Comparative analysis of micromorphological characters in two distantly related Mexican oaks, Quercus conzattii and Q. eduardii (Fagaceae), and their hybrids

Interspecific hybridization occurs with high frequency in the genusQuercus, but few studies have analyzed and compared micromorphological characters in putative parental species and their hybrids.Quercus eduardii andQ. conzattii are two Mexican black oak species that, although distantly related, have formed at least one population of hybrid origin, where individuals with intermediate macromorphology are present. The purpose of this investigation was to analyze the degree of differentiation in micromorphological characters between the two species and to assess the expression of these characters in individuals with intermediate macromorphology. Foliar trichomes, epicuticular waxes, stomata, and pollen grains, were examined using scanning electron microscopy (SEM) in the three types of individuals (Q. conzatti, Q. eduardii, and intermediates). Trichome density was quantified with light microscopy. Types of trichomes present, length of trichome arms, types of epicuticular wax on the leaf surfaces, and the position of stomata with respect to the foliar surface were characters useful to differentiate betweenQ. conzattii andQ. eduardii. Plants with intermediate macromorphology displayed a pattern of micromorphological characters that were identical to one parental species (Q. conzattii), or extreme or novel relative to both species.     

Taxonomic identity of Quercus coccifera L. in the Iberian Peninsula is maintained in spite of widespread hybridisation, as revealed by morphological, ISSR and ITS sequence data

Interspecific introgression is a well-known phenomenon in oaks whose ecological and evolutionary consequences, although relevant, are still unclear. We investigated molecular variation and any evidence for hybridisation in nine natural populations of kermes oak (Quercus coccifera L.) from the Iberian Peninsula. Additive patterns in 59 nrDNA ITS sequences revealed that hybrid individuals showing intermediate genotypes between kermes and holm (Q. ilex L.) oaks are very frequent, although intermediate morphotypes are uncommon. Bayesian analysis of ISSR fingerprinting patterns indicated extensive gene-flow among Q. coccifera populations and neighbouring Quercus taxa. Introgression appeared to be an active mechanism in the pair Q. coccifera–Q. ilex, but no conclusive evidence supporting hybridisation between Q. coccifera and other co-occurring Quercus species was found. The role of canalisation in the maintenance of stable morphological characters in the face of extensive introgression is discussed.     

Transgressive character expression in hybrid zones between the native invasives Tithonia tubaeformis and Tithonia rotundifolia (Asteraceae) in Mexico

Natural hybridization frequently promotes gene introgression among closely related species in sympatric populations, producing complex patterns of morphological variation. Therefore, a detailed understanding of the dynamics of interspecific gene flow and its morphological patterns is of widespread interest. We tested if introgressive hybridization promotes an increase in transgressive characters in comparison with the parental species. A sunflower species complex occurring in Mexico formed by two native invasive species, Tithonia tubaeformis and Tithonia rotundifolia, was analyzed using 46 morphological characters (leaf, flower and fruit) in five hybrid zones (N = 150 individuals) and two pure sites for each parental species (N = 80 individuals). In general, T. tubaeformis differed significantly from T. rotundifolia in all the examined characters, except six foliar and one inflorescence character. Morphological characters support the hypothesis of hybridization in this complex, even though both species remain morphological distinct in mixed stands. Individual hybrids appear to be a mosaic of parent-like (24.8 % of traits), intermediate (26.1 %) and transgressive (37.8 %) phenotypes (the remaining 11.3 % of the traits did not differ significantly from both parental species). Our results suggest that individuals from the same parental species were more similar among themselves than to putative hybrids, indicating occasional hybridization with segregation in hybrid types or backcrossing to parents. Evidence indicates a unidirectional pattern of gene flow toward T. rotundifolia.     

Variation in morphological traits in a recent hybrid zone between closely related Quercus liaotungensis and Q. mongolica (Fagaceae)

Aims Hybridization usually leads to gene introgression between related species in hybrid zones, associated with complex patterns of morphological variation. Nevertheless, previous studies have tended to ignore the effects of geographic variation in hybridization rates on species taxonomy. This study aims to investigate the variation of morphological traits between two sympatric and taxonomically confused oak species, Quercus liaotungensis and Q. mongolica, and reveal the effects of hybridization rates on morphological traits and the taxonomic boundary.Methods We used seven microsatellite loci to evaluate species status and measured 15 morphological traits in 26 trees in the recent hybrid zone between Q. liaotungensis and Q. mongolica, and we characterized the differences between the two oak species and their hybrids for the investigated traits.Important findings Molecular analyses indicated that 74% of 78 sampled maternal trees were hybrids between Q. liaotungensis and Q. mongolica although the observed morphological variation suggested that they had remained distinct species. Across all of the differentiated leaf and reproductive traits, the hybrids expressed patterns similar to Q. liaotungensis, which may suggest dominant expression of parental characters. These results are consistent with our expectation that hybrids will be difficult to distinguish from parental species in a recent hybrid zone.     

Community structure of canopy arthropods associated to Quercus crassifolia×Quercus crassipes complex

Quercus crassifolia and Q. crassipes are dominant species in temperate forests of central Mexico and hybridize between each other when they occur in sympatry. Oak canopies contain a considerable portion of arthropod diversity and the hybrid zones can provide new habitats to epiphyte fauna. We tested if the establishment of hybrids in contact zones with their parental hosts increases the species diversity of canopy arthropods assuming that hybrid trees constitute new genotypes of potential new habitats to small organisms. We examined the effect of hybridization on some community structure parameters (diversity, composition, similarity and density of arthropod fauna) of canopy arthropods compared to their parental species in a hybrid zone located in central Mexico. We employed 17 leaf morphological traits and six diagnostic RAPD primers to identify parental and hybrid plants. The RAPDs marker showed unidirectional introgression towards Q. crassifolia, and were detected hybrid (F₁), backcrosses and introgression individual trees. In total, 30 oak canopies were fogged during rainy and dry season. We recognized 532 taxa of arthropods belonging to 22 orders associated with tree canopies. The taxonomic status of host‐trees may be an important factor in the arthropod community structure and that seasonality (dry and rainy) is not a factor that could modify their organization. Trees of Q. crassipes registered the highest densities of arthropod fauna followed by hybrid hosts (F₁); trees originated by backcrosses towards Q. crassifolia registered a significant less arthropod density than F₁ hybrids; and trees of Q. crassifolia had the lowest density. Hybrid plants and Q. crassipes individuals had higher diversity (H′) of arthropods than Q. crassifolia plants. Hybrid plants had also more rare species in both seasons in comparison with parental species. This study suggests that hybrid oaks act as a center of biodiversity by accumulating arthropods of both parental and different species including a considerable number of rare species.     

Widespread hybridization and bidirectional introgression in sympatric species of coral reef fish

Coral reefs are highly diverse ecosystems, where numerous closely related species often coexist. How new species arise and are maintained in these high geneflow environments have been long‐standing conundrums. Hybridization and patterns of introgression between sympatric species provide a unique insight into the mechanisms of speciation and the maintenance of species boundaries. In this study, we investigate the extent of hybridization between two closely related species of coral reef fish: the common coral trout (Plectropomus leopardus) and the bar‐cheek coral trout (Plectropomus maculatus). Using a complementary set of 25 microsatellite loci, we distinguish pure genotype classes from first‐ and later‐generation hybrids, identifying 124 interspecific hybrids from a collection of 2,991 coral trout sampled in inshore and mid‐shelf reefs of the southern Great Barrier Reef. Hybrids were ubiquitous among reefs, fertile and spanned multiple generations suggesting both ecological and evolutionary processes are acting to maintain species barriers. We elaborate on these finding to investigate the extent of genomic introgression and admixture from 2,271 SNP loci recovered from a ddRAD library of pure and hybrid individuals. An analysis of genomic clines on recovered loci indicates that 261 SNP loci deviate from a model of neutral introgression, of which 132 indicate a pattern of introgression consistent with selection favouring both hybrid and parental genotypes. Our findings indicate genome‐wide, bidirectional introgression between two sympatric species of coral reef fishes and provide further support to a growing body of evidence for the role of hybridization in the evolution of coral reef fishes.     

Sympatric genome size variation and hybridization of four oak species as determined by flow cytometry genome size variation and hybridization

The Quercus species serve as a powerful model for studying introgression in relation to species boundaries and adaptive processes. Coexistence of distant relatives, or lack of coexistence of closely relative oak species, introgression may play a role. In the current study, four closely related oak species were found in Zijinshan, China. We generated a comprehensive genome size (GS) database for 120 individuals of four species using flow cytometry‐based approaches. We examined GS variability within and among the species and hybridization events among the four species. The mean GSs of Q. acutissima, Q. variabilis, Q. fabri, and Q. serrata var. brevipetiolata were estimated to be 1.87, 1.92, 1.97, and 1.97 pg, respectively. The intraspecific and interspecific variations of GS observed among the four oak species indicated adaptation to the environment. Hybridization occurred both within and between the sections. A hybrid offspring was produced from Q. fabri and Q. variabilis, which belonged to different sections. The GS evolutionary pattern for hybrid species was expansion. Hybridization between the sections may be affected by habitat disturbance. This study increases our understanding of the evolution of GS in Quercus and will help establish guidelines for the ecological protection of oak trees.     

Leaf morphological analyses in four European oak species (Quercus) and their hybrids: A comparison of traditional and geometric morphometric methods

Abstract

In this study, leaf morphology was assessed in a mixed oak stand (western France) using two geometric morphometric (landmark and outline) datasets and one dataset of 19 leaf measures. Adult oaks (817 oaks), comprising four white oak species (Quercus petraea, Q. robur, Q. pubescens and Q. pyrenaica), were sampled for DNA extraction and genetic analysis (nuclear microsatellites). Leaf morphology was assessed on 336 oaks, comprising pure species and hybrids as determined by genetic assignment. This comparative study of oak leaf morphology, based on the use of two free size geometric morphometric methods and a set of leaf measurements, combined with the genetic assignment of individuals to pure species or hybrids, provided information about the differences among species and the intermediate leaf morphology of their hybrids.     

Structure of herbivore communities in two oak (Quercus spp.) hybrid zones

Summary We examined patterns of density and species diversity for leaf-mining Lepidopterans and gall-forming Hymenopterans in two oak (Quercus spp.) hybrid zones: Quercus depressipes x Q. rugosa and Q. emoryi x Q. coccolobifolia. In both species complexes, hybrid hosts typically supported significantly lower densities and species diversity of parasites than did parental types. This contradicts the findings of Whitham (1989) that suggested that hybrid hosts may act as parasite sinks both in ecological and evolutionary time. We discuss features of hybrid zones that are likely to influence patterns of herbivory.     

SIGNIFICANT ROLE FOR HISTORICAL EFFECTS IN THE EVOLUTION OF REPRODUCTIVE ISOLATION: EVIDENCE FROM PATTERNS OF INTROGRESSION BETWEEN THE CYPRINID FISHES,LUXILUS CORNUTUS AND LUXILUS CHRYSOCEPHALUS

Samples of Luxilus cornutus, Luxilus chrysocephalus, and their hybrids were collected along hypothesized routes of dispersal from Pleistocene refugia to examine the significance of geographic variation in patterns of introgression between these species. Patterns of allozyme and mitochondrial DNA (mtDNA) variation were generally consistent with those from previous studies. Tests of Hardy-Weinberg equilibrium revealed significant deficiencies of heterozygotes in all samples, indicating some form of reproductive isolation. Mitochondrial DNAs of each species were not equally represented in F₁ hybrids; however, this bias was eliminated when the two largest samples were excluded from the analysis. Backcross hybrids exhibited biased mtDNA introgression, as samples from Lake Erie (eastern) and Lake Michigan (western) drainages showed significant excesses of mtDNAs from L. chrysocephalus and L. cornutus, respectively, relative to frequencies of diagnostic allozyme markers. The extent and direction of allozyme and mtDNA introgression was quantified by calculating isolation index values from morphologically “pure” individuals of each species from each locality. Analysis of variance of these measures identified limited introgression of allozyme variants with no geographic pattern, but significant differences in direction of mtDNA introgression between drainages (i.e., postglacial dispersal route). Association between patterns of mtDNA introgression and dispersal route across the latitudinal width of the contact zone is best explained by genetic divergence during past isolation of ancestral populations from these drainages. These results identify a significant role for historical effects in the evolution of reproductive isolation and the process of speciation.     

Effect of hybridization of the Quercus crassifolia×Quercus crassipes complex on the community structure of endophagous insects

In a previous study, we showed that the geographic proximity of hybrid plants to the allopatric areas of parental species increases their morphological and genetic similarity with them. In the present work, we explored whether the endophagous fauna of hybrid plants show the same pattern. We studied the canopy species richness, diversity and composition of leaf-mining moths (Lepidoptera: Tischeridae, Citheraniidae) and gall-forming wasps (Hymenoptera: Cynipidae) associated with two species of red oaks (Quercus crassifolia and Quercus crassipes) and their interspecific hybrid (Quercus×dysophylla Benth pro sp.) in seven hybrid zones in central Mexico, during four seasons in 2 years. The study was conducted on 194 oak trees with known genetic status [identified by leaf morphology and molecular markers (random amplified polymorphic DNAs)], and the results indicate a bidirectional pattern of gene flow. Hybrid plants supported intermediate levels of infestation of gall-forming and leaf-mining insects compared to their putative parental species. The infestation level of leaf-mining insects varied significantly following the pattern: Q. crassifolia>hybrids>Q. crassipes, whereas the gall-forming insects showed an inverse pattern. A negative and significant relationship was found between these two types of insect guilds in each host taxa, when the infestation percentage was evaluated. It was found that 31.5% (n=11) of the endophagous insects were specific to Q. crassipes, 22.9% (n=8) to Q. crassifolia, and 8.6% (n=3) to hybrid individuals. The hybrid bridge hypothesis was supported in the case of 25.7% (n=9) of insects, which suggests that the presence of a hybrid intermediary plant may favor a host herbivore shift from one plant species to another. Greater genetic diversity in a hybrid zone is associated with greater diversity in the endophagous community. The geographic proximity of hybrid plants to the allopatric site of a parental species increases their similarity in terms of endophagous insects and the Eje Neovolcánico acts as a corridor favoring this pattern. Electronic Supplementary Material Supplementary material is available for this article at     

The micromorphologies of seed coats and petal trichomes of the taxa of Viola subsect. Boreali-Americanae (Violaceae) and their utility in discerning orthospecies from hybrids

Viola subsect.Boreali-Americanae, confined to North America including northern México, has long been considered one of the most taxonomically difficult temperate groups of the angiosperms. Hybridization, followed in some localities by introgression, has made it difficult for systematists to discern the “core” species by using only standard macromorphological characters. Analyses that employed scanning electron microscopy (SEM) of seed coats and petal trichomes generated new characters. SEM of the surface of seeds of each of the orthospecies revealed a suite of primary and secondary sculpture characters unique to each species that are correlated with a suite of macromorphological characters. In a number of species, SEM of petal trichomes provided additional unique characters. SEM of the surface of seed coats enabled, also, the identification of instances of hybridization by revealing blends of structures of the primary and secondary sculpture typical of different orthospecies and instances of putative introgression by revealing deviations in surface structures from those typical of orthospecies. A procedure based on iterative examinations of macromorphology and micromorphology, which was developed in this research, enable the discernment of hybrids and putative introgressants. Combining the micromorphological and macromorphological data with ecological and phytogeographical data enabled the delimitation of sixteen orthospecies.     

Trichomes and photosynthetic pigment composition changes: responses of <Emphasis Type="Italic">Quercus ilex </Emphasis>subsp. <Emphasis Type="Italic">ballota</Emphasis> (Desf.) Samp. and<Emphasis Type="Italic"> Quercus coccifera</Emphasis> L. to Mediterranean stress conditions

Sun and shade leaves of two Mediterranean Quercus species, Quercus ilex subsp. ballota (Desf.) Samp. and Quercus coccifera L., were compared by measuring leaf optical properties, photosynthetic pigment composition and photosystem II efficiency. The presence of trichomes in the adaxial (upper) leaf surface of Q. ilex subsp. ballota seems to constitute an important morphological mechanism that allows this species to maintain a good photosystem II efficiency during the summer. Q. coccifera has almost no trichomes and seems instead to develop other physiological responses, including a smaller light-harvesting antenna size, higher concentrations of violaxanthin cycle pigments and a higher (zeaxanthin + antheraxanthin)/(violaxanthin + antheraxanthin + zeaxanthin) ratio. Q. coccifera was not able to maintain a good photosystem II efficiency up to the end of the summer. In Q. ilex subsp. ballota leaves, natural loss or mechanical removal of adaxial-face leaf trichomes induced short-term decreases in photosystem II efficiency. These changes were accompanied by de-epoxidation of violaxanthin cycle pigments, suggesting that the absence of trichomes would trigger physiological responses in this species. Our data have revealed different patterns of response of Q. ilex subsp. ballota and Q. coccifera facing the stress conditions prevailing in the Mediterranean area.     

Differential effects of host plant hybridization on herbivore community structure and grazing pressure on forest canopies

Interspecific hybridization in plants is known to have ecological effects on associated organisms. We examined the differences in insect herbivore community structure and grazing pressure on tree canopy leaves among natural hybrids and their parental oak species. We measured leaf traits, herbivore community structure, and grazing pressure on leaves of two oak species, Quercus crispula and Q. dentata, and their hybrids. The concentration of nitrogen in canopy leaves was greater in hybrids and in Q. dentata than in Q. crispula. The concentration of total phenolics was lower in hybrids than in Q. crispula. The concentration of condensed tannin was greater in hybrids than in Q. crispula. Relative herbivore abundance and species richness were greater on oak hybrids than on either parental species; herbivore species diversity and composition on hybrids were close to those on Q. crispula. Herbivore grazing pressure was lower on hybrids and Q. dentata than on Q. crispula. There was a negative correlation between herbivore grazing pressure and leaf nitrogen, suggesting that interspecific variation among oak taxa in herbivore pressure may be explained by leaf nitrogen; variation in herbivore community structure among oak taxa is likely to be controlled by polygenic leaf traits. Differing responses of (1) herbivore community structure and (2) herbivore grazing pressure to host plant hybridization may play important roles in regulating herbivore biodiversity in cool‐temperate forest canopies.     

Human disturbance causes the formation of a hybrid swarm between two naturally sympatric fish species

Most evidence for hybrid swarm formation stemming from anthropogenic habitat disturbance comes from the breakdown of reproductive isolation between incipient species, or introgression between allopatric species following secondary contact. Human impacts on hybridization between divergent species that naturally occur in sympatry have received considerably less attention. Theory predicts that reinforcement should act to preserve reproductive isolation under such circumstances, potentially making reproductive barriers resistant to human habitat alteration. Using 15 microsatellites, we examined hybridization between sympatric populations of alewife (Alosa pseudoharengus) and blueback herring (A. aestivalis) to test whether the frequency of hybridization and pattern of introgression have been impacted by the construction of a dam that isolated formerly anadromous populations of both species in a landlocked freshwater reservoir. The frequency of hybridization and pattern of introgression differed markedly between anadromous and landlocked populations. The rangewide frequency of hybridization among anadromous populations was generally 0–8%, whereas all landlocked individuals were hybrids. Although neutral introgression was observed among anadromous hybrids, directional introgression leading to increased prevalence of alewife genotypes was detected among landlocked hybrids. We demonstrate that habitat alteration can lead to hybrid swarm formation between divergent species that naturally occur sympatrically, and provide empirical evidence that reinforcement does not always sustain reproductive isolation under such circumstances.     

Hybridization and introgression in a mixed population of the intertidal seaweeds Fucus evanescens and F. serratus

The introduced Fucus evanescens (hermaphroditic) and the native F. serratus (dioecious) have been in secondary contact along the Danish coast of the Kattegat Sea for 60–100 years and dioecious hybrids have been observed at Blushøj for several years. Hybridization in Fucus is unusual because it appears to always involve a hermaphroditic and a dioecious parental pair. We determined the degree and spatial patterns of introgression for 286 individuals using 10 microsatellite loci and cpDNA. Hybrids accounted for nearly 13% of the population, yet parental species were well differentiated (F_ST = 0.633). The presence of F. evanescens chloroplasts in 100% of F₁ hybrids revealed asymmetrical hybridization. Fucus evanescens cpDNA was observed in 50% of introgressed and 5.4% of pure F. serratus, but no F. serratus cpDNA was found in F. evanescens. In contrast, nuclear DNA introgression was symmetrical with an equal amount (≈1.5%) of genes introgressed into each parental species. Survivorship and viability data suggest selection against hybrids in the hybrid zone.     

Osmotic stress responses of individual white oak (Quercus section, Quercus subgenus) genotypes cultured in vitro

White oaks (Quercus section, Quercus subgenus) are widely distributed in Europe. Quercus petraea (sessile oak), an economically important species is predicted to be affected by climate change. Q. pubescens (pubescent oak) and Q. virgiliana (Italian pubescent oak) are economically less important, drought tolerant species. Frequent hybridization of white oaks was observed and currently the introgression of Q. pubescens and Q. virgiliana in non-mediterranean regions of Europe has been reported. Our goal was to use tissue cultures established from individual trees of the above taxa and their putative hybrids, all present in the forest stand of Síkf?kút LTER Research Area (NE Hungary) as simple experimental model systems for studying drought/osmotic stress tolerance. Tissue cultures are more suitable models for such studies, than seedlings, because they are genetically identical to the parent plants. Polyethylene glycol (PEG6000) treatments were used for this purpose. The identification of taxa was based on leaf morphological traits and microsatellite analysis and showed that Q. petraea is genetically distinct to all other taxa examined. We established six callus lines of Quercus. As expected, in Q. petraea cultures PEG6000 induced severe loss of fresh weight and the ability to recover after removal of the osmoticum, which was not characteristic for Q. pubescens and Q. virgiliana. Putative hybrids exhibited an intermediate response to osmotic stress. Activity gels showed the increase of single-strand preferring (SSP) nuclease and no significant change of guaiacol-peroxidase activities in drought-sensitive genotypes/cultures and no significant increase of SSP nuclease activities accompanied with increases of guaiacol-peroxidase activities in drought-tolerant ones. This indicates that drought/osmotic stress tolerance is associated to increased capacity of scavenging reactive oxygen species and hence less susceptibility to DNA damage. Our results confirm that tissue cultures of oak are suitable model systems for studying drought/osmotic stress responses.     

Hybridization and divergence in multi‐species oak (Quercus) communities

Oaks (Quercus: Fagaceae) commonly interbreed yet retain their morphological, genetic and ecological distinctiveness. Post‐zygotic isolation mechanisms, such as ecologically dependent selection on adaptive loci, may therefore limit introgression. To test this hypothesis, we quantified hybridization and genetic divergence across the contact zone of four red oaks (Quercus section Lobatae) in the Great Lakes region of North America using a suite of 259 amplified fragment length polymorphisms and 27 genic and genomic microsatellite markers. First, we identified hybrids using genetic structure analysis and confirmed the reliability of our assignments via simulations. Then, we identified candidate loci for species maintenance with three complementary tests for selection and obtained partial gene sequences linked to an outlier locus and three other loci. We detected evidence of recent hybridization among all species and considerable gene flow between Q. ellipsoidalis and Q. velutina. Overall, c. 20% of Q. velutina had recent ancestry from Q. ellipsoidalis, whereas nearly 30% of Q. ellipsoidalis had a Q. velutina ancestor. Most loci were negligibly to weakly differentiated among species, but two gene‐linked microsatellites deviated significantly from neutral expectations in multiple, complementary outlier tests. Both outlier loci were located in the same 15‐cM bin on an existing Q. robur linkage map, a region under divergent selection in other oak species. Adaptive loci in this highly differentiated genomic region may contribute to ecological divergence among species and limit introgression.