Genetic analysis of native and introduced populations of Taeniatherum caput-medusae (Poaceae): implications for biological control
S.J. Novak1,2 and R. Sforza3
Summary Genetic analysis of both native and introduced populations of invasive species can be used to exam ine population origins and spread. Accurate delineation of an invasive species’ source populations can contribute to the search for specific and effective biological control agents. Medusahead, Tae- niatherum caput-medusae (L.) Nevski, a primarily self-pollinating Eurasian annual grass that was introduced in the western USA in the late 1800s, is now widely distributed in California, Idaho, Nevada, Oregon, Utah and Washington. The goal of our current research is to assess introduction dynamics and range expansion of this grass in the western USA, and to identify source populations in the native range to facilitate the search for potential biocontrol agents. Across introduced populations, nine multilocus genotypes were detected, and we suggest a minimum of seven separate introduction events of T. caput-medusae in the western USA. Although range expansion appears to have occurred primarily on a local level, several introduced populations appear to be composed of admixtures of introduced genotypes. None of the native populations analysed to date possess the exact multilocus genotypes detected in introduced populations. We have recently begun screening Eurasian popula tions using intersimple sequence repeat (ISSR) genetic markers to determine whether this polymerase chain reaction–based technique can provide a higher degree of resolution for the identification of source populations.
Keywords: invasive grass, multilocus genotypes, multiple introductions.
Introduction (Novak and Mack, 2001, 2005; Lavergne and Molof sky, 2007). Additionally, genetic analysis of native and Experimental analyses of both native and introduced introduced populations can be used to examine popula populations of invasive species can be used to assess tion origins and spread (Roderick and Navajas, 2003). various ecological, genetic and evolutionary aspects of Accurate delineation of an invasive species’ source invasion and the invasion process (Hierro et al., 2005; populations (or regions) can contribute to the search for Novak, 2007). For instance, comparison of the level biological control agents. Indeed, the identification of and structure of genetic diversity within and among areas of origin may reduce the economic cost of pros native and introduced populations can be used to de pecting for agents and may result in the development termine whether the distribution of a species in its new of more specific and effective biological control agents range stems from single or multiple introduction events (Goolsby et al., 2006a). Taeniatherum caput-medusae (L.) Nevski, a mem ber of the tribe Triticeae in the grass family, is consi 1 Current address: CSIRO European Laboratory, Campus International de Baillarguet, 34988 Montferrier-sur-Lez, France. dered a noxious weed in many western US states (e.g. 2 Permanent address: Boise State University, Department of Biology, Colorado, California, Oregon, Nevada and Utah). The 1910 University Dr., Boise, ID, 83725-1515, USA. grass was first collected in the USA in Roseburg, Or 3 USDA-ARS, European Biological Control Laboratory, Campus Inter egon in 1887 (Fig. 1), and its collection history is well national de Baillarguet, 34988 Montferrier-sur-Lez, France. Corresponding author: S.J. Novak
422 Genetic analysis of native and introduced populations of Taeniatherum caput-medusae (Poaceae)
Figure 1. Chronology of the spread of Taeniatherum caput-medusae in the western USA. This chronol- ogy was reconstructed using published accounts (see text), herbarium specimens and historical
rangeland in the western USA (Young, 1992; Miller et been recognized (Frederiksen, 1986), but only T. caput- al., 1999). It primary occurs in areas disturbed by over medusae ssp. asperum is believed to have been intro grazing and fire in the 25–100 cm annual precipitation duced into the USA (Young, 1992). Recently, foreign zone, and it can become the dominant plant species at exploration was carried out for identifying candidates for certain sites (Hironaka, 1961; Dahl and Tisdale, 1975; biological control, and several plant pathogens were de Young, 1992). Ominously, the species has probably not scribed, including the fungi, Ustilago phrygica Magnus yet reached its ecological limit. If its ecological require and Tilletia bornmuelleri Magnus (Siegwart et al., 2003; ments approximate those of Bromus tectorum L., it has Widmer and Sforza, 2004). A preliminary host range the potential to spread widely in the Great Basin of the screening with U. phrygica, a systematic smut fungi that USA and beyond. Different methods have been tried to was collected in Turkey and attacks T. caput-medusae, control T. caput-medusae (burning, grazing, competi has been conducted (Sforza et al., 2004). tion and herbicides), and all have generally resulted in Because natural enemy pressure can vary across failure (Horton, 1991). genotypes (Evans and Gomez, 2003), populations and re The native range of T. caput-medusae includes gions, the enemy release hypothesis is best tested by com much of Eurasia, where three distinct subspecies have paring introduced populations with native populations
423 XII International Symposium on Biological Control of Weeds that were the source of the invasion. When implement pulations were brought back to the United States De ing biological control programs to reverse the release partment of Agriculture Agricultural Research Service from natural enemies, knowledge of source populations European Biological Control Laboratory (USDA-ARS- or regions would increase the probability of finding EBCL) in Montpellier, France, on an official authoriza highly specialized enemies (Goolsby et al., 2006b). In tion (04LR011) granted by the French government. Seeds addition, evaluating the efficacy of candidate biologi were stored in a quarantine greenhouse at the EBCL until cal control agents should be done on the full range of further use. genotypic diversity present within the introduced range (Gaskin et al., 2005), through the evaluation of specific polymerase chain reaction (PCR) primers when screen Enzyme electrophoresis ing populations (Marrs et al., 2006). The level and structure of genetic diversity within The overall goals of our current research are to as and among populations of T. caput-medusae in its sess introduction dynamics and range expansion of invasive range in the western USA is based on the T. caput-medusae in the western USA and to identify analysis of 1663 individuals from 45 populations. In source populations in the species’ native range to fa the laboratory, one seed from each individual in a po cilitate the search for potential biocontrol agents. This pulation was germinated on moistened filter paper in research specifically addresses the following questions. a Petri dish and harvested approximately 7 days after (1) How many multilocus genotypes occur in western germination. Enzyme electrophoresis was conducted US populations of T. caput-medusae, and what is their generally following the methods of Soltis et al. (1983), geographic distribution? (2) What does the distribution with modifications described by Novak et al. (1991). of these genotypes indicate about range expansion of The 15 enzymes employed in this study were resolved this species in its new territory? (3) How many geno with enzyme electrophoresis using four buffer systems types occur in native range populations, and what is (1, 6, 8 and 9), and these 15 enzymes were genetically their geographic distribution? (4) Can source popula encoded by 29 loci. Because T. caput-medusae is a dip tions for the invasion of the grass in the western USA loid with low genetic diversity, the genetic basis of all be identified? allozyme variation observed was easily inferred based on known subunit structure and compartmentalization of these enzymes (Weeden and Wendel, 1989). Methods and materials Sampling of plant material ISSR analysis One objective of our sampling has been to collect Five of the 49 Eurasian populations of T. caput- plant material from populations across the entire geo medusae mentioned above were selected for a prelimi graphic distribution of T. caput-medusae, in both its nary analysis using intersimple sequence repeat (ISSR) invasive and native ranges. Another objective has been genetic markers: one population from Morocco, Spain, to obtain population samples at or near localities where France, Greece (Crete) and Turkey. For each popula the plant was first collected, or reported, during its in tion, five seeds were randomly selected from each of vasion of the western USA (Fig. 1). Samples have been three plants located 5, 18 and 25 m along the transect collected from a total of 45 populations in the states of from which they were sampled. In the EBCL quaran California, Idaho, Nevada, Oregon, Utah and Washing tine greenhouse, seeds were germinated in Petri dishes ton, with several early collection localities represented. with distilled water at 25°C, 80% relative humidity and Two groups of native range samples have been 16:8 h light/dark. Ten days after germination, leaves included in this study. The first group consisted of were removed from the plants and frozen at -20°C. 23 populations, with 22 of these populations being ac Total genomic DNA was extracted from frozen leaf cessions obtained from the USDA Plant Introduction material using DNeasy Plant Mini Kits according to Laboratory in Pullman, WA: 12 populations from Tur the manufacturer’s instructions (Qiagen Inc., Valen key, seven from Afghanistan, two from Iran and one cia, CA). After extraction, DNA was amplified with from Kazakhstan. The personnel of the USDA Plant the PCR using six ISSR primers decribed by Wolfe Introduction Laboratory variously classified these ac et al. (1998). Primer names and sequences are provi cessions to each of the three subspecies of T. caput- ded in Table 1. DNA amplifications were performed medusae. Only one of these 23 populations originated in 20 µl final reaction volumes containing 1 U of Taq in Europe: Sterea Hellas, Greece. The second group DNA polymerase (Qiagen Inc.), 1´ buffer (Qiagen), consisted of 49 populations collected in August or Sep 1 mM MgCl2, 0.2 mM of each deoxyribonucleotide tember, 2002 and 2003, from across the grasses’ native triphosphates, 0.5 µM of a single primer and 2 µl of the range in Eurasia. For most populations, intact spikes template DNA. Amplifications were performed using were collected from 30 to 40 individual plants along the GeneAmp PCR System 9700 (Applied Biosys a transect at approximately 1-m intervals and placed tems, Forest City, CA) as follows: 94°C for 3 min, then in separate envelopes. Seeds from the 49 Eurasian po 35 cycles at 94°C for 30s, 45°C for 45 s and and 72°C
424 Genetic analysis of native and introduced populations of Taeniatherum caput-medusae (Poaceae)
Table 1. Identity and nucleotide sequences of the ISSR primers used in this preliminary analysis of Taeniatherum caput-medusae from its native range. ISSR primers used in this study were described by Wolfe et al. (1998). The utility of each primer, based on the criteria described in the text, is indicated: Y yes, N no. Primer Primer sequence Utility
ISSR-17898A (CA)7–AC Y
ISSR-17898B (CA)7–GT N
ISSR-17899A (CA)7–AG Y
ISSR-17899B (CA)7–GG Y
ISSR-814 (CT)8–TG Y ISSR-HB15 GTG–GTG–GTG–GC N
for 1 1/2 min. A final extension was performed at 72°C observed in the Palouse region of eastern Washington. for 7 min. Amplified products were electrophoresed on The multilocus genotypes detected in Pullman, Malloy 1.0% agarose gels. Gels were stained with ethidium Prairie and Salt Creek appear to be restricted to just a bromide, and DNA fragments were visualized with a single population. Heterozygous multilocus genotypes UV transilluminator and photographed. were found in two different populations: White Bird, ID contained two heterozygous genotypes and one of Data analysis these genotypes was also detected at Emigrant Hill, OR. The level of polymorphisms within introduced popula Allozyme multilocus genotypes were identified tions was low: Only 17 of 45 populations (37.8%) con from enzyme electrophoresis data, and these genotypes tained two or more multilocus genotypes. Furthermore, were used to assess introduction dynamics and spread of these 17 polymorphic populations, only three con of T. caput-medusae in the western USA and to identify tained three or more multilocus genotypes. source populations of the grass in its native range. Al lozyme multilocus genotypes are defined as the com Multilocus genotypes in the native range posite genotype over all loci examined and therefore are designated based on the identity of alleles at each Two distinct categories of multilocus genotypes scored enzyme locus. Populations were defined as ge were detected within the 23 Eurasian populations. The netically polymorphic if they contained two or more first group of genotypes were quite distinct and diffe multilocus genotypes. As part of our preliminary anal red from those detected in the western USA at multiple ysis of native populations of T. caput-medusae using loci. These genotypes were present in seven populati ISSR genetic markers, bands were not scored; however, ons from Turkey, and all populations from Afghanistan, we did qualitatively assess each primer to determine Iran and Kazakhstan. Based on their different enzyme its utility for future analysis. Specifically, primers were banding patterns, plus their larger seed size, these pop evaluated based on whether they (1) did not generate ulations probably all consisted of T. caput-medusae bands in control reactions, (2) generated clear, distinct, ssp. crinitum. The enzyme multilocus genotypes de darkly stained bands and (3) were polymorphic among tected in the remaining populations in Turkey and the test populations. one from Greece were similar to those observed in the western USA, and these populations were tentatively Results assigned to T. caput-medusae ssp. asperum. However, none of the multilocus genotypes present in these Multilocus genotypes in the 23 native populations was an exact match to those pre introduced range viously detected in the introduced range. Multilocus genotypes are named based on the populations in which they were first found. A total of Preliminary ISSR analysis nine multilocus genotypes were detected among all Of the six ISSR primers that were screened in our 45 populations: seven homozygous multilocus geno preliminary analysis of native populations of T. caput- types and two genotypes with one or two heterozygous medusae, four met our selection criteria and will prove loci (unpublished data, not shown). The seven homo useful in future studies of both native and introduced zygous genotypes were first detected in Roseburg, OR, populations (Table 1). Samples from the five countries Steptoe Butte, WA, Rattlesnake Station, ID, Ladd Can (populations) display different DNA banding patterns yon, OR, Pullman, WA, Malloy Prairie, WA and Salt with primer ISSR-17899A (Fig. 2), although no variabil Creek, UT. Five different multilocus genotypes were ity was detected among individuals within populations.
425 XII International Symposium on Biological Control of Weeds
Figure 2. Photograph of DNA banding patterns obtained using ISSR primer 17899A. Note the different banding patterns for populations of Taeniatherum caput-medusae from different countries. Contents of the lanes on this gel are as follows: 1 1 kb ladder; 2– 4 France; 5 and 6, Greece; 7 and 8, Morocco; 9–11, Spain; 12–14, Turkey; 15 PCR control; 16 extraction control.
Discussion introduction of this grass is ongoing. Our results for T. caput-medusae join a growing body of information Introduction dynamics and spread indicating that, for invasive plant species, multiple in in the western USA troduction may be the rule rather than the exception (Novak and Mack, 2005) and may contribute to inva The level of genetic diversity observed across and siveness (Allendorf and Lundquist, 2003; Lavergne within western US populations of T. caput-medusae and Molofsky, 2007; Novak, 2007). is lower than the mean value reported for other self- The low level of polymorphisms observed within pollinating plant species (Hamrick and Godt, 1990) but introduced populations of T. caput-medusae indicates similar to that of other invasive plants that exhibit a uni that gene flow or dispersal among these populations is parental mode of reproduction such as selfing (Novak low. Thus, we conclude that spread or range expansion et al., 1991). Yet, despite its lack of genetic diversity, of the species has occurred mostly at the local or re at least at the loci examined in this study, this species gional level and certainly has not been widespread. is now invasive over much of the semi-arid portions of However, the detection of populations that appear to be the western USA. admixtures of different introduced genotypes, as seen at The occurrence and geographic distribution of mul several locations in the western USA (data not shown), tilocus genotypes can provide insights into introduction suggests that intermixing of genotypes can take place dynamics and spread of invasive species (Novak and if multiple introductions have occurred within the same Mack, 2001, 2005). Multilocus genotype results for region. Moreover, the detection of heterozygous mul western US populations of T. caput-medusae are con tilocus genotypes suggests that plants with different sistent with the pattern often associated with multiple genotypes and potentially originating from different introductions. Based on just the number of homozygous introduction events have recently mated. Such hybrid multilocus genotypes detected across all populations, ization events have been suggested to contribute to we suggest a minimum of seven independent founder increased invasiveness in introduced species (Ellstrand events. This conclusion is bolstered by the observation and Schierenbeck, 2000). that four of the localities where these genotypes were detected are at or near early collection sites of the plant: Roseburg (1887), Steptoe Butte (1901), Rattlesnake Source populations Station (1930) and Ladd Canyon (1944). Although the multilocus genotyes observed in po The detection of five different multilocus genotypes pulations from Greece and several from Turkey are in eastern Washington suggests that multiple introduc similar to those of the western USA, no exact matc tions can occur within a relatively small geographic hes were found among native populations. Thus, our area. Because the plant was not collected in Utah until allozyme analysis did not reveal the source populations 1988, the detection of a unique multilocus genotype in (or regions) for the introduction of T. caput-medusae Salt Creek, Utah may be evidence for a relatively re in the USA, but the data clearly excludes many of the cent introduction event. If so, these data indicate that southwest and central Asian locations from serving as
426 Genetic analysis of native and introduced populations of Taeniatherum caput-medusae (Poaceae) source populations. These results probably stem from References insufficient sampling in the native range, especially Europe: Only one of 23 native populations was from Allendorf, F.W. and Lundquist, L.L. (2003) Introduction: Europe, and the other 22 populations were collected population biology, evolution, and control of invasive from southwest or central Asia. species. Conservation Biology 17, 24–30. Additional analysis of European populations will be Burdon, J.J. and Marshall, D.R. (1981) Biological control required before source populations, and introduction and the reproductive mode of weeds. Journal of Applied Ecology 18, 649–658. dynamics of the invasion of T. caput-medusae in the Dahl, B.E. and Tisdale, E.W. (1975) Environmental factors western USA can be more confidently described. To related to medusahead distribution. 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