A molecular phylogeny of the genus Gagea (Liliaceae) in Germany inferred from non-coding chloroplast and nuclear DNA sequences

The systematics of the mainly yellow flowered Gagea species complex (Liliaceae) has long been considered difficult because only a few phenotypic features within this genus and as a result of hypothesized interspecific hybridisation. A molecular phylogenetic study of seven Gagea species (G. bohemica, G. lutea, G. minima, G. pomeranica, G. pratenis, G. spathacea and G. villosa) from Germany has been undertaken, based on plastid DNA sequences (trnL(UAA)-trnF(GAA), psbA-trnH) and on the nuclear ribosomal internal transcribed spacer (ITS). Sequence divergence within the Gagea species ranges up to 15.5% for psbA-trnH, 22.0% for trnL-trnF and 23.7% for ITS (ITS1 + 5.8S rRNA + ITS2). Two subspecies of Gagea bohemica: G. bohemica subsp. saxatilis and G. bohemica subsp. bohemica are characterized by trnL-trnF data and morphological features. Analysis of the ITS region shows that G. pomeranica represents a hybrid of G. pratensis and G. lutea. Lloydia serotina was initially used as an outgroup species, but it was placed within the investigated Gagea species in the psbA-trnH and the trnL-trnF phylogenetic tree.     

Phylogenetic relationships of Gagea Salisb. (Liliaceae) in Italy, inferred from molecular and morphological data matrices

All known Italian Gagea species (23) representing seven out of 14 sections were included in this study. Morphological, molecular and combined data support the basal position of G. trinervia (Anthericoides). According to the molecular data, the sections Anthericoides, Lloydia, Gagea, Minimae and Spathaceae are very well supported; representatives of the sections Didymobulbos and Fistulosae clustered together in the same clade. In this clade, several near-related species groups (series) were recognised. According to 35 morpho-anatomical, ontogenetic and karyological data the section Gagea forms a well-defined monophyletic clade. Within this clade G. tisoniana, G. pusilla and G. cfr. paczoskii are near related. The position of the critical putative hybrid taxa G. luberonensis and G. polidorii is discussed. New series Solenarium (Dulac) Peruzzi and Tison, Occidentales (A. Terracc.) Peruzzi and Tison and Saxatiles (A. Terracc.) Peruzzi and Tison are proposed.     

Hybridity as a main evolutionary force in the genus Gagea Salisb. (Liliaceae)

Abstract

The important role of hybridity in the evolution of the genus Gagea is becoming clear, through morphological, karyological and molecular evidences. Several species were recently inferred or hypothesized to be of hybrid origin; especially in sect. Didymobulbos, sect. Fistulosae and sect. Gagea. Representative case studies, such as G. luberonensis J.-M. Tison, G. polidorii J.-M. Tison and G. pomeranica Ruthe, are presented and discussed.

Since many Gagea species show very reduced or null sexual reproduction vs. a massive vegetative propagation, there are several problems in distinguishing hybrids from hybridogenous species in this genus. The actual trend is to consider them at specific level because of their ability to stabilize, at least through vegetative propagation by bulblets and/or bulbils.     

Reproductive isolation vs. interbreeding between Gagea lutea (L.) Ker Gawl. and G. pratensis (Pers.) Dumort. (Liliaceae) and their putative hybrids in Mecklenburg‐Western Pomerania (Germany)

Mecklenburg‐Western Pomerania is the main range of two hybridogeneous Gagea taxa, G. megapolitana Henker and G. pomeranica Ruthe, which have the same two parental species, G. lutea (L.) Ker. Gawl. and G. pratensis (Pers.) Dumort. We assessed the degree of reproductive isolation vs. interbreeding between these taxa using data from field observations, crossing experiments and pollen characteristics for nine hybrid, four G. lutea and five G. pratensis populations. Pollen viability was highest in 6x G. lutea and lowest in G. pratensis (most probably 5x), with intermediate figures for the studied hybrids (5x–7x). Despite the assumed anorthoploid states, sexual reproduction (though sometimes very rare) was recorded for all populations in the field and/or in experiments. The crossing experiments revealed that all taxa are also able to hybridize. However, there were differences in the directions (i.e. role as maternal vs. paternal parent) as well as the success of the crossings: the primary hybridization G. lutea x G. pratensis resulted in more seeds than the reverse combination (17.5 vs. 3.3%), but the seed set was highest in backcrosses of the hybrids with G. lutea pollen (41.2%). These differences can be explained by overlap of flowering times, and reduced fertilities due to ploidy levels. The study showed that the taxa of the G. lutea‐pratensis hybrid complex are not yet reproductively isolated but can interbreed and will generate hybrids of higher ranks, forming a hybrid swarm, most probably leading to introgression via backcrosses with G. lutea.     

Equisetum (Equisetaceae) species or hybrids? ISSR fingerprinting profiles help improve diagnoses based on morphology and anatomy

The applicability of “inter-simple sequence repeats” (ISSR)-PCR as a molecular character complex for differentiation of Equisetum taxa is described with a special focus on the detection of hybrids. All Central European Equisetum species and the four most frequent hybrids are considered: E. arvense, E. fluviatile, E. telmateia, E. palustre, E. sylvaticum, E. pratense, E. × litorale, E. ramosissimum, E. hyemale, E. variegatum, E. × moorei, E. × trachyodon and E. × meridionale. The ISSR banding patterns are highly typical for each species. The positions of the clusters of hybrid taxa prove their hybrid origin and enable identification of the parental species. Genetic similarities of populations suggest that in some species vegetative reproduction predominates (e.g. E. arvense), whereas for others (e.g. E. telmateia) sexual reproduction seems to be more frequent. In addition to the molecular analyses, the characteristics of stem cross sections of the four hybrids and their parental species are shown and discussed.     

Molecular evidence for hybridization between invasive <Emphasis Type="Italic">Solidago canadensis</Emphasis> and native <Emphasis Type="Italic">S</Emphasis>. <Emphasis Type="Italic">virgaurea</Emphasis>

Hybridization between alien and native species is biologically very important and could lead to genetic erosion of native taxa. Solidago × niederederi was discovered over a century ago in Austria and described by Khek as a natural hybrid between the alien (nowadays regarded also as invasive) S. canadensis and native S. virgaurea. Although interspecific hybridization in the genus Solidago is considered to be relatively common, hybrid nature of S. × niederederi has not been independently proven using molecular tools, to date. Because proper identification of the parentage for the hybrid Solidago individuals solely based on morphological features can be misleading, in this paper we report an additive polymorphism pattern expressed in the ITS sequences obtained from individuals representing S. × niederederi, and confirm the previous hypothesis that the parental species of this hybrid are S. canadensis and S. virgaurea. Additionally, based on variability at the cpDNA rpl32-trnL locus, we showed that in natural populations hybridization occurs in both directions.     

Morphological and molecular investigations of <Emphasis Type="Italic">Gagea</Emphasis> (Liliaceae) in southeastern Kazakhstan with special reference to putative altitudinal hybrid zones

The most important center of speciation in the genus Gagea is thought to be in Central Asia. Here, we focus on species diversity in southeastern Kazakhstan (around Almaty, Ili-Alatau range of the Western Tian-Shan mountains). We studied an elevational transect, reaching from lowland steppes to the alpine zone (500–2750 m a. s. l.), and carried out detailed morphological and molecular investigations for populations of Gagea spp. Nine species were identified in different altitudinal zones; one of these (Gagea almaatensis) is described as new to science. We could detect two altitudinal contact zones between closely related species: G. filiformis and G. granulosa (sect. Minimae), and G. almaatensis and G. kuraminica (sect. Gagea). Morphological and molecular investigations (ITS data and cpDNA networks) indicate ongoing hybridization of co-occurring G. filiformis into G. granulosa and putative bidirectional hybridization events between G. almaatensis and G. kuraminica.     

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%).     

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.     

A pre‐Miocene Irano‐Turanian cradle: Origin and diversification of the species‐rich monocot genus Gagea (Liliaceae)

The Irano‐Turanian (IT) floristic region is considered an important center of origin for many taxa. However, there is a lack of studies dealing with typical IT genera that also occur in neighboring areas. The species‐rich monocot genus Gagea Salisb. shows a center of diversity in IT region and a distribution in adjacent regions, therefore representing a good study object to investigate spatial and temporal relationships among IT region and its neighboring areas (East Asia, Euro‐Siberia, Himalaya, and Mediterranean). We aimed at (a) testing the origin of the genus and of its major lineages in the IT region, (b) reconstructing divergence times, and (c) reconstructing colonization events. To address these problems, sequences of the ribosomal DNA internal transcribed spacer (ITS) region of 418 individuals and chloroplast intergenic spacers sequences (psbA‐trnH, trnL‐trnF) of 497 individuals, representing 116 species from all sections of the genus and nearly its entire distribution area were analyzed. Divergence times were estimated under a random molecular clock based on nrITS phylogeny, which was the most complete data set regarding the representation of species and distribution areas. Ancestral distribution ranges were estimated for the nrITS data set as well as for a combined data set, revealing that Gagea most likely originated in southwestern Asia. This genus first diversified there starting in the Early Miocene. In the Middle Miocene, Gagea migrated to the Mediterranean and to East Asia, while migration into Euro‐Siberia took place in the Late Miocene. During the Pleistocene, the Arctic was colonized and Gagea serotina, the most widespread species, reached North America. The Mediterranean basin was colonized multiple times from southwestern Asia or Euro‐Siberia. Most of the currently existing species originated during the last 3 Ma.     

Molecular and phytochemical evidence for the taxonomic integrity of Salix alba, S. fragilis, and their hybrid S. × rubens (Salicaceae) in mixed stands in SE Germany

AFLP fingerprinting, nrDNA sequencing, and HPLC of phenolic compounds from bark samples was used to assess the intensity of hybridisation in two mixed stands of Salix alba, S. fragilis, and their hybrid S. × rubens (Salicaceae) along the Danube in the vicinity of Regensburg (SE Germany). AFLP analyses of 130 individuals from two mixed and two pure strands of the parental species resulted in a matrix of 337 polymorphic loci that was analysed with Bayesian cluster algorithms implemented in the software programmes Structure and NewHybrids. The results, together with sequence polymorphisms of the nrDNA ITS1 + 5.8S region, indicate that the three taxa are distinctly separated and that only a few F2-hybrids and no backcrosses to the parental species are present in the surveyed mixed stands. These findings were corroborated by HPLC studies of the phenolic compounds from bark samples that were carried out for the same individuals. A multivariate statistical analysis of relative peak areas of chromatograms (PCA, principal component analysis) indicates the clear separation of parental and the intermediate position of S. × rubens individuals.     

Comparison of the internal transcribed spacer region (ITS) of the ribosomal RNA genes in wild and cultivated two and six-rowed barleys (Hordeum vulgare L.)

The ITS region of the ribosomal RNA genes from two and six-rowed cultivated barley (Hordeum vulgare subsp. distichon and H. v. subsp. hexastichon, respectively), and its two and six-rowed wild relatives (H. v. subsp. spontaneum and H. v. subsp. agriocrithon, respectively) was isolated and sequenced. The entire ITS region is 598 bp in the two-rowed taxa (H. v. subsp. distichon and H. v. subsp. spontaneum) and 599 bp in the six-rowed ones (agriochriton and hexastichon). The ITS1 is 217 bp in the six-rowed barleys (H. v. subsp. agriochriton and H. v. subsp. hexastichon) and 218 bp in the two-rowed barleys (H. v. subsp. distichon and H. v. subsp. spontaneum). The 5.8S region is 163 bp in all studied H. vulgare taxa. The ITS2 region is 217 bp in the two-rowed barleys (H. v. subsp. distichon and H. v. subsp. spontaneum) and 219 bp in the six rowed ones (H. v. subsp. hexastichon and H. v. subsp. agriochriton). The ITS sequence data of the studied taxa and that of three other wild Hordeum species (H. murinum, H. marinum and H. chilense) were aligned and a phylogeny tree was reconstructed using the Lasergene Program. H. v. subsp. spontaneum was appeared as the ancestor of all other H. vulgare taxa.     

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.     

Genotypical and multiple phenotypical traits discriminate Salix × rubens Schrank clearly from its parent species

Most studies on Salix hybrids concerning the diversity in a hybrid complex included typical morphological characteristics of leaves, buds, twigs and flowers for comparison with genotypic traits. Our analyses are based on a set of phenotypical traits of 19 clones of the S. alba/S. fragilis-aggregate which includes characteristics as phenology, growth, and composition of secondary leaf compounds, for the first time additional to morphological traits. Three clearly distinct groups (S. alba L., S. fragilis L., and S. × rubens Schrank) could be identified based on phenotypical traits and ITS1 and 5.8S nrDNA sequences. S. × rubens revealed additivities at variable ITS1 and 5.8S positions as well as intermediate morphology and secondary compound pattern, but was characterised by significant earlier foliation start. This property leads to a better utilization of the vegetation period and may explain the detected better growth compared to the parent species and therefore also may have contributed to the widespread occurrence of S. × rubens in Central Europe.     

Polymorphic Sites in ITS1-5.8S rDNA-ITS2 Region in Hybridogenic Genus × <Emphasis Type="Italic">Elyhordeum</Emphasis> and Putative Interspecific Hybrids <Emphasis Type="Italic">Elymus</Emphasis> (Poaceae: Triticeae)

The genus Elymus L. is a complicated aggregate of ecological and geographical races, species, subspecies, varieties, and hybrids. We suggest that comparative analysis of intragenomic polymorphism of internal transcribed spacers ITS1 and ITS2 of 35S rRNA genes in the supposed hybrids and their possible “parents” can be one of the approaches to verification of hybrid origin of the samples collected in nature to confirm or reject the hypotheses about their possible “parents.” Polymorphic sites (PS) in ITS of 23 Elymus species, as well as in two supposed interspecific Elymus hybrids and in a supposed intergeneric hybrid between Elymus × Hordeum determined as × Elyhordeum sp., were analyzed in the work. We collected all hybrids in the Altai. There were 2 and 5 PS in two samples of E. dahuricus and 1 and 4 PS in two studied samples of E. schrenkianus in the ITS1-5.8S rDNA-ITS2 region. From 0 to 4 (modes 0 and 3) PS were detected in 32 samples relating to 21 tetraploid Elymus species. More PS (14) were found in the × Elyhordeum sp. sample. A large number of single nucleotide substitutions were found in 5.8S rRNA in × Elyhordeum. It was shown that about half of them do not change the secondary structure of the 5.8S rRNA molecule, so these molecules probably retain the ability to work as a component of large subunit of a ribosome. On the other hand, the absence or weakening of 5.8S rDNA homogenization in × Elyhordeum indirectly suggests that a significant part of 5.8S rDNA is not transcribed. Paradoxically, ITS sequences of × Elyhordeum sp. are less polymorphic than 5.8S rDNA. There are no ITS sequences derived from Hordeum among × Elyhordeum ITS sequenced by Sanger method. No traces of the H subgenome and a subgenome originating from Agropyron (P-subgenome) are seen in the Alt 10–278 plant genome (a chimera, combining the morphological traits of Elymus, Elytrigia, and Agropyron). In this plant, as well as in the supposed intersectional hybrid Alt 11–60 distinguished by a mosaic of the traits typical for the E. caninus × E. mutabilis species, only 4 and 5 PS, respectively, are detected when sequencing by Sanger method. The comparison of ITS sequences of the supposed Elymus Alt 10–278 hybrid and its probable “parents” demonstrates that one of the species of the Elymus macrourus kinship circle, as well as the Elytrigia geniculata, could be one of its ancestors. The comparison of the ITS sequence of the supposed parental species with ITS of Alt 11–60 samples and five PS of the supposed Alt 11–60 hybrid does not contradict the hypothesis that this is an intersectional hybrid of the first generation that emerged with the involvement of E. caninus and E. mutabilis common in the Altai.     

Molecular evidence for the hybridity ofIlex × wandoensis and the phylogenetic study of KoreanIlex based on ITS sequence data

In this study, internal transcribed spacer (ITS) regions within the nuclear ribosomal DNA of KoreanIlex were analyzed in order to investigate any molecular evidence thatI. × wandoensis could serve as a putative hybrid betweenI. cornuta andI. integra. We also sought to clearly identify taxonomic relationships and problems caused by consecutive external morphological characters among taxa in the genus. We sequenced 20 clones fromI. × wandoensis and found these individuals displayed intra-genomic polymorphisms within ITS regions. The analysis of the clones clearly demonstrated the presence of discrete sequences from bothI. cornuta andI. integra, thereby confirmingI. × wandoensis is a species that was formed by crossingI. cornuta andI. integra. Lastly, the subgenusIlex, which contains the evergreen species, failed to form a monophyletic group in a strict consensus tree that was prepared based upon ITS regions.I. crenata var.microphylla in the subgenus KoreaIlex, which has presented taxonomic problems previously, formed an independent clade within the consensus tree, thereby showing distinction fromI. crenata.) Genetic discontinuity ofI. macropoda andI. Macropoda for.pseudo-macropoda individuals couldn't be confirmed.     

Morphological evaluation of the Pinus kesiya complex (Pinaceae)

The main aim of this study is to compare all taxa from the Pinus kesiya complex and related P. tabuliformis using statistical, ordination and discrimination techniques focusing on ten most discriminating morphological traits. Special emphasis was placed on the recently described taxa Pinus densata subsp. tibetica and P. × naxiorum (=P. yunnanensis × P. densata). Population comparisons of the particular main geographic distribution areas of Pinus kesiya, P. densata subsp. tibetica and P. yunnanensis were also conducted separately within each mentioned taxon using the same data. Pinus densata subsp. tibetica proved to be sufficiently morphologically differentiated from subsp. densata as well as from other species of the P. kesiya complex and P. tabuliformis. The recently originating hybrid P. × naxiorum appears to be intermediate between its parents. The unique shrubby taxon P. densata var. pygmaea is clearly closer to P. densata than to P. yunnanensis, to which it has been classified in Chinese floras. Populations of four distant Pinus kesiya geographic distribution areas lack substantial differences that would support the recognition of infraspecific taxa such as subsp. insularis or var. langbianensis. Pinus yunnanensis is very similar to P. kesiya, with only one trait, leaf length, being significantly different.