Heredity 72 (1994) 412—419 Received 8 October 1993 Genetical Society of Great Britain

Geographical origin of an introduced pest, bonariensis (Kuschel), determined by RAPD analysis

C. LENNEY WILLIAMS, S. L. GOLDSON*t, D. B. BAIRDt & D. W. BULLOCK Centre for Molecular Biology, and Veterinary Sciences Group, Lincoln University, P.O. Box 84, Canterbury, New Zealand and AgResearch, P.O. Box 60, Canterbury, New Zealand

TheArgentine stem , (Kuschel) (Coleoptera: ), is an important introduced pasture pest in New Zealand. In this study geographical populations of this were analysed using polymerase chain reaction-based randomly amplified polymorphic DNA (RAPD), in an attempt to determine the geographical origin of the pest. Morphologically indistinguishable individuals were collected from nine South American, five New Zealand and one Australian populations. Ten primers were screened for usefulness, two of which revealed signifi- cant, scorable polymorphisms between these populations. The results indicated that the sampled New Zealand L. bonariensis populations originated from the east coast of South America.

Keywords:geneticvariation, introduced species, Listronotus bonariensis, populations, RAPD.

Introduction founding events and selection pressure (Baker & Stebbins, 1965). TheArgentine stem weevil Listronotus bonariensis It is probable that L. bonariensis arrived in New (Kuschel) (Coleoptera: Curculionidae) is an introduced Zealand (and Australia) less than 100 years ago, and pest (Goldson & Emberson, 1980) attacking improved given that 2—3 generations a year are possible (Kelsey, graminaceous pasture throughout New Zealand 1958) only about 200—250 generations can have (Prestidge eta!., 1991). The damage caused by this pest elapsed since its establishment. Given that the New is estimated to range from SNZ 78—251 million Zealand environment resembles that of South annually (Prestidge et a!., 1991). Native to South America, it is thought that selection pressures in the America, L. bonariensis is thought to have been native and colonized habitats would be comparable. accidentally introduced to New Zealand and Australia; That, together with the short time since introduction, it was first reported in New Zealand in 1927 (Marshall, suggests genetic similarity between the introduced 1937) and, with the advent of pastoral grazing, quickly population and the population of origin might be high. achieved pest status. However, founder effect, random genetic drift and/or Elucidation of genetic variation in geographical recurrent introductions could have altered the colon- populations can be an important aspect of pest studies ized populations' genetic make-up. (Kambhampati et al., 1990; Roehrdanz & Johnson, In order to define the origin of pest populations it is 1988), providing insight into the geographical origin of necessary to demonstrate genetic similarities between colonized populations (Kambhampati et a!., 1991). colonized and potential source populations. This type Within the native habitat, the extent of genetic varia- of study can use numerous tecimiques including tion between geographical populations depends on phenotypic variation, allozyme data, DNA data several factors, including gene flow between popula- (restriction fragment length polymorphisms, sequence tions and time since separation (Hartl, 1980; analysis), morphometrical analysis, or behavioural Templeton et a!., 1990). Genetic differences within the differences. With small , one common difficulty introduced population can be the result of the genetic encountered with DNA analysis is lack of material. In variation in the founder population, the number of this study, this problem was overcome using poiy- merase chain reaction-based randomly amplified poly- morphic DNA (RAPDs) (Welsh & McClelland, 1990; *Correspondence Williams, eta!., 1990). 412 GEOGRAPHICAL ORIGIN OF AN INSECT PEST 413

This relatively new technique offers advantages with respect to the amount of material needed and the fact DNA isolation that nothing need be known about the genomic organi- DNAwas isolated from individual . Each weevil zation of the organism. The technique has generated was ground under liquid nitrogen and incubated in iso- useful results in the identification of unique individuals lation buffer (8.5 mrvt Tris pH 8, 25 m EDTA pH 8, (Smith et al., 1992), detection of genetic variability 0.5 per cent SDS, 25 ifiM /3-mercaptoethanol and (Chalmers et a!., 1992; Landry et a!., 1993), race 0.5 mg m1' Proteinase K) at 50°C for 1 h with occa- identification (Crowhurst et al., 1991) and generation sional inverting. Following phenol:chloroform washes, of linkage maps (Paran et al., 1991). The technique is the DNA was precipitated with 100 per cent ethanol becoming widely used (see Hedrick 1992 for a review and washed with 70 per cent ice-cold ethanol, then air of various current applications), and a review of poten- dried and suspended in TE (10 mi Tris pH 8.0, 0.1 tial applications has been given by Hadrys et al. (1992). mM EDTA) containing 25 pg m1' RNase A. The samples were incubated at 37°C for 30 mm, then diluted with 690 1u1 H20. 5 1ul of this DNA (approxi- Materials and methods mately 30 ng) was used as a template for RAPD reactions. Each weevil yielded enough DNA for Sample collection approximately 145 RAPD reactions. Adult L. bonariensis were collected from nine climati- cally diverse localities in South America between Primerselection September 1989 and February 1990 (Fig. 1). Methods of collection and transportation have been discussed Theprimers used were 10-bp oligonucleotides of elsewhere (Goidson et al., 1990). Samples were then random sequence (Oligos Etc.,Inc., Guilford, frozen at —100°Cuntil use. Adult weevils were also Connecticut). Ten primers were initially screened using collected from five localities in New Zealand in pooled samples of DNA from each population, as January and April 1992 (Fig. 2) and one locality in follows. 20 p1 of DNA from each individual weevil Australia (Flaxley, South Australia) in December 1991. within each population were pooled. 5 1ul (30 ng) were

Welisford

Ruakura

Reporoa

Lincoln

Gore

0 600km ______0 200kni Fig.1 South American Listronotus bonariensis collection sites. Fig.2 New Zealand Listronotus bonariensis collection sites. 414 C. L. WILLIAMS ETAL. then taken from each pooled sample as templates for quots (5 1u 1) of the amplification products were then the RAPD reaction. This resulted in one reaction for electrophoresed at 65 V for 2 h through 1.4 per cent each population and made it possible to select useful SeaKem LE Agarose in TBE (89 mi Tris, 89 mM primers. Primers were designated as useful if they boric acid, 2 mrvi EDTA) containing 0.25 ig m1' generated few, distinct bands which showed poly- ethidium bromide and visualized using UV light. morphisms between populations. Primers that gener- Based on this protocol, the DNA from 12 individ- ated many bands (> 15 per sample) were designated as uals from each of the nine populations (except Brazil, unscorable and not pursued. Of the 10 primers initially in which case six individuals were used) was amplified screened, two were selected as potentially useful. by the two useful primers. This resulted in a banding These were primer 267/2(5'-ACATAGACGG-3')and pattern for each individual and also resulted in a popu- primer GT09 (5'-TCTGCCGTGA-Y). lation profile for each population. A negative control (without template DNA) was included in each set of RAPD reactions amplified. The same sample amplified with reactions and electrophoresis the same primer and under the same reaction condi- RAPDreactions were carried out based on the tions in different tubes on the same day or on different methods developed by Williams et at. (1990) and days resulted in the same bands (not shown). Amplifi- Welsh and McCIelland (1990). The 25-ul reactions cation profiles were similar to those of DNA extracted contained 0.4 mrvt dNTPs, lx Taq polymerase buffer, 1 from individual L. bonariensis wing muscles (not unit Taq polymerase, 30 ng DNA and either primer shown). 267/2 (200 nrvi) or primer GTO9 (600 nM). Each reac- tion was overlaid with a drop of silicon oil. The reaction profile was 45 cycles of 94°C for 1 Scoringof bands mm, 36°C for 1 mm, 72°C for 2 mm. The samples were Onlydistinct bands were included in the data set. Each then held at 72°C for 5 mm. Amplifications were individual was scored for the presence (1) or absence carried out on a Hybaid thermal reactor and were (0) of every such band. This was done by initially scor- all carried out on the same thermocycler to eliminate ing against a molecular weight marker (BRL 1 kb the possibility of machine to machine variation. Au- ladder) and assigning tentative identification numbers

Fig. 3 Population profiles generated by primer GTO9. The amplifications and electrophoresis were carried out as described in Materials and methods. In each lane are the amplification pro- ducts of one individual from each of the labelled populations. The lanes marked (—)arethe negative controls. The molecular weight marker (MW) is the BRL 1 kb ladder. Population profiles from (a) Ruakura, (b) Uruguay, (c) Lin- -MW coln, and (d) Bariloche. GEOGRAPHICAL ORIGIN OF AN INSECT PEST 415

1636—

1016—

SI?—

Fig. 4 Population profiles generated by primer 267/2. The amplification and electrophoresis were carried out as described in Materials and methods. In each lane are the amplification pro- ducts of one individual from each of the labelled populations. The lanes marked (—)arethe negative controls. The molecular weight marker (MW) is the BRL 1 kb ladder. Population profiles from (a) Ruakura, (b) Uruguay, (c) Lin- coln, and (d) Bariloche. MW MW -Mw to each band based on approximate molecular weight. Nei and Li band sharing (Nei & Li, 1979), again to These band identification numbers were verified by provide cross-checks on interpretation. These individ- running an aliquot of representative individuals from ual similarity coefficients were reduced to population each gel that contained the band of interest side by side similarities by averaging individuals within populations. on a subsequent gel (verification gels not shown). The similarity coefficients presented in Table 1 used Bands that ran at the same molecular weight were the band-sharing measure of similarity (Hedrick, 1985, scored as the same band. and references therein). The Nei and Li measure of band sharing was calculated from population frequen- cies to give the similarity between two populations. Dataanalysis This measure was determined in two ways, using posi- Thepresence/absence data matrix was analysed using tive frequencies only, and using both positive and nega- the SAS procedure DISCRIM (Parker, 1987) (similar to tive frequencies. Kambhampati et a!., 1992). This procedure generated Dendrograms were constructed from the similarity ordered canonical discriminant functions for the matrices by hierarchical cluster analysis with an variables (bands). The means of three canonical discri- average link option using the GENSTAT program minant functions for each of the sampled populations (Genstat 5 committee, 1987). The similarity matrices were plotted. The first two canonical discriminant were also analysed in Genstat by principal coordinate functions were selected as the x and y axes. The third analysis to generate a simpler display of the rela- canonical discriminant function was selected as the z tionship between populations than the dendrogram. axis, perpendicular to the plane of the page, and is represented in Fig. 5 by relative size of the letters Results representing the plotted values. The data were also analysed by cluster analysis and PrimerGTO9 generated 28 bands and primer 267/2 principal coordinate analysis to verify that the results generated 34 bands from the 174 L. bonariensis did not depend on the type of analysis used. Similarity studied. The number of bands generated for each indi- matrices were calculated from the data on band vidual varied from 1 to 12. All distinct bands were presence or absence for each individual. Genetic included in the analysis. distance or similarity were determined by Jacquard Primer GTO9 resulted in one prominent band of similarity, genetic distance (Gower, 1985), band approximately 800 bp in all the New Zealand popula- sharing (Hedrick, 1985, and references therein), and tions, along with many other bands detectable at 416 C. L. WILLIAMS ETAL.

a

7 BL 6 5 ci 0 a)3a)

Z RN Fig. 5 A 3D distribution of sampled cc0 BR Listronotus bonariensis populations 0 generated by the SAS procedure DISCRIM. 0n -1 The abbreviations used are: AS Ct)—2 BB (Australia), BA (Buenos Aires), BB U- G (Bahia Blanca), BL (Bariloche), BR —. (Brazil), G (Gore), K (Ruakura), L — (Lincoln), LS (La Serena), P (Reporoa), _'; RN (Rio Negro), SC (South Chile), U Sc (Uruguay), W (Welisford), Z —6 I I I I I I I (Mendoza). —3 —2 —1 0 1 2 3 4 5 6 7 Second canonical variate score

various molecular weights (e.g. Fig. 3, a and c). The Buenos Aires was slightly displaced from the New Australian population also generated a similar banding Zealand/Australia/Uruguay grouping. South Chile was pattern (not shown). Conversely, the South American split from the rest of the sampled populations by populations generated quite diverse population pro- canonical discriminant function 2 and Bariloche was files (e.g. Fig. 3, b and d). Using GTO9, there was an split from the rest of the populations by canonical apparent similarity between the New Zealand, the discriminant functions 1 and 2. Australian and the Uruguayan profiles (Fig. 3, a and c, The population similarity matrix calculated by compared to b). The Buenos Aires profile was also reducing the full shared fragment similarity matrix similar to these (not shown). between individuals is given in Table 1. The New Primer 26 7/2 resulted in two prominent bands Zealand and Australian populations are most similar to (1600 bp and 700 bp) in the New Zealand (Fig. 4, a each other and the Uruguayan population and to a and c) and Australian (not shown) populations. Other slightly lesser degree to the Buenos Aires population. fragments were also generated, some individual- On the leading diagonal are values for within popula- specific and others present in several individuals. tion similarity, which range from 0.388 to 0.647. Four Again, the South American population profiles were of the six introduced populations (Lincoln, Ruakura, quite distinct from one another (e.g. Fig. 4, b and d). As Welisford and Australia) exhibit a higher degree of with primer GTO9, primer 267/2 generated population within population similarity than the native popula- profiles for the Uruguay and Buenos Aires popula- tions. This reduction in intra-population variability is tions, which closely resembled the New Zealand and consistent with some sort of genetic 'bottleneck', such Australian profiles (Fig. 4, a and c, compared to b). as a colonization event. The mean canonical variate scores for each popula- An example of the dendrograms generated using the tion from the discriminant analysis (using the SAS proce- band frequencies for each population is given in Fig. 6. dure DIscRIM) of the presence/absence data matrix are All five populations from New Zealand, the Australian plotted in Fig. 5. The mean scores for the New Zealand population and Uruguay and Buenos Aires cluster as a and Australian populations clustered in the vicinity of distinct and separate group. This cluster was observed the means for the Uruguay and Buenos Aires popula- in all five dendrograms generated, with a minor amount tions (Fig. 5), supporting the initial impression based of branch swapping noted. The clustering of the South on visual examination of the gels. The population from American populations in some cases reflected the Gore was slightly displaced from this cluster. In terms geographical distance separating the populations. For of the third canonical discriminant function (the z axis) example, the Buenos Aires and Uruguay populations GEOGRAPHICAL ORIGIN OF AN INSECT PEST 417

Lincoln N Wellsford Uruguay 00 Ruakura 00 - Reporoa CC Buenos Aires '4-00 Gore C00C C) C N C Australia 00 C C C Bahia Blanca N N N Rio Negro N N 00 N Brazil CC South Chile La Serena —C — 2-1 00 NO fl r— Bariloche -1 N r)C Mendoza

1 00 90 80 70 60 50 -CNt/ 00 C rC\ \ CC C it CCCCC % similarity N00 N Fig.6 Dendrogram of Listronotus bonariensis populations Nr)\C eN N tfl C Lf)NN N c generated by average linkage cluster analysis using Nei and CCCCCC Li similarity based on shared fragments. For this dendro- F-I gram, only positive matches were counted. C( C\ O r 00 N tr N '-ICN N -C N rN CCCCCCC- cluster together and are only about 60 km apart. The 00 r)CrNC\ C N populations from Bahia Blanca and Rio Negro also C f)'f C\N m-N rnN m - r cluster together and are less than 100 km apart. There CC C C C C C C C also were clusters of populations that were separated N 00 C 00 N C N by fairly large distances. For example, the population N C C GO rV N from Brazil clustered with the population from South CCCCC CCC Chile. C N 00 N .C N r C\ C N 00 C 0000 N GO 'N Discussion C C C mNC C C C C C C C Thebands generated using the RAPD technique are t c 00 m 'NN '-4,C'-NN 00 N N \C C c clearly genetic markers, albeit anonymous. The specific C C C C C NrnrflNNC C C C C C C interpretation of these markers is open to discussion; however, for the purposes of this study they have 11 c N 'f N 0000 N 00 C proved useful. N Or '.C 00 '( C C\ '€) C\ C r N m't The profiles generated by these primers indicated dCCCCCCCCCCC© that the populations in South America are distinct from each other. This result suggests that (within the limits of co5 CO NN 00 0000 N'r '.C- 'r00 r'C rN rm —C tflC tr Ct NNr the technique) most of these populations are to some CC C C C C C C C C C C C C extent genetically isolated, consistent with the geo- 00 graphical and climatic barriers that separate them. 00 '0 C 00 N m N C Nm N'-'C00 C m oc N Lfl N -4 I/ Within New Zealand, most of the populations C CC C C C C C C C C mrrC C C C C sampled generated very similar profiles. This suggests n CO that within the limits of sampling, there is a high degree of genetic similarity between populations throughout the country. 'Eoc co - - CI Q 0) ) The population from Australia generated a very similar profile to the New Zealand populations, indi- .Cenm L) cating a strong genetic similarity between the weevils 418 C. L. WILLIAMS ETAL. sampled in these countries. This suggests either that As an initial screen of the genetic makeup of the one of these countries was colonized by L. bonariensis sampled L. bonariensis populations, RAPDs have from the other, or that both countries were colonized provided new and useful information which will assist from a single source. further research on this important agricultural pest. The New Zealand and Australian profiles closely The results presented here demonstrate the use of the resembled the profiles from the east coast of South RAPD technique, which may have wide application in America (Uruguay and Buenos Aires), indicating that studies of other populations. there was significant genetic similarity between these populations. It should be noted that the Buenos Aires and Uruguay collection sites were only about 60 km Acknowledgements across the Rio de Ia Plata from each other. Weare grateful to Ken Chalmers and Srinivas Kamb- The populations from Brazil and South Chile hampati for their insights into data analysis. We also clustered together in all of the dendrograms generated would like to thank Gail Timmerman and John (e.g. Fig. 6). This clustering suggested that, despite the Marshall for initially providing aliquots of primers, and vast distance separating them, these populations were Gary Barker, Paul Addison, Han Eerens and Steve genetically similar (although not to the extent of the Valentine for sample collection. This research was New Zealand group). To our knowledge, this is the first funded from a grant to the New Zealand Pastoral Agri- reported study on the population genetics of L. culture Research Institute, Ltd. by the New Zealand bonariensis, and the data suggest that there may have Foundation for Research, Science and Technology. been some migration or colonizing events between these two populations. Further interpretation of the relationships between the South American populations References may be possible using these data, but is beyond the BAKER,H. G. AND STEBBINS, 0. L. (eds) 1965. The Genetics of focus of this study. Colonizing Species. Academic Press, Inc., New York. The dendrograms clearly showed that the South CHALMERS, K. J., WAUGH, R., SPRENT, J. I., SIMONS, A. J. AND POWELL, American populations clustering with the New w. 1992. Detection of genetic variation between and within Zealand/Australian populations are Buenos Aires and populations of Gliricidia sepium and G. maculata using Uruguay. 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