPs in 57 ac- synonymous with Miscanthidium and Sclerostachya cessions of Saccharum and some Saccharum complex (Clayton and Renvoize 1986). It seems that the Saccharum members; they found no variation using 2 chloroplast complex is a group of plants with their own evolutionary probes that covered approximately 20% of the wheat chlo- history. However, it remains unclear as to what has been roplast genome, although a single fragment was missing the role of the processes of hybridization and chromosome for the single Erianthus and Miscanthus species studied. doubling in the evolution of this complex, though there has In addition, nine mitochondrial probes yielded ten differ- been much speculation. In addition, it is unclear what is ent pattern types, although no phylogenetic hypothesis was the main reproductive mode in nature: vegetative or sex- generated from their data. Sobral et al. (1994) studied chlo- ual. The taxonomy of Saccharum has been and still is com- roplast RFLPs using 32 accessions representing eight gen- plex and controversial. Some of the controversy may stem era and 19 species with 15 restriction enzymes and 12 rice from the use of morphological traits as such traits may be chloroplast probes that covered the rice chloroplast ge- difficult to score in polyploid species that can interbreed. nome entirely. Sixty-two mapped restriction site mutations Furthermore, it is clear that human activities over the past (18 informative) placed the accessions into nine clades: 10,000 years or more have played a crucial role in the dis- seven from within the Saccharinae, maize, and sorghum. persal, selection, and vegetative propagation of particular Maternal lineages of Saccharum, Narenga, Sclerostachya, genotypes of sugarcane and its relatives, including inter- and Miscanthus were shown to form a monophyletic group specific and intergeneric hybrids (Brandes 1928; Artsch- displaying little variation, whereas Erianthus and Eccoi- wager and Brandes 1958; Bellwood 1985). Studies using lopus were shown to be different from other Saccharum leaf flavonoids (Williams and Harborne 1974; Daniels et complex members. Because the level of polymorphism de- al. 1980) and leaf waxes (Smith and Martin-Smith 1978) tected by previous studies was very low, we sequenced po- suggest that a large amount of variability is present in some tentially polymorphic regions in the chloroplast genome to species (notably S. spontaneum and Erianthus species). allow a finer level of phylogenetic resolution within the Studies using the tools of molecular systematics in the Saccharum complex and herein report the results. Saccharum complex were pioneered by Waldron et al. (1974). They used/3-amylase isoenzymes as genetic mark- ers in Saccharum and its relatives and found that collec- tion site was correlated with banding pattern, which was Materials and methods proposed to indicate widespread occurrence of hybridiza- tion. The limited existing body of molecular data support Plant materials a recent common ancestry for sorghum and sugarcane. Plant accessions and their origins are listed in Table 1. These acces- Hamby and Zimmer (1988) used nuclear rRNA sequences sions were chosen to represent a wide range of species, and they dif- of taxa within Poales to infer phylogeny. Their phyloge- fer in their origin and chromosome numbers; some have been stud- netic hypothesis showed that Sorghum and Saccharum ied previously (Sobral et al. 1993). were very closely related; in fact these had the smallest Primer design pairwise genetic distance (d=0.00611) of any two genera sampled. This is interesting because Sorghum is thought To study cytoplasmic DNA sequence variation we chose regions in the genome that were highly conserved though interspersed with to have radiated from Africa whereas Saccharum has been polymorphic regions. We constructed primers to the conserved re- suggested to have radiated from Southeastern Asia. Cur- gions that would amplify across regions that were expected to be rent taxonomy suggests that the genetic distance from Sac- polymorphic based on previous work (Zurawski et al.