A Comparison of Non-Destructive Visceral Swab and Tissue Biopsy Sampling Methods for Genotyping-by-Sequencing in the Freshwater Mussel Fusconaia askewi.

Limiting harm to organisms caused by genetic sampling is an important consideration for rare species, and a number of non-destructive sampling techniques have been developed to address this issue in freshwater mussels. Two methods, visceral swabbing and tissue biopsies, have proven to be effective for DNA sampling, though it is unclear as to which method is preferable for genotyping-by-sequencing (GBS). Tissue biopsies may cause undue stress and damage to organisms, while visceral swabbing potentially reduces the chance of such harm. Our study compared the efficacy of these two DNA sampling methods for generating GBS data for the unionid freshwater mussel, the Texas pigtoe (Fusconaia askewi). Our results find both methods generate quality sequence data, though some considerations are in order. Tissue biopsies produced significantly higher DNA concentrations and larger numbers of reads when compared with swabs, though there was no significant association between starting DNA concentration and number of reads generated. Swabbing produced greater sequence depth (more reads per sequence), while tissue biopsies revealed greater coverage across the genome (at lower sequence depth). Patterns of genomic variation as characterized in principal component analyses were similar regardless of the sampling method, suggesting that the less invasive swabbing is a viable option for producing quality GBS data in these organisms.

Genetic structure in Louisiana Iris species reveals patterns of recent and historical admixture.

PREMISE: When divergent lineages come into secondary contact, reproductive isolation may be incomplete, thus providing an opportunity to investigate how speciation is manifested in the genome. The Louisiana Irises (Iris, series Hexagonae) comprise a group of three or more ecologically and reproductively divergent lineages that can produce hybrids where they come into contact. We estimated standing genetic variation to understand the current distribution of population structure in the Louisiana Irises. METHODS: We used genotyping-by-sequencing techniques to sample the genomes of Louisiana Iris species across their ranges. We sampled 20 populations (n = 632 individuals) across 11,249 loci and used Entropy and PCA models to assess population genetic data. RESULTS: We discovered evidence for interspecific gene flow in parts of the range. Our analysis revealed patterns of population structure at odds with widely accepted nominal taxonomy. We discovered undescribed hybrid populations, designated as belonging to the I. brevicaulis lineage. Iris nelsonii shared significant ancestry with only one of the purported parent species, I. fulva, evidence inconsistent with a hybrid origin. CONCLUSIONS: This study provides several key findings important to the investigation of standing genetic variation in the Louisiana Iris species complex. Compared to the other nominal species, I. brevicaulis contains a large amount of genetic diversity. In addition, we discovered a previously unknown hybrid zone between I. brevicaulis and I. hexagona along the Texas coast. Finally, our results do not support the long-standing hypothesis that I. nelsonii has mixed ancestry from three parental taxa.

Asymmetric introgression between fishes in the Red River basin of Texas is associated with variation in water quality.

When ecologically divergent taxa encounter one another, hybrid zones can form when reproductive isolation is incomplete. The location of such hybrid zones can be influenced by environmental variables, and an ecological context can provide unique insights into the mechanisms by which species diverge and are maintained. Two ecologically differentiated species of small benthic fishes, the endemic and imperiled prairie chub, Macrhybopsis australis, and the shoal chub, Macrhybopsis hyostoma, are locally sympatric within the upper Red River Basin of Texas. We integrated population genomic data and environmental data to investigate species divergence and the maintenance of species boundaries in these two species. We found evidence of advanced-generation asymmetric hybridization and introgression, with shoal chub alleles introgressing more frequently into prairie chubs than the reciprocal. Using a Bayesian Genomic Cline framework, patterns of genomic introgression were revealed to be quite heterogeneous, yet shoal chub alleles were found to have likely selectively introgressed across species boundaries significantly more often than prairie chub alleles, potentially explaining some of the observed asymmetry in hybridization. These patterns were remarkably consistent across two sampled geographic regions of hybridization. Several environmental variables were found to significantly predict individual admixture, suggesting ecological isolation might maintain species boundaries.

Effect of preservation on fish morphology over time: Implications for morphological studies.

It has long been recognized that the process of preserving biological specimens results in alterations of body shape, though detailed studies examining the degree to which morphological changes occur throughout the preservation process are lacking. We utilize geometric morphometric analyses, an increasingly common tool for examining shape variation in a wide variety of biological disciplines, to examine the effects of formalin and ethanol preservation on the body shape of 10 freshwater fish species over time: from fresh specimens to eight weeks after preservation. We found significant changes in body shape among fresh and formalin fixed specimens. Furthermore, changes in body shape continue to occur after subsequent ethanol preservation. Two fish species collected at multiple localities show significant morphological differences for a limited number of morphometric characters. However, the significance, or lack thereof, often changed inconsistently from one stage of preservation to another. We conclude that morphometric analyses would ideally be performed on fresh specimens. However, recognizing that this is not always feasible, it is important to be aware of the morphometric changes that can occur during preservation.

Integrating Bayesian genomic cline analyses and association mapping of morphological and ecological traits to dissect reproductive isolation and introgression in a Louisiana Iris hybrid zone.

Hybrid zones provide unique opportunities to examine reproductive isolation and introgression in nature. We utilized 45,384 single nucleotide polymorphism (SNP) loci to perform association mapping of 14 floral, vegetative and ecological traits that differ between Iris hexagona and Iris fulva, and to investigate, using a Bayesian genomic cline (BGC) framework, patterns of genomic introgression in a large and phenotypically diverse hybrid zone in southern Louisiana. Many loci of small effect size were consistently found to be associated with phenotypic variation across all traits, and several individual loci were revealed to influence phenotypic variation across multiple traits. Patterns of genomic introgression were quite heterogeneous throughout the Louisiana Iris genome, with I. hexagona alleles tending to be favoured over those of I. fulva. Loci that were found to have exceptional patterns of introgression were also found to be significantly associated with phenotypic variation in a small number of morphological traits. However, this was the exception rather than the rule, as most loci that were associated with morphological trait variation were not significantly associated with excess ancestry. These findings provide insights into the complexity of the genomic architecture of phenotypic differences and are a first step towards identifying loci that are associated with both trait variation and reproductive isolation in nature.

QTL mapping reveals the genetic architecture of loci affecting pre- and post-zygotic isolating barriers in Louisiana Iris.

BACKGROUND: Hybridization among Louisiana Irises has been well established and the genetic architecture of reproductive isolation is known to affect the potential for and the directionality of introgression between taxa. Here we use co-dominant markers to identify regions where QTL are located both within and between backcross maps to compare the genetic architecture of reproductive isolation and fitness traits across treatments and years. RESULTS: QTL mapping was used to elucidate the genetic architecture of reproductive isolation between Iris fulva and Iris brevicaulis. Homologous co-dominant EST-SSR markers scored in two backcross populations between I. fulva and I. brevicaulis were used to generate genetic linkage maps. These were used as the framework for mapping QTL associated with variation in 11 phenotypic traits likely responsible for reproductive isolation and fitness. QTL were dispersed throughout the genome, with the exception of one region of a single linkage group (LG) where QTL for flowering time, sterility, and fruit production clustered. In most cases, homologous QTL were not identified in both backcross populations, however, homologous QTL for flowering time, number of growth points per rhizome, number of nodes per inflorescence, and number of flowers per node were identified on several linkage groups. CONCLUSIONS: Two different traits affecting reproductive isolation, flowering time and sterility, exhibit different genetic architectures, with numerous QTL across the Iris genome controlling flowering time and fewer, less distributed QTL affecting sterility. QTL for traits affecting fitness are largely distributed across the genome with occasional overlap, especially on LG 4, where several QTL increasing fitness and decreasing sterility cluster. Given the distribution and effect direction of QTL affecting reproductive isolation and fitness, we have predicted genomic regions where introgression may be more likely to occur (those regions associated with an increase in fitness and unlinked to loci controlling reproductive isolation) and those that are less likely to exhibit introgression (those regions linked to traits decreasing fitness and reproductive isolation).

Differential response of the homoploid hybrid species Iris nelsonii (Iridaceae) and its progenitors to abiotic habitat conditions.

PREMISE OF THE STUDY: Homoploid hybrid speciation involves the evolution of reproductive isolation between a hybrid lineage and its progenitors without a change in chromosome number. Ecological divergence presumably plays a large role in the stabilization of hybrid lineages, as all homoploid hybrid species described to date are reported to be ecologically divergent from their progenitors. However, the described ecological divergence in most systems is anecdotal and has not been empirically tested. METHODS: We assessed the vegetative response of Iris nelsonii, a homoploid hybrid species, and its three progenitor species, I. brevicaulis, I. fulva, and I. hexagona, to different abiotic conditions (i.e., varied sunlight availability and flooding conditions) that largely characterize the habitats of these four species in their natural habitats in Louisiana, USA. KEY RESULTS: The species differed in their responses to the water-level treatment for many of the response variables, including rhizome weight, ramet growth, plant height, and two principal components used to characterize the data. The species differed in their response to the light-level treatment for root allocation and the principal component used to characterize plant size. Iris nelsonii significantly differed from its progenitors, including its most closely related progenitor species, in response to many of the treatments. CONCLUSIONS: The differential response to abiotic habitat conditions of I. nelsonii suggests that this species is ecologically divergent from its progenitor species.

Hybrid fitness across time and habitats.

There has been considerable debate about the role of hybrids in the evolutionary process. One question has involved the relative fitness of hybrid versus non-hybrid genotypes. For some, the assumption of lower hybrid fitness continues to be integral to their concept of species and speciation. In contrast, numerous workers have suggested that hybrid genotypes might demonstrate higher relative fitness under various environmental settings. Of particular importance in deciding between these opposing hypotheses are long-term analyses coupling ecological and genetic information. Although currently rare, such analyses have provided a test of the fitness of hybrid genotypes across generations and habitats and their role in adaptation and speciation. Here we discuss examples of these analyses applied to viruses, prokaryotes, plants and Darwin's Finches.

Geographical variation in postzygotic isolation and its genetic basis within and between two Mimulus species.

The aim of this study is to investigate the evolution of intrinsic postzygotic isolation within and between populations of Mimulus guttatus and Mimulus nasutus. We made 17 intraspecific and interspecific crosses, across a wide geographical scale. We examined the seed germination success and pollen fertility of reciprocal F(1) and F(2) hybrids and their pure-species parents, and used biometrical genetic tests to distinguish among alternative models of inheritance. Hybrid seed inviability was sporadic in both interspecific and intraspecific crosses. For several crosses, Dobzhansky-Muller incompatibilities involving nuclear genes were implicated, while two interspecific crosses revealed evidence of cytonuclear interactions. Reduced hybrid pollen fertility was found to be greatly influenced by Dobzhansky-Muller incompatibilities in five out of six intraspecific crosses and nine out of 11 interspecific crosses. Cytonuclear incompatibilities reduced hybrid fitness in only one intraspecific and one interspecific cross. This study suggests that intrinsic postzygotic isolation is common in hybrids between these Mimulus species, yet the particular hybrid incompatibilities responsible for effecting this isolation differ among the populations tested. Hence, we conclude that they evolve and spread only at the local scale.

Transmission ratio distortion results in asymmetric introgression in Louisiana Iris.

BACKGROUND: Linkage maps are useful tools for examining both the genetic architecture of quantitative traits and the evolution of reproductive incompatibilities. We describe the generation of two genetic maps using reciprocal interspecific backcross 1 (BC1) mapping populations from crosses between Iris brevicaulis and Iris fulva. These maps were constructed using expressed sequence tag (EST)- derived codominant microsatellite markers. Such a codominant marker system allowed for the ability to link the two reciprocal maps, and compare patterns of transmission ratio distortion observed between the two. RESULTS: Linkage mapping resulted in markers that coalesced into 21 linkage groups for each of the reciprocal backcross maps, presumably corresponding to the 21 haploid chromosomes of I. brevicaulis and I. fulva. The composite map was 1190.0-cM long, spanned 81% of the I. brevicaulis and I. fulva genomes, and had a mean density of 4.5 cM per locus. Transmission ratio distortion (TRD) was observed in 138 (48.5%) loci distributed in 19 of the 21 LGs in BCIB, BCIF, or both BC1 mapping populations. Of the distorted markers identified, I. fulva alleles were detected at consistently higher-than-expected frequencies in both mapping populations. CONCLUSIONS: The observation that I. fulva alleles are overrepresented in both mapping populations suggests that I. fulva alleles are favored to introgress into I. brevicaulis genetic backgrounds, while I. brevicaulis alleles would tend to be prevented from introgressing into I. fulva. These data are consistent with the previously observed patterns of introgression in natural hybrid zones, where I. fulva alleles have been consistently shown to introgress across species boundaries.

Asymmetric introgressive hybridization among louisiana iris species.

In this review, we discuss findings from studies carried out over the past 20+ years that document the occurrence of asymmetric introgressive hybridization in a plant clade. In particular, analyses of natural and experimental hybridization have demonstrated the consistent introgression of genes from Iris fulva into both Iris brevicaulis and Iris hexagona. Furthermore, our analyses have detected certain prezygotic and postzygotic barriers to reproduction that appear to contribute to the asymmetric introgression. Finally, our studies have determined that a portion of the genes transferred apparently affects adaptive traits.

Adaptation by introgression.

Both selective and random processes can affect the outcome of natural hybridization. A recent analysis in BMC Evolutionary Biology of natural hybridization between an introduced and a native salamander reveals the mosaic nature of introgression, which is probably caused by a combination of selection and demography.

The genetic architecture of reproductive isolation in Louisiana irises: hybrid fitness in nature.

Negative epistasis in hybrid genomes commonly results in postzygotic isolation between divergent lineages. However, some genomic regions may be selectively neutral or adaptive in hybrids and thus may potentially cross species barriers. We examined postzygotic isolation between ecologically similar species of Louisiana Iris: Iris brevicaulis and I. fulva to determine the potential for adaptive introgression in nature. Line-cross analyses allowed us a general overview of the gene action responsible for fitness-related traits. We then used a QTL mapping approach to detect genomic regions responsible for variation in these traits. Although hybrid classes suffered reduced fitness for many traits, hybrid means were equivalent to at least one of the parental species in overall estimates of maternal and paternal fitness during the two years of the field study. The genetic architecture underlying the fitness-related traits varied across field site and year of the study, thus emphasizing the importance of the environment in determining the degree of postzygotic isolation and potential for introgression across natural hybrid zones.

Review. Genetic exchange and the origin of adaptations: prokaryotes to primates.

Data supporting the occurrence of adaptive trait transfer (i.e. the transfer of genes and thus the phenotype of an adaptive trait through viral recombination, lateral gene transfer or introgressive hybridization) are provided in this review. Specifically, we discuss examples of lateral gene transfer and introgressive hybridization that have resulted in the transfer or de novo origin of adaptations. The evolutionary clades in which this process has been identified include all types of organisms. However, we restrict our discussion to bacteria, fungi, plants and animals. Each of these examples reflects the same consequence, namely that the transfer of genetic material, through whatever mechanism, may result in adaptive evolution. In particular, each of the events discussed has been inferred to impact adaptations to novel environmental settings in the recipient lineage.

The genetic architecture of reproductive isolation in Louisiana irises: pollination syndromes and pollinator preferences.

In animal-pollinated plants, pollinator preferences for divergent floral forms can lead to partial reproductive isolation. We describe regions of plant genomes that affect pollinator preferences for two species of Louisiana Irises, Iris brevicaulis and Iris fulva, and their artificial hybrids. Iris brevicaulis and I. fulva possess bee and bird-pollination syndromes, respectively. Hummingbirds preferred I. fulva and under-visited both I. brevicaulis and backcrosses toward this species. Lepidopterans preferred I. fulva and backcrosses toward I. fulva, but also under-visited I. brevicaulis and I. brevicaulis backcrosses. Bumblebees preferred I. brevicaulis and F1 hybrids and rarely visited I. fulva. Although all three pollen vectors preferred one or the other species, these preferences did not prevent visitation to other hybrid/parental classes. Quantitative trait locus (QTL) mapping, in reciprocal BC1 mapping populations, defined the genetic architecture of loci that affected pollinator behavior. We detected six and nine QTLs that affected pollinator visitation rates in the BCIb and BCIf mapping populations, respectively, with as many as three QTLs detected for each trait. Overall, this study reflects the possible role of quantitative genetic factors in determining (1) reproductive isolation, (2) the pattern of pollinator-mediated genetic exchange, and thus (3) hybrid zone evolution.

Ecological divergence associated with mating system causes nearly complete reproductive isolation between sympatric Mimulus species.

Speciation often involves the evolution of numerous prezygotic and postzygotic isolating barriers between divergent populations. Detailed knowledge of the strength and nature of those barriers provides insight into ecological and genetic factors that directly or indirectly influenced their origin, and may help predict whether they will be maintained in the face of sympatric hybridization and introgression. We estimated the magnitude of pre- and postzygotic barriers between naturally occurring sympatric populations of Mimulus guttatus and M. nasutus. Prezygotic barriers, including divergent flowering phenologies, differential pollen production, mating system isolation, and conspecific pollen precedence, act asymmetrically to completely prevent the formation of F(1) hybrids among seeds produced by M. guttatus (F(1)g), and reduce F(1) hybrid production among seeds produced by M. nasutus (F(1)n) to only about 1%. Postzygotic isolation is also asymmetric: in field experiments, F(1)g but not F(1)n hybrids had significantly reduced germination rates and survivorship compared to parental species. Both hybrid classes had flower, pollen, and seed production values within the range of parental values. Despite the moderate degree of F(1)g hybrid inviability, postzygotic isolation contributes very little to the total isolation between these species in the wild. We also found that F(1) hybrid flowering phenology overlapped more with M. guttatus than M. nasutus. These results, taken together, suggest greater potential for introgression from M. nasutus to M. guttatus than for the reverse direction. We also address problems with commonly used indices of isolation, discuss difficulties in calculating meaningful measures of reproductive isolation when barriers are asymmetric, and propose novel measures of prezygotic isolation that are consistent with postzygotic measures.

The genetic architecture of reproductive isolation in Louisiana irises: flowering phenology.

Despite the potential importance of divergent reproductive phenologies as a barrier to gene flow, we know less about the genetics of this factor than we do about any other isolating barrier. Here, we report on the genetic architecture of divergent flowering phenologies that result in substantial reproductive isolation between the naturally hybridizing plant species Iris fulva and I. brevicaulis. I. fulva initiates and terminates flowering significantly earlier than I. brevicaulis. We examined line crosses of reciprocal F1 and backcross (BC1) hybrids and determined that flowering time was polygenic in nature. We further defined quantitative trait loci (QTL) that affect the initiation of flowering in each of these species. QTL analyses were performed separately for two different growing seasons in the greenhouse, as well as in two field plots where experimental plants were placed into nature. For BCIF hybrids (BC1 toward I. fulva), 14 of 17 detected QTL caused flowering to occur later in the season when I. brevicaulis alleles were present, while the remaining 3 caused flowering to occur earlier. In BCIB hybrids (BC1 toward I. brevicaulis), 11 of 15 detected QTL caused flowering to occur earlier in the season when introgressed I. fulva alleles were present, while the remaining 4 caused flowering to occur later. These ratios are consistent with expectations of selection (as opposed to drift) promoting flowering divergence in the evolutionary history of these species. Furthermore, epistatic interactions among the QTL also reflected the same trends, with the majority of epistatic effects causing later flowering than expected in BCIF hybrids and earlier flowering in BCIB hybrids. Overlapping QTL that influenced flowering time across all four habitat/treatment types were not detected, indicating that increasing the sample size of genotyped plants would likely increase the number of significant QTL found in this study.

Detecting adaptive trait introgression between Iris fulva and I. brevicaulis in highly selective field conditions.

The idea that natural hybridization has served as an important force in evolutionary and adaptive diversification has gained considerable momentum in recent years. By combining genome analyses with a highly selective field experiment, we provide evidence for adaptive trait introgression between two naturally hybridizing Louisiana Iris species, flood-tolerant Iris fulva and dry-adapted I. brevicaulis. We planted reciprocal backcross (BC1) hybrids along with pure-species plants into natural settings that, due to a flooding event, favored I. fulva. As expected, I. fulva plants survived at much higher rates than I. brevicaulis plants. Backcross hybrids toward I. fulva (BCIF) also survived at significantly higher rates than the reciprocal backcross toward I. brevicaulis (BCIB). Survivorship of BCIB hybrids was strongly influenced by the presence of a number of introgressed I. fulva alleles located throughout the genome, while survivorship in the reciprocal BCIF hybrids was heavily influenced by two epistatically acting QTL of opposite effects. These results demonstrate the potential for adaptive trait introgression between these two species and may help to explain patterns of genetic variation observed in naturally occurring hybrid zones.

Loci affecting long-term hybrid survivorship in Louisiana irises: implications for reproductive isolation and introgression.

Iris fulva and I. brevicaulis are long-lived plant species known to hybridize where they coexist in nature. Year-to-year survival contributes significantly to overall fitness for both species and their hybrid derivatives, and differences in hybrid survivability may have important consequences to interspecific gene flow in nature. We examined the genetic architecture of long-term survivorship of reciprocal backcross I. fulva x I. brevicaulis hybrids in a common-garden, greenhouse environment. Differences in mortality were found between the two backcross (BC1) hybrid classes, with hybrids crossed toward I. fulva (BCIF) revealing twice the mortality of those hybrids backcrossed toward I. brevicaulis (BCIB). Using genomic scans on two separate genetic linkage maps derived from the reciprocal hybrid populations, we found that hybrid survivorship is influenced by several genetic regions. Multiple interval mapping (MIM) revealed four quantitative trait loci (QTLs) in BCIF hybrids that were significantly associated with survivorship. Introgressed I. brevicaulis DNA increased survivorship at three of the four QTLs. For the fourth QTL, introgressed I. brevicaulis DNA was associated with decreased survivorship. No QTLs were detected in BCIB hybrids; however, single-marker analysis revealed five unlinked loci that were significantly associated with survivorship. At all five markers, survivorship was positively associated with introgressed I. fulva DNA. The present findings have important implications for the evolutionary dynamics of naturally occurring hybrid zones. Regions of the genome that increase survivorship when in a heterozygous (i.e., hybrid) state should have an increased likelihood of passing across species boundaries, whereas those that decrease survivorship will be less likely to introgress.