Founder events influence structures of Aspergillus flavus populations

Summary In warm regions, agricultural fields are occupied by complex Aspergillus flavus communities composed of isolates in many vegetative compatibility groups (VCGs) with varying abilities to produce highly toxic, carcinogenic aflatoxins. Aflatoxin contamination is reduced with biocontrol products that enable atoxigenic isolates from atoxigenic VCGs to dominate the population. Shifts in VCG frequencies similar to those caused by the introduction of biocontrol isolates were detected in Sonora, Mexico, where biocontrol is not currently practiced. The shifts were attributed to founder events. Although VCGs reproduce clonally, significant diversity exists within VCGs. Simple sequence repeat (SSR) fingerprinting revealed that increased frequencies of VCG YV150 involved a single haplotype. This is consistent with a founder event. Additionally, great diversity was detected among 82 YV150 isolates collected over 20 years across Mexico and the United States. Thirty‐six YV150 haplotypes were separated into two populations by Structure and SplitsTree analyses. Sixty‐five percent of isolates had MAT1‐1 and belonged to one population. The remaining had MAT1‐2 and belonged to the second population. SSR alleles varied within populations, but recombination between populations was not detected despite co‐occurrence at some locations. Results suggest that YV150 isolates with opposite mating‐type have either strongly restrained or lost sexual reproduction among themselves.


Introduction
In warm agricultural areas, fungi belonging to Aspergillus section Flavi frequently infect and contaminate maize with aflatoxins Probst et al., 2010). Aflatoxins are naturally occurring, highly toxic and carcinogenic mycotoxins that can induce acute and/or chronic health detriments, including death, to both humans and animals Monson et al., 2015;Xu et al., 2018). The most prevalent and toxic aflatoxin, B 1 , is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC, 2002). Aflatoxin B 1 is strictly regulated in most developed nations (FAO, 2004;Wu and Khlangwiset, 2010), with the enforcement of these regulations limiting commercialization and consumption of contaminated commodities. These limitations may cause a severe economic loss for farmers, processors and consumers (Wu and Khlangwiset, 2010;Bui-Klimke et al., 2014). However, in nations where regulations are either poorly enforced or non-existent, humans and domestic animals are continuously exposed to aflatoxins (Gong et al., 2008;Shephard, 2008;Hoffmann et al., 2018;Voth-Gaeddert et al., 2018). This results in losses in human health and livestock productivity that have a proportionately greater economic impact than that seen in the developed world (Udomkun et al., 2017;Nishimwe et al., 2019).
Aflatoxin-producing fungi interacting with various crops are highly diverse in terms of community structure at the species and genotype level, toxin production potentials and adaptation to diverse cropping systems (Mehl et al., 2012). The most studied species is A. flavus because this is the most common causal agent of aflatoxin contamination Klich, 2007). Aspergillus flavus has two prominent morphotypes, the S and L, which differ in both morphology and physiology. The S morphotype produces numerous, small sclerotia (diameter < 400 μm) and consistently high levels of aflatoxin B 1 , while the L morphotype produces fewer, larger sclerotia (diameter > 400 μm) and variable levels of aflatoxin B 1 (Cotty, 1989;Probst et al., 2012;Singh and Cotty, 2019). Variation in the aflatoxin-producing potential of fungal communities has a large impact on the extent to which crops become contaminated (Probst et al., 2007;Cotty et al., 2008;Bandyopadhyay et al., 2016).
Each A. flavus morphotype is subdivided into numerous vegetative compatibility groups (VCGs). Members of the same VCG are isogenic for all vegetative incompatibility (vic) loci and form heterokaryons when their physiologically complementary hyphae anastomose (Bayman and Cotty, 1991;Leslie, 1993). Membership in a VCG is typically determined with functional vegetative compatibility analyses (VCA) using nitrate non-utilizing (nit) mutants Cotty, 1991, 1993). Within each of the four A. flavus VCGs previously examined, variation among isolates is best explained by clonal descent with mutation over large numbers of generations Cotty, 2010, 2015). For other A. flavus VCGs, members are more closely related to each other than to individuals within other VCGs (Papa, 1986;Bayman and Cotty, 1993).
Aspergillus is an anamorph genus, and A. flavus has been considered an asexual species (Papa, 1986;Cotty et al., 1994;Klich, 2007;Islam et al., 2018). However, functional mating-type idiomorphs of A. flavus have been characterized (Ramirez-Prado et al., 2008), and the possibility of sexual recombination under field conditions suggested following induction of low fertility ascomata under laboratory conditions between isolates of opposite mating-type and different VCGs (Horn et al., 2009a(Horn et al., , 2009b(Horn et al., , 2014Olarte et al., 2012). In addition, sexual reproduction in sclerotia may occur in the field if sclerotia incubated for 4 months in the laboratory are allowed to come in contact with natural A. flavus populations in the field (Horn et al., 2016). Those findings were detected in experiments conducted in fields in Georgia, USA. On the other hand, in agricultural fields in Arizona and Texas, there is no detectable recombination among sympatric VCGs co-existing across extensive areas (Grubisha and Cotty, 2010). Results from studies examining aflatoxinproducing fungal communities associated with maize in Mexico , Kenya (Islam et al., 2018) and in several nations in sub-Saharan Africa (Adhikari et al., 2016) suggest communities of aflatoxinproducing fungi are (i) highly stable, (ii) shaped predominantly by clonal reproduction and mutation, and (iii) participate in little or no sexual recombination. The frequency of sexual recombination within A. flavus populations in nature remains unknown (Fedorova et al., 2009;Horn et al., 2009aHorn et al., , 2011Horn et al., , 2016Kwon-Chung and Sugui, 2009;Dyer and O'Gorman, 2012).
In a 3-year study of A. flavus L morphotype populations associated with maize production in Sonora, Mexico, 136 VCGs were identified (Ortega-Beltran and . However, VCG composition varied by year and no VCG was consistently dominant across years. Certain VCGs had drastic spatio-temporal 'shifts', that is, had drastic changes in frequencies over single years in both multiple fields and multiple agro-ecosystems. The most prominent shift was a change in the frequency of VCG SON003 (Ortega-Beltran and . Subsequently, VCG SON003 was identified as previously described YV150, and SON003 is now referred to as YV150 (Mehl and Cotty, 2010) in the current work. YV150, originally characterized from cotton field soil in Yuma Valley, Arizona, in 1987, has been collected for over 20 years from crops and soils across an extensive geographic area ranging from Nayarit, Mexico to Arkansas, USA (South to North) and from Arizona to Georgia, USA (West to East). Variability for genetic markers among members of YV150 has not been previously described. The increase in the relative frequency of YV150 observed in 2006 in Sonora was hypothesized to be a result of a founder event in which genetic variation was lost when a small number of individuals from the larger population disproportionately established the population that developed the previous summer (Ortega-Beltran and . However, no genetic evidence to test this hypothesis was developed and it remains unclear if shifts in YV150 frequencies in Sonora resulted from rapid increases of one or a few YV150 clones or a sudden increase by the VCG as a whole. In the latter case, the diversity of YV150 in 2006 would reflect the overall diversity of YV150. The causes for dominance and subsequent drastic decline of YV150 in Sonora, Mexico, also are unknown. However, the adaptability of members of VCG YV150 is reflected in repeated detection of this VCG for over 20 years and the frequent association of members of the VCG with maize and cotton production in North America. The objectives of the present work were (i) to assess genetic diversity within 82 isolates of VCG YV150 from Mexico and the United States collected over 20 years using 24 microsatellite markers for A. flavus previously shown to be useful for quantifying variability within a VCG Cotty, 2010, 2015;Ortega-Beltran et al., 2016) and (ii) to determine if members of this VCG from spatially and temporally distinct populations possess clonal or recombinant population structures. The evidence gathered suggests swings in incidences of VCGs reflect influences of founder events. In addition, VCG YV150 was found to be similar to previously examined VCGs Cotty, 2010, 2015;Ortega-Beltran et al., 2016) in the diversity of SSR haplotypes but distinct in having the diversity distributed across isolates containing one or the other mating-type idiomorph. Another important finding is that YV150 is composed of two divergent populations, one containing the MAT1-1 idiomorph and the other MAT1-2. Previously it was emphasized that distributions of A. flavus VCGs are best described from samples collected over multiple years representing multiple sites distributed across large areas (Ortega-Beltran et al., 2015). Dominance by a single haplotype of a VCG during 1 year, across a large area, in the presence of multiple haplotypes of the same VCG reported in the current work could not have been detected with less intensive sampling.

Mating-type idiomorph characterization in VCG YV150
Each of the 82 VCG YV150 isolates (Table 1; Fig. 1) produced a single amplicon (Ramirez-Prado et al., 2008), either a PCR product 395 bp in length associated with MAT1-1 or a PCR product 273 bp in length associated with MAT1-2 ( Table 2). All 48 isolates from Mexico contained the MAT1-1 idiomorph. Both MAT1-1 and MAT1-2 isolates were detected in Arizona and Texas. The three isolates in a priori population SO are MAT1-2. The YV150 tester pair was developed from an isolate from Arizona cotton field soil collected in 1987 and is MAT1-2. A tester pair was developed from two maize soil isolates from Sonora collected in 2006, both of them are MAT1-1. All 82 isolates formed heterokaryons (complement the nitrate auxotrophy) with one or both isolates from both tester pairs regardless of the isolate mating-type. Thus, mating-type alleles in A. flavus do not function as vegetative compatibility genes in a manner similar to that reported for Neurospora crassa (Perkins, 1988).

Microsatellite genotyping and allelic diversity
For each of the 82 YV150 isolates, 23 loci produced single peaks in the expected range and were relatively easy to score. Locus AF26 was not amplified in any YV150 isolate, regardless of the idiomorph they contained, and was excluded from analyses. Only one locus was monomorphic across all isolates (AF66) (Supplementary Table 1). Over 20% of isolates (20 total) were arbitrarily selected to verify results by re-amplification. Uncertain alleles were triple-checked.

Determination of a posteriori populations
The network generated in SplitsTree assigned isolates into two clusters (Fig. 2), considered as a posteriori populations (Table 3). MAT1-2 isolates-all from the United States-composed a single group, while MAT1-1 composed another group, regardless of geographical origin and year of isolation. Principal coordinates analysis (PCoA) results also yielded two populations (data not shown) identical to those detected with SplitsTree. In PCoA, the first two axes accounted for 99.48% (94.65% and 4.83%, first and second axis respectively) of the genetic variation. Only 0.25% of the variation was explained by the third axis.

Genotypic diversity
Out of 36 haplotypes detected in YV150, 32 were represented by a single isolate (Table 3). The MAT1-1 and MAT1-2 populations had 14 and 11 polymorphic loci, respectively. Isolates with MAT1-2 had up to 14 alleles in a polymorphic locus compared with six in isolates with MAT1-1 (Table 3). Linkage disequilibrium, non-random association among pairs of loci due to lack of recombination, within clone-corrected haplotypes of these two populations was highly significant (P < 0.001, in both cases; Table 3).

Diversity indices
All haplotypes were still detected within the YV150 population when isolates originating from Sonora soil 2006 were excluded. Only one haplotype was detected during 2006 across the examined area of Sonora and therefore the Shannon-Wiener diversity index, H (Begon et al., 1996), was 1.0 for that year (Table 4). That haplotype was detected in other years and locations. In contrast, in the same year, around 1000 km away, each of the 10 YV150 isolates detected in Arizona soil had a distinct haplotype; that population had an H value of 0.077, indicating very high diversity. The seven isolates recovered from Sonora soil in 2007 and 2008 were composed of six haplotypes and also had high diversity with an H of 0.028 (Table 4).

Population structure
Assessment of genetic composition was conducted using Structure (Pritchard et al., 2000) for clone-corrected YV150 haplotypes. In the Structure algorithm, the initial conditions were eight a priori populations (Table 1). The number of genetic clusters was not clearly indicated with values of the log probability (ln P(D)) among 20 successive runs for each K (Evanno et al., 2005). However, the delta K value clearly indicated that the most likely number of genetic clusters (K) was two (ΔK = 1612) (Fig. 3). Delta K was obtained using Structure Harvester (Earl and VonHoldt, 2012), which follows the Evanno method (Evanno et al., 2005). There were nine loci in which the same allele occurred in both populations (Supplementary  Table 1). However, all MAT1-1 haplotypes belong to one cluster and all MAT1-2 haplotypes belong to the other (Fig. 4). We did not detect SSR haplotypes associated with both mating types (Fig. 4). Analyses of the partitioning of the genetic variance revealed that 74.5% of the variation was attributed to differences between populations (P < 0.0001) and only 25.5% was due to intra mating-type variation (Table 5). Genetic relationships of YV150 with other VCGs with the same range Members of VCGs OD02, MR17, CRG136, and YV36 have been previously subjected to genetic studies in our laboratory Cotty, 2010, 2015;Ortega-Beltran et al., 2016). For the five VCG comparison (i.e., OD02, MR17, CRG136, YV36, and YV150), 20 SSR loci were used out of the set of 24 SSR loci (Grubisha and Cotty, 2009). There were four SSR loci that either did not amplify or were highly polymorphic and for that reason were excluded. Locus AF26 did not amplify in any of the isolates of YV150, as mentioned above. Locus   Fig 2. The output of SplitsTree using the neighbour net and phitest approaches (Huson and Bryant, 2006). Out of 36 haplotypes, two a posteriori populations were determined: MAT1-1 and MAT1-2 populations. [Color figure c a n b e v i e w e d a t wileyonlinelibrary.com] Table 3. Descriptive statistics of Aspergillus flavus VCG YV150 a posteriori populations.
A posteriori populations as determined using SplitsTree (Huson and Bryant, 2006).  AF18 did not amplify in any of the isolates of OD02, as described previously (Grubisha and Cotty, 2010). Loci AF48 and AF64 were highly polymorphic in OD02, as described previously (Grubisha and Cotty, 2010). The second network generated in SplitsTree revealed that members of each VCG and of each of the two matingtype idiomorphs of YV150 comprised single clusters (Fig. 5).

Discussion
Populations of the Aspergillus flavus L morphotype resident in agricultural fields are mosaics of many VCGs (Bayman and Cotty, 1991;Horn and Greene, 1995;Ortega-Beltran and Cotty, 2018). SSR haplotypes vary among members of each VCG and linkage equilibrium exists among members of each of the four VCGs previously examined Cotty, 2010, 2015;Ortega-Beltran et al., 2016). Linkage equilibrium among unlinked SSR loci suggests that genetic exchange within VCGs occurs (Grubisha and Cotty, 2015). SSR loci are not in equilibrium between VCGs, and recombination has not been detected among the five VCGs examined to date (Fig. 5) Cotty, 2010, 2015;Ortega-Beltran et al., 2016). Frequencies of individual VCGs within a single field can change several fold between seasons (Bayman and Cotty, 1991), and significant shifts in frequencies of several different VCGs have occurred across an entire region (Ortega-Beltran and . Each shift occurred in a single year, in many maize fields within four agro-ecosystems distributed over large areas of Sonora, Mexico. The shifts were attributed to founder events, possibly during colonization of organic matter associated with crop development, magnified by the rapid, copious ] averaged across 20 simulations for each K (data not shown) was used to calculate the optimal number of populations, delta K, using Structure Harvester (Earl and VonHoldt, 2012), following the Evanno method (Evanno et al., 2005).    . The presence of a single haplotype in YV150 and the high frequency of YV150 in the Aspergillus flavus community as a whole are both consistent with rapid population expansion from a founder event within YV150. While there is no data from 2005 to distinguish an extreme bottleneck due to selection from one due to a founder event, it is usefully modelled as a founder event because of its similarity to what is observed when treating crops with atoxigenic biocontrol strains in which the new population on annual crops has greatly reduced variability from the soil population and primarily reflects the biocontrol haplotype/VCG itself (Bandyopadhyay et al., 2016;Ortega-Beltran and Cotty, 2018). The use of atoxigenic L morphotype genotypes as active ingredients of biocontrol products causes shifts in A. flavus population composition, so that the applied genotypes compose most of A. flavus associated with treated crops Bandyopadhyay et al., 2016). These induced changes in population structure result in lower crop aflatoxin content. SSR haplotypes of atoxigenic A. flavus active ingredients are stable over multiple years of biocontrol applications (Grubisha and Cotty, 2015). In both population shifts caused by atoxigenic strain use in agriculture and in the natural event examined in the current work, individual SSR haplotypes dominate the populations after the shift has occurred. However, differences are apparent between the natural founder events described in the current and previous studies (Grubisha and Cotty, 2015;Ortega-Beltran and Cotty, 2018) and founder events induced by atoxigenic strain biocontrol applications. Atoxigenic strain biocontrol products have residual influence, and atoxigenic strains frequently exceed 90% of the A. flavus soil population 1 year after treatment and over 30% 3 years after treatment (Cotty, 2000;Cotty et al., 2007). In contrast, all the natural population fluctuations discussed here quickly dissipated to background levels by the season following high detection (Ortega-Beltran and . The natural founder events were all detected in the soil. However, natural dispersal over such a large area must involve aerial dispersal of conidia (Brown and Hovmøller, 2002;Isard et al., 2005;Aylor, 2018), colonization of above-ground organic resources, and incorporation into the soil either by cultivation or natural processes. Thus, the population shifts detected in the soil occurred in the spring or summer after the founder event. Failure of the natural population shifts to persist as long as those from an atoxigenic biocontrol application may be attributable to smaller sizes of the A. flavus communities over much of the examined area, a less-extreme initial shift, and possibly removal or degradation of crop debris such as stalk and cobbs, which harbour long-term A. flavus reservoirs (Ashworth et al., 1969;Jaime-Garcia and Cotty, 2004;Cotty, 2010, 2013). Colonized organic matter may have a greater long-term impact on A. flavus populations than the ephemeral thin-walled A. flavus conidia. The short-term influence of the founder events described here should caution against discontinuing biocontrol applications even after extensive population modification has been achieved.
In total, 82 isolates of A. flavus VCG YV150 were examined by SSR characterization in the current study, and 41 of these are the single haplotype that participated in the founder event detected in 2006 Sonoran soil. The remaining 41 YV150 isolates represented 35 SSR haplotypes that had been collected over 20 years across large regions of the United States and Mexico (Table 1). This level of SSR haplotype diversity (i.e., few haplotypes represented by multiple isolates) is common among members of all VCGs examined to date Cotty, 2010, 2015;Ortega-Beltran et al., 2016). The great diversity of A. flavus populations is shaped by clonal reproduction and mutation-driven evolution (Islam et al., 2018).
Members of a VCG may be considered to descend from the same clonal lineage and to be more similar to each other than to members of other VCGs (Bayman and Cotty, 1993;Leslie, 1993;Horn and Greene, 1995;Grubisha and Cotty, 2010). However, unlike other VCGs examined in detail by SSR, diversity in YV150 was distributed across isolates containing one or the other of the mating-type idiomorphs with 35.3% of haplotypes associated with the MAT1-1 idiomorph and 64.7% with the MAT1-2 idiomorph. There are other aflatoxin-producing VCGs reported to contain isolates with opposite matingtype idiomorphs, but in those VCGs only a single member of the less common idiomorph has been identified (Ramirez-Prado et al., 2008;Horn et al., 2009b;Sweany et al., 2011).
Structure, SplitsTree, and PCoA analyses of SSR data from the current study indicate that the YV150 haplotypes represent two distinct populations, one containing MAT1-1 and the other MAT1-2 (Figs. 2 and 4). Introgression of haplotypes associated with one mating-type population into the other was not detected with Structure, suggesting the absence of hybridization between populations (Pritchard et al., 2000) despite their co-occurrence in some areas. Analysis of molecular variance (AMOVA) results also indicate a lack of recombination between isolates with opposite mating-type (Table 5). The divergence between the MAT1-1 and MAT1-2 populations (Figs. 2 and 5) is similar to divergences among all four previously examined VCGs (Fig. 5). However, there is no evidence that the separation of the two populations is a result of either characteristics or activities of the mating-type idiomorphs. From the current results, it is not clear if the distinct structures of the MAT1-2 and MAT1-1 populations derived through divergence from a common ancestral population with both idiomorphs or from two separate ancestral populations developing vegetative compatibility. Knowledge of the molecular characteristics of het loci, which dictates self-identify within a VCG, is currently insufficient to address this.
Vegetative compatibility has frequently been used to characterize variability in A. flavus populations (Bayman and Cotty, 1991;Horn and Greene, 1995). As in the present study, most studies determine membership in a VCG with a functional assay that pairs complementary nitrate auxotrophs with a nitrate non-utilizing (nit) mutant from each isolate being evaluated. Complementation of the nit mutants indicates membership in the VCG. Previously, four VCGs with many members identified in this manner and collected from sympatric populations distributed over large areas have been examined with an SSR population genetic approach Cotty, 2010, 2015;Ortega-Beltran et al., 2016). In each case, recombination was not detected between members of different VCGs, but gene flow and allelic equilibrium were detected among individuals within each VCG. Each VCG also contained only a single mating-type idiomorph. In the current study, VCG YV150 differs from this trend. Although auxotroph complementation puts isolates containing MAT1-1 into the same VCG as isolates containing MAT1-2, the population genetic analyses indicate that recombination has not occurred recently between MAT1-2 and MAT1-1 populations in the field.
The current results suggest that each mating-type idiomorph of VCG YV150 has a predominantly clonal reproduction mode and that recombination between YV150 isolates with opposite mating-type is rare, if occurring at all. In predominantly clonally reproducing pathogens, recombination and genetic exchange is not ruled out but is considered too rare to have a significant influence on population clonal structure Ayala, 2012, 2017). If recombination occurred between YV150 isolates with opposite mating-type idiomorph, there could have been two non-mutually exclusive scenarios (Grubisha and Cotty, 2010). The first one is that microsatellite loci would be re-arranged in the resulting progeny. The second is that new arrangements of microsatellite loci would not occur, but recombination would be noticed by detecting infiltration of haplotypes associated with MAT1-1 into MAT1-2, or vice versa. Neither of those scenarios was detected in the current study.
Significant research efforts have revealed the sexual cycle of A. flavus and other aflatoxin-producing species both in the laboratory and after field release of sclerotia produced and incubated in the laboratory (Horn et al., 2009a(Horn et al., , 2009b(Horn et al., , 2014(Horn et al., , 2016Olarte et al., 2012). Highly artificial conditions were necessary to observe low-frequency recombination events. Thus, the epidemiological significance of A. flavus recombination in nature remains unknown (Fedorova et al., 2009;Kwon-Chung and Sugui, 2009;Horn et al., 2009aHorn et al., , 2011Horn et al., , 2016Dyer and O'Gorman, 2012). Over a decade ago, it was suggested that if recombination occurred in A. flavus, such an event would be more likely to occur between isolates belonging to the same VCG (Ehrlich et al., 2007;Fedorova et al., 2009). Sexual reproduction was not detected in A. parasiticus when members of a VCG with opposite mating-type were paired under artificial conditions (Horn et al., 2009b). Thus, there are several lines of evidence that indicate sexual recombination is rare in A. flavus including in vitro pairings (Horn et al., 2016), observations of undetectable recombination among VCGs (Grubisha and Cotty, 2010) and linkage disequilibria in several populations (Grubisha and Cotty, 2010;Islam et al., 2018; Table 3).
The MAT1-1 and MAT1-2 populations differ in geographic distribution. MAT1-2 isolates were not found in Mexico. Factors favouring dominance of MAT1-1 isolates in Mexico are unknown, although all were associated with maize production. MAT1-1 and MAT1-2 are sympatric in both Arizona and Texas (Table 2). MAT1-2 was isolated only from cotton fields in the United States. Cropping systems and rotations influence compositions of A. flavus populations (Horn and Greene, 1995;Jaime-Garcia and Cotty, 2010) and isolates belonging to different VCGs differ in abilities to colonize diverse substrates Cotty, 2010, 2013). Planting of cotton or maize may thus influence frequencies in the environment of isolates belonging to the two populations of YV150. Around 120 000 ha of cotton are planted annually in Mexico (SIACON, 2010;FAO, 2016), and most of the production occurs in Tamaulipas and Sinaloa. Comparison of A. flavus communities associated with maize and cottonseed in Tamaulipas and Sinaloa may allow rigorous testing of influences of crops on the prevalence of isolates of the two YV150 populations.
In the current study, clonal reproduction and isolation similar to that seen between VCGs were detected between isolates harbouring different idiomorphs in the same VCG. Genetic isolation exists among sympatric VCGs co-distributed across large regions of Arizona and Texas (Grubisha and Cotty, 2010) and in VCG YV36 to which the atoxigenic biocontrol agent Aspergillus flavus AF36 belongs (Grubisha and Cotty, 2015;Ortega-Beltran et al., 2016) and this is also the case for the MAT1-1 and MAT1-2 populations of YV150.

Experimental procedures
Aspergillus flavus isolates and assignment into VCG YV150. YV150 is one of the first VCGs characterized in our laboratory and has been subjected to several studies, both published and unpublished. After completing the characterization of communities associated with maize in Sonora at the VCG level (Ortega-Beltran and Cotty, 2018), we investigated whether VCGs from the collection in Arizona (which include VCGs from several areas in the United States) were also present in Sonora. The results of that investigation revealed that YV150 and SON003 are the same VCG. There were over a hundred thousand A. flavus isolates examined during the period in which the isolates were collected, 1988-2008. Eighty-two isolates belonging to A. flavus VCG YV150 from different years, substrates, and geographic origins were used in the current study (Table 1; Fig. 1). Isolates from Sonoran maize field soils collected in 2006, 2007 and 2008 (46 isolates) (Ortega-Beltran and Cotty, 2018) and one isolate from the United States were previously reported (Ehrlich and Cotty, 2004;Mehl and Cotty, 2010). The remainder of YV150 isolates were collected from crop and soil samples from Mexico (two isolates) and the United States (33 isolates) using previously described methods (Cotty, 1997;Probst et al., 2010). Soil and maize samples from Mexico were imported into the United States under an APHIS Permit to Move Live Plant Pests and Noxious Weeds, USDA-APHIS-PPQ 526, and maintained at the USDA-ARS Aflatoxin Research Laboratory in the School of Plant Sciences, University of Arizona, Tucson. A single isolate from maize kernels was obtained from a native maize landrace accession collected in 2004. Isolates from Mexico, from both maize and maize field soils, and from Arizona cotton field soils from 2006 were recovered by dilution plate technique on modified rose Bengal agar (Cotty, 1994). Cottonseed isolates from 1991 through 1993 were obtained by surface-disinfecting seeds with 95% ethanol and plating directly into 5-2 agar (Cotty, 1989). The assignment of isolates into VCG YV150 was conducted using nit mutants in VCA on complementation agar (Cotty and Taylor, 2003) following previously described protocols (Ortega-Beltran and Cotty, 2018). In 2006, a total of 27 maize fields within four agro-ecosystems encompassing 200 km 2 and at elevations ranging from 6 to over 2100 m above sea level were sampled, and YV150 was detected in each maize field (Ortega-Beltran and . From 26 fields, we arbitrarily selected one YV150 isolate. From the remaining field, we selected all 11 YV150 detected in that field. All evaluated YV150 isolates produce aflatoxins.

Microsatellite genotyping
Isolates were genotyped using 24 microsatellite markers for A. flavus (Grubisha and Cotty, 2009). DNA isolation, multiplex-PCR, and microsatellite genotyping were conducted as previously described Cotty, 2009, 2010;Callicott and Cotty, 2015). In order to assess the consistency of the data, over 20% of isolates were subjected to at least three independent PCR and genotyping assays for all loci.

Population genetic analyses
Isolates were placed into eight a priori populations based on year of isolation and geographic origin (Table 1). Haplotypes, allele frequencies and evenness among isolates with MAT1-1 and MAT1-2 were assessed independently with GENODIVE 2.0b11 (Meirmans and Van Tienderen, 2004). Evenness indicates haplotype distribution within each population.
A Cavalli-Sforza chord distance matrix obtained with GENODIVE was used to generate a phylogenetic tree using SplitsTree 4.8 (Huson and Bryant, 2006). Recombination and genetic distance among YV150 haplotypes were evaluated with the neighbour-net approach and the phi-test of SplitsTree. This approach uses a jackknife strategy and repeats the phi-test after each individual is removed and subsequently replaced. A posteriori populations were then determined. The covariance matrix of the Φ PT distance matrix was used to perform a PCoA in GENALEX (Peakall and Smouse, 2006) and a posteriori populations were confirmed.
Partitioning of genetic variance was calculated considering isolates with MAT1-1 as one population and isolates with MAT1-2 as a different population with a hierarchical AMOVA using Arlequin 3.5 (Excoffier and Lishcer, 2010). Significance was based on 10 200 permutations. Loci AF64 and AF48 were removed from the MAT1-2 population because of allelic variability (eight and 14 alleles respectively). Increased variability may be the result of loci under selection (Grubisha and Cotty, 2010). No loci were removed for the MAT1-1 population since the maximum number of alleles in any given locus was six.
Linkage disequilibrium between pairs of polymorphic loci of a posteriori populations was estimated using MULTILOCUS 1.3b (Agapow and Burt, 2001). Statistical significance was determined by 1000 permutations. The most variable loci within each a posteriori population were excluded. Linkage disequilibrium was estimated using the index of association ( r d ) standardized for the included loci. Significance was based on 1000 randomizations and compared the observed value of r d to that of expected under the null hypothesis of random mating (Agapow and Burt, 2001).
Genetic structure was assessed with Bayesian clustering program Structure 2.2.3 (Pritchard et al., 2000) to define genetic groups within VCG YV150. Haplotypes were assigned to K populations using the admixture model and default parameters. Markov Chain Monte Carlo (MCMC) simulations were run for K = 1-8 (eight a priori populations; Table 1). Simulations (20 total) were run with a burn-in length of 100 000 MCMC generations followed by 1 million MCMC iterations for each K. The optimal number of populations was obtained using Structure Harvester (Earl and VonHoldt, 2012), which calculates the rate of change in the log probability of data between successive runs of K (Evanno et al., 2005).
A second phylogenetic tree was generated by assessing recombination and genetic distance between YV150 haplotypes and haplotypes of VCGs OD02, MR17, CRG136, (Grubisha and Cotty, 2010) and YV36 (Grubisha and Cotty, 2015) (including the haplotype of AF36, the active ingredient fungus of the aflatoxin biocontrol product Aspergillus flavus AF36 Prevail®). A Cavalli-Sforza chord distance matrix obtained with GEN-ODIVE was used to generate a phylogenetic tree using SplitsTree as above.

Diversity indices
Shannon-Wiener diversity index (H) was calculated for both haplotypes and isolates of diverse YV150 populations as follows: (i) all except those found in Sonora soil 2006, (ii) those found in Sonora soil 2006, (iii) those found in Arizona soil 2006, and (iv) those found in Sonora soil 2007 and Sonora soil 2008. The formula used was H = − P S i = 1 Pi × lnPi where Pi is the proportion for the ith haplotype and S is the total number of haplotypes in YV150 detected in the current study (Begon et al., 1996). The same formula was used to calculate H for isolates but in this case, Pi is the proportion for the ith isolate and S is the total number of isolates of YV150 used in the current study.