Identification of hybrids in broad-leaved Potamogeton species (Potamogetonaceae) in China using nuclear and chloroplast DNA sequence data

Hybridization is relatively frequent in the pondweed genus Potamogeton. A total of five putative hybrids of broad-leaved Potamogeton in China were collected in our recent investigations. We used internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast rbcL gene sequences to confirm the origins of the putative hybrids. Using ITS sequence additivity, we confirmed that the five putative hybrids were P. × anguillanus Koidzumi (P. wrightii × P. perfoliatus), P. × malainoides Miki (P. distinctus × P. wrightii), P. distinctus × P. nodosus, P. nodosus × P. wrightii, and P. distinctus × P. gramineus. The latter four hybrids are new records for China, and P. distinctus × P. gramineus is a new hybrid combination in Potamogeton. We found a new genotype of P. perfoliatus in northeast China. Hybrids between the new and a common genotype of P. perfoliatus were found in Central China. The maternal parents of the six hybrids were confirmed by chloroplast rbcL gene sequence data. The hybrids P. × anguillanus and P. distinctus × P. gramineus are reciprocal hybrids. P. × anguillanus has multiple origins from different populations. P. distinctus × P. gramineus has multiple origins within a single population.     

Nuclear and chloroplast DNA sequences data support the origin of Potamogeton intortusifolius J.B. He in China as a hybrid between P. perfoliatus Linn. and P. wrightii Morong

Internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast rbcL gene sequence data were used to test the hypothesis that natural populations of Potamogeton intortusifolius J.B. He in China originated from hybridization between P. perfoliatus Linn. and P. wrightii Morong. Based on ITS sequences data, P. intortusifolius possessed heterozygous rDNA genotypes which confirmed the hybrid origin of P. intortusifolius. Chloroplast rbcL gene sequences of P. intortusifolius from Yichang population revealed the same chloroplast haplotype as P. perfoliatus and the samples of P. intortusifolius from Weinan population had the same chloroplast haplotype as P. wrightii, which indicated that both putative parental species had been the maternal parent and that the two populations of P. intortusifolius had independent origins. This study confirms P. intortusifolius as a reciprocal hybrid. Because P. × intortusifolius in China has the same hybrid origin as P. × anguillanus Koidz. in Japan, it is suggested that P. × intortusifolius should be a synonym of P. × anguillanus.     

Maternal effects and ecological divergence in aquatic plants: a case study in natural reciprocal hybrids between Potamogeton perfoliatus and P. wrightii

When parental taxa are adapted to different habitats, hybrid genotypes are often highly heterogeneous, such that habitat or ecological factors influence hybrid fate and ecological performance. Trait expression in hybrids is not always intermediate between the parents, but may instead be either parental‐like or extreme (transgressive) depending on genetic control of the phenotypes. Maternal effects arising from interspecific interaction between cytoplasmic and nuclear genomes are widely recognized as playing a role in character expression of natural hybrids. Such interaction often leads to hybrid sterility or inviability. When hybrids are viable, however, cytonuclear interaction may contribute to hybrid persistence through its influence on trait expression. To date, maternal influence on hybrid performance has been examined primarily in experimentally produced hybrids, or in natural hybrids without identification of the cross direction owing to difficulty in obtaining species‐specific molecular markers. In aquatic plants, many hybrids persist by extensive clonal growth and are important components of aquatic communities. Many such hybrids are known in Potamogeton (pondweeds), the largest aquatic genus. Because Potamogeton species are ecologically highly diverse and maternal lineages are readily distinguished using molecular markers, natural hybrids of Potamogeton are well‐suited for studies of maternal effects, especially those affecting vegetative performance. As a case study, we have focused on maternal effects on drought tolerance and depth distribution in the natural hybrid P. × anguillanus derived from the closely related species P. perfoliatus and P. wrightii.     

A dynamic evolution of chromosome in subgenus Potamogeton revealed by physical mapping of rDNA loci detection

In this study, we analyzed 12 species in the subgenus Potamogeton (8 tetraploids and 2 diploids, 2 putative tetraploid hybrids) at the chromosomal level, including counting the chromosome number and physically mapping the rDNA. The extent of variation in the chromosome number and rDNA loci was determined in the Potamogeton species on both the inter- and intra-specific level. Moreover, one of the hybrid sets (P. perfoliatus ♂?×?P. wrightii ♀) was picked for performing artificial pollination for producing F1 generation adults and for sequential morphological character analysis and FISH detection. After comparing the parental species and natural hybrids from three different geographic locations, the intraspecific variations of rDNA loci were revealed in sampled plants; in addition, comparisons between artificial F1 and natural hybrids (P.?×?intortusifolius) showed a rapid change of 45S rDNA loci in response to the interspecific hybridization. We also compared our results concerning rDNA patterns with phylogenetic documents to derive complementary clues to karoytype evolution and interspecific relationships. Based on frequent hybridizations and active clonal reproduction with weak genetic selection, the rDNA chromosomal repatterning, such as the gain or loss of rDNA loci together with rDNA movements, might be one trend of chromosome evolution in this genus.     

Phylogenetic relationships and hybrid origin of Potamogeton species (Potamogetonaceae) distributed in China: insights from the nuclear ribosomal internal transcribed spacer sequence (ITS)

Hybridization and polyploidization are important evolutionary processes in higher plants and have greatly enriched the diversity of the genus Potamogeton (Potamogetonaceae). To study the phylogenetic relationships and hybrid origin of Potamogeton species, 35 accessions representing 20 species, including diploids, tetraploids and hexaploids, and three hybrids were collected in China and their ribosomal internal transcribed spacers (ITS) were cloned, sequenced and statistically analyzed. The data showed that ITS sequences were informative to analyze the phylogeny of Potamogeton, and the phylogenetic tree revealed that Potamogeton species examined could be mainly divided into two groups (Group I and II), corresponding to subgenus Potamogeton and subgenus Coleogeton, respectively. Then, the evolutionary mechanism on the polyploidy of Potamogeton species was discussed. P. natans probably was an allotetraploid and one of its parent might result from aneuploidy change of species with 2n=28. P. hubeiensis might be derived from the hybridization between P. octandrus and P. cristatus. We suggested that both P. lucens and P. maackianus probably were allotetraploids, and P. obtusifolius might be a diploid hybrid between P. compressus and P. pusillus. Moreover, P. malainoides might have undergone biased concerted evolution toward one of its parent P. wrightii, and P. intortusifolius might be a synonymy of P. × anguillanus.     

Potamogeton×exilis (P. alpinus×P. natans), a new hybrid pondweed from Finland

A Potamogeton hybrid recently discovered in Finland was proven to be a new taxon, which has a different parentage than the most similar P.×vepsicus (=P. natans×praelongus) described from Russia. Based on molecular and morphological investigation, the new hybrid was identified as P. alpinus×natans and is here described as P.×exilis nothosp. nov. The hybrid is known only from several nearby localities in a single river system. All discovered colonies almost certainly originate from a single hybridization event that occurred in the upper part of the river system. Other localities downstream have been established through fragmentation and dispersal of stems and rhizomes of this sterile hybrid. A detailed morphological comparison of the new hybrid with similar hybrids and species is provided.     

First record of Potamogeton×salicifolius for Italy,with isozyme evidence for plants collected in Italy and Sweden

Abstract

Potamogeton×salicifolius, a hybrid between P. lucens and P. perfoliatus, is identified as a new taxon for Italy. This is the first record of this hybrid from southern Europe. The Italian sample was studied in cultivation and compared with a living specimen of P.×salicifolius from Sweden, where the hybrid is rather widespread. In addition to morphological features, the most compelling evidence for the hybrid origin of these plants came from the isozyme analysis. The additive “hybrid” banding patterns of the five enzyme systems studied indicate inheritance from P. lucens and P. perfoliatus. The distribution of this hybrid in Europe coincides with the areas most severely affected by the Late Pleistocene glaciation. The relationships between environmental conditions, history of the habitat and rise of hybrids are discussed.     

Barriers to interspecific hybridization between Juglans nigra L. and J. regia L species

Juglans nigra and Juglans regia are phylogenetically divergent species. Despite the economic interest in Juglans?×?intermedia (J. nigra?×? J. regia), walnut hybridization is rare under natural conditions and still difficult using controlled pollination. Here, we evaluated some reproductive mechanisms that may prevent successful natural hybridization. The study of flowering phenology of 11 J. nigra and 50 J. regia trees growing in a plantation provided information regarding the opportunity for interspecific crosses. Variation in flower size, pollen quality of putative donors, and variation in seed yield and rate of hybrid production among putative maternal trees were examined. DNA fingerprinting and parentage analyses based on nine microsatellites permitted the identification of hybrids and hybridogenic parent. Our data indicated that overlap occurred between the staminate flowering of protogynous J. regia and the beginning of pistillate flowering of protogynous J. nigra. Differences in floral size were computed between walnut species. Only three hybrids among 422 offspring of eleven J. nigra progenies were identified. Interspecific hybridization involving pollination of one early-flowering-protogynous J. nigra by three protogynous J. regia trees was detected. The correct development of J. regia male gametophytes, high pollen viability (86.5 %), and germination (57.6 %) ruled out the possibility that low pollen quality contributed to depressed hybrid production. Our findings indicated that these two species tended to remain reproductively isolated. The substantial disjunction in flowering time and additional prezygotic barriers such as differences in floral size and conspecific pollen advance may affect interspecific gene flow between J. regia and J. nigra.     

Reduced clonal reproduction indicates low potential for establishment of hybrids between wild and cultivated strawberries (Fragaria vesca × F. × ananassa)

The genus Fragaria (Rosaceae) contains 24 species, including hybrid species such as the garden strawberry (Fragaria × ananassa Duch.). Natural hybridization between Fragaria species has repeatedly been reported, and studies on the hybridization potential between F. × ananassa and its wild relatives have become increasingly important with the outlook for genetically modified garden strawberries. In Europe, a candidate species for hybridization with garden strawberries is the common woodland strawberry (Fragaria vesca L.). Although a previous field survey indicated that the potential for hybridization between F. vesca and F. × ananassa is low, it is not clear whether the lack of natural hybrids is caused by known pre- and postzygotic barriers, or whether hybrid plants lack the fitness to establish in natural F. vesca populations. We grew different F. vesca and F. vesca × F. × ananassa hybrid clones with and without competition in a greenhouse and assessed biomass production, clonal reproduction, and sexual reproduction of plants. While some hybrid clones exceeded F. vesca in biomass production, general clonal reproduction was much lower and delayed in hybrids. Furthermore, hybrids were sterile. These results demonstrate a mechanism by which the general lack of F. vesca × F. × ananassa hybrids in natural habitats can be explained, in addition to the known low hybridization potential between garden and woodland strawberries. We conclude that hybrids have a competitive disadvantage against co-occurring F. vesca plants due to inferior and delayed clonal reproduction, and that the potential for hybrid establishment under natural conditions is low.     

Evidence for the hybrid origin of<Emphasis Type="Italic">potamogeton ×cooperi</Emphasis> (<Emphasis Type="Italic">Potamogetonaceae</Emphasis>): Traditional morphology-based taxonomy and molecular techniques in concert

The identity of plants morphologically intermediate betweenPotamogeton crispus andP. perfoliatus from two recently discovered sites, one in Moravia, Czech Republic and another in Wales, United Kingdom, was investigated with molecular markers. Evidence from restriction fragment length polymorphism analysis of the nuclear internal transcribed spacer region of ribosomal DNA and of thetrnK-trnQ chloroplast DNA intergenic spacer confirmed the morphology-based determination of two putative hybrid samples asP. ×cooperi. The hybrids showed the ITS variants of both parental taxa, consistent with the expected biparental inheritance of nuclear DNA. The chloroplast DNA markers indicateP. crispus as the female parent in both hybridization events. The hybrid origin of another dubious sample was excluded by the molecular data, in accordance with previous detailed morphological examination. This plant represented an extreme, narrow-leaved form ofP. perfoliatus, imitatingP. ×cooperi in some characters. The results of the molecular analyses are discussed in relation to the morphology of the plants. They underline that somePotamogeton hybrids could indeed be identified by careful and detailed morphological examination and also that these identifications were reliable and confirmed by molecular markers. This study exemplifies that long-term taxonomic expertise usually generates very well-founded specific questions suitable for straightforward treatment by appropriate molecular methods. The process and ecological implications of hybrid formation are also discussed     

Using DNA‐based techniques to identify hybrids among linear‐leaved Potamogeton plants collected in China

Abstract It is well known that interspecific hybrids occur in the genus Potamogeton. The linear‐leaved Potamogeton species commonly have highly variable morphological characteristics. Their hybrids often show similar vegetative characters to their parental species and their identification based solely on morphology is not always conclusive. In order to clarify whether there are any hybrids from the linear‐leaved Potamogeton plants collected in China, we used internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast rbcL gene sequences to identify the hybrids. Using ITS sequence additivity, we identified four hybrids, namely P. orientalis (P. pusillus×P. oxyphyllus), P. pusillus×P. berchtoldii, P. foliosus×P. octandrus, and P. cristatus×P. octandrus. The latter three hybrids should be considered as new hybrids in Potamogeton. The maternal parents of the four hybrids were confirmed using chloroplast rbcL gene sequences.     

Interspecific hybridization in the genus Hieracium s. str.: evidence for bidirectional gene flow and spontaneous allopolyploidization

Although reticulation has indisputably played an important role in the evolutionary history of the genus Hieracium s. str. (Asteraceae), convincingly documented cases of recent interspecific hybridization are very rare. Here we report combined evidence on recent hybridization between two diploid species, Hieracium alpinum and H. transsilvanicum. The hybrid origin of the plants from the Romanian Eastern Carpathians was supported by additive patterns of nuclear ribosomal DNA polymorphism (ITS), an intermediate position of hybrid plants in principal coordinate analysis based on amplified fragment length polymorphism phenotypes (AFLP), and additivity at one allozyme locus. Flow cytometric analyses and chromosome counting showed that two hybrids were diploid (2n ~ 2x ~ 18) while one was surprisingly tetraploid (2n = 4x = 36). To our knowledge, this is the first record of spontaneous polyploidization following interspecific crossing in the genus. Allozyme data, especially the presence of unbalanced heterozygosity at one locus, suggest the origin of this tetraploid via a triploid bridge with subsequent backcrossing to H. alpinum. According to PCR-RFLP analyses of the trnT-trnL intergenic spacer, all H. ×krasani hybrids examined had the H. alpinum haplotype while H. transsilvanicum served as a pollen donor. The hybrids occurred at the locality with abundant H. alpinum plants where paternal H. transsilvanicum was missing. Previously reported instances of interspecific hybridization between the same parental taxa showed an opposite direction of crossing and relative abundance of parental taxa. This suggests that the direction of hybridization might be influenced by the frequency of parental taxa at the locality.     

Clonal growth architecture and spatial dynamics of 10 species of the genus <Emphasis Type="Italic">Potamogeton</Emphasis> across different habitats in Kashmir Valley,India

The plasticity in clonal architecture may enable plants to effectively respond to environmental constraints and to enhance species ecological niche breadth but its role in plant tolerance to water flow is poorly documented. The present study was carried out to determine whether the clonal architecture varies with respect to water flow in 10 species of the genus Potamogeton colonizing habitats differing by flow conditions. For these 10 species, the traits describing clonal architecture were measured on individuals sampled in natural sites and plasticity in clonal architecture was examined in a common garden growth experiment. The clonal growth architecture did not vary significantly in the species which inhabit either standing (P. lucens, P. natans, and P. pusillus) or running water (P. amblyphyllus and P. berchtoldii). However, the species inhabiting both standing as well as running waters (P. crispus, P. nodosus, P. pectinatus, P. perfoliatus, and P. wrightii) showed considerable and significant variation in clonal growth architecture across these habitats. Transplantation experiment revealed that clonal architecture observed between the plants under different conditions is plastic and not due to genetic differentiation. The present study demonstrated that plasticity in the clonal architecture may enable these species to inhabit stressful conditions of flowing water.     

Two new spontaneous hybrids of American hard pines from Pinus sect. Trifoliae (Pinaceae) found in the unique Russian Sochi Arboretum

In the 1990s, an extraordinarily abundant live collection of the genus Pinus, including 49 species, 4 subspecies, and 7 interspecific hybrids, found in the Sochi Arboretum in Russia on the northeast coast of the Black Sea, was taxonomically revised. Among these taxa, two previously unknown interspecific hybrids of American hard pine species from P. sect. Trifoliae had arisen spontaneously in culture. These are described here as the new hybrid species Pinus × transamericana (P. muricata × P. rigida) and Pinus × critchfieldii (P. patula × P. taeda). All known natural or artificial interspecific hybrids within P. sect. Trifoliae are listed, and hybridization between American hard pine species is discussed. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Chloroplast evidence for the multiple origins of the hybrid Potamogeton × fluitans

Previous studies of Potamogeton × fluitans Roth. have supported the putative origin of this taxon as a result of hybridization between Potamogeton lucens L. and Potamogeton natans L. In the British Isles P. × fluitans is restricted to only a few sites, while both its parents are relatively common. Previous studies have shown that most of these hybrid populations are monomorphic, with the exception of the Moors River, in Hampshire and Dorset, where multiple genotypes have been identified. This is despite the fact that here the hybrid grows in the absence of one its parents, P. lucens. In this study, species-specific differences in the TrnL (UAA) chloroplast region, a maternally inherited marker, were used to identify the original seed-bearing parent of the hybrids within this river system. It was shown that both species have been maternal parents in the creation of this hybrid population.     

POLLEN TRANSFER BY NATURAL HYBRIDS AND PARENTAL SPECIES IN AN IPOMOPSIS HYBRID ZONE

Models of hybrid zones differ in their assumptions about the relative fitnesses of hybrids and the parental species. These fitness relationships determine the form of selection across the hybrid zone and, along with gene flow, the evolutionary dynamics and eventual outcome of natural hybridization. We measured a component of fitness, export and receipt of pollen in single pollinator visits, for hybrids between the herbaceous plants Ipomopsis aggregata and I. tenuituba and for both parental species. In aviary experiments with captive hummingbirds, hybrid flowers outperformed flowers of both parental species by receiving more pollen on the stigma. Although hummingbirds were more effective at removing pollen from anthers of I. aggregata, hybrid flowers matched both parental species in the amount of pollen exported to stigmas of other flowers. These patterns of pollen transfer led to phenotypic stabilizing selection, during that stage of the life cycle, for a stigma position intermediate between that of the two species and to directional selection for exserted anthers. Pollen transfer between the species was high, with flowers of I. aggregata exporting pollen equally successfully to conspecific and I. tenuituba flowers. Although this study showed that natural hybrids enjoy the highest quality of pollinator visits, a previous study found that I. aggregata receives the highest quantity of pollinator visits. Thus, the relative fitness of hybrids changes over the life cycle. By combining the results of both studies, pollinator-mediated selection in this hybrid zone is predicted to be strong and directional, with hybrid fitness intermediate between that of the parental species.     

Genomic relationships between hexaploid Helianthus resinosus and diploid Helianthus annuus (Asteraceae)

Genus Helianthus comprises diploid and polyploid species. An autoallopolyploid origin has been proposed for hexaploid species but the genomic relationships remain unclear. Mitotic and meiotic studies in annual Helianthus annuus (2n = 2x = 34) and perennial Helianthus resinosus (2n = 6x = 102) as well as the F₁ hybrids between both species were carried out. Chromosome counting confirmed the hybrid origin of the latter plants and their tetraploid condition. Bivalents in hybrids ranged from 12 to 28 ( $ \bar{x} $  = 20.8). Univalents, trivalents and quadrivalents were also observed. Meiotic products comprised dyads, triads and normal tetrads and pollen grains were heterogeneous in size. These observations suggest the occurrence of 2n pollen in addition to the expected n. Genomic in situ hybridization (GISH) of total H. annuus DNA on H. resinosus chromosomes rendered weak but uniform signals; similar hybridization pattern was observed using three other annual species. Hybridization with H. annuus probe performed on root tip cells of F₁ H. annuus × H. resinosus hybrids revealed 17 chromosomes with a strong hybridization signal. GISH in hybrid meiocytes distinguished chromosomes from parental species and revealed autosyndetic pairing of H. resinosus chromosomes, allosyndetic pairing in bivalents, trivalents and quadrivalents, and the presence of univalents derived from parents, H. annuus and H. resinosus. Results obtained from classical and molecular cytogenetics do not support H. annuus as a direct ancestor of H. resinosus. The occurrence of allosyndetic pairing and the relatively high fertility of the F₁ hybrids point to the possibility that useful genes could be transferred from H. resinosus to cultivate sunflower, although the effective rate of recombination has not been evaluated. GISH method proved effective to recognize parental chromosomes in H. annuus × H. resinosus progeny.     

Panicum shastense (gramineae), a sterile hybrid between P. Pacificum and P. Scribnerianum

Natural and synthetic hybrids are reported betweenPanicum, scribnerianum Nash and two other species,P. pacificum Hitchc. & Chase andP. occidentale Scribn. & Merr. The hybrids are morphologically intermediate between the parents, and those in whichP. pacificum is one parent strongly resemble the rareP. shastense Scribn. & Merr. All the parent plants studied cytologically had regular meiosis and a gametic number ofn = 9 chromosomes. The hybrids usually showed good pairing of chromosomes in meiosis, but irregularities such as univalents and lagging chromosomes were common. In the hybrids generally less than 7% of the pollen grains stained with cotton blue in lactophenol, and the plants set no seed, whereas the parent plants generally had over 90% pollen stainability and a high percentage of seed set. Pollen fromP. shastense, like that of the hybrids, was 3–4% stainable.Panicum shastense is believed to be based on a first generation sterile hybrid and the nameP. xshastense is proposed.     

Molecular characterization of two natural hybrids from the Iberian Peninsula: <Emphasis Type="Italic">Ranunculus × luizetii</Emphasis> and <Emphasis Type="Italic">R. × peredae</Emphasis> (Ranunculaceae)

Two nothospecies, Ranunculus × luizetii and R. × peredae (Ranunculaceae), were analyzed and discussed. For this purpose, Amplified Fragment Length Polymorphism (AFLP) markers, nuclear rDNA sequences (ITS1, 5.8S and ITS2) and pollen viability were conducted. The profiles of these hybrid samples were compared to their putative progenitors. Several additive polymorphic sites detected in the ITS sequences of the hybrid samples (R. × luizetii and R. × peredae) also confirmed their derived origins from ribotypes of their parental taxa (R. parnassiifolius subsp. parnassiifolius × R. pyrenaeus; R. amplexicaulis × R. cabrerensis subsp. cabrerensis, respectively). Despite the lack of exclusive AFLP markers reported in both hybrids, presumably due to effects of introgression, the concerted evolution of many rDNA polymorphisms towards either of the parental ribotypes indicated their ancient origin. Pollen fertility estimation in R. × luizetii presented a mean value of 60.58%, which showed that hybrid samples are well established and fertile. However, a larger difference was observed in R. × peredae, where the mean value of pollen fertility was very low (18.91%).