Bulletin of Entomological Research (2002) 92, 17–24 DOI: 10.1079/BER2001141

Mitochondrial DNA sequence divergence among (: ) clones from cultivated and non-cultivated hosts: haplotype and host associations

J.A. Anstead1, J.D. Burd2 and K.A. Shufran2* 1Department of Entomology and Plant Pathology, 127 Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA: 2USDA- ARS, Plant Science and Water Conservation Research Laboratory, 1301 N. Western Road, Stillwater, OK 74075, USA

Abstract

A 1.0 kb region of the mitochondrial cytochrome oxidase subunit I gene from the greenbug , Schizaphis graminum (Rondani), was sequenced for 24 field collected clones from non-cultivated and cultivated hosts. Maximum likelihood, maximum parsimony and neighbour-joining phylogenies were estimated for these clones, plus 12 previously sequenced clones. All three tests produced trees with identical topologies and confirmed the presence of three clades within S. graminum. Clones showed no relationship between biotype and mtDNA haplotype. At least one biotype was found in all three clades, suggesting exchange among clades of genetic material conditioning for crop virulence, or the sharing of a common ancestor. However, there was a relationship between host and haplotype. Clade 1 was the most homogenous and contained 12 of 16 clones collected from cultivated hosts and five of the six collected from johnsongrass, Sorghum halepense, a congener of cultivated sorghum, S. bicolor. Four of the six clones collected from Agropyron spp. were found in clade 2. Clade 3 contained two clones from wheat, Triticum aestivum, and four from non-cultivated hosts other than Agropyron spp. A partitioning of populations by mtDNA haplotype and host suggests the occurrence of host adapted races in Schizaphis graminum.

Introduction Barrion, 1987). Diehl & Bush (1984) recommended the division of the term biotype into five categories depending The term biotype has been defined in a number of on the mechanisms underlying biotype differentiation; (i) different ways; a group recognized by biological function non-genetic polyphenisms; (ii) polymorphic or polygenic rather than by morphological characters (Eastop, 1973), or variation within populations; (iii) geographic races; (iv) host an infraspecific category of populations with similar races; and (v) species. In , biotypes have been most genetic composition for a biological attribute (Saxena & commonly defined on the basis of overcoming plant resistance (planthoppers: Claridge & Hollander, 1980), and their ability to utilize different hosts (: Porter et al., *Author for correspondence 1997; Birkle & Douglas, 1999; Sunnucks et al., 1997). Of the Fax: 1 (405) 624 4142 recognized insect biotypes on agricultural hosts E-mail: [email protected] approximately 50% are aphids (Saxena & Barrion, 1987). 18 J.A. Anstead et al.

The greenbug, Schizaphis graminum (Rondani) differences are assumed to be related to the biotypic status of (Hemiptera: Aphididae), is an aphid pest of wheat, Triticum each population. aestivum, sorghum, Sorghum bicolor, barley, Hordeum vulgare MtDNA gene sequences are particularly attractive for (all Poaceae), and a number of other graminaceous crops in developing phylogenies of recently diverged taxa because the USA. It reproduces primarily by parthenogenesis south they evolve rapidly (ten times faster than genomic DNA of approximately 35°N, and is holocyclic north of this (Miyata et al., 1982)) and they have higher likelihood of latitude. It is somewhat unique among aphids in that it tracking a short internode than a nuclear-autosomal-gene causes direct damage to crop hosts and often kills the entire tree (Moore, 1995). The gene sequences are maternally plant. Host plant resistance has been used in an attempt to inherited and are not recombinant, making the construction control Schizaphis graminum damage, however, its of phylogenetic trees easier. It should be noted, however, that effectiveness has been compromised by the occurrence of using a mitochondrial gene sequence does not guarantee that virulent biotypes (for review, see Porter et al., 1997). With the the correct phylogeny will be generated. Cases of shared exception of biotype D, which was characterized on the basis ancestral polymorphisms and multiple substitutions at a of insecticide resistance, biotypes A–K have been single nucleotide site can occur (Simon et al., 1994). characterized by their ability to damage certain resistant The majority of past studies have concentrated on S. plants (Porter et al., 1997). Each biotype is defined by its graminum populations from cultivated hosts or laboratory ‘virulence profile’, i.e. a set of defined host plants shows a clones. Schizaphis graminum has a large host range, including specific pattern of damage (virulence) and resistance 70 species of grasses in 29 genera (Michels, 1986), most of (avirulence) against it. While the distribution of S. graminum which are non-cultivated species. Wild and weedy grass biotypes is known to some extent (Dumas & Mueller, 1986; hosts are considered important oversummering hosts for Peters et al., 1997), their origin and evolutionary status have S. graminum. There is a period during the summer when remained opaque (Porter et al., 1997). Under the system of wheat is not available and sorghum is an unsuitable host. classification of Diehl & Bush (1984), greenbug biotypes During this time period, S. graminum may be found on could be described as a case of polymorphic variation within grasses (Daniels, 1961). Schizaphis graminum was found populations, host race, or possibly even within species. oversummering on 23 grass species in the Texas panhandle To ascertain whether resistant crop varieties were from 1953 to 1959 with western wheat grass, Agropyron responsible for selection and occurrence of S. graminum smithii (Poaceae), being the most important host (Daniels, biotypes, Porter et al. (1997) extensively reviewed the 1960). However, it is known that certain clones or literature concerning relationships between hosts, resistant populations cannot use the full complement of available genes and biotypes. No correlation was found between the hosts (Dahms et al., 1954; Anstead, 2000). introduction of wheat cultivars and the emergence of The purpose of this study was to measure the genetic biotypes virulent to them and only a weak correspondence variation of S. graminum clones collected from non-cultivated between the release of resistant sorghum cultivars and the grass hosts in the Great Plains using an mtDNA marker. DNA characterization of new biotypes resistant to them. They sequences of the COI gene in the mtDNA were used to instead proposed that S. graminum populations might be estimate the degree of relatedness between these clones. These comprised of a complex of host-adapted races that evolved were compared with the laboratory clones used by Shufran et on non-cultivated hosts. al. (2000), together with a clone from South Carolina and a To test the above hypothesis, Shufran et al. (2000) clone from Syria. A phylogeny was constructed using these conducted a phylogenetic analysis using sequence data from sequences, which allowed the inference of evolutionary the mtDNA cytochrome oxidase subunit I (COI) gene of all relationships . The relationships between phylogeny, biotype published biotypes plus several other unique clones. Their and host plant were examined, in particular to look for any results revealed three clades within S. graminum, each with a evidence of host-adapted races. The genetic variation within significant amount of divergence between them. Distances in some biotypes was also estimated. percent sequence differentiation were 4% (between clades 1 and 2), 6.2% (between clades 1 and 3) and 6.8% (between clades 2 and 3). Clade 1 contained the ‘agricultural biotypes’ Materials and methods (C, E, J, K and I). Clade 2 contained F, G and the New York Insect collections (NY) clone. The NY clone was collected in 1958 and may represent an example of biotype A (Shufran et al., 2000). Schizaphis graminum collected from non-cultivated and Clade 3 contained B, the Canada wild rye (CWR) clone (an cultivated grasses in Oklahoma, Kansas and Colorado in a unpublished biotype collected from Elymus canadensis concurrent study were used for genetic analysis (Anstead, (Poaceae) and a clone collected from wheat in Europe (EUR). 2000). Collection dates and locations are given in table 1. Biotype H fell outside the rest of these clades with the Clonal colonies were established and their biotype outgroup, Schizaphis rotundiventris (Signoret) (Hemiptera: determined (Anstead, 2000), after which a sub-sample was Aphididae). This study supported the findings of Powers et stored at 80°C for later genetic analysis. A clone from al. (1989) which suggested that biotypes were probably host South Carolina, USA (SC), obtained from Stewart Gray, races and diverged prior to the beginnings of human USDA-Agricultural Research Service, Ithaca, New York, was agriculture. However, both studies only examined a single also included. This clone showed susceptible reactions on example of each biotype, therefore ignoring the possibility of ‘Custer’, CI17882, ‘Largo’, and GRS1201 wheat and variation within biotypes. For example, it was shown that ‘Wintermalt’ barley, and resistant reactions on ‘Amigo’ biotype E populations in the field were comprised of wheat and ‘Post 90’ barley. However, the reaction on DS28A multiple clonal lineages (Shufran et al., 1992). This is a wheat was inconclusive (J.D. Burd, unpublished data). problem common to many studies of aphid biotypes, where Based on reactions to these host differentials, the SC clone is single populations of each biotype are compared and any unique when compared to previously published biotypes. Divergence of Schizaphis graminum on non-cultivated hosts 19

Table 1. Collection data for Schizaphis graminum isolates, excluding those of Shufran et al. (2000)*. Clade Date State or Closest town Host Biotype or Genbank country clone Accession no. designation 3 31/VII/98 Oklahoma Balko Sorghum halepense Non-cultivated I AF285896 3 7/VIII/98 Oklahoma Guymon Chloris verticillata Non-cultivated I AF285898 3 6/VIII/98 Oklahoma Guymon Echinochloa crusgalli Non-cultivated I AF285899

1 1/VI/99 Oklahoma Marshall S. halepense Non-cultivated I AF285894 1 13/VII/99 Oklahoma Marshall S. halepense Non-cultivated E AF285895 1 21/VII/99 Oklahoma Redrock S. halepense Non-cultivated I AF285897 1 1/VI/99 Kansas Hays Agropyron smithii Non-cultivated I AF285901 1 1/VII/99 Oklahoma Sumner S. bicolor Non-cultivated E AF285902 1 10/IX/99 Kansas Hays Triticum aestivum Cultivated I AF285906 1 21/VII/99 Oklahoma Sumner S. halepense Non-cultivated I AF285912 1 8/X/99 Colorado Prospect valley A. intermedium Non-cultivated WWG AF285915 1 6/III/00 Syria Tel Hayda T. aestivum Cultivated SYR AF285916 1 XII/95 S. Carolina ? T. aestivum Cultivated SC AF285907 1 4/VIII/98 Kansas S. Haven S. bicolor Cultivated I AF285908 1 10/VIII/99 Kansas Winona T. aestivum Cultivated I AF285909 1 13/XI/99 Kansas Hays T. aestivum Cultivated I AF285910 1 1/VI/99 Kansas Hays Bromus tectorum Non-cultivated G AF285911 1 21/VII/99 Oklahoma Sumner S. bicolor Cultivated I AF285913 1 21/VII/99 Oklahoma Sumner S. halepense Non-cultivated I AF285914

2 1/VI/99 Kansas Hays B. tectorum Non-cultivated I AF285893 2 1/VI/99 Kansas Hays A. smithii Non-cultivated K AF285905 2 10/VIII/99 Colorado Mead A. intermedium Non-cultivated G AF285900 2 1/VI/99 Kansas Hays A. smithii Non-cultivated G AF285903 2 1/VI/99 Kansas Hays A. smithii Non-cultivated G AF285904

Isolates without * designation are in the order as they appear (top to bottom) in the cladograms in figs 1 and 2.

However, it may have a similar virulence profile as the NY by Shufran et al. (2000). The PCR products were direct or the CWR clones. The Syrian (SYR) clone was collected on sequenced by the Recombinant DNA/Protein Resource 6 March 2000, from wheat in Tel Hayda by M. El Bouhssini Facility (Department of Biochemistry and Molecular Biology, (International Center for Agricultural Research in the Dry Oklahoma State University, Stillwater, Oklahoma) using a Areas, Aleppo Syria). Because it was preserved in 95% Perkin-Elmer (Applied Biosystems) model 373 XL DNA ethanol, no biotype information exists for the SYR clone. Sequencing System incorporating an ABI-373 Automated Twelve laboratory clones used by Shufran et al. (2000) were DNA Sequencer. also included. Unfortunately the provenance of some of these clones is unclear. The laboratory colonies of biotypes F, Sequence analysis H and J were from the original field collections and their origin is known (Porter et al., 1982; Puterka et al., 1988; Sequences were aligned using the multiple alignment Beregovoy & Peters, 1994). The origins of the Canada wild program (MAP) (Huang, 1994). The alignment had a rye, New York and European clones are also known. mismatch score of 15, a gap open penalty of 30, and a gap However, the samples of biotypes B, C, E, G, I and K came extension penalty of 3. Alignments were first carried out on from laboratory colonies of uncertain origin. each clone separately to provide a complete sequence for each clone. The sequences were then aligned. The 1043 bases that had sequence data for both strands DNA extraction, PCR amplification and sequencing were used in the phylogenetic reconstruction. This is less DNA was extracted from between one and three than the 1200 bases used by Shufran et al. (2000), but individuals of each clone using the procedure of Black et al. produced the same phylogenetic trees as with the longer (1992). Amplification was carried out according to the sequence. The sequenced region stretched from the E2 procedures of Shufran et al. (2000), except 1.5 U Taq DNA domain to the M12 domain (Lunt et al., 1996). Schizaphis polymerase (Gibco-BRL) was used in each reaction. rotundiventris (Shufran et al., 2000) was used as the outgroup Amplification of a PCR product of the correct size was in all tests. The analysis was carried out using the MEGA confirmed by electrophoresing 10 µl of the reaction mixture statistical package (Kumar et al., 1993) and PAUP version in a 1X TAE, 1.2% agarose gel and staining with ethidium 4.0b2 (written by David Swofford). We used the same bromide. Once the appropriate band was detected, statistical analyses as Shufran et al. (2000). Distances were contaminants such as primer-dimers and amplification estimated by the method of Tamura & Nei (1993) with a primers were removed from the remaining 40 µl of the gamma correction factor of = 0.3 (estimated by maximum reaction using the Wizard PCR Prep Kit (Promega, Madison, likelihood procedure in PAUP), because there were unequal Wisconsin). The product was sequenced using the rates in the number and types of transitions and amplification primers and a set of internal primers designed transversions. Distances were also estimated using the 2- 20 J.A. Anstead et al. parameter method (Kimura, 1980). A dendrogram produced four of the five clones collected from johnsongrass, Sorghum with neighbour-joining (NJ) analysis (Saitou & Nei, 1987) halepense (Poaceae). Clade 2 contained four of the six clones was based on the above Tamura & Nei (1993) distances with collected from Agropyron spp. Clade 3 contained three clones 1000 bootstrap replications. Maximum parsimony (MP) from three separate grass species. analysis was performed by the bootstrapping method (1000 replications) with heuristic search and using a 95% majority Discussion rule consensus. A maximum likelihood (ML) dendrogram was produced according to the method of Hasegawa et al. Our results confirm that there are three clades (COI (1985). All sequences were submitted to GenBank and have haplotypes) within S. graminum. There were only low levels the consecutive accession numbers, AF285893–AF285916. of mtDNA variation within each clade. Biotype H was grouped outside the rest of the clones and may represent a separate Schizaphis species. However other events, such as a Results mitochondrial colonization that affected only this clone, Among the S. graminum clones there were 128 variable could account for this divergence. Mitochondrial sweeps sites. Of these, 85 were third codon substitutions (66%). There have been used to explain unusual divergence in mtDNA were 98 silent substitutions and 28 replacement substitutions. sequences in Anopheles dirus Peyton & Harrison (Diptera: There were substitutions in all the COI regions sequenced but Culicidae) (Walton et al., 2000) and have been linked with only the NY isolate contained a substitution (lysine to the spread of symbionts such as Wolbachia (Rickettsiaceae) glutamic acid) linked to the function of an active site (Lunt et (Shoemaker et al., 1999), although Wolbachia is unknown in al., 1996). Divergence between clones was measured using aphids. both Kimura 2-parameter distances and Tamura-Nei gamma The SC clone was grouped with the agricultural clones, distances. Both measurements produced similar results, as might be expected, as it was collected from wheat. The therefore only Tamura-Nei gamma distances are mentioned WWG clone (which represents a new and undescribed in the text. Schizaphis rotundiventris differed from S. graminum biotype) was also grouped in this clade. Divergence within by 12.7–15.9%. Biotype H differed from the rest of S. S. graminum (excluding biotype H) was over 5%. Generally graminum by 6.5–8.5%. The maximum divergence within the aphids have low mtDNA divergence: divergence of only species (excluding biotype H) was 6.3%. Divergence within 0.4% was found in a study using the COI gene, cited as biotypes was also estimated (table 2). The topologies of trees evidence against the hypothesis that there were host races in produced by maximum parsimony, maximum likelihood and the pea aphid, Acyrthosiphon pisum (Harris) (Aphididae) neighbour joining analysis were identical, and therefore only (Boulding, 1998). Sitobion miscanthi (Takahashi) and Sitobion the maximum likelihood results are shown (figs 1 and 2). Our avenae (Fabricius) (Aphididae) differ by only 1.5% sequence results revealed three clades. Genetic diversity within each divergence in the COI gene (Sunnucks & Hales, 1996). clone is shown in table 3. Clements et al. (2000) used sequences from the cytochrome The phylogeny generated is shown according to biotype oxidase subunit II (COII) gene and elongation factor-1 alpha in fig. 1. Biotype I was present in all clades. Biotypes G and (EF-1) to examine variation within the Myzus persicae K were present in clades 1 and 2. The two new collections of (Sulzer) complex (Hemiptera: Aphididae). The low variation biotype E were in the same clade as the laboratory clone of within this complex led them to conclude that M. nicotianae biotype E. A unique clone collected from Agropyron smithii Blackman and M. persicae were synonymous (all COII (Anstead, 2000) was in clade 1 and will be referred to as the sequences were identical for all M. persicae) clones. Allozyme western wheatgrass (WWG) clone. variation is also very low in all aphids including Schizaphis Figure 2 shows the same phylogeny but according to graminum (Black et al., 1992). The comparatively large host, when known. Clade 1 contained all but one of the field amount of variability within S. graminum is most likely a collected clones from wheat and sorghum. It also contained result of large host range. Its high diversity was probably primarily driven by selection on non-cultivated hosts. Within clade 1, however, there is the possibility that Table 2. Range of per cent differentiation in nucleotide sequence divergence may be driven by selection produced by within Schizaphis graminum biotypes, estimated by two methods. agricultural practices. Some of the divergence is also likely Biotype n Kimura 2- parameter Tamura-Nei gamma to be due to unrecognized species within S. graminum, with distance distances ( = 0.3) biotype H an obvious candidate. Molecular clocks for mitochondrial genes in E 3 0.10–0.87 0.11–0.91 give a substitution rate of approximately 2% per million G 4 0.10–3.05 0.12–3.52 years (Brower, 1994; Juan et al., 1996). Using this rate, the I 15 0.00–4.89 0.00–5.87 distances between clades (2–4%) indicate they have not K 2 2.15 2.34 shared a common mitochondrial ancestor for between one

Table 3. Per cent maximum differentiation in nucleotide sequence within and between clades of Schizaphis graminum, estimated by two methods. Within clades Between clades

1 2 3 1 and 2 2 and 3 1 and 3 Kimura 2- parameter distance 1.07 1.2 1.16 2.05 3.77 3.66 Tamura-Nei gamma distances ( = 0.3) 1.11 1.27 1.24 2.21 4.47 4.28 Divergence of Schizaphis graminum on non-cultivated hosts 21

ROT* ROT H* Triticum aestivum* B* B* 96 EUR* Triticum aestivum* CWR* Clade 96 Elymus canadensis* Clade 100 I 3 100 Sorghum halepense 3 I Chloris verticillata 98 I 98 Echinochloa crusgalli 100 I* 100 I* K* K* J* Triticum aestivum* I Sorghum halepense E Sorghum halepense 96 I 96 Sorghum halepense I Agropyron smithii 100 E 100 Sorghum bicolor 99 I 99 Triticum aestivum 100 I 100 Sorghum halepense C* Clade C* Clade WWG 1 Agropyron smithii 1 98 SYR 98 Triticum aestivum 100 E* 100 E* SC Triticum aestivum 97 I 97 Sorghum bicolor 100 I 100 Triticum aestivum I Triticum aestivum G Bromus tectorum I Sorghum bicolor I Sorghum halepense F* Poa compressa* 100 NY* 100 NY* Scale G* Scale G* 100 1% 100 I Clade 1% Bromus tectorum Clade divergence K 2 Divergence Agropyron smithii 2 95 G 95 Agropyron intermedius 100 G 100 Agropyron smithii Agropyron smithii

Fig. 1. Maximum likelihood tree of Schizaphis graminum isolates Fig. 2. Maximum likelihood tree of Schizaphis graminum isolates by biotype, produced from nucleotide sequences from a 1043 bp by host, produced from nucleotide sequences from a 1043 bp portion of the COI gene. For both distance/neighbour-joining portion of the COI gene. For both distance/neighbour-joining and maximum parsimony analysis, 1000 bootstrap replications and maximum parsimony analysis, 1000 bootstrap replications were performed. The percentage of replications supporting each were performed. The percentage of replications supporting each branch are shown. The top value represents neighbour-joining, branch are shown. The top value represents neighbour-joining, while the bottom number represents maximum parsimony while the bottom number represents maximum parsimony (*from Shufran et al. (2000); ROT, S. rotundiventris, EUR, Europe; (*from Shufran et al. (2000); ROT, S. rotundiventris, NY, New CWR, Canada wild rye; SC, South Carolina; NY, New York; SYR, York). Syria; WWG, intermediate wheatgrass). and two million years. A previous study of mitochondrial 21 clones in this clade. Clade 1 may comprise those DNA restriction patterns (Powers et al., 1989) showed a Schizaphis graminum clones able to exploit crops and utilize divergence level indicating 300,000–600,000 years had close relatives such as Sorghum halepense. passed since biotypes B and C shared a common ancestor. Four of the five clones added to clade 2 were collected Both studies indicate there was substantial divergence from two closely related Agropyron species. This suggests within this species long before the beginnings of wheat clade 2 may be divergent as a result of host specialization. cultivation in the fertile crescent over 10,000 years ago The WWG clone was unable to survive or reproduce on (Zohary & Hopf, 1988). However, because mtDNA is Elymus canadensis (Anstead, 2000). Clade 3 contained single inherited in a strictly maternal lineage, this result could have clones collected from S. halepense, Chloris verticillate and been caused in one of two ways; geographical isolation or Echinichloa crusgalli (Poaceae). Further collections would be sympatric isolation on separate hosts. needed to see if further clones from these hosts partitioned There is no evidence that these clades diverged as a result into this clade. of geographical isolation. Clade 1 contains clones from Evidence for host-adapted races has been found in other Kansas, Oklahoma, Idaho, Colorado and Syria, clade 2 aphid species. In one of the best documented examples contains clones from Ohio, Wisconsin, Kansas and Colorado sympatric races of the pea aphid Acyrthosiphon pisum were and clade 3 Oklahoma, Colorado and Germany. The found on separate hosts. These races were found to be correlation between host genus and clade provides some genetically divergent and reproductively isolated (Via, 1999; evidence that these clades could have diverged on separate Via et al., 2000). Host races have also been identified in Aphis hosts. Of the 17 clones in clade 1 that host data are available gossypii Glover (Hemiptera: Aphididae). Vanlerberghe- for, 14 were from wheat, sorghum or Sorghum halepense, a Masutti & Chavigny (1998) found A. gossypii populations congener of cultivated sorghum. There were two clones from were genetically different on cucurbits and non-cucurbits. wheat and one from S. halepense outside this clade. Clade 1 Using the (GATA)4 probe De Barro et al. (1995a) showed was the most homogeneous despite containing the most Sitobion avenae populations collected from wheat had clones. There was only 1.1% sequence divergence among the significantly different fingerprints than those collected from 22 J.A. Anstead et al. adjacent non-cultivated grasses. In a similar study they gene is the possibility that some sequences may be found different and diagnostic RAPD–PCR banding patterns transposed out of the mitochondria DNA into nuclear from S. avenae collected from wheat and cocksfoot (De Barro genomic DNA (Sunnucks & Hales, 1996). Patterns of et al., 1995b). Host restricted forms (biotypes) of Therioaphis evolution are different in mtDNA and nuclear genes and this trifolii (Monell) (Hemiphera: Aphididae) have also been could lead to the generation of an incorrect phylogeny. identified from lucerne and clover (Sunnucks et al., 1997). However none of the sequences in this study showed the There was no segregation of Schizaphis graminum into ‘numerous ambiguities’ found in PCR products derived clades according to biotype. Biotype I was present in all from whole-aphid DNA when transposed mtDNA three clades and biotypes G and K were present in two sequences were present (Sunnucks & Hales, 1996). clades. There are a number of reasons why this could occur. There are a number of studies that looked at nuclear There may be exchange of genetic material between clades markers in S. graminum. Random amplified polymorphic conditioning for virulence to crops. Alternatively, three DNA polymerase chain reaction (RAPD–PCR) was used to clades may have shared virulence genes originating from a detect DNA polymorphisms in four aphid species including common ancestor. Finally, it is conceivable that if there were greenbugs (Black et al., 1992). Differences were detectable a selective advantage for virulence, virulence could have between and within greenbug biotypes. The length of the emerged independently in each clade as a result of intergenic spacer (IGS) region of the rRNA cistron has been convergent evolution. It should be noted, however, that such used as a molecular fingerprinting probe to study diversity an advantage has not been demonstrated. within and between greenbug populations. Shufran et al. The present system of assigning biotypes is a purely (1991) used it to show that most of the variation in a phenetic classification and as such appears to have no greenbug population could be found from sampling a phylogenetic importance. This was confirmed by estimates single leaf. In a similar study using the intergenic spacer of divergence within biotypes (table 2). Biotypes G, K and I region as a marker, 93.1% of the clonal diversity was found had divergences within them higher than within the three to be present in a single field of overwintering greenbugs clades. Of the biotypes from which there were multiple (Shufran & Wilde, 1994). Black (1993) found differences in clones, only E had a lower divergence than that of the clades. the intergenic spacer subrepeat structure which were This was, however, based on only three clones. The addition variable enough to separate biotypes. He found some of sequences from further biotype E collections would variation within biotype E, but concluded that each biotype probably add to this divergence. tested (B, C and E) probably evolved from a single Many previous authors have treated biotypes as discrete population or maternal lineage. Shufran et al. (1992), populations, even when variation was found within a however, showed biotype E populations in the field were biotype. Black (1993) found variation in the sub-repeat comprised of many clonal lineages. These studies indicate a structure of the rDNA intergenic spacer within biotypes of fundamental problem when looking for differences between S. graminum, but concluded that each biotype tested (B, C biotypes using a particular marker. Most used very few and E) probably evolved from a single population or populations, Black (1993) used only one clone of biotype B, maternal lineage. There still exists an assumption that a F, G and H, two of biotype C and three of biotype E. These pattern of virulence to resistant crops means something in samples are not large enough to distinguish normal evolutionary terms; e.g. ‘the development of biotype F is variation between clones from differences between driven by native grasses’ (Kindler & Hays, 1999). Whilst biotypes. some biotypes may be associated with particular hosts, it is Despite the limitations of this study, the results offer not accurate to say their formation is driven by them. Our further evidence to support the theory that S. graminum has results show that whilst biotype designations are useful to also evolved host adapted races as first suggested by Porter researchers, farmers and breeders, they should not be et al. (1997). Only one of the three clades of S. graminum treated as an evolutionary unit or be given taxonomic status. (clade 1) appears to be adapted to crop hosts. Possible This study showed there was greater diversity amongst mechanisms for reduced gene flow between S. graminum S. graminum clones collected from non-cultivated hosts than clones in the same geographical location include mate choice among those collected from cultivated hosts. Non-cultivated and an anholocyclic life cycle. There is evidence that these hosts may act as reservoirs for both genetic and biotypic mechanisms exist in S. graminum: In one study biotype E diversity (i.e. genes for conditioning virulence). The males, when given a choice, strongly preferred biotype E presence of biotype I in each of the three clades suggests females and biotype C males preferred biotype C females there is exchange of virulence genes among the clades. The (Eisenbach & Mittler, 1987). Under an 11 h photoperiod, mechanism, which would account for gene flow between biotypes C, E and I clones readily produced large numbers races, is the holocyclic portion of the S. graminum life cycle. of sexuals, G and F clones produced lower numbers of Sexual reproduction and overwintering as eggs occurs in the sexuals, and biotypes B, H and J clones produced no sexuals USA on grasses such as Poa pratensis (Poaceae) (Wadley, (Puterka & Peters, 1990; Ullah & Peters, 1996). Ideally, a 1931). Hence, it is probable that virulence genes present in more thorough phylogeographical survey, incorporating populations on non-cultivated grasses could be transferred additional markers (in particular genomic markers), will be into populations commonly found on crops. Resistance carried out to explore fully the relationships between host, screening against S. graminum from non-cultivated hosts biotype and genotype. It would also be interesting to see if would be of interest and value to plant breeders developing there were differences in the endosymbionts present in each new sources of resistance in crops. clade as Buchnera in particular has been found to confer This study has limitations, primarily because it used only advantages in aphids feeding on particular hosts by a single marker and looked at S. graminum from a limited providing biochemical pathways for the generation of geographical area. Another potential drawback with the COI essential amino acids (see Moran, 2001). Divergence of Schizaphis graminum on non-cultivated hosts 23

Acknowledgements De Barro, P.J., Sherratt, T.N., Carvalho, G.R., Nicol, D., Iyengar, A. & Maclean, N. (1995a) Geographic and microgeographic The authors wish to thank Dr Stewart Gray, (USDA-ARS, genetic differntiation in two aphid species over southern Ithaca, New York) for providing the South Carolina clone and England using the multilocus (GATA) probe. Molecular Dr Mustapha El Bouhssini (International Center for 4 Ecology 4, 375–382. Agricultural Research in the Dry Areas, Aleppo, Syria) for De Barro, P.J., Sherratt, T.N., Brookes, C.P. David, O. & providing specimens from Syria. We acknowledge the Recombinant DNA/Protein Resource Facility for DNA Maclean, N. (1995b) Spatial and temporal genetic variation sequencing and oligonucleotide synthesis. Dr Kris Giles and in British field populations of the grain aphid Sitobion Dr Jack Dillwith (Oklahoma State University, Stillwater, avenae (F.) 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