Salsola tragus - Canadian Science Publishing

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Molecular markers indicate two cryptic, genetically divergent populations of Russian thistle (Salsola tragus) in California1 Frederick J. Ryan and Debra R. Ayres

Abstract: Genetic variability among accessions of Russian thistle (Salsola tragus L.) from California was investigated using allozymes and DNA-based molecular markers. Aspartate aminotransferase and 6-phosphogluconate dehydrogenase displayed two multienzyme phenotypes that were widespread in plants throughout the state. Random amplified polymorphic DNA analysis was conducted on samples of the two isoenzymic phenotypes collected throughout California, as well as additional accessions from France and Turkey and Salsola paulsenii Litv. Six primers produced 23 polymorphic bands. Analysis of the patterns of bands by calculation of simple matching coefficients and cluster analysis confirmed the genetic distinctness of the two isoenzymic phenotypes of S. tragus; S. paulsenii was markedly different from both types. Mean fruit weights from plants grown under similar conditions were different between the two types as well. These results and preliminary cytological analysis together suggest that the two types are actually two different species of Salsola, only one of which has been previously recognized. Analysis of the DNA-based markers suggests that one of the genetic entities may be closely related to Salsola found in Europe, while the area of origin of the second entity is currently obscure. Key words: allozyme, genetic diversity, RAPD assay, Salsola tragus, Salsola paulsenii. Résumé : Les auteurs ont examiné la variabilité génétique parmi différentes accessions du chardon de Russie (Salsola tragus L.) venant en Calfornie, en utilisant des allozymes et des marqueurs moléculaires basés sur l’ADN. L’aspartate aminotransférase et la 6-phosphogluconate déshydrogénase montrent deux phénotypes à enzymes multiples qui sont largement répandus dans ces plantes, sur l’ensemble de l’état. Les auteurs ont conduit une analyse du polymorphisme aléatoire de l’ADN amplifié sur des échantillons de deux phénotypes isozymiques récoltés sur l’ensemble de la Californie, ainsi que d’accessions supplémentaires venant de France et de Turquie et du Salsola paulsenii Litv. Six amorçes ont produit 23 bandes polymorphiques. L’analyse du patron de ces bandes, par simple calcul des coéficients de correspondance et par analyse des regroupements, confirme la distinction génétique des deux phénotypes isozymiques du S. tragus; le S. paulsenii étant nettement différents de ces deux types. Les poids moyens des fruits provenant de plantes cultivées sous les mêmes conditions diffèrent entre les deux types également. Ces résultats, ainsi que des analyses cytologiques préliminaires, pris ensemble, suggèrent que les deux types seraient dans les faits deux espèces distinctes de Salsola, dont une seule des deux a été antérieurement reconnue. L’analyse des marqueurs basés sur l’ADN suggère qu’une des entités génétiques pourrait être étroitement voisine des Salsola trouvés en Europe, alors que la région d’origine de la seconde demeure obscure pour le moment. Mots clés : allozyme, diversité génétique, essais RAPD, Salsola tragus, Salsola paulsenii. [Traduit par la Rédaction]

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Introduction Russian thistle or tumbleweed (Salsola tragus L., Chenopodiaceae (1)), now widespread throughout North America, is believed to have been introduced into North Dakota from Eurasia in the 1870s (2). It spread rapidly throughout the western United States owing to plant dispersion by wind,

perhaps aided by railroad shipment of cattle (3). It was first reported in California in 1893 in Lancaster (3), and its appearance throughout the state can be traced through florae and herbarium specimens. Jepson (4) in 1914 stated that S. tragus, called Salsola kali L. var. tenuifolia G. F. W. Mey. in that reference, was “only sparingly established in California” and gave the following as dates of its appearance: Lan-

Received April 1, 1999 F.J. Ryan2 and D.R. Ayres.3 United States Department of Agriculture, Agriculture Research Service, Aquatic Weed Research Laboratory and Section of Plant Biology, University of California, Davis, CA 95616, U.S.A. 1

Mention of a brand name does not constitute a guarantee or warranty by the U.S. Department of Agriculture and is not an endorsement over other similar products. 2 Author to whom all correspondence should be sent at the following address: United States Department of Agriculture, Agriculture Research Service, Horticultural Crops Research Laboratory, 2021 South Peach Avenue, Fresno, CA 93727, U.S.A. (e-mail: [email protected]). 3 Present address: Division of Evolution and Ecology, University of California, One Shields Avenue, Davis, CA 95616, U.S.A. Can. J. Bot. 78: 59–67 (2000)

J:\cjb\cjb78\cjb-01\B99-160.vp Wednesday, March 08, 2000 10:25:11 AM

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caster (1890) [sic], Bakersfield (1895), Stanislaus County (1903), Antioch (1900), Salinas Valley (1910), and Solano County (1911). The number of introductions, species involved, and their taxonomic status in North America are unsettled questions. Mosyakin (5) identified the following species present in North America: S. kali L., S. kali L. subsp. kali, S. kali L. subsp. pontica (Pallas) Mosyakin, S. tragus, Salsola paulsenii Litv., Salsola collina Pallas, Salsola soda L., and Salsola vermiculata L. Salsola tragus was recognized (5) as the correct name for the widespread species, and the problem of synonymy in this taxon was discussed. The following have been used as synonyms: Salsola australis R. Br., Salsola iberica Sennen and Pau, S. kali L. var. tenuifolia Tausch., Salsola pestifer A. Nelson, and S. kali L. subsp. ruthenica (Iljin) Soó. Keys have been prepared to identify the different species of Salsola present in North America (5, 6, 7). Mosyakin (5) noted that S. tragus and S. paulsenii occurred widely in California, whereas S. kali subsp. pontica, S. soda, and S. vermiculata had been found in a limited number of localities in that state. The Jepson manual Higher plants of California (1) reported that S. tragus and S. paulsenii have widespread distribution within California, while S. soda and S. vermiculata have restricted ranges. Other Salsola species were not noted. Beatley (8) recognized two species of Salsola in the deserts of southern California and the Great Basin, S. iberica and S. paulsenii, the barbwire Russian thistle. The latter could be distinguished from the former by its spiny character even in the juvenile form. Beatley (8) observed that in southern Nevada, S. iberica was abundant at elevations above 6000 ft (1 ft = 0.3048 m), S. paulsenii below 4000 ft, and hybrids and mixed stands were present in the intermediate elevations, although these were not described. In Utah, S. pestifer, S. paulsenii, and S. collina were reported in 1969 (9), as well as a newly discovered Salsola species that was termed “the hybrid” or Salsola X, found in a restricted area of the state. A systematic study of Salsola species in Utah (6) characterized S. pestifer as well as two varieties of S. paulsenii, one with a spinose perianth tip and a second with a lax perianth tip. The latter was widely distributed in Nevada and was in the Central Valley of California. The hybrid was studied as well. Chromosome numbers for all taxa were reported (6) and are summarized in Table 1. The species in this study could not be differentiated by their flavonoid complement, determined by two-dimensional paper chromatography. Chromosome number for Salsola species in North America and some distinguishing characters, based on information in the keys, are given in Table 1. Crompton and Bassett (7) found no evidence of hybridization under field conditions among the three species established in Canada, S. pestifer, S. collina, and S. kali. Crompton and Bassett (7) described S. pestifer as wind-pollinated, producing seed autogamously and allogamously. All Salsola species in North America are annuals, except for the perennial S. vermiculata (5). Two lepidopterous agents were introduced to control S. tragus in California, Coleophora parthenica Meyrick in 4

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1973 and Coleophora klimeschiella Toll in 1977 (11). Although both agents became established, neither apparently had a measurable effect on the populations of the plants. This was ascribed to the effects of native hymenoptera on these moths and to asynchrony between their life histories and that of their host plants (12). Coleophora parthenica originated in Egypt, Pakistan, and Turkey, while C. klimeschiella was from Pakistan (13), but the taxonomic relationship of the host Salsola in these regions to the species found in California is not known. Molecular markers can provide objective measures of phenotypic and genetic differentiation. The limitations of isoenzymes and the advantages of DNA-based markers in studies of population structure and differentiation have been the subject of much interest (14–18). The random amplified polymorphic DNA (RAPD) assay (19, 20) is useful because it does not depend on sequence information and requires only limited amounts of DNA that may be available from dried material. The RAPD assay was used to characterize populations of the introduced aquatic weed Hydrilla verticillata (L.f.) Royle in North America and from 44 accessions throughout its range in Asia, Europe, and Africa (21). Plants were identified from locations in Asia with the greatest similarity to those of the present infestations in the United States, suggesting potential locations for biocontrol agents most closely adapted to the biotypes of concern. This work was undertaken to use isoenzymes and RAPD markers to define the population of S. tragus within California and to find genetically similar populations in the native range in Europe and Asia. The ultimate goal of this research is to allow selective matching of compatible biocontrol agents from overseas with the predominant Salsola species presently found in California and in western North America.

Material and methods Plant material Stands of S. tragus were sampled from populations along roadsides. Usually, a minimum of five individuals were sampled at 10m intervals where practical. Very large stands were sampled at approximately 400-m intervals. The location of the collection sites in California are shown in Fig. 1.4 Branches were clipped and placed in plastic bags on ice until return to the laboratory. Samples were stored at 4°C and processed within 2 weeks of collection. When it was not possible to isolate DNA within this time, samples were dried at room temperature and stored; material from overseas was dried before shipment.

Allozyme analysis Plant tissue (150 mg) was ground in a mortar and pestle with liquid nitrogen then in 1 mL of 50 mM Tris-HCl, pH 7.8, with 10% glycerol and 1 mM phenylmethylsulfonyl fluoride, 20 mM 2mercaptoethanol and 10% ethanol. Samples were centrifuged at 7700 × g for 5 min and kept at 4°C. Slab gels, 14 × 16 × 0.75 cm, contained 7.5% total acrylamide, and the buffers were those for nondenaturing electrophoresis (22). In a preliminary survey the following isoenzyme systems were examined using the staining procedure of Wendell and Weeden (23): aconitate hydratase (EC 4.2.1.3), alcohol dehydrogenase (EC 1.1.1.1), aspartate aminotransferase (AAT) (EC 2.6.1.1), glucose 6-phosphate isomerase

The complete list of accessions and allozyme analysis may be obtained from the Depository of Unpublished Data, CISTI, National Research Council of Canada, Montreal Road, Ottawa, ON K1A 0S2, Canada. © 2000 NRC Canada



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Table 1. Summary of distinguishing characters of adventive Salsola species in North America. Species

Chromosome no.

Characters

Reference

S. soda S. kali

2n = 18 2n = 36

5

S. kali subsp. kali S. kali subsp. pontica S. paulsenii

Not given Not given 2n = 36

S. paulsenii lax form

2n = 54

Salsola X

2n = 54

S. tragus S. pestifer (synonym for S. tragus) S. collina S. vermiculata

2n = 36 2n = 36

Restricted to saline habitat in San Francisco Bay. No spinose apex on perianth; fruiting perianth 6–7 mm in diam. Maritime saline habitat. Perianth segments with rigid subspinose apex and distinct midvein. Perianth segments with weak apex and obscure midvein. Spinose apex on perianth; fruiting perianth 7–12 mm in diam. Stems red to purple, fruiting perianth 8–9 mm in diam. Lax apex on perianth; fruiting perianth less than 8 mm in diam. Plants yellow-green without red stems. Found in restricted area in Utah (1972); fruiting perianth 3–4 mm in diam. Plants yellow-green without red stems No columnar beak on perianth; perianth less than 8–10 mm in diam. Stems red or purple at maturity; fruiting perianth 3–6 mm in diam. Plants blue-green with red stems; fruiting perianth 2 mm in diam. Perianth segments wingless. Perennial; perianth segments with pubescent apex

2n = 18 2n = 18

5 5 5 5 6, 7 6 6 5 7, 10 6 5 5

Fig. 1. Location of collection sites within California. The following sites were not located on the map to maintain legibility: 24, 26, 28, 29, 31, 32, 33, 34, 54, 55, 56, 57, 58, 60, and 61. 䉭, type A without type B; ⵧ, type B without type A; (䊉) both type A and type B.

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Table 2. Primers producing polymorphic bands with accessions of S. tragus. Primer*

Sequence

Size of polymorphic bands (bp)

OP-C-18 UBC-724 OP-G-12

TGA GTG GGT G CTC CCT CCT C CAG CTC ACG A

OP-G-11 OP-F-09

TGC CCG TCG T CCA AGC TTC C (GATA)4

1380, 955, and 810 1775, 1015, and 920 1500, 1100, 1000, 660, 425, and 360 1780, 1280, 745, 680, and 460 800 1645, 1110, 940, and 670

Fig. 2. Allozyme patterns in Salsola tragus. (1) 6-phosphogluconate dehydrogenase. (2) aspartate aminotransferase. In each case, type A is on the left, type B is on the right. The analysis was conducted on four plants from site 19.

*Primers designated OP were from Operon Technologies, while the one designated UBC was from the University of British Columbia Protein Service Unit.

(EC 5.3.1.9), NAD-isocitrate dehydrogenase (EC 1.1.1.41), malate dehydrogenase (EC 1.1.1.37), malic enzyme (EC 1.1.1.40), phosphoglucomutase (EC 5.4.2.2), 6-phosphogluconate dehydrogenase (6-P-glu-DH) (EC 1.1.1.44), shikimate dehydrogenase (EC 1.1.1.25), and superoxide dismutase (EC 1.15.1.1). The following were stained according to the methods described by Vallejos (24): succinate dehydrogenase (EC 1.3.99.1) and xanthine dehydrogenase (EC 1.1.1.204). Interpretation of allozyme patterns was based on the reported quaternary structure of these enzymes (25).

DNA isolation Isolation of DNA was by the method of Saghai-Maroof et al. (26). Fresh (100 mg) or dried (50 mg) plant material was ground in a mortar and pestle with liquid nitrogen then 1 mL of grinding buffer (50 mM Tris-HCl, pH 8.0, with 0.70 M NaCl, 10 mM EDTA, 1% hexadecyltrimethylammonium bromide (CTAB), 0.1% 2-mercaptoethanol) was added, and material was ground to a slurry. This was incubated at 65°C for 1 h then centrifuged at 7700 × g for 10 min. The supernatant was extracted with an equal volume (600 µL) of chloroform – isoamyl alcohol (24:1) and DNA was precipitated by the addition of 50 µL 2.5 M sodium acetate and 500 µL isopropanol to the aqueous phase. The suspension was kept at –20°C for at least 2 h. The DNA was recovered by centrifugation at 7700 × g for 5 min, dried, then redissolved in 200 µL of 10 mM Tris-HCl, 1 mM EDTA, pH 7.8 (TE buffer). Five units of DNAse-free RNAse were added, and the mixture was kept at 35°C for 20 min. DNA was precipitated by the addition of 20 µL 2.5 M sodium acetate and 500 µL ethanol. DNA was precipitated from ethanol at least twice and stored at –20°C. DNA was quantified in solution in TE buffer from the absorbance at 260 nm (27).

RAPD amplification and analysis Screening of primers and initial RAPD amplifications were carried out in a Perkin-Elmer (Norwalk, Conn.) Model 4800 thermocycler. Amplifications during the second year and later of the project were done on an Ericomp DeltaCycler I (San Diego, Calif.) thermocycler using the same temperature profiles as on the PE thermocycler but with times adjusted so that effective times within the cycle were approximately equal to those of the Perkin-Elmer instrument. This was necessary because the Perkin-Elmer thermocycler responded to block temperature while the Ericomp instrument determined timing for each step from a temperature probe within a tube in the block. A test amplification of the same DNA samples with the same primers on the two instruments resulted in indistinguishable products so the data from the two instruments were pooled for analysis. For amplification, 15 ng genomic DNA were used in a 25-µL reaction volume. Other components were as follows: 2.5 µL PerkinElmer buffer II; 4 mM MgCl2; 200 µM dATP, dCTP, dGTP, and dTTP; 10 pmol primer; and 1 U of AmpliTaq DNA polymerase

(Perkin-Elmer). The mixture was overlaid with 30 µL mineral oil. For decameric primers, amplification proceeded through 40 cycles of 94°C (30 s), 37°C (30 s), and 72°C (1 min) in the Perkin-Elmer thermal cycler, while in the Ericomp thermocycler these parameters were adjusted to 94°C (1 s), 37°C (20 s), and 72°C (30 s) with the fastest ramping time. For hexadecameric primers, the annealing temperature was 45°C. Sixty decameric primers (kits C, G, and F, Operon Technologies, Alameda, Calif.) were screened to find those that produced strongly reacting repeatable polymorphic bands. An additional 50 decameric primers were obtained from the University of British Columbia Nucleic Acid – Protein Service Unit, Vancouver, British Columbia, Canada (primers 702–798, even numbers). In addition, five hexadecameric primers (four base-repeating units) synthesized at the Protein Structure Laboratory, University of California, Davis, were screened as well. From these 115 primers, those producing polymorphic bands are listed in Table 2. After amplification, reaction products were separated by gel electrophoresis on 2% agarose gel (NuSieve 3:1, FMC, Rockland, Maine) in 89 mM Tris, 89 mM boric acid, 2 mM ethylenediaminetetraacetic acid, pH 8.0. Molecular weight standards were the HindIII digest of Lambda DNA and the 50- to 2500-bp markers, both from FMC Corporation (Rockport, Maine). The molecular weights of the bands are given in the legend to Fig. 3. Electrophoresis was at 45 V for 2 h. Gels were stained with ethidium bromide at 0.5 µg·L–1 and photographed in a transilluminator under UV light. Molecular weights were assigned to amplified bands and a presence–absence (binary) matrix was constructed for the polymorphic bands. Binary data were analyzed using the NTSYS-pc program, version 1.80 of Rohlf (28). Similarities between individuals were calculated from simple matching coefficients, in which presence and absence of a band have equal weight, and the resulting matrix of similarity was further analyzed by unweighted pair-group method, arithmetic average (UPGMA), to produce a dendrogram. The goodness of fit between the original similarity matrix and the dendrogram was determined from the correlation between the similarity matrix and the cophenetic matrix using the NTSYS-pc program. Wright’s fixation index, Fst, was approximated from the RAPDs presence–absence matrix for the accessions of type A plants from California in Fig. 4, using the RAPDFST program (29). © 2000 NRC Canada



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Fig. 3. Amplified products from RAPD assays. Primers were (A) UBC-724, (B) OP-C-18, (C) OP-G-11, and (D) OP-G-12. The samples were site 32 plants 6–10 (lanes 1–5); site 44 plants 1–4 (lanes 6–9). Lanes 3–5 are type B, the remainder are type A. The positions of the polymorphic bands are shown on bars next to the gel photographs. The first lane of each primer set contains molecular mass markers. These are 2600 and 2300 (both bright), 1000, 700, 500 (faster of the two bands), 400, and 300 base pairs.

Fruit characters and plant habit For these measurements, collections were made from plants that were still in their original locations to prevent selective losses of fruit during tumbling. Fruit, i.e., the seed, seed coat, and attached calyxes, sometimes described as the perianth, were collected on the dates and at the locations indicated in Table 3 and stored at 4°C in sealed plastic bags. Intact fruit from a single site (N = 150) were individually weighed. To test whether the means of the weights from the pooled values for types A and B were significantly different, a two-sample t test for unequal variances was used. Analysis of variance was used to test differences among means within collections of fruit of type A or type B. Means were separated using Fisher’s protected LSD test. The diameter of fruiting perianths was determined by placing the fruit on 1-mm ruled graph paper. Diameter of individual fruits was estimated to the nearest 0.5 mm. Fifty fruit of each type were measured. Statistical analyses were conducted as for fruit weights. Plants were observed at weekly to monthly intervals during the growing season from 1996 through 1998 at site 20. Observations at other sites were irregular throughout this period.

Results Allozyme analysis Thirteen enzyme systems were screened for polymorphisms in small populations of plants from northern California. In the initial screening, isoenzymes were examined for four to six individuals from two or three geographically distant populations, e.g., Davis and Cameron Park, 100 km apart. The majority of isoenzymes were monomorphic for the populations screened (alcohol dehydrogenase, superoxide dismutase, malic enzyme, glucose 6-phosphate dehydrogenase), or did not produce interpretable results (shikimate dehydrogenase, xanthine dehydrogenase, aconitate hydratase, succinate dehydrogenase). Possible polymorphisms were detected in the allozyme patterns for malate dehydrogenase, glucose phosphate isomerase, and phosphoglucomutase. Aspartate aminotransferase (AAT) and 6-phosphogluconate dehydrogenase (6-P-glu-DH), however, revealed robust allelic

differences in the study populations. In accord with the purpose of this study, we limited further investigation to these two isozymes. Two covarying patterns for AAT and 6-P-gluDH activity were observed (Fig. 2); the patterns are called type A and type B for purposes of further discussion. Both enzymes are dimeric and generally display two loci (23). The faster locus for AAT in type A had two alleles, manifest as three evenly spaced bands of activity, while the slower locus had only a single allele. Thus there were four major bands for AAT for type A. The pattern for type B indicated a single allele at both loci for AAT, giving 2 major bands with the slower locus identical to that for type A. Two loci of 6-P-glu-DH were present in type A; the slower had a single allele with a mobility identical to that of the slower locus of type B. The faster locus appeared to have two alleles present as two principal bands with a less active band between them. There was only a single allele at this locus in type B. Over a 2.5-year period, 519 plants from 65 different locations were screened for patterns of AAT and 6-P-glu-DH activities sites (Fig. 1 and unpublished data). Plants of type A represented 62.3% of the total. Those of type B were 27.6% of the total, while those with aberrant enzymic phenotypes represented 6.6% of the total. The remainder of the plants were subsequently identified as S. paulsenii, for instance, those at site 42. The ranges of types A and B are similar throughout the Central Valley and southern California. Type A without B was found at 41 sites, while type B without A was at 14, and both types were present together at 10 sites (Fig. 1). Occasionally plants were found that showed aberrant allozyme patterns, particularly during late season screening, e.g., sites 55 and 62. These patterns were characterized by additional bands in zymograms of either polymorphic allozyme superimposed on the pattern of type A. Subsequent resampling of a larger number of plants at these locations near the beginning of the next growing season failed to find the aberrant types. It was concluded that these aberrant © 2000 NRC Canada



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Fig. 4. Dendrogram generated by UPGMA from simple matching coefficients from RAPD analysis of Salsola tragus and Salsola paulsenii. For material from California, designation is by accession code with a number indicating the individual plants. Plants were from the following sites in California indicated by the numbers in Fig. 1: SN2, 32; SN1, 33; BP, 38; LW, 39; AV, 40; S.p., 42; LA, 45; RC, 52; CP, 53; ED, 55;FO, 56; RS, 60; YC, 62. FR designates plants from site 16 grown in Davis and RD designates plants from Redding. France: F-CN, Carnon; F-GR, Grau du Roi, both near Montpellier. Turkey: T-ES, Eynisahan; T-IP, Isparta; T-KK, Kirka; TYS, Yakasinek. An asterisk indicates 21 identical individuals from the sites below. The number in parentheses is the number of plants from that site: 56 (4), 22 (5), 41 (4), 52 (4), 33 (2), 62 (2).

forms were induced by environmental conditions or the growth stage of the plants. It has been noted that the AAT system may show artefactual bands (p. 36 in (23)). RAPD assay Screening of 110 decameric primers and 5 hexadecameric primers allowed selection of 5 decameric primers and 1 hexa-

decameric primer that were strongly reacting and produced polymorphic bands among the accessions. The primers, their sequences, and sizes of the polymorphic amplified products are given in Table 2. The amplified products from primers UBC-724, OP-C18, OP-G11, and OP-G12 with a number of accessions known to be type A or type B by isoenzymic analysis are shown in Fig. 3. For the assay, DNA samples © 2000 NRC Canada



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from 98 plants from California, Turkey, and France, as well as plants identified as S. paulsenii were amplified with the reactive primers, and the resultant 23 bands were scored for presence or absence. Fifty-eight RAPD phenotypes were identified in the material surveyed. The cophenetic correlation of this dendrogram with the original data was 0.87, indicating a good fit to the original data (28). Multivariate analysis of RAPD similarity revealed three clusters: one inclusive of all type A individuals determined from isoenzymic analysis, another of all type B individuals, and a third of the accessions identified as S. paulsenii. Additionally, these clusters included accessions that were not characterized by isoenzyme analysis, specifically, dried plant material from France and Turkey. Among the 67 individuals in the general type A cluster, there were 53 RAPD phenotypes. There was evidence of population structure, i.e., the partitioning of genetic variation within populations, for type A plants from California. For instance, individuals from Fresno County, Redding, and Laws (FR, RD, and LW, respectively, in Fig. 4) showed a high degree of within-site similarity. Approximation of Fst, the reduction in heterozygosity due to genetic drift in subdivided populations, for the California accessions of type A in Fig. 4 using the RAPDFST program (29) gave a value of 0.550 ± 0.120, which suggests a structured population. Plants from Turkey fell within two clusters with a single outlier, but all were associated with type A. Accessions from France fell within a single cluster, with the exception of two plants, and were also associated with type A. Plants from site 42, identified as S. paulsenii, formed a cluster distinct from the type A and type B groupings, although it appeared to be more closely associated with type A. Plants identified as type B from the allozyme data (sites 22, 32, 33, 52, 56, and 62) were included in a single cluster, with only 3 RAPD phenotypes for 24 individuals, and this cluster was distinct from all other accessions. Variation within type B was found only at sites 32 and 33. Fruit characters and habit of plants Fruit, collected from plants of known type at several different times and from several locations, differed in appearance by type (Fig. 5). Those of type A had a folded calyx, while fruit of type B had a flat calyx. The mean weights were different between the two types (Table 3). The distributions of weights were sometimes not normal for type A (data not shown), with substantial numbers of low-weight fruit present. The mean weights of fruits of the same type from the same location were significantly different for collections made in different years (Table 3). These differences presumably represent the effects of local climatic conditions on the plants, since collections were made at the end of the growing season. Plants of the two types at site 20, however, were in very nearly a common garden situation, growing on the same soil type and experiencing identical climatic conditions in a ruderal setting. The fruit of type A were consistently heavier than those of type B, suggesting a genetic basis for the differing phenetic character. The values of fruit diameter were different between type A and type B (Table 3) The diameters of the fruiting perianth of both type A and type B are within the range of

65 Fig. 5. Fruit of type A (A) and type B (B) plants. In the lateral view (left side), the folded calyxes of type A (arrow 1) are contrasted with the flattened calyxes of type B (arrow 2). In the view from the top (right side), the more prominent veins in the calyxes of the type B fruit can be noted (arrow 3).

3 to 6 mm for S. pestifer given by Crompton and Bassett (7; Table 1).

Discussion The presence of two groups within Salsola tragus with such strong and consistent differences was unexpected, since the most recent flora of the state (1) indicated only a single widespread species with the characters attributed to Salsola tragus. Allozyme analysis indicates that type A and type B have some common alleles, but the patterns of AAT and 6-Pglu-DH are characteristic of and invariant for each type, suggesting no genetic interaction between the two types. Within each type, variability was detected with the RAPD assay that was not seen in the two isoenzyme systems, with type A more variable than type B. The consistent presence of more alleles per locus in type A than in type B suggests a difference in ploidy between the two types. Species with chromosome numbers of 2n = 18 and 2n = 36 are known within Salsola, and there are indications of higher ploidy as well (5, 6; Table 1). Preliminary observations on chromosome numbers in root tips from seedlings from this study indicate that type A has 2n = 36, while type B has 2n = 18 (John Bailey, University of Leicester, U.K., personal communication). Fruit weights and diameters were different between the two types under similar conditions in the field. The shape of the calyx in the fruit was different between the two types. Finally, the two types have different chromosome numbers. The presence of so many differences in phenetic characters between the two types strongly suggests that what we have termed types are actually two cryptic species. From the data on fruit character, plant habit, and chromosome numbers we conclude that type A is actually S. tragus, while type B is a previously undescribed taxon. The fruit character of type B may have been noted as variants within S. tragus. A preliminary assessment of the distribution of the two types in western North America has been conducted by examining specimens at the Herbarium at the California Acad© 2000 NRC Canada



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Can. J. Bot. Vol. 78, 2000 Table 3. Mean weights and perianth diameters of fruit of Salsola tragus. Type

Site

A UC Davis Airport A Site 20, Fresno A Site 20, Fresno A Site 20,* Fresno A Site 23, Fresno A Site 23, Fresno B Site 20, Fresno B Site 20, Fresno B Site 20, Fresno B Site 32 Summary Mean of all type A Mean of all type B t statistic (unequal sample variance)

Year 1994 1997 1998 1997 1997 1998 1996 1997 1998 1997

Mean weight ± SD (mg) 2.65±0.63a 2.00±0.63b 2.49±0.76c 1.85±0.78bd 2.03±0.53be 1.76±0.30a 1.32±0.39b 1.64±0.54c 1.61±0.36c Weight ± SD (mg) 2.20±0.74 (N = 750) 1.58±0.44 (N = 600) 19.26 (p < 0.0001)

Mean diam. ± SD (mm) 3.5±1.1a 3.2±1.0ab

3.0±1.1b 5.1±1.1a 4.5±1.2b 4.8±1.4ab Diam. ± SD (mm) 3.2±1.1 (N = 150) 4.8±1.2 (N = 150) –12.99 (p < 0.0001)

Note: Type was assigned from patterns of aspartate aminotransferase and 6-phosphogluconate dehydrogenase after electrophoresis. For determinations of weight, 150 fruit were used, while 50 fruit were used for diameter measurements. For both measurements, means within each type followed by different letters are significantly different (p < 0.05) according to Fisher’s protected LSD test. *Site approximately 100 m from site 20.

emy of Sciences, San Francisco. Collections of material from Arizona, Washington State, Nevada, and Oregon contained examples of type A and type B, as judged by the fruit characters, suggesting that both are widespread in western North America. There is evidence of population structure within the type A plants in California. The Fst value of 0.55 suggests local populations are genetically distinct. The Salsola species under consideration are wind-pollinated, have hermaphroditic flowers with the possibility of autogamy, an annual life cycle, and seeds that are dispersed by gravity, all of which are associated with the maintenance of population genetic structure (30). Plant mobility may also contribute to population genetic structure by a founder effect if the population within an area is descended from a small number of parents. Stalling et al. (31) determined that plants of S. iberica travelled between 60 and 4069 m in a 6-week period on summer fallow and wheat stubble fields of eastern Washington, distributing an average of 3.56 × 104 seeds per plant. Given this mobility and relatively high reproductive effort, many individuals in an area may be descended from a single parent. For instance, the plants sampled at 400-m intervals at site 39 (Fig. 3, LW1 to 4) were assigned to a single cluster in the dendogram suggesting they were closely related. Allozymes were used to delimit genetic variation and deduce the population structure of S. komarovi Iljin, widespread in coastal areas of Asia but with restricted habitat (32). Differentiation at the local level was attributed to low levels of gene flow among populations owing to physical barriers. This species showed a good deal more variation at the allozyme level than do either type A or type B of Salsola tragus, judging from the level of enzyme polymorphism. The population structure of different species of Salsola within their native ranges in Europe and Asia is not known at all. Differential saline tolerance among accessions of S. kali from northern Europe (33) suggests some genetic differentiation has occurred. One goal of this research was to determine similarities between populations of S. tragus in California and accessions

from abroad to aid in the search for effective biocontrol agents. The results of the RAPD assay suggest that the type A of Salsola tragus is similar to plants collected both in France and in Turkey. Salsola kali subsp. pontica, native to the maritime coast of southern Europe and the Mediterranean, is established on the east coast of the U.S. (5) but has not been reported in California; its affinity to type A is not currently known. Many other species of Salsola, however, are present in Europe, Asia, or North Africa and may be similar to the type A plants from California. At present, no plant material from outside the U.S. has been found that is similar to type B. The large genetic difference between type A and type B is noteworthy for its implications for biocontrol. If an effective biocontrol agent that was specific for one of the types were introduced, as may have been the case with Coleophora species in California, then suppression of one type may allow expansion of the second type. Nothing is known of the relative susceptibility of the two types to control by Coleophorid insects or of the relative efficacy of other biocontrol agents. Further work is needed to assess the susceptibility of both types to biocontrol agents. Possible differences in susceptibility to Uromyces salsolae Reichardt have been noted between types A and B of Salsola from California, and detailed investigations are being pursued (W.L. Bruckart, United States Department of Agriculture, Agriculture Research Service, Ft. Detrick, Md., personal communication).

Acknowledgements The authors thank Dr. James Young, USDA, Agriculture Research Service, Reno, Nev., Dr. Jodie Holt, University of California, Riverside, Calif., Dr. Michael Pitcairn, California Department of Food and Agriculture, and two reviewers from this journal who provided helpful comments on the manuscript. Dr. Pitcairn provided helpful suggestions during the course of the work. Dennis Margosan, USDA Agriculture Research Service, Horticultural Crops Research Labora© 2000 NRC Canada



Ryan and Ayres

tory, Fresno, prepared Figs. 2 and 4. The efforts of Dr. R. Sobhian and Dr. Lloyd Knudson, USDA Agriculture Research Service Biocontrol Laboratory, Montpellier, France, in providing samples from France and Turkey are gratefully acknowledged. This work was supported by contract 94-0537 from the California Department of Food and Agriculture.

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