Ambiguous species boundaries: Hybridization and morphological variation in two closely related Rubus species along altitudinal gradients

Although hybridization frequently occurs among plant species, hybrid zones of divergent lineages formed at species boundaries are less common and may not be apparent in later generations of hybrids with more parental‐like phenotypes, as a consequence of backcrossing. To determine the effects of dispersal and selection on species boundaries, we compared clines in leaf traits and molecular hybrid index along two hybrid zones on Yakushima Island, Japan, in which a temperate (Rubus palmatus) and subtropical (Rubus grayanus) species of wild raspberry are found. Leaf sinus depth in the two hybrid zones had narrower clines at 600 m a.s.l. than the molecular hybrid index and common garden tests confirmed that some leaf traits, including leaf sinus depth that is a major trait used in species identification, are genetically divergent between these closely related species. The sharp transition in leaf phenotypic traits compared to molecular markers indicated divergent selection pressure on the hybrid zone structure. We suggest that species boundaries based on neutral molecular data may differ from those based on observed morphological traits.     

Hybridization and speciation

Hybridization has many and varied impacts on the process of speciation. Hybridization may slow or reverse differentiation by allowing gene flow and recombination. It may accelerate speciation via adaptive introgression or cause near‐instantaneous speciation by allopolyploidization. It may have multiple effects at different stages and in different spatial contexts within a single speciation event. We offer a perspective on the context and evolutionary significance of hybridization during speciation, highlighting issues of current interest and debate. In secondary contact zones, it is uncertain if barriers to gene flow will be strengthened or broken down due to recombination and gene flow. Theory and empirical evidence suggest the latter is more likely, except within and around strongly selected genomic regions. Hybridization may contribute to speciation through the formation of new hybrid taxa, whereas introgression of a few loci may promote adaptive divergence and so facilitate speciation. Gene regulatory networks, epigenetic effects and the evolution of selfish genetic material in the genome suggest that the Dobzhansky–Muller model of hybrid incompatibilities requires a broader interpretation. Finally, although the incidence of reinforcement remains uncertain, this and other interactions in areas of sympatry may have knock‐on effects on speciation both within and outside regions of hybridization.     

Reduced male fertility is common but highly variable in form and severity in a natural house mouse hybrid zone

Barriers to gene flow between naturally hybridizing taxa reveal the initial stages of speciation. Reduced hybrid fertility is a common feature of reproductive barriers separating recently diverged species. In house mice (Mus musculus), hybrid male sterility has been studied extensively using experimental crosses between subspecies. Here, we present the first detailed picture of hybrid male fertility in the European M. m. domesticus-M. m. musculus hybrid zone. Complete sterility appears rare or absent in natural hybrids but a large proportion of males (~30%) have sperm count or relative testis weight below the range in pure subspecies, and likely suffer reduced fertility. Comparison of a suite of traits related to fertility among subfertile males indicates reduced hybrid fertility in the contact zone is highly variable among individuals and ancestry groups in the type, number, and severity of spermatogenesis defects present. Taken together, these results suggest multiple underlying genetic incompatibilities are segregating in the hybrid zone, which likely contribute to reproductive isolation between subspecies.     

Dispersal and selection mediate hybridization between a native and invasive species

Hybridization between native and non-native species has serious biological consequences, but our understanding of how dispersal and selection interact to influence invasive hybridization is limited. Here, we document the spread of genetic introgression between a native (Oncorhynchus clarkii) and invasive (Oncorhynchus mykiss) trout, and identify the mechanisms influencing genetic admixture. In two populations inhabiting contrasting environments, non-native admixture increased rapidly from 1984 to 2007 and was driven by surprisingly consistent processes. Individual admixture was related to two phenotypic traits associated with fitness: size at spawning and age of juvenile emigration. Fish with higher non-native admixture were larger and tended to emigrate at a younger age―relationships that are expected to confer fitness advantages to hybrid individuals. However, strong selection against non-native admixture was evident across streams and cohorts (mean selection coefficient against genotypes with non-native alleles (s) = 0.60; s.e. = 0.10). Nevertheless, hybridization was promoted in both streams by the continuous immigration of individuals with high levels of non-native admixture from other hybrid source populations. Thus, antagonistic relationships between dispersal and selection are mediating invasive hybridization between these fish, emphasizing that data on dispersal and natural selection are needed to fully understand the dynamics of introgression between native and non-native species.     

Robertsonian translocations in free-living populations of the house mouse in Belgium

Mice with Robertsonian (Rb) translocations have been discovered in some 45 populations in Europe. In Belgium, we have observed either Rb(4.12) in a heterozygous or homozygous state (2 n = 39 and 38 respectively), or Rb(4.12) homozygous with a heterozygous Rb(5.10), or both fusions in a homozygous state (2 n = 37 and 36, respectively). Some locations are highly polymorphic and hétérozygotes are found frequently. These observations suggest that the Rb population in Belgium could be of recent origin and probably is the result of introgression from Alsace (France) or Germany.     

Introgressive hybridization and natural selection in Darwin's finches

Introgressive hybridization, i.e. hybridization with backcrossing, can lead to the fusion of two species, but it can also lead to evolution of a new trajectory through an enhancement of genetic variation in a new or changed ecological environment. On Daphne Major Island in the Galápagos archipelago, ~1–2% of Geospiza fortis finches breed with the resident G. scandens and with the rare immigrant species G. fuliginosa in each breeding season. Previous research has demonstrated morphological convergence of G. fortis and G. scandens over a 30‐year period as a result of bidirectional introgression. Here we examine the role of hybridization with G. fuliginosa in the evolutionary trajectory of G. fortis. Geospiza fuliginosa (~12 g) is smaller and has a more pointed beak than G. fortis (~17 g). Genetic variation of the G. fortis population was increased by receiving genes more frequently from G. fuliginosa than from G. scandens (~21 g). A severe drought in 2003–2005 resulted in heavy and selective mortality of G. fortis with large beaks, and they became almost indistinguishable morphologically from G. fuliginosa. This was followed by continuing hybridization, a further decrease in beak size and a potential morphological fusion of the two species under entirely natural conditions.     

Variation in fluctuating asymmetry levels across a Robertsonian polymorphic zone of the house mouse

Fluctuating asymmetry (FA) levels were assessed within the Barcelona Robertsonian polymorphic zone of Mus musculus domesticus as a measure of developmental instability (DI). This zone is characterized by populations with a reduced diploid number (2n = 27–39) surrounded by others with standard karyotype (2n = 40). Mice were distributed into four classes according to mean diploid number at each collecting site, and differences in their FA levels were studied. Three different FA indices were calculated on interlandmark distances from two major morphogenetic units of the mandible: the teeth and muscular areas. Three kinds of analysis were performed: trait‐by‐trait, considering the two regions separately and pooling all traits. In univariate analyses few differences were detected between classes. Nevertheless, when characters were pooled, differences in FA values were found between one Robertsonian group and standard mice, and between several Robertsonian classes. Teeth area traits showed higher FA levels in Rb I mice, i.e. those geographically close to the standard populations. However, FA levels decreased in more introgressed Robertsonian groups and showed significant differences with Rb I. Muscular area traits showed lower DI levels in the Robertsonian mice, especially those with an intermediate mean diploid number (Rb II). When all traits were pooled, FA levels increased in Rb I, whilst the other Robertsonian groups showed similar, or even lower, FA values to those of the standard mice. The higher FA levels found in Rb I suggest an increase in the disruption of genetic coadaptation. Furthermore, in Rb I the presence of metacentrics is more recent than in more introgressed populations; we therefore suggest that Rb I have had less time to restore genetic coadaptation. In addition, the teeth region of the mandible seems to be more sensitive to genomic stress than the muscular region. In the light of these results we suggest that hybrids should be separated into groups and that mandible morphogenetic units be differentiated when using FA in hybrid zones to analyse DI.     

Chromosomal variation in the house mouse

Although the standard karyotype of the western house mouse, Mus musculus domesticus , consists entirely of acrocentric chromosomes, there are 97 distinct 'populations' that are characterized by various combinations of metacentric chromosomes that have arisen by Robertsonian (Rb) fusions and whole-arm reciprocal translocations (WARTs). In this review we discuss the processes behind the origin and fixation of these rearrangements and then present a unified list of all known metacentric populations and evaluate their phylogenetic relationships. Eleven independent phylogeographical 'systems', each consisting of 2–25 metacentric populations, were identified in Scotland, Denmark, Northern Europe–Northern Switzerland, Southern Switzerland, Northern Italy, Croatia, Spain, Central–Southern Italy, Peloponnesus, Mainland Greece and Madeira. There are six isolated metacentric populations that do not belong to any of these systems. To generate phylogenies of the metacentric populations within each system, we determined those outcomes with the fewest steps regarding accumulation of metacentrics by Rb fusions, WARTs and zonal raciation and taking into account geographical proximity. These phylogenies should be viewed as working hypotheses that will be refined with further chromosomal and molecular data and improvements in methods of phylogenetic reconstruction. The list of metacentric populations and our phylogenies are also published electronically and can be accessed at http://www.studenec.ivb.cz/Projects/RobertsonianMice/ .  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 535–563.     

Differential introgression from a sister species explains high FST outlier loci within a mussel species

Scanning genomes for loci with high levels of population differentiation has become a standard of population genetics. F_ST outlier loci are most often interpreted as signatures of local selection, but outliers might arise for many other reasons too often left unexplored. Here, we tried to identify further the history and genetic basis underlying strong differentiation at F_ST outlier loci in a marine mussel. A genome scan of genetic differentiation has been conducted between Atlantic and Mediterranean populations of Mytilus galloprovincialis. The differentiation was low overall (F_ST = 0.03), but seven loci (2%) were strong F_ST outliers. We then analysed DNA sequence polymorphism at two outlier loci. The genetic structure proved to be the consequence of differential introgression of alleles from the sister‐hybridizing species Mytilus edulis. Surprisingly, the Mediterranean population was the most introgressed at these two loci, although the contact zone between the two species is nowadays localized along the Atlantic coasts of France and the British Isles. A historical contact between M. edulis and Mediterranean M. galloprovincialis should have happened during glacial periods. It proved difficult to disentangle two hypotheses: (i) introgression was adaptive, implying edulis alleles have been favoured in Mediterranean populations, or (ii) the genetic architecture of the barrier to edulis gene flow is different between the two M. galloprovincialis backgrounds. Five of the seven outliers between M. galloprovincialis populations were also outliers between M. edulis and Atlantic M. galloprovincialis, which would support the latter hypothesis. Differential introgression across semi‐permeable barriers to gene flow is a neglected scenario to interpret outlying loci that may prove more widespread than anticipated.     

Reproductive isolation among two interfertile Rhododendron species: low frequency of post-F1 hybrid genotypes in alpine hybrid zones

Hybrids between the acid-loving species Rhododendron ferrugineum and the basic soil species Rhododendron hirsutum occur on soils of intermediate pH in the European Alps. Material from two hybrid zones ~500 m apart, and also nearby populations of each parent species, was surveyed for presence/absence of 31 random amplified polymorphic DNA markers that distinguish parents. Based on morphological assessment, the material comprised 51 putative hybrids, 18 putative R. ferrugineum individuals and 26 putative R. hirsutum plants. RAPD data were analysed using a Bayesian approach implemented by the program newhybrids , and also by principal coordinates analysis. The identity of all R. ferrugineum plants examined was confirmed; however, of the putative R. hirsutum individuals examined, two were certainly and 11 possibly hybrid derivatives. Among all hybrid derivatives examined, about half were designated as F₁s or a similar class, otherwise backcrosses to R. hirsutum appeared to be common whereas other hybrid classes were rare and backcrosses to R. ferrugineum possibly absent. Despite this, artificially generated seed of F₂ class and backcrosses in each direction showed greater viability than one parent ( R. hirsutum ). Introgression from R. ferrugineum was also detected in a population that from morphology appeared to contain only R. hirsutum . Hence, the direction of backcrossing might be highly asymmetric within hybrid zones, causing unidirectional gene flow from R. ferrugineum into R. hirsutum . Conversely, the rarity of backcrosses to R. ferrugineum , F₂s and later hybrid generations, which might be due to phenology effects and habitat-mediated selection, could play a part in restricting gene flow towards R. ferrugineum .     

Spatial variation in gene flow across a hybrid zone reveals causes of reproductive isolation and asymmetric introgression in wall lizards*

Hybrid zones provide insights into the evolution of reproductive isolation. Sexual selection can contribute to the evolution of reproductive barriers, but it remains poorly understood how sexual traits impact gene flow in secondary contact. Here, we show that a recently evolved suite of sexual traits that function in male-male competition mediates gene flow between two lineages of wall lizards (Podarcis muralis). Gene flow was relatively low and asymmetric in the presence of exaggerated male morphology and coloration compared to when the lineages share the ancestral phenotype. Putative barrier loci were enriched in genomic regions that were highly differentiated between the two lineages and showed low concordance between the transects. The exception was a consistently low genetic exchange around ATXN1, a gene that modulates social behavior. We suggest that this gene may contribute to the male mate preferences that are known to cause lineage-assortative mating in this species. Although female choice modulates the degree of reproductive isolation in a variety of taxa, wall lizards demonstrate that both male-male competition and male mate choice can contribute to the extent of gene flow between lineages.     

Effect of Robertsonian translocations on sperm head form in the house mouse

Sperm morphology reflects a long process of adaptation to external conditions and the barriers encountered before ova fertilization can take place; however, not all morphological variation found in gametes can be explained by the effects of these selective forces, as the genetic component may also contribute to the establishment of different gametic features. In north‐eastern Spain, there is a wide Robertsonian system of Mus musculus domesticus, where individuals with 2n ranging from 27 to 40 chromosomes have been described. To elucidate the effect of the karyotype on sperm head form, a comparative analysis between different chromosomal groups of mice from this zone was carried out. Sperm heads from eight St (2n = 40) and 24 Rb (2n = 30–39) males were processed for scanning electron microscopy and analysed using geometric morphometric techniques. Canonical variate analyses showed substantial shape differences between St and Rb mice in the ventral spur region and between Rb groups in the post‐acrosomal region. Significant differences in sperm head size were also detected between chromosomal groups. Structural disorders related to spermatogenesis, genetic alterations, and epistatic interactions among loci are probably involved in the relationship between the phenotypic variation of the sperm head and Rb translocations. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 878–889.     

Contrasting patterns of hybridization in large house spiders (Tegenaria atrica group, Agelenidae)

The integrity of species is not fixed and may vary geographically. Here we investigate the geographic distributions and interactions of species in the Tegenaria atrica group (Araneae: Agelenidae). Detailed mapping of T. saeva and T. gigantea in England and Wales shows them to be broadly allopatric in southern England with a tightly defined, and possibly long-standing, narrow zone of parapatry in central southern England. In the north of England (Yorkshire), by contrast, the species are broadly sympatric as a result of recent range expansions. GIS techniques are used to map the species distributions and to quantify, we believe for the first time, the intimacy of interspecific interactions. The extent and nature of hybridization in these two areas is examined through regression and multivariate analyses of morphology. We show that the relative incidence of hybridization is much greater in Yorkshire than within the parapatric zone in the south. Clear patterns of asymmetric introgression are observed in both northern and southern England, with a greater impact of T. gigantea on T. saeva than vice versa. We find no sign of morphological reproductive character displacement at the zone of parapatry that might indicate reinforcement, although we cannot exclude more subtle effects, for example via cuticular pheromones. The integrity of these two species seems to be breaking down in northern England, a process that might gain momentum as the gene pools become more similar.     

Seeking signatures of reinforcement at the genetic level: a hitchhiking mapping and candidate gene approach in the house mouse

Reinforcement is the process by which prezygotic isolation is strengthened as a response to selection against hybridization. Most empirical support for reinforcement comes from the observation of its possible phenotypic signature: an accentuated degree of prezygotic isolation in the hybrid zone as compared to allopatry. Here, we implemented a novel approach to this question by seeking for the signature of reinforcement at the genetic level. In the house mouse, selection against hybrids and enhanced olfactory‐based assortative mate preferences are observed in a hybrid zone between the two European subspecies Mus musculus musculus and M. m. domesticus, suggesting a possible recent reinforcement event. To test for the genetic signature of reinforcing selection and identify genes involved in sexual isolation, we adopted a hitchhiking mapping approach targeting genomic regions containing candidate genes for assortative mating in mice. We densely scanned these genomic regions in hybrid zone and allopatric samples using a large number of fast evolving microsatellite loci that allow the detection of recent selection events. We found a handful of loci showing the expected pattern of significant reduction in variability in populations close to the hybrid zone, showing assortative odour preference in mate choice experiments as compared to populations further away and displaying no such preference. These loci lie close to genes that we pinpoint as testable candidates for further investigation.     

Strong premating divergence in a unimodal hybrid zone between two subspecies of the house mouse

Although selection against hybridization is expected to generate prezygotic divergence in unimodal hybrid zones, such a pattern has been seldom described. This study aims to better understand how prezygotic mechanisms may evolve in such zones. We investigated prezygotic divergence between populations of two subspecies of mice (Mus musculus musculus and M. m. domesticus) located at the edges of their unimodal hybrid zone in Denmark, and we developed an original multiple-population choice-test design, which allows assessment of within and between subspecies variation. Our study demonstrates that a strong assortative preference characterises one of the two subspecies (musculus) and that urinary signals are involved in this subspecies recognition. Taking into account the specific genetic and geographical characteristics of the Danish hybrid zone, we discuss the influence of the above pattern on its fate and the mechanisms that could have favoured this prezygotic divergence, among which the role of recombined populations constituting the core of the zone.     

A hybrid zone dominated by fertile F1s: maintenance of species barriers in Rhododendron

Isolating barriers between interbreeding sympatric or parapatric interfertile species are maintained by processes that occur within their hybrid zones. Although the effects of intrinsic selection on hybrid fitness are well known, less is understood about extrinsic fitness variation. At Tiryal Dag, northeast Turkey, Rhododendron x sochadzeae (R. ponticum x caucasicum) forms large populations in which neither segregation nor backcrossing occur, in habitats intermediate between those of its parents. Using single-copy species-specific random amplified polymorphic DNA and inter simple sequence repeat markers, it was determined that most or all R. x sochadzeae plants are F1s, and that there are many separate genets present. Hand pollination and germination experiments showed that R. x sochadzeae plants can produce viable seed of F2s or backcrosses in either direction. Furthermore, adult backcrosses have been observed in habitats atypical for R. x sochadzeae. From this, all non-F1 hybrid derivatives appear to be eliminated in the hybrid zone at Tiryal Dag as a result of postgermination selection. This absence of post-F1 hybrid derivatives apparently prevents introgression. This type of hybrid population is here termed an F1-dominated hybrid zone (F1DZ), and also occurs in Encelia. The observed dominance of F1s within a narrow habitat range is best explained by habitat-mediated superiority of F1s over all other genotype classes. Therefore, habitat-mediated selection against the second hybrid generation might be preventing interspecific gene flow in R. x sochadzeae. F1DZ formation is postulated to require the formation of F1s in quantity, habitat-mediated superiority in F1s, and highly specific habitat conditions.     

Many a slip: dissecting the causes of reproductive isolation in two species of Tegenaria spiders (Agelenidae)

Key to our understanding of the mechanisms underlying the process of speciation is the determination of the nature of the barriers to gene flow between related taxa. Species that show zero gene flow in sympatry or parapatry are of little use in this respect. In the present study, we used two closely‐related species of large house spider, Tegenaria saeva and Tegenaria gigantea, which hybridize to a limited extent along a natural contact zone in southern Britain. The species are apparently indistinguishable with respect to habitat utilization and phenology. Laboratory crosses using individuals from both allopatric and parapatric populations suggest that, although male and female courtship, as well as web and cuticular‐borne pheromones, are conserved between the species, mechanical difficulties are experienced during interspecific copulation. Copulation bouts are, on average, significantly shorter during interspecific matings because of these difficulties, and are probably not sufficiently long for effective sperm transfer to take place. In the two cases of successful interspecific crossing, and in subsequent F₁ and backcross generations, there are few indications of differential fertility, fecundity or viability, suggesting little post‐zygotic incompatibility. The high success rate of crosses between F₁ hybrids and both parental species underlines the principally mechanical barrier to gene flow between these taxa. Once this is breached, there appears to be little impediment to continuing introgression, which could, in some geographical areas at least, ultimately lead to the fusion of the two species. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 355–367.