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Article

Frequency of Outcrossing and Isolation Distance in Faba Beans (Vicia faba L.)

by
Kedar N. Adhikari
1,*,
Lucy Burrows
1,
Abdus Sadeque
1,
Christopher Chung
2,
Brian Cullis
2 and
Richard Trethowan
1
1
Plant Breeding Institute, School of Life and Environmental Science, The University of Sydney, Narrabri, NSW 2390, Australia
2
Centre for Biometrics and Data Science for Sustainable Primary Industries, National Institute for Applied Statistics Research Australia, The University of Wollongong, Wollongong, NSW 2522, Australia
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(7), 1893; https://doi.org/10.3390/agronomy13071893
Submission received: 29 May 2023 / Revised: 30 June 2023 / Accepted: 10 July 2023 / Published: 17 July 2023
(This article belongs to the Section Crop Breeding and Genetics)
Browse Figures
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Abstract

:
Faba beans (Vicia faba L.) constitute a partially outcrossing species requiring an isolation distance to maintain genetic purity when more than one variety is grown in field conditions. This information is crucial for seed growers and faba bean breeders. A study was conducted at the University of Sydney’s Plant Breeding Institute, Narrabri, over two years to examine the extent of natural outcrossing using a creamy white flower characteristic as a morphological marker, which is controlled by a single recessive gene. The white-flowered genotype (IX225c) was grown in paired rows of 150 m length in four directions from a central 480 m2 plot of the normal flowered genotype PBA Warda. A beehive was placed in the central plot at the flowering time and natural pollination was allowed. At maturity, seed samples were taken from the white-flowered genotype at designated intervals along each axis and 100 seeds from each sample were grown in the glasshouse/birdcage to the 4–5 leaf stage and the proportion of plants displaying a stipule spot pigmentation (normal flower color and spotted stipule are linked) was used to determine the percentage of outcrossing. Maximum outcrossing of 2.28% occurred where both genotypes were grown side by side (0 m) and the degree of outcrossing decreased as the distance along each axis from the central plot increased. At a 6 m distance, the outcrossing was less than 1%; however, on occasion, it increased to 1% beyond a distance of 100 m, indicating the volatile and unpredictable nature of bee flights. Distance had a major effect on outcrossing but the direction and its interaction had no effect. The results suggest that to limit outcrossing to below 0.5%, a distance of more than 150 m between plots of different faba beans cultivars would be required. It also indicated that Australian faba bean genotypes are mostly self-fertile and a relatively narrow isolation distance will ensure self-fertilization in seed production and breeding programs.

1. Introduction

Faba beans (Vicia faba L.) are believed to have originated in the Middle East and have been grown extensively around the world since the pre-historic period, spreading through Europe, North Africa and Central Asia [1,2,3]. Over 2000 years ago, the faba bean expanded into China through traders and to South America during the Columbian period, while it was introduced to Australia and North America only in the later part of the 20th century [2]. Faba beans are a protein rich grain mostly consumed for food in Middle Eastern countries and also used for animal feed. Globally, its annual production is above 5.5 million tons, of which the Australian share is over 0.5 million tons from 269,000 ha [4].
Faba beans are a crop with a mixed mating system and fertilization can occur through self-pollination as well as cross-pollination. In fact, pollination of faba beans can occur in three ways: auto-pollination (without any aid), pollination by insects with the pollen from the same flower through flower tripping (self-pollination) and pollination by pollen transfer from one flower to another by pollinators (cross-pollination) [5,6]. In such species, outcrossing occurs when two or more varieties are grown in proximity in field conditions. It is, therefore, necessary to grow faba beans in isolation to maintain the genetic purity of varieties [7]. However, limited information is available on the extent of natural outcrossing and the isolation distance required to prevent the outcrossing of faba beans. The rate of outcrossing in faba beans varies from 7–82% depending upon the genotype, environment and planting arrangement [8]. Pope and Bond [9] found that outcrossing decreased with increasing distance from the pollen source, ranging from 17% at 0.9 m, 1.25% at 92 m and 0.59% at 184 m. Similarly, Bond and Poulsen [10] reported an outcrossing of 4–84% and Gottschalk [11] reported 40%. Suso et al. [12] studied faba bean outcrossing in two locations and reported 65% in Cordoba (Spain) and 33% in Rennes (France). These large differences in the outcrossing rate apparently depend on genetic and environmental factors as well as methods of measurement. In insect-pollinated crops, pollinator abundance, diversity, activity and foraging behavior represent the major ecological factors potentially affecting the outcrossing rate [13].
The isolation of lines to maintain genetic purity is critical to breeding programs and seed production companies because a minimum distance must be maintained between lines in an open field to avoid outcrossing [14,15]. To determine the level of outcrossing, it is crucial to define the isolation distance between two varieties [16]. Cross-pollination in faba beans occurs through insects such as honeybees (Apis millifera) and bumblebees (Bombus terrestris) in Europe. In northern Africa, solitary bees (Eucera pulveracea), bumblebees and honeybees are the main pollinators [17]. However, this process is facilitated by honeybees alone in Australia as there are no other effective insect pollinators including bumblebees [18]. As pollinators are the main source of pollen movement, pollinator foraging activities influence outcrossing rates [19]. Conflicting reports are available on the role of honeybees in Australia. Neither pollination nor fertilization was a limiting factor on seed yield in faba beans in northern New South Wales [20] but a yield increase was reported by placing beehives in the vicinity of crops in South Australia [21]. In another study conducted in the Riverina (NSW, Australia), Sommerville [22] found a 25% increase in yield using honeybees and recommended two hives/ha to maximize the yield. This contrasts with an earlier report in southern Australia that faba beans flower in late winter and early summer when feral honeybees are actively seeking pollen, hence the cost of placing bee hives can be avoided [23]. Bishop and Nakagawa [24] also reported a reduction in faba bean yield of 33% without bee pollination. Bishop et al. [25] suggested that pollination of faba beans in the field depends on pollinator populations and environmental conditions including weather. It has been observed that the European source of germplasm is largely outcrossing, whereas the ICARDA and the Australian germplasm is largely self-pollinating [26]. Earlier reports also indicated that outcrossing differs from genotype to genotype [27,28].
The level of outcrossing in many crops can be characterized by environmental variation in different growing regions [8,12]. Therefore, specific isolation distances need to be developed for specific regions, but no such information is available for faba beans in Australia. The results from previous research of outcrossing in faba beans have limited geographic applicability to other growing areas worldwide [29]. The majority of such research was conducted in Europe and may not be relevant to Australian conditions due to differences in pollinators and genotypes.
This experiment was, therefore, conducted to assess the extent of natural outcrossing when two varieties are grown side by side and to estimate the impact of distance on outcrossing to identify an effective isolation distance for producing genetically pure seeds. Information was also sought on whether the wind direction had any role in outcrossing.

2. Materials and Methods

2.1. Plant Material

Two phenotypically distinct faba bean genotypes, PBA Warda and IX225C displaying different flower colors of normal and white, respectively, were chosen for the study (Figure 1). These genotypes were maintained in the University of Sydney’s faba bean breeding program at Narrabri, NSW, Australia through selfing for several generations and were assumed to be homozygous for their respective flower color. Genotype IX225C has a creamy white flower (Figure 2a) which is controlled by either one of the two complementary recessive genes (zt1 or zt2) [30,31] and PBA Warda has normal flower color with a black dot on the wing petal (Figure 2b). The latter trait is linked to a stipulate spot pigmentation which is visible from the seedling stage.

2.2. Planting Arrangements and Observations

The experiment was conducted at the University of Sydney’s Plant Breeding Institute, Narrabri, NSW, Australia (30°16′26.7″ S 149°48′34.0″ E), over two seasons. The creamy white flower color from IX225C (a breeding line) was used as a phenotypic marker in this study. In both years, PBA Warda with a normal flower color (a popular faba bean variety for this region) was planted in a central plot of 24 m × 20 m using a tine seeder (Figure 1). Two 150 m rows at 50 cm spacing were sown to IX225C in four directions perpendicular to the central plot. The remaining field area was filled with field pea (Pisum sativum L.) in the first year and chickpea (Cicer arietinum L.) in the second year. Both crops are self-pollinated and do not attract pollinators. A beehive was placed in the central plot of faba beans to facilitate bee activity for pollination throughout the flowering period. Flowering occurred 4–5 days earlier in IX225C than PBA Warda; however, faba beans are an indeterminate crop and flowering persists for at least six weeks giving ample time for cross pollination.
At maturity, seed samples were taken from the four perpendicular arms at specified distances from the central plot. Samples were initially taken from each arm at 0, 1, 2 and 3 m and then every 3 m for the first 30 m, every 5 m from 30–60 m and every 10 m thereafter. Four to five plants were harvested at each distance point in each arm from both rows and the seed bulked to provide at least 200 seeds per sample.
After harvesting, 100 seeds from each sample were grown in metal trays covered with potting mix in a glasshouse over winter and the remaining seeds were kept aside. The seedlings were grown to the 4–5 leaf stage or until a stipule spot pigmentation (SSP), an early-morphological marker for a colored flower, was distinctly visible (Figure 3). SSP is an extra floral nectary located at the base of each faba bean leaf to attract pollinators [32]. This character is found in commonly grown faba bean genotypes and is linked to the normal flower color with a distinct black spot on its winged petal [33]. As the seed samples were taken from IX225C with the creamy white flower color, any progenies displaying SSP in the sample must have been outcrossed with pollen from the central PBA Warda plot; those plants without SSP were, therefore, regarded as self-pollinated. Although SSP is linked to normal flower color with a distinct black spot, delinking of these traits was reported in some genotypes [34]. To confirm whether the traits were still linked in the lines in this experiment, the entire 28 samples of the west arm along with two parents were grown to flowering and recorded for SSP and their flower color. There was a 100% match of SSP with the flower color, thus confirming the traits were still linked in the genotype of our study. Therefore, all the remaining samples were grown only to the 4–5 leaf stage until the SSP was clearly visible. The seeds from each sample were grown under natural light at an ambient temperature. Not all 100 seeds in each sample emerged or grew successfully and the outcrossing percentage was determined based on the available plants. However, if the sample size was less than 50 plants, more seeds were taken from the reserve seed lot and planted to achieve approximately 100 plants per sample.

2.3. Statistical Analysis

The experiment is considered as an observational study since the sampling from a population at a fixed point has an independent variable that is not manipulated by the researcher. In this context, the direction is used to capture the effect of wind on outcrossing which is naturally occurring. As a geometric entity with the use of distances, we can view it as a spatial process. Hence, it is appropriate to view for each of the two years a realization of a spatial process: namely the pollination of a faba bean flower by honeybees. An outcrossing event occurs when a flower in extended arms is pollinated by pollen originating from the central plot (Figure 1). Each pollination event can be recorded in so-called polar coordinates, (r, θ), where r is the Euclidean distance from a reference point, being the edge of the central plot where two genotypes intersect at each arm and θ is the angle set to 0 for the easterly direction and measured in radians. For example, 90 deg = π/2 radians = North. The sampling grid used for both years consisted of 112 (28 × 4) combinations of (r, θ). To address the aims of the experiment, we sought to model the probability of a pollination event as a function of r and θ, the latter constrained by the limited nature of the sampling grid. Notwithstanding this, empirical evidence for an anisotropic spatial process was provided by fitting the following generalized linear mixed model (GLMM), using the symbolic notation of Wilkinson and Rogers [35],
g(µ) = 1 + r + Theta + r:Theta + spl(r) + spl(r):Theta+
Year + Year:Direction + Year:Direction:Distance
where g(·) is the link function, set to the logit link; Theta, Year, Direction and Distance which are categorical factors with 4, 2, 4 and 28 levels, respectively, and underlined terms are fitted as random effects. The two terms Theta and Direction are completely aliased but we include them as separate terms to distinguish between anatomical and plot factors. The inclusion of the spl(·) terms models the relationship of the logit probability of pollination using a cubic smoothing spline but is functionally dependent on Theta, see Verbyla et al. [36] for details. Assuming all random effects follow a Gaussian distribution, we fit (1) using Penalized Quasi-Likelihood (PQL) as described by Breslow and Clayton [37], in ASReml-R (Butler et al. [38]).

3. Results

A total of 14,405 seeds were evaluated for outcrossing over the two years from 203 samples, each capturing the outcrossing frequency for a unique year by a combination of θ by r. The outcrossing occurred throughout the experimental plot at different levels. There were some outliers and, to aid in the fitting process, three extreme outliers with outcrossing percentages of 32.64%, 31.11% and 23.33% were removed. The two largest outliers had low germination rates (49 and 45%) which supported their removal from the analysis. A further eight samples did not have sufficient seed. Although they were tested, being low in number to determine the outcrossing rate, these samples were also removed from the analysis. Certain additional points were flagged as possible outliers but were kept in the analysis with caution. They had outcrossing rates of 8.33% (2016, South, 130 m) and 5.08% (2016, South, 80 m).
Figure 4 presents a scatter plot and smoothed response of the empirical logit [39] of outcrossing versus the Euclidean distance for each direction. This plot suggests that there is a strong non-linear relationship between outcrossing and Euclidean distance but that there appears to be little evidence for this to be influenced by direction. There are also several instances of outcrossing frequencies at larger distances, indicating the unpredictable nature of this phenomenon.
Table 1 presents the PQL estimates of the variance components of the random model terms that realize variation in the response due to the model terms. These are also expressed as a percentage of the total variance on the underlying (logistic) scale. One of the larger components (19.58) illustrates the strength of the non-linear relationship between an outcrossing event and Euclidean distance. Table 2 presents a summary of the strength of the anatomical terms in the fit of GLMM. There is a significant effect of distance (r) (p = 0.031) but there is little evidence of anisotropy in the spatial process; in other words, the direction (Theta) was not significant (p = 0.277) and neither was the interaction of distance (r) or direction (Theta). However, these results need to be considered with caution given the minimal sampling of directions and the level of unexplained variation in the data (see Table 1). While fitting the GLMM, the values 25.9 and 52.59 are associated with the non-linearity of the relationship between logit outcrossing and distance, respectively.
Figure 5 presents fitted cubic smoothing splines of the relationship between the probability of outcrossing against the Euclidean distance for each level of Theta. Although the terms associated with the interaction between r (Distance) and Theta (Direction) are not statistically significant (Table 2), it is interesting and useful to present the cubic smoothing splines for each direction (Figure 5) as the easterly direction appears quite different (low) from the other three directions. Hence, a conservative approach to determining a safe exclusion distance would be to use the relationship between outcrossing and distance for the westerly direction as it has the highest BLUP (Best Linear Unbiased Predictor) for out-crossing, or, the mean of the north, west and south directions, ignoring the easterly direction which has the flattest curve. If we take the outcrossing means for each direction from the model, then outcrossing rates of N: 0.482%, E: 0.209%, W: 0.643% and S: 0.498% are observed. A simple average ignoring the east direction gives an outcrossing value of 0.541%. Alternatively, as the northern curve has the highest outcrossing at higher distances, a conservative approach would be to find a distance where the expected out-crossing drops below 0.5%. In this case, 118 m between different faba beans plots would be recommended.
Figure 6 presents the same plot as Figure 5 but with added confidence intervals depicting the range of estimates for the results. The width of the confidence interval is due to the high degree of variability in the data, reinforcing the suggestion of a conservative approach for a safe exclusion distance. The curves suggest that the majority of outcrossing occurs at low values of r, i.e., closer to the pollen source with the humps indicating that, on occasion, wind or other factors may cause bees to fly at longer distances. The west direction has a slightly higher predicted outcrossing than the other directions.

3.1. Distance

The outcrossing occurred in all directions in the experimental field with higher rates closer to the central plot. The highest outcrossing of 2.28% was found in samples at zero m from the pollen source, i.e., when two genotypes were grown side by side and the proportion decreased as the distance increased (Figure 7). However, less than 1% of outcrossing persisted over the entire distance of 150 m. In this experiment, pollen flow was monitored only one way, i.e., from the central plot of PBA Warda to extended arms with the genotype IX225c (Figure 2). The highest outcrossing frequency in individual samples was up to 8% at 18 m from the pollen source in one year at the western side; yet at 15 m and 21 m on the same direction, the frequencies were 3% and 2%, respectively. The magnitude of outcrossing was randomly fluctuating from 0 to 8% with no clear pattern but it was in a decreasing trend as the distance increased from the pollen source. Within 3 m from the pollen source, outcrossing decreased to 2% and it was about 1.6% at 18 m after which it fluctuated between 0 and 1% (Figure 7). There was 1% outcrossing at 130 m and it persisted to 0.3% at the edge of the experiment, at 150 m, while it was almost zero at 40, 50, 60, 120 and 140 m. When two genotypes were grown side by side, i.e., at 0 m, the outcrossing rate ranged from 0–6% in different directions. It ranged from 2.41–4.81% in the west, north and south and none in the east in one year; yet in the second year, it was none in the west, north and south but 5.97% in the east.

3.2. Wind Speed and Direction

Wind direction and speed were obtained from the meteorological station at Narrabri West Post Office located about 11 km from the experimental plots. Flowering began towards the end of June and reached a peak throughout July. Therefore, more emphasis was given to the July wind patterns. At 9 a.m., the dominant wind direction was from the east; the wind was generally calm and 17–24% of the time the wind speed was less than 20 km/h. The other dominant wind was from the southeast [40]. However, this changed significantly in the afternoon (Figure 8). At 3 p.m., the dominant wind was from the west followed by the northwest and southwest. Wind patterns for June and August were similar to July and not presented here.

4. Discussion

The degree of outcrossing decreased with increasing distance from the pollen source. This is similar to reports from other outcrossing experiments in different crops [9,10,11,14,15]. Prior to these studies, it was assumed the honeybees would fly in any direction to gather pollen and nectar in the plot. Palmer et al. [41] suggested that outcrossing may be restricted by behavioral patterns of pollinators when selecting and visiting flowers. However, the current result clearly shows that distance was the major factor in the distribution and extent of outcrossing. The direction, although not significant, would have played a minor role in outcrossing. The bulk of flowering in the field trial occurred in mid-July at a time when the average wind direction was east and south-east at speeds up to 30 km/h and 20 km/h, respectively (Figure 4). Almost 50% of the wind assessments during July were in both the east and south-east directions. These prevailing wind directions and speeds were also observed in June and August. Based on the prevailing wind, we expected more outcrossing in the west and north-western plots than in the east. This was observed: the maximum outcrossing was recorded on the western and southern sides and the least on the eastern side. Although outcrossing was solely driven by honeybees, the wind or sun direction could have played a role by altering bees’ flight paths.
Various factors influencing outcrossing were reported in the past including environmental variables, such as location and temperature, and the activity and behaviors of pollinators [12,15]. Although the effect of wind on pollinators and the consequences of outcrossing has not been widely researched, the physical challenges wind places on bees should influence outcrossing. Wind may not be a carrier of pollen as faba bean pollen grains are sticky and heavy [32]. Faba bean pollen size ranges from 27–38 μm depending upon genotypes [42] similar to another outcrossing species Brassica with 30–40 µm [43].
Honeybees use the sun as a compass for flight: even when the sun is hidden by a cloud, bees can correctly find the position of the sun from the pattern of polarized light [44,45]. It was further confirmed by Evangelista et al. [46] that bees can find their directional information from the sun’s polarized light. Australia is situated in the southern hemisphere and during winter months, the sun sits longer on the western horizon and faba bean flowers become more receptive after mid-day. It is speculated that bees are swept by the current of a strong north-westerly wind. Larger activity in the north can be explained by calm days where the bees are attracted by the warmth of the sun’s rays from the north. Large amounts of activity in the westerly direction could be due to easterly morning winds. Honeybees can potentially gain more sunlight and extra hours for foraging to the western side of their hives. This may explain why more outcrossing occurred in the western direction in both years. However, the direction was not significant in this study and more or less directional outcrossing makes no difference.
Wind presents a highly variable and physical challenge to the flight stability of bees. Drag forces on the body and wings increase in windy conditions, decreasing the overall control of flight movement [47]. A study of bumblebees (Bombus sp.) found flight approach paths changed from multidirectional to unidirectional during windy conditions, as well as limiting the turning angles for landing on flowers [47]. High wind speeds significantly hampered honeybee flights; they visited fewer flowers and were reluctant to take off [48]. There are no bumblebees present in Australia and pollination of faba beans is explicitly performed by honeybees. Honeybees prefer foraging in a single species and the same row and do not take long flights providing sufficient nectar and pollen are available [49], thus restricting outcrossing.
Earlier, it was recommended that faba bean crops grown for seed should be at least 500 m away from any other faba bean crop to prevent outcrossing [2]. However, the current study, based on field experiments over two years, has consistently shown that outcrossing is extremely low at these distances. The maximum outcrossing found at a single location was only 8% and the mean outcrossing was less than 3% when two genotypes were grown side by side. It decreased significantly with distance and at 18 m was less than 1%, although this low level persisted out to 150 m from the pollen source.
Similarly to the majority of outcrossing studies, particularly of faba beans, the distance was a major factor in this study. Although the outcrossing occurred in the entire field in this experiment, it was less than 1% and there was a clear trend of decreased outcrossing with increased distance from the pollen source. A low level of outcrossing (0.3%) was found even at 150 m from the pollen source, indicating possible outcrossing at even larger distances. However, this is a very low frequency and in a cross-pollinated crops, such as faba beans, this degree of outcrossing should be permissible.
Some studies showed that outcrossing persists over a range of distances; however, at intermittent frequencies [50]. This is consistent with the current results as the outcrossing rate occurs intermittently with no clear pattern. Taber III [50] suggests that intermittent frequencies are linked to the crop orientation and pollinator patterns and behaviors.

4.1. Why Was Outcrossing Low?

In earlier studies, the outcrossing of faba beans was reported to be as high as 84% when two genotypes were grown side by side and it decreased with the increasing distance [9,10,11]. However, in the current study, the highest magnitude was only 6% and this decreased sharply to less than 1% within 6 m of the pollen source. We explored possible reasons why observed outcrossing was low in this study.

4.1.1. Floral Synchrony

Various studies concluded that floral synchrony affects the level of outcrossing. Floral synchronization between two varieties is essential to maximize pollination and therefore outcrossing proportions [51]. The timing and duration at which flowering occurs influence the synchronicity and the flowering overlap [52]. A study of outcrossing in sorghum found high rates of outcrossing when floral synchrony was high [51]. In conjunction with synchronicity, another important factor is the floral display size [53]. The aggregation of the entire floral display, particularly the number of open flowers, causes variation in the visitation of pollinators, and therefore outcrossing [41]. Generally, faba beans display numerous flowers simultaneously in order to attract pollinators. High inflorescence numbers, rather than the quantity of nectar production, may be selected by pollinators, thus influencing outcrossing [41]. Since both faba bean genotypes had a similar flowering time and kept flowering simultaneously for more than a month, the synchronization of flowering should not have been a problem in this study.

4.1.2. Density of Pollinators

The management of bees during trials represents another understudied aspect of pollinator behavior. Sommerville [22] identified that the placement of hives and temperature influenced pollinator activities: he observed that bee activity increased in elevated and warm areas leading to a 25% yield increase. Later studies also reported that bee activities increased at elevated temperatures leading to a higher rate of outcrossing [6]. Although this study did not estimate yield, hive placement can play a significant role in bees’ flight paths. A stocking rate of two hives/ha is considered adequate for faba beans [22]. The current experiment had an area of over 10 ha and had only one hive. It might be argued that the distance required was too extensive and outcrossing was therefore restricted to shorter distances. However, this large area was not entirely sown to faba beans. When the actual area of faba bean cultivation was considered, only 1080 m2 or 1/10th of a hectare was sown to faba beans. The remaining area was filled with field peas in the first year and chickpeas in the second year, both of which do not attract honeybees as they are fully self-pollinated crops. Thus, the density of honeybees might not be the cause of the low outcrossing observed in this study. Furthermore, natural pollination in the field is favored in Australian conditions as the majority of flowering occurs towards the end of winter, corresponding to when honeybees are in search of pollen [21]. Therefore, the beehive number is unlikely to have influenced outcrossing significantly as enough bees come to forage in the field naturally.

4.1.3. Genotypic Differences

Previous studies on outcrossing were mainly from Europe where auto fertility in the germplasm is low. Due to low auto fertility, European faba beans experience high rates of outcrossing while the Australian-bred lines show high rates of auto fertility [26]. A high rate of auto fertility was reported in Middle Eastern germplasm [28] which has the ICARDA source. The Australian germplasm is mainly derived from the ICARDA source, and hence we expect a high rate of auto-fertility. Both the genotypes studied were developed in Australia and both show very high rates of auto fertility as they were grown in bee-proof screenhouses for several generations and no sterility was observed. In Australia, all early segregating lines in the breeding program are grown in bee-proof enclosures without any pollination aid. This might have indirectly selected for auto fertility in Australian bred germplasm. This is in contrary to European lines which do not set pods without manual tripping when grown in the absence of pollinators.

4.1.4. Plant Density

Although the experimental area was large, there were only two rows of faba beans planted in four directions from the central plot where a beehive was located. Due to the low number of flowers in this area, bees might have restricted foraging to the central plot. Bees were observed departing from a plant when the perceived return is low, leading to fewer flowers subsequently visited, ultimately limiting the amount of pollen transferred between varieties. Additionally, the majority of the pollen grains are deposited on the initial flowers during a trip [54]. This implies that there may not have been enough plants to attract bees in these extended rows as there were field peas in one year and chickpeas in the second year in the remaining area. Both are self-pollinated crops and are not attractive to bees.

4.2. Future Studies

As outcrossing was found to occur up to the edge of the experimental field (150 m), future experimental designs will need to increase the distance from the origin to establish an outer limit to outcrossing. Instead of planting only two rows in extended arms, the whole area can be planted with the creamy white flowering genotype or some other genotype with a phenotypic marker and more samples taken can be taken throughout the area. However, conducting such a large-scale experiment will require significantly more resources than used in this study. Furthermore, increasing the pollinator density and better monitoring of their activities will provide a better understanding of how far bees travel while foraging and their flying patterns.

5. Conclusions

The findings of this study provide guidance on the isolation distance that different varieties of faba bean should be grown in to avoid outcrossing. It was expected that the bees would fly in any direction to pollinate the faba beans and that distance would be the major factor. The results were as expected but the lower frequency of outcrossing than expected can mostly be attributed to the high rate of auto fertility in the chosen germplasm. Despite the exclusivity of bees as the pollinators, wind would also have a role in pollination as it may influence bee flight paths and behaviors. The faba bean is an outcrossing species and a certain degree of outcrossing is expected and permissible in faba beans. Since less than 1% outcrossing was observed at 150 m from the pollen source, it can be assumed that a safe isolation distance for growing faba bean seeds need not be more than 200 m between varieties. Further studies of larger experimental fields and better monitoring of wind and pollinator activities will further improve our understanding of outcrossing in faba beans.

Author Contributions

Conceptualization, K.N.A.; Methodology, K.N.A.; Software, B.C.; Formal analysis, C.C. and B.C.; Investigation, K.N.A., L.B. and A.S.; Data curation, A.S.; Writing – original draft, L.B., K.N.A. and A.S.; Writing – review & editing, K.N.A. and R.T.; Supervision, K.N.A. and R.T.; Project administration, K.N.A.; Funding acquisition, K.N.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Grains Research and Development Corporation (grant number: UA00163).

Data Availability Statement

The data are available from the University of Sydney’s research database and can be made available by contacting the main and corresponding author (KNA).

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Field trial layout for determining the outcrossing rate in faba beans using genotypes viz., PBA Warda (normal flower) and IX225c (white flower) at the University of Sydney’s Plant Breeding Institute, Narrabri, NSW, Australia.
Figure 1. Field trial layout for determining the outcrossing rate in faba beans using genotypes viz., PBA Warda (normal flower) and IX225c (white flower) at the University of Sydney’s Plant Breeding Institute, Narrabri, NSW, Australia.
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Figure 2. Faba bean flowers with a creamy white color from IX225C (a) and normal flowers with a distinct black dot on the wing petal from PBA Warda (b).
Figure 2. Faba bean flowers with a creamy white color from IX225C (a) and normal flowers with a distinct black dot on the wing petal from PBA Warda (b).
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Figure 3. Faba bean flowers with a creamy white flower and no stipule pigmentation (a) and flowers with a normal flower color with a black dot on the wing petal and stipule pigmentation (b). Yellow arrow shows the stipule on both pictures without and with the stipule, respectively.
Figure 3. Faba bean flowers with a creamy white flower and no stipule pigmentation (a) and flowers with a normal flower color with a black dot on the wing petal and stipule pigmentation (b). Yellow arrow shows the stipule on both pictures without and with the stipule, respectively.
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Figure 4. Scatter plot and smooth response of the empirical logit of the probability of outcrossing versus Euclidean distance for each direction.
Figure 4. Scatter plot and smooth response of the empirical logit of the probability of outcrossing versus Euclidean distance for each direction.
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Figure 5. Fitted cubic smoothing splines of the relationship between the probability of outcrossing against Euclidean distance for each level of Theta (direction).
Figure 5. Fitted cubic smoothing splines of the relationship between the probability of outcrossing against Euclidean distance for each level of Theta (direction).
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Figure 6. Fitted cubic smoothing splines of the relationship between the probability of outcrossing pollination against Euclidean distance with confidence intervals for each level of Theta.
Figure 6. Fitted cubic smoothing splines of the relationship between the probability of outcrossing pollination against Euclidean distance with confidence intervals for each level of Theta.
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Figure 7. Mean outcrossing % after combining all directions within a distance (the data were combined because the direction was not significant).
Figure 7. Mean outcrossing % after combining all directions within a distance (the data were combined because the direction was not significant).
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Figure 8. Rose of wind direction versus wind speed in km/h at Narrabri West Post Office. Data obtained from the Bureau of Meteorology, Copyright © Commonwealth of Australia 2021.
Figure 8. Rose of wind direction versus wind speed in km/h at Narrabri West Post Office. Data obtained from the Bureau of Meteorology, Copyright © Commonwealth of Australia 2021.
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Table
Table 1. Penalized Quasi-Likelihood estimates of the variance components of the random model terms. These are also expressed as a percentage of the total variance on the underlying (logistic) scale. The polar coordinates, (r, θ), are represented by r and Theta; terms with spl were fitted as a spline term to capture non-linearity.
Table 1. Penalized Quasi-Likelihood estimates of the variance components of the random model terms. These are also expressed as a percentage of the total variance on the underlying (logistic) scale. The polar coordinates, (r, θ), are represented by r and Theta; terms with spl were fitted as a spline term to capture non-linearity.
Term#EffectsPQL (% of Total)
Site21.61
Site:Direction80.25
spl(r)2619.58
spl(r):Theta1040.27
Site:Direction:Distance12425.9
units!R-52.39
Table
Table 2. Summary of probability values for incremental Wald type pivots for fixed effects in the generalized liner mixed model.
Table 2. Summary of probability values for incremental Wald type pivots for fixed effects in the generalized liner mixed model.
TermDegree of Freedom Wald Statistic Pr (Chisq)
(Intercept)1352.290
r14.640.031
Theta33.860.277
r:Theta32.230.525
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Adhikari, K.N.; Burrows, L.; Sadeque, A.; Chung, C.; Cullis, B.; Trethowan, R. Frequency of Outcrossing and Isolation Distance in Faba Beans (Vicia faba L.). Agronomy 2023, 13, 1893. https://doi.org/10.3390/agronomy13071893

AMA Style

Adhikari KN, Burrows L, Sadeque A, Chung C, Cullis B, Trethowan R. Frequency of Outcrossing and Isolation Distance in Faba Beans (Vicia faba L.). Agronomy. 2023; 13(7):1893. https://doi.org/10.3390/agronomy13071893

Chicago/Turabian Style

Adhikari, Kedar N., Lucy Burrows, Abdus Sadeque, Christopher Chung, Brian Cullis, and Richard Trethowan. 2023. "Frequency of Outcrossing and Isolation Distance in Faba Beans (Vicia faba L.)" Agronomy 13, no. 7: 1893. https://doi.org/10.3390/agronomy13071893

APA Style

Adhikari, K. N., Burrows, L., Sadeque, A., Chung, C., Cullis, B., & Trethowan, R. (2023). Frequency of Outcrossing and Isolation Distance in Faba Beans (Vicia faba L.). Agronomy, 13(7), 1893. https://doi.org/10.3390/agronomy13071893

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