Putative Hybrid Individuals Research Articles

How we look: European wild mouflon and feral domestic sheep hybrids.

Hybridization between wild animals and feral individuals from closely related domestic species can occur when they share the same habitat. Such events are generally regarded as a threat to the genetic integrity and survival of established wild populations. The aim of this study is to confirm a hybridization between European mouflon and feral sheep on the Adriatic island Dugi Otok, where a group of individuals phenotypically indicative of a possible introgression was photographed. Using an ovine medium-density SNP array genotypes, we provided genetic evidence of recent gene admixture between the two species. The ADMIXTURE analysis suggests that two hybrid individuals had 82.2% and 94.1% of their ancestry attributable to feral sheep, respectively, with the rest of the mouflon in origin. NEWHYBRIDS analysis estimated the combined probabilities for the putative hybrid individuals belonging to one of the hybrid classes at 87.0% and 90.1%, respectively. The main phenotypic differences in the hybrids were observed in the shape of the horns, a stronger horn circumference than in the mouflon and weaker than for the sheep, pronounced horn rings, a shorter tail, and intermediate color. We therefore recommend constant monitoring of the purity of the mouflon population to maintain its genetic integrity. In addition, all feral sheep should be removed from the wild, and released sheep raised in semi-wild conditions should be better controlled.

Comparative genomics of two Empidonax flycatchers reveal candidate genes for bird song production.

Whole-genome level comparisons of sister taxa that vary in phenotype against a background of high genomic similarity can be used to identify the genomic regions that might underlie their phenotypic differences. In wild birds, this exploratory approach has detected markers associated with plumage coloration, beak and wing morphology, and complex behavioral traits like migration. Here, we use genomic comparisons of two closely related suboscine flycatchers (Empidonax difficilis and E. occidentalis) and their hybrids to search for candidate genes underlying their variation in innate vocal signals. We sequenced the genomes of 20 flycatchers that sang one of two species-specific parental song types and 14 putative hybrid individuals with intermediate song types. In the resulting genomic comparisons, we found six areas of high differentiation that may be associated with variation in non-learned songs. These narrow regions of genomic differentiation contain a total of 67 described genes, three of which have been previously associated with forms of language impairment and dyslexia in humans, and 18 of which are known to be differentially expressed in the song nuclei regions of the avian brain compared to adjacent parts of the avian brain. This 'natural experiment' therefore may help identify loci associated with song differences that merit further study across bird lineages with both learned and innate vocalizations.

Morphological and molecular evidence of the hybrid origin of Crepidiastrum ×muratagenii in Korea

The plant “Hong-do-go-deul-ppae-gi” has been considered as Crepidiastrum × muratagenii, a hybrid between C. denticulatum and C. lanceolatum, based on its morphological traits and geographical distribution. To reveal the hybrid origin of Hong-do-go-deul-ppae-gi, we examined additional morphological traits of this plant and its putative parents (C. denticulatum, C. lanceolatum, C. platyphyllum) and analyzed one nuclear ribosomal internal transcribed spacer (ITS) region and four chloroplast regions (trnT-L, trnL-F, rpl16 intron, and rps16 intron). As a result of examining the morphological traits, putative hybrid individuals were classified into three types based on the habit, cauline leaf, outer phyllary, and achene beak traits. A molecular analysis found that the ITS sequences of Type 1 and Type 2 individuals showed additive species-specific sites of C. denticulatum and C. lanceolatum. Plastid sequences of Type 1 and Type 2 individuals showed C. denticulatum and C. lanceolatum sequences, respectively. However, Type 3 individuals had ITS and plastid sequences corresponding to C. denticulatum. Accordingly, Type 1 and Type 2 individuals not only share morphological traits with C. denticulatum and C. lanceolatum but also show additive species-specific sites for C. denticulatum and C. lanceolatum, and not C. platyphyllum, supporting its origin as a hybrid between C. denticulatum and C. lanceolatum. Type 3 had morphological traits similar to other hybrid types but was distinguished with respect to outer phyllaries and demonstrated some resemblance to C. denticulatum. In a molecular analysis, Type 3 was found to be identical with regard to the sequence of C. denticulatum and was judged to be an ecological variation of C. denticulatum.

Novel Post-Glacial Haplotype Evolution in Birch—A Case for Conserving Local Adaptation

Despite constituting the western-most edge of the population distributions for several native European plants, Ireland has largely been left out of key Europe-wide phylogeographic studies. This is true for birch (Betula pubescens Ehrh. and Betula pendula Roth), for which the genetic diversity has yet to be mapped for Ireland. Here we used eight cpDNA markers (two Restriction Fragment Length Polymorphism (RFLP) and six Simple Sequence Repeat (SSR)) to map the genetic diversity of B. pubescens, B. pendula, and putative hybrid individuals sampled from 19 populations spread cross most of the island of Ireland. Within Ireland, 11 distinct haplotypes were detected, the most common of which (H1) was also detected in England, Scotland, France, and Norway. A moderate level of population structuring (GST = 0.282) was found across Ireland and the genetic diversity of its northern populations was twice that of its southern populations. This indicates that, unlike other native Irish trees, such as oak and alder, post-glacial recolonization by birch did not begin in the south (i.e., from Iberia). Rather, and in agreement with palynological data, birch most likely migrated in from eastern populations in Britain. Finally, we highlight Irish populations with comparatively unique genetic structure which may be included as part of European genetic conservation networks.

Crossability of Putative Hybrids of <i>Pinus sylvestris</i> and <i>Pinus mugo</i> with Their Parents

Artificial hybridization experiment with the Pinus sylvestris and Pinus mugo putative hybrid individuals and their parents revealed a selective nature of their crossability conditioned by the identical chloroplast DNA (cpDNA) haplotypes of the crossed individuals. Efficiency of hybridization between hybrid individual H1 of P. sylvestris haplotype and P. sylvestris paternal tree was relatively high, as evidenced by 78.03% of filled seeds. On the contrary, the crossing of H1 individual with P. mugo was unsuccessful. Likewise, the crossability of the H2 individual of P. mugo haplotype with the paternal tree P. mugo resulted in 31.15% of filled seeds. A relatively high crossability was characteristic also for the H1 and H2 hybrid trees of different cpDNA haplotypes. A conclusion has been drawn postulating weakened reproduction barrier between hybrid swarm individuals and the paternal species P. sylvestris and P. mugo. Differentiation of the putatively hybrid individuals from the parental species is accompanied by the variation in cpDNA inheritance.

Reinforced evidence on partial compatibility between Pinus sylvestris and Pinus mugo and on maternal inheritance of chloroplast DNA in the Pinus mugo × Pinus sylvestris cross

Abstract The crossability relationship between Scots pine (Pinus sylvestris L.) and mountain dwarf pine (Pinus mugo Turra) was tested under field conditions using an artificial pollination approach. There was partial compatibility between the parental species, as evidenced by the amount of filled seeds in their reciprocal crossings and in control variants from self-pollination, controlled intraspecific outcrossing and open pollination of mother trees. The crossability degree in P. sylvestris × P. mugo was characterised by the index 0.15, and the reciprocal crossing by the index 0.18. Crossability of P. sylvestris and P. mugo with their putative hybrid individuals was much higher; the number of filled seeds was comparable with that of the control variants. The reciprocal crossings of P. sylvestris and P. mugo species were highlighted by the opposite inheritance of their chloroplast DNA (cpDNA). The paternal cpDNA inheritance in P. sylvestris × P. mugo and maternal cpDNA inheritance in P. mugo × P. sylvestris was repeatedly confirmed using the cpDNA trnV-trnH/HinfI marker as well as the newly developed partial cpDNA trnV-trnH/AseI marker. The nature of the latter is described in terms of the nucleotide sequence.

Molecular evidence for natural hybridization betweenLigularia nelumbifoliaandCremanthodium stenoglossum(Asteraceae, Senecioneae)

Natural hybridization occurred frequently in the sunflower family. To date, however, no study on natural hybridization involving in Ligularia and Cremanthodium has been reported. Here, we presented the molecular evidence for natural hybridization between Ligularia nelumbifolia (Bureau & Franch.) Hand.-Mazz. and Cremanthodium stenoglossum Ling & S.W.Liu. Four nuclear DNA regions were sequenced to test the natural hybridization hypothesis, and three chloroplast DNA regions were sequenced to determine the direction of hybridization. Analyses of the investigated DNA data suggested that all of the putative hybrid individuals were derived from hybridization between L. nelumbifolia and C. stenoglossum and that bidirectional hybridization occurred. Moreover, sympatric Ligularia tsangchanensis (Franch.) Hand.-Mazz. and Ligularia virgaurea (Maxim.) Mattf. ex Rehder & Kobuski were not apparently involved in the hybridization. Although NewHybrids analysis showed that all the putative hybrid individuals were F1class, a low frequency of backcrossing to C. stenoglossum might exist in the hybrid swarm. In such a case, hybrids might serve as a bridge facilitating gene flow between L. nelumbifolia and C. stenoglossum, and hybrid speciation is unlikely to happen for these hybrid individuals without asexual reproduction. Given the poorly resolved phylogenetic relationship between Ligularia and Cremanthodium, the occurrence of natural hybridization between L. nelumbifolia and C. stenoglossum might provide new insights into the recircumscription and redelimitation of these two genera.

Breakdown of species boundaries in Mandevilla: floral morphological intermediacy, novel fragrances and asymmetric pollen flow.

Phenotypic intermediacy is an indicator of putative hybrid origin and has provided the main clues to discovering hybrid plants in nature. Mandevilla pentlandiana and M.laxa (Apocynaceae) are sister species with clear differences in floral phenotype and associated pollinator guilds: diurnal Hymenoptera and nocturnal hawkmoths, respectively. The presence of individuals with intermediate phenotypes in a wild population raises questions about the roles of visual and olfactory signals (i.e. corolla morphology and floral fragrances) as barriers to interbreeding, and how the breakdown of floral isolation occurs. We examined phenotypic variation in a mixed Mandevilla population, analysing the chemical composition of floral fragrances, characterising floral shape through geometric morphometrics and assessing individual grouping through taxonomically relevant traits and an unsupervised learning algorithm. We quantified the visitation frequencies of floral visitors and tracked their foraging movements using pollen analogues. The presence of morphologically intermediate individuals and pollen analogue movement suggested extensive hybridisation between M.laxa and M.pentlandiana, along with asymmetrical rates of backcrossing between these putative hybrids and M.laxa. Floral volatiles from putative hybrid individuals showed a transgressive phenotype, with additional compounds not emitted by either parental species. Our results suggest the presence of a hybrid swarm between sympatric M.pentlandiana and M.laxa and indicate that initial hybridisation events between these parental species are rare, but once they occur, visits between putative hybrids and M.laxa are common and facilitate continued introgression.

On the origin of orphan hybrids between Aquilegia formosa and Aquilegia flavescens.

We report the investigation of an Aquilegia flavescens × A. formosa population in British Columbia that is disjunct from its parents—the latter species is present locally but ecologically separated, while the former is entirely absent. To confirm hybridity, we used multivariate analysis of floral characters of field-sampled populations to ordinate phenotypes of putative hybrids in relation to those of the parental species. Microsatellite genotypes at 11 loci from 72 parental-type and putative hybrid individuals were analysed to assess evidence for admixture. Maternally inherited plastid sequences were analysed to infer the direction of hybridization and test hypotheses on the origin of the orphan hybrid population. Plants from the orphan hybrid population are on average intermediate between typical A. formosa and A. flavescens for most phenotypes examined and show evidence of genetic admixture. This population lies beyond the range of A. flavescens, but within the range of A. formosa. No pure A. flavescens individuals were observed in the vicinity, nor is this species known to occur within 200 km of the site. The hybrids share a plastid haplotype with local A. formosa populations. Alternative explanations for this pattern are evaluated. While we cannot rule out long-distance pollen dispersal followed by proliferation of hybrid genotypes, we consider the spread of an A. formosa plastid during genetic swamping of a historical A. flavescens population to be more parsimonious.

Increased divergence in floral morphology strongly reduces gene flow in sympatric sexually deceptive orchids with the same pollinator

In sexually deceptive orchids, pollinator specificity is the main factor responsible for species isolation. The two sexually deceptive orchids Ophrys chestermanii and Ophrys normanii occur sympatrically on Sardinia and attract the same pollinator, males of Bombus vestalis, by producing the same odour bouquets. Since gene flow between these two species has been shown to be almost absent, their genetic distinctness seems to be preserved by other forms of reproductive barrier than pollinator specificity. The aim of this study is to investigate the nature and strength of these isolation barriers which apparently halt gene flow between these two orchids that share the same pollinator. Morphological measurements, combined with observations of cross-pseudocopulations, revealed that, in sympatry, the longer caudicles of O. normanii and the smaller and differently shaped stigmatic cavity in O. chestermanii are the main factors preventing gene flow between these two sympatric species. In contrast, other pre- or post-mating barriers seem to be weak or absent, as the two species have overlapping flowering time and we were able to rear hybrid offspring from interspecific manual crosses. At the same time, genetic analysis of orchid plantlets collected at sympatric sites detected the presence of only two putative hybrid individuals. Thus, the probability of natural cross-pollination between O. normanii and O. chestermanii seems to be strongly limited by divergence in their floral morphology. For some traits, this divergence was found increased between sympatric populations.

A last stand in the Po valley: genetic structure and gene flow patterns in Ulmus minor and U. pumila.

Ulmus minor has been severely affected by Dutch elm disease (DED). The introduction into Europe of the exotic Ulmus pumila, highly tolerant to DED, has resulted in it widely replacing native U. minor populations. Morphological and genetic evidence of hybridization has been reported, and thus there is a need for assessment of interspecific gene flow patterns in natural populations. This work therefore aimed at studying pollen gene flow in a remnant U. minor stand surrounded by trees of both species scattered across an agricultural landscape. All trees from a small natural stand (350 in number) and the surrounding agricultural area within a 5-km radius (89) were genotyped at six microsatellite loci. Trees were morphologically characterized as U. minor, U. pumila or intermediate phenotypes, and morphological identification was compared with Bayesian clustering of genotypes. For paternity analysis, seeds were collected in two consecutive years from 20 and 28 mother trees. Maximum likelihood paternity assignment was used to elucidate intra- and interspecific gene flow patterns. Genetic structure analyses indicated the presence of two genetic clusters only partially matching the morphological identification. The paternity analysis results were consistent between the two consecutive years of sampling and showed high pollen immigration rates (∼0·80) and mean pollination distances (∼3 km), and a skewed distribution of reproductive success. Few intercluster pollinations and putative hybrid individuals were found. Pollen gene flow is not impeded in the fragmented agricultural landscape investigated. High pollen immigration and extensive pollen dispersal distances are probably counteracting the potential loss of genetic variation caused by isolation. Some evidence was also found that U. minor and U. pumila can hybridize when in sympatry. Although hybridization might have beneficial effects on both species, remnant U. minor populations represent a valuable source of genetic diversity that needs to be preserved.

Hybridization betweenMauremys japonicaandMauremys reevesiiInferred by Nuclear and Mitochondrial DNA Analyses

The Japanese Pond Turtle, Mauremys japonica, and the Reeves' Pond Turtle, Mauremys reevesii, are common aquatic geoemydid species in Japan. The former is endemic to Japan, whereas the latter is distributed widely throughout East Asia, China, Korea, Taiwan, and Japan. Although the Japanese populations of M. reevesii were generally considered to be indigenous, it was suggested recently that Japanese M. reevesii is derived from multiple artificial introductions from elsewhere in Asia. Putative hybrid individuals, with intermediate morphological characters, have been observed in the wild. Furthermore, captive congeneric individuals have produced viable offspring that attained sexual maturity. To clarify the extent of hybridization between these species, we examined nuclear DNA and mitochondrial DNA genetic markers of putative hybrids collected in the field. We found that most of the animals assayed had nuclear DNA derived from both species. In addition, several individuals originated from hybrid mothers implying that hybridization is indeed occurring in the wild and that female hybrids are fertile and reproduce. Our results indicate an ongoing process of introgression of introduced M. reevesii alleles into the gene pool of native populations of M. japonica.

Hybridization betweenCalopteryx splendensandC. haemorrhoidalisconfirmed by morphological and genetic analyses

Hybridization between Calopteryx haemorrhoidalis and any of its congeners has not been reported until now. We observed spontaneous matings between male C. splendens and female C. haemorrhoidalis at a locality in Central Italy, together with some putative hybrid individuals that had a mixed phenotype. Here, we report the morphological and molecular characterization of five suspected hybrids collected from this population during 2001 (n=1), 2012 (n=2) and 2013 (n=2). A discriminant analysis based on 13 morphological variables correctly separated both parental species (with 100% assignation success) and classified the hybrid from 2001 as splendens phenotype and those from 2012 and 2013 as haemorrhoidalis. Genotype data (microsatellite loci) was used to confirm the hybrid origin of these specimens, although there were differences between the individual from 2001 and those from 2012 and 2013; the 2001 individual had alleles that were present in both parent species, suggesting it is an F1 hybrid, but the indivi...

Serological characteristics of some putative hybrid individuals from a Pinus sylvestris x Pinus mugo hybrid swarm population

The antigenic properties of some putative hybrid individuals from a hybrid swarm population were studied. All the plants were to a major or lesser degree intermediate between three pure taxa, i.e. <em>P. sylvestris, P. mugo</em> and <em>P. uliginosa</em>. The plants are, however, not intermediate in general, but are sin-Mar to one species in part of their antigenic properties (enzymatic systems) and to the other one in the rest. It means that there is no common trend in the variation of the antigenic characters (proteins) and they behave independently.

한국산 쑥속(Artemisia L.) 산흰쑥절(sect. Absinthium)의 분류학적 재검토

The morphological characters and scientific names of Korean Artemisia sect. Absinthium were reviewed based on the type specimen, the original description, observation of the living materials and voucher specimens. The form of the habitat, the structure of underground, the leaf shape and capitulum features were valuable characters in identifying the taxa. The sect. Absinthium in Korea was classified as three taxa of A. sieversiana, A. lagocephala, A. fauriei. The illustration and description of each taxa were provided. Particularly, A. fauriei in this section with hair on receptacle and A. fukudo in sect. Abrotanum without hair on receptacle grow in mixed groups on the West shore of Korea. Two species with populations which grow in mixed groups, was observed as being putative hybrid individuals based on morphological characters.

Is a new invasive herb emerging? Molecular confirmation and preliminary evaluation of natural hybridization between the invasive Sphagneticola trilobata (Asteraceae) and its native congener S. calendulacea in South China

Sphagneticola trilobata is an invasive herb in South China. Here, we reported some morphological intermediates between S. trilobata and the indigenous congener S. calendulacea in the field for the first time, which were suspected to be hybrids. Morphological and molecular methods were used to test this hybrid origin hypothesis, and eco-physiological parameters were measured to evaluate the hybrid’s invasion potential. The hybrid origin hypothesis was confirmed by analysis of microsatellite and nrITS sequences. Bayesian assignment analysis based on six microsatellite loci genotypes separated the studied individuals into three groups, which corresponded to the two parent species and the putative hybrids that were identified on the basis of leaf morphology. The hybrid individuals were categorized as F1 hybrids with membership coefficients (Qi values) close to 0.5. Phylogenetic analysis based on nrITS data showed that the putative hybrid individuals possessed two types of species-level divergent clones, and were inseparate with S. trilobata clade and S. calendulacea clade, respectively. The maternally-inherited chloroplast marker atpB-rbcL demonstrated that the invasive species functioned as the pollen donors. Ecophysiological studies on photosynthesis, specific leaf area (SLA), leaf nutrient concentration, and construction cost in the parental species and the hybrid demonstrated that the hybrid showed slightly lower or equivalent growth potential compared to the invasive species and significant higher energy-use efficiency than both parents. As such, it seems that a new invasive herb is emerging. As a consequence of this hybridization, the native species S. calendulacea may be disappearing rapidly, which presents a major concern for conservation efforts.

Bayesian analysis of hybridization and introgression between the endangered european mink (Mustela lutreola) and the polecat (Mustela putorius)

Human-mediated global change will probably increase the rates of natural hybridization and genetic introgression between closely related species, and this will have major implications for conservation of the taxa involved. In this study, we analyse both mitochondrial and nuclear data to characterize ongoing hybridization and genetic introgression between two sympatric sister species of mustelids, the endangered European mink (Mustela lutreola) and the more abundant polecat (M. putorius). A total of 317 European mink, 114 polecats and 15 putative hybrid individuals were collected from different localities in Europe and genotyped with 13 microsatellite nuclear markers. Recently developed Bayesian methods for assigning individuals to populations and identifying admixture proportions were applied to the genetic data. To identify the direction of hybridization, we additionally sequenced mtDNA and Y chromosomes from 78 individuals and 29 males respectively. We found that both hybridization and genetic introgression occurred at low levels (3% and 0.9% respectively) and indicated that hybridization is asymmetric, as only pure polecat males mate with pure European mink females. Furthermore, backcrossing and genetic introgression was detected only from female first-generation (F1) hybrids of European mink to polecats. This latter result implies that Haldane's rule may apply. Our results suggest that hybridization and genetic introgression between the two species should be considered a rather uncommon event. However, the current low densities of European mink might be changing this trend.

DNA evidence for the hybridization of wild turtles in Taiwan: possible genetic pollution from trade animals

Field surveys in Taiwan have uncovered turtles presumed to be hybrids based on their intermediate morphology. We sequenced a mitochondrial (ND4) and nuclear (R35) gene of two putative hybrid individuals, along with representatives of the potential parental species (Mauremys mutica, M. reevesii, M. sinensis), to determine their genetic identity. Based on our data, both individuals are hybrids, with independent, recent origins resulting from the mating of a female M. reevesii and a male M. sinensis. Since we question whether the highly traded M. reevesii is endemic to Taiwan, this hybridization could represent human-mediated genetic pollution. We also discuss the implications of our findings on turtle conservation in Taiwan.

Hybridization between the threatened plant, Lespedeza leptostachya Englem. and its co-occurring congener Lespedeza capitata Michx.: morphological and molecular evidence

Natural hybridization is common in the genus Lespedeza. No hybrids between Lespedeza leptostachya Englem. and Lespedeza capitata Michx. are formally recognized in any of the current floras, however observations in the field suggest that hybridization might occur in many of their shared habitats. Putative hybrids were compared to L. leptostachya and L. capitata using morphological measurements and screened for the presence of species-specific trnL-F gene region (cpDNA) and the ITS gene region (nrDNA). A discriminate analysis of 10 morphological measurements identified the hybrids as intermediate to both parents with two PCA axes explaining 99% of the variation between taxa. The presence of hybrids was confirmed by genetic markers with individuals morphologically identified as hybrids having cpDNA trnL-F genotypes identical to L. leptostachya and the ITS (nrDNA) phenotypes in most cases contain the ITS genotype of both parents, however, some putative hybrid individuals contained the ITS genotype of only one parents. Those individuals with L. leptostachya ITS and trnL-F could be a case of misclassification, but the presence of both L. capitata ITS genotypes and L. leptostachyatrnL-F genotypes suggest segregation has occurred, which may result from either selfing or backcrossing.

Edraianthus × lakusicii (Campanulaceae) a new intersectional natural hybrid: morphological and molecular evidence

The genus Edraianthus A.DC. has a center of distribution in the Balkans and it is one of the most prominent groups of endemic plants in this region. During our recent fieldtrip to Mt. Lovcen (Montenegro), putative hybrid individuals were encountered, morphologically intermediate between two sympatric taxa, E. tenuifolius (E. sect. Edraianthus) and E. wettsteinii subsp. lovcenicus (E. sect. Uniflori). Multivariate morphometric and molecular analyses were carried out to investigate the occurrence of hybridization between these two species. As a result, a new nothospecies is described here, Edraianthus 9 lakusicii V. Stevanovic ´ & D. Lakusic ´, a natural hybrid between E. tenuifolius and E. wettsteinii subsp. lovcenicus. At present, this hybrid is known only from the single locality of Mt. Lovcen. Its population size is estimated to be \50 mature individuals and the estimated ''area of occupancy'' is smaller than 1 km 2 .