Tracking the progression of speciation: variable patterns of introgression across the genome provide insights on the species delimitation between progenitor–derivative spruces (Picea mariana × P. rubens)

The genic species concept implies that while most of the genome can be exchanged somewhat freely between species through introgression, some genomic regions remain impermeable to interspecific gene flow. Hence, interspecific differences can be maintained despite ongoing gene exchange within contact zones. This study assessed the heterogeneous patterns of introgression at gene loci across the hybrid zone of an incipient progenitor–derivative species pair, Picea mariana (black spruce) and Picea rubens (red spruce). The spruce taxa likely diverged in geographic isolation during the Pleistocene and came into secondary contact during late Holocene. A total of 300 SNPs distributed across the 12 linkage groups (LG) of black spruce were genotyped for 385 individual trees from 33 populations distributed across the allopatric zone of each species and within the zone of sympatry. An integrative framework combining three population genomic approaches was used to scan the genomes, revealing heterogeneous patterns of introgression. A total of 23 SNPs scattered over 10 LG were considered impermeable to introgression and putatively under diverging selection. These loci revealed the existence of impermeable genomic regions forming the species boundary and are thus indicative of ongoing speciation between these two genetic lineages. Another 238 SNPs reflected selectively neutral diffusion across the porous species barrier. Finally, 39 highly permeable SNPs suggested ancestral polymorphism along with balancing selection. The heterogeneous patterns of introgression across the genome indicated that the speciation process between black spruce and red spruce is young and incomplete, albeit some interspecific differences are maintained, allowing ongoing species divergence even in sympatry. The approach developed in this study can be used to track the progression of ongoing speciation processes.     

Outlier loci highlight the direction of introgression in oaks

Loci considered to be under selection are generally avoided in attempts to infer past demographic processes as they do not fit neutral model assumptions. However, opportunities to better reconstruct some aspects of past demography might thus be missed. Here we examined genetic differentiation between two sympatric European oak species with contrasting ecological dynamics (Quercus robur and Quercus petraea) with both outlier (i.e. loci possibly affected by divergent selection between species or by hitchhiking effects with genomic regions under selection) and nonoutlier loci. We sampled 855 individuals in six mixed forests in France and genotyped them with a set of 262 SNPs enriched with markers showing high interspecific differentiation, resulting in accurate species delimitation. We identified between 13 and 74 interspecific outlier loci, depending on the coalescent simulation models and parameters used. Greater genetic diversity was predicted in Q. petraea (a late‐successional species) than in Q. robur (an early successional species) as introgression should theoretically occur predominantly from the resident species to the invading species. Remarkably, this prediction was verified with outlier loci but not with nonoutlier loci. We suggest that the lower effective interspecific gene flow at loci showing high interspecific divergence has better preserved the signal of past asymmetric introgression towards Q. petraea caused by the species' contrasting dynamics. Using markers under selection to reconstruct past demographic processes could therefore have broader potential than generally recognized.     

Genetic validation and characterization of RAPD markers differentiating black and red spruces: molecular certification of spruce trees and hybrids

 Picea mariana (black spruce) and P. rubens (red spruce) are closely related species which are difficult to differentiate morphologically. RAPD markers differentiating black and red spruces have been previously identified. In the present study, genetic validity of these markers was determined using samples representing range–wide provenances. Their applicability for certifying genetic identity of individual black, red trees and their hybrids from several sympatric and allopatric locations was demonstrated. These diagnostic fragments of both red and black spruce were present at a frequency of over 0.95 in allopatric provenances, but at a lower frequency in some sympatric provenances (0.43–1.00). Natural populations of red spruce exhibiting typical red spruce phenotype contained black spruce diagnostic RAPD fragments and black spruces growing in bogs with typical bog black spruce morphology, contained red spruce-specific RAPD markers. Some major RAPD markers were cloned and sequenced. The results reveal an extremely high degree of identity between the random primer and the primer binding sites on the genome. Amplification of black and red spruce genomic DNA with designed primers flanking the species-diagnostic RAPD markers indicates that most of RAPD markers used to differentiate black spruce from red spruce are not species specific since these sequences were detected in several spruce species using a more sensitive detection method. Received June 17, 2002; accepted August 5, 2002 Published online: February 4, 2003     

Genetic architecture and genomic patterns of gene flow between hybridizing species of Picea

Hybrid zones provide an opportunity to study the effects of selection and gene flow in natural settings. We employed nuclear microsatellites (single sequence repeat (SSR)) and candidate gene single-nucleotide polymorphism markers (SNPs) to characterize the genetic architecture and patterns of interspecific gene flow in the Picea glauca × P. engelmannii hybrid zone across a broad latitudinal (40–60 degrees) and elevational (350–3500 m) range in western North America. Our results revealed a wide and complex hybrid zone with broad ancestry levels and low interspecific heterozygosity, shaped by asymmetric advanced-generation introgression, and low reproductive barriers between parental species. The clinal variation based on geographic variables, lack of concordance in clines among loci and the width of the hybrid zone points towards the maintenance of species integrity through environmental selection. Congruency between geographic and genomic clines suggests that loci with narrow clines are under strong selection, favoring either one parental species (directional selection) or their hybrids (overdominance) as a result of strong associations with climatic variables such as precipitation as snow and mean annual temperature. Cline movement due to past demographic events (evidenced by allelic richness and heterozygosity shifts from the average cline center) may explain the asymmetry in introgression and predominance of P. engelmannii found in this study. These results provide insights into the genetic architecture and fine-scale patterns of admixture, and identify loci that may be involved in reproductive barriers between the species.     

Population genetic structure of Picea engelmannii, P. glauca and their previously unrecognized hybrids in the central Rocky Mountains

Areas of geographic overlap between potentially hybridizing species provide the opportunity to study interspecific gene flow and reproductive barriers. Here we identified hybrids between Picea engelmannii and P. glauca by their genetic composition at 17 microsatellite markers, and determined the broad-scale geographic distribution of hybrids in the central Rocky Mountains of North America, a geographic region where hybrids and isolation between species had not previously been studied. Parameter estimates from admixture models revealed considerable variation in ancestry within and among collection sites, suggesting that within this area of geographic overlap, the interaction of the two species varies extensively. The results document a previously unrecognized patchy distribution of hybrids between P. engelmannii and P. glauca, including locations where hybrids were not known or expected to exist. Further, the ancestry of many hybrids was consistent with multiple generations of hybridization, with probable directional backcrossing to P. engelmannii, suggesting a relatively porous species boundary. The identification and characterization of hybridization between these spruce in this region raises the question of what factors maintain barriers to gene flow in these long-lived forest trees. The current research lays the groundwork for future study of the ecological and evolutionary contexts of their hybridization, as well as of differential introgression and permeability of species boundaries.     

Limited gene exchange between two sister species of leaf beetles within a hybrid zone in the Alps

Analysing genomic variation within and between sister species is a first step towards understanding species boundaries. We focused on two sister species of cold‐resistant leaf beetles, Gonioctena quinquepunctata and G. intermedia, whose ranges overlap in the Alps. A previous study of DNA sequence variation had revealed multiple instances of mitochondrial genome introgression in this region, suggesting recent hybridization between the two species. To evaluate the extent of gene exchange resulting from these hybridization events, we sampled individuals of both species inside and outside the hybrid zone and analysed genomic variation among them using RAD‐seq markers. Individual levels of introgression in the nuclear genome were estimated first by defining species‐specific SNPs (displaying a fixed difference between species) a priori and second by using model‐based methods. Both types of analyses indicated little gene exchange, if any, between species at the level of the nuclear genome. Whereas the first method suggested slightly more gene flow, we argue that it has likely overestimated introgression in the phylogeographic context of this study. We conclude that strong intrinsic barriers prevent genetic exchange at the level of the nuclear genome between the two species. The apparent discrepancy observed between introgression occurring in the nuclear and mitochondrial genomes could be explained by selection acting in favour of the latter. Also, these results have consequences for the phylogeographic study of each species, since we can assume that genetic diversity in the overlapping portion of their ranges is not the product of introgression.     

Species-specific nuclear and chloroplast single nucleotide polymorphisms to distinguish Picea glauca, P. mariana and P. rubens

 Picea rubens (red spruce) and P. mariana (black spruce) are closely related species which are difficult to differentiate morphologically. They are sympatric with P. glauca (white spruce) in the northern portion of their ranges. In order to identify potential interspecific polymorphisms, the chloroplast trnK intron and rpl33-psaJ-trnP region were sequenced, and the nuclear-encoded ITS region of the rDNA repeat was partially sequenced. Thirteen chloroplast and 12 nuclear candidate interspecific single nucleotide polymorphisms (SNPs) were identified. The species-specificity of several SNPs was determined by surveying DNAs amplified from trees representing range-wide provenance tests; these included 46 red spruce from 11 provenances, 84 black spruce from 30 provenances and 90 white spruce from 22 provenances. Two SNPs (1 chloroplast and 1 nuclear), which distinguish black spruce from red and white spruce, were consistent among 96–100% of the trees surveyed. Five SNPs (4 chloroplast and 1 nuclear), which distinguish white spruce from red and black spruce, were consistent among 100% of surveyed trees. These species-specific SNPs were used to identify anonymous spruce samples in a blind test, and their utility for small amounts of tissue, as little as single needles, was demonstrated. Scoring these SNPs is much less labor intensive than previous molecular methods for taxa differentiation (restriction fragment length polymorphisms or random amplified polymorphic DNAs), therefore they can be applied to large population studies. Received: 16 December 1998 / Accepted: 5 January 1999     

Natural hybridization between black spruce and red spruce

Using species-specific random amplified polymorphic DNA (RAPD) markers and morphological characters, natural hybridization between the closely related black spruce Picea mariana (Mill.) B.S.P. and red spruce P. rubens Sarg. was evaluated in natural populations from north-eastern North America. Sampling included populations from both areas of allopatry and also 14 populations from part of the area of sympatry located in the province of Québec and covering several thousands of square kilometres. Classification results from RAPD species-specific markers and from a discriminant function based on morphology were compared. Molecular analysis of the allopatric populations indicated a small amount of interspecific gene leakage with no asymmetric directionality to introgression. A high occurrence of hybrid/introgressant individuals was observed within sympatric populations, suggesting weak reproductive isolation. As expected, the detection of such individuals was more efficient using molecular markers than with morphological traits. The hybrid zone appeared extensive with variable species structure and, in some stands, the main component composed of hybrid/introgressant trees. Implications for the genecology and genetic management of these species are discussed.     

Bidirectional cytoplasmic and nuclear introgression in the New World cottons Gossypium barbadense and G. Hirsutum (Malvaceae)

Patterns of interspecific cytoplasmic (plastid and mitochondrial) and nuclear introgression are typically asymmetrical: cytoplasmic gene flow or “capture” is frequently observed without evidence of nuclear introgression. In contrast, nuclear introgression without concomitant cytoplasmic introgression has rarely been demonstrated. Gossypium barbadense L. and G. hirsutum L. have large indigenous ranges in the New World semiarid tropics, with an extensive area of sympatry in the Caribbean and Central America. Numerous accessions of both species were surveyed for diagnostic cpDNA restriction sites. These data, in conjunction with previous information on nuclear markers, lead to several conclusions: 1) introgression between G. hirsutum and G. barbadense is bidirectional for both nuclear and cytoplasmic genes; 2) patterns of introgression between the two species are not symmetrical—in G. barbadense, introgression of G. hirsutum alleles is largely restricted to modem cultivars and is uncommon in areas of sympatry; in contrast, introgression of G. barbadense alleles into G. hirsutum is relatively common in areas of sympatry and is rare in modem cultivars; 3) nuclear introgression is geographically more widespread and more frequently detected than cytoplasmic introgression. Several mechanisms may underlie the differential patterns of cytoplasmic and nuclear gene flow observed, including differential fitness of infraspecific and interspecific cytonuclear combinations and selection against female function in interspecific backcrosses. Possible explanations for the observed asymmetrical patterns of introgression include differences in population sizes combined with phenological differences that bias interspecific pollen transfer.     

Continent‐wide population genomic structure and phylogeography of North America’s most destructive conifer defoliator,the spruce budworm (Choristoneura fumiferana)

The spruce budworm, Choristoneura fumiferana, is presumed to be panmictic across vast regions of North America. We examined the extent of panmixia by genotyping 3,650 single nucleotide polymorphism (SNP) loci in 1975 individuals from 128 collections across the continent. We found three spatially structured subpopulations: Western (Alaska, Yukon), Central (southeastern Yukon to the Manitoba–Ontario border), and Eastern (Manitoba–Ontario border to the Atlantic). Additionally, the most diagnostic genetic differentiation between the Central and Eastern subpopulations was chromosomally restricted to a single block of SNPs that may constitute an island of differentiation within the species. Geographic differentiation in the spruce budworm parallels that of its principal larval host, white spruce (Picea glauca), providing evidence that spruce budworm and spruce trees survived in the Beringian refugium through the Last Glacial Maximum and that at least two isolated spruce budworm populations diverged with spruce/fir south of the ice sheets. Gene flow in the spruce budworm may also be affected by mountains in western North America, habitat isolation in West Virginia, regional adaptations, factors related to dispersal, and proximity of other species in the spruce budworm species complex. The central and eastern geographic regions contain individuals that assign to Eastern and Central subpopulations, respectively, indicating that these barriers are not complete. Our discovery of previously undetected geographic and genomic structure in the spruce budworm suggests that further population modelling of this ecologically important insect should consider regional differentiation, potentially co‐adapted blocks of genes, and gene flow between subpopulations.     

Ecological differentiation and habitat unsuitability maintaining a ground beetle hybrid zone

Exogenous selection via interactions between organisms and environments may influence the dynamics of hybrid zones between species in multiple ways. Two major models of a hybrid zone allowed us to hypothesize that environmental conditions influence hybrid zone dynamics in two ways. In the first model, an environmental gradient determines the mosaic distribution at the boundary between ecologically differentiated species (mosaic hybrid zone model). In the second model, a patch of unsuitable habitat traps a hybrid zone between species whose hybrids are unfit (tension zone model). To test these, we examined the environmental factors influencing the spatial structure of a hybrid zone between the ground beetles Carabus maiyasanus and C. iwawakianus using GIS‐based quantification of environmental factors and a statistical comparison of species distribution models (SDMs). We determined that both of the hypothetical processes can be important in the hybrid zone. We detected interspecific differences in the environmental factors in presence localities and their relative contribution in SDMs. SDMs were not identical between species even within contact areas, but tended to be similar within the range of each species. These results suggest an association between environments and species, and provide evidence that ecological differentiation between species plays a role in the maintenance of the hybrid zone. Contact areas were characterized by a relatively high temperature, low precipitation, and high topological wetness. Thus, the contact areas were regarded as being located in an unsuitable habitat with a drier climate, where those populations are likely to occur in patches with limited precipitation concentrated. A comparison of spatial scales suggests that exogenous selection via environmental factors may be weaker than endogenous selection via genitalic incompatibility.     

Nuclear introgression without mitochondrial introgression in two turtle species exhibiting sex‐specific trophic differentiation

Despite the presence of reproductive barriers between species, interspecific gene introgression has been documented in a range of natural systems. Comparing patterns of genetic introgression in biparental versus matrilineal markers can potentially reveal sex‐specific barriers to interspecific gene flow. Hybridization has been documented in the freshwater turtles Graptemys geographica and G. pseudogeographica, whose ranges are largely sympatric. Morphological differentiation between the species is restricted to females, with female G. geographica possessing large heads and jaws compared to the narrow heads of G. pseudogeographica females. If hybrid females are morphologically intermediate, they may be less successful at exploiting parental feeding niches, thereby limiting the introgression of maternally inherited, but not biparental, molecular markers. We paired sequence data with stable isotope analysis and examined sex‐specific genetic introgression and trophic differentiation in sympatric populations of G. geographica and G. pseudogeographica. We observed introgression from G. pseudogeographica into G. geographica at three nuclear loci, but not at the mitochondrial locus. Analysis of ?¹⁵N and ?¹³C was consistent with species differences in trophic positioning in females, but not males. These results suggest that ecological divergence in females may reduce the opportunity for gene flow in this system.     

Cytophotometric differentiation of high elevation spruces: physiological and ecological implications

Red and black spruce and their hybrids can be determined by morphological indices; however, the criteria are somewhat subjective and increasingly difficult to use at higher elevations. Although the chromosome number is identical (2n = 24), red spruce has twice as much nuclear DNA (48 pg) than black spruce (24 pg) and thus the species and their hybrids can also be separated by cytophotometry. This is relevant to spruce decline studies because black spruce is much more resistant to high elevation environmental stresses, both natural and anthropogenic. It also has implications for the effect of climatic changes on the composition of high elevation spruce-fir forests because red spruce can outcompete black spruce under more mesic conditions. Four elevation transects sampling spruce on the east and west sides of Mount Washington (New Hampshire) and Camels Hump (Vermont) and a single transect on the southwest side of Whiteface Mountain (New York) were made to investigate the degree of hybridization and introgression between these two species. A positive correlation was found between increased elevation and increased black spruce genes on Mount Washington and Camels Hump. Pure black spruce was found on Mount Washington from 1356 m to 1582 m. No pure black or red spruce was found on Camels Hump although the proportion of red spruce alleles was significantly greater on Camels Hump. All trees sampled at all elevations on Whiteface Mountain were pure red spruce. Thus the proportion of black spruce alleles in high elevation spruce populations decreases from east to west. This closely parallels the increase in spruce decline which increases from east to west.     

Cytophotometric Differentiation Of HIGH Elevation Spruces: Physiological and Ecological Implications

Red and black spruce and their hybrids can be determined by morphological indices; however, the criteria are somewhat subjective and increasingly difficult to use at higher elevations. Although the chromosome number is identical (2n = 24), red spruce has twice as much nuclear DNA (48 pg) than black spruce (24 pg) and thus the species and their hybrids can also be separated by cytophotometry. This is relevant to spruce decline studies because black spruce is much more resistant to high elevation environmental stresses, both natural and anthropogenic. It also has implications for the effect of climatic changes on the composition of high elevation spruce-fir forests because red spruce can outcompete black spruce under more mesic conditions. Four elevation transects sampling spruce on the east and west sides of Mount Washington (New Hampshire) and Camels Hump (Vermont) and a single transect on the southwest side of Whiteface Mountain (New York) were made to investigate the degree of hybridization and introgression between these two species. A positive correlation was found between increased elevation and increased black spruce genes on Mount Washington and Camels Hump. Pure black spruce was found on Mount Washington from 1356 m to 1582 m. No pure black or red spruce was found on Camels Hump although the proportion of red spruce alleles was significantly greater on Camels Hump. All trees sampled at all elevations on Whiteface Mountain were pure red spruce. Thus the proportion of black spruce alleles in high elevation spruce populations decreases from east to west. This closely parallels the increase in spruce decline which increases from east to west.     

Fungal evolutionary genomics provides insight into the mechanisms of adaptive divergence in eukaryotes

Fungi are ideal model organisms for dissecting the genomic bases of adaptive divergence in eukaryotes. They have simple morphologies and small genomes, occupy contrasting, well‐identified ecological niches and tend to have short generation times, and many are amenable to experimental approaches. Fungi also display diverse lifestyles, from saprotrophs to pathogens or mutualists, and they play extremely important roles in both ecosystems and human activities, as wood decayers, mycorrhizal fungi, lichens, endophytes, plant and animal pathogens, and in fermentation or drug production. We review here recent insights into the patterns and mechanisms of adaptive divergence in fungi, including sources of divergence, genomic variation and, ultimately, speciation. We outline the various ecological sources of divergent selection and genomic changes, showing that gene loss and changes in gene expression and in genomic architecture are important adaptation processes, in addition to the more widely recognized processes of amino acid substitution and gene duplication. We also review recent findings regarding the interspecific acquisition of genomic variation and suggesting an important role for introgression, hybridization and horizontal gene transfers (HGTs). We show that transposable elements can mediate several of these genomic changes, thus constituting important factors for adaptation. Finally, we review the consequences of divergent selection in terms of speciation, arguing that genetic incompatibilities may not be as widespread as generally thought and that pleiotropy between adaptation and reproductive isolation is an important route of speciation in fungal pathogens.     

Maintaining their genetic distance: Little evidence for introgression between widely hybridizing species of Geum with contrasting mating systems

Within the plant kingdom, many genera contain sister lineages with contrasting outcrossing and inbreeding mating systems that are known to hybridize. The evolutionary fate of these sister lineages is likely to be influenced by the extent to which they exchange genes. We measured gene flow between outcrossing Geum rivale and selfing Geum urbanum, sister species that hybridize in contemporary populations. We generated and used a draft genome of G. urbanum to develop dd‐RAD data scorable in both species. Coalescent analysis of RAD data from allopatric populations indicated that the species diverged 2–3 Mya, and that historical gene flow between them was extremely low (1 migrant every 25 generations). Comparison of genetic divergence between species in sympatry and allopatry, together with an analysis of allele frequencies in potential parental and hybrid populations, provided no evidence of contemporary introgression in sympatric populations. Cluster‐ and species‐specific marker analyses revealed that, apart from four early‐generation hybrids, individuals in sympatric populations fell into two genetically distinct groups that corresponded exactly to their morphological species classification with maximum individual admixture estimates of only 1–3%. However, we did observe joint segregation of four putatively introgressed SNPs across two scaffolds in the G. urbanum population that was associated with significant morphological variation, interpreted as tentative evidence for rare, recent interspecific gene flow. Overall, our results indicate that despite the presence of hybrids in contemporary populations, genetic exchange between G. rivale and G. urbanum has been extremely limited throughout their evolutionary history.     

High‐density interspecific genetic linkage mapping provides insights into genomic incompatibility between channel catfish and blue catfish

Catfish is the leading aquaculture species in the United States. The interspecific hybrid catfish produced by mating female channel catfish with male blue catfish outperform both of their parent species in a number of traits. However, mass production of the hybrids has been difficult because of reproductive isolation. Investigations of genome structure and organization of the hybrids provide insights into the genetic basis for maintenance of species divergence in the face of gene flow, thereby helping develop strategies for introgression and efficient production of the hybrids for aquaculture. In this study, we constructed a high‐density genetic linkage map using the hybrid catfish system with the catfish 250K SNP array. A total of 26 238 SNPs were mapped to 29 linkage groups, with 12 776 unique marker positions. The linkage map spans approximately 3240 cM with an average intermarker distance of 0.25 cM. A fraction of markers (986 of 12 776) exhibited significant deviation from the expected Mendelian ratio of segregation, and they were clustered in major genomic blocks across 15 LGs, most notably LG9 and LG15. The distorted markers exhibited significant bias for maternal alleles among the backcross progenies, suggesting strong selection against the blue catfish alleles. The clustering of distorted markers within genomic blocks should lend insights into speciation as marked by incompatibilities between the two species. Such findings should also have profound implications for understanding the genomic evolution of closely related species as well as the introgression of hybrid production programs in aquaculture.