Over the past 25 years, the Louisiana Irises have emerged as an ideal system in which to ask questions regarding speciation, hybridization, and adaptation . Drawing on previous studies examining the evolution, ecology, and genetics of Irises [4, 9, 15, 26, 29], we mapped QTL for 11 traits important for pre- and post-zygotic isolation using two maps derived from reciprocal backcrosses based on co-dominant EST-SSR markers . Given the high degree of collinearity of the two maps, we have been able to compare the location of QTL across both maps for the first time. Despite the presence of multiple reproductive isolating barriers, the Louisiana Iris genome appears tolerant to introgression at multiple loci [20, 31] and many of the markers used in this study have shown evidence of transmission ratio distortion with a bias towards introgression of I. fulva alleles, while I. brevicaulis alleles are under represented .
Understanding the genetic architecture underlying traits important to reproductive isolation and hybrid fitness allows us to develop hypotheses regarding which genetic regions are important for maintaining species distinctions and which may provide a selective advantage when allowed to introgress through hybridization in nature. Here we discuss the genetic relationship among 11 traits that affect pre- and post-zygotic isolation between two closely related species, I. brevicaulis and I. fulva inferred by QTL mapped using collinear, reciprocal-backcross genetic maps.
Previous studies have shown that flowering time in natural populations of I. fulva and I. brevicaulis acts as a strong pre-zygotic isolating barrier, with only a small proportion of the latest flowering I. fulva overlapping with the earliest flowering I. brevicaulis for approximately 2 weeks in late-April and early May [19, 21]. Across both maps, we found 13 QTL for flowering time. At all eight loci detected in the BCIB population that were associated with variation in flowering time, individuals with introgressed I. fulva alleles flowered earlier than those with the I. brevicaulis allele, and conversely, for all 5 flowering time QTL detected in the BCIF population, introgression of the I. brevicaulis alleles resulted in an increase in the time to flowering. Two pairs of these QTL overlap on homologous linkage groups 4 and 13 in both backcross maps (Figure1, Table 1). These overlapping QTL suggest the potential for allelic differences at the same locus affecting flowering time between I. brevicaulis and I. fulva, although the confidence intervals for each QTL are large, encompassing many genes, leaving the possibility that each overlapping QTL may actually represent different loci affecting flowering time. Within each backcross population, several QTL from different years or treatments overlapped, indicating that there may be loci responding to specific developmental factors (e.g. first vs. second year post transplant) or environmental cues (e.g. wet vs. dry). For example, QTL detected in the dry site in both 2006 and 2007 overlap in both mapping populations (LG 1 in the BCIF population and LG 4 in the BCIB population; Figure1), suggesting that these loci may function to control flowering time in dry conditions. Another pair of overlapping QTL on linkage group eight in the BCIB mapping population were identified using data collected from both the wet and the dry plots in 2006 that may represent either a common locus responding to particular environmental conditions experienced in 2006 or, perhaps, a locus that controls variation in flowering during the first year.
Genetic studies in model systems have shown that flowering time is a complex trait responding to both endogenous and environmental cues, with loci that promote and delay flowering interacting to establish proper timing . The 2006 phenological data using a dominant Iris retroelement (IRRE) marker system was previously analyzed  and although most of the QTL that were identified in that study had effect directions consistent with what is found in this study (i.e. I. brevicaulis alleles cause later floral transition, I. fulva alleles cause earlier floral transition), several QTL with opposite effect directions (e.g. I. brevicaulis alleles that cause earlier floral transition, I. fulva alleles that cause later floral transition) were identified  that we were unable to detect in this study. This may be attributed to the fact that their study had slightly larger sample sizes and identified a greater overall number of flowering time QTL .
Flood tolerance and long-term survival
The habitat commonly associated with Louisiana Irises fluctuates dramatically both throughout the year, and year-to-year, as water levels and temperatures fluctuate. Under changing conditions, it is expected that plants will have environment dependent responses that may appear under stressful conditions (e.g. flood, drought). We evaluated data for both long-term survival in mildly fluctuating conditions as well as survival in extreme flooding conditions in the backcross mapping populations using data from a transplanted field plot that experienced standard environmental fluctuations after 3 years and another plot that experienced abnormally strong flooding. Only two survival QTL were detected: one QTL associated with variation in flood tolerance and one QTL associated with long-term survival. As would be predicted from the habitat associations of the two species [20, 33], introgression of the I. brevicaulis allele into the I. fulva genetic background at either of these loci resulted in decreased survivorship. Two QTL linked to increased survival in the BCIF mapping population that were identified in a previous study were not recovered here . As in the previous studies that analyzed both survival in the greenhouse and the flood survival data using dominant markers, no loci affecting survival were identified in the BCIB populations in this study .
The BCIB mapping population exhibits higher pollen sterility (32.3 % mean sterility; range 0.58 – 100 % sterile) relative to the BCIF population (7.56 % mean sterility; range 0.18-66.7 % sterile). These BCIB values contrast with the parents used to generate the crosses as Ib 25, If 174, and the F1 hybrid all had pollen sterility less than 10 %. We detected two QTL in the BCIB mapping population in which introgression of the heterospecific (I. fulva) allele resulted in a decrease in the proportion of sterile pollen grains and one QTL in the BCIF mapping population in which introgression of the heterospecific (I. brevicaulis) allele resulted in an increase in pollen sterility. The location of the QTL on BCIB LG 3 associated with a decrease in sterility also corresponds to a region with significant transmission ratio distortion (TRD) whereby I. fulva alleles were overrepresented in the BCIB mapping population , consistent with heterozygosity being favored in this region. The other two QTL associated with sterility do not correspond with regions exhibiting TRD. A potential explanation for the increase in fertility in the BCIB individuals associated with the introgression of either of the two I. fulva loci is that homozygosity in I. brevicaulis may have negative effects (i.e. due to inbreeding depression). Both iris species demonstrate a mixed-mating reproductive strategy, but I. brevicaulis demonstrates lower levels of inbreeding than does I. fulva[21, 34]. This pattern of mating would be predicted to result in more heterozygosity in I. brevicaulis, and indeed this species does demonstrate higher levels of heterozygosity relative to I. fulva[30, 35]. Given that a proportion of the heteroyzgosity in these species involves deleterious recessive alleles, we would predict that our crossing design would uncover more deleterious alleles in I. brevicaulis than in I. fulva. Specifically, both backcross populations would have higher levels of homozygosity than would be present in the progeny of natural outcrossing individuals, potentially revealing loci that cause sterility when homozygous . Alternatively, the fact that each backcross population was created from a different F1 individual (F12 and F13) may contribute to differential levels of sterility in the backcross populations. Interspecific incompatibilities between the species could also explain the increase in sterility in the BCIB mapping population, although evidence supporting either negative interactions between heterospecific nuclear genes or cytonuclear incompatibilities has not been found . Further crossing experiments among and within I. fulva and I. brevicaulis individuals should help to elucidate the potential causes for pollen sterility.
Growth traits affecting fitness
It is well documented that I. brevicaulis and I. fulva occur in different habitats, indicating that they vary in physiological attributes [9, 15, 19, 33]. In addition, they also differ in vegetative and floral traits that may affect fitness [26, 27, 35]. The interplay between genetic pathways controlling physiology and architecture are likely important for controlling variation in these traits . For example, the ability to generate carbon and nutrient stores may affect the ability of plants to produce more branching points, but this trait is also controlled by genes important for determining the location and frequency of branch production . While developmental pathways controlling branching (number of growth points produced, number of nodes per inflorescence), the transition to flowering (inflorescence present/absent), and fruit production all have unique downstream genetic components, these pathways are also dependent to some extent on physiological processes such as resource accumulation [38–41]. Therefore, it would be expected that some QTL for these traits would be independent and specific to the downstream pathway involved, but some affecting physiology may be shared.
Relative to the previous analysis using dominant markers (i.e. ), far fewer QTL were detected in this study (~1/3 as many for BCIB and ~1/2 as many for BCIF). Interestingly, in very few instances were overlapping QTL for the same growth trait identified across study year and habitat (BCIB LG 4, BCIF LG 5, LG 11), however in several cases, overlapping QTL for different growth traits were identified. The limited number of overlapping QTL detected in this study could potentially result from the independent genetic architecture underlying these traits. Alternatively, the small sample size for some of these traits limits our power to detect QTL, so QTL of small effect that may actually be overlapping are not detected. As such, the degree of overlap reported herein is likely a conservative estimate of QTL overlap.
In the plants examined for this study, the number of growth points produced before the flowering season increased in both backcross populations from 2006 to 2007 as the newly planted rhizomes became more established . Although the increased number of growth points translated into a higher likelihood of flowering, this increase in the number of new growth points actually resulted in a lower proportion of growth points that produced an inflorescence . This phenomenon may be explained by the co-occurrence of QTL on BCIF LG 8 where introgression of I. brevicaulis alleles results in a decrease in the number of growth points produced by weight and an increase in the proportion of growth points that produce an inflorescence (Figure1).
QTL for two traits, whether or not an inflorescence is formed (flowering) and the number of nodes produced per inflorescence (branching), are found in the same regions across several individual linkage groups. This can be seen on linkage groups BCIB LG 11 and BCIF LG 3. In both cases, the heterospecific allele is associated with decreases in the trait values. On LG 11, two QTL detected in the BCIB population are in a homologous position with a QTL detected in the BCIF population associated with variation in the number of floral nodes per inflorescence, indicating that I. brevicaulis has an allele in this region that increases inflorescence and floral node production in both genetic backgrounds. A similar pattern is seen on the end of LG 2 where QTL were detected in which the I. fulva allele is associated with increased inflorescence production (in the BCIB population) and higher numbers of floral nodes per inflorescence (in the BCIF population). It is possible that a few genetic regions controlling resource acquisition explain QTL for both of these traits.
One interesting pattern is that all of the QTL in this study associated with the number of flowers per node have negative additive effects, even those identified in both populations on homologous regions of LGs 1 and 19. Usually a second flower is produced at a node only after flowers at all other nodes have fully developed . Therefore, the trait ‘number of flowers per node’ is likely to be affected by whether or not there is enough energy to produce flowers at all nodes and then begin to produce secondary flowers at nodes. In parental populations, I. fulva produces more flowers per node  and we do not have an explanation for why all of the QTL identified in this study, independent of cross direction, have negative additive effects unless heterozygosity at each of these loci results in decreased trait values.
Overall genomic architecture of pre-zygotic isolation and hybrid fitness
Introgression of traits in a hybrid zone is dependent on the genetic architecture underlying traits affecting isolation [43, 44]. If QTL underlying traits that contribute to isolation are dispersed throughout the genome, a greater proportion of the genome will be linked to these loci, decreasing the potential for the introgression of beneficial QTL while maintaining species boundaries. However, when these factors are clustered, the likelihood of introgression across the genome is increased, especially if there are positive fitness effects of the donor allele on the recurrent parent . Additionally, introgression is influenced by the effect size and direction of alleles at clustered loci. Most of the QTL identified in this study are relatively evenly distributed across the genome. In this study, flowering time and sterility are the primary traits affecting isolation. The flowering time QTL are distributed throughout the genome, but introgression of any of the alleles identified in this study would have the effect of decreasing reproductive isolation between the parental taxa, weakening this key species isolation barrier. The effect of this shift on fitness is complicated and likely differs depending on other genetic and environmental variables. On BCIB LG 4, QTL for four different traits are located in a relatively small region. These traits include flowering time, sterility, and fruit set – all of which have the potential to affect reproductive isolation and fitness. In this region, introgression of the heterospecific allele increases the fitness of the introgressed individual in that it increases the number of nodes per inflorescence and fruit production while decreasing sterility, suggesting that this region may likely introgress across species boundaries in nature, although this introgression may have the added effect of decreasing isolation through flowering time. One region of the genome that would likely experience selection against introgression is on LG 9 where I. brevicaulis alleles in the I. fulva background increase sterility and are linked to additional QTL decreasing fitness (fewer inflorescences per growth point and fewer flowers per node). However, QTL increasing sterility are not widespread in this study, suggesting that this trait only restricts introgression in a small portion of the genome.