Genetic diversity within and among 42 native populations of Bromus tectorum (cheatgrass) was characterized within two regions, the eastern Mediterranean and the western Mediterranean. Two hypotheses  were tested for the genetic diversity of these populations: 1) populations from the eastern Mediterranean are more genetically diverse compared with populations to the west, a potential consequence of the species’ westward dispersal with the spread of agriculture and 2) populations across the Mediterranean contain comparable genetic diversity but display high genetic differentiation, a potential consequence of both regions having served as refugia during glacial advances in the late Quaternary Period.  Populations in the eastern Mediterranean possess 16 polymorphic loci and 37 multilocus genotypes.  In contrast, populations from the western Mediterranean include a subset of these polymorphic loci (9) and fewer multilocus genotypes (19), consistent with the dispersal of B. tectorum with the east-west Holocene spread of agriculture.  Among the 19 multilocus genotypes identified in populations from the western Mediterranean, 13 are undetected among eastern Mediterranean populations.  Average genetic diversity within populations from the eastern Mediterranean is nonetheless comparable to the genetic diversity in populations from the Iberian Peninsula, whereas diversity is lowest in the populations from southern France.  Our results suggest a prominent role for agriculture in the grass’s western spread, although glacial history and environmental heterogeneity also could have influenced the grass’s genetic diversity.  The exceptionally high level of self-pollination (> 99%) in B. tectorum has contributed to preserving the genetic signature associated with the species’ biogeographical history across the Mediterranean Region.  

Methods

Collection protocol

Collection of samples involved the removal of mature caryopses (hereafter referred to as seeds) from individuals in each of 42 populations in the Mediterranean Region (Supplemental Information Table 1). Samples were obtained from three populations each in Jordan and Syria, 11 populations in Israel, 14 populations in France, seven populations in Spain, and four populations in Portugal.  Collection locations were selected to enhance the resolution of the genetic diversity within populations and the genetic structure within and among populations across this portion of the native range.  Collections were made in disturbed sites, e.g. pastures and agricultural fields, roadsides, and archeological ruins (see Supplemental Information Table 1).  Each population was sampled from an area of at least 500 m2, and we collected seeds (mature panicles) from 25-35 plants from each population (with two exceptions: Shinshar, SY, N = 12; Sisteron, FR, N = 20).  To minimize inadvertently collecting full siblings in this highly self-pollinating plant species (Novak and Mack 2016), individuals were collected 1-2 m apart.  In low density populations, the entire population was sampled. To account for seed dormancy (Allen and Meyer 2002), seeds were stored at room temperature in paper envelopes for at least three months before being germinated for analysis.

Allozyme analysis

Allozymes are single-locus, codominant molecular markers; consequently, the genotype of individuals (i.e., homozygous versus heterozygous) can be inferred from enzyme banding patterns at each scored locus.  Although allozymes underestimate genetic diversity compared to other markers (Rowe et al. 2017), their use here allows these results to be compared directly to the findings of our previous analyses of 51 native populations of B. tectorum (Novak and Mack 1991) and 318 invasive populations in North America (Novak and Mack 2016).  In addition, our extensive use of allozymes has optimized the procedures for this species.  Finally, allozymes are a relatively rapid method for gathering population genetic data; the genotypes of approximately 30 individuals at each of 25 allozyme loci could be characterized per day.

In preparation for allozyme analysis, seeds were germinated on moist filter paper and allowed to grow for 7-10 days. Harvesting occurred when seedlings were between 2 and 8 cm tall.  Allozyme analysis was performed using the methods of Soltis et al. (1983) as modified by Novak et al. (1991) and Schachner et al. (2008).  The 15 enzymes assayed in this study were coded for by 25 putative loci (Supplemental Information Table 2).  As reported repeatedly (e.g., Valliant et al. 2007; Huttanus et al. 2011; Pawlak et al. 2015), the genotype found most frequently in the native and introduced range is referred to as the Most Common Genotype (MCG) (Supplemental Information Table 2).  Allele nomenclature for B. tectorum initially followed Novak and Mack (1993) but has been updated based on the results of the new analyses reported here (see below).    

Data analysis

Alleles for every individual in a population were recorded at each of the 25 loci and analyzed using BIOSYS-1 (Swofford and Selander 1989), following methods of Novak et al. (1991), Huttanus et al. (2011), and Pawlak et al. (2015).  To answer the main questions of this study, the 42 populations of B. tectorum we analyzed were grouped into two geographic regions, the eastern Mediterranean (populations from Israel, Jordan, and Syria) and the western Mediterranean (populations from Spain, Portugal, and southern France).  In addition, the populations from Spain and Portugal were further grouped into the Iberian Peninsula Region. Genetic diversity was described using the following parameters: the number of alleles per locus (A), the number of polymorphic loci (P), the percentage of polymorphic loci (%P; a locus is polymorphic if the most common allele ≤ 99%), expected mean heterozygosity (Hexp), the mean observed heterozygosity (Hobs), and the number of homozygous multilocus genotypes (#MLGs).  Expected mean heterozygosity was computed using Nei’s (1978) unbiased estimate method.  Mean observed heterozygosity (Hobs) was obtained through the direct count method, and the statistical significance of deviations from random mating were analyzed using a Chi-square test. Allele combinations were identified and evaluated by determining the number and frequency of distinct homozygous MLGs for each population.  

Allelic frequencies, Wright’s fixation index (F = 1 - Hobs/Hexp) and Wright’s F-statistics (F_IT and F_ST) were computed for all polymorphic loci (Wright 1965, 1978) using BIOSYS-1 (Swofford and Selander 1989). Variance components in Wright’s F-statistics (1978) were employed to calculate Nei’s gene (allelic) diversity statistics (Nei 1973, 1977); the diversity partitioned within populations (HS) and diversity partitioned among populations (DST) were summed to obtain the value of the total gene diversity (HT). Proportion of the total gene diversity partitioned among-populations (GST) was calculated as GST = DST/HT.  Means of the diversity statistics were compared to the means of previously analyzed populations in the native range of B. tectorum (Novak and Mack 1993; Novak and Mack 2016).

Nei’s (1978) unbiased genetic-identity coefficients (I) were computed to evaluate genetic similarity within and among populations from the eastern and western Mediterranean and all pair-wise comparisons (I = 1.0 denotes genetically identical populations, and I = 0.0 indicates complete genetic differentiation among populations). Phenograms for the 17 populations from the eastern Mediterranean Region, the 25 populations from the western Mediterranean Region and all 42 populations from the study area were constructed using the un-weighted pair-group method with arithmetic averaging (UPGMA) algorithm in BIOSYS-1, based on values of genetic identity (I) (Swofford and Selander 1989).  The UPGMA phenograms were used to examine the genetic relationships among all 42 populations, as well as among the populations from the eastern and western Mediterranean regions.