Mitochondrial DNA sequence divergence among greenbug (Homoptera: Aphididae) biotypes: evidence for host-adapted races

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Insect Molecular Biology (2000) 9(2), 179 –184

Mitochondrial DNA sequence divergence among greenbug (Homoptera: Aphididae) biotypes: evidence for host-adapted races

Blackwell Science, Ltd

K. A. Shufran,1 J. D. Burd,1 J. A. Anstead2 and G. Lushai3 1

USDA-ARS, Plant Science and Water Conservation Research Laboratory, Stillwater, OK, USA, 2Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, USA, 3University of Southampton, School of Biological Sciences, Biodiversity and Ecology Division, Southampton, UK Abstract The full complement of known greenbug, Schizaphis graminum (Rondani), biotypes found in the USA were subjected to a molecular phylogenetic analysis based on a 1.2-kb portion of the cytochrome oxidase I mitochondrial gene. In addition to these nine biotypes (B, C, E, F, G, H, I, J and K), a probable isolate of the enigmatic biotype A (NY), a ‘new biotype’ collected from Elymus canadensis (L.) (CWR), and an isolate from Germany (EUR) were included. Schizaphis rotundiventris (Signoret) was included as an outgroup. Genetic distances among S. graminum biotypes ranged from 0.08% to 6.17% difference in nucleotide substitutions. Neighbour-joining, maximum parsimony and maximum likelihood analyses all produced dendrograms revealing three clades within S. graminum. Clade 1 contained the ‘agricultural’ biotypes commonly found on sorghum and wheat (C, E, K, I, plus J) and there were few substitutions among these biotypes. Clade 2 contained F, G and NY, and Clade 3 contained B, CWR and EUR, all of which are rarely found on crops. The rarest biotype, H, fell outside the above clades and may represent another Schizaphis species. S. graminum biotypes are a mixture of genotypes belonging to three clades and may have diverged as host-adapted races on wild grasses. Keywords: biotype, cytochrome oxidase I gene, hostadapted races, Schizaphis graminum, molecular phylogenetics. Received 4 October 1999; accepted 22 November 1999. Correspondence: Dr Kevin A. Shufran, USDA-ARS, 1301 N. Western Rd, Stillwater, OK 74075, USA. Tel.: (405) 624 – 4141 × 240; fax: (405) 624 – 4142; e-mail: [email protected]

© 2000 Blackwell Science Ltd

Introduction Approximately 50% of the recognized insect biotypes on agricultural crops are aphids (Saxena & Barrion, 1987). One such aphid, the greenbug, Schizaphis graminum (Rondani) (Homoptera: Aphididae) consists of multiple biotypes that have hindered its management by host plant resistance (see Porter et al., 1997 for a comprehensive review). Since 1961, eleven greenbug biotypes have been described and given the letter designations A–K. All designations (except D, which was erroneously based on insecticide resistance) were based on plant responses (dead or alive) to greenbug feeding on an array of host plant differentials containing various resistance genes (Porter et al., 1997). The ability of a greenbug biotype to kill a plant with a specific resistance gene or genes is known as virulence. Although the occurrence of greenbug biotypes in the USA is well documented, their origin and evolution are still controversial subjects (Porter et al., 1997). Various hypotheses have been proposed and include mutations (Starks & Schuster, 1976), selection by resistant cultivars (e.g. Eisenbach & Mittler, 1987), introductions (Blackman, 1981), sexual recombination (Puterka & Peters, 1989), and exploitation of crops by pre-existing biotypes (Porter et al., 1997). The utilization of modern, molecular genetic techniques helped gain new insights into understanding this problem. Three independent studies, focused on different regions of the genome of a few greenbug biotypes, yielded similar results. Some biotypes were found to be distinct genetic entities that probably diverged through years of reproductive isolation, predating the world history of wheat (Triticum aestivum L.) cultivation (about 10 000 years ago). Using restriction enzyme patterns of mtDNA, Powers et al. (1989) estimated a nucleotide sequence divergence of 1.2% and 1.0% between biotypes B vs. C, and B vs. E, respectively. Biotypes C and E were more closely related, with only about 0.17% sequence divergence. Based on these estimates and the use of a molecular clock, they suggested biotypes B and C shared a common ancestral mitochondrial genome approximately 0.3–0.6 million years ago during the Pleistocene era. Black et al. (1992) demonstrated the usefulness of random 179

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amplified polymorphic DNA (RAPD) to detect genetic polymorphisms among greenbug biotypes. All biotypes tested (B, C, E, F, G, H and I) could easily be differentiated by RAPD profiles, except biotypes C and E which were virtually identical. Using Southern analysis, Black (1993) also found biotype-specific patterns in the repeat structure of the intergenic spacer (IGS) region of the ribosomal RNA cistron. Similarly with mtDNA, biotypes B, C, E, F, G and H all showed substantial divergence and were easily separable, except C and E. He concluded that each biotype began as a single clone or population, followed by a lengthy period of reproductive isolation, and that this hypothesis was supported by the results of Powers et al. (1989). The above studies concluded that biotypes C and E (holocyclic biotypes) were so closely related that they were indistinguishable. Biotype B (an anholocyclic biotype) was the most divergent and zero to little variation within biotypes was found (based on mtDNA, RAPD, or certain regions of rDNA). These techniques were useful in providing markers for population studies. The above studies also provided supporting evidence that the evolution of at least some greenbug biotypes could be attributed to host-race formation independent of or prior to human agricultural practices (Porter et al., 1997). While the above studies were informative about the relationships among some biotypes, none included the full complement of known biotypes today, nor could the genetic markers used be analysed in a phylogenetic context. Lacking from all was the enigmatic biotype A. Wood (1961) described the first greenbug biotype and gave it the designation B when he discovered a population that killed resistant DS-28 A wheat. Biotype A then referred to that portion of the population that was unable to injure DS-28 A and predated the discovery of biotype B. However, the three above molecular studies (Powers et al., 1989; Black et al., 1992; Black, 1993), could not determine whether biotypes arose independently of one another, or if they were selected from or diverged from existing biotypes. Since these publications, two additional biotypes have been described, J (Beregovoy & Peters, 1995) and K (Harvey et al., 1997). In the present study we examined mtDNA nucleotide sequences and estimated the degree of genetic relatedness among all known greenbug biotypes, including a probable sample of biotype A, a grass collected isolate with unique virulence characteristics, an isolate from Germany, and S. rotundiventris (Signoret) as an outgroup. We attempted to infer the phylogenetic relationships among biotypes by constructing a phylogeny based on these DNA sequences. We discuss that phylogeny with respect to feeding reactions on host–plant differentials, ecology, and reproductive strategy. This is the first comprehensive study, and the first to identify phylogenetic, evolutionary relationships among all identified S. graminum biotypes.

Figure 1. Maximum likelihood tree of greenbug biotypes, isolates and S. rotundiventris produced from nucleotide sequences from a 1.2-kb portion of the COI gene. For both distance/neighbour-joining and maximum parsimony analyses, 1000 bootstrap replications were performed. The percentage of replications supporting each branch are shown. The top value represents neighbour-joining, while the bottom number represents maximum parsimony.

Results and discussion Silent and replacement substitutions were found between S. rotundiventris and S. graminum, as well as among the twelve S. graminum biotypes and isolates. Among the S. graminum biotypes and isolates tested (S. rotundiventris excluded), there were 123 variable sites; ninety-five were silent substitutions and twenty-eight were replacement substitutions. Transition to transversion ratios ranged from 0 to 7.33. The majority (74%) of transitions were thymine– cytosine substitutions, while the bulk of the transversions were adenine–thymine (59%) and adenine–cytosine (25%). The third codon position was biased towards adenine (38%) and thymine (46%). Genetic divergence was detected among greenbug biotypes and isolates. 1000 bootstrap replications were performed in the maximum parsimony, maximum likelihood, and distance/neighbour-joining analyses. Cladograms produced by all three methods had identical topologies, therefore we only present the maximum likelihood tree (Fig. 1). Three distinct clades were identified, which we herein refer to as 1, 2 and 3 for simplicity and to avoid confusion with the biotype letter designations. Clade 1 contained the predominant ‘agricultural’ biotypes infesting sorghum and wheat, i.e. C, E, I and K. Biotype J was also included, which was found on wheat in Idaho but is a nonvirulent biotype (Beregovoy & Peters, 1995). © 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 179 –184

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Table 1. Sequence analysis of a 1.2-kb portion of the cytochrome oxidase I gene of Schizaphis graminum biotypes and isolates, plus S. rotundiventris. Below diagonal: Kimura 2-parameter distances. Above diagonal: Tamura-Nei gamma distances (α = 0.23).

B C E F G H I J K NY CWR EUR S. rot.

B

C

E

F

G

H

I

J

K

NY

CWR

EUR

S. rot.

* 0.0407 0.0380 0.0389 0.0461 0.0542 0.0380 0.0434 0.0371 0.0434 0.0042 0.0076 0.0902

0.0525 * 0.0042 0.0239 0.0309 0.0542 0.0059 0.0093 0.0034 0.0282 0.0415 0.0451 0.0921

0.0482 0.0044 * 0.0213 0.0282 0.0515 0.0034 0.0067 0.0008 0.0256 0.0389 0.0424 0.0892

0.0504 0.0269 0.0236 * 0.0118 0.0506 0.0230 0.0265 0.0204 0.0093 0.0398 0.0434 0.0921

0.0648 0.0371 0.0333 0.0129 * 0.0617 0.0300 0.0335 0.0274 0.0093 0.0470 0.0488 0.0999

0.0768 0.0765 0.0712 0.0716 0.0976 * 0.0514 0.0533 0.0506 0.0589 0.0524 0.0588 0.0844

0.0472 0.0061 0.0034 0.0256 0.0354 0.0697 * 0.0084 0.0025 0.0274 0.0388 0.0424 0.0892

0.0556 0.0097 0.0070 0.0302 0.0400 0.0728 0.0088 * 0.0059 0.0309 0.0442 0.0478 0.0940

0.0466 0.0034 0.0008 0.0225 0.0320 0.0692 0.0025 0.0061 * 0.0247 0.0380 0.0415 0.0883

0.0590 0.0330 0.0295 0.0098 0.0098 0.0897 0.0315 0.0362 0.0283 * 0.0443 0.0461 0.0970

0.0043 0.0536 0.0492 0.0514 0.0660 0.0726 0.0483 0.0567 0.0476 0.0601 * 0.0084 0.0892

0.0078 0.0593 0.0547 0.0570 0.0682 0.0848 0.0536 0.0624 0.0530 0.0623 0.0087 * 0.0921

0.1594 0.1624 0.1550 0.1689 0.1969 0.1389 0.1509 0.1645 0.1515 0.1825 0.1535 0.1619 *

Clade 2 contained biotypes F and G, and the NY isolate (Fig. 1). Within Clade 2, biotype F was distinct, while biotype G and the NY isolate were more similar to each other. Biotype F is known as the ‘bluegrass’ biotype and it has been suggested that it represents the population Wood (1961) referred to as biotype A (Kindler & Spomer, 1986). Our results support this suggestion, because of its relatedness to the NY isolate that was collected in 1958. Both biotype F and the NY isolate were collected in the Great Lakes region, Ohio and Wisconsin, respectively. Although originally collected from wheat in 1986, biotype G is considered rare and has only accounted for a maximum of 2–3% of greenbugs surveyed on crops since that time (Bush et al., 1987; Ullah, 1993). All three members of Clade 2 are extremely virulent to wheat and sorghum, however they do not generally occur on these crops. Clade 3 included biotype B, and the EUR and CWR isolates and was distinct from Clades 1 and 2. Members of Clade 3 were very similar and could not be resolved (Fig. 1). Genetic distances among biotypes and isolates were estimated [ Tamura & Nei (1993) with a gamma correction, and the 2-parameter method (Kimura, 1980)] and the results supported the above cladogram. In the following discussion we concentrate on 2-parameter distances (Table 1, below diagonal) to allow for later comparisons with other aphid studies utilizing the cytochrome oxidase I (COI). Tamura-Nei gamma distances tended to be greater, especially between clades and the outgroup (Table 1, above diagonal). Schizaphis rotundiventris differed from S. graminum by 8.44% (H) to 9.99% (G) in nucleotide sequence divergence (Table 1). Schizaphis graminum biotypes and isolates tested showed a substantial degree of divergence in their mtDNA sequences. The most divergent biotype was H, which ranged from 5.06% (F) to 6.17% (G) difference in nucleotide sequence from the remaining biotypes and isolates. Biotype H also grouped separately from all others and did not reside in Clades 1, 2 or 3, © 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 179 –184

instead falling outside the S. graminum species (Fig. 1). Sexual morphs have never been induced in biotype H, therefore it is considered an anholocyclic biotype, which may account for its high degree of divergence. Even though it was found on wheat, biotype H has never been collected again since it was initially discovered in 1986 (Peters et al., 1997). Black (1993) also concluded that biotype H was the most divergent among those tested, based on restriction digests of rDNA. Biotypes B and J have also been reported to be anholocyclic. However, we have no information whether sexual reproduction in the CWR or EUR isolate can be induced. The NY isolate is androcyclic, i.e. only males were produced when it was exposed to conditions that normally trigger the production of oviparae and males in other biotypes (Cathy Sue Katsar and Stewart Gray, USDA-ARS, Ithaca, NY, personal communication). Our results based on sequence divergence in the COI gene, combined with earlier studies of mtDNA (Powers et al., 1989), rDNA (Black, 1993) and RAPD (Black et al., 1992), lead us to conclude that S. graminum biotypes are probably host-adapted races. Rather than ten unique biotypes (based on plant response), there are only three clades within S. graminum. There was a greater amount of genetic diversity among clades than among members within clades. The agricultural biotypes (Clade 1) found predominantly on sorghum, represent a genetically homogeneous assemblage of individuals that are able to exploit this ephemeral, domesticated host. Because biotype H is so divergent from other biotypes, we question its species identity. We found as much or more divergence between biotype H and other S. graminum biotypes and isolates than between Acyrthosiphon species (Boulding, 1998), as well as between Uroleucon species (Moran et al., 1999). Based on our results, S. graminum biotypes are probably host-adapted races that may have evolved on wild grasses as suggested by Porter et al. (1997). However,

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our study was limited because only one individual laboratory clone of each biotype was tested, therefore we have no estimate of mtDNA variation within single biotypes. Multiple genotypes probably comprise single biotypes in S. graminum, because fifty-six unique clones (maternal lineages) were found among sixty-one field collected biotype E individuals (Shufran et al., 1992). While our results are compelling, there are limitations of analysing mtDNA markers. Because of their maternal inheritance, they are subject to divergence at higher rates than nuclear markers. In aphids, this problem is exasperated by parthenogenetic reproduction whereby mtDNA haplotypes are inherited strictly along matriarchal lineages. The phylogenetic patterns detected in this study could easily have arisen by geographical isolation of matriarchal lineages, instead of sympatric isolation on different hosts. We recognize this problem and the need to develop nuclear markers to verify the relationships discovered using mtDNA sequences. However, in an ongoing study of greenbugs on wild grasses, we have thus far found at least one biotype I individual belonging to each of the three phylogenetic clades (J. A. Anstead, unpublished data). This suggests exchange among biotypes of nuclear genes that condition virulence. Also, out of four greenbug isolates collected from johnsongrass, Sorghum halepense (L.) Pers., all but one carried the Clade 1 (agricultural biotype) COI haplotype (J. A. A. unpublished data). This supports genetic fingerprinting results which showed that individual greenbug clones utilize both wild S. halepense and cultivated S. bicolor (K. A. Shufran, unpublished data). Research currently being conducted in our laboratory on the biotypic and mtDNA diversity of greenbugs associated with noncultivated grasses will continue to yield answers to further elucidate the enigma of S. graminum biotypes. Experimental procedures Insect material Twelve greenbug biotypes/isolates were analysed. Biotypes B, C, E, F, G, H, I and K were obtained from clonal colonies maintained at this facility. The biotypic status of each colony had been recently confirmed by Monte Andersen (USDA-ARS, Stillwater, OK) through host differential reactions. Biotype J individuals used in this study were obtained from a frozen (– 80°C) sample taken from the original colony as described by Beregovoy & Peters (1995). A colony (designated NY) obtained from Stewart Gray (USDA-ARS, Ithaca, NY) was also analysed. This isolate was originally collected in Madison, Wisconsin in 1958 and was maintained by continuous parthenogenetic reproduction at Cornell University on barley (Hordeum vulgare L.) from 1959 to the present. The NY isolate is significant because it may represent a living example of the enigmatic biotype ‘A’, a designation given to populations avirulent to DS-28 A wheat at the time biotype B was reported (Wood, 1961). Further evidence, suggesting that the NY isolate may be an example of biotype A, is based on reactions by

host–plant differentials. The NY isolate is avirulent to DS-28 A, ‘Amigo’, ‘Insave’ rye, ‘Post’ barley, and PI 426756 (Dave Porter, USDA-ARS, Stillwater, OK, unpublished data). Another isolate (designated CWR), collected by J. D. Burd from Canada wild rye (Elymus canadensis L.) in Stillwater, OK during 1997 was also tested. The CWR isolate is unique because it does not fit the virulence profile of any other known biotype and produces the opposite host reaction as biotype G, i.e. it is virulent to GRS 1201 wheat, but avirulent to Amigo wheat (J. D. Burd, unpublished data). Another greenbug isolate of unknown biotype (designated EUR for European) collected from wheat in Rostock, Germany (provided by Thomas Thieme, BTL, Sagerheid, Germany) was also included. Finally, we used S. rotundiventris (Signoret) (collected from Tokyo, Japan, identified, and provided by Takema Fukatsu, National Institute of Bioscience and Technology, Tsukuba, Japan) as an outgroup. S. rotundiventris was preserved in ethanol.

DNA extraction DNA was extracted from ten individuals of each aphid clone (except a single S. rotundiventris was used and the DNA extracted using the procedures of Black et al., 1992) by grinding them with a sterile Teflon pestle in a 1.5-ml microcentrifuge tube in 100 µl extraction buffer (0.1 M NaCl, 0.2 M sucrose, 0.1 M Tris-HCl pH 8.0, 0.05 M EDTA, 0.5% SDS). The pestle was washed with an additional 100 µl of extraction buffer, the tube capped, and then incubated at 65°C for 30 min. To precipitate proteins, 30 µl of 8 M potassium acetate was added, the tube placed on ice for 30 min, and then spun in a microcentrifuge at >10 000 g for 15 min. The supernatant was removed, transferred to a clean 1.5 ml tube, and extracted once with an equal volume of phenol : chloroform (1 : 1). DNA was then precipitated by adding 2.5 volumes of 100% ethanol and kept at – 80°C for 30 min, and then spun in the microcentrifuge for 15 min. The DNA pellet was washed with 70% ethanol, air dried, and suspended in 50 µl of TE (pH 8.0).

Amplification, cloning and sequencing A 1.4-kb fragment of the cytochrome oxidase I (COI) gene was amplified using the primers C1-J-1718 (5′-GGAGGATTTGGA AAT TGAT TAGT TCC-3′) and L2-N-3014 (5′-TCCAATGCACT AATCTGCCATATTA-3′) (Simon et al., 1994). Reactions were carried out in 50 µl volumes of 1× PCR reaction buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl) (Gibco-BRL); 3.0 mM MgCl2; 0.2 mM dNTPs (Promega); 100 ng total genomic template DNA; 0.4 µM each primer; and 2.0 U Taq DNA polymerase (Gibco-BRL) added at the first primer annealing step. An MJ Research PTC-100 Thermal Controller with ‘Hot-Bonnet’ was used to amplify the COI gene with the following programme steps: (1) 96°C 5 min; (2) 94°C 30 s; (3); 55°C 30 s; (4) 72°C 1 min 30 s; (5) cycle to step 2, twenty-nine times; (6) 72°C 5 min; (7) 4°C hold. The amplified PCR products were visualized by electrophoresing 30 µl of the reaction mixture in 1.2% low melting point agarose gels and staining with ethidium bromide. The appropriate bands were extracted using the Wizard PCR Prep Kit (Promega, Madison, WI) and cloned into the pGEM-T Easy vector (Promega, Madison, WI) according to the manufacturer’s instructions. The cloned inserts were sequenced by the Recombinant DNA /Protein Resource Facility (Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK) using an ABI-373 A Automated DNA Sequencing System. Besides the T7 and SP6 sequencing sites provided in the vector, we also designed © 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 179 –184

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Mitochondrial DNA divergence among greenbug biotypes two oligonucleotides (4861: 5′-ATAAAAT TAAATCAAATCCC-3′ and 4862: 5′-ATGTTGAAATTATTGATCC-3′) and used them as internal primers to sequence across the COI cloned insert. A third oligonucleotide was developed to fully sequence the S. rotundiventris insert (5660: 5′-AGCTGGTGCTAT TACTATAT TAC - 3′), because primer 4862 did not work for this species.

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review. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

References Sequence analysis Sequences were aligned using the MAP program (Huang, 1994). This was a multiple sequence alignment with a mismatch score of –15, a gap open penalty of 30, and a gap extension penalty of 3. After initial computer alignments of all forward and reverse sequences, we manually corrected for discrepancies and gaps were removed by referring to the printed sequence output. Distance, maximum parsimony and maximum likelihood methods were used in phylogeny reconstruction. Schizaphis rotundiventris was used as the outgroup in all tests. The MEGA statistical package (Kumar et al., 1993) and PAUP version 4.0b2 (written by David Swafford) were used to conduct the analyses. Distances were estimated by the method of Tamura & Nei (1993) with a gamma correction factor of α = 0.23 (estimated by maximum likelihood procedure in PAUP), because there were unequal rates in the number and types of transitions and transversions. To allow comparisons with other aphid studies using the COI gene, we also estimated distances using the 2-parameter method (Kimura, 1980). A dendrogram produced with neighbour-joining (NJ) analysis (Saitou & Nei, 1987) was based on the above calculated Tamura & Nei (1993) distances, with 1000 bootstrap replications. Maximum parsimony (MP) analysis was performed by bootstrapping method (1000 replications) with heuristic search, using a 95% majority rule consensus. There were no gaps included in the analyses. A maximum likelihood (ML) dendrogram was produced conforming to the method of Hasegawa et al. (1985). All sequences used to construct the phylogeny were submitted to GenBank and have the following accession numbers: AF220511, AF220523.

Acknowledgements We thank Dr Thomas Thieme (BTL, Sagerheid, Germany) for providing the sample of greenbug from Germany, and Dr Takema Fukatsu (National Institute of Bioscience and Technology, Tsukuba, Japan) for providing the specimens of S. rotundiventris. Dr Stewart Gray (USDA-ARS, Ithaca, NY) provided the New York isolate. Dr David Porter (USDA-ARS, Stillwater, OK) provided information on biotypic status and host–plant differentials, and Monte Anderson (USDA-ARS, Stillwater, OK) maintained and verified the laboratory biotypes. We acknowledge the Recombinant DNA /Protein Resource Facility (Oklahoma State University, Stillwater, OK) for DNA sequencing and oligonucleotide synthesis, as well assistance with primer design. Dr William C. Black IV (Colorado State University, Fort Collins, CO) is thanked for providing assistance with data analysis, and for peer review of an earlier draft of the manuscript. Dr Nancy Moran (University of Arizona, Tucson, AZ) and Dr Robert Foottit (Agriculture and Agri-Food Canada, Ottawa, Ontario) are also thanked for providing peer © 2000 Blackwell Science Ltd, Insect Molecular Biology, 9, 179 –184

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