MOLECULAR ECOLOGY AND EVOLUTION Host Race Evolution in Schizaphis graminum (Hemiptera: Aphididae): Nuclear DNA Sequences KEVIN A. SHUFRAN1 USDAÐARS, Wheat, Peanut, and Other Field Crops Research Unit, 1301 N. Western Road, Stillwater, OK 74075 Downloaded from https://academic.oup.com/ee/article-abstract/40/5/1317/420303 by guest on 13 October 2019 Environ. Entomol. 40(5): 1317Ð1322 (2011); DOI: http://dx.doi.org/10.1603/EN11103 ABSTRACT The greenbug aphid, Schizaphis graminum (Rondani) was introduced into the United States in the late 1880s, and quickly was established as a pest of wheat, oat, and barley. Sorghum was also a host, but it was not until 1968 that greenbug became a serious pest of it as well. The most effective control method is the planting of resistant varieties; however, the occurrence of greenbug biotypes has hampered the development and use of plant resistance as a management technique. Until the 1990s, the evolutionary status of greenbug biotypes was obscure. Four mtDNA cytochrome oxidase subunit I (COI) haplotypes were previously identiÞed, suggesting that S. graminum sensu lato was comprised of host-adapted races. To elucidate the current evolutionary and taxonomic status of the greenbug and its biotypes, two nuclear genes and introns were sequenced; cytochrome c (CytC) and elongation factor 1-␣ (EF1-␣). Phylogenetic analysis of CytC sequences were in complete agreement with COI sequences and demonstrated three distinct evolutionary lineages in S. graminum. EF1-␣ DNA se- quences were in partial agreement with COI and CytC sequences, and demonstrated two distinct evolutionary lineages. Host-adapted races in greenbug are sympatric and appear reproductively isolated. Agricultural biotypes in S. graminum likely arose by genetic recombination via meiosis during sexual reproduction within host-races. The 1968 greenbug outbreak on sorghum was the result of the introduction of a host race adapted to sorghum, and not selection by host resistance genes in crops. KEY WORDS biotypes, cytochrome c, elongation factor 1-␣, greenbug, host-plant resistance The greenbug, Schizaphis graminum (Rondani), was The sudden appearance and wide range of the “sor- Þrst discovered in Virginia in 1882 and by 1890 large ghum biotype”, or biotype C as described by Harvey scale outbreaks on wheat, Triticum aestivum L., and and Hackerott (1969), was thought to be a result of an oat, Avena sativa L., began to occur in Texas and introduction of a genotype specializing on that host Oklahoma, as well as other states including Missouri, (Eastop 1973, Blackman 1981). Biotype C was consid- Illinois, and Indiana (Webster and Phillips 1912). It ered morphologically distinct from previous popula- remained mostly relegated to these hosts until 1968 tions (biotype B) damaging to wheat (Blackman 1981, when grain sorghum, Sorghum bicolor (L.) Moench, Fargo et al. 1986, Inayatullah et al. 1987). This was an was infested and injured over a large portion of the early indication that S. graminum sensu lato might be United States (Harvey and Hackerott 1969). Previ- comprised of evolutionary divergent host races or ously, sorghum had been reported as a host, but green- subspecies; however, this theory mostly was ignored bug had not been a signiÞcant pest. until biotypes were examined for variability by using At the time of the 1968 outbreak on sorghum, the molecular genetics approaches. A simplistic model of greenbug biotype phenomenon was well established biotype evolution, whereby biotype E evolved from C, (Porter et al. 1997). The occurrence of greenbug bio- and C evolved from B was generally accepted (In- types capable of injuring and killing resistant wheat ayatullah 1985). and sorghum was thought to be the result of selection Subsequent molecular ecology studies cast doubt pressure placed on single or major host plant resis- on this theory and instead proposed that S. graminum tance genes in a manner analogous to insecticide re- biotypes represented host-adapted races and were the sistance (Teetes 1980) and this theory persisted well products of hundreds if not thousands of years of into the late 20th century (Eisenbach and Mittler reproductive isolation on different hosts (Powers et al. 1987a, de Ponti and Mollema 1992, Lazar et al. 1995). 1989, Black et al. 1992, Black 1993). These studies led to a new theory of the evolution and occurrence of S. graminum biotypes, in which biotypes existed inde- pendently of agriculture and were maintained on The United States Department of Agriculture is an equal oppor- tunity provider and employer. noncultivated hosts (Porter et al. 1997). This new 1 Corresponding author, e-mail: [email protected]. paradigm was supported by sequencing of mtDNA 0046-225X/11/1317Ð1322$04.00/0 ᭧ 2011 Entomological Society of America 1318 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 5 Table 1. Schizaphis graminum biotypes and mtDNA haplotype trons, cytochrome c (CytC) and elongation factor 1-␣ associations from which nDNA was sequenced (EF1-␣), were polymerase chain reaction (PCR) am- pliÞed and sequenced. mtDNA Biotype Reference Haplotype A 640 bp PCR amplicon of the CytC was produced and direct sequenced using the primers cytC-C-5Ј I C (Shufran et al. 2000) Ј Ј E (Shufran et al. 2000) (5 -AAGTGTGCYCARTGCCACAC-3 ) (Palumbi E-OK (Shufran and Puterka 2011) 1996) and cytC-B-3Ј (5Ј-CATCTTGGTGCCGGGG- I (Shufran et al. 2000) ATGTATTTCTT-3Ј) (Palumbi 1996). Amplicons J (Shufran et al. 2000) were puriÞed using the Wizard SV Gel and PCR K (Shufran et al. 2000) II F (Shufran et al. 2000) Clean-Up System (Promega, Madison, WI). A 1.2-kbp G (Shufran et al. 2000) amplicon of the EF1-␣ was produced using the primers NY (Shufran et al. 2000) EF2Ð3Ј (5Ј-ATGTGAGCAGTGTGGCAATCCAA-3Ј) Downloaded from https://academic.oup.com/ee/article-abstract/40/5/1317/420303 by guest on 13 October 2019 ?-OK (Shufran and Puterka 2011) (Palumbi 1996) and EFS175 (5Ј-GGAAATGGG- III B (Shufran et al. 2000) Ј B-OK (Shufran and Puterka 2011) AAAAGGCTCCTTCAAGTAYGCYTGGG-3 ) (Mo- Paspalum vaginatum (Shufran and Puterka 2011) ran et al. 1999). The EF1- ␣ amplicon was then cloned H H (Shufran et al. 2000) using the pGEM ÐT Easy Vector kit (Promega, Mad- ison, WI) and sequenced with the T7 and SP6 primers. DNA sequencing was conducted with 5ϫ to 6ϫ cov- cytochrome oxidase subunit I (COI), which deter- erage with BigDye-terminated reactions analyzed on mined S. graminum populations had three mtDNA an ABI model 3730 DNA Analyzer (Recombinant COI haplotypes (Shufran et al. 2000, Anstead et al. DNA/Protein Core Facility, Department of Biochem- 2002). These mtDNA sequencing results were consis- istry and Molecular Biology, Oklahoma State Univer- tent with the results of restriction fragment length sity, Stillwater, OK). Internal primers EF4 and EF5 polymorphism (RFLP) analysis of mtDNA (Powers et were used to complete sequencing (von Dohlen et al. al. 1989) and rDNA (Black 1993) and supported the 2002) DNA sequences were assembled and aligned by theory that S. graminum was comprised of host- the ClustalW method (Thompson et al. 1994) using adapted races or subspecies. However, studies of in- the LaserGene Ver. 8.0.2 software suite (DNASTAR, trabiotype variation demonstrated that biotypes ex- Inc., Madison, WI). Default alignment parameters isted independent of COI haplotypes (Anstead et al. were used; gap penalty 15.0, gap length penalty 6.66, 2002). Single biotypes were found in more than one delay divergent sequences 30%, and DNA transition COI haplotype. weight 0.5. Phylogenetic analyses were conducted us- The source of biotypic diversity was shown to be a ing the MEGA5 statistical software package (Tamura product of recombination of virulence genes during et al. 2011). Maximum likelihood (ML) method with meiosis, which occurs during the holocyclic life cycle 1,000 bootstraps was used based on the Tamura and phase of S. graminum (Puterka and Peters 1989, 1990, Nei (1993) model, with uniform substitution rates 1995). Crosses between and within biotypes resulted among sites, all sites (gaps and/or missing data) used, in new biotypic variation in the offspring. Multiple and the ML heuristic method Nearest-Neighbor-In- biotypes found among aphids with different COI hap- terchange. Completed sequences were submitted to lotypes (Anstead et al. 2002) suggest there was gene GenBank with accession numbers: CytC, JF719744 - ßow between host races or homologous virulence JF719757; and EF1-␣, JF968544 - JF968556. Voucher genes existed between host races that were reproduc- specimens of S. graminum biotypes (sisters of speci- tively isolated. To answer this question, DNA se- mens used for DNA analysis) were deposited with the quence variation in the intron and coding regions of Museum of Entomology, FL State Collection of Ar- two nuclear genes was analyzed using the same S. thropods, Gainesville, FL. graminum populations of Shufran et al. (2000), plus four additional clones. Results In total, 518 nucleotides were sequenced from S. Materials and Methods graminum CytC. No nucleotide variation was found in Ten S. graminum biotypes (B, C, E, F, G, H, I, J, K, the coding region, i.e., the Þrst 80 bases, and all sub- and NY), preserved at Ϫ80ЊC from Shufran et al. stitutions, deletions, and insertions occurred in the (2000), three biotypes (E, B, and an unknown, des- 438-bp intron that followed. Phylogenetic analysis ignated as “?”) collected from wheat at Okeene, OK with maximum likelihood produced a dendrogram (Shufran and Puterka 2011), and the Paspalum vagi- with three major clades (Fig. 1a), although the clade natum Sw. (AKA Florida or P) biotype (Nuessly et al. containing biotypes C, E, E-OK, I, K and J was not well 2008) were used in the current study. The mtDNA supported by bootstrap analysis. Neighbor-joining COI haplotype associated with each is shown in Table and maximum parsimony analysis produced essen- 1. The prepGEM Insect DNA extraction kit (ZyGEM tially identical results. Each clade corresponded to Corp.